Antimicrobial activity study of new quinazolin-4(3h)-ones against Staphylococcus aureus and Streptococcus pneumoniae

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Abbreviations: PBP – penicillin-binding protein; MRSA – methicillin-resistant Staphylococcus aureus; PBP2a – penicillin-binding protein; ATP – adenosine triphosphate, MIC – minimum inhibitory concentration; DMSO – dimethyl sulfoxide; DMF – dimethylformamide; MIB – meat infusion broth; MIA – meat infusion agar; AC – atypical colonies; TC – typical colonies; NMR – nuclear magnetic resonance; TLC – thin layer chromatography; NA – nucleic acid; FnBPs – fibronectin-binding proteins

 

INTRODUCTION

Currently, multi-resistance of pathogenic bacteria to antimicrobial agents used in medical practice, is a serious public health problem [1-6]. As a rule, the formation of resistance occurs in the course of antibiotic therapy, especially in the departments with more intensive use of this group drugs. Clinical studies have established the dominance of antibiotic-resistant strains in the structure of nosocomial infections. Thus, there is a need to search for new antibacterial substances characterized by high efficacy, low toxicity and insensitive to the suppressing action of pathogens [7–9].

It has been proven that Staphylococcus aureus and Streptococcus pneumonia are the most common and express various virulence factors. They are pathogens of a wide range of diseases in humans and animals, have the greatest resistance to antibiotics among gram-positive microorganisms [2, 10–14].

The emergence of Staphylococcus aureus resistance to β-lactam antibiotics, as well as to other antimicrobial agents, limits its use in medicine due to the following factors: its mutation and selection, the acquisition of new genetic material from other resistant organisms during the processes of transformation, transduction and conjugation, implying a change in the adhesive properties of the cell surface. It is known that functioning of ATP-dependent efflux pumps, which are carrier proteins that push antimicrobial agents out of the cell, contributes to the resistance formation of Staphylococcus aureus and Streptococcus pneumoniae to fluoroquinolones and the drugs of the tetracycline group [15–18].

Quinazolin-4(3H)-one and its derivatives, which are condensed heterocyclic nitrogen-containing compounds, are known as a promising class of substances exhibiting antibacterial, antifungal, anti-tuberculosis, and antiviral kinds of activity [3]. Its dependence on the nature and number of quinazolinone nucleus substituents has been described. It was found out that the compounds of this group have a pharmacological effect against Staphylococcus aureus, Streptococcus pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli [3, 5, 19].

It has been proven that quinoline derivatives, which are the basis of the quinazolinones structure, inhibit the DNA synthesis, promoting the cleavage of bacterial DNA gyrase and type IV topoisomerase, resulting in the death of a bacterial cell [20–24]. The ability of compounds of the quinazolinone series, similar to β-lactam antibiotics used to prevent pathogenic processes in the body caused by Staphylococcus aureus and Streptococcus pneumoniae, to participate in the irreversible serine acylation of the active center of transpeptidase – penicillin-binding protein (PBP), catalyzing the formation of peptidaregine, an essential component of the bacterial cell wall, has been described. As a result of the formation of a stable lactam-acyl-enzyme complex, transpeptidase and carboxypeptidase kinds of the enzyme activity are inhibited, leading to the death of the pathogen.

A unique ability of quinazolinones, realized in synergy with piperacillin and tazobactam, to form bonds with the allosteric site of penicillin-binding protein 2a (PBP2a) of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci, which cannot be inhibited by β-lactams, has been established [15, 25–27]. The possibility of the joint use of quinazoline derivatives with chloramphenicol to increase its intracellular concentration in pathogenic strains applying efflux pumping systems to resist the action of antimicrobial drugs, has been proven [28, 29]. Probably, during their passage, quinazolinone, having a lower polarity, binds to an efflux pump to a greater extent, undergoes an outflow more easily and facilitates the penetration of an antibiotic into a microbial cell with a constant concentration [16–18, 20, 21, 30].

The uniqueness of the structure of quinazolin-4 (3H)-one new derivatives, the possibility of using it together with other antimicrobial agents in order to increase their pharmacological effect and prevent the resistance emergence to them, creates the need for a comprehensive study of their activity.

THE AIM of the research is to study the antimicrobial activity of quinazolin-4 (3H)-one derivatives against Staphylococcus aureus and Streptococcus pneumoniae in vitro as well as to assess the effect of their structural changes on the biological activity of the analyzed substances, the lipophilicity of their molecules to predict the ability of inducing their resistance by the mechanism of the active outflow.

MATERIALS AND METHODS

Research objects

The objects of the study were new derivatives of quinazolin-4 (3H)-one.

The chemical structure of new quinazolinone compounds can be described by the general formula shown in Fig. 1. The yield and physicochemical properties of the new substances are presented in Table 1.

