Microwave-Assisted Synthesis, Lipophilicity and In Vitro Antimicrobial Activity of Hydrazide-Hydrazones of Phenylacetic Acid

Abstract

Microwave-assisted synthesis was applied to obtain fifteen hydrazide-hydrazones of phenylacetic acid. The chemical structure of the synthesized compounds was confirmed on the basis of the analysis of the IR, ¹H NMR and ¹³C NMR spectra. Experimental logP values for all obtained acylhydrazones were established with the use of chromatographic methods. The synthesized compounds were tested for potential antimicrobial activity in in vitro conditions against a panel of microorganisms, which included eight strains of Gram-positive bacteria and six strains of Gram-negative bacteria as well as six strains of yeasts belonging to Candida spp. The assays we performed revealed significant antibacterial activity of obtained hydrazide-hydrazones of phenylacetic acid. Some of the tested compounds possessed much higher activity than that of reference antimicrobial agents towards Gram-positive bacterial strains. It is also worth mentioning that some of synthesized compounds possessed significant activity towards MRSA strain—methicillin-resistant Staphylococcus aureus ATCC 43300 and MSSA strain—methicillin susceptible Staphylococcus aureus ATCC 6538 and ATCC 29213.

1. Introduction

Medicines that possess in their chemical structure acylhydrazone moiety are widely used in the prevention of many diseases, which include, among others, Chagas disease (nitrofurazone), tuberculosis (nitrofurantoin), peptic ulcer disease (furazolidone), malignant hyperthermia (azumolene) and malignant neuroleptic syndrome (dantrolene). They are also effective in the treatment of diarrhea (nifuroxazide, furazolidone), and some of them show antihemorrhagic effects (carbazochrome). In addition, they can be used as potential anticancer substances [1,2,3,4,5,6,7,8,9]. The chemical structures of hydrazide-hydrazones that are used in medicine are presented in Figure 1.

Hydrazide-hydrazone moiety can also be found in many compounds that possess a wide spectrum of antimicrobial activities, which include mainly antibacterial and antifungal properties [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28].

One of the methods for the synthesis of hydrazide-hydrazones is chemical synthesis, which is assisted with microwave radiation. It is based on the heating of appropriate hydrazides of carboxylic acid with corresponding aldehydes or ketones in the microwave synthesizer under specific pressure and temperature conditions and for a specified time. This method, unlike the conventional method (heating the reactants at reflux temperature or mixing the reactants at room temperature), is much faster and also produces compounds with higher yields [13,15,29,30,31,32,33].

Microwave-assisted chemical synthesis has many advantages. It allows shorter reaction time, and often the yield of compounds synthesized by this method is higher than in the case of other synthesis methods [13,15,29,30,31,32,33].

Bearing in mind the facts presented above, we performed microwave-assisted synthesis of novel hydrazide-hydrazones of phenylacetic acid and evaluated them for in vitro antimicrobial activity with the aim of obtaining compounds with significant antimicrobial properties.

2. Materials and Methods

2.1. Chemistry

The reagents and solvents that were used in this research were purchased from Sigma-Aldrich Co. (Saint Louis, MI, USA), Merck Co. (Darmstadt, Germany) and Polish Chemical Reagents POCh (Gliwice, Poland). These chemicals were used without further purification. Microwave-assisted synthesis of hydrazide-hydrazones was performed with the use of the CEM Discover system 908010 synthesizer (CEM Co., Matthews, NC, USA). Thin-layer chromatography (TLC) was applied to check the purity of synthesized hydrazide-hydrazones and to monitor the progress of performed reactions. Aluminum plates were covered with silica gel (aluminum oxide 60 F-254, Merck Co., Darmstadt, Germany), and chloroform-ethanol mixture 10:1 (v/v) was used as a mobile phase. Irradiation with UV light (λ = 254 nm) was used to detect the spots on the chromatograms. The Nicolet 6700 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA) in the ATR mode was used to register the IR spectra of synthesized acylhydrazones. The ¹H NMR and ¹³C NMR spectra were registered with the Bruker Avance 300 and 600 apparatus (Bruker BioSpin GmbH, Ettlingen, Germany). The melting points of obtained compounds were measured with the use of the Fisher–Johns apparatus (Fisher Scientific, Hampton, NH, USA). An elemental analysis of synthesized compounds was performed with the use of a Perkin Elmer 2400 series II CHNS/O analyzer (Waltham, MA, USA).

Detailed Procedure for the Synthesis of Hydrazide-Hydrazones of Phenylacetic Acid (2–16)

An amount of 0.001 mole of hydrazide of phenylacetic acid (1) was placed in a tube appropriate for microwave synthesis and dissolved in 4 mL of ethanol (96%). Subsequently, 0.0011 mole of appropriate substituted aromatic aldehyde was added, and the content of the tube was subjected to microwave radiation (300 W of power, for 7 min at 155 °C). After that, the precipitate was filtered off under reduced pressure and dried. In the case of compounds 9, 10, 11 and 16, the precipitate appeared after 24 h. The obtained compounds were re-crystalized from ethanol (96%) (Scheme 1).

N′-[(E)-(5-bromo-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (2)

Color: light yellow; M.p.: 198–200 °C; yield: 85%; for C₁₅H₁₃BrN₂O₂ (333.18) calculated: C: 54.07%, H: 3.93%, N: 8.41%; found: C: 54.21%, H: 3.91%, N: 8.44%. IR (cm⁻¹): 3186 (NH), 3064 (CH, arom.), 2950, 2907 (CH, aliph.), 1662 (C=O), 1603 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.57 (s, 2H, CH₂), 6.87 (t, 2H, ArH, J = 6 Hz), 7.22–7.28 (m, 1H, ArH), 7.29–7.34 (m, 1H, ArH), 7.37–7.42 (m, 1H, ArH), 7.75 (d, 1H, ArH, J = 6 Hz), 7.81 (d, 1H, ArH, J = 6 Hz), 8.24 (s, 1H, ArH), 8.38 (s, 1H, =CH), 11.43 (s, 1H, OH), 11.93 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.44 (CH₂), 110.86, 119.08, 127.15, 128.83, 129.57, 133.95, 135.84, 138.93 (10C_ar), 144.99 (=CH), 155.95, 156.77 (2C_ar), 167.04 (C=O).

N′-[(E)-(3,5-dibromo-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (3)

Color: light yellow; M.p.: 222–226 °C; yield: 95%; for C₁₅H₁₂Br₂N₂O₂ (412.08) calculated: C: 43.72%, H: 2.94%, N: 6.80%; found: C: 43.86%, H: 2.93%, N: 6.77%. IR (cm⁻¹): 3182 (NH), 3064, 3025 (CH, arom.), 2957, (CH, aliph.), 1660 (C=O), 1603 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.62 (s, 2H, CH₂), 7.27–7.28 (m, 1H, ArH), 7.32–7.34 (m, 4H, ArH), 7.80–7.81 (m, 2H, ArH), 8.33 (s, 1H, =CH), 12.26 (s, 1H, OH), 12.51 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.19 (CH₂), 110.82, 111.59, 121.38, 127.23, 128.87, 129.62, 132.56, 135.94, 141.17 (11C_ar), 146.51 (=CH), 153.99 (C_ar), 167.26 (C=O).

