ANTIMICROBIAL ACTIVITY OF NOVEL 1-[(2,4-(DI-TERT-BUTYLPHENOXY))- 3-DIALKYLAMINO-2-PROPANOL] DERIVATIVES

Introduction. The microbial biofilm-forming ability is one of the major aspects of the emerging issue of antibiotic resistance, which makes them tolerant to antibiotics and host defense systems and other external stresses, thus contributing to persistent chronic infections. A series of relevant studies confirmed the high efficiency of aminopropanol derivatives as potential antibacterial and antifungal agents. This present study was aimed to evaluate the antimicrobial activity of new 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives on the planktonic bacterial/fungal cells and biofilms. Material and methods. The minimum inhibitory concentrations (MIC) of the new compounds were determined by a standard method, along with their effects on biofilms estimated via the gentian violet adsorption-desorption assay.Results. The КVM-219 compound showed the most pronounced effect on planktonic bacterial and fungal cells. The MIC values ranged between 0.78 μg/mL to 12.5 μg/mL, depending on the microbial strain. The KVM-316 compound exhibited the strongest inhibitory effect on biofilms, thus preventing their formation by S. aureus (96.1%), E. coli (57.2%), and P. aeruginosa (96.1%). Conclusions. The 15 newly synthesized 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives revealed marked antibacterial and antifungal effects on planktonic microorganisms. Most of these compounds showed a strain-specific inhibition of biofilm formation by at least 50% for S. aureus 222, E. coli 311, P. aeruginosa 449 and C. glabrata 404 strains.


INTRODUCTION
Over the decades, antimicrobial agents have been considered the primary suppressing means for bacterial infections. However, their irrational use led to the emergence and spread of antimicrobialresistant strains (1,2). The antimicrobial drug resistance has led to a decrease of infection prevention and control measures and lowered the therapeutic effectiveness thus resulting in a prolonged patient's hospital stay and increased treatment costs. The antibiotic resistance has been regarded nowadays as a major threat to public internal safety across many countries. Therefore, a global action plan to overcome antimicrobial resistance has been developed by World Health Assembly (3). In 2017, WHO presented a list of 12 bacterial species that pose a threat to human health, which are classified into three categories of pathogens, namely critical, high and medium priority, according to the urgency of need for new antibiotics (4). The goal of the global action plan is to ensure, for as long as possible, continuity of successful treatment and prevention of infectious diseases by effective and safe medicines that are quality-assured, used in a responsible way and accessible to all who need them. To achieve this goal, five strategic objectives have been set out: to improve awareness and understanding of antimicrobial resistance; to strengthen knowledge through surveillance and research; to reduce the incidence of infection; to optimize the use of antimicrobial agents; to develop the economic case for sustainable investment that takes account of the needs of all countries; and to increase investments in new medicines, diagnostic tools, vaccines and other interventions (3). Considering all of the above mentioned, the most promising approach is to search for potentially novel antimicrobial agents for combatting antimicrobial resistance.
The microbial biofilm-forming ability is one of the major aspects of the emerging issue of antibiotic resistance, which makes them tolerant to antibiotics and host defence systems and other external stresses, thus contributing to persistent chronic infections (5). Several studies confirmed the high efficiency of aminopropanol derivatives as potential antibacterial and antifungal agents, which actually drew our interest to compounds of this group (6,7,8).

Bacterial strain and growth conditions
The study was conducted on the gram-positive

Chemicals
The new derivatives of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] were first synthesised at the Institute of Organic Chemistry NAS of Ukraine. These compounds were synthesized by using the same procedure (9). Its general structural formula is shown in Figure 1.

Minimum Inhibitory Concentration (MIC) determination
Synthesized compounds antimicrobial activity (I -XV) was tested by the twofold serial dilution method (10, 11) against gram-positive (S. aureus ATCC 25923) and gram-negative (E. coli ATCC 25922, P. aeruginosa ATCC 27853) bacteria, and yeasts (C. albicans NTCC 885/653). Inoculum density was 1-2×10 5 CFU/mL culture medium (bacteria) and 1-2×10 4 CFU/mL (yeasts). The 96well microtiter plates with bacterial cultures were incubated for 18-24 h, while yeasts -for 24-48 h at 35-37°C. Mueller-Hinton broth and Saburo dextrose broth were used for minimal inhibitory concentration (MIC) determination. The lowest compound concentration inhibiting the microbial growth was considered as the MIC. All assays were performed in triplicate for control of culture growth (as a positive) and cultural media (as a negative).

