Design, synthesis, and in vitro antimicrobial activity of hydrazide–hydrazones of 2-substituted acetic acid

Article abstract of DOI:10.1111/cbdd.12820

In this study, 30 hydrazide–hydrazones of phenylacetic (3–10) and hydroxyacetic acid (11–32) were synthesized by the condensation reaction of appropriate 2-substituted acetic acid hydrazide with different aromatic aldehydes. The obtained compounds were characterized by spectral data and evaluated in vitro for their potential antimicrobial activities against a panel of reference strains of micro-organisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi belonging to the Candida spp. The results from our antimicrobial assays indicated that among synthesized compounds 3–32, especially compounds 6, 14, and 26 showed high bactericidal activity (MIC?=?0.488–7.81?μg/ml) against reference Gram-positive bacteria, and in some cases, their activity was even better than that of commonly used antibiotics, such as cefuroxime or ampicillin.

Full text of DOI:10.1111/cbdd.12820

Received Date : 03-May-2016
Revised Date : 06-Jul-2016
Accepted Date : 09-Jul-2016
Article type : Research Article
Design, synthesis and in vitro antimicrobial activity
of hydrazide-hydrazones of 2-substituted acetic acid
Łukasz Popiołek1* and Anna Biernasiuk2
1Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin,
4A Chodźki Street, 20-093 Lublin, Poland, *e-mail: lukasz.popiolek@umlub.pl
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of
Lublin, 1 Chodźki Street, 20-093 Lublin, Poland
KEYWORDS
hydrazide-hydrazones, antimicrobial activity, MIC, MBC, Gram-positive bacteria
ABSTRACT
In this study 30 hydrazide-hydrazones of phenylacetic (3-10) and hydroxyacetic acid (11-32)
were synthesized by the condensation reaction of appropriate 2-substituted acetic acid
hydrazide with different aromatic aldehydes. The obtained compounds were characterized by
spectral data and evaluated in vitro for their potential antimicrobial activities against a panel
of reference strains of microorganisms, including Gram-positive bacteria, Gram-negative
bacteria and fungi belonging to the Candida spp. The results from our antimicrobial assays
indicated that among synthesized compounds 3-32, especially compounds 6, 14 and 26
showed high bactericidal activity (MIC = 0.488 – 7.81 μg/ml) against reference Gram-
positive bacteria and in some cases their activity was even better than that of commonly used
antibiotics, like cefuroxime or ampicillin.
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12820
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INTRODUCTION
The treatment of bacterial and fungal infectious diseases remains a challenging problem
because of the increasing number of multi-drug resistant pathogens. Despite the fact that a
wide variety of drugs are being used in dealing with infections, still there is a need for the
development of new effective antibacterial and antifungal agents [1].
Hydrazide-hydrazone compounds are not only intermediates in synthesis and drug
development but they are also very effective organic compounds on their own [2, 3]. It is well
known from the literature that this class of compounds possesses diverse biological and
pharmacological properties, such as antiviral [4], anti-inflammatory [5, 6], antiamoebic [7],
antiprotozoal [8] and anticancer activities [9-12]. The hydrazide-hydrazone derivatives also
play an essential role as antimicrobial agents by virtue of their highly reactive azomethine
group (-NH-N=CH-) in their structure [13-19].
Isoniazid (isonicotinic acid hydrazide) is characterized by a very high antitubercular
activity towards Mycobacterium tuberculosis [20, 21]. Maccari et al. and Coelho et al.
synthesized new isoniazid hydrazide-hydrazones by reacting isonicotinic acid hydrazide with
various aldehydes and ketones [22, 23]. Antimicrobial activity assays revealed that these
compounds had significant inhibitory activity against various strains of M. tuberculosis,
including isoniazid-resistant strains [22, 23]. Buu-Hoi et al. synthesized some hydrazide-
hydrazones with antitubercular activity that were reported to have lower toxicity than
hydrazides because of the blockage of NH2 group [24].
Recently we reported interesting results concerning the synthesis and antibacterial activity
of hydrazide-hydrazones [25-27]. It is worth to stress that in our previous research hydrazide-
hydrazones of substituted benzoic acids showed significant activity against Gram-postitive
bacteria
[28].
Especially
N-[((2-hydroxy-3,5-diiodophenyl)methylidene]-3-
methoxybenzhydrazide displayed activity much higher than commonly used
chemotherapeutics against Bacillus spp. [28]. The above mentioned findings further support
the growing importance of the synthesis of hydrazide-hydrazones derivatives.
Encouraged by above facts and in continuation of our work in describing novel
biologically active compounds, we hereby report the synthesis, spectral studies and
antimicrobial evaluation of new hydrazide-hydrazone derivatives of 2-substituted acetic
acids.
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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, Germany) and
1
13
are uncorrected. The H NMR and C NMR spectra were recorded on the Bruker Avance
300 apparatus (Bruker BioSpin GmbH, 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 multiplet 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. USA), 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
analyser (PG, Gdańsk, Poland). The results of elemental analysis (C, H, N) were within ±
0.4% of the calculated values. ClogP values of synthesized compounds were calculated using
HyperChemTM 8.0.8 [29].
Preparation of 2-phenylacetic acid hydrazide (1)
The compound was prepared according to a procedure from the literature [30].
CAS Registry Number: 937-39-3. Analytical and spectral data is consistent with that reported
1
in the literature [30]. Yield: 83%; m.p.: 116-117°C. ClogP = 0.80; H NMR (DMSO-d6) δ
(ppm) = 3.49 (s, 2H, CH2), 3.76 (s, 2H, NH2), 7.29-7.43 (m, 5H, ArH), 8.19 (s, 1H, NH); 13C
NMR (DMSO) δ (ppm) = 40.1 (CH2), 128.4, 129.2, 129.4, 134.8 (6Car), 173.9 (C=O).
Analysis for C8H10N2O (150.18) Calculated: C: 63.98%, H: 6.71%, N: 18.65%; Found: C:
63.94%, H: 6.73%, N: 18.70%.
Preparation of 2-hydroxyacetic acid hydrazide (2)
The compound was prepared according to a procedure from the literature [31].
CAS Registry Number: 3530-14-1. Analytical and spectral data is consistent with those
1
reported in the literature [31]. Yield: 63%; m.p.: 122-123°C. ClogP = -1.60. H NMR
(DMSO-d6) δ (ppm) = 3.90 (s, 2H, NH2), 4.21 (s, 2H, CH2), 5.30 (s, 1H, OH), 8.84 (s, 1H,
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NH); 13C NMR (DMSO) δ (ppm) = 61.2 (CH2), 171.0 (C=O). Analysis for C2H6N2O2 (90.08)
Calculated: C: 26.67%, H: 6.71%, N: 31.10%; Found: C: 26.71%, H: 6.68%, N: 31.14%.
