Synthesis and antimicrobial activity of amino acid and peptide derivatives of mycophenolic acid.

The series of 16 novel amino acid and peptide mycophenolic acid (MPA) derivatives was obtained as potential antibacterial agents. Coupling of MPA with respective amines was optimized with condensing reagents such as EDCI/DMAP and T3P/TEA. Amino acid analogs were received both as methyl esters and also with the free carboxylic group. The biological activity of the products was tested on five references bacterial strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923. Peptide derivatives proved to be the most versatile ones, their MIC values relative to most strains was lower than MPA alone. It has been noted that the activity of amino acid derivatives depends on the configuration at the chiral center in the amino acid unit and methyl esters indicated better antimicrobial activity than analogs with free carboxylic group.

Introduction

Mycophenolic acid 1 (MPA) possesses interesting scope of biological properties including antimicrobial, antifungal, antiviral, immunosuppressive and antitumor features. MPA 1 (Fig. 1 ) was discovered in 1896 by Italian physician Bartolomeo Gosio, who collected the fungus from damaged maize and called it Penicillium glaucum [1], [2], [3]. It was found that the fungus exhibited antimicrobial activity against anthrax bacterium [4]. Although MPA 1 occurred to be the first antibiotic isolated in pure and crystalline form, further studies were not continued for a long time [5].

Fig. 1

Structure of MPA [1].

In the 1970s, the biochemical causes of immunodeficiency were investigated in children, and inosine 5′-monophosphate dehydrogenase (IMPDH) was established as an enzyme responsible for the undesired immune response in autoimmune diseases. Studies towards a suitable inhibitor were started, followed by a successful clinical trial with the compound under the trade name Cell Cept 2 (Fig. 2 ), which was approved for use in kidney transplantation by the US Food and Drug Administration in 1995 [6].

Fig. 2

Structures of MMF 2 and MPS 3[9].

MPA 1 is a competitive and reversible inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH), predominantly isoforms II, which is present in tumor cells and in activated lymphocytes [7], [8].

MPA 1 is an active substance of immunosuppressive drugs for the prevention of acute and chronic rejection of allogenic organ transplants. So far, there are clinically applied two derivatives of MPA: mycophenolate mofetil 2 (MMF) known as CellCept produced by Roche and mycophenolate sodium 3 (MPS) (Fig. 2) under the trade name Myfortic manufactured by Novartis [9], [10], [11].

MPA 1 possesses significant antimicrobial properties e.g. against Anthrax bacterium [4], Botrytis cinerea [12]), antifungal like against Rhizoctonia Solani [13], antiviral for instance against Dengue virus [14], avian reoviruses (ARV) [15], coronaviruses (MERS_CoV) [16] and rotavirus [17]. Mycophenolic acid 1 exhibits also a wide spectrum of anticancer properties [18], [19], [20].

Recent studies in 2015 showed that the combination of rapamycin with MPA significantly improves clinical symptoms (erythema, edema, rash and rash) at atopic dermatitis, also reduced epidermal exfoliation, edema and cellulite [21].

On the other hand, multidrug resistance is also a significant problem in treatment of bacterial infections, and structural modifications of MPA could provide promising antimicrobial agents.

Taking into account such a large number of positive aspects of the MPA, we attempted to modify its structure towards microbiological activity. We optimized synthesis of several new amino acid and peptide MPA derivatives by conjugation with various condensation reagents.

New synthesized compounds have been tested on five reference strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923.

Results and discussion

Chemistry

According to research in molecular modeling, polar group at the end of the side chain of MPA 1 interacts with Ser 276 of IMPDH and is one of the crucial factors for maintenance of its activity [7]. In literature were reported amide MPA analogs bearing glycine [22], [23], [24] alanine [22] valine, glutamic acid, leucine and phenylalanine [3] moieties as potential anticancer and immunosupressive agents. In order to obtain an amide bond between the MPA molecule and the amino acid, a very large amount of condensing agents was checked, for example: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) with pyridine, N,N′-dicyclohexylcarbodiimide (DCC) with NMM O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) with N-methylomorpholine (NMM) or method of mixed anhydrides (isobutyl chloroformate). Unfortunately, none of the above methods occurred to be effective, since low conversion of substrates or difficulties with purification of target amides [3]. EDCI (1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide) was successfully used as a condensing reagent in the presence of DMAP without any racemization suppressant in the synthesis of optically active amino acid derivatives, where amino group of chiral amino acid is acylated [25], [26], [27]. This method proved to be suitable for the synthesis of most of compounds 11–17. Amino acid MPA derivatives 11–17 were obtained by means of a condensation reagent 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI) in the presence of 4-(dimethylamino)pyridine (DMAP) acting as a base in anhydrous N,N-dimethylformamide (DMF) (Scheme 1 ).

Scheme 1

Synthesis of amino acid derivatives of MPA 11–17 using the EDCI/DMAP method.

In case of derivatives of MPA with isoleucine 14 or malonate 17 moiety the best results were received using the T3P/Et3N procedure, where triethylamine acts as a base and propanephosphonic acid anhydride (T3P) is a condensing reagent (Scheme 2 ).

Scheme 2

Synthesis of amino acid derivatives of MPA 14, 17[30] using the T3P/TEA method.

The method using T3P in the presence of TEA provides amides in high yields with very low epimerization and is particularly well-suited for the coupling of racemization prone acids and amines, including relatively non-nucleophilic anilines, with easy reaction setup and product isolation [28], [29]. Finally, synthesis of compounds 14 and 17 [30] was optimized using T3P/TEA method. The results of the synthesis of MPA analogs 11–17 are given in Table 1 .

Table 1

Yields of obtained compounds 11–17.

Compound	Compound no	Yield [%]
MPA-Asp(OMe)-OMe	11	73
MPA-Thr-OMe	12	71
MPA-D-Thr-OMe	13	70
MPA-Ile-OMe	14	68
MPA-Arg(NO2)-OMe	15	70.5
MPA-D-Arg(NO2)-OMe	16	66
MPA-Mal-(OMe)2	17	45

In order to investigate the influence of methyl ester on activity against chosen microorganisms, methyl esters were hydrolyzed with lithium hydroxide monohydrate to produce derivatives with free carboxylic group 18–23 (Scheme 3 ). The yields of the obtained analogs were presented in Table 2 .

