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Research Article

Hydrazide-hydrazones of 3-methoxybenzoic acid and 4-tert-butylbenzoic acid with promising antibacterial activity against Bacillus spp

Łukasz PopiołekDepartment of Organic Chemistry and Correspondence[email protected]
&
Anna BiernasiukDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, Lublin, Poland
Pages 62-69 | Received 22 Jan 2016, Accepted 04 Mar 2016, Published online: 13 Apr 2016

Abstract

A series of 28 hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acid were synthesized and screened in vitro against the panel of reference strains of bacteria and fungi with the use of the broth microdilution method according to EUCAST and CLSI guidelines. Five of the synthesized compounds were found to exhibit high bacteriostatic or bactericidal activity against Gram-positive bacteria. The antimicrobial activity of compounds 13, 14, and 16 against Bacillus spp. was higher than that of commonly used antibiotics, like cefuroxime or ampicillin.

Introduction

One of the main reasons why the infectious microbial diseases still remain pressing problems worldwide is the widespread excessive use of antibacterial agents, which leads to the development of bacteria more resistant to commonly used antibioticsCitation1. Consequently, there is still need to explore safe and potent chemotherapeutic agents. It is desirable to discover drugs with improved potency, wide activity spectrum and low toxicityCitation2.

In many reports, besides being useful building blocks for the synthesis of a variety of heterocyclic rings and in drug developmentCitation3,Citation4, hydrazide-hydrazones are considered to be good scaffolds for different pharmaceutical applicationsCitation5–11 and have a significant role to play as antimicrobial agentsCitation12–16. The biological activity associated with these compounds is attributed to the presence of the highly reactive azomethine group (–CO–NH–N=CH–). Moreover, some widely used chemotherapeutic drugs such as nitrofurazone and furazolidone are known to contain this group ()Citation17–19.

Figure 1. Chemotherapeutic drugs containing hydrazide–hydrazone moiety: nitrofurazone (a) and furazolidone (b).

Figure 1. Chemotherapeutic drugs containing hydrazide–hydrazone moiety: nitrofurazone (a) and furazolidone (b).

Additionally, as we have reported earlier, we synthesized some hydrazide–hydrazone derivatives which were shown to exhibit antimicrobial activityCitation20–22. Some of these compounds had interesting in vitro activity against Gram-positive bacteria. Especially N-[2–(3-bromo-4-methoxyphenyl)ethenyl]furan-2-carboxamide and N-[2–(3-bromo-4-methoxyphenyl)ethenyl]thiophene-2-carboxamide which showed the significant inhibitory activity towards Bacillus cereus ATCC 10876Citation20.

Considering the above-mentioned facts and in continuation of our research of new chemotherapeutic agents, in this paper, we would like to present synthesis and in vitro antimicrobial evaluation of new hydrazide-hydrazones which exhibit significant antibacterial activity towards Gram-positive bacteria, especially against Bacillus spp.

Materials and methods

Chemistry

All required chemicals and solvents were purchased from Sigma-Aldrich (Munich, Germany) and Merck Co. (Darmstadt, Germany) and used without further purification. Melting points were determined with Fisher-Johns blocks (Fisher Scientific, Schwerte, Germany) and are uncorrected. The 1H NMR and 13C NMR spectra were recorded on the Bruker Avance 300 apparatus (Bruker BioSpin GmbH, Munich, Germany) in DMSO-d6 with TMS as the internal standard. The chemical shifts are expressed on the δ (ppm) scale with the use of TMS as the standard reference. The coupling constants (J) are given in Hertz. Spin multiples are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and b (broad). The progress of the reaction and purity of obtained compounds were monitored by TLC using precoated aluminum sheet 60 F254 plates (Merck Co., Kenilworth, NJ), in a CHCl3/C2H5OH (10:1, v/v) solvent system. The spots were detected by exposure to the UV lamp at 254 nm. The elemental analysis of obtained compounds was carried out with the AMZ 851 CHX analyzer (PG, Gdańsk, Poland). The results of elemental analysis (C, H, and N) were within ± 0.4% of the calculated values. C log P values of synthesized compounds were calculated using HypeChemTM 8.0.8Citation23.

Preparation of 3-methoxybenzoic acid hydrazide (1)

The compound was prepared according to a procedure from the literatureCitation24.

CAS registry number: 5785-06-8. Analytical and spectral data is consistent with those reported in the literatureCitation24. Yield: 85%; m.p.: 93–95 °C. 1H NMR (DMSO-d6) δ (ppm) = 3.80 (s, 3H, CH3), 4.26 (s, 2H, NH2), 7.10–7.12 (m, 1H, ArH), 7.41–7.43 (m, 1H, ArH), 7.49–7.53 (m, 2H, ArH), 8.81 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.1 (CH3), 113.3, 118.7, 122.1, 128.7, 134.2, 160.5 (6Car), 166.9 (C=O). Analysis for C8H10N2O2 (166.18) Calculated: C: 57.82%, H: 6.07%, N: 16.86%; Found: C: 57.93%, H: 6.03%, N: 16.89%.

Preparation of 4-tert-butylbenzoic acid hydrazide (2)

The compound was prepared according to a procedure from the literatureCitation25.

CAS Registry Number: 43100-38-5. Analytical and spectral data is consistent with those reported in the literatureCitation25. Yield: 78%; m.p.: 121–123 °C. 1H NMR (DMSO-d6) δ (ppm) = 1.36 (s, 9H, 3 × CH3), 4.16 (s, 2H, NH2), 7.45–7.47 (d, 2H, ArH, J = 6 Hz), 7.80–7.82 (d, 2H, ArH, J = 6 Hz), 8.28 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 34.3 (Ct-butyl), 126.1, 131.4, 131.7, 153.6 (6Car), 167.4 (C=O). Analysis for C11H16N2O (192.26) Calculated: C: 68.72%, H: 8.39%, N: 14.57%; Found: C: 68.77%, H: 8.43%, N: 14.63%.