Preparation of hydrazide-hydrazones (3-32)
General procedure
The solution of 0.01 mole of adequate 2-substituted acetic acid hydrazide (1 or 2) and
appropriate substituted aromatic aldehyde (0.011 mole) was heated under reflux for 3h. After
that it was cooled and the formed precipitate was filtered off and recrystallized from ethanol.
2-phenyl-N-[1H-pyrrol-2-ylmethylidene]acethydrazide (3)
1
CAS Registry Number: 67973-81-3. Yield: 80%; m.p.: 210-212°C; ClogP = 2.13; H NMR
(DMSO-d6) δ (ppm) = 3.98 (s, 2H, CH2), 6.10-6.14 (m, 2H, ArH), 6.41-6.45 (m, 2H, ArH),
6.94-6.97 (m, 1H, ArH), 7.23-7.35 (m, 2H, ArH), 7.83 (s, 1H, ArH), 8.06 (s, 1H, =CH), 11.07
13
(s, 1H, NH), 11.27 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 40.9 (CH2), 112.85. 120.8,
128.4, 129.2, 129.4, 134.8 (8Car), 135.0 (=CH), 144.9, 148.3 (2Car), 168.3 (C=O). Analysis
for C13H13N3O (227.26) Calculated: C: 68.70%, H: 5.77%, N: 18.49%; Found: C: 68.81%, H:
5.73%, N: 18.53%.
N-[(5-chlorofuran-2-yl)methylidene]-2-phenylacethydrazide (4)
1
CAS Registry Number: 406702-39-4. Yield: 80%; m.p.: 150-152°C; ClogP = 2.44; H NMR
(DMSO-d6) δ (ppm) = 3.91 (s, 2H, CH2), 6.64-6.66 (m, 2H, ArH), 6.97-6.98 (m, 2H, ArH),
7.28-7.33 (m, 2H, ArH), 7.81 (s, 1H, ArH), 8.03 (s, 1H, =CH), 11.40 (s, 1H, NH); 13C NMR
(DMSO) δ (ppm) = 27.5 (CH2), 109.8, 115.7, 116.3, 126.9, 128.4, 128.8, 129.5, 130.0 (8Car),
135.9 (=CH), 149.6, 167.1 (2Car), 172.7 (C=O). Analysis for C13H11ClN2O2 (262.69)
Calculated: C: 59.44%, H: 4.22%, N: 10.66%; Found: C: 59.49%, H: 4.26%, N: 10.62%.
N-[(5-iodofuran-2-yl)methylidene]-2-phenylacethydrazide (5)
1
CAS Registry Number: 343592-07-4. Yield: 79%; m.p.: 157-159°C; ClogP = 2.74; H NMR
(DMSO-d6) δ (ppm) = 3.91 (s, 2H, CH2), 6.81-6.85 (m, 2H, ArH), 7.25-7.33 (m, 4H, ArH),
13
7.81 (s, 1H, ArH), 8.00 (s, 1H, =CH), 11.38 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 28.1
(CH2), 115.2, 116.7, 126.9, 128.6, 128.8, 129.5, 130.0 (7Car), 136.0 (=CH), 154.6, 154.7,
167.1 (3Car), 172.6 (C=O). Analysis for C13H11IN2O2 (354.14) Calculated: C: 44.09%, H:
3.13%, N: 7.91%; Found: C: 44.12%, H: 3.15%, N: 7.88%.
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N-[(5-nitrofuran-2-yl)methylidene]-2-phenylacethydrazide (6)
1
CAS Registry Number: 324577-71-1. Yield: 93%; m.p.: 186-188°C; ClogP = 0.27; H NMR
(DMSO-d6) δ (ppm) = 3.96 (s, 2H, CH2), 7.21-7.36 (m, 4H, ArH), 7.77-7.81 (m, 2H, ArH),
7.94 (s, 1H, ArH), 8.19 (s, 1H, =CH), 11.83 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 27.9
(CH2), 115.3, 126.9, 127.2, 128.7, 128.9, 129.6, 130.1, 131.4 (8Car), 135.6 (=CH), 152.2,
168.3 (2Car), 173.2 (C=O). Analysis for C13H11N3O4 (273.24) Calculated: C: 57.14%, H:
4.06%, N: 15.38%; Found: C: 57.19%, H: 4.02%, N: 15.42%.
N-[(2-bromo-5-fluorophenyl)methylidene]-2-phenylacethydrazide (7)
1
Yield: 58%; m.p.: 161-163°C; ClogP = 4.29; H NMR (DMSO-d6) δ (ppm) = 4.02 (s, 2H,
CH2), 7.20-7.63 (m, 4H, ArH), 7.71-7.73 (m, 1H, ArH), 7.74-7.77 (m, 2H, ArH), 8.29-8.30
(d, 1H, ArH, J = 3 Hz), 8.53 (s, 1H, =CH), 11.69 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) =
27.6 (CH2), 118.0, 118.3, 118.8, 119.2, 126.8, 127.2, 128.8, 129.6, 129.9 (10Car), 135.5
(=CH), 136.1, 167.3 (2Car), 173.1 (C=O). Analysis for C15H12BrFN2O (335.17) Calculated:
C: 53.75%, H: 3.61%, N: 8.36%; Found: C: 53.81%, H: 3.60%, N: 8.39%.
N-[(2-chloro-3-methoxyphenyl)methylidene]-2-phenylacethydrazide (8)
1
Yield: 95%; m.p.: 197-199°C; ClogP = 3.62; H NMR (DMSO-d6) δ (ppm) = 3.88 (s, 3H,
CH3), 3.99 (s, 2H, CH2), 7.18-7.35 (m, 4H, ArH), 7.37-7.39 (m, 1H, ArH), 7.50-7.53 (m, 1H,
13
ArH), 7.58-7.61 (m, 2H, ArH), 8.42 (s, 1H, =CH), 11.60 (s, 1H, NH); C NMR (DMSO) δ
(ppm) = 27.9 (CH2), 56.8 (CH3), 113.4, 121.5, 124.1, 126.8, 128.4, 129.2, 129.4, 135.0
(11Car), 150.00 (=CH), 157.6 (Car), 168.3 (C=O). Analysis for C16H15ClN2O2 (302.76)
Calculated: C: 63.47%, H: 4.99%, N: 9.25%; Found: C: 63.51%, H: 4.97%, N: 9.28%.
