Synthesis of promising antimicrobial agents: hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid

Article abstract of DOI:10.1111/cbdd.13639

In this research, we synthesized and evaluated for in vitro antimicrobial activity a new series of hydrazide-hydrazones obtained from 5-nitrofuran-2-carboxylic acid. New compounds were identified and characterized by spectral methods (¹H NMR and ¹³C NMR). All tested hydrazide-hydrazones proved to be promising antimicrobial agents. Antimicrobial activity and antifungal activity of new derivatives of 5-nitrofuran-2-carboxylic acid were revealed in many cases to be higher than the activity of reference substances (nitrofurantoin, cefuroxime and ampicillin).

Full text of DOI:10.1111/cbdd.13639

DR ŁUKASZ POPIOŁEK (Orcid ID : 0000-0002-3250-3257)
DR ANNA BIERNASIUK (Orcid ID : 0000-0002-2844-9876)
Article type : Research Article
Synthesis of promising antimicrobial agents:
hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid
Łukasz Popiołek1*, Bernadetta Rysz1, Anna Biernasiuk2, Monika Wujec1
1 Department of Organic Chemistry, Faculty of Pharmacy with Medical Analytics Division,
Medical University of Lublin, 4A Chodźki Street, 20-093 Lublin, Poland;
2 Department of Pharmaceutical Microbiology, Faculty of Pharmacy with Medical Analytics
Division, Medical University of Lublin, 1 Chodźki Street, 20-093 Lublin, Poland;
*corresponding author: lukasz.popiolek@umlub.pl
ABSTRACT
In this research we synthesized and evaluated for in vitro antimicrobial activity a new series of
hydrazide-hydrazones obtained from 5-nitrofuran-2-carboxylic acid. New compounds were
13
identified and characterized by spectral methods (1H NMR and C NMR). All tested hydrazide-
hydrazones proved to be promising antimicrobial agents. Antimicrobial and antifungal activity of
new derivatives of 5-nitrofuran-2-carboxylic acid revealed in many cases to be higher than the
activity of reference substances (nitrofurantoin, cefuroxime and ampicillin).
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/CBDD.13639
This article is protected by copyright. All rights reserved

GRAPHICAL ABSTRACT
1. INTRODUCTION
Discovery of novel antimicrobial agents represents an up to-date research topic due to the
increasing number of microbial strains resistant to commonly used chemotherapeutics (Coates,
2002). Based on the above fact, there is a considerable need for discovery of new compounds
which will act as antimicrobial agents (Coates, 2002; Moellering, 2011).
A significant part among thousands of new biologically active compounds synthesized each
year by medicinal chemists around the world belongs to the group of hydrazide-hydrazones
(Rollas, 2007; Bala, 2013; Popiołek, 2017A). This is due to the fact that this group of compounds
possesses very interesting and broad spectrum of biological properties (Rollas, 2007; Bala, 2013;
Popiołek, 2017), which include antimicrobial (Rollas, 2002; Metwally, 2006; Backes, 2014;
Manikandan, 2017; Zha, 2017), anticancer (Kumar, 2012; Nasr, 2014, 2018; Nikolova-
Mladenova, 2017; Tantak, 2017; Sreenivasulu, 2019), anti-inflammatory (Sondhi, 2006; Gökçe,
2009; Moldovan, 2011), anticonvulsant (Dehestani, 2018), and analgesic activity (Navidpour,
2014).
In our previous research a series of hydrazide-hydrazones showed very promising antimicrobial
and antifungal activities (Popiołek, 2016A, 2016B, 2017B, 2017C, 2018A). For instance, a series
of nitrofurazone analogues containing hydrazide-hydrazone moiety synthesized by our group
displayed, depending on the compounds and bacterial or fungal strains, higher antimicrobial
activity than ciprofloxacin, nitrofurantoin or cefuroxime, which were used as reference substances
(Popiołek, 2017C). In that research we used different hydrazides of carboxylic acids and
condensed them with 5-nitro-2-furaldehyde (Popiołek, 2017C). In our current research we decided
to modify previous research and we used 5-nitrofuran-2-carboxylic acid hydrazide and condensed
it with various aliphatic and aromatic aldehydes. The data obtained allow us to investigate the
influence of different substituents on the antimicrobial activity of hydrazide-hydrazones. In
addition to this, in our earlier study hydrazide-hydrazones of 3-hydroxy-2-naphthoic acid also
displayed significant anticancer activity against human renal cell adenocarcinoma (769-P) and
This article is protected by copyright. All rights reserved