Preparation of hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acids (3–30)

General procedure

To the solution of 0.01 mole of adequate carboxylic acid hydrazide: 3-methoxybenzhydrazide (1) or 4-tert-butylbenzhydrazide (2) in 10 mL of ethanol, an appropriate substituted aromatic aldehyde (0.011 mole) was added. The mixture was heated under reflux for 3 h. Then it was allowed to cool and the formed precipitate was filtered off and recrystallized form methanol (3–16) or ethanol (17–30).

N-[(2,3-dimethoxyphenyl)methylidene]-3-methoxybenzhydrazide (3)

CAS registry number: 351365-58-7. Yield: 79%; m.p.: 169–171 °C. C log P = 2.67. 1H NMR (DMSO-d6) δ (ppm) = 3.80 (s, 3H, CH3), 3.84 (s, 6H, 2 × CH3), 7.11–7.18 (m, 3H, ArH), 7.42–7.53 (m, 4H, ArH), 8.75 (s, 1H, =CH), 11.86 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.8 (CH3), 56.2 (CH3), 61.7 (CH3), 111.3, 114.7, 118.0, 120.3, 124.9, 128.2, 130.1, 135.2, 143.8 (10Car), 148.4 (=CH), 153.1, 159.6 (2Car), 169.2 (C=O). Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.90%, H: 5.80%, N: 8.95%.

N-[(2,4-Dimethoxyphenyl)methylidene]-3-methoxybenzhydrazide (4)

CAS Registry Number: 329720-10-7. Yield: 54%; m.p.: 137–139 °C. C log P  = 2.67. 1H NMR (DMSO-d6) δ (ppm) = 3.83 (s, 6H, 2 × CH3), 3.87 (s, 3H, CH3), 6.63–6.66 (m, 2H, ArH), 7.13–7.16 (m, 1H, ArH), 7.40–7.52 (m, 3H, ArH), 7.80–7.82 (m, 1H, ArH), 8.72 (s, 1H = CH), 11.68 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.1 (CH3), 56.5 (CH3), 56.8 (CH3), 100.3, 107.1, 113.5, 117.5, 118.9, 123.6, 128.4, 129.7, 133.5 (9Car), 150.7 (=CH), 160.2, 160.9, 162.8 (3Car), 163.7 (C=O) 56.1 (CH3), 56.5 (CH3), 56.8 (CH3), 100.3, 107.1, 113.5, 117.5, 118.9, 123.6, 128.4, 129.7, 133.5 (9Car), 150.7 (=CH), 160.2, 160.9, 162.8 (3Car), 163.7 (C=O). Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.91%, H: 5.78%, N: 8.79%.

N-[(3,4-Dimethoxyphenyl)methylidene]-3-methoxybenzhydrazide (5)

CAS registry number: 1396397-12-8. Yield: 66%; m.p.: 138–140 °C. C log P  = 2.67. 1H NMR (DMSO-d6) δ (ppm) = 3.81 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.84 (s, 3H, CH3), 7.02–7.05 (d, 1H, ArH, J = 9 Hz), 7.15–7.20 (m, 2H, ArH), 7.34 (s, 1H, ArH), 7.42–7.50 (m, 3H, ArH), 8.39 (s, 1H, =CH), 11.71 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.1 (CH3), 56.7 (CH3), 56.8 (CH3), 110.1, 113.5, 114.1, 118.9, 121.1, 122.6, 128.4, 129.4, 133.5 (9Car), 148.7 (=CH), 149.8, 150.5, 160.2 (3Car), 163.7 (C=O). Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.92%, H: 5.74%, N: 8.94%.

N-[(3-Ethoxy-4-hydroxyphenyl)methylidene]-3-methoxybenzhydrazide (6)

CAS registry number: 362711-54-4. Yield: 52%; m.p.: 172–174 °C. C log P  = 2.98. 1H NMR (DMSO-d6) δ (ppm) = 1.35–1.39 (t, 3H, CH3, J = 6 Hz), 3.83 (s, 3H, CH3), 4.04–4.11 (q, 2H, CH2, J = 6 Hz), 6.84–6.87(d, 2H, ArH, J = 9 Hz), 7.07–7.17 (m, 2H, ArH), 7.29–7.30 (m, 1H, ArH), 7.41–7.49 (m, 3H, ArH), 8.33 (s, 1H, =CH), 9.50 (s, 1H, OH), 11.63 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 15.2 (CH3), 55.8 (CH3), 64.3 (CH3), 111.2, 113.2, 115.9, 118.5, 120.2, 123.2, 126.1, 130.1, 135.5, 147.7, 148.8 (11Car), 149.7 (=CH), 159.6 (Car), 163.1 (C=O). Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.90%, H: 5.79%, N: 8.87%.

3-Methoxy-N-[(4-methoxyphenyl)methylidene]benzhydrazide (7)

CAS registry number: 341945-25-3. Yield: 62%; m.p.: 152–154 °C. C log P  = 2.92. 1H NMR (DMSO-d6) δ (ppm) = 3.79 (s, 3H, CH3), 3.80 (s, 3H, CH3), 6.92–6.94 (d, 2H, ArH, J = 6 Hz), 7.11–7.13 (m, 1H, ArH), 7.41–7.44 (t, 1H, ArH, J = 3 Hz), 7.49 (s, 1H, ArH), 7.52–7.54 (d, 2H, ArH, J = 6 Hz), 7.57–7.59 (m, 1H, ArH), 8.39 (s, 1H, =CH), 11.06 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.0 (CH 3), 56.1 (CH3), 113.5, 114.3, 118.9, 122.6, 127.1, 128.4, 129.1 133.5 (10Car), 149.3 (=CH), 160.1, 160.2 (2Car), 163.7 (C=O). Analysis for C16H16N2O3 (284.31) Calculated: C: 67.59%, H: 5.67%, N: 9.85%; Found: C: 67.64%, H: 5.65%, N: 9.83%.