Scheme 3

Synthesis of amino acid analogues of MPA with free carboxylic group.

Table 2

Yields of carboxylic acid analogs 18–23.

Compound	Compound no	Yield [%]
MPA-Asp-OH	18	87
MPA-Thr-OH	19	88
MPA-D-Thr-OH	20	86
MPA-Ile-OH	21	80
MPA-Arg(NO2)-OH	22	87
MPA-D-Arg(NO2)-OH	23	89

The next step was to obtain pentapeptide derivatives containing in their structure tuftsin and retro-tuftsin 24, 27, 28. The protected tuftsin and retro-tuftsin derivatives, and pentapeptides, were synthesized by the mixed anhydride method with isobutyl chloroformate and N-methylmorpholine (NMM) in anhydrous DMF [31], [32], [33], [34], [35], [36], [37]. Tuftsin is known as an natural immunomodulator of a wide range of biological properties, occurring in human blood and other mammals, is able to stimulate certain white blood cells (monocytes, macrophages, and neutrophils). This tetrapeptide provides also antitumor, antimicrobial, anticoagulant and analgesic properties [38], [39], [40], [41].

Despite of such advantageous properties of tuftsin, the peptide is not stable, its half-life in blood is 16 min. It is hydrolyzed by leucine aminopeptidase, carboxypeptidase B, proteinase and subtilisin to tripeptides such as H-Lys-Pro-Arg-OH and H-Thr-Lys-Pro-OH. The resulting compounds inhibit the activity of tuftsin [42], but tuftsin analogs with biologically active compounds such as MPA could improve its metabolic properties. Another way to stabilize the tuftin molecule is to attach another amino acid to the ε-amino group of lysine, thus forming a pentapeptide, which could be also implemented in designed conjugates.

In the course of optimization of the consecutive stages towards final peptides we applied condensation reagents such as EDCI, EEDQ, or T3P. In the case of tuftsin analog 24 the most effective one occurred to be T3P procedure in the presence of TEA (Scheme 4 ), whereas compounds bearing retro-tuftsin 27, 28 were optimized with EDCI in the presence of DMAP (Scheme 5 ). Mycophenolic acid and peptides were coupled in anhydrous DMF at 0 °C to produce MPA derivatives 25, 29, 30 in reasonable yields. The results of these experiments are shown in Table 3 . The Fmoc protective group was removed with 20–30% diethylamine solution towards target compounds 26, 31, 32. Fmoc removal yields were within the range of 80–85%.

Scheme 4

Synthesis of pentapeptide analog of MPA 26 containing in its structure tuftsin.

Scheme 5

Synthesis pentapeptides analogs of MPA 31, 32 containing in their structures retro-tuftsin.

Table 3

Yields of obtained conjugates of MPA and Fmoc-protected peptides 25, 29, 30.

Compound	Compound no	Yield [%]
MPA-T-β-Ala	25	53
MPA-RT-Gly	29	58
MPA-RT- β-Ala	30	57

Microbiological results

The present study evaluated the effect of the obtained compounds 11–23, 26, 31, 32 on five reference strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923. Selected compounds differ in the structure of the skeleton to be able to see if any of its components are of particular importance for antibacterial activity.

The micro dilution method in the broth gave the possibility of determining the minimum concentration of microbial growth inhibitor (MIC) within a narrow and precise concentration range. By carrying out a 96-well microplate study, the analysis of results may consist of a visual assessment of the degree of turbidity in a given well or a spectrophotometer (OD600) [43], [44]. When testing water-insoluble products (compounds with arginine), incompletely dispersible, may interfere with reading of absorbance, it is advisable to use colored growth indicators of microorganisms. The activity of sparingly soluble compounds may be erroneous.

In the presented work, a redox indicator - resazurin was used as a measure of the presence of live microorganisms present in tested compounds. Resazurin is subjected to reduction of activity by active metabolic microorganisms by changing the color from blue (resazurin) to pink (resorufin) and the intensity of color correlates with the size of the population. It has been found in this and other works that the use of resazurin as an indicator of metabolic activity of bacteria increases the sensitivity of detection [45].

For standard antibiotics and MPA for E. coli it was possible to determine MIC90 (bold type in Table 4 ), for the remaining compounds for all tested bacteria MIC50 was determined.

Table 4

Inhibitory concentrations of test compounds for selected microorganisms (μg/ml).

Compound	MIC range (ug/ml)	MIC [ug/ml]
		Gram-positive bacteria		Gram-negative bacteria
		S. aureus MSSA ATCC 25923	S. aureus MRSA ATCC 43300	A. baumannii ATCC 17978	E.coli ATCC 8739	K. pneumoniae ATCC 700603(ESBL)	P. aeruginosa ATCC 27853
MPA	5.9–3000	>>187.5	750	128	750	128	>>1500.0
11	0.125–256	32	32	256	256	256	NI
12	0.125–256	8	32	256	256	256	NI
13	5.9–3000	NI	NI		>>1500.0	NI	>>1500.0
14	0.125–256	16	256	256	256	256	NI
15	0.125–256	32	64	128	256	64	NI
16	5.9–3000	NI	NI		>>375.0	NI	>>1500.0
17	0.125–256	256	256	256	256	128	NI
18	5.9–3000	NI	NI		>>750.0	NI	>>1500.0
19	5.9–3000	NI	NI		>>750.0	>>375.0	>>1500.0
20	5.9–3000	NI	NI		>>1500.0	NI	>>1500.0
21	5.9–3000	NI	>>3000	NI	>>375.0	>>187.5	>750.0
22	5.9–3000	NI	NI		>>750.0	NI	NI
23	5.9–3000	NI	NI		>>1500.0	NI	>>1500.0
26	0.125–256	32	32	128	128	32	NI
31	0.125–256	64	64	128	256	16	NI
32	0.125–256	32	32	128	256	32	NI
Kanamycin	2–1000	7.8	125	NI	15.6	NI	1500
Ampicillin	0.125–256	0.125	128	2	8	128	256

The value in bold type - MIC, is the lowest concentration causing complete inhibition of growth or decreasing the number of bacterial population by over 90%; another - MIC50.

Value with the “>” sign - concentration inhibiting growth by 10–50% relative to positive control.

Value with the sign “>>” - concentration causing very poor growth inhibition (less than 10% of control).