N′-[(E)-(5-chloro-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (4)

Color: white; M.p.: 194 °C; yield: 52%; for C₁₅H₁₃ClN₂O₂ (288.73) calculated: C: 62.40%, H: 4.54%, N: 9.70%; found: C: 62.50%, H: 4.55%, N: 9.67%. IR (cm⁻¹): 3625 (OH), 3189 (NH), 3067 (CH, arom.), 2970 (CH, aliph.), 1664 (C=O), 1603 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.57 (s, 2H, CH₂), 6.92 (t, 2H, ArH, J = 6 Hz, J = 12 Hz), 7.22–7.27 (m, 1H, ArH), 7.29–7.35 (m, 2H, ArH), 7.62 (d, 1H, ArH, J = 6 Hz), 7.68 (d, 1H, ArH, J = 6 Hz), 8.24 (s, 1H, ArH), 8.39 (s, 1H, =CH), 11.43 (s, 1H, OH), 11.93 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.44 (CH₂), 118.63, 121.07, 127.15, 128.83, 129.57, 131.15, 135.84, 139.0 (10C_ar), 145.11 (=CH), 155.53, 156.36 (2C_ar), 167.03 (C=O).

N′-[(E)-(3,5-dichloro-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (5)

Color: white; M.p.: 282 °C; yield: 94%; for C₁₅H₁₂Cl₂N₂O₂ (323.17) calculated: C: 55.75%, H: 3.74%, N: 8.67%; found: C: 55.56%, H: 3.75%, N: 8.69%. IR (cm⁻¹): 3625 (OH), 3182 (NH), 3070 (CH, arom.), 2950 (CH, aliph.), 1660 (C=O), 1595 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.62 (s, 2H, CH₂), 7.22–7.30 (m, 2H, ArH), 7.32–7.36 (m, 2H, ArH), 7.58–7.60 (m, 1H, ArH), 7.64–7.66 (m, 1H, ArH), 8.25 (s, 1H, ArH), 8.36 (s, 1H, =CH), 11.63 (s, 1H, OH), 12.23 (s, 1H, NH). ¹³C NMR (150 MHz, DMSO-d₆): 41.23 (CH₂), 121.18, 121.87, 123.35, 127.23, 128.87, 129.62, 130.64, 135.56, 140.28 (11C_ar), 146.41 (=CH), 152.59 (C_ar) 167.26 (C=O).

N′-[(E)-(3-bromo-5-chloro-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (6)

Color: white; M.p.: 210–212 °C; yield: 76%; for C₁₅H₁₂BrClN₂O₂ (367.62) calculated: C: 49.01%, H: 3.29%, N: 7.62%; found: C: 49.20%, H: 3.30%, N: 7.70%. IR (cm⁻¹): 3182 (NH), 3067 (CH, arom.), 2956 (CH, aliph.), 1662 (C=O), 1594 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.62 (s, 2H, CH₂), 7.24–7.28 (m, 2H, ArH), 7.31–7.36 (m, 3H, ArH), 7.68–7.70 (m, 1H, ArH), 7.71–7.73 (m, 1H, ArH), 8.33 (s, 1H, =CH), 12.26 (s, 1H, OH), 12.48 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.19 (CH₂), 111.22, 120.78, 123.70, 127.24, 128.88, 129.62, 133.40, 135.54, 141.20 (11C_ar), 146.61 (=CH), 153.60 (C_ar), 167.27 (C=O).

N′-[(E)-(5-chloro-2-hydroxy-3-iodophenyl)methylidene]-2-phenylacetohydrazide (7)

Color: cream; M.p.: 222–224 °C; yield: 37%; for C₁₅H₁₂ClIN₂O₂ (414.62) calculated: C: 43.45%, H: 2.92%, N: 6.76%; found: C: 43.56%, H: 2.93%, N: 6.77%. IR (cm⁻¹): 3167 (NH), 3027 (CH, arom.), 2917 (CH, aliph.), 1656 (C=O), 1599 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.62 (s, 2H, CH₂), 7.26–7.28 (m, 1H, ArH), 7.32–7.34 (m, 4H, ArH), 7.68 (d, 1H, ArH, J = 6 Hz), 7.83 (d, 1H, ArH, J = 6 Hz), 8.26 (s, 1H, =CH), 12.26 (s, 1H, OH), 12.68 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.18 (CH₂), 87.04, 119.36, 124.05, 127.24, 128.88, 129.61, 130.59, 135.55, 138.98 (11C_ar), 146.71 (=CH), 156.12 (C_ar), 167.25 (C=O).

N′-[(E)-(2-hydroxy-3,5-diiodophenyl)methylidene]-2-phenylacetohydrazide (8)

Color: light yellow; M.p.: 240–242 °C; yield: 82%; for C₁₅H₁₂I₂N₂O₂ (506.08) calculated: C: 35.60%, H: 2.39%, N: 5.54%; found: C: 35.53%, H: 2.38%, N: 5.52%. IR (cm⁻¹): 3299 (OH), 3052 (CH, arom.), 2930 (CH, aliph.), 1662 (C=O), 1612 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.61 (s, 2H, CH₂), 7.27–7.28 (m, 1H, ArH), 7.32–7.34 (m, 4H, ArH), 7.88 (d, 1H, ArH, J = 6 Hz), 8.03 (d, 1H, ArH, J = 6 Hz), 8.23 (s, 1H, =CH), 12.25 (s, 1H, OH), 12.71 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.15 (CH₂), 82.52, 88.11, 120.75, 127.23, 128.87, 129.61, 135.57, 139.23, 146.58 (11C_ar), 146.87 (=CH), 156.95 (C_ar), 167.23 (C=O).

N′-[(E)-(2-bromo-3-hydroxy-4-methoxyphenyl)methylidene]-2-phenylacetohydrazide (9)

Color: white; M.p.: 184–186 °C; yield: 61%; for C₁₆H₁₅BrN₂O₃ (363.21) calculated: C: 52.91%, H: 4.16%, N: 7.71%; found: C: 52.98%, H: 4.14%, N: 7.69%. IR (cm⁻¹): 3678 (OH), 3254 (NH), 3064 (CH, arom.), 2941 (CH, aliph.), 1652 (C=O), 1593 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.53 (s, 2H, CH₂), 3.88 (s, 3H, OCH₃), 7.08 (t, 1H, ArH, J = 6 Hz), 7.21–7.27 (m, 2H, ArH), 7.30–7.35 (m, 3H, ArH), 7.41 (d, 1H, ArH, J = 6 Hz), 7.46 (d, 1H, ArH, J = 6 Hz), 8.32 (s, 1H, =CH), 11.45 (s, 1H, OH), 11.72 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.74 (CH₂), 56.74 (OCH₃), 111.58, 112.12, 117.83, 126.84, 128.70, 129.83, 136.22, 142.65, 144.22, 146.05 (12C_ar), 149.79 (=CH), 166.88 (C=O).