 

Figure 1 – General formula of quinazolin-4 (3H)-one derivatives

 

Table 1 – Chemical structure of new quinzolin-4 (3H)-one derivatives

Сompound	R1	R2	R3	Yield, %	Mp., °С
VMA-10-10	H	H	4-dimethylaminophenyl	67	261–264
VMA-10-18	Н	Н	4-methoxyphenyl	61	228–229
VMA-10-21	Н	Н	4-phenylpiperazin-1-yl	73	222–224
VMA-13-05	Н	Н	β-naphthyl	56	199–201
VMA-17-01	H	H	phenylamino	83	156–158
VMA-17-04	Н	СН3	phenylamino	72	222–224
VMA-13-17	Br	H	NHC(NH)NH2	89	242–244

 

Synthesis of new derivatives of quinzolin-4(3H)-one

The synthesis of new derivatives was carried out according to the classical scheme of the nucleic bases alkylation with alkyl halides in anhydrous dimethylformamide (DMF) in the presence of a potassium carbonate excess. NMR¹H spectra were recorded on a BrukerAvance 400 spectrometer (400 MHz) in DSMO-d6, tetramethylsilane as the internal standard. The spectra were interpreted using the ACD/HNMR PredictorPro 3.0 licensed program from Advanced Chemistry Development (Canada). The melting points were measured in glass capillaries on a Mel-Temp 3.0 instrument (Laboratory Devices Inc., USA). The purity and individuality of the compounds were monitored by the TLC method.

N-[4-(Dimethylamino)phenyl]-2-[4-oxo-3(4H)-quinazolinyl] acetamide (Laboratory code: VMA-10-10).

A mixture of 2.0 g (13.7 mmol) of quinazolin-4(3H)-one, 4.0 g (28.9 mmol) of anhydrous potassium carbonate and 50 ml of DMF is stirred at the temperature of 100-105°C for 30 min., then 3.2 g (15.1 mmol) of 2-Chloro-N-[4-(dimethylamino)phenyl]acetamide is added and stirred at the same temperature for 1 hour. After that, the mixture is cooled down to room temperature and filtered.

The filtrate is kept at the temperature of 0–5°C within 24 hours. The separated precipitate is filtered off, washed with cold DMF, water, and dried in air. It is recrystallized from DMF to get 2.95 g of the VMA-10-10compound, the yield is 67%, the mp. is 261–264°C.

The NMR¹H spectrum, δ, ppm, is the following: 2.78 s (6H, CH₃). 4.76 s (2H, CH₂); 6.63 d (8 Hz, 2H, phenyl); 7.34 d (8 Hz, 2H, phenyl); 7.51 t (7 Hz, 1H, H⁶); 7.66 d (8 Hz, 1H, H⁸); 7.78 t (7 Hz, 1H, H⁷); 8.09 d (8 Hz, 1H, H⁵); 8.29 s (1H, H²); 10.08 s (1H, NH).

The rest of the compounds are obtained in the same way.

N-(4-Methoxyphenyl)-2-[4-oxo-3(4H)-quinazolinyl]acetamide (Laboratory code: VMA-10-18). The NMR¹H spectrum, δ, ppm is the following: 3.72 s (3H, OCH₃); 4.85 s (2H, CH₂); 7.51 d (8 Hz, 2H, phenyl); 6.90 d (8 Hz, 2H, phenyl); 7.57 t (7 Hz, 1H, H⁶); 7.73 d (8 Hz, 1H, H⁸); 7.86 t (7 Hz, 1H, H⁷); 8.16 d (8 Hz, 1H, H⁵); 8.37 s (1H, H²); 10.31 s (1H, NH).

3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]quinazolin-4(3H)-one (Laboratory code: VMA-10-21). The NMR¹H spectrum, δ, ppm is as follows: 3.14-3.32 m (4H, piperazine); 3.62-3.78 m (4H, piperazine); 5.01 s (2H, CH₂); 6.96-7.01 m (2H, phenyl); 7.23-7.29 m (3H, phenyl); 7.55 t (7.5 Hz, 1H, H⁶); 7.71 d (8 Hz, 1H, H⁸); 7.86 t (7.5 Hz, 1H, H⁷); 8.17 d (8 Hz, 1H, H⁵); 8.26 s (1H, H²).

N-(2-Naphthyl)-2-[4-oxo-3(4H)-quinazolinyl] acetamide (Laboratory code: VMA-13-05). The NMR¹H spectrum, δ, ppm is as follows: 5.81 s (2H, CH2); 7.55-8.89 m (11H, H⁵, H⁶, H⁷, H⁸, naphthyl); 8.42 s (1H, H²).

N-Phenyl-2-[4-oxo-3 (4H)-quinazolinyl]acetamide (Laboratory code: VMA-17-01). The NMR¹H spectrum, δ, ppm is as follows: 5.67 s (2H, CH₂); 7.54-7.77 m (5H, H⁶, H⁸, phenyl); 7.87 t (1H, 8 Hz, H⁷); 8.07–8.19 m (3H, H⁵, phenyl); 8.39 s (1H, H²).