“NI” - no inhibitory effect at the compound concentrations used or stimulation of growth.

Inhibitory concentrations of tested compounds 11–23, 26, 31, 32 for selected microorganisms (μg/ml) is shown in Table 4. Figure S-1 (See electronic supplementary information) compares MIC values [μg/mL] of compounds and standard drug against different bacteria species.

The scope of the examined derivatives MPA was chosen individually. The methodological basis for further elaboration in the implementation and interpretation of minimal inhibitory concentrations is EUCAST, supplemented by the recommendations of the Scientific Societies, data from the publication and own experience [46].

Investigated compounds exerted a variety of effects depending on the species of bacteria. It was demonstrated that most of tested bacterial species (except for Acinetobacter baumannii for which MIC MPA was the same, and P. aeruginosa, where no activity was observed) were inhibited by compound 26 (MIC 32–128 μg/ml) as compared to MPA (128- ≥1500 μg/ml). In case of compound 17 the inhibition of K. pneumoniae was comparable to the control effect of ampicillin (128 μg/ml). The MIC50 for this compound is at MIC50 of MPA. The MIC50 for compound 15 is lower than MIC50 of MPA. For the remaining bacteria there is no clear activity (at the concentration limit). Compounds 11, 12, 13, 15, 26, 31, 32 exhibited the greatest activity against S. aureus, but the MSSA strain was more sensitive than MRSA. In case of analog 21, for S. aureus MSSA, the inhibitory force (MIC50 = 8 μg/ml) is significant if compared to MPA (MIC50 » 187.5 μg/ml), which causes very poor growth inhibition. In case of K. pneumoniae for compounds 11, 12, 14 MIC50 is equal to 256 μg/ml (concentration limit) and is higher than for MPA (MIC50 = 128 μg/ml). These modifications do not affect the improvement of antibacterial activity for this bacterium. Conversely, compounds 15 and 26, 31, 32 indicated lower MIC, (MIC50 = 64, 16, 32, 32 μg/ml respectively) and show enhanced antimicrobial effects compared to MPA. By analyzing the results it can be concluded that MPA modifications such as 11, 12, 15, 26, 31, 32 are promising for S. aureus, while 15, 26, 31, 32 modifications are promising for K. pneumoniae. There was no bacteriostatic effect of the tested compounds on Pseudomonas aeruginosa. The remaining compounds 18–23 caused very poor growth inhibition (<10% control) or no inhibitory effect on the growth of bacteria. The bactericidal effect was not found for any MPA derivative. Compounds 17–23 exhibit poor activity against all groups of microorganisms, no susceptibility was observed.

Structure-activity analysis

Analyzing the results, it can be seen that the peptide derivatives of MPA 26, 31, 32 have better activity than the parent compound 1 itself. In case of strains K. pneumoniae ATCC 700603 (ESBL) and S. aureus MRSA ATCC 43300 their values MIC were lower (16–32 for K. pneumoniae ATCC 700603 (ESBL), 32–64 for S. aureus MRSA ATCC 43300) than for the antibiotics themselves: kanamycin and ampicillin. A slightly better microbiological activity for S. aureus MRSA strains revealed derivatives 26, 32 containing in his peptidic structure β-alanine moiety. There was no difference in effect between tuftsin skeleton and retro-tuftsin.

Microbiological activity of amino acid esters depends both on substituent and configuration in amino acid moiety. It can be clearly seen, that in case of threonine 12, 13 and arginine 15, 16 D enantiomer is definitely less active against all strains. Although d-amino acids can enhance stability of the active substance due to its improved resistance against proteases [47], obtained results suggest that natural configuration of the tested compounds is privileged against investigated bacterial strains.

Deprotection of methyl esters 18–23 changed activity considerably. We observed a significant advantage of derivatives protected by methyl ester over those with a free carboxylic group. This is noticeable for all strains of bacteria, and could be explained by better cell membrane penetration due to lower polarity [19].

Summary

We have received 16 new MPA derivatives using EDCI and T3P condensation reagents. The obtained compounds were tested on five different bacterial cell lines. By analyzing the results it can be concluded that MPA modifications such as 11, 12, 15, 26, 31, 32 are promising for S. aureus, while 15, 26, 31, 32 modifications are promising for K. pneumoniae. It was noted that the compounds with the free carboxyl group 17–23 exhibit poor activity against all groups of microorganisms. Microbiological activity of amino acid esters depends both on substituent and configuration in amino acid moiety. It was found that derivatives of D configuration 13, 16 were much less active than those of L configuration 12, 15.

These results confirm that structure modifications of MPA derivatives can be designed towards antimicrobial properties.

Experimental section

All reactions were performed in inert atmosphere with magnetic stirring. DMF was purified by distillation from benzene/water. The reactions were monitored by TLC on Merck F254 silica gel precoated plates. The following solvent systems (by volume) were used for TLC development: CH2Cl2:MeOH:CH3COOH (15:1:0.1, v/v/v) (A), CH2Cl2:MeOH (50:1, v/v) (B), CHCl3:MeOH:NH3 (9:1:0.2, v/v/v) (C), CHCl3:MeOH:NH3 (5:1:0.2, v/v/v) (D).1H NMR and 13C NMR spectra were taken on the camera Varian Unity 500 Plus in CDCl3, CD3OD or DMSO. Mass spectra were performed at the Laboratory of Mass Spectrometry MALDI-TOF on the matrix DHB (BIFLEX III Bruker).

Conditions of chromatographic HPLC separation and detection of examined compounds (11–24, 27, 28): column - Poroshell EC-C18 (3.0 × 150 mm), 2.7 μm, Agilent Technologies; column temperature - 40 °C; injection volume - 2 μL; flow rate - 0.4 mL/min; eluents: (A) 0.1% HCOOH in water, (B) 0.1% HCOOH in ACN/MeOH (1:1, v/v); gradient program (Table 5 ):

Table 5

Gradient program for HPLC.

Time [min]	% A	% B
0	90	10
20	0	100
30	0	100

Post time - 10 min; UV–Vis detection; wavelengths UV: 254 nm; Vis: 580 nm; peak width > 0.1 min (2s); ESI MS detection.