N′-[(E)-(4-hydroxy-3-iodo-5-methoxyphenyl)methylidene]-2-phenylacetohydrazide (10)

Color: cream; M.p.: 210–214 °C; yield: 71%; for C₁₆H₁₅IN₂O₃ (410.21) calculated: C: 46.85%, H: 3.69%, N: 6.83%; found: C: 46.98%, H: 3.70%, N: 6.85%. IR (cm⁻¹): 3628 (OH), 3182 (NH), 3056 (CH, arom.), 2999, 2965 (CH, aliph.), 1643 (C=O), 1606 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.54 (s, 2H, CH₃), 3.88 (s, 3H, OCH₃), 7.21–7.27 (m, 2H, ArH), 7.28–7.34 (m, 3H, ArH), 7.54 (d, 1H, ArH, J = 18 Hz), 7.83 (s, 1H, ArH), 8.06 (s, 1H, =CH), 11.32 (s, 1H, OH), 11.55 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.66 (CH₂), 56.56 (OCH₃), 84.89, 109.53, 126.80, 128.68, 129.48, 129.81, 130.55, 136.38, 142.06, 145.84 (12C_ar), 147.72 (=CH), 166.84 (C=O).

N′-[(E)-(3-ethoxy-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide (11)

Color: cream; M.p.: 134–136 °C; yield: 83%; for C₁₇H₁₈N₂O₃ (298.34) calculated: C: 68.44%, H: 6.08%, N: 9.39%; found: C: 68.37%, H: 6.10%, N: 9.41%. IR (cm⁻¹): 3307 (OH), 3181 (NH), 3083 (CH, arom.), 2969 (CH, aliph.), 1659 (C=O), 1604 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 1.34 (t, 3H, CH₃, J = 6 Hz), 3.57 (s, 2H, CH₂), 4.05 (q, 2H, CH₂, J = 6 Hz), 6.80–6.84 (m, 1H, ArH), 6.97–7.01 (m, 1H, ArH), 7.10 (d, 1H ArH, J = 6 Hz), 7.21–7.28 (m, 2H, ArH), 7.30–7.35 (m, 3H, ArH), 8.42 (s, 1H, =CH), 11.37 (s, 1H, OH), 11.84 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 15.21 (CH₃), 41.45 (CH₂), 64.59 (OCH₂), 115.63, 119.47, 121.35, 127.13, 128.70, 128.83, 129.59, 129.83, 135.91, 141.40 (12C_ar), 147.50 (=CH), 166.79 (C=O).

N′-[(E)-(2-hydroxy-3-nitrophenyl)methylidene]-2-phenylacetohydrazide (12)

Color: orange; M.p.: 176 °C; yield: 67%; for C₁₅H₁₃N₃O₄ (299.28) calculated: C: 60.20%, H: 4.38%, N: 14.04%; found: C: 60.11%, H: 4.39%, N: 14.08%. IR (cm⁻¹): 3484 (OH), 3177, 3107 (NH), 3077 (CH, arom.), 2948 (CH, aliph.), 1663 (C=O), 1614 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.62 (s, 2H, CH₂), 7.10–7.15 (m, 1H, ArH), 7.23–7.28 (m, 1H, ArH), 7.28–7.36 (m, 5H, ArH), 7.89–7.99 (m, 1H, ArH), 8.50 (s, 1H, =CH), 11.61 (s, 1H, OH), 12.17 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.30 (CH₂), 119.62, 122.10, 126.96, 127.23, 128.87, 129.61, 135.40, 138.08, 138.96 (11C_ar), 146.33 (=CH), 151.94 (C_ar), 167.23 (C=O).

N′-[(E)-(2-hydroxy-5-nitrophenyl)methylidene]-2-phenylacetohydrazide (13)

Color: yellow; M.p.: 232–234 °C; yield: 83%; for C₁₅H₁₃N₃O₄ (299.28) calculated: C: 60.20%, H: 4.38%, N: 14.04%; found: C: 60.38%, H: 4.36%, N: 14.01%. IR (cm⁻¹): 3626 (OH), 3286 (NH), 3068 (CH, arom.), 2932 (CH, aliph.), 1655 (C=O), 1603 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.59 (s, 2H, CH₂), 7.08 (t, 1H, ArH, J = 6 Hz, J = 12 Hz), 7.22–7.28 (m, 2H, ArH), 7.32–7.36 (m, 2H, ArH), 8.13–8.17 (m, 1H, ArH), 8.28 (s, 1H, ArH), 8.51 (s, 1H, =CH), 8.54–8.57 (m, 1H, ArH), 11.54 (s, 1H, OH), 12.03 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.45 (CH₂), 117.55, 121.87, 124.27, 127.17, 128.84, 129.58, 135.78, 140.36 (10C_ar), 143.76 (=CH), 162.25, 162.97 (2C_ar), 167.23 (C=O).

N′-[(E)-(4-hydroxy-3-nitrophenyl)methylidene]-2-phenylacetohydrazide (14)

Color: orange; M.p.: 180 °C; yield: 87%; for C₁₅H₁₃N₃O₄ (299.28) calculated: C: 60.20%, H: 4.38%, N: 14.04%; found: C: 60.25%, H: 4.39%, N: 14.08%. IR (cm⁻¹): 3580 (OH), 3284 (NH), 3056 (CH, arom.), 2943 (CH, aliph.), 1661 (C=O), 1622 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.54 (s, 2H, CH₂), 7.18–7.27 (m, 2H, ArH), 7.29–7.34 (m, 3H, ArH), 7.87–7.92 (m, 1H, ArH), 7.96 (s, 1H, ArH), 8.15–8.17 (m, 1H, ArH), 8.18 (s, 1H, =CH), 11.42 (s, 1H, OH), 11.66 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.64 (CH₂), 120.13, 124.13, 126.84, 128.69, 129.87, 132.92, 136.21, 141.13 (10C_ar), 144.94 (=CH), 153.43, 153.72 (2C_ar), 167.02 (C=O).