N-Phenyl-2-[4-oxo-3(4H)-quinazolinyl]propanamide (Laboratory code: VMA-17-04). The NMR¹H spectrum, δ, ppm is as follows: 1.53 d (3H, 7 Hz, CH₃) 5.49 q (1H, 7 Hz, CH); 7.56–7.80 m (5H, H⁶, H⁸, phenyl); 7.85 t (1H, 8 Hz, H⁷); 8.06–8.19 m (3H, H⁵, phenyl); 8.40 s (1H, H²).

N- [6-Bromoquinazolin-3 (4H) -yl] acetylguanidine (Laboratory code: VMA-13-17). The NMR¹H spectrum, δ, ppm is as follows: 4.37 s (2H, CH2); 7.47 br. s (4H, NH); 7.60 d (1H, 8 Hz, H⁸); 7.90 d (1H, 8 Hz, H⁷); 8.17 s (1H, H²); 8.28 s (1H, H⁵).

Test cultures

A primary microbiological screening of the antimicrobial activity of the synthesized compounds in order to identify the lead compound, was carried out using cultures of Staphylococcus aureus and Streptococcus pneumoniae isolated from sick patients provided by the clinical diagnostic laboratory, City Clinical Hospital No. 3 n. a. S.M. Kirov, Astrakhan. The studies were approved by the Ethics Committee of Astrakhan State Medical University of the Ministry of Health of Russia (protocol No. 6 dated November 27, 2018).

Research methods

The analysis of substances with the assigned codes – VMA-10-10, VMA-10-18, VMA-10-21, VMA-13-05, VMA-17-01, VMA-17-04, VMA-13-17 – was carried out in vitro by the serial dilutions method in accordance with the requirements of the international standard ISO 20776-1:2006¹ and the National Standard GOST R ISO 20776-1-2010², identical to the international one.

The determination of the microorganism’s sensitivity to quinazolinone derivatives was carried out by the macro method (test tube) in the medium of meat infusion broth (MIB) prepared in accordance with GOST 20729-75.

Preparation process of working solutions

The working solution was prepared by dissolving a 4 mg sample of the test substance in 0.5 ml of dimethyl sulfoxide (DMSO), followed by adding 4.5 ml of a physiological solution to it. The choice of the solvent was carried out in accordance with the Methodological Recommendations “Sensitivity determination of microorganisms to antibacterial drugs”³, as well as taking into account the solubility of the compounds under study, with a preliminary assessment of DMSO effect on the strains of the microorganisms used [33]. It was found out that the compounds under study are insoluble in water, slightly soluble in 40 and 90% ethyl alcohol, and freely soluble in DMSO. A series of solutions with an exponentially decreasing concentration was obtained from the resulting initial solution: 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 μg/ml. A solution of ceftriaxone (JSC Sintez, Kurgan, P N000750/01) with the concentration equivalent to the process solution was used as a reference drug. Process solutions were introduced into 1 ml test tubes.

Inoculum preparation

Inoculum preparation was carried out in accordance with the requirements for the method of direct suspension of morphologically similar colonies collected using a sterile bacteriological loop in a sterile isotonic solution.

Methodology

Suspensions of Staphylococcus aureus and Streptococcus pneumoniae, diluted in a liquid nutrient medium to 10⁶ cfu/ml, were added 1 ml each into the test tubes with the solutions of the studied substances.

The inoculations in the tubes closed with sterile cotton-gauze stoppers, were incubated for 24 hours at the temperature of + 37°C. At the end of the incubation period, they were visually assessed in the transmitted light. In the control tubes, in which a native culture had been grown without adding a reference drug or test compounds, complete turbidity of the culture medium indicating an intensive growth of the culture, was noted.

The determination of the minimum inhibitory concentration (MIC) of the test substance assumed the establishment of the lowest substance concentration at which there had been no bacterial growth. That was evidenced by the absence of turbidity of the solution, which was recorded visually.

Assessment of microorganisms growth

The assessment of the bacteria viability was carried out according to the value of the lowest concentration of the test substance that prevents the visible growth of bacteria, or, in other words, the minimum inhibitory concentration. The inoculation of 0.05 ml of the precipitate obtained by centrifuging the contents of each tube of the series at 1500 rpm for 10 min and separating the supernatant, was carried out on the meat infusion agar (MIA) placed in Petri dishes. The nutrient medium was prepared by dissolving the dry agar followed by autoclaving. After the inoculation, the Petri dishes were placed in a thermostat. The analysis of the characteristic growth was performed 24 hours after the incubation of the inoculation at the temperature of +37°C [33].

The determination of the antimicrobial activity of the test substances hypothesized a sixfold reproduction of the selected analysis method [31-32]. The absence of the culture growth was taken for the bactericidal effect manifested by the substance, while the inhibition of the culture growth, its intermittent growth, and the formation of single colonies indicated a bacteriostatic effect.