N-[(3-Ethoxy-4-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (8)

CAS registry number: 396655-61-1. Yield: 53%; m.p.: 157–159 °C. C log P  = 3.01. 1H NMR (DMSO-d6) δ (ppm) = 1.34–1.39 (t, 3H, CH3, J = 6 Hz, J = 9 Hz), 3.82 (s, 3H, CH3), 3.83 (s, 3H, CH3), 4.03–4.10 (q, 2H, CH2, J = 6 Hz), 7.02–7.05 (d, 1H, ArH, J = 9 Hz), 7.15–7.22 (m, 2H, ArH), 7.33–7.34 (m, 1H, ArH), 7.41–7.50 (m, 3H, ArH), 8.38 (s, 1H, =CH), 11.70 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 15.2 (CH3), 55.8 (CH3), 55.9 (CH3), 64.2 (CH2), 109.7, 112.0, 113.3, 117.8, 120.2, 122.3, 127.4, 130.1, 135.4, 148.6 (10Car), 148.7 (=CH), 151.3, 159.7 (2Car), 163.2 (C=O). Analysis for C18H20N2O4 (328.36) Calculated: C: 65.84%, H: 6.14%, N: 8.53%; Found: C: 65.88%, H: 6.16%, N: 8.47%.

N-[(4-Hydroxy-3,5-dimethoxyphenyl)methylidene]-3-methoxybenzhydrazide (9)

CAS registry number: 544661-09-8. Yield: 67%; m.p.: 149–151 °C. C log P  = 2.38. 1H NMR (DMSO-d6) δ (ppm) = 3.83 (s, 6H, 2 × CH3), 3.84 (s, 3H, CH3), 6.99 (s, 2H, ArH), 7.14–7.18 (m, 1H, ArH), 7.42–7.49 (m, 3H, ArH), 8.34 (s, 1H, =CH), 8.93 (s, 1H, OH), 11.69 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.2 (CH3), 56.5 (2 × CH3), 105.1, 113.3, 120.2, 124.9, 130.0, 135.1, 137.9, 139.4 (10Car), 148.6 (=CH), 149.1, 155.3 (2Car), 159.6 (C=O). Analysis for C17H18N2O5 (330.33) Calculated: C: 61.81%, H: 5.49%, N: 8.48%; Found: C: 61.87%, H: 5.46%, N: 8.52%.

N-[(4-Propoxyphenyl)methylidene]-3-methoxybenzhydrazide (10)

CAS registry number: 351365-16-7. Yield: 68%; m.p.: 96–98 °C. C log P  = 4.13. 1H NMR (DMSO-d6) δ (ppm) = 0.96–1.01 (t, 3H, CH3, J = 9 Hz, J = 6 Hz), 1.69–1.81 (m, 2H, CH2), 3.83 (s, 3H, CH3), 3.96–4.01 (t, 2H, CH2, J = 6 Hz), 7.00–7.03 (d, 2H, ArH, J = 9 Hz), 7.14–7.17 (m, 1H, ArH), 7.41–7.50 (m, 3H, ArH), 7.65–7.68 (d, 2H, ArH, J = 9 Hz), 8.40 (s, 1H, =CH), 11.69 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 10.8 (CH3), 22.5 (CH2), 30.5 (CH2), 55.8 (CH3), 69.5 (CH2), 113.2, 115.2, 117.3, 120.2, 127.2, 129.2, 130.2, 135.4 (10Car), 148.2 (=CH), 159.6, 160.8 (2Car), 163.1 (C=O). Analysis for C18H20N2O3 (312.36) Calculated: C: 69.21%, H: 6.45%, N: 8.97%; Found: C: 69.26%, H: 6.42%, N: 8.94%.

3-Methoxy-N-{[4-(trifluoromethyl)phenyl]methylidene}benzhydrazide (11)

CAS registry number: 710291-60-4. Yield: 65%; m.p.: 122–124 °C. C log P  = 4.06. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 7.17–7.20 (m, 1H, ArH), 7.33–7.39 (m, 1H, ArH), 7.41–7.53 (m, 2H, ArH), 7.81–7.84 (d, 2H, ArH, J = 9 Hz), 7.94–7.97 (d, 2H, ArH, J = 9 Hz), 8.54 (s, 1H, =CH), 12.03 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.8 (CH3), 111.9, 113.4, 118.1, 119.3, 120.4, 126.2, 126.3, 128.1, 130.2, 135.0, 138.8 (11Car), 146.5 (=CH), 159.7 (Car), 163.5 (C=O). Analysis for C16H13F3N2O2 (322.28) Calculated: C: 59.63%, H: 4.07%, N: 8.69%; Found: C: 59.68%, H: 4.11%, N: 8.73%.

N-[(2-Chloro-3-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (12)

Yield: 97%; m.p.: 204–206 °C. C log P  = 3.44. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 3.89 (s, 3H, CH3), 7.17–7.23 (m, 2H, ArH), 7.37–7.54 (m, 4H, ArH), 7.61–7.63 (d, 2H, ArH, J = 9 Hz), 8.90 (s, 1H, =CH), 12.06 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.8 (CH3), 56.8 (CH3), 113.4, 113.5, 118.1, 118.7, 120.7, 122.1, 128.3, 130.2, 133.2, 134.9 (10Car), 144.5 (=CH), 155.4, 159.7 (2Car), 163.3 (C=O). Analysis for C16H15ClN2O3 (318.75) Calculated: C: 60.29%, H: 4.74%, N: 8.79%; Found: C: 60.33%, H: 4.71%, N: 8.84%.