N′-[(E)-(2-hydroxy-3,5-dinitrophenyl)methylidene]-2-phenylacetohydrazide (15)

Color: yellow; M.p.: 232–234 °C; yield: 79%; for C₁₅H₁₂N₄O₆ (344.28) calculated: C: 52.33%, H: 3.51%, N: 16.27%; found: C: 52.50%, H: 3.50%, N: 16.31%. IR (cm⁻¹): 3487 (OH), 3162 (NH), 3084 (CH, arom.), 2993 (CH, aliph.), 1659 (C=O), 1615 (C=N); ¹H NMR (600 MHz, DMSO-d₆): 3.64 (s, 2H, CH₂), 7.22–7.29 (m, 2H, ArH), 7.32–7.36 (m, 3H, ArH), 8.60 (s, 1H, =CH), 8.67–8.69 (m, 1H, ArH), 8.75–8.79 (m, 1H, ArH), 11.64 (s, 1H, OH), 12.39 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.22 (CH₂), 122.83, 123.35, 125.13, 127.26, 128.69, 129.62, 129.85, 135.49, 137.79 (11C_ar), 144.95 (=CH), 158.35 (C_ar), 167.49 (C=O).

2-phenyl-N′-[(E)-(2,3,4-trihydroxyphenyl)methylidene]acetohydrazide (16)

CAS Number: 1935707-89-3; Color: white; M.p.: 222–224 °C; yield: 52%; for C₁₅H₁₄N₂O₄ (286.28) calculated: C: 62.93%, H: 4.93%, N: 9.79%; found: C: 62.85%, H: 4.95%, N: 9.78%. ¹H NMR (600 MHz, DMSO-d₆): 3.55 (s, 2H, CH₂), 6.37 (d, 1H, ArH, J = 12 Hz), 6.77 (d, 1H, ArH, J = 6 Hz), 7.22–7.35 (m, 5H, ArH), 8.24 (s, 1H, =CH), 8.48 (s, 1H, OH), 9.46 (s, 1H, OH), 11.32 (s, 1H, OH), 11.70 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO-d₆): 41.37 (CH₂), 108.06, 111.18, 121.50, 127.10, 128.82, 129.58, 133.12, 136.02 (10C_ar), 147.82 (=CH), 149.13, 149.43 (2C_ar), 166.43 (C=O).

2.2. Lipophilicity

The reversed-phase thin-layer chromatography on 10 × 20 cm RP18 F₂₅₄ plates (E. Merck, Darmstadt, Germany) was applied to determine the experimental lipophilicity of the synthesized hydrazide-hydrazones of phenylacetic acid 2–16 (

Table 1. The logP values from the literature [34] and the calculated R_M0 values for the reference substances.

	RM0	S	r2	φ
Acetonitrile-Water
2-aminophenol	1.1768	−0.02	0.9964	54.37
salicylamide	1.2269	−0.03	0.9970	44.77
4-dimethylaminebenzaldehyde	1.5319	−0.02	0.9843	62.20
eugenol	2.0104	−0.03	0.9879	62.44
thymol	2.4306	−0.04	0.9890	68.33
phenyl salicylate	2.9514	−0.04	0.9873	79.68
1,4-Dioxane-Water
2-aminophenol	1.0844	−0.02	0.9916	47.21
salicylamide	0.9964	−0.02	0.9957	45.60
4-dimethylaminebenzaldehyde	1.6406	−0.03	0.9928	56.20
eugenol	1.9741	−0.03	0.9940	66.90
thymol	2.5440	−0.04	0.9828	69.90
phenyl salicylate	3.6480	−0.05	0.9940	73.84
Methanol-Water
2-aminophenol	0.9785	−0.02	0.9975	58.90
salicylamide	1.1377	−0.02	0.9817	56.60
4-dimethylaminebenzaldehyde	2.1143	−0.03	0.9891	75.02
eugenol	2.7002	−0.04	0.9937	75.91
thymol	3.2151	−0.04	0.9957	80.56
phenyl salicylate	4.2259	−0.05	0.9724	88.04

φ is the amount of organic modifier in the mobile phase. R_M0 and S are the intercept and slope of the linear regression equation, respectively. r² is the correlation coefficient.

).

Our analysis was performed in a horizontal Teflon chamber with an eluent distributor (DS Chromdes Lublin, Poland), and the chromatograms were developed at the distance of 9 cm from the origin of the plate at a temperature of 24 ± 0.5 °C. The spots of tested compounds 2–16 were located under ultraviolet illumination (λ = 254 nm).

The reference substances with known lipophilicity [34] (2-aminophenol, logP 0.62; salicylamide, logP 1.28; 4-dimethylaminebenzaldehyde, logP 1.81; eugenol, logP 2.27; thymol, logP 3.3; phenyl salicylate, logP 3.8) were obtained from Sigma-Aldrich Co. (Saint Louis, MI, USA) or Merck Co. (Darmstadt, Germany). The solvents we used had been purchased from Polish Chemical Reagents POCh (Gliwice, Poland) and employed without any further modification. The mobile phases used in this experiment were prepared by mixing appropriate amounts of water and adequate polar modifier (50–75% of acetonitrile, 50–70% of 1,4-dioxane and 60–85% of methanol).

Hydrazide-hydrazones of phenylacetic acid (2–16) and the reference substances were dissolved in methanol to obtain concentrations of 2.0 mg/mL and volumes of 0.2 μL. Then they were applied onto the RP-TLC plates.

The R_M values were calculated for the standard and for the tested compounds (2–16) on the basis of the obtained retardation coefficient (R_F) values with the use of the following known chromatographic formula:

$${\mathrm{R}}_{\mathrm{M}}=\mathrm{l}\mathrm{o}\mathrm{g}{\displaystyle \frac{(1-{\mathrm{R}}_{\mathrm{F}})}{{\mathrm{R}}_{\mathrm{F}}}}$$

The R_M0 values equivalent to the retention of the compound extrapolated to pure water as a mobile phase were calculated on the basis of the equation R_M = R_M0 − Sφ, where φ was the volume fraction of the organic modifier in the mobile phase. The calibration curves for further lipophilicity estimation (logP_EXP) were obtained by correlating the calculated R_M0 values for six reference substances with their logP values. The experimental lipophilicity of tested substances 2–16 was calculated on the basis of the established calibration equations and their R_M0 values.

2.3. Microbiology

In Vitro Antimicrobial Assay

The in vitro antimicrobial assays of obtained acylhydrazones 2–16 were performed according to EUCAST [35] and CLSI [36] standards. A detailed description of these procedures is provided in the Supplementary Materials and in earlier articles published by our research group [37,38,39].

3. Results

3.1. Chemistry

In our current research, we managed to synthesize fifteen hydrazide-hydrazones of phenylacetic acid (2–16). Hydrazide of phenylacetic acid (1) was used as a starting compound, which produced corresponding acylhydrazones of this acid 2–16 in condensation reactions with substituted appropriate aromatic aldehydes. Microwave-assisted synthesis consisted of the heating of reagents in the microwave synthesizer in specific conditions (power of microwave—300 W, time of the reaction—7 min, temperature—155 °C) (Scheme 1).