N-[(2-chloro-5-nitrophenyl)methylidene]-2-phenylacethydrazide (9)
CAS Registry Number: 349457-86-9. Yield: 90%; m.p.: 193-195°C; ClogP = -1.53; 1H NMR
(DMSO-d6) δ (ppm) = 4.03 (s, 2H, CH2), 7.20-7.29 (m, 1H, ArH), 7.31-7.34 (m, 1H, ArH),
7.81-7.82 (d, 1H, ArH, J = 3 Hz), 7.84-7.85 (d, 1H, ArH, J = 3 Hz), 8.20-8.25 (m, 2H, ArH),
8.38 (s, 1H, =CH), 8.62-8.63 (d, 1H, ArH, J = 3 Hz), 8.66-8.67 (d, 1H, ArH, J = 3 Hz), 11.79
13
(s, 1H, NH); C NMR (DMSO) δ (ppm) = 27.8 (CH2), 122.0, 124.7, 128.2, 129.2, 129.4,
134.7, 135.5, 139.1, 145.9 (10Car), 148.2 (=CH), 155.1, 159.4 (2Car), 168.3 (C=O). Analysis
for C15H12ClN3O3 (317.73) Calculated: C: 56.70%, H: 3.81%, N: 13.23%; Found: C: 56.74%,
H: 3.83%, N: 13.20%.
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N-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]-2-phenylacethydrazide (10)
1
CAS Registry Number: 341973-80-6. Yield: 91%; m.p.: 156-158°C; ClogP = 2.57; H NMR
(DMSO-d6) δ (ppm) = 3.79 (s, 3H, CH3), 3.81 (s, 3H, CH3), 3.97 (s, 2H, CH2), 6.94 (s, 2H,
ArH), 7.18-7.33 (m, 4H, ArH), 7.85 (s, 1H, ArH), 8.09 (s, 1H, =CH), 8.87 (s, 1H, OH), 11.29
13
(s, 1H, NH); C NMR (DMSO) δ (ppm) = 28.9 (CH2), 56.8 (2xCH3), 107.4, 128.1, 128.4,
129.2, 129.4, 134.8, 137.4 (10Car), 147.0 (=CH), 148.6 (2Car), 168.3 (C=O). Analysis for
C17H18N2O4 (314.34) Calculated: C: 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.99%, H:
5.74%, N: 8.94%.
2-hydroxy-N-[1H-pyrrol-2-ylmethylidene]acethydrazide (11)
1
Yield: 46%; m.p.: 186-188°C; ClogP = -0.27; H NMR (DMSO-d6) δ (ppm) = 3.92 (s, 2H,
CH2), 5.49 (s, 1H, OH), 6.10-6.13 (m, 1H, ArH), 6.39-6.41 (m, 1H, ArH), 6.87-6.90 (m, 1H,
13
ArH), 8.20 (s, 1H, =CH), 10.90 (s, 1H, NH), 11.45 (s, 1H, NH); C NMR (DMSO) δ (ppm)
= 61.7 (CH2), 109.6, 113.6, 122.8, 127.5 (4Car), 140.9 (=CH), 168.0 (C=O). Analysis for
C7H9N3O2 (167.16) Calculated: C: 50.29%, H: 5.43%, N: 25.14%; Found: C: 50.33%, H:
5.41%, N: 25.18%.
N-[(5-chlorofuran-2-yl)methylidene]-2-hydroxyacethydrazide (12)
CAS Registry Number: 1883135-55-4. Yield: 47%; m.p.: 150-152°C; ClogP = 0.04; 1H NMR
(DMSO-d6) δ (ppm) = 3.97 (s, 2H, CH2), 5.63 (s, 1H, OH), 6.65-6.66 (d, 1H, ArH, J = 3 Hz),
6.95-6.96 (d, 1H, ArH, J = 3 Hz), 8.22 (s, 1H, =CH), 11.31 (s, 1H, NH); 13C NMR (DMSO) δ
(ppm) = 61.7 (CH2), 109.9, 116.1, 136.6, 137.6 (4Car), 149.7 (=CH), 168.9 (C=O). Analysis
for C7H7ClN2O3 (202.59) Calculated: C: 41.50%, H: 3.48%, N: 13.83%; Found: C: 41.54%,
H: 3.49%, N: 13.85%.
2-hydroxy-N-[(5-iodofuran-2-yl)methylidene]acethydrazide (13)
1
Yield: 67%; m.p.: 177-179°C; ClogP = 0.34; H NMR (DMSO-d6) δ (ppm) = 3.97 (s, 2H,
CH2), 5.63 (s, 1H, OH), 6.79-6.80 (d, 1H, ArH, J = 3 Hz), 6.83-6.85 (m, 1H, ArH), 8.20 (s,
13
1H, =CH), 11.21 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 61.7 (CH2), 96.4, 116.5, 122.7,
136.4 (4Car), 154.8 (=CH), 168.9 (C=O). Analysis for C7H7IN2O3 (294.05) Calculated: C:
28.59%, H: 2.40%, N: 9.53%; Found: C: 28.65%, H: 2.38%, N: 9.56%.
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2-hydroxy-N-[(5-nitrofuran-2-yl)methylidene]acethydrazide (14)
1
CAS Registry Number: 89792-34-7. Yield: 96%; m.p.: 200-202°C; ClogP = -2.13; H NMR
(DMSO-d6) δ (ppm) = 4.03 (s, 2H, CH2), 5.72 (s, 1H, OH), 7.21-7.22 (d, 1H, ArH, J = 3 Hz),
7.78-7.79 (d, 1H, ArH, J = 3 Hz), 8.38 (s, 1H, =CH), 11.73 (s, 1H, NH); 13C NMR (DMSO) δ
(ppm) = 61.9 (CH2), 114.8, 116.2, 135.3, 151.7 (4Car), 153.1 (=CH), 168.7 (C=O). Analysis
for C7H7N3O5 (213.15) Calculated: C: 39.44%, H: 3.31%, N: 19.71%; Found: C: 39.49%, H:
3.29%, N: 19.75%.