Statistical processing of research results

Statistical processing of the research results was carried out using the following software packages: Microsoft Office Excel 2007 (Microsoft, USA), BIOSTAT 2008 Professional 5.1.3.1. (“Analyst-Soft” Inc., USA). When processing the results obtained, a parametric method with the determination of the Student’s t-test with the Bonferroni correction was used. The differences in the comparison groups were assessed at the constantly chosen significance level of p ≤ 0.05.

RESULTS AND DISCUSSION

The analysis of the antimicrobial activity of the substances with codes VMA-10-10, VMA-10-18, VMA-10-21, VMA-13-05, VMA-17-01, VMA-17-04, VMA-13-17 showed that their manifestation depends on the multiplicity of dilution and a type of a pathogenic microorganism.

The experimental data obtained are summarized in Tables 2–5.

 

Table 2 – Indicators of visual assessment of compounds activity against growth of Staphylococcus aureus (MIB medium)

Series (compounds, drugs)	Concentration, μg/ml
	128	64	32	16	8	4	2	1	0.5	0.25
DMSO	++	++	+++	+++	+++	+++	+++	+++	+++	+++
Ceftriaxone	–	–	–	–	–	+	++	+++	+++	+++
VMA-10-10	++	++	++	++	++	++	+++	+++	+++	+++
VMA-10-18	+	+	+	+	+++	+++	+++	+++	++++	++++
VMA-10-21	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
VMA-13-05	–	–	–	++	++	++	+++	+++	+++	+++
VMA-17-01	–	–	–	++	++	+++	+++	+++	+++	+++
VMA-17-04	–	–	–	–	++	++	++++	++++	++++	++++
VMA-13-17	+	+	++	++	++	++	+++	+++	+++	+++

Note: “–” – full medium transparency; “+ –” – incomplete medium transparency; “+” – weak growth; ++ – moderate growth; +++ – intensive growth

 

The growth pattern analysis of Staphylococcus aureus and Streptococcus pneumoniae in the meat infusion broth and on the meat infusion agar with DMSO showed a moderate growth of microorganisms at the concentration of 128 and 64 μg/ml, as well as an intensive growth in the concentration range from 32 to 0.25 μg/ml.

During the visual control of Staphylococcus aureus cultures in the meat infusion broth, the signs of growth in the test tubes with ceftriaxone were observed at low drug concentrations – 2–0.5 μg/ml. A moderate growth of the culture was observed in the presence of the VMA-10-10 compound in the concentration range of 128-4 μg/ml and in the case of VMA-17-01 – in the concentration range of 16–8 μg/ml. The intensive development of cells, accompanied by strong turbidity of the nutrient medium, the formation of flakes and abundant sediment, were observed in test tubes at the concentrations of the compound VMA-10-21 in the range of 128–0.25 μg/ml.

A significant culture growth was also recorded in the test tubes with substances VMA-17-04 with a concentration of 4–0.25 μg/ml and VMA-13-17 with its content of 2–0.25 μg in 1 ml of the solution.

Table 3 shows the results of inoculating Staphylococcus aureus on a solid nutrient medium – meat infusion agar.

 

Table 3 – Indicators of visual assessment of compounds activity against growth of Staphylococcus aureus (MIA medium)

Series (compounds, drugs)	n	Concentration, μg/ml
		128	64	32	16	8	4	2	1	0.5	0.25
DMSO	6	++	++	+++	+++	+++	+++	+++	+++	+++	+++
Ceftriaxone	6	–	–	+AC	+AC	+AC	+AC	+++AC	+++AC	+++AC	+++AC
VMA-10-10	6	+++AC	+++AC	+++AC	+++AC	+++AC	++++AC	++++AC	++++AC	++++AC	++++AC
VMA-10-18	6	+ AC	+АC	+ AC	+AC	+++AC	+++TC	+++TC	+++TC	++++TC	++++TC
VMA-10-21	6	++AC	+++TC	+++TC	+++TC	++++TC	++++TC	++++TC	++++TC	++++TC	++++TC
VMA-13-05	6	–	–	+AC	+++AC	++++AC	++++AC	++++AC	++++AC	++++AC	++++AC
VMA-17-01	6	–	–	–	++ AC	++ AC	+++ TC	+++ TC	+++ TC	+++ TC	+++ TC
VMA-17-04	6	–	–	–	–	++ AC	++AC	++++ AC	++++ AC	++++ AC	++++AC
VMA-13-17	6	+ AC	+ AC	++ AC	++ AC	++ AC	++AC	+++ TC	+++ TC	+++ TC	+++TC

Note: “–” – no colonies; “+” – single colonies; “++” – ≤ 50%, “+++” – ≤ 75%; “++++” – ≤ 100% of colonizating the Petri dish area; AC – atypical colonies; TC – typical colonies

 

The Table 3 data indicate that in the presence of the control, ceftriaxone at the concentrations of 128–64 μg/ml, the growth of the culture is completely suppressed, while at its content of 32–4 μg in 1 ml of the solution, the growth of single colonies of the pathogen is observed.