Among synthesized compounds 2–16, acylhydrazones 2–6, 13 and 16 [40,41,42,43,44,45,46] are known in scientific literature, but they were evaluated for the first time against a wide panel of microorganisms.

The yields of the performed condensation reactions were in the range of 37–95%. The acylhydrazone 7—N′-[(E)-(5-chloro-2-hydroxy-3-iodophenyl)methylidene]-2-phenylacetohydrazide was synthesized with the lowest yield (37%), whereas compound 3—N′-[(E)-(3,5-dibromo-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide was synthesized with the highest yield (95%).

The melting points of synthesized compounds 2–16 were within the range of 134–282°C. The lowest melting temperature (134–136 °C) was recorded for acylhydrazone 11: N′-[(E)-(3-ethoxy-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide, whereas compound 5: N′-[(E)-3,5-dichloro-2-hydroxyphenyl)methylidene]-2-phenylacetohydrazide, was characterized by the highest one (282 °C).

The IR, ¹H NMR and ¹³C NMR spectroscopies were used to establish the chemical structures of the obtained acylhydrazones of phenylacetic acid 2–16. Examples of IR, ¹H NMR and ¹³C NMR spectra are presented in Supplementary Materials (Figures S1–S9).

3.2. Lipophilicity

It is well known that chromatographic methods allow fast and reproducible determination of experimental lipophilicity. In our research, we applied the standardization procedure with six reference substances (range of lipophilicity of 0.62–3.80) [34]. Their logP values were in high correlation with their R_M0 values in three organic modifiers, i.e., acetonitrile, 1,4-dioxane and methanol solvent systems (

Table 2. The R_M0 values of the synthesized acylhydrazones of phenylacetic acid 2–16.

Compound No	Acetonitrile-Water				1,4-Dioxane-Water				Methanol-Water
	RM0	S	r2	φ	RM0	S	r2	φ	RM0	S	r2	φ
2	2.7793	−0.04	0.9722	70.86	3.1301	−0.05	0.9803	64.17	4.0434	−0.05	0.9931	83.00
3	3.1071	−0.04	0.9792	76.11	3.7815	−0.06	0.9717	67.54	4.8978	−0.06	0.9913	87.08
4	2.4385	−0.04	0.9849	68.03	3.2336	−0.05	0.9927	61.91	3.9436	−0.05	0.9862	80.80
5	2.8437	−0.04	0.9872	73.71	3.8941	−0.06	0.9997	65.10	4.7399	−0.06	0.9886	84.77
6	2.9758	−0.04	0.9918	74.58	3.9957	−0.06	0.9955	65.42	4.7418	−0.04	0.9983	86.00
7	3.1485	−0.04	0.9863	76.34	4.0481	−0.06	0.9978	66.01	4.8895	−0.05	0.9913	87.24
8	3.3686	−0.04	0.9747	79.02	4.0034	−0.06	0.9846	68.82	5.0271	−0.06	0.9979	88.90
9	2.0621	−0.04	0.9916	58.52	2.4781	−0.04	0.9939	58.01	3.1511	−0.04	0.9866	76.25
10	2.1722	−0.04	0.9873	61.40	2.6718	−0.05	0.9926	59.00	3.5089	−0.04	0.9943	78.19
11	2.1966	−0.04	0.9834	62.39	2.4623	−0.04	0.9957	58.57	3.3256	−0.06	0.9932	77.28
12	2.3122	−0.04	0.9858	62.26	2.7184	−0.05	0.9985	60.10	3.3190	−0.06	0.9913	78.44
13	2.5229	−0.04	0.9916	63.57	2.9981	−0.05	0.9932	59.95	3.5991	−0.05	0.9902	79.83
14	2.1061	−0.04	0.9838	59.31	2.4686	−0.04	0.9988	58.77	3.1517	−0.04	0.9891	76.62
15	0.8263	−0.02	0.9903	40.06	1.0641	−0.02	0.9882	45.35	2.4159	−0.04	0.9783	65.56
16	1.2273	−0.03	0.9732	43.00	1.0413	−0.03	0.9948	45.60	1.7280	−0.03	0.9809	63.13

φ is the amount of organic modifier in the mobile phase. R_M0 and S are the intercept and slope of the linear calibration equation, respectively. r² is the correlation coefficient.

). On the basis of this fact, the appropriate calibration curves for a further lipophilicity study were calculated:

(1)

acetonitrile: logP_EXP = (R_M0 − 0.6348)/0.5749; r² = 0.9568

(2)

1,4-dioxane: logP_EXP = (R_M0 − 0.2591)/0.7899; r² = 0.9098

(3)

methanol: logP_EXP = (R_M0 − 0.1883)/1.0124; r² = 0.9568

The obtained coefficients of determination (r²) for the mentioned equations were above 0.90 for three organic modifiers (i.e., acetonitrile, 1,4-dioxane and methanol). Similarly, for our six reference compounds, the correlations between the R_F and R_M0 values were sufficiently high (r² ≥ 0.97) with all solvents used.

Better correlations (i.e., r² ≥ 0.99 for compounds 7 or 16) were calculated for methanol and 1,4-dioxane solvent systems, providing accuracy for further lipophilicity determination (Table 2).

The logP_EXP values (experimental lipophilicity) of the fifteen tested substances 2–16 were calculated on the basis of the above calibration equations and respective R_M0 values (

Table 3. The logP_EXP values of the synthesized hydrazide-hydrazones of phenylacetic acid 2–16.

Compound Number	LogPacetonitrile	LogP1.4-dioxane	LogPmethanol
2	3.73	3.63	3.81
3	4.30	4.46	4.65
4	3.14	3.77	3.71
5	3.84	4.60	4.50
6	4.07	4.73	4.50
7	4.37	4.80	4.64
8	4.76	4.74	4.78
9	2.48	2.81	2.93
10	2.67	3.05	3.28
11	2.72	2.79	3.10
12	2.92	3.11	3.09
13	3.28	3.47	3.37
14	2.56	2.80	2.93
15	0.33	1.02	2.20
16	1.03	0.99	1.52

).

3.3. Microbiology

In antibacterial activity screening, we used a panel of microorganisms (eight strains of Gram-positive bacteria, six strains of Gram-negative bacteria and six strains of yeasts belonging to Candida spp.—

Table 4. The activity data of studied compounds 2–9 expressed as MIC (MBC or MFC) [µg/mL] and {MBC/MIC or MFC/MIC} values against the reference strains of bacteria and fungi.