N-[(2-bromo-5-fluorophenyl)methylidene]-2-hydroxyacethydrazide (15)
1
Yield: 70%; m.p.: 187-189°C; ClogP = 1.89; H NMR (DMSO-d6) δ (ppm) = 4.02 (s, 2H,
CH2), 5.64 (s, 1H, OH), 7.21-7.30 (m, 1H, ArH), 7.61-7.68 (m, 1H, ArH), 7.70-7.76 (m, 1H,
ArH), 8.74 (s, 1H, =CH), 11.63 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 61.8 (CH2),
118.5, 119.5, 135.4, 140.9 (4Car), 145.2 (=CH), 160.3, 163.5 (2Car), 169.3 (C=O). Analysis
for C9H8BrFN2O2 (275.07) Calculated: C: 39.30%, H: 2.93%, N: 10.18%; Found: C: 39.35%,
H: 2.90%, N: 10.21%.
N-[(2-chloro-3-methoxyphenyl)methylidene]-2-hydroxyacethydrazide (16)
1
Yield: 77%; m.p.: 165-167°C; ClogP = 1.22; H NMR (DMSO-d6) δ (ppm) = 3.88 (s, 3H,
CH3), 4.00 (s, 2H, CH2), 5.61 (s, 1H, OH), 7.16-7.20 (m, 1H, ArH), 7.31-7.38 (m, 1H, ArH),
7.48-7.55 (m, 1H, ArH), 8.83 (s, 1H, =CH), 11.58 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) =
56.8 (CH3), 61.7 (CH2), 113.9, 118.7, 121.8, 133.4, 144.0 (5Car), 144.2 (=CH), 155.4 (Car),
169.1 (C=O). Analysis for C10H11ClN2O3 (242.66) Calculated: C: 49.50%, H: 4.57%, N:
11.54%; Found: C: 49.50%, H: 4.59%, N: 11.52%.
N-[(2-chloro-5-nitrophenyl)methylidene]-2-hydroxyacethydrazide (17)
1
Yield: 95%; m.p.: 232-233°C; ClogP = -0.5; H NMR (DMSO-d6) δ (ppm) = 4.05 (s, 2H,
CH2), 5.70 (s, 1H, OH), 7.81-7.84 (d, 1H, ArH, J = 9 Hz), 8.19-8.25 (m, 1H, ArH), 8.64-8.65
(d, 1H, ArH, J = 9 Hz), 8.87 (s, 1H, =CH), 11.78 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) =
61.8 (CH2), 121.5, 125.6, 132.0, 133.7, 137.8, 142.0 (6Car), 147.1 (=CH), 169.5 (C=O).
Analysis for C9H8ClN3O4 (257.63) Calculated: C: 41.96%, H: 3.13%, N: 16.31%; Found: C:
41.93%, H: 3.14%, N: 16.35%.
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2-hydroxy-N-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]acethydrazide (18)
1
Yield: 61%; m.p.: 112-114°C; ClogP = -0.26; H NMR (DMSO-d6) δ (ppm) = 3.79 (s, 3H,
CH3), 3.81 (s, 3H, CH3), 3.97 (s, 2H, CH2), 5.57 (s, 1H, OH), 6.91-6.92 (d, 2H, ArH, J = 3
13
Hz), 8.24 (s, 1H, =CH), 8.86 (s, 1H, OH), 11.04 (s, 1H, NH); C NMR (DMSO) δ (ppm) =
56.5 (2xCH3), 61.7 (CH2), 104.7, 104.9, 124.8, 125.0, 138.3, 142. (6Car), 148.5 (=CH), 168.4
(C=O). Analysis for C11H14N2O5 (254.24) Calculated: C: 51.97%, H: 5.55%, N: 11.02%;
Found: C: 51.95%, H: 5.57%, N: 11.06%.
N-[(3-chloro-4-methoxyphenyl)methylidene]-2-hydroxyacethydrazide (19)
1
Yield: 85%; m.p.: 231-232°C. ClogP = 1.22. H NMR (DMSO-d6) δ (ppm) = 3.90 (s, 3H,
CH3), 3.97 (s, 2H, CH2), 5.59 (s, 1H, OH), 7.18-7.24 (m, 1H, ArH), 7.56-7.62 (m, 1H, ArH),
7.73-7.76 (m, 1H, ArH), 8.30 (s, 1H, =CH), 11.20 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) =
56.7 (CH3), 61.7 (CH2), 113.4, 122.0, 128.0, 128.2, 128.4, 142.1 (6Car), 146.4 (=CH), 168.7
(C=O). Analysis for C10H11ClN2O3 (242.66) Calculated: C: 49.50%, H: 4.57%, N: 11.54%;
Found: C: 49.55%, H: 4.55%, N: 11.58%.
N-[(3,5-dibromo-4-hydroxyphenyl)methylidene]-2-hydroxyacethydrazide (20)
1
Yield: 94%; m.p.: 258-260°C; ClogP = 2.26; H NMR (DMSO-d6) δ (ppm) = 3.98 (s, 2H,
CH2), 5.59 (s, 1H, OH), 7.82-7.83 (d, 2H, ArH, J = 3 Hz), 8.22 (s, 1H, =CH), 11.29 (s, 1H,
OH), 11.40 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 61.7 (CH2), 112.7, 129.3, 130.8,
131.0, 140.9 (5Car), 144.9 (=CH), 152.6 (Car), 168.8 (C=O). Analysis for C9H8Br2N2O3
(351.98) Calculated: C: 30.71%, H: 2.29%, N: 7.96%; Found: C: 30.76%, H: 2.27%, N:
7.99%.
N-[(3-bromo-4-methoxyphenyl)methylidene]-2-hydroxyacethydrazide (21)
1
Yield: 94%; m.p.: 237-239°C; ClogP = 1.50; H NMR (DMSO-d6) δ (ppm) = 3.89 (s, 3H,
CH3), 3.90 (s, 2H, CH2), 5.58 (s, 1H, OH), 7.14-7.20 (t, 1H, ArH, J = 9 Hz), 7.59-7.65 (m
1H, ArH), 7.87-7.89 (m, 1H, ArH), 8.29 (s, 1H, =CH), 11.20 (s, 1H, NH); 13C NMR (DMSO)
δ (ppm) = 56.9 (CH3), 61.7 (CH2), 111.6, 113.3, 128.7, 128.9, 130.9, 131.2 (6Car), 146.2
(=CH), 168.7 (C=O). Analysis for C10H11BrN2O3 (287.11) Calculated: C: 41.83%, H: 3.86%,
N: 9.76%; Found: C: 41.87%, H: 3.84%, N: 9.79%.