An intensive growth is recorded on the Staphylococcus aureus meat infusion agar when using VMA-10-10 at the concentrations of 128–0.25 μg/ml and VMA-10-21 at 64–0.25 μg/ml. No growth of colonies was observed in the concentration ranges of 128–16 μg/ml of the substance VMA-17-04, 128–64 μg/ml – VMA 13-05, 128–32 μg/ml – VMA-17-01. The results indicate the ability of these compounds to inhibit the development of Staphylococcus aureus and, as a consequence, to exhibit a pronounced antimicrobial activity against the pathogen.

Table 4 shows the results of Streptococcus pneumoniae inoculations on a liquid nutrient medium (meat infusion broth).

 

Table 4 – Indicators of visual assessment of compounds activity against growth of Streptococcus pneumoniae (MIB medium)

Series (compounds, drugs)	Concentration, μg/ml
	128	64	32	16	8	4	2	1	0.5	0.25
DMSO	++	++	+++	+++	+++	+++	+++	+++	+++	+++
Ceftriaxone	–	–	–	–	–	+	+	+	+	+
VMA-10-10	+ –	+ –	+ –	+	+	++	++	+++	+++	+++
VMA-10-18	+	+	+	+	+++	+++	+++	+++	++++
VMA-10-21	+	+++	+++	+++	+++	+++	+++	+++	+++	+++
VMA-13-05	–	–	–	+	+ –	++	+++	+++	+++	+++
VMA-17-01	–	–	+ –	+ –	+	++	++	+++	+++	+++
VMA-17-04	–	–	+	+	+	+	++	++	+++	+++
VMA-13-17	+	+	+	+	+	+	+++	+++	+++	+++

Note: “–” – full medium transparency; “+ –” – incomplete medium transparency; “+” – weak growth; ++ – moderate growth; +++ – intensive growth

 

During the visual control of Streptococcus pneumonia inoculations on the meat infusion broth, the signs of growth in the test tubes with ceftriaxone were observed at the concentration of 4–0.25 μg/ml. A moderate growth of the culture was observed in the presence of the VMA-10-21 compound in the concentration range of 64–0.25 μg/ml, and of the VMA-10-18 substance – at its content of 8–0.25 μg in 1 ml. Lower values were set for the VMA-13-17, VMA-13-05 derivatives – 2–0.25 μg/ml and for VMA-10-10, VMA-17-01 derivatives – 1–0.25 μg/ml.

A complete transparency of the medium was observed in the tubes with quinazolinone derivative VMA-13-05 at the concentration of 128–32 μg/ml, of compounds VMA-17-01 and VMA-17-04 – in the content of the active ingredient of 128–64 μg in 1 ml. The results obtained indicate a pronounced antipneumococcal activity of the substances.

Table 5 shows that the culture of Streptococcus pneumoniae gives a heavy growth on the MIA in the presence of VMA-10-10, VMA-10-18 compounds at the concentrations of 4–0.25 μg/ml, in the presence of VMA-13-05 substances – at 8–0.25 μg/ml and in the presence of the VMA-17-04 derivative – at the concentrations of 2–0.25 mg/ml. The results obtained indicate the lack of sensitivity of the pathogen to these substances in the given dilution.

 

Table 5 – Indicators of visual assessment of compounds activity against growth of Streptococcus pneumoniae (MIA medium)

Series (compounds, drugs)	n	Concentration, μg/ml
		128	64	32	16	8	4	2	1	0.5	0.25
DMSO	6	++	++	+++	+++	+++	+++	+++	+++	+++	+++
Ceftriaxone	6	–	–	–	+АC	+АC	+АC	++АC	++АC	++АC	++АC
VMA-10-10	6	++АC	++АC	++АC	++АC	++АC	++++АC	++++АC	++++АC	++++АC	++++АC
VMA-10-18	6	++АC	++АC	++ АC	++ АC	+++АC	+++TC	+++TC	+++TC	++++TC	++++TC
VMA-10-21	6	+АC	+TC	++TC	++TC	+++TC	+++TC	+++TC	+++TC	++++TC	++++TC
VMA-13-05	6	–	–	–	–	++++АC	++++АC	++++АC	++++АC	++++АC	++++АC
VMA-13-17	6	+ АC	+ АC	++ АC	++ АC	++ АC	++ АC	+++ TC	+++ TC	+++ TC	+++ TC
VMA-17-04	6	–	–	+ АC	+АC	++ АC	++ АC	++++ TC	++++ TC	++++ TC	++++ TC
VMA-17-01	6	–	–	+АC	++ АC	++ АC	+++ TC	+++ TC	+++ TC	+++ TC	+++ TC

Note: “–” – no colonies; “+” – single colonies; “++” – ≤ 50%, “+++” – ≤ 75%; “++++” – ≤ 100% of colonizating the Petri dish area; AC – atypical colonies; TC – typical colonies

 

When the content of VMA-13-05 is at the concentration of 128–16 μg/ml, the growth of the pathogenic strain colonies is not observed. This is similar to the effects of VMA-17-04 and VMA-17-01 in the concentration range of 128–64 μg/ml. Consequently, in this content in the solution, the substances are characterized by a high antimicrobial activity against Streptococcus pneumoniae.