General procedure for the preparation amino acid analogues of MPA 11–23

Amino acid methyl ester hydrochloride 4–10 (0.181 mmol), MPA 1 (0.166 mmol) and DMAP (0.270 mmol) were dissolved in 2 mL anhydrous DMF. Then the solution cooled to 0 °C in an ice bath, then added (0.271 mmol) of EDCI (in case of 7, 10 0.271 mmol of T3P in 50% DMF was used [30]). Stirring was continued under nitrogen at 0 °C for 2 h and then at room temperature. The progress of the reaction was controlled by TLC. After completion of the synthesis, the solvent was evaporated under reduced pressure using a vacuum pump. The product was purified by preparative thin layer chromatography. Structures of synthesized derivatives 11–17 were established by spectroscopic methods (1H NMR, 13C NMR) and MS, HPLC-MS.

Hydrolysis of the esters 11–16 was carried out by removing the methyl ester from the carboxylic group. Methyl ester 11–16 (0.18 mmol) was dissolved in 1.8 ml of methanol. Then, was added 0.013 g (0.54 mmol) of lithium hydroxide monohydrate dissolved in 1.18 ml of water. The mixture was stirred on a magnetic stirrer for 24 h at room temperature. The solution was acidified with 2 N HCl and extracted with ethyl acetate (EtOAc). The extract was dried with MgSO4, evaporated and purified by preparative thin layer chromatography with respective solvent system A-D to produce compounds 18–23.

Dimethyl N-mycophenoyl-L-asparaginate 11

MPA-Asp(OMe)-OMe (11): 1H NMR (DMSO-d 6) δ ppm: 1.71 (s, 3H), 2.06 (s, 3H), 2.099 (m, 2H), 2.14 (m, 2H), 2.60 (m, 1H, β-D3), 2.68 (dd, J = 16.3 Hz, J = 6.3 Hz, 1H, β-D3), 3.27 (d, J = 6.8 Hz, 2H, d), 3.58 (s, 3H, -COOCH3), 3.59 (s, 3H, -COOCH3), 3.68 (s, 3H), 4.54 (dd, J = 14.3 Hz, J = 6.9 Hz, 1H, α-D2), 5.09 (t, J = 6.5 Hz, 1H), 5.22 (s, 2H), 8.32 (d, J = 12.8 Hz, 1H, D-NH);

13C NMR (DMSO-d 6) δ ppm: 11.48, 16.42, 22.85, 34.26, 35.24, 36.05 (β-D3), 48.81 (α-D2), 52.10 (COOCH3), 52.55 (COOCH3), 61.05, 69.030, 107.37, 116.17, 122.90, 123.06, 134.28, 146.21, 153.44, 162.99, 170.65, 170.86, 171.67, 172.22;

MS m/z calculated for C23H29NO9 463.18; found 462.18 [M+H]+.

Rf = 0.5 (B).

89.15% pure by HPLC.

N-mycophenoyl-l-threonine methyl ester 12

MPA-ThrOMe (12): 1H NMR (CDCl3-d1) δ ppm: 1.18 (d, J = 6.4 Hz, 3H, γ-T4), 1.83 (s, 3H), 2.16 (s, 3H), 2.33 (m, 2H), 2.40 (m, 2H), 3.4 (d, J = 6.8 Hz, 2H), 3.75 (s, 3H), 3.77 (s, 3H, -COOCH3), 4.31 (m, 1H, β-T3), 4.59 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H, α-T2), 5.21 (s, 2H), 5.27 (t, J = 6.6 Hz, 1H), 6.34 (d, J = 8.7 Hz, 1H, T-NH);

13C NMR (CDCl3-d1) δ ppm: 11.56, 16.25, 19.84 (γ-T4), 22.68, 34.90, 34.98, 52.57 (COOCH3), 57.07 (β-T3), 61.05, 68.04 (α-T2), 70.02, 106.45, 116.73, 122.19, 122.95, 134.39, 144.13, 153.64, 163.59, 171.61, 173.48;

MS m/z calculated for C22H29NO8 435.19; found 436.20 [M+H]+.

Rf = 0.5 (A).

99.77% pure by HPLC.

N-mycophenoyl-d-threonine methyl ester 13

MPA-D-ThrOMe (13): 1H NMR (DMSO-d 6) δ ppm: 0.98 (d, J = 6.4 Hz, 3H, γ-T4), 1.72 (s, 3H), 2.05 (s, 3H), 2.12 (m, 2H), 2.19 (m, 2H), 3.26 (d, J = 6.8 Hz, 2H), 3.59 (s, 3H), 3.67 (s, 3H, -COOCH3), 4.02 (m, 1H, β-T3), 4.21 (dd, J = 8.5 Hz, J = 3.5 Hz, 1H, α-T2), 5.1 (t, J = 6.5 Hz, 1H), 5.21 (s, 2H), 7.94 (d, J = 8.5 Hz, 1H, T-NH);

13C NMR (CDCl3) δ ppm: 11.56, 16.25, 19.84 (γ-T4), 22.68, 34.90, 34.98, 52.57 (COOCH3), 57.07 (β-T3), 61.05, 68.04 (α-T2), 70.02, 106.45, 116.73, 122.19, 122.95, 134.39, 144.13, 153.64, 163.59, 171.61, 173.48;

MS m/z calculated for C22H29NO8 435.19; found 436.11 [M+H]+.

Rf = 0.5 (A).

99.72% pure by HPLC.

N-mycophenoyl-l-isoleucine methyl ester 14

MPA-Ile-OMe (14): 1H NMR (DMSO-d 6) δ ppm: 0.76 (m, 6H, δ-I5, ε-I6), 1.08 (m, 1H, γ-I4), 1.30 (m, 1H, γ-I4), 1.66 (m, 1H, β-I3), 1.718 (s, 3H), 2,07 (s, 3H), 2.1 (m, 2H), 2.19 (m, 2H), 3.27 (d, J = 7.4 Hz, 2H), 3.583 (s, 3H), 3.67 (s, 3H, COOCH3), 4.13 (dd, J = 7.8 Hz, J = 6.8 Hz, 1H, α-I2), 5.06 (t, J = 6.6 Hz, 1H), 5.231 (s, 2H), 8.06 (d, J = 8.1 Hz, 1H, I-NH);

13C NMR (DMSO-d 6) δ ppm: 11.51, 11.51 (δ-I5), 15.84 (ε-I6), 16.50, 22.85, 25.21 (γ-I4), 34.11 (β-I3), 35.36, 36.71, 51.96 (COOCH3), 56.60 (α-I2), 61.06, 69.06, 107.40, 116.39, 122.90, 122.92, 134.42, 146.21, 153.17, 163.02, 170.60, 172.50, 172.67;

MS m/z calculated for C24H33NO7 447.23; found 448.24 [M+H]+.