Species		MIC (MBC or MFC) [µg/mL] and {MBC/MIC or MFC/MIC} Values of the Studied Compounds and Positive Controls
		2	3	4	5	6	7	9	CIP/VA */ NY **	NIT	CFX	APC
Gram-positive bacteria	Staphylococcus aureus ATCC 43300	1000 (>1000) {>1}	-	1000 (>1000) {>1}	-	-	-	-	0.24 (0.24) {1}	7.81 (15.62)	0.49	nd
	Staphylococcus aureus ATCC 6538	1000 (>1000) {>1}	-	1000 (>1000) {>1}	-	1000 (>1000) {>1}	-	-	0.24 (0.24) {1}	15.62 (15.62)	nd	nd
	Staphylococcus aureus ATCC 29213	1000 (>1000) {>1}	1000 (>1000) {>1}	-	-	250 (>1000) {>4}	-	-	0.48 (0.48) {1}	nd	nd	nd
	Staphylococcus epidermidis ATCC 12228	-	500 (>1000) {>2}	-	-	62.5 (>1000) {>16}	-	-	0.12 (0.12) {1}	3.91 (7.81)	0.24	nd
	Enterococcus faecalis ATCC 29212	250 (>1000) {>4}	-	250 (>1000) {>4}	-	31.25 (>1000) {>32}	-	1000 (>1000) {>1}	0.98 * (1.95) {2}	nd	nd	nd
	Micrococcus luteus ATCC 10240	1000 (>1000) {>1}	250 (>1000) {>4}	250 (>1000) {>4}	250 (>1000) {>4}	31.25 (>1000) {>32}	-	250 (>1000) {>4}	0.98 (1.95) {2}	62.5 (62.5)	0.98	nd
	Bacillus subtilis ATCC 6633	-	-	-	-	500 (>1000) {>2}	-	-	0.03 (0.03) {1}	3.91 (3.91)	15.62	62.5
	Bacillus cereus ATCC 10876	1000 (>1000) {>1}	500 (>1000) {>2}	-	-	250 (250) {1}	-	-	0.06 (0.12) {2}	7.81 (15.62)	31.25	nd
Gram-negative bacteria	Bordetella bronchiseptica ATCC 4617	-	-	-	-	-	-	-	0.98 (0.98) {1}	125 (>1000)	nd	nd
	Klebsiella pneumoniae ATCC 13883	-	-	-	-	-	-	-	0.12 (0.24) {2}	15.62 (31.25)	nd	nd
	Proteus mirabilis ATCC 12453	-	-	-	-	-	-	-	0.03 (0.03) {1)	62.5 (125)	nd	nd
	Salmonella typhimurium ATCC 14028	-	-	-	-	-	-	-	0.06 (0.06) {1}	31.25 (62.5)	nd	nd
	Escherichia coli ATCC 25922	-	-	-	-	-	-	-	0.004 (0.008) {2}	7.81 (15.62)	nd	nd
	Pseudomonas aeruginosa ATCC 9027	-	-	-	-	-	-	-	0.48 (0.98) {2}	nd	nd	nd
Fungi	Candida auris CDC 311903	1000 (>1000) {>1}	-	-	-	-	-	-	0.48 ** (0.48) {1}	na	na	na
	Candida albicans ATCC 2091	1000 (>1000) {>1}	-	-	-	1000 (>1000) {>1}	-	-	0.24 ** (0.24) {1}	na	na	na
	Candida albicans ATCC 10231	1000 (>1000) {>1}	1000 (>1000) {>1}	1000 (>1000) {>1}	-	500 (>1000) {>2}	1000 (>1000) {>1}	-	0.48 ** (0.48) {1}	na	na	na
	Candida parapsilosis ATCC 22019	500 (>1000) {>2}	500 (>1000) {>2}	500 (>1000) {>2}	-	1000 (>1000) {>1}	1000 (>1000) {>1}	1000 (>1000) {>1}	0.24 ** (0.48) {2}	na	na	na
	Candida glabrata ATCC 90030	1000 (>1000) {>1}	-	-	-	1000 (>1000) {>1}	-	-	0.24 ** (0.48) {2}	na	na	na
	Candida krusei ATCC 14243	-	-	-	-	-	-	-	0.24 ** (0.24) {1}	na	na	na

The standard antimicrobial agents used as positive controls: ciprofloxacin (CIP), nitrofurantoin (NIT), cefuroxime (CFX) and ampicillin (APC) for bacteria (except enterococci), vancomycin (VA *) for enterococci and nystatin (NY **) for fungi; “-” no activitiy; nd, not determined; na, not applicable.

and

Table 5. The activity data of studied compounds 10–16 expressed as MIC (MBC or MFC) [µg/mL] and {MBC/MIC or MFC/MIC} values against the reference strains of bacteria and fungi.

Species		MIC (MBC or MFC) [µg/mL] and {MBC/MIC or MFC/MIC} Values of the Studied Compounds and Positive Controls
		10	11	12	13	14	15	16	CIP/VA * /NY **	NIT	CFX	APC
Gram-positive bacteria	Staphylococcus aureus ATCC 43300	-	15.62 (>1000) {>64}	250 (>1000) {>4}	15.62 (>1000) {>64}	-	-	1.95 (1.95) {1}	0.24 (0.24) {1}	7.81 (15.62)	nd	nd
	Staphylococcus aureus ATCC 6538	-	15.62 (>1000) {>64}	250 (>1000) {>4}	1.95 (>1000) {>512}	500 (>1000) {>2}	1000 (>1000) {>1}	7.81 (31.25) {4}	0.24 (0.24) {1}	15.62 (15.62)	0.98	nd
	Staphylococcus aureus ATCC 29213	-	15.62 (>1000) {>64}	250 (>1000) {>4}	15.62 (>1000) {>64}	125 (>1000) {>8}	-	7.81 (7.81) {1}	0.48 (0.48) {1}	nd	nd	nd
	Staphylococcus epidermidis ATCC 12228	-	62.5 (>1000) {>16}	62.5 (>1000) {>16}	7.81 (>1000) {>128}	31.25 (>1000) {>32}	500 (>1000) {>2}	3.91 (3.91) {1}	0.12 (0.12) {1}	3.91 (7.81)	0.24	nd
	Enterococcus faecalis ATCC 29212	-	125 (>1000) {>8}	500 (>1000) {>2}	500 (>1000) {>2}	1000 (>1000) {>1}	125 (>1000) {>8}	1000 (>1000) {>1}	0.98 * (1.95) {2}	nd	nd	nd
	Micrococcus luteus ATCC 10240	500 (>1000) {>2}	31.25 (>1000) {>32}	125 (>1000) {>8}	31.25 (>1000) {>32}	500 (>1000) {>2}	500 (>1000) {>2}	125 (250) {2}	0.98 (1.95) {2}	62.5 (62.5)	0.98	nd
	Bacillus subtilis ATCC 6633	-	250 (>1000) {>4}	125 (250) {2}	250 (>1000) {>4}	250 (>1000) {>4}	1000 (>1000) {>1}	500 (1000) {2}	0.03 (0.03) {1}	3.91 (3.91)	15.62	62.5
	Bacillus cereus ATCC 10876	-	62.5 (>1000) {>16}	125 (>1000) {>8}	250 (>1000) {>4}	1000 (>1000) {>1}	-	500 (500) {1}	0.06 (0.12) {2}	7.81 (15.62)	31.25	nd
Gram-negative bacteria	Bordetella bronchiseptica ATCC 4617	-	-	-	-	-	-	1000 (>1000) {>1}	0.98 (0.98) {1}	125 (>1000)	nd	nd
	Klebsiella pneumoniae ATCC 13883	-	-	-	-	-	-	31.25 (31.25) {1}	0.12 (0.24) {2}	15.62 (31.25)	nd	nd
	Proteus mirabilis ATCC 12453	-	-	-	-	-	-	500 (500) {1}	0.03 (0.03) {1)	62.5 (125)	nd	nd
	Salmonella typhimurium ATCC 14028	-	-	-	-	-	-	500 (500) {1}	0.06 (0.06) {1}	31.25 (62.5)	nd	nd
	Escherichia coli ATCC 25922	-	-	-	-	-	-	500 (1000) {2}	0.004 (0.008) {2}	7.81 (15.62)	nd	nd
	Pseudomonas aeruginosa ATCC 9027	-	-	-	-	-	-	500 (500) {1}	0.48 (0.98) {2}	nd	nd	nd
Fungi	Candida auris CDC 311903	-	-	-	-	-	-	-	0.48 ** (0.48) {1}	na	na	na
	Candida albicans ATCC 2091	-	-	-	-	-	-	1000 (>1000) {>1}	0.24 ** (0.24) {1}	na	na	na
	Candida albicans ATCC 10231	-	-	-	-	-	-	1000 (>1000) {>1}	0.48 ** (0.48) {1}	na	na	na
	Candida parapsilosis ATCC 22019	-	-	-	-	-	-	-	0.24 ** (0.48) {2}	na	na	na
	Candida glabrata ATCC 90030	-	-	-	-	-	-	-	0.24 ** (0.48) {2}	na	na	na
	Candida krusei ATCC 14243	-	-	-	-	-	-	-	0.24 ** (0.24) {1}	na	na	na