human hepatocellular carcinoma (HepG2) and proved to be safe towards normal reference cell
lines (rat cardiomyocytes - H9c2 and green monkey kidney cells - GMK), which is very important
in designing novel antimicrobial agents (Popiołek, 2018B).
Motivated by the above facts and findings and in continuation of our ongoing efforts towards
with the searching for antimicrobial agents, in this research we have designed, synthesized and in
vitro evaluated for antimicrobial activity a new series of hydrazide-hydrazones of
5-nitrofuran-2-carboxylic acid.
2. MATERIALS AND METHODS
2.1 General
Reagents and solvent used in this research were purchased from Sigma-Aldrich (Munich,
Germany) and Merck Co. (Darmstadt, Germany) and were used without further purification.
Melting points were determined on Fisher-Johns blocks melting point apparatus (Fisher Scientific,
1
13
Germany) and left 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. Chemical shifts are reported in ppm (δ) with the use of TMS as the standard reference.
The coupling constants (J) are given in Hertz. The progress of the reaction and purity of the
obtained compounds were monitored by TLC using pre-coated 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 the 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.
2.2 Synthesis of 5-nitrofuran-2-carboxylic acid hydrazide (2)
This compound was synthesized according to the procedure described earlier (Ahmad, 2016). In
two-neck round-bottom flask equipped with condenser, thermometer and magnetic stirrer 0.002
mole (0.34g) of methyl ester of 5-nitrofuran-2-carboxylic acid was placed and 5 mL of anhydrous
ethanol was added. Subsequently, the flask was equipped with ice bath and the content was stirred
for 15 minutes, until the ester was dissolved. Next 100 µl of 100% hydrazine hydrate was added
and the content was stirred for 30 minutes. The temperature of the reaction mixture should be
This article is protected by copyright. All rights reserved

around 0°C. After that formed precipitate was filtered off and dried. Obtained compound was re-
crystallized from ethanol.
1
CAS Number: 5469-78-3; Yield: 73%; M.p.: 160ºC; H NMR (DMSO-d6) δ (ppm): 4.71 (s, 2H,
NH2), 7.36-7.37 (d, 1H, ArH, J = 3 Hz), 7.74-7.76 (d, 1H, ArH, J = 6 Hz), 10.21 (s, 1H, NH). 13C
NMR (DMSO-d6) δ (ppm): 113.9, 115.2, 148.2, 152.0 (4Car), 156.1 (C=O); For C5H5N3O4
(171.11) Calculated: C%: 35.10, H%: 2.95, N%: 24.56; Found: C%: 35.22, H%: 2.93, N%: 24.61.
2.3 Synthesis of hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid (3 – 21)
0.0012 Mole (0.2g) of hydrazide of 5-nitrofuran-2-carboxylic acid (2) and 5 mL of ethanol (96%)
was placed in a round bottom flask. Hydrazide was heated under reflux until it completely
dissolved in ethanol. Then 0.00132 mole of appropriate aldehyde was added and the content of the
flask was heated under reflux for 2 hours. After that, the solution was allowed to cool and the
formed precipitate was filtered off and re-crystallized from ethanol.
This article is protected by copyright. All rights reserved