Rf = 0.46 (B).

98.37% pure by HPLC.

N-mycophenoyl-l-arginine methyl ester 15

MPA-Arg(NO )OMe (15): 1H NMR (CDCl3) δ ppm: 1.58 (m, 3H, γ-R4, β-R3), 1.77 (s, 3H), 1.84 (m, 1H, β-R3a), 2.10 (s, 3H), 2.22 (m, 2H), 2.34 (m, 2H), 3.23 (m, 2H, δ-R5), 3.32 (d, J = 6.5 Hz, 2H), 3.70 (s, 3H), 3.72 (s, 3H, -COOCH3), 4.56 (m, 1H, α-R2), 5.16 (s, 2H), 5.21 (t, J = 6.6 Hz, 1H), 8.64 (m, 1H, R-NH);

13C NMR (CDCl3) δ ppm: 11.5, 16.12, 22.60, 24.12 (β-R3), 30.60 (γ-R4), 34.70, 34.99, 40.35 (δ-R5), 50.68 (α-R2), 52.68 (COOCH3), 60.99, 70.12, 106.29, 116.96, 121.95, 122.90, 134.24, 144.26, 153.45, 159.27, 163.60, 172.66, 172.92;

MS m/z calculated for C24H33N5O9 535.55; found 535.23 M+.

Rf = 0.4 (A).

100% pure by HPLC.

N-mycophenoyl-d-arginine methyl ester 16

MPA-D-Arg(NO )OMe (16): 1H NMR (DMSO-d 6) δ ppm: 1.47 (m, 3H, γ-R4, β-R3) 1.65 (m, 1H, β-R3), 1.72 (s, 3H), 2,06 (s, 3H), 2.11 (m, 2H), 2.14 (m, 2H), 3.10 (m, 2H, δ-R5), 3.27 (d, J = 6.7 Hz, 2H), 3.59 (s, 3H), 3.67 (s, 3H, -COOCH3), 4.16 (q, J = 13.2 Hz, 1H, α-R2), 5.10 (t, J = 6.6 Hz, 1H), 5.22 (s, 2H, b), 8.18 (d, J = 7.5 Hz, 1H, R-NH);

13CNMR (CDCl3) δ ppm: 11.5, 16.12, 22.60, 24.12 (β-3R), 30.60 (γ-R4), 34.70, 34.99, 40.35 (δ-R5), 50.68 (α-R2), 52.68 (COOCH3), 60.99, 70.12, 106.29, 116.96, 121.95, 122.90, 134.24, 144.26, 153.45, 159.27, 163.60, 172.66, 172.92;

MS: m/z calculated for C24H33N5O9 535.55; found 535.45 M+.

Rf = 0.46 (A).

100% pure by HPLC.

Dimethyl N-mycophenoylomalonate 17

NMR and MS data are presented in our recent publication [30].

Rf = 0.6 (B).

99.5% pure by HPLC.

N-mycophenoyl-l-aspartic acid 18

MPA-Asp-OH (18): 1H NMR (DMSO-d 6) δ ppm: 1.71 (s, 3H), 2.06 (s, 3H), 2.12 (m, 4H), 2.49 (m, 1H, β-D3), 2.63 (dd, J = 15.7 Hz, J = 6.3 Hz, 1H, β-D3), 3.27 (d, J = 6.6 Hz, 2H), 3.67 (s, 3H), 4.35 (dd, J = 14.1 Hz, J = 7.0 Hz, 1H, α-D2), 5.07 (t, J = 6.5 Hz, 1H), 5.21 (s, 2H), 7.94 (d, J = 7.7 Hz, 1H, D-NH);

13C NMR (DMSO-d 6) δ ppm: 11.48, 16.44, 22.97, 34.43, 35.39, 36.86 (β-D3), 50.01 (α-D2), 61.03, 66.97, 107.47, 115.72, 122.97, 133.96, 146.27, 161.06, 170.88, 171.49, 171.89, 173.36;

MS m/z calculated for C23H29NO9 435.47; found 436.16 [M+H]+.

Rf = 0 in A-D solvent systems.

99.49% pure by HPLC.

N-mycophenoyl-l-threonine 19

MPA-ThrOH (19): 1H NMR (400 MHz, DMSO-d 6) δ ppm: 0.97 (d, J = 6.3 Hz, 3H, γ-T4), 1.74 (s, 3H), 2.08 (s, 3H), 2.16 (m, 2H), 2.25 (m, 2H), 3.29 (d, J = 6.7 Hz, 2H), 3.69 (s, 3H), 3.99 (m, 1H, β-T3), 4.08 (dd, J = 8.2 Hz, J = 3.5 Hz, 1H, α-T2), 5.12 (t, J = 6.4 Hz, 1H), 5.23 (s, 2H), 7.66 (d, J = 8.2 Hz, 1H, T-NH), 7.95 (s, 1H, COOH);

13C NMR (400 MHz, CDCl3) δ ppm: 11.49, 16.45, 20.39 (γ-T4), 21.60, 34.49, 35.50, 55.36 (β-T3), 61.06, 66.32 (α-T2), 69.04, 106.45, 107.41, 116.03, 122.97, 123.21, 134.34, 146.21, 162.71, 163.08, 170.81, 172.34, 172.52;

MS m/z calculated for C21H27NO8 421.44; found 422.57 [M+H]+.

Rf = 0.39 (D).

98.65% pure by HPLC.

N-mycophenoyl-d-threonine 20

MPA-D-ThrOH (20): 1H NMR (DMSO-d 6) δ ppm: 0.93 (d, J = 8.6 Hz, 3H, γ-T4), 1.72 (s, 3H), 2.06 (s, 3H), 2.11 (m, 2H), 2.17 (m, 2H), 3.26 (d, J = 6.7 Hz, 2H), 3.67 (s, 3H), 3.94 (m, 1H, β-T3), 4.03 (m, 1H, α-T2), 5.09 (t, J = 6.5 Hz, 1H), 5.22 (s, 2H), 7.56 (d, J = 8 Hz, 1H, T-NH);

13C NMR (CDCl3) δ ppm: 11.49, 16.45, 20.39 (γ-T4), 21.60, 34.49, 35.50, 55.36 (β-T3), 61.06, 66.32 (α-T2), 69.04, 106.45, 107.41, 116.03, 122.97, 123.21, 134.34, 146.21, 162.71, 163.08, 170.81, 172.34, 172.52; MS m/z calculated for C21H27NO8 421.44; found 422.37 [M+H]+.