The standard antimicrobial agents used as positive controls: ciprofloxacin (CIP), nitrofurantoin (NIT), cefuroxime (CFX) and ampicillin (APC) for bacteria (except enterococci), vancomycin (VA *) for enterococci and nystatin (NY **) for fungi; “-” no activitiy; nd, not determined; na, not applicable.

).

Our results show varied antimicrobial bioactivity of tested compounds (2–16). Gram-positive bacteria, especially the strains belonging to Staphylococcus, were the most sensitive to these substances. Among them, compound 16 had the highest activity against all staphylococci with a very strong bactericidal effect (MIC = 1.95–7.81 µg/mL, MBC = 1.95–31.25 µg/mL and MBC/MIC = 1–4). Moreover, the next substance 13 indicated similar activity at MIC in the range from 1.95 to 15.62 µg/mL and very strong or strong activity (but with bacteriostatic effect) towards these bacteria. Compound 11 was also noteworthy due to its strong bioactivity (MIC = 15.62 µg/mL) against all Staphylococcus aureus strains and good activity (MIC = 62.5 µg/mL) towards Staphylococcus epidermidis ATCC 12228. Its effect, similar to that of substance 13, was bacteriostatic. The remaining Gram-positive bacteria (Micrococcus luteus ATCC 10240, Enterococcus faecalis ATCC 29212 and Bacillus spp.) were slightly less sensitive to substances 11, 13 and 16 (MIC = 31.25–1000 µg/mL), which indicated good and moderate activity of the substances or their mild effect on E. faecalis ATCC 29212 (compound 16). Other compounds, namely 6, 12 and 14, mainly showed bioactivity at MIC = 31.25–1000 µg/mL and MBC = 250–>1000 µg/mL against these microorganisms, especially against S. epidermidis ATCC 12228 (good activity at MIC = 31.25–62.5 µg/mL). S. aureus ATCC 43300, insensitive to substances 6 and 14, was exceptional in this respect. In turn, other compounds had weaker (MIC = 125–1000 µg/mL) or absent (MIC > 1000 µg/mL) antibacterial effects. Moreover, all compounds except one, i.e., hydrazide-hydrazone 7, inhibited the growth of Micrococcus luteus ATCC 10240 in the MIC range from 31.25 to 1000 µg/mL. Additionally, substance 7 showed no activity towards any cocci or bacilli (Table 4 and Table 5).

In the case of the reference Gram-negative bacteria, only compound 16 showed some bioactivity. The rods from the Enterobacterales family and Pseudomonas aeruginosa ATCC 9027 were sensitive to this substance at MIC = 31.25–500 µg/mL and MBC = 31.25–1000 µg/mL. The bioactivity of compound 16 with a bactericidal effect (MBC/MIC = 1–2) was good towards Klebsiella pneumoniae ATCC 13883 or moderate against other rods. Bordetella bronchiseptica ATCC 4617 was the least susceptible to this substance (MIC = 1000 µg/mL and MBC > 1000 µg/mL). All remaining compounds showed no activity towards Gram-negative microorganisms (Table 4 and Table 5).

Our data presented in Table 4 and Table 5 also indicate potential antifungal activity of the tested compounds towards yeasts. Among them, substances 2, 3, 4, 6, 7, 9 and 16 showed moderate and mild anticandidal effect or no bioactivity. The minimal concentrations of selected substances that inhibited the growth of Candida spp. ranged from 500 to 1000 µg/mL. The MBC values exceeded 1000 µg/mL in each case. Two strains, Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019, were most sensitive to these compounds. On the other hand, Candida krusei ATCC 14243 was unsusceptible to all tested substances. The other compounds had no antifungal activity (Table 4 and Table 5).

4. Discussion

4.1. Chemistry

Spectral methods (IR, ¹H NMR and ¹³C NMR) were applied to establish the chemical structure of synthesized hydrazide-hydrazones of phenylacetic acid 2–16. In the ¹H NMR spectra of acylhydrazones, the singlet signal for proton of =CH group can be found at δ 8–9 ppm. The signal for the carbon atom of the =CH group in the ¹³C NMR spectra is often found in the range of δ 145–160 ppm [11]. Additionally, compounds from this group show, among others, a signal for a carbonyl group (C=O) at around 1650 cm⁻¹ in the IR spectra [11].

In the case of synthesized hydrazide-hydrazones of phenylacetic acid (2–16) in the ¹H NMR spectra, we found singlet signals for the proton of the =CH group in the range of δ 8.06–8.60 ppm. In the ¹³C NMR spectra signals for the carbon atom of the =CH group were found at δ 143.76–149.79 ppm. In the IR spectra of acylhydrazones of phenylacetic acid (2–16), a signal for a carbonyl group (C=O) appeared in the range of 1643–1664 cm⁻¹.