N-[(3-Chloro-4-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (13)

Yield: 81%; m.p.: 148–150 °C. C log P  = 3.44. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 3.92 (s, 3H, CH3), 7.15–7.18 (m, 1H, ArH, J = 9 Hz), 7.23–7.26 (d, 1H, ArH, J = 9 Hz), 7.42–7.51 (m, 3H, ArH), 7.66–7.70 (m, 1H, ArH), 7.81–7.82 (d, 1H, ArH, J = 3 Hz), 8.38 (s, 1H, =CH), 11.84 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.0 (CH3), 56.8 (CH3), 113.5, 115.5, 118.9, 122.6, 123.3, 125.5, 127.4, 128.4, 130.7, 133.5 (10Car), 148.7 (=CH), 156.2, 160.2 (2Car), 163.3 (C=O). Analysis for C16H15ClN2O3 (318.75) Calculated: C: 60.29%, H: 4.74%, N: 8.79%; Found: C: 60.35%, H: 4.78%, N: 8.83%.

N-[(3-Bromo-4-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (14)

CAS registry number: 904253-53-8. Yield: 71%; m.p.: 149–151 °C. C log P  = 3.71. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 3.91 (s, 3H, CH 3), 7.15–7.23 (m, 2H, ArH), 7.41–7.51 (m, 3H, ArH), 7.70–7.73 (d, 1H, ArH, J = 9 Hz), 7.95–7.97 (t, 1H, ArH, J = 6 Hz), 8.37 (s, 1H, =CH), 11.84 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.8 (CH3), 56.9 (CH3), 111.7, 113.3, 118.3, 120.1, 128.8, 130.1, 131.3, 136.1 (10Car), 146.2 (=CH), 157.7, 159.7 (2Car), 163.9 (C=O). Analysis for C16H15BrN2O3 (363.20) Calculated: C: 52.91%, H: 4.16%, N: 7.71%; Found: C: 52.96%, H: 4.18%, N: 7.67%.

N-[(2-Bromo-3-hydroxy-4-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (15)

CAS registry number: 1798917-09-5. Yield: 97%; m.p.: 212–214 °C. C log P  = 2.64. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 3.89 (s, 3H, CH3), 7.09–7.18 (m, 2H, ArH), 7.42–7.53 (m, 4H, ArH), 8.78 (s, 1H, =CH), 9.65 (s, 1H, OH), 11.94 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 56.0 (CH3), 56.8 (CH3), 108.1, 113.5, 115.0, 118.9, 119.9, 122.6, 126.2, 128.4, 133.5 (9Car), 144.8 (=CH), 145.0, 148.9, 160.2 (3Car), 163.6 (C=O). Analysis for C16H15BrN2O4 (379.20) Calculated: C: 50.68%, H: 3.99%, N: 7.39%; Found: C: 50.71%, H: 4.01%, N: 7.43%.

N-[((2-Hydroxy-3,5-diiodophenyl)methylidene]-3-methoxybenzhydrazide (16)

CAS registry number: 339011-17-5. Yield: 94%; m.p.: 205–207 °C. C log P  = 5.58. 1H NMR (DMSO-d6) δ (ppm) = 3.84 (s, 3H, CH3), 7.19–7.22 (m, 1H, ArH), 7.45–7.55 (m, 3H, ArH), 7.88–7.89 (d, 1H, ArH, J = 3 Hz), 8.05–8.06 (d, 1H, ArH, J = 3 Hz), 8.44 (s, 1H, =CH), 12.51 (s, 1H, OH), 12.94 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.8 (CH3), 82.6, 88.3, 113.5, 118.5, 120.4, 120.8, 130.3, 133.9, 139.1, 146.9 (10Car), 147.7 (=CH), 157.1, 159.7 (2Car), 163.2 (C=O). Analysis for C15H12I2N2O3 (522.08) Calculated: C: 34.51%, H: 2.32%, N: 5.37%; Found: C: 34.56%, H: 2.30%, N: 5.39%.

4-Tert-butyl-N-[(2,3-dimethoxyphenyl)methylidene]benzhydrazide (17)

CAS registry number: 325798-99-0. Yield: 87%; m.p.: 216–218 °C. C log P  = 4.55. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 3.80 (s, 3H, CH3), 3.84 (s, 3H, CH3), 7.11–7.15 (m, 2H, ArH), 7.47–7.49 (m, 1H, ArH), 7.53–7.56 (d, 1H, ArH, J = 6 Hz), 7.86–7.89 (d, 1H, ArH, J = 9 Hz), 11.83 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 56.2 (CH3), 61.7 (CH3), 115.1, 117.4, 124.9, 125.7, 127.9, 122.3, 131.0, 132.8, 143.5 (10Car), 148.4 (=CH), 153.1, 154.9 (2Car), 163.9 (C=O). Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%, H: 7.11%, N: 8.23%; Found: C: 70.63%, H: 7.08%, N: 8.25%.

4-Tert-butyl-N-[(2,4-dimethoxyphenyl)methylidene]benzhydrazide (18)

CAS registry number: 328921-38-6. Yield: 65%; m.p.: 198–200 °C. C log P  = 4.55. 1H NMR (DMSO-d6) δ (ppm) = 1.31 (s, 9H, 3 × CH3), 3.82 (s, 3H, CH3), 3.86 (s, 3H, CH3), 6.63–6.65 (m, 2H, ArH), 7.51–7.54 (d, 2H, ArH, J = 9 Hz), 7.80–7.83 (m, 1H, ArH), 7.85–7.86 (d, 2H, ArH, J = 9 Hz), 8.72 (s, 1H, =CH), 11.66 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 34.3 (Ct-butyl), 56.0 (CH3), 56.8 (CH3), 100.3, 107.1, 117.5, 125.8, 129.7, 130.3, 132.9 (9Car), 150.7 (=CH), 152.96, 160.9, 162.8 (3Car), 164.3 (C=O). Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%, H: 7.11%, N: 8.23%; Found: C: 70.61%, H: 7.09%, N: 8.26%.