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N-[(3-bromo-4-hydroxyphenyl)methylidene]-2-hydroxyacethydrazide (22)
1
Yield: 40%; m.p.: 190-192°C; ClogP = 1.46; H NMR (DMSO-d6) δ (ppm) = 3.96 (s, 2H,
CH2), 5.57 (s, 1H, OH), 6.97-7.01 (m, 1H, ArH), 7.45-7.50 (m, 1H, ArH), 7.76-7.82 (m, 1H,
ArH), 8.24 (s, 1H, =CH), 10.77 (s, 1H, OH), 11.12 (s, 1H, NH); 13C NMR (DMSO) δ (ppm)
= 61.7 (CH2), 110.2, 116.9, 127.3, 127.5, 128.1, 131.7 (6Car), 146.6 (=CH), 168.6 (C=O).
Analysis for C9H9BrN2O3 (273.08) Calculated: C: 39.58%, H: 3.32%, N: 10.26%; Found: C:
39.63%, H: 3.30%, N: 10.29%.
N-[(2-bromo-3-hydroxy-4-methoxyphenyl)methylidene]-2-hydroxyacethydrazide (23)
1
Yield: 65%; m.p.: 216-218°C; ClogP = 1.25; H NMR (DMSO-d6) δ (ppm) = 3.87 (s, 3H,
CH3), 3.97 (s, 2H, CH2), 5.56 (s, 1H, OH), 7.03-7.09 (m, 1H, ArH), 7.35-7.44 (m, 1H, ArH),
13
8.68 (s, 1H, =CH), 9.65 (s, 1H, OH), 11.43 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 56.7
(CH3), 61.7 (CH2), 111.4, 112.3, 118.1, 126.5, 143.1, 144.2 (6Car), 147.1 (=CH), 168.7
(C=O). Analysis for C10H11BrN2O4 (303.11) Calculated: C: 39.62%, H: 3.66%, N: 9.24%;
Found: C: 39.66%, H: 3.64%, N: 9.26%.
N-[(2,3-difluorophenyl)methylidene]-2-hydroxyacethydrazide (24)
1
Yield: 51%; m.p.: 179-181°C; ClogP = 1.24; H NMR (DMSO-d6) δ (ppm) = 4.02 (s, 2H,
CH2), 5.64 (s, 1H, OH), 7.76-7.90 (m, 2H, ArH), 8.02-8.05 (m, 1H, ArH), 8.47 (s, 1H, =CH),
11.48 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 61.7 (CH2), 118.8, 122.0, 124.7, 124.8,
125.6, 135.5 (6Car), 139.6 (=CH), 169.1 (C=O). Analysis for C9H8F2N2O2 (214.17)
Calculated: C: 50.47%, H: 3.77%, N: 13.08%; Found: C: 50.54%, H: 3.79%, N: 13.05%.
2-hydroxy-N-{[4-(trifluoromethyl)phenyl]methylidene}acethydrazide (25)
1
Yield: 74%; m.p.: 142-144°C; ClogP = 1.84; H NMR (DMSO-d6) δ (ppm) = 4.01 (s, 2H,
CH2), 5.90 (s, 1H, OH), 7.51-7.53 (d, 2H, ArH, J = 6 Hz), 7.65-7.67 (d, 2H, ArH, J = 6 Hz),
8.41 (s, 1H, =CH), 10.18 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 61.9 (CH2), 124.5,
127.2, 129.5, 130.2, 135.9 (6Car), 148.6 (=CH), 168.7 (C=O). Analysis for C10H9F3N2O2
(246.18) Calculated: C: 48.79%, H: 3.68%, N: 11.30%; Found: C: 48.83%, H: 3.70%, N:
11.33%.
2-hydroxy-N-[(2-hydroxy-3,5-diiodophenyl)methylidene]acethydrazide (26)
1
Yield: 94%; m.p.: 248-250°C; ClogP = 2.76; H NMR (DMSO-d6) δ (ppm) = 4.06 (s, 2H,
CH2), 5.72 (s, 1H, OH), 7.77-7.78 (d, 1H, ArH, J = 3 Hz), 8.03-8.04 (d, 1H, ArH, J = 3 Hz),
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13
8.42 (s, 1H, =CH), 11.99 (s, 1H, OH), 12.90 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 61.5
(CH2), 82.5, 88.2, 120.7, 139.2, 146.8 (5Car), 147.6 (=CH), 157.0 (Car), 169.2 (C=O).
Analysis for C9H8I2N2O3 (445.98) Calculated: C: 24.24%, H: 1.81%, N: 6.28%; Found: C:
24.28%, H: 1.80%, N: 6.30%.
N-[(3-ethoxy-2-hydroxyphenyl)methylidene]-2-hydroxyacethydrazide (27)
1
Yield: 97%; m.p.: 207-209°C; ClogP = 0.76; H NMR (DMSO-d6) δ (ppm) = 1.32-1.36 (t,
3H, CH3, J = 6 Hz), 4.00-4.08 (q, 2H, CH2, J = 9 Hz, J = 6 Hz), 4.03 (s, 2H, CH2), 5.65 (s,
1H, OH), 6.77-6.85 (m, 1H, ArH), 6.96-7.05 (m, 1H, ArH), 7.18-7.20 (d, 1H, ArH, J = 6 Hz),
8.59 (s, 1H, =CH), 11.13 (s, 1H, OH), 11.50 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 13.8
(CH3), 61.9 (CH2), 64.5 (CH2), 118.7, 120.1, 120.4, 122.2, 146.9, 147.9 (6Car), 149.4 (=CH),
168.7 (C=O). Analysis for C11H14N2O4 (238.24) Calculated: C: 55.46%, H: 5.92%, N:
11.76%; Found: C: 55.51%, H: 5.90%, N: 11.79%.
N-[(3-ethoxy-4-methoxyphenyl)methylidene]-2-hydroxyacethydrazide (28)
1
Yield: 61%; m.p.: 172-174°C; ClogP = 0.79; H NMR (DMSO-d6) δ (ppm) = 1.33-1.37 (t,
3H, CH3, J = 6 Hz), 3.80 (s, 3H, CH3), 3.98 (s, 2H, CH2), 4.01-4.08 (q, 2H, CH2, J = 6 Hz, J
= 9 Hz), 5.58 (s, 1H, OH), 6.97-7.02 (m, 1H, ArH), 7.12-7.14 (m, 1H, ArH), 7.23-7.26 (m,
13
1H, ArH), 8.28 (s, 1H, =CH), 11.08 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 13.8 (CH3),
56.8 (CH3), 61.9 (CH2), 64.5 (CH2), 111.9, 114.5, 121.8, 128.9 (4Car), 147.9 (=CH), 149.2,
151.7 (2Car), 168.7 (C=O). Analysis for C12H16N2O4 (252.27) Calculated: C: 57.13%, H:
6.39%, N: 11.10%; Found: C: 57.18%, H: 6.37%, N: 11.07%.