Table 6 shows the average results of assessing the antibacterial action of the most active substances against the strains of Staphylococcus aureus and Streptococcus pneumoniae.

 

Table 6 – Average results of antibacterial action of the most active substances against Staphylococcus aureus and Streptococcus pneumoniae strains

Series (compounds, drugs)	Concentration, μg/ml
	128	64	32	16	8	4	2	1	0.5	0.25
Ceftriaxone	0	0	0	0	0	18.1±2.3	18.8±2.2	19.3±2.2	22.5±3.6	22.7±3.2
Against Staphylococcus aureus strains
VMA-13-05	0	0	0	29.5±2.4 ***	32.1±3.1 ***	38.4±3.8 **	59.4±4.7 ***	65.3±4.2 ***	65.8±5.6 ***	68.3±5.4 ***
VMA-17-01	0	0	0	28.3±2.1 ***	33.8±3.7 ***	39.9±4.2 **	64.4±4.3 ***	65.7±4.1 ***	65.6±6.6 ***	69.3±6.1 ***
VMA-17-04	0	0	0	0	27.3±3.1 ***	28.1±2.8 *	78.4±5.9 ***	81.3±7.1 ***	83.6±7.3 ***	85.2±6.5 ***
Against Streptococcus pneumoniae strains
VMA-13-05	0	0	0	14.3±1.8 ***	16.4±2.1 ***	26.3±1.8 *	61.3±4.8 ***	63.8±5.6 ***	66.4±5.2 ***	71.6±6.9 ***
VMA-17-01	0	0	12.3±1.8 ***	14.9±2.0 ***	15.7±1.9 ***	25.9±1.8 *	27.3±2.0 *	56.4±4.6 ***	62.3±4.9 ***	68.3±6.0 ***
VMA-17-04	0	0	10.2±1.3 ***	12.7±1.8 ***	12.8±1.4 ***	13.2±1.9	26.2±1.9 *	28.6±2.2 **	53.8±5.2 ***	55.7±5.2 ***

Note: * – p <0.05; ** – p <0.01; *** – p <0.001 – by reference to the indicators of the antibacterial ceftriaxone action

 

The analysis of the average results of the antibacterial action of the most active substances against pathogenic microorganisms, makes it possible to conclude the following. The bactericidal activity of the compounds VMA-13-05, VMA-17-01 and VMA-17-04 is comparable to the action of ceftriaxone at the concentrations of 128 and 64 μg/ml; their bactericidal activity against Staphylococcus aureus manifests itself at the concentration of 32 μg/ml. When analyzing the antimicrobial action of the most active quinazoline compounds in subsequent concentrations, it was found out that the bactericidal activity of VMA-13-05, VMA-17-01 and VMA-17-04 statistically significantly decreases in proportion to the decrease of the substances concentration in relation to the reference drug – ceftriaxone.

The heterocyclic nature of quinazolinone compounds determines their ability to inhibit a PBP2a activity due to the formation of hydrogen bonds with the amino acids of the allosteric enzyme site: lysine, glutamine and asparagine. As a result of this interaction, an active site, where the carbonyl group and the nitrogen atom of another molecule of the antimicrobial agent are covalently bound to the carboxyl and amino groups of lysine and arginine, is opened. The enzyme is suppressed and, therefore, the biosynthesis of the bacterial cell wall is blocked [37-40]. The analysis of various substituents effect in the molecule of quinazolinone derivatives made it possible to identify the functional groups and structural fragments that take part in the formation of a chemical bond with the amino acid residues of the enzyme, due to which the pharmacological effect of the substances is probably realized. The studies of the relationship between the structure and activity of quinazolinone derivatives have shown that the presence of a substituted aromatic ring at position 3 and a methyl group is essential for the compound to exhibit the antimicrobial activity [34]. In this case, the quinazolinone compounds containing a phenyl radical are characterized by a higher binding affinity than the substances with a methyl group, which can be explained by an increase in the number of hydrophobic bonds with amino acids of the active site [35]. It has been shown that the substituent in the phenyl ring also has a significant effect on the antibacterial activity. Methoxy, methyl, hydroxy groups, as well as bromine and chlorine atoms, increase the antimicrobial effect [24]. It has been proven that the combination of two or more biologically active fragments in one molecule also contributes to an increase in the antibacterial effect due to a change in the degree of polarity of the drug molecule [1].