N-(2-methylpropylidene)-5-nitrofuran-2-carbohydrazide (3)
1
Yield: 10%; M.p.: 163°C; H NMR (DMSO-d6) δ (ppm): 1.07-1.09 (d, 6H, 2xCH3, J = 6 Hz),
2.55-2.59 (m, 1H, CH), 7.49-7.50 (d, 1H, ArH, J = 3 Hz), 7.72-7.74 (d, 1H, ArH, J = 6 Hz), 7.78-
13
7.79 (d, 1H, =CH, J = 3 Hz), 11.80 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm): 19.9 (2xCH3),
31.6 (CH), 113.8, 116.7, 147.7, 152.1 (4Car), 152.9 (=CH), 159.3 (C=O); For C9H11N3O4 (225.20)
Calculated: C%: 48.00, H%: 4.92, N%: 18.66; Found: C%: 48.10, H%: 4.91, N%: 18.71.
N-(2,2-dimethylpropylidene)-5-nitrofuran-2-carbohydrazide (4)
1
Yield: 72%; M.p.: 184°C; H NMR (DMSO-d6) δ (ppm): 1.10 (s, 9H, 3xCH3), 7.48-7.50 (d, 1H,
ArH, J = 6 Hz), 7.74 (s, 1H, =CH), 7.77-7.79 (d, 1H, ArH, J = 6 Hz), 11.76 (s, 1H, NH); 13C NMR
(DMSO-d6) δ (ppm): 27.5 (3xCH3), 35.29 (Ct-butyl), 113.3, 121.1, 146.6, 152.2 (4Car), 156.6
(=CH), 161.8 (C=O); For C10H13N3O4 (239.23) Calculated: C%: 50.21, H%: 5.48, N%: 17.56;
Found: C%: 50.34, H%: 5.47, N%: 17.51.
N-[3-(methylsulphanyl)propylidene]-5-nitrofuran-2-carbohydrazide (5)
Yield: 47%; M.p.: 126°C; 1H NMR (DMSO-d6) δ (ppm): 2.09 (s, 3H, CH3), 2.57-2.61 (t, 2H, CH2,
J = 6 Hz), 2.68-2.74 (q, 2H, CH2, J = 6 Hz), 7.50-7.51 (d, 1H, ArH, J = 3 Hz), 7.78-7.79 (d, 1H,
ArH, J = 3 Hz), 7.79-7.83 (t, 1H, =CH, J = 9 Hz, J = 3 Hz), 11.95 (s, 1H, NH); 13C NMR (DMSO-
d6) δ (ppm): 14.6 (SCH3), 30.3 (CH2), 33.6 (CH2), 115.4, 123.6, 138.2, 150.1 (4Car), 154.8 (=CH),
156.2 (C=O); For C9H11N3O4S (257.27) Calculated: C%: 42.02, H%: 4.31, N%: 16.33; Found:
C%: 41.96, H%: 4.30, N%: 16.29.
5-nitro-N-(pentylidene)furan-2-carbohydrazide (6)
1
Yield: 14%; M.p.: 140°C; H NMR (DMSO-d6) δ (ppm): 0.88-0.93 (t, 3H, CH3, J = 6 Hz, J = 9
Hz), 1.28-1.40 (m, 2H, CH2), 1.43-1.53 (m, 2H, CH2), 2.25-2.32 (q, 2H, CH2, J = 9 Hz), 7.48-7.50
(d, 1H, ArH, J = 6 Hz), 7.77-7.79 (t, 1H, =CH, J = 6 Hz), 7.78-7.80 (d, 1H, ArH, J = 6 Hz), 11.85
(s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 14.2 (CH3), 22.2 (CH2), 28.5 (CH2), 32.2 (CH2),
113.3, 116.8, 146.6, 147.7 (4Car), 152.8 (=CH), 155.1 (C=O); For C10H13N3O4 (239.23)
Calculated: C%: 50.21, H%: 5.48, N%: 17.56; Found: C%: 50.35, H%: 5.46, N%: 17.51.
This article is protected by copyright. All rights reserved