2-hydroxy-N-[(4-propoxyphenyl)methylidene]acethydrazide (29)
1
Yield: 81%; m.p.: 122-124°C; ClogP = 1.52; H NMR (DMSO-d6) δ (ppm) = 0.96-1.00 (t,
3H, CH3, J = 6 Hz), 1.68-1.81 (m, 2H, CH2), 3.94-3.98 (t, 2H, CH2, J = 6 Hz), 4.00 (s, 2H,
CH2), 5.58 (s, 1H, OH), 6.96-7.06 (m, 2H, ArH), 7.55-7.60 (m, 2H, ArH), 8.65 (s, 1H, =CH),
13
11.06 (s, 1H, NH); C NMR (DMSO) δ (ppm) = 10.6 (CH3), 21.4 (CH2), 61.9 (CH2), 71.7
(CH2), 115.1, 127.4, 128.8 (5Car), 148.6 (=CH), 159.5 (Car), 167.7 (C=O). Analysis for
C12H16N2O3 (236.27) Calculated: C: 61.00%, H: 6.83%, N: 11.86%; Found: C: 61.05%, H:
6.85%, N: 11.84%.
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2-hydroxy-N-{[4-(pyrrolidin-1-yl)phenyl]methylidene}acethydrazide (30)
1
Yield: 81%; m.p.: 256-258°C; ClogP = 1.55; H NMR (DMSO-d6) δ (ppm) = 1.94-19.8 (m,
4H, 2xCH2-pyrrolidine), 3.26-3.29 (m, 4H, 2xCH2-pyrrolidine), 3.93 (s, 2H, CH2), 5.53 (s, 1H, OH),
13
6.53-6.58 (m, 2H, ArH), 7.41-7.48 (m, 2H, ArH), 8.20 (s, 1H, =CH), 11.09 (s, 1H, NH); C
NMR (DMSO) δ (ppm) = 25.4 (2Cpyrrolidine), 47.7 (2Cpyrrolidine), 61.7 (CH2), 111.9, 121.4,
128.6, 128.9, 144.9 (5Car), 148.9 (=CH), 149.3 (Car), 167.9 (C=O). Analysis for C13H17N3O2
(247.29) Calculated: C: 63.14%, H: 6.93%, N: 16.99%; Found: C: 63.17%, H: 6.91%, N:
17.02%.
2-hydroxy-N-{[4-(piperidin-1-yl)phenyl]methylidene}acethydrazide (31)
1
Yield: 76%; m.p.: 160-162°C; ClogP = 1.28; H NMR (DMSO-d6) δ (ppm) = 1.53-1.58 (m,
6H, 3xCH2-piperidine), 3.23-3.25 (m, 4H, 2xCH2-piperidine), 3.94 (s, 2H, CH2), 5.55 (s, 1H, OH),
13
6.91-6.99 (m, 2H, ArH), 7.43-7.49 (m, 2H, ArH), 8.23 (s, 1H, =CH), 10.93 (s, 1H, NH); C
NMR (DMSO) δ (ppm) = 23.4, 25.1, 49.7 (5Cpiperidine), 61.9 (CH2), 110.8, 124.6, 129.7 (5Car),
148.6 (=CH), 152.5 (Car), 168.7 (C=O). Analysis for C14H19N3O2 (261.32) Calculated: C:
64.35%, H: 7.33%, N: 16.08%; Found: C: 64.39%, H: 7.37%, N: 16.11%.
2-hydroxy-N-[quinolin-2-ylmethylidene]acethydrazide (32)
1
Yield: 87%; m.p.: 219-221°C. ClogP = 1.45. H NMR (DMSO-d6) δ (ppm) = 4.16 (CH2),
5.73 (s, 1H, OH), 7.61-7.66 (m, 1H, ArH), 7.71-7.88 (m, 1H, ArH), 7.99-8.14 (m, 3H, ArH),
8.38-8.42 (m, 1H, ArH), 8.57 (s, 1H, =CH), 11.73 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) =
61.9 (CH2), 117.9, 126.9, 127.6, 128.0, 129.7, 130.1, 136.3, 141.8 (8Car), 143.4 (=CH), 151.5
(Car), 168.7 (C=O). Analysis for C12H11N3O2 (229.23) Calculated: C: 62.87%, H: 4.84%, N:
18.33%; Found: C: 62.92%, H: 4.83%, N: 18.30%.
Microbiology
In vitro antimicrobial assay
The examined compounds 3-32 were screened in vitro for antibacterial and antifungal
activities using the broth microdilution method according to European Committee on
Antimicrobial Susceptibility Testing (EUCAST) [32] and Clinical and Laboratory Standards
Institute guidelines [33] against a panel of reference and clinical or saprophytic strains of
microorganisms, including Gram-positive bacteria (Staphylococcus aureus ATCC 25923,
Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 6538, Staphylococcus
epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876,
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Micrococcus luteus ATCC 10240), Gram-negative bacteria (Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Bordetella
bronchiseptica ATCC 4617, Salmonella typhimurium ATCC 14028, Pseudomonas
aeruginosa ATCC 9027) and fungi belonging to yeasts (Candida albicans ATCC 10231,
Candida parapsilosis ATCC 22019).
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 [34, 35]. 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) effect of the
tested compounds [36].
All the experiments were repeated three times (n = 3) and representative data are
presented. The detailed procedure for the in vitro antimicrobial assay is presented in
Supplementary Materials.
The statistical analysis of obtained antimicrobial assays results is presented in Table 1S. in
Supplementary Materials. Minimal Inhibitory Concentration (MIC) values in this table are
presented as ‘Mode MIC’ - most frequent value in the data set of MIC values and ‘Mean MIC
± SE’ - standard error (SE) is the standard deviation of the sampling distribution of the mean
of MIC values.
RESULTS AND DISCUSSION
Chemistry
The main aim of this study was to synthesize new hydrazide-hydrazones (3-32) and to
investigate theirs in vitro antimicrobial activities. The synthesis of hydrazide intermediate (1,
2) and target compounds (3-32) was performed according to the reactions outlined in Scheme
1.