The mechanism of the substances interaction with DNA gyrase has been described. It also depends on the substituents nature determining the polarity of the molecule, its ability to form various chemical bonds with the enzyme. In this case, the death of a bacterial cell is known to be mediated by a violation of the DNA synthesis during the DNA gyrase inhibition involved in the reduction (negative supercoiling) of a nucleic acid (NA) molecule, with a quinazolinone derivative [37]. It has been established that its effect can be explained by the formation of an intermediate complex “DNA-topoisomerase-quinazolinone” due to the donor-acceptor interaction of the carbonyl group oxygen atom of the antimicrobial agent and the phosphate group of DNA, nitrogen with guanine and NA asparagine, and the substituents of the quinazolinone molecule with its non-polar groups. Binding to the active site of the enzyme occurs due to the hydrogen bonds of the quinazolinone derivative with the amino acid residues of serine and arginine [37].

The possibility of the quinazolinone derivatives interaction with peptidoglycan precursors cannot excluded. That leads to the inhibition of its polymerization (transglycosylation) and the subsequent stage of cross-linking (transpeptidation). The bactericidal effect of the drug is realized during the formation of an intermediate complex “quinazolinone – peptidoglycan derivative”, as a result of which depolarization of the membrane occurs, its permeability increases, leakage of potassium ions and cytoplasmic ATP occurs resulting in the cell death [41, 42].

The idea of the efflux pumps functioning increases a number of requirements for the investigated antimicrobial substances, in the form of a combination of high efficacy with resistance to outflow. One of the options for achieving it can be the dissipation of the membrane potential [29, 34]. It has been proven that the presence of a keto group, a benzyl radical and nitrogen atom in the quinazolinone structure, contributes to a decrease in lipophilicity; covalently bound bromine in the quinazoline core; methoxyphenyl and methyl substituents, on the contrary, increase hydrophobicity [35, 36]. The saturation of the quinazolinone derivatives molecules by the centers that reduce hydrophobicity, suggests an insignificant degree of binding to efflux proteins and, as a consequence, a low probability of resistance to these substances from the point of view of the efflux theory [5, 7, 28].

The analysis of the results obtained shows that the compound VMA-17-04, and, to a lesser extent, VMA-13-05, are active against Staphylococcus aureus and have a bacteriostatic effect. The structure of the substance VMA-13-05 contains a naphthyl substituent, which makes the molecule more lipophilic and, as a result, increases its penetration into the cell membrane of the pathogenic culture. The polarity of VMA-17-04, due to the amide group associated with the quinazolinone moiety and the benzene ring, causes an increase in the interaction degree of the electron donor center in the form of a nitrogen atom with the active sites of enzymes that catalyze the DNA replication and protein synthesis.

The assessment of the test compounds antimicrobial activity against Streptococcus pneumoniae shows the manifestation of the bacteriostatic effect of the VMA-13-05 derivatives. The VMA-17-04 and VMA-17-01 compounds are characterized by a weakly expressed antimicrobial effect.

The VMA-10-10 substance has practically no effect on Staphylococcus aureus and Streptococcus pneumoniae.

Probably, the difference of the membrane components of gram-positive bacteria in the chemical composition can be the reason for the unequal manifestation of the pharmacological activity of the VMA-17-04 and VMA-13-05 substances in relation to the pathogens. The presence of the quinazolinone derivatives in the molecules differing from their substituents in the structure, determines the difference in the mechanism of their binding to the substances of the pathogens cell membrane acting as adhesives, which are one of the virulence factors of these microorganisms. It has been established that the main role in the adhesion process of Streptococcus pneumonia, is played by collagen-binding and fibronectin-binding proteins, lipoteichoic acid, as well as surface phosphoryl-choline, which is a part of teichoic acid with choline-binding proteins attached to it. The adhesive activity of Staphylococcus aureus is carried out due to fibrinogen-binding protein, the molecules of which are bound to the peptidoglycan of the cell wall, collagen adhesin, extracellular protein, fibronectin-binding proteins, teichoic acid, as well as staphylococcal haptoglobin receptor residues, consisting of 145 amino acid residues [43].

The nature of the substituents in the molecule determines the varying degrees of lipophilicity of the compounds, which, according to Gibbonson, is an important property of the substance that characterizes its solubility in the bacterial membrane, and the degree of binding to efflux proteins or pump substrates. The hydrophobicity of derivatives serves as a factor that reduces the recognition and transport of antimicrobial agents by a suction pump, which is especially important in the search for the inhibitors of their outflow [29]. Although the lipophilicity of the VMA-13-05 structure suggests better binding to the efflux pump protein, which can lead to the emergence of resistance in Staphylococcus aureus and Streptococcus pneumoniae due to a decrease in the concentration of the antimicrobial agent, it cannot serve as an objective characteristic of this process without additional data obtained by an alternative methods analysis.

CONCLUSION

Thus, among the synthesized derivatives of quinazolin-4(3H)-one, the substances that exhibit a pronounced antimicrobial activity against Staphylococcus aureus (VMA-17-04) and Streptococcus pneumoniae (VMA-13-05), have been identified. This is apparently due to the effect of the lipophilic site of their molecules on the manifestation of the antimicrobial action. The results obtained in the course of this study, determine the prospects for further research of the antimicrobial properties of new quinazoline-4(3H)-one compounds in order to increase their pharmacological effect and prevent the development of pathogenic microorganisms’ resistance.