4-Tert-butyl-N-[(3,4-dimethoxyphenyl)methylidene]benzhydrazide (19)

CAS registry number: 328921-37-5. Yield: 79%; m.p.: 214–216 °C. C log P  = 4.55. 1H NMR (DMSO-d6) δ (ppm) = 1.31 (s, 9H, 3 × CH3), 3.81 (s, 3H, CH3), 3.82 (s, 3H, CH3), 7.02–7.04 (d, 1H, ArH, J = 6 Hz), 7.19–7.22 (m, 1H, ArH), 7.35 (s, 1H, ArH), 7.51–7.55 (d, 2H, ArH, J = 9 Hz), 7.84–7.86 (d, 2H, ArH, J = 6 Hz), 8.39 (s, 1H, =CH), 11.69 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 34.30 (Ct-butyl), 56.8 (CH3), 56.9 (CH3), 110.1, 114.1, 122.1, 125.8, 129.4, 130.3, 132.8 (9Car), 148.7 (=CH), 149.7, 150.5, 152.9 (3Car), 164.3 (C=O). Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%, H: 7.11%, N: 8.23%; Found: C: 70.62%, H: 7.14%, N: 8.20%.

4-Tert-butyl-N-[(3-ethoxy-4-hydroxyphenyl)methylidene]benzhydrazide (20)

CAS registry number: 525562-83-8. Yield: 76%; m.p.: 216–217 °C. C log P  = 4.86. 1H NMR (DMSO-d6) δ (ppm) = 1.31 (s, 9H, 3 × CH3), 1.34–1.39 (t, 3H, CH3, J = 9 Hz, J = 6 Hz), 4.04–4.11 (q, 2H, CH2, J = 6 Hz, J = 9 Hz), 6.84–6.87 (d, 1H, ArH, J = 9 Hz), 7.06–7.10 (m, 1H, ArH), 7.30 (s, 1H, ArH), 7.52–7.55 (d, 2H, ArH, J = 9 Hz), 7.82–7.85 (d, 2H, ArH, J = 9 Hz), 8.32 (s, 1H, =CH), 9.50 (s. 1H, OH), 11.61 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 15.2 (CH3), 31.4 (3 × CH3), 35.1 (Ct-butyl), 64.3 (CH2), 110.7, 115.9, 122.5, 125.7, 126.2, 127.9, 131.3, 147.6 (10Car), 148.6 (=CH), 149.6, 154.9 (2Car), 163.3 (C=O). Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%, H: 7.11%, N: 8.23%; Found: C: 70.63%, H: 7.09%, N: 8.26%.

4-Tert-butyl-N-[(4-methoxyphenyl)methylidene]benzhydrazide (21)

CAS registry number: 328921–35-3. Yield: 98%; m.p.: 196–198 °C. C log P  = 4.80. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 3.81 (s, 3H, CH3), 7.01–7.04 (d, 2H, ArH, J = 9 Hz), 7.52–7.55 (d, 2H, ArH, J = 9 Hz), 7.66–7.69 (d, 2H, ArH, J = 9 Hz), 7.83–7.86 (d, 2H, ArH, J = 9 Hz), 8.39 (s, 1H, =CH), 11.67 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 55.8 (CH3), 114.8, 125.7, 127.4, 127.9, 129.1, 131.3 (10Car), 147.9 (=CH), 154.9, 161.2 (2Car), 163.3 (C=O). Analysis for C19H22N2O2 (310.39) Calculated: C: 73.52%, H: 7.14%, N: 9.03%; Found: C: 73.58%, H: 7.11%, N: 9.06%.

4-Tert-butyl-N-[(3-ethoxy-4-methoxyphenyl)methylidene]benzhydrazide (22)

CAS registry number: 394685-88-2. Yield: 95%; m.p.: 206–208 °C. C log P  = 4.89. 1H NMR (DMSO-d6) δ (ppm) = 1.31 (s, 9H, 3 × CH3), 1.34–1.39 (t, 3H, CH3, J = 6 Hz, J = 9 Hz), 3.81 (s, 3H, CH3), 4.03–4.10 (q, 2H, CH2, J = 6 Hz, J = 9 Hz), 7.02–7.04 (d, 1H, ArH, J = 6 Hz), 7.18–7.21 (m, 1H, ArH), 7.33 (s, 1H, ArH), 7.52–7.55 (d, 2H, ArH, J = 9 Hz), 7.83–7.86 (d, 2H, ArH, J = 9 Hz), 8.37 (s, 1H, =CH), 11.67 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 15.2 (CH3), 31.4 (3 × CH3), 35.2 (Ct-butyl), 55.9 (CH3), 64.2 (CH2), 109.6, 112.0, 122.2, 125.7, 127.5, 127.9, 131.3, 148.2 (10Car), 148.7 (=CH), 151.2, 154.9 (2Car), 163.4 (C=O). Analysis for C21H26N2O3 (354.44) Calculated: C: 71.16%, H: 7.39%, N: 7.90%; Found: C: 71.21%, H: 7.37%, N: 7.94%.

4-Tert-butyl-N-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]benzhydrazide (23)

CAS registry number: 524730-78-7. Yield: 69%; m.p.: 236–238 °C. C log P  = 4.26. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 3.82 (s, 6H, 2 × CH3), 6.98 (s, 2H, ArH), 7.53–7.55 (d, 2H, ArH, J = 6 Hz), 7.82–7.85 (d, 2H, ArH, J = 9 Hz), 8.33 (s, 1H, =CH), 8.92 (s, 1H, OH), 11.66 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.1 (Ct-butyl), 56.5 (2 × CH3), 105.0, 125.1, 125.7, 127.9, 131.3, 138.3 (9Car), 147.8 (=CH), 148.8, 152.9 (3Car), 164.3 (C=O). Analysis for C20H24N2O4 (356.41) Calculated: C: 67.40%, H: 6.79%, N: 7.86%; Found: C: 67.47%, H: 6.76%, N: 7.89%.