Rf = 0.46 (D).

99.02% pure by HPLC.

N-mycophenoyl-l-isoleucine 21

MPA-Ile-OH (21): 1H NMR (DMSO-d 6) δ ppm: 0.77 (d, J = 7.3 Hz, 3H, δ-I5), 0.79 (t, J = 7.5 Hz, 3H, ε-I6) 1.071 (m, 2H, γ-I4), 1.66 (m, 1H, β-I3), 1.72 (s, 3H), 2.05 (s, 3H), 2.05 (m, 2H), 2.09 (m, 2H), 3.26 (d, J = 6.7 Hz, 2H), 3.66 (s, 3H), 4.05 (m, 1H, α-I2), 5.07 (t, J = 6.5 Hz, 1H), 5.21 (s, 2H), 7.81 (d, J = 8.5 Hz, 1H, I-NH);

13C NMR (DMSO-d 6) δ ppm: 11.49, 11.77 (δ-I5), 16.04 (ε-I6), 16.50, 22.92, 25.15 (γ-I4), 34.29 (β-I3), 35.52, 36.84, 57.07 (α-I2), 61.03, 69.0, 107.41, 115.79, 122.97, 123.28, 134.20, 146.22, 154.06, 163.07, 170.86, 172.22, 174.12;

MS: m/z calculated for C23H31NO7 433.49; found 434.38 [M+H]+.

Rf = 0.674 (D).

100% pure by HPLC.

N-mycophenoyl-l-arginine 22

MPA-Arg(NO )OH (22): 1H NMR (400 MHz, DMSO-d 6) δ ppm: 1.53 (m, 3H, γ-R4, β-R3) 1.68 (m, 1H, β-R3), 1.73 (s, 3H), 2.07 (s, 3H), 2.14 (m, 2H), 2.18 (m, 2H), 3.13 (m, 2H, δ-R5), 3.28 (d, J = 6.7 Hz, 2H), 3.68 (s, 3H), 4.06 (m, 1H, α-R2), 5.11 (t, J = 6.3 Hz, 1H), 5.23 (s, 2H), 7.86 (d, J = 7.3 Hz, 1H, R-NH), 8.52 (s, 1H, COOH);

13C NMR (400 MHz, DMSO-d 6) δ ppm: 11.48, 16.46, 21.65, 22.96 (β-R3), 29.23 (γ-R4), 34.53, 35.51, 40.57 (δ-R5), 52.71 (α-R2), 61.03, 69.00, 107.41, 122.97, 123.42, 134.17, 146.22, 159.74, 163.09, 170.91, 172.08, 172.59;

MS: m/z calculated for C23H31N5O9 521.52; found 522.47 [M+H]+.

Rf = 0.22 (D).

100% pure by HPLC.

N-mycophenoyl-d-arginine 23

MPA-D-Arg(NO )OH (23): 1H NMR (DMSO-d 6) δ ppm: 1.48 (m, 3H, γ-R4, β-R3) 1.62 (m, 1H, β-R3), 1.72 (s, 3H), 2.04 (s, 3H), 2.10 (m, 2H), 2.15 (m, 2H), 3.12 (m, 2H, δ-R5), 3.25 (d, J = 6.3 Hz, 2H), 3.66 (s, 3H), 3.96 (q, J = 13 Hz, 1H, α-R2), 5.06 (t, J = 6.4 Hz, 1H), 5.20 (s, 2H), 8.20 (d, J = 8.17 Hz, 1H, R-NH);

13C NMR (DMSO-d 6) δ ppm: 11.48, 16.46, 21.65, 22.96 (β-R3), 29.23 (γ-R4), 34.53, 35.51, 40.57 (δ-R5), 52.71 (α-R2), 61.03, 69.00, 107.41, 122.97, 123.42, 134.17, 146.22, 159.74, 163.09, 170.91, 172.08, 172.59;

MS: m/z calculated for C23H31N5O9 521.52; found 522.39 [M+H]+.

Rf = 0.195 (D).

99.06% pure by HPLC.

General procedure for the preparation tuftsin/retro-tuftsin derivatives 24, 27 and 28

The procedure for the synthesis of compounds 24, 27 and 28 by the mixed anhydride method has been published previously [31], [32], [33], [34], [35], [36], [37]. The Boc-protecting groups was removed by treatment with TFA.

General procedure for the preparation MPA-Thr-Lys(FmocβAla)-Pro-Arg(NO2)-ThrOCH325

Peptide (0.055 mmol), MPA (0.05 mmol) and TEA (0.026 mmol) were dissolved in 1 ml of anhydrous DMF. The solution was then cooled to 0 °C in an ice bath, followed by the addition of (0.103 mmol) of T3P in 50% DMF. The reaction mixture was stirred in a spherical flask filled with inert gas (nitrogen) at 0 °C for 2 h and then at room temperature for 48 h. The progress of the reaction was controlled by TLC plates. After completion of the synthesis, the solvent was distilled off under reduced pressure using a vacuum pump. The product was purified using preparative thin-layer chromatography.

MPA-Thr-Lys(FmocβAla)-Pro-Arg(NO2)-ThrOCH325

MS m/z calculated for C58H76N10O16 1155.25; found 1155.54 M+.

Rf = 0.5 (C).

General procedure for the preparation MPA-Thr-Lys(βAla)-Pro-Arg(NO2)-ThrOCH326

Compound 25 (0.0286 mmol) was treated with 1 ml of chloroform followed by the addition of 0.2–0.3 ml of diethylamine (DEA). The deprotection reaction was carried out at room temperature for 12–24 h and controlled by TLC. Then, solvent was distilled off under reduced pressure using a vacuum pump. The product was purified using preparative thin-layer chromatography.