In this study the hydrazides of phenylacetic acid (1) and glycolic acid (2) were obtained by
the reaction of commercially available ethyl esters with hydrazine hydrate [30, 31]. Proton
1
assignments in H NMR spectra for compounds 1 and 2 showed signals at δ 3.76 ppm (1),
3.90 ppm (2) for NH2 group and δ 8.19 ppm (1), 8.84 ppm (2) for NH group. Compounds 1
and 2 showed also the disappearance of the characteristic signals for the ethyl group, which
also successfully confirmed the formation of appropriate hydrazides (1, 2). The treatment of
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obtained hydrazides (1, 2) with appropriate substituted aldehydes yielded the final products
(3-32).
The elemental analysis was performed to confirm the purity of compounds. The
1
1
compounds (3-32) were characterized by H NMR and 13C NMR spectral studies. The H
NMR spectra of all hydrazide-hydrazones (3-32) confirmed the proposed structures. In all the
compounds the NH peaks of hydrazone appeared at δ 10.18 – 11.83 ppm. The peak for =CH
group was observed at δ 8.00 – 8.87 ppm, confirming the formation of final products. In the
13C NMR spectra the peaks for carbonyl carbon (C=O) were present in the range δ 168.0 –
173.2 ppm (Scheme 1.). The signals for other aliphatic and aromatic residues in 1H NMR and
13C NMR appeared in their respective range. Moreover the elemental analyses were
consistent with their assigned structures. All obtained physico-chemical data for synthesized
compounds is given in Experimental Section.
Among the synthesized derivatives (3-32) only compound 3 [37], 6 [38] and 14 [39] have
any reference in the literature. Structures of compounds 4, 5, 9, 10 and 12 are known, but
there are no references reporting their use, synthesis and physico-chemical characterization.
In addition to this our 22 synthesized hydrazide-hydrazones: 7, 8, 11, 13 and 15-32 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 study indicated that among the examined compounds 3-10 (hydrazide-
hydrazones of phenylacetic acid) only compound 6 had inhibitory effect on the growth of
reference strains of microorganisms. On the basis of minimal inhibitory concentration (MIC)
values presented in Table 1, obtained by the broth microdilution method, it was shown that
tested compound 6 exhibited very strong activity against all tested reference Gram-positive
bacteria with MIC = 0.488 – 7.81 µg/ml and MBC = 0.488 – 15.62 µg/ml. This compound
showed bactericidal effect towards these bacteria (MBC/MIC = 1 – 4). The bacteria
belonging to B. subtilis ATTC 6633 were the most sensitive to this compound (MIC = MBC
= 0.488 µg/ml, MBC/MIC = 1). It is worth stressing that the activity of compound 6 against
B. subtilis ATTC 6633 was 32 times higher than the activity of cefuroxime (MIC = 15.63
µg/ml), whereas the activity against B. cereus ATCC 10876 was 8 times higher than activity
of cefuroxime (MIC = 31.25 µg/ml) and 16 times higher than the activity of ampicillin (MIC
= 62.5 µg/ml).
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The tested compound 6 was found also to be active against reference Gram-negative
bacteria (Bordetella spp. ATTC, Pseudomonas spp. ATTC and rod-shaped bacteria of the
family Enterobacteriaceae) with MIC = 31.25 – 62.5 µg/ml, MBC = 31.25 – 250 µg/ml and
bactericidal effect (MBC/MIC = 1 – 4). Among them, Escherichia coli ATTC 25922 was
especially susceptible to compound 6 (MIC = MBC = 31.25 µg/ml, MBC/MIC = 1) (Table
1). It is worth to add that according to the study recently performed by Palace-Berl et al. N-
[(5-nitrofuran-2-yl)methylidene]-2-phenylacethydrazide (6) shows also promising activity
against Trypanosoma cruzi in comparison to benznidazole [38].
The results of our study indicated that among the examined hydrazide-hydrazones of
hydroxybenzoic acid (11-32), substance 14 showed the widest spectrum of antimicrobial
activity against all tested reference bacteria and yeasts. This compound showed very strong
bactericidal effect towards Staphylococcus spp. ATTC and Bacillus spp. ATTC (MIC = 1.95
– 7.81 µg/ml and MBC = 3.91 – 15.62 µg/ml). The activity of this compound against B.
subtilis ATCC 6633 (MIC = 7.81 µg/ml) was 2 times higher than the activity of cefuroxime
against this bacterium (MIC = 15.63 µg/ml). In the case of B. cereus ATCC 10876 the values
of MIC of compound 14 (MIC = 7.81 µg/ml) were 4 times lower than the MIC of cefuroxime
(MIC = 31.25 µg/ml) and 8 times lower than the MIC of ampicillin (MIC = 62.5 µg/ml). In
both cases this compound showed the bactericidal effect (MBC/MIC = 1 – 2).
In turn, minimum concentration of 14, which inhibited growth of M. luteus ATTC 10240
was 62.5 µg/ml and MBC = 125 µg/ml. Gram-negative rods from Enterobacteriaceae family
were also susceptible to this substance at concentrations from 7.81 µg/ml (S. typhimurium
ATTC 14028 and E. coli ATTC 25922) to 62.5 µg/ml (P. mirabilis ATTC 12453). In
addition, compound 14 possessed mild bioactivity against P. aeruginosa ATTC 9027 and
yeasts belonging to Candida spp. ATTC (MIC = 1000 µg/ml) (Table 1). It is worth to
mention
that
according
to
Hossack
2-hydroxy-N-[(5-nitrofuran-2-
yl)methylidene]acethydrazide (14) shows also antibacterial activity against Proteus vulgaris
urinary tract infections in rats comparable to nitrofurantoin [39].
Compound 26 exhibited very high inhibitory effect on the growth of all tested reference
strains of Gram-positive bacteria with MIC = 3.91 – 7.81 µg/ml and MBC = 7.81 – 125
µg/ml. This compound showed very strong activity towards these bacteria with bactericidal
effect against them, with the exception of micrococci (bacteriostatic activity – MBC/MIC =
16). Compound 26 showed also good bioactivity towards reference yeasts Candida spp. (MIC
= 125 µg/ml, MBC = 500 – 1000 µg/ml and MBC/MIC = 4 – 8) (Table 1).
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Moreover, compounds 16 and 20 exhibited good or moderate and 22 – moderate or mild
activity towards all reference Gram-positive bacteria. The remaining newly synthesized
compounds had some inhibitory effect on the growth of reference strains of bacteria and
yeasts or were inactive against these microorganisms (Table 1).