4-Tert-butyl-N-[(4-propoxyphenyl)methylidene]benzhydrazide (24)

CAS registry number: 544689-51-2. Yield: 73%; m.p.: 196–198 °C. C log P  = 6.01. 1H NMR (DMSO-d6) δ (ppm) = 0.96–1.01 (t, 3H, CH3, J = 9 Hz, J = 6 Hz), 1.32 (s, 9H, 3 × CH3), 1.69–1.80 (m, 2H, CH2), 3.96–4.00 (t, 2H, CH2, J = 6 Hz), 7.00–7.03 (d, 2H, ArH, J = 9 Hz), 7.52–7.55 (d, 2H, ArH, J = 9 Hz), 7.64–7.67 (d, 2H, ArH, J = 9 Hz), 7.83–7.86 (d, 2H, ArH, J = 9 Hz), 8.39 (s, 1H, =CH), 11.66 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 10.85 (CH3), 21.46 (CH2), 30.5 (CH2), 31.4 (3 × CH3), 35.2 (Ct-butyl), 69.5 (CH2), 115.2, 125.7, 127.2, 127.9, 129.1, 131.3 (10Car), 147.8 (=CH), 154.9, 160.7 (2Car), 163.3 (C=O). Analysis for C21H26N2O2 (338.44) Calculated: C: 74.52%, H: 7.74%, N: 8.28%; Found: C: 74.58%, H: 7.72%, N: 8.31%.

4-Tert-butyl-N-{[4-(trifluoromethyl)phenyl]methylidene}benzhydrazide (25)

Yield: 74%; m.p.: 198–200 °C. C log P  = 5.94. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 7.55–7.57 (d, 2H, ArH, J = 6 Hz), 7.81–7.89 (m, 4H, ArH), 7.94–7.97 (d, 2H, ArH, J = 9 Hz), 8.52 (s, 1H, =CH), 12.00 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH 3), 35.2 (Ct-butyl), 124.6 (CF3), 125.8, 126.2, 128.1, 129.5, 130.2, 132.9, 135.9 (11Car), 149.3 (=CH), 152.9 (Car), 164.3 (C=O). Analysis for C19H19F3N2O (348.36) Calculated: C: 65.51%, H: 5.50%, N: 8.04%; Found: C: 65.57%, H: 5.53%, N: 8.01%.

4-Tert-butyl-N-[(2-chloro-3-methoxyphenyl)methylidene]benzohydrazide (26)

Yield: 92%; m.p.: 259–261 °C. C log P  = 5.32. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 3.89 (s, 3H, CH3), 7.20–7.23 (d, 1H, ArH, J = 9 Hz), 7.37–7.42 (t, 1H, ArH, J = 6 Hz, J = 9 Hz), 7.54–7.57 (d, 2H, ArH, J = 9 Hz), 7.61–7.63 (d, 1H, ArH, J = 6 Hz), 7.87–7.90 (d, 2H, ArH, J = 9 Hz), 8.89 (s, 1H, =CH), 12.03 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 36.8 (CH3), 114.6, 119.7, 122.8, 125.8, 128.0, 128.3, 132.3, 142.3 (10Car), 149.9 (=CH), 155.4, 157.6 (2Car), 164.0 (C=O). Analysis for C19H21ClN2O2 (344.83) Calculated: C: 66.18%, H: 6.14%, N: 8.12%; Found: C: 66.23%, H: 6.11%, N: 8.16%.

4-Tert-butyl-N-[((3-chloro-4-methoxyphenyl)methylidene]benzohydrazide (27)

CAS registry number: 328921-99-9. Yield: 75%; m.p.: 227–229 °C. C log P  = 5.32. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH 3), 3.92 (s, 3H, CH3), 7.23–7.26 (d, 1H, ArH, J = 9 Hz), 7.53–7.56 (d, 2H, ArH, J = 9 Hz), 7.66–7.69 (d, 1H, ArH, J = 9 Hz), 7.81–7.86 (m, 3H, ArH), 8.37 (s, 1H, =CH), 11.80 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 56.8 (CH3), 113.5, 122.12, 125.7, 127.8, 128.3, 128.4, 130.1, 133.4 (10Car), 147.9 (=CH), 155.9, 156.2 (2Car), 164.2 (C=O). Analysis for C19H21ClN2O2 (344.83) Calculated: C: 66.18%, H: 6.14%, N: 8.12%; Found: C: 66.23%, H: 6.12%, N: 8.15%.

N-[(3-Bromo-4-methoxyphenyl)methylidene]-4-tert-butylbenzohydrazide (28)

CAS registry number: 328921-88-6. Yield: 79%; m.p.: 234–236 °C. C log P  = 5.59. 1H NMR (DMSO-d6) δ (ppm) = 1.31 (s, 9H, 3 × CH3), 3.91 (s, 3H, CH3), 7.19–7.22 (d, 1H, ArH, J = 9 Hz), 7.53–7.55 (d, 2H, ArH, J = 6 Hz), 7.63–7.65 (d, 1H, ArH, J = 6 Hz), 7.83–7.86 (d, 2H, ArH, J = 9 Hz), 7.96–7.97 (d, 1H, ArH, J = 9 Hz), 8.36 (s, 1H, =CH), 11.80 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 56.9 (CH3), 111.7, 113.3, 125.7, 126.9, 127.9, 128.9, 129.1, 131.3 (10Car), 147.2 (=CH), 152.9, 157.2 (2Car), 164.3 (C=O). Analysis for C19H21BrN2O2 (389.29) Calculated: C: 58.62%, H: 5.44%, N: 7.20%; Found: C: 58.68%, H: 5.40%, N: 7.25%.