N-(hexylidene)-5-nitrofuran-2-carbohydrazide (7)
1
Yield: 37%; M.p.: 130°C; H NMR (DMSO-d6) δ (ppm): 0.86-0.90 (t, 3H, CH3, J = 6 Hz), 1.28-
1.33 (m, 4H, 2xCH2), 1.45-1.55 (m, 2H, CH2), 2.24-2.32 (q, 2H, CH2, J = 9 Hz), 7.48-7.49 (d, 1H,
ArH, J = 3 Hz), 7.77-7.78 (d, 1H, ArH, J = 3 Hz), 7.79-7.80 (t, 1H, =CH, J = 3 Hz), 11.83 (s, 1H,
NH); 13C NMR (DMSO-d6) δ (ppm): 14.0 (CH3), 22.9 (CH2), 27.2 (CH2), 30.0 (CH2), 31.0 (CH2),
115.4, 123.6, 150.1, 154.8 (4Car), 156.2 (=CH), 163.3 (C=O); For C11H15N3O4 (253.25)
Calculated: C%: 52.17, H%: 5.97, N%: 16.59; Found: C%: 51.25, H%: 5.95, N%: 16.60.
N-(heptylidene)-5-nitrofuran-2-carbohydrazide (8)
1
Yield: 45%; M.p.: 138°C; H NMR (DMSO-d6) δ (ppm): 0.85-0.89 (t, 3H, CH3, J = 6 Hz), 1.26-
1.34 (m, 6H, 3xCH2), 1.47-1.51 (m, 2H, CH2), 2.24-2.31 (q, 2H, CH2, J = 9 Hz), 7.48-7.50 (d, 1H,
ArH, J = 6 Hz), 7.76-7.79 (t, 1H, =CH, J = 6 Hz, J = 3 Hz), 7.78-7.79 (d, 1H, ArH, J = 3 Hz),
11.84 (s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 14.4 (CH3), 22.5 (CH2), 26.3 (CH2), 28.8
(CH2), 31.5 (CH2), 32.5 (CH2), 113.9, 116.8, 147.7, 152.2 (4Car), 152.9 (=CH), 155.1 (C=O); For
C12H17N3O4 (267.28) Calculated: C%: 53.92, H%: 6.41, N%: 15.72; Found: C%: 53.79, H%: 6.42,
N%: 15.66.
5-nitro-N-(octylidene)furan-2-carbohydrazide (9)
1
Yield: 19%; M.p.: 124°C; H NMR (DMSO-d6) δ (ppm): 0.84-0.89 (t, 3H, CH3, J = 9 Hz, J = 6
Hz), 1.27-1.30 (m, 8H, 4xCH2), 1.47-1.52 (m, 2H, CH2), 2.24-2.31 (q, 2H, CH2, J = 9 Hz), 7.48-
7.50 (d, 1H, ArH, J = 6 Hz), 7.76-7.79 (t, 1H, =CH, J = 6 Hz, J = 3 Hz), 7.78-7.79 (d, 1H, ArH, J
13
= 3 Hz), 11.83 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm): 14.4 (CH3), 22.5 (CH2), 26.4 (CH2),
28.9 (CH2), 29.1 (CH2), 31.7 (CH2), 32.5 (CH2), 113.9, 116.8, 147.7, 152.2 (4Car), 152.9 (=CH),
155.1 (C=O); For C13H19N3O4 (281.31) Calculated: C%: 55.50, H%: 6.81, N%: 14.94; Found:
C%: 55.68, H%: 6.79, N%: 14.96.
5-nitro-N-(nonylidene)furan-2-carbohydrazide (10)
1
Yield: 24%; M.p.: 120°C; H NMR (DMSO-d6) δ (ppm): 0.84-0.88 (t, 3H, CH3, J = 6 Hz), 1.26-
1.32 (m, 10H, 5xCH2), 1.47-1.52 (m, 2H, CH2), 2.24-2.31 (q, 2H, CH2, J = 6 Hz, J = 9 Hz), 7.48-
7.49 (d, 1H, ArH, J = 3 Hz), 7.76-7.79 (t, 1H, =CH, J = 6 Hz, J = 3 Hz), 7.78-7.89 (d, 1H, ArH, J
13
= 3 Hz), 11.83 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm): 14.5 (CH3), 22.6 (CH2), 26.4 (CH2),
27.6 (CH2), 29.1 (CH2), 29.2 (CH2), 31.7 (CH2), 32.5 (CH2), 113.9, 116.8, 147.7, 152.2 (4Car),
This article is protected by copyright. All rights reserved