4.2. Lipophilicity

Lipophilicity is a factor that significantly affects the biological activity of drugs. It plays a very important role in the interaction of the drug with the receptor, determining the toxicity and solubility of the substance. Lipophilicity influences pharmacokinetic processes in living organisms, especially the distribution process, i.e., the transportation of a drug across biological membranes [47]. The knowledge of lipophilicity parameters is therefore very important, both for the research devoted to searching for new drug structures with the required biological activity and for understanding the correct mechanisms of pharmacokinetics and distributing drugs already present on the market.

In the case of methanol–water solvent systems (Table 2 and Table 3), the highest logP_EXP values of the tested hydrazide-hydrazones of phenylacetic acid 2–16 were calculated, with the exception of substances (4–7, and 13). The lower logP_EXP values were found for acetonitrile (except for acylhydrazones 1 and 16).

For all solvent systems used in our experiments, the highest logP_EXP values were calculated for compounds 3, 5, 6, 7 and 8 containing two halogen substituents (2-hydroxyphenyl) in ortho- and para-positions. Due to this fact, it can be stated that the position types of the halogen atoms in a 2-hydroxyphenyl ring did not affect the lipophilicity of these five acylhydrazones (3, and 5–8).

In the case of 2-hydroxynitrophenyl-substituted acylhydrazones (compounds 12, 13 and 14), differences were clearly seen. Hydrazide-hydrazone 13, which was substituted with a 2-hydroxy-5-nitrophenyl, displayed the highest logP values for all solvent systems used in our experiments, which illustrates the importance of positional isomerism in the lipophilicity of these compounds. The acylhydrazone 2 substituted with 5-bromo-2-hydroxyphenyl showed similar logP values to compound 4 with 5-chloro-2-hydroxyphenyl substituent (Table 2 and Table 3).

When compounds 9, 10 and 11 are compared, the 2-hydroxy-3-ethoxyphenyl-substituted hydrazide-hydrazone of phenylacetic acid (11) was found to be lipophilic, just like compound 10, which possessed a 3-methoxy-4-hydroxy-5-iodophenyl or acylhydrazone 9 substituted with a 2-bromo-3-hydroxy-5-methoxyphenyl. Such a situation was registered in the case of all solvent systems used. Bearing in mind all the tested acylhydrazones of phenylacetic acid 2–16, the lowest lipophilicity was shown for compounds 15 and 16 substituted with the 2-hydroxy-3,5-dinitrophenyl and 2,3,4-trihydroxyphenyl, respectively (Table 2 and Table 3).

4.3. Microbiology

On the basis of the presented antimicrobial activity assays in vitro, it can be stated that in some cases, the antibacterial activities of tested hydrazide-hydrazones 2–16 were better or equal to those of commonly used antibacterial agents, which we employed as reference substances (nitrofurantoin, cefuroxime or ampicillin).

Acylhydrazone 16 showed four times greater activity (MIC = 1.95 µg/mL) than nitrofurantoin (MIC = 7.81 µg/mL) towards methicillin-resistant Staphylococcus aureus ATCC 43300 strain. The same compound again displayed the same activity as nitrofurantoin against Staphylococcus epidermidis ATCC 12228 (MIC = 3.91 µg/mL). Additionally, its activity against these bacterial stains was bactericidal (MBC/MIC = 1).

The highest activity against Staphylococcus aureus ATCC 6538 (MSSA strain), a methicillin-susceptible strain was shown for compounds 11, 13 and 16. The minimal inhibitory concentration value towards this strain for compound 11 (MIC = 15.62 µg/mL) was equal to the activity of nitrofurantoin (MIC = 15.62 µg/mL). Then, compounds 13 (MIC = 1.95 µg/mL) and 16 (MIC = 7.81 µg/mL) showed an activity level that was, respectively, eight times and two times higher against this bacterium than the activity of nitrofurantoin (MIC = 15.62 µg/mL).

It is also worth underlining the activity of the tested hydrazide-hydrazones towards Micrococcus luteus ATCC 10240. The antibacterial activity of compounds 6, 11 and 13 (MIC = 31.25 µg/mL) was two times higher than the activity of nitrofurantoin (MIC = 62.5 µg/mL).

It can be stated that the highest antibacterial activity was manifested by hydrazide-hydrazones of phenylacetic acid, which were substituted with 3-bromo-5-chloro-2-hydroxyphenyl, 3-ethoxy-2-hydroxyphenyl, 2-hydroxy-5-nitrophenyl and 2,3,4-trihydroxyphenyl substituents from the hydrazone part of the compound.

The antibacterial activity towards Gram-positive bacteria falls when beside the hydroxyl group in position two in the phenyl ring two identical halogen atoms (compounds 3 and 5) are also present. When only one halogen atom is present, the antibacterial activity rises (acylhydrazones 2 and 4). Where two halogen atoms are present in the chemical structure of hydrazide-hydrazone, the activity rises (substance 6), but this fact is not observed when one of these atoms is iodine (compound 7).

The presence of the OH group in position two in the phenyl ring in the acylhydrazone of phenylacetic acid is crucial for the antibacterial activity. When this group is placed in a different position, the activity against Gram-positive bacteria is much lower (compound 10). The significant rise in the activity is especially seen when more than one hydroxyl group (compound 16) is present in the structure of tested acylhydrazones 2–16. The presence of more than one OH group is also crucial to the activity against Gram-negative bacterial strains (acylhydrazone 16).

In the case of the nitro group in the structure of tested acylhydrazones (12, 13, 14), its presence increases the antibacterial activity against Gram-positive bacteria. The introduction of two NO₂ groups lowers the activity (substance 15).

In general, the highest antibacterial activity was shown by hydrazide-hydrazones with more than one hydroxyl group (16) and without halogen atoms (11). A lower activity was displayed by the compounds with one NO₂ group (12, 13, 14) and two NO₂ groups. Finally, the lowest activity was shown by acylhydrazones with one or two halogen atoms (2–5, 7).

5. Conclusions

In this paper, we described the microwave-assisted synthesis and chemical structure determination of fifteen hydrazide-hydrazones of phenylacetic acid 2–16. Experimental lipophilicity of the obtained compounds was determined with the use of chromatographic methods. The antimicrobial activity results in vitro showed a potential antimicrobial effect of the tested hydrazide-hydrazones 2–16. Among them, substances 6, 11–14, and 16 manifested the greatest activity towards reference Gram-positive bacteria, especially against Staphylococcus spp. with a very strong bactericidal effect of substance 16 (MIC = 1.95–7.81 µg/mL). The remaining compounds showed varied and weaker activities. It seems practical to use our selected compounds in the prevention and treatment of some infections in the future.