FUNDING

This work was carried out within the framework of the state assignment of the Ministry of Health of the Russian Federation in terms of conducting research on the topic “Search and development of promising compounds with antibacterial activity among pyrimidine derivatives for the creation of drugs” 48.2-2021.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHORS’ CONTRIBUTION

Marina A. Samotrueva – research concept and design, research planning, critical intellectual content review, final approval of manuscript for publication; Alexander A. Ozerov – scheme development of derivatives synthesis, obtaining data on physicochemical properties and spectral characteristics of substances, manuscript editing, its final approval for publication; Alla A. Starikova – data collection, text writing, chemical substantiation of ongoing processes based on structures of investigated substances, preparation of manuscript draft; Narmina Mutallimaga-kyzy Gabitova – carrying out microbiological research, assessment, substantiation and statistical processing of data obtained; Daria V. Merezhkina – implementation of quinazoline derivatives synthesis; Alexandra A. Tsibizova – data collection, assessment, substantiation and statistical processing of data obtained; Ivan N. Tyurenkov – research planning, research methodology, manuscript editing, assessment of results obtained by microbiological methods; final approval of manuscript for publication.

 

¹ CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. Wayne, PA: Clinicaland Laboratory Standards Institute; 2015.

² National Standard GOST R ISO 20776-1-2010 Clinical laboratory research and in vitro diagnostic test systems. Investigation of the sensitivity of infectious agents and assessment of the functional characteristics of products for the study of sensitivity to antimicrobial agents. Part 1. Reference method for laboratory study of the activity of antimicrobial agents against fast-growing aerobic bacteria that cause infectious diseases. Russian

³ Methodical instructions 4.2.1890-04. Determination of the sensitivity of microorganisms to antibacterial drugs: Guidelines. M.: Federal Center for State Sanitary and Epidemiological Supervision of the Ministry of Health of Russia, 2004: 91 p. Russian

About the authors

Marina A. Samotrueva

Аstrakhan State Medical University

Email: ms1506@mail.ru
ORCID iD: 0000-0001-5336-4455

Doctor of Sciences (Medicine), Professor, Head of the Department of Pharmacognosy, Pharmaceutical Technology and Biotechnology

Russian Federation, 121, Bakinskaya Str., Astrakhan, Russia, 414000

Alexander A. Ozerov

Volgograd State Medical University; Volgograd Medical Research Center

Email: prof_ozerov@yahoo.com
ORCID iD: 0000-0002-4721-0959

Doctor of Sciences (Chemistry), Professor, Head of the Department of Pharmaceutical and Toxicological Chemistry

Russian Federation, 1, Pavshikh Bortsov Sq., Volgograd, Russia, 400131; 1, Pavshikh Bortsov Sq., Volgograd, Russia, 400131

Alla A. Starikova

Аstrakhan State Medical University

Author for correspondence.
Email: alhimik.83@mail.ru
ORCID iD: 0000-0002-5210-5248

Assistant, Department of Chemistry, Faculty of Pharmacy

Russian Federation, 121, Bakinskaya Str., Astrakhan, Russia, 414000

Narmina Mutallimaga-kyzy Gabitova

Astrakhan State Medical University; Scientific Research Institute for the Study of Leprosy

Email: narmina85@inbox.ru
ORCID iD: 0000-0002-3867-8330

Assistant, Department of Pharmacognosy, Pharmaceutical Technology and Biotechnology; Junior Researcher

Russian Federation, 121, Bakinskaya Str., Astrakhan, Russia, 414000; 3, Nikolay Ostrovsky Ave., Astrakhan, Russia, 414057

Daria V. Merezhkina

Volgograd State Medical University

Email: merezhkinad@mail.ru
ORCID iD: 0000-0002-9848-7149

Postgraduate student, Department of Pharmaceutical and Toxicological Chemistry

Russian Federation, 1, Pavshikh Bortsov Sq., Volgograd, Russia, 400131

Alexandra A. Tsibizova

Аstrakhan State Medical University

Email: sasha3633@yandex.ru
ORCID iD: 0000-0002-9994-4751

Candidate of Sciences (Pharmacy), Associate Professor, Department of Pharmacognosy, Pharmaceutical Technology and Biotechnology

Russian Federation, 121, Bakinskaya Str., Astrakhan, Russia, 414000

Ivan N. Tyurenkov

Volgograd State Medical University; Volgograd Medical Research Center

Email: fibfuv@mail.ru
ORCID iD: 0000-0001-7574-3923

Doctor of Sciences (Medicine), Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pharmacology and Pharmacy of the Institute of Continuous Medical and Pharmaceutical Education, the Faculty of Advanced Training of Physicians

Russian Federation, 1, Pavshikh Bortsov Sq., Volgograd, Russia, 400131; 1, Pavshikh Bortsov Sq., Volgograd, Russia, 400131

References

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