MPA-Thr-Lys(βAla)-Pro-Arg(NO2)-ThrOCH326

MPA-T-βAla (26): 1H NMR (400 MHz, DMSO-d 6) δ ppm: 0.97 (d, J = 6.3 Hz, 3H, γ-T4), 1.33 (m, 2H, γ-K4), 1.38 (m, 3H, δ-K, β-K3), 1.47 (m, 2H, γ-R4) 1.55 (m, 1H, β-R3), 1.63 (m, 1H, β-K3), 1.71 (m, 1H, β-R3), 1.74 (s, 3H), 1.78 (m, 1H, β-P3), 1.88 (m, 2H, γ-P4, γ-P4), 2.04 (m, 1H, β-P3), 2.08 (s, 3H), 2.16 (m, 2H), 2.27 (m, 2H), 2.46 (m, 2H, α-βA2), 2.89 (q, J = 7.3 Hz, 2H, β-βA3), 3.02 (m, 2H, ε-K6), 3.15 (m, 2H, δ-R5), 3.30 (d, J = 7.0 Hz, 2H), 3.5 (m, 1H, δ-P5), 3.65 (s, 3H, COOCH3), 3.65 (m, 1H, δ-P5), 3.70 (s, 3H), 3.91 (m, 1H, β-T3), 4.18 (m, 2H, α-T2, α-R2), 4.35 (m, 1H, α-P2), 4.47 (m, 1H, α-K2), 5.13 (t, J = 6.4 Hz, 1H), 5.25 (s, 2H), 7.79 (m, 2H, α-KNH, α-TNH), 78.10 (m, 1H, ε-KNH), 8.33 (d, J = 7.2 Hz, 1H, α-RNH);

13C NMR (400 MHz,CD3OD-d4) δ ppm: 10.07, 14.95, 18.82 (γ-T4), 22.18 (γ-K4), 22.24, 24.81 (γ-P4), 28.21 (γ-R4), 28.45 (β-R3), 29.03 (δ-K5), 30.46 (β-P3), 31.39 (β-K3), 34.33, 35.16, 35.88 (α-βA2), 38.54 (β-βA3), 40.18 (ε-K6), 42.15 (δ-R5), 46.92 (δ-P5), 48.48 (α-R2, α-K2), 51.49 (OCH3), 58.63 (α-T2), 60.08 (α-P2), 60.22, 67.10 (β-T3), 69.47, 106.33, 116.53, 122.24, 123.01, 133.82, 145.28, 153.28, 163.44, 170.77 (T1), 171.13 (K1),171.25 (βA1), 172.15, 172.41 (R1), 173.04 (P1), 174.67;

MS m/z calculated for C42H64N10O14 933.02; found 933.47 M+.

Rf = 0.37 (D).

94.8% pure by HPLC.

General procedure for the preparation peptide analogue of MPA and retro-tuftsin 29, 30

Peptide 27, 28 (0.0289 mmol), MPA (0.0212 mmol) and DMAP (0.0289 mmol) were dissolved in 1 ml of anhydrous DMF. The solution was then cooled to 0 °C in an ice bath, then of EDCI was added. The whole was stirred with magnetic stirrer in a spherical flask under inert gas (nitrogen) at 0 °C for 2 h and then at room temperature for 48 h. The progress of the reaction was controlled by TLC plates. After completion of the synthesis, the solvent was distilled off under reduced pressure using a vacuum pump. The product was purified using preparative thin-layer chromatography.

MPA-Arg(NO2)-Pro-Lys(FmocGly)-ThrOCH329

MS m/z calculated for C56H72N10O16 1141.23; found 1141.65 M+.

Rf = 0.474 (C).

MPA-Arg(NO2)-Pro-Lys(FmocβAla)-ThrOCH330

MS m/z calculated for C58H76N10O16 1155.25; found 1155.58 M+.

Rf = 0.2 (D).

Synthesis of peptide analogue of MPA and retro-tuftsin 31, 32

Compounds 29, 30 (0.017 mmol) was treated with 1 mL of chloroform followed by the addition of 0.2–0.3 mL of diethylamine (DEA). The deprotection reaction was controlled by TLC plates. The solvent was distilled off under reduced pressure using a vacuum pump. The product was purified using preparative thin-layer chromatography.

MPA-Arg(NO2)-Pro-Lys(Gly)-ThrOCH331

MPA-RT-Gly (31): 1H NMR (400 MHz, DMSO-d 6) δ ppm: 1.06 (d, J = 6.6 Hz, 3H, γ-T4), 1.29 (m, 2H, γ-K4), 1.38 (m, 3H, δ-K5, β-R3), 1.46 (m, 3H, β-K3, γ-R4), 1.65 (m, 2H, β-K3, β-R3), 1.73 (s, 3H), 1.81 (m, 2H, γ-P4, β-P3), 1.88 (m, 1H, γ-P4), 1.98 (m, 1H, β-P3), 2.06 (s, 3H), 2.13 (m, 2H), 2.19 (m, 2H), 3.07 (m, 2H, ε-K6), 3.11 (m, 2H, δ-R5), 3.26 (d, J = 6.7 Hz, 2H), 3.36 (m, 2H, α-G2), 3.51 (m, 1H, δ-P5), 3.60 (m, 1H, δ-P5), 3.63 (s, 3H, COOCH3), 3.68 (s, 3H), 4.10 (m, 1H, β-T3), 4.26 (m, 1H, α-T2), 4.28 (m, 1H, α-K2), 4.34 (m, 2H, α-P2), 4.45 (m, 1H, α-R2), 5.12 (t, J = 6.5 Hz, 1H), 5.21 (s, 2H), 7.82 (d, J = 8.4 Hz, 1H, α-TNH), 7.97 (m, 3H, α-RNH, α-KNH, ε-KNH);

13C NMR (400 MHz,CD3OD-d4) δ ppm: 10.04, 14.99, 18.97 (γ-T4), 22.59, 24.65 (γ-K4), 28.43 (γ-P4), 29.22 (γ-R4), 31.05 (β-R3), 33.94 (δ-K5), 35.05 (β-P3), 38.65 (β-K3), 40.53, 42.17, 42.78 (ε-K6), 48.47 (δ-R5), 50.77 (δ-P5), 51,47 (α-R2), 53.28 (OCH3), 56.93 (α-K2), 57.81 (α-T2), 60.11 (α-P2), 60.15, 66.99 (β-T3), 69.33, 106.38, 122.41, 123.16, 133.57, 145.29, 159.56, 163.41, 171, 171.13 (T1), 172.56 (P1), 172.97 (K1), 173.25 (R1), 174.23 (G1);

MS m/z calculated for C41H62N10O14 918.99; found 919.45 M+.