Quantitative biofilm assay
The anti-biofilm activity of the tested compounds was determined by using the microtiter plate for biofilm formation assay described by O'Toole (12). The overnight cultures were diluted 100fold with fresh TSB medium (bacteria) or Saburo dextrose broth (yeasts). Cell suspensions (100 µL) were transferred into individual wells of sterile polystyrene 96-well plate. The anti-biofilm effect was estimated by growing strains in media with or without test compounds (2.0×MIC) at 37 o C for 24 hours. After incubation, the media were discarded, and plates were rinsed thrice with distillate water to remove nonadherent cells. Adherent cells were stained for 10-15 min with 0.1% crystal violet. The dye was extracted with ethanol for 15 min to quantify biofilm formation. The optical density was measured at 630 nm via the Absorbance Microplate Reader (model ELx800, BioTek, USA). The measurements were performed in six replications and repeated for at least three times; the values were then averaged.
Data are presented as M±m, where M is the mean value and m is the standard error of the mean.
Thus, according to the study results the most pronounced inhibitory effect on biofilms formation were found for the IV compound, with a 96.1% decrease in S. aureus biofilm mass, 57.2% − E. coli and 96.1% − P. aeruginosa.

DISCUSSIONS
The comparative study of the antibacterial and antifungal activity of 1-[(2,4-(di-tert-buty-lphenoxy))-3-dialkylamino-2-propanol] derivatives with various substituents in the molecular structure showed that substitutes close to the amino fragment might affect the antimicrobial activity of the tested compounds (tab. 2).
Upon the insertion of a nitro group at the 4-position to the benzylic radical, viz. the XIII compound, it showed a reduced antifungal activity (MIC 20.0 μg/mL) in comparison with XII compound (2.5 μg/mL).
The replacement of one of the methyl groups for cyclohexyl in the molecule of the V compound led to a decrease of its antifungal activity (MIC 12.5 μg/mL), while its inhibitory effect towards S. aureus remained nearly unchanged (MIC 3.12 μg/mL) compared with the I compound (MIC 2.5 μg/mL).
The introduction of the chlorine at the 4-position led to completely different consequences: the MIC value of the VІ compound showed a two-fold decrease in comparison with the MIC values of the I and V compounds (S. aureus − 1.56-3.12 μg/mL; C. albicans − 25.0-12.5 μg/mL). However, this decrease was not statistically significant.
The addition of N-methylpyrrolidine fragment to the amino group allowed to increase the antibacterial and antifungal activity of the VII derivative by 3.2 and 2.4 times, respectively (compared with I and VIII compounds). The MIC values towards S. aureus and C. albicans decreased to 0.78 μg/mL and 1.56 μg/mL, respectively.
The replacement of hexamethylenamine pyrrolidine radical in the XI derivative was accompanied by a decreased inhibitory activity (compared to the VII compound), whereas the MIC values towards S. aureus and C. albicans were 5.0-7.5 μg/mL. This present study demonstrated that both tested derivatives 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] were able to suppress the gram-positive and gram-negative bacteria in fungal (C. glabrata) planktonic microorganisms and biofilms formation processes. The comparative structure-activity analysis showed that the inhibitory effect depended not only on the molecular structure and position of the substituents of 2020 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives, but also on the microbial strain used.

CONCLUSIONS
1. The studies on planktonic microorganisms demonstrated that the newly synthesized derivatives of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] have antibacterial and antifungal effects. This research showed that the anti-biofilm effects of the most evaluated compounds could specifically reduce the biofilm formation ability of S. aureus 222, E. coli 311, P. aeruginosa 449 and C. glabrata 404 by at least 50%, depending on the nature of the substituents used in their molecules.
2. The newly synthesized derivatives of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] represent a promising class of chemical compounds, which might lead to the development of novel antimicrobial agents intended for the treatment of many infectious diseases. Further researches are required to study the broad activity spectrum of compounds with the most pronounced antimicrobial action, as well as their antibacterial and antifungal mechanisms, acute toxicity and efficacy in vivo.