152.9 (=CH), 155.1 (C=O); For C14H21N3O4 (295.33) Calculated: C%: 56.94, H%: 7.17, N%:
14.23; Found: C%: 56.98, H%: 7.16, N%: 14.26.
N-[(2-bromophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (11)
CAS Number: 1243683-04-6; Yield: 81%; M.p.: 244-245°C; 1H NMR (DMSO-d6) δ (ppm): 7.42-
7.47 (t, 1H, ArH, J = 6 Hz, J = 9 Hz), 7.57-7.59 (d, 1H, ArH, J = 6 Hz), 7.65-7.68 (m, 1H, ArH),
7.75-7.77 (d, 1H, ArH, J = 6 Hz), 7.81-7.82 (d, 1H, ArH, J = 3 Hz), 7.92-7.94 (m, 1H, ArH), 8.46
(s, 1H, =CH), 12.42 (s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 113.9, 117.3, 122.7, 126.9,
129.9, 131.6, 133.5, 136.8, 147.6, 148.3 (10Car), 152.3 (=CH), 153.5 (C=O); For C12H8BrN3O4
(338.11) Calculated: C%: 42.63, H%: 2.38, N%: 12.43; Found: C%: 42.59, H%: 2.37, N%: 12.46.
N-[(3-bromophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (12)
CAS Number: 1243683-04-6; Yield: 91%; M.p.: 173-175°C; 1H NMR (DMSO-d6) δ (ppm): 7.57-
7.58 (d, 1H, ArH, J = 3 Hz), 7.66-7.73 (m, 4H, ArH), 7.81-7.82 (d, 1H, ArH, J = 3 Hz), 8.47 (s,
13
1H, =CH), 12.34 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm): 115.4, 123.6, 123.9, 125.7, 129.7,
129.9, 130.6, 136.4, 150.1, 154.8 (10Car), 156.2 (=CH), 160.1 (C=O); For C12H8BrN3O4 (338.11)
Calculated: C%: 42.63, H%: 2.38, N%: 12.43; Found: C%: 42.69, H%: 2,38, N%: 12.40.
N-[(4-bromophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (13)
1
CAS Number: 913480-93-0; Yield: 68%; M.p.: 204°C; H NMR (DMSO-d6) δ (ppm): 7.30-7.33
(d, 2H, ArH, J = 9 Hz), 7.50-7.55 (m, 1H, ArH), 7.57-7.59 (d, 1H, ArH, J = 6 Hz), 7.81-7.83 (d,
13
1H, ArH, J = 6 Hz), 7.94-7.98 (m, 1H, ArH), 8.75 (s, 1H, =CH), 12.37 (s, 1H, NH); C NMR
(DMSO-d6) δ (ppm): 113.9, 116.6, 117.4, 125.5, 126.9, 142.9, 147.4, 152.3 (10Car), 153.3 (=CH),
160.7 (C=O); For C12H8BrN3O4 (338.11) Calculated: C%: 42.63, H%: 2.38, N%: 12.43; Found:
C%: 42.59, H%: 2.38, N%: 12.39.
N-[(2-chlorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (14)
1
Yield: 79%; M.p.: 240°C; H NMR (DMSO-d6) δ (ppm): 7.45-7.50 (m, 2H, ArH), 7.55-7.56 (m,
1H, ArH), 7.58-7.60 (d, 1H, ArH, J = 6 Hz), 7.82-7.83 (d, 1H, ArH, J = 3 Hz), 8.02-8.05 (m, 1H,
ArH), 8.91 (s, 1H, =CH), 12.50 (s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 113.4, 113.9, 117.3,
121.6, 129.5, 133.3, 135.5, 147.5, 148.8, 152.3 (10Car), 153.3 (=CH), 156.9 (C=O); For
This article is protected by copyright. All rights reserved

C12H8ClN3O4 (293.66) Calculated: C%: 49.08, H%: 2.75, N%: 14.31; Found: C%: 49.15, H%:
2,74, N%: 14,33.
N-[(3-chlorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (15)
1
CAS Number: 125273-90-7; Yield: 67%; M.p.: 158-160°C; H NMR (DMSO-d6