Rf = 0.454 (C).

86% pure by HPLC.

MPA-Arg(NO2)-Pro-Lys(βAla)-ThrOCH332

MPA-RT-βAla (32): 1H NMR (400 MHz, DMSO-d 6) δ ppm: 1.04 (d, J = 7.0 Hz, 3H, γ-T4), 1.27 (m, 2H, γ-K4), 1.38 (m, 3H, δ-K5, β-R3), 1.548 (m, 3H, β-K3, γ-R4), 1.65 (m, 2H, β-K3, β-R3), 1.73 (s, 3H), 1.82 (m, 2H, γ-P4, β-P3), 1.89 (m, 1H, γ-P4), 2.00 (m, 1H, β-P3), 2.07 (s, 3H), 2.10 (m, 2H), 2.16 (m, 2H), 2.44 (t, J = 6.8 Hz, 2H, α-βA2), 2.95 (t, J = 5.3 Hz, 2H, β-βA3), 3.03 (m, 2H, ε-K6), 3.13 (m, 2H, δ-R5), 3.27 (d, J = 6.8 Hz, 2H), 3.52 (m, 1H, δ-P5b), 3.615 (m, 1H, δ-P5a), 3.623 (s, 3H, COOCH3), 3.686 (s, 3H, c), 4.102 (m, 1H, β-T3), 4.25 (m, 1H, α-T2), 4.28 (m, 1H, α-K2), 4.34 (m, 2H, α-P2), 4.44 (m, 1H, α-R2), 5.11 (t, J = 6.9 Hz, 1H), 5.22 (s, 2H), 7.85 (d, J = 8.4 Hz, 1H, α-TNH), 8.04 (m, 3H, α-RNH, α-KNH, ε-KNH);

13C NMR (400 MHz,CD3OD-d4) δ ppm: 10.06, 14.98, 18.98 (γ-T4), 22.31, 24.66 (γ-K4), 28.30 (γ-P4), 29.22 (γ-R4), 31.00 (β-R3), 31.96 (β-βA3), 33.95 (δ-K5), 35.06 (β-P3), 36.06 (α-A2), 38.66 (β-K3), 40.56, 42.151, 46.868 (ε-K6), 48.33 (δ-R5), 50.85 (δ-P5), 51.80 (α-R2), 53.29 (OCH3), 56.93 (α-K2), 57.81 (α-T2), 60.18, 67.01 (β-T3), 69.37, 106.37, 116.03, 122.37, 123.12, 133.63, 145.31, 159.56, 163.42, 170.96, 170.99 (T1), 172.51 (P1), 172.98, 173.26 (K1), 174.24 (R1, βA1);

MS m/z calculated for C42H64N10O14 933.02; found 933.47 M+.

Rf = 0.3 (D).

100% pure by HPLC.

Microbiological activity evaluation

Bacterial strains

Susceptibility testing was performed for five reference strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923. All bacteria were routinely passaged and cultured in MHB liquid medium and MHA solid medium.

Preparing the compounds for testing

At the beginning of the experiment the effect of solvents DMSO ((at concentrations ranging from 0.01% to 12.5%) on bacterial cells viability was investigated by measurement OD600 and by plating on a solid medium. The bacterial growth after 18–20 h incubation was compared to control strain without DMSO. No impact of DMSO on bacterial cells was determined at concentration below 6.0%. The research was done in triplicate. The stock solution for conjugates of mycophenolic acid was prepared in 6% DMSO. Serial dilutions were made with water and the growth medium to a final concentration of 3000 μg/l. The solutions were frozen and thawed only once and then discarded.

Determination of minimal-inhibitory concentrations (MICs)

The screening sensitive bacteria to MPA derivatives was performed with MICs tests. MICs were determined by the microdilution method in Mueller Hinton Broth II (MHB II) on 96-well polystyrene microtiterplates according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [48], [49].

The cell bacterial cultures from the logarithmic growth phase were diluted in a medium at a ratio 1: 1000) and them suspension was diluted 50-fold in saline to received McFarland 0.5 scale.

On 96-well titration plates 100 μl of MHB II medium was applied. Then, 100 μl of the compound solution was added to each of the first wells in each column and serial double dilutions were made. In case of compounds 11, 12, 14, 15, 17 and 26, 31, 32 were in range from 256.0 μg/ml to 0.125 μg/ml in case of compound 21 were in the range from 3000.0 μg/ml to 5.9 μg/ml. Two conventional antibiotics: ampicillin (Sigma) or kanamycin (Sigma) was used as a control. For ampicillin the range of concentration was from 256.0 to 0.125 μg/ml, for kanamycin from 1000.0 to 2.0 μg/ml. Finally, 100 μl of suitable bacterial suspension was inoculated to each well to achieve 104 cells/ml. Inoculated plates were incubated at 35 ± 1 °C for 18–20 h and MICs were recorded. Evaluation was performed spectrophotometrically (PlateReader AF2200 UV/VIS and Fluorescence Microplate Reader from Eppendorf) OD600 and visually.

MICs determined by spectrophotometric techniques were defined as the smallest concentration of the compound above which inhibition of bacterial cell growth was expressed as MIC50 and MIC90 (50% and 90%, respectively, of bacterial growth arrest).

For confirmation results the resazurin –based assay was also used. Resazurin is an oxidation-reduction indicator used for the evaluation of cell growth [45], [49] - pink or uncolored indicates growth and blue inhibition of growth.

Resinazurin (10 μl of 0.015% aqueous solution) was added to each well. Samples were incubated at 37 °C for 2 h (in the dark) and measurements of absorbance and visual evaluation of color according to control were made.

The lowest concentration at which color change occurred is taken as the MIC value. MBC value was not possible to determine in each case.

Statistical analysis of results

MIC values were determined in 3 replicates in 3 independent experiments. Data were collected and statistically tested (ANOVA parametric test and Kruskal-Willis nonparametric test). Statistical work has been done using RStudio and using the Microsoft Excel 2010 in all calculations, assuming p < 0.05 as statistically significant.