The influence of substituents on the antimicrobial activity of synthesized compounds (3-
32) is presented in Figure 1.
We would like to underline that strong activity of three synthesized compounds, in some
cases even better than that of commonly used antibiotics, against Gram-positive is crucial due
to the fact that these bacteria are common causes of severe infections. This significant
activity also presents an opportunity for searching new hydrazide-hydrazone analogues as
potential antimicrobial agents.
CONCLUSIONS
In this study 30 hydrazide hydrazones of phenylacetic (3-10) and hydroxyacetic acid (11-
32) were synthesized and evaluated in vitro for their potential antimicrobial activities. Our
results indicated that among newly synthesized compounds 3-32, especially compounds 6, 14
and 26 showed high bactericidal activity against reference bacteria. These substances
exhibited very strong or strong bactericidal effect towards Gram-positive and Gram-negative
bacteria, respectively. Thus, these hydrazide-hydrazones may be regarded as precursor
compounds for searching for new hydrazide-hydrazones showing significant antimicrobial
activity.
CONFILCT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
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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Compound
Compound
R1
R2
R1
R2
No
3
No
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
OH
pyrrol-2-yl
5-chlorofuran-2-yl
5-iodofuran-2-yl
5-nitrofuran-2-yl
2-Br-5-F-Ph
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
4-OH-3,5-diOCH3-Ph
3-Cl-4-OCH3-Ph
2,3-diBr-4-OH-Ph
3-Br-4-OCH3-Ph
3-Br-4-OH-Ph
4
5
6
7
8
2-Cl-3-OCH3-Ph
2-Cl-5-NO2-Ph
4-OH-3,5-diOCH3-Ph
pyrrol-2-yl
2-Br-3-OH-4-OCH3-Ph
2,3-diF-Ph
9
10
11
12
13
14
15
16
17
4-CF3-Ph
2-OH-3,5diI-Ph
3-OC2H5-2-OH-Ph
3-OC2H5-4-OCH3-Ph
4-OC3H7-Ph
OH
5-chlorofuran-2-yl
5-iodofuran-2-yl
5-nitrofuran-2-yl
2-Br-5-F-Ph
OH
OH
OH
4-pyrrolidinyl-Ph
4-piperidinyl-Ph
quinolin-2-yl
OH
2-Cl-3-OCH3-Ph
2-Cl-5-NO2-Ph
OH
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Scheme 1. Synthesis route to new hydrazide-hydrazone derivatives (3-32).
Figure 1. The influence of substituents on the antimicrobial activity of hydrazide-hydrazone
derivatives.
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Table 1. The activity data of hydrazide-hydrazones expressed as MIC (MBC/MFC) [µg/ml] against the reference strains of bacteria and fungi.
Species
MIC (MBC/MFC) [µg/ml] of the tested compounds
6
14
16
19
20
22
24
26
27
30
31
CIP
CFX
APC
FLU
Staphylococcus
aureus
ATCC 25923
Staphylococcus
aureus
ATCC 6538
Staphylococcus
aureus
ATCC 43300
Staphylococcus
epidermidis
ATCC 12228
Micrococcus
luteus
1.95
(3.91)
7.81
(7.81)
125
(250)
31.25
(500)
500
1000
7.81
1000
(>1000)
-
-
-
0.488
0.49
nd
na
(>1000) (>1000) (7.81)
125
(500)
7.81
(15.62)
7.81
(7.81)
31.25
(125)
1000
(>1000)
7.81
(7.81)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.244
0.244
0.122
0.976
0.031
0.061
0.976
0.98
nd
nd
nd
na
na
na
na
na
na
na
250
(500)
3.91
(3.91)
3.91
(7.81)
62.5
(250)
1000
(>1000)
7.81
(15.62)
-
500
(>1000)
1.95
(1.95)
1.95
(3.91)
250
(500)
1000
(>1000)
3.91
1000
0.24
0.98
15.63
31.25
nd
nd
(7.81) (>1000)
500
(>1000)
7.81
(15.62)
62.5
(125)
500
1000
7.81
(125)
125
(>1000)
nd
(>1000) (>1000)
ATCC 10240
Bacillus
subtilis
ATCC 6633
Bacillus
62.5
(125)
7.81
(7.81)
0.488
(0.488)
7.81
(7.81)
250
(500)
1000
(1000)
500
(1000)
-
-
500
(>1000)
nd
62.5
(125)
3.91
7.81
500
1000
500
(500)
7.81
(7.81)
cereus
-
-
62.5
nd
(15.62) (15.62)
(1000) (>1000)
ATCC 10876
Bordetella
bronchiseptica
ATCC 4617
Klebsiella
62.5
(125)
62.5
(250)
1000
(>1000)
500
(>1000)
-
-
-
-
1000
(>1000)
31.25
(62.5)
15.62
(31.25)
pneumoniae
ATCC 13883
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.122
0.030
nd
nd
nd
nd
na
na
Proteus
31.25
62.5
-
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mirabilis
ATCC 12453
Salmonella
typhimurium
ATCC 14028
Escherichia
coli
ATCC 25922
Pseudomonas
aeruginosa
ATCC 9027
Candida
albicans
ATCC 10231
Candida
parapsilosis
ATCC 22019
(62.5)
(62.5)
31.25
(62.5)
7.81
(7.81)
1000
(>1000)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.061
0.004
0.488
na
nd
nd
nd
na
na
nd
nd
nd
na
na
na
na
31.25
(31.25)
7.81
250
500
(500)
(7.81) (>1000)
62.5
(250)
1000
-
-
-
-
-
-
-
-
na
(>1000)
1000
(>1000)
1000
-
500
1000
1000
1000
125
125
(250)
500
500
0.976
1.953
(>1000) (>1000) (>1000) (>1000) (>1000) (1000)
(>1000) (>1000)
1000
1000
500
1000
1000
(>1000) (>1000)
125
(500)
250
(500)
250
250
-
-
na
(>1000) (>1000) (>1000)
(>1000) (1000)
The standard compounds used as positive control for bacteria: CPX – Ciprofloxacin; CFX – Cefuroxime; APC – Ampicillin and for fungi: FLU –
Fluconazole.
na - not applicable; nd – not determined; ‘-‘ – no activity; MIC – Minimal Inhibitory Concentration; MBC – Minimal Bactericidal Concentration;
Compounds with bactericidal effect (MBC/MIC ≤ 4) are marked in bold.
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This article is protected by copyright. All rights reserved.