N-[((2-Bromo-3-hydroxy-4-methoxyphenyl)methylidene]-4-tert-butylbenzhydrazide (29)

Yield: 95%; m.p.: 212–214 °C. C log P  = 4.52. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 3.89 (s, 3H, CH3), 7.09–7.12 (d, 1H, ArH, J = 9 Hz), 7.50–7.56 (t, 3H, ArH, J = 9 Hz), 7.86–7.89 (d, 2H, ArH, J = 9 Hz), 8.78 (s, 1H, =CH), 9.65 (s, 1H, OH), 11.92 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.4 (3 × CH3), 35.2 (Ct-butyl), 56.5 (CH3), 111.4, 112.2, 118.9, 125.7, 126.5, 127.9, 131.3, 144.2 (10Car), 147.8 (=CH), 150.1, 154.8 (2Car), 163.8 (C=O). Analysis for C19H21BrN2O3 (405.28) Calculated: C: 56.31%, H: 5.22%, N: 6.91%; Found: C: 56.35%, H: 5.20%, N: 6.95%.

4-Tert-butyl-N-[(2-hydroxy-3,5-diiodophenyl)methylidene]benzohydrazide (30)

CAS registry number: 325990-74-7. Yield: 79%; m.p.: 250–252 °C. C log P  = 7.28. 1H NMR (DMSO-d6) δ (ppm) = 1.32 (s, 9H, 3 × CH3), 7.57–7.59 (d, 2H, ArH, J = 6 Hz), 7.88 (s, 1H, ArH), 7.89–7.91 (d, 2H, ArH, J = 6 Hz), 8.05–8.06 (d, 1H, ArH, J = 3 Hz), 8.44 (s, 1H, =CH), 12.46 (s, 1H, OH), 12.99 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 31.3 (3 × CH3), 35.3 (Ct-butyl), 82.9. 88.3, 120.8, 125.9, 128.1, 129.8, 139.5 (9Car), 147.9 (=CH), 148.2, 155.9, 157.1 (3Car), 164.3 (C=O). Analysis for C18H18I2N2O2 (548.16) Calculated: C: 39.44%, H: 3.31%, N: 5.11%; Found: C: 39.48%, H: 3.35%, N: 5.08%.

Microbiology

In vitro antimicrobial assay

The examined compounds 3–30 were screened in vitro for antibacterial and antifungal activities with the use of the broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST)Citation26 and Clinical and Laboratory Standards Institute guidelinesCitation27 against a panel of reference strains of microorganisms, including Gram-positive bacteria (Staphylococcus aureus ATCC 6538, S. aureus ATCC 25923, S. aureus ATCC 43300, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876, and Micrococcus luteus ATCC 10240), Gram-negative bacteria (Bordetella bronchiseptica ATCC 4617, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 9027) and against the fungi belonging to yeasts (Candida albicans ATCC 2091, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019). These micro-organisms came from American Type Culture Collection (ATCC), routinely used for the evaluation of antimicrobials. All the used microbial cultures were first subcultured on a suitable medium at 30–35 °C for 18–48 h.

The surfaces of Mueller–Hinton agar (for bacteria) and RPMI 1640 with MOPS (for fungi) were inoculated with the suspensions of bacterial or fungal species. The microbial suspensions were prepared in sterile saline with an optical density of McFarland standard scale 0.5 – approximately 1.5 × 108 CFU (Colony Forming Units)/ml for bacteria, and 5 × 105 CFU/ml) for fungi.

The samples containing the examined compounds were dissolved in 1 ml dimethyl sulfoxide (DMSO). Furthermore, the bacterial and fungal suspensions were put onto Petri dishes with the solid media containing 2 mg/ml of the tested compounds and incubated under appropriate conditions. The inhibition of microbial growth was evaluated by the comparison with a control culture prepared without any sample tested. Ciprofloxacin, cefuroxime, ampicillin (Sigma, St. Louis, MO) were used as a reference antibacterial compounds and fluconazole (Sigma) as reference antifungal compound.

Subsequently minimal inhibitory concentration (MIC) of the compounds was examined by the microdilution broth method, with the use of their two-fold dilutions in Mueller–Hinton broth (for bacteria) and RPMI 1640 broth with MOPS (for fungi), prepared in 96-well polystyrene plates. The final concentrations of the compounds ranged from 2000 to 0.98 μg/ml. The microbial suspensions were prepared in sterile 0.85% NaCl with an optical density of 0.5 McFarland standard. Next 2 μl of each bacterial or fungal suspensions were added to each well containing 200 μl broth and various concentrations of the examined compounds. After incubation, the MIC was assessed spectrophotometrically as the lowest concentration of the samples showing complete bacterial or fungal growth inhibition. Appropriate DMSO, growth and sterile controls were carried out. The medium with no tested substances was used as controlCitation28,Citation29.

The minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) is defined as the lowest concentration of the compounds that is required to kill a particular bacterial or fungal species. MBC or MFC were determined by removing 20 μl of the culture using for MIC determinations from each well and spotting onto appropriate agar medium. The plates were incubated. The lowest compound concentrations with no visible growth observed were assessed as bactericidal or fungicidal concentrations. All the experiments were repeated three times and representative data were presentedCitation28,Citation29.

In this study, no bioactivity was defined as a MIC > 1000 μg/ml, mild bioactivity as a MIC in the range 501–1000 μg/ml, moderate bioactivity with MIC from 126 to 500 μg/ml, good bioactivity as a MIC in the range 26–125 μg/ml, strong bioactivity with MIC between 10 and 25 μg/ml and very strong bioactivity as a MIC < 10 μg/ml. The MBC/MIC or MFC/MIC ratios were calculated in order to determine bactericidal/fungicidal (MBC/MIC ≤ 4, MFC/MIC ≤ 4) or bacteriostatic/fungistatic (MBC/MIC > 4, MFC/MIC > 4) effects of the tested compoundsCitation30.

Results and discussion

Chemistry

Titled compounds were synthesized according to the stages presented in Scheme 1. First hydrazides of 3-methoxybenzoic acid (1) and 4-tert-butylbenzoic acid (2) were synthesized with the use of the methods described earlierCitation24,Citation25 by the reaction of commercially available methyl esters of above mentioned carboxylic acids with hydrazine hydrate. Then the synhtesized hydrazides were subjected to the condensation reactions with corresponding substituted aromatic aldehydes what allowed us to obtain appropriate hydrazide-hydrazone derivatives (3–30). The reactions were carried out by heating substrates in ethanol under reflux. The structures of all synthesized (1–30) compounds were fully confirmed by means of 1H NMR, 13C NMR data and elemental analysis. Appropriate spectral and physico-chemical properties are presented in the Experimental section.

Scheme 1. Synthesis scheme of hydrazide-hydrazone derivatives (3–30).

Scheme 1. Synthesis scheme of hydrazide-hydrazone derivatives (3–30).

To the best of our knowledge among the synthesized derivatives (3–30) only compound 5Citation24 has any reference in the literature. As for compounds 1–4, 7–11, 14–24, 27, 28 and 30 their structures are known, but there are no references reporting their use, synthesis and physico-chemical characterization. Moreover, our synthesized hydrazide-hydrazones 12, 13, 25, 26, and 29 are new and their structures and synthesis have not been reported in the literature so far.

In vitro antimicrobial activity against a panel of reference strains

The results of our in vitro antimicrobial activity study indicated, that synthesized compounds 3–6, 8–12, 15, and 17–24 had no inhibitory effect on the growth of the reference strains of Gram-positive and Gram-negative bacteria and yeasts belonging to fungi. To the contrary, compounds 13, 14, 16, 23, and 30 had significant inhibitory effect on the growth of the reference strains of Gram-positive bacteria.

On the basis of the MIC values obtained with the broth microdilution method (), we can conclude that Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 10876 reveal the highest vulnerability to synthesized hydrazide-hydrazone derivatives (13, 14, 16, 23, and 30).

Table 1. The activity data expressed as MIC (μg/ml) against the reference strains of Gram-positive bacteria.

In the case of Bacillus subtilis ATCC 6633, the activity of compound 16 (MIC=1.95 μg/ml) was almost 10 times higher than cefuroxime (MIC=15.63 μg/ml), whereas for compounds 13 and 14, their values of MIC 3.91 μg/ml and 7.81 μg/ml, respectively, were almost five and two times lower than the MIC of cefuroxime. Compound 30 showed the same value of MIC (15.63 μg/ml) as cefuroxime against B. subtilis ATCC 6633. It is also worth to add that compounds 14 and 23 (MIC=31.25 μg/ml) had bactericidal effects (MBC/MIC ≤ 4), other compounds (13, 16, and 30) showed bacteriostatic effects (MBC/MIC > 4) against this pathogen.

As for the Bacillus cereus ATCC 10876, compounds 13, 14, and 16 were the most active against this bacterium. Their activity (MIC=7.81 μg/ml) was fourfold higher than that of cefuroxime (MIC=31.25 μg/ml) and eight-fold times higher than for ampicillin (MIC=62.5 μg/ml). Compound 23 inhibited the growth of B. cereus ATCC 10876 with the same strength as ampicillin. In addition, compound 16 showed bactericidal effect (MBC/MIC ≤ 4) against this microorganism.

Considering M. luteus ATCC 10240, compounds 16 and 30 with MIC values of 3.91 μg/ml and 15.63 μg/ml, respectively, were the most active with bacteriostatic effect against this pathogen. Compounds 13 and 14 didn’t show any activity against this microorganism.

The activity of synthesized hydrazide-hydrazones (13, 14, 16, 23, and 30) against Staphylococcus spp. can be assessed as very strong to good (MIC=7.81 – 125 μg/ml) or in some cases as moderate (MIC > 125 μg/ml). The most active compound with strong bactericidal effect against three strains of S. aureus (ATCC 25923, ATCC 6538, and ATCC 43300) was N-[(3-bromo-4-methoxyphenyl)methylidene]-3-methoxybenzhydrazide (14) with a MIC value of 15.62 μg/ml. Lower values of MIC parameter against these bacteria were showed by compounds: 13 (MIC=7.81 μg/ml) and 16 (MIC=7.81–62.5 μg/ml) but with bacteriostatic effect. Compounds 23 and 30 had the highest MICs against these pathogens (125–500 μg/ml). The lowest value of MIC against Staphylococcus epidermidis ATCC 12228 was showed by compounds 13, 14, and 16 (MIC=15.62 μg/ml) with bactericidal (14) and bacteriostatic (13 and 16) effect.

It is worth to stress that the most promising conclusion of this research is sensitiveness of Bacillus spp. to synthesized hydrazide-hydrazones (13, 14, 16, 23, and 30). This susceptibility, especially against Bacillus cereus, is crucial in the development of new hydrazide-hydrazones as antimicrobial agents due to the fact that these bacteria are common causes of serious infections like, myocarditis or pneumoniaCitation31,Citation32.

Conclusions

In conclusion, we synthesized 28 hydrazide-hydrazones of two substituted benzoic acids using the condensation reaction of appropriate benzoic acid hydrazides with different substituted aromatic aldehydes. All prepared compounds were subjected to in vitro antimicrobial activity assays. Our screening results indicated that five of the newly synthesized compounds 13, 14, 16, 23, and 30 showed high bacteriostatic or bactericidal activity against Gram-positive bacteria. Antimicrobial activity of compounds 13, 14, and 16 against Bacillus spp. was higher than that of the commonly used antibiotics like, cefuroxime or ampicillin. Bacillus subtilis ATCC 6633 (MIC=1.95–31.25 μg/ml) proved to be the most sensitive strain of bacteria to the tested compounds. The promising results presented in this article may result in the future development of new hydrazide-hydrazones analogs as potential antimicrobial agents.

Declaration of interest

The authors report that they have no conflicts of interest. This project was partially supported by research grant for young scientists granted to Dr Łukasz Popiołek (MNmb25). Dr Łukasz Popiołek is awarded with the Fellowship for Young Scientists with Outstanding Scientific Achievements from the Medical University of Lublin (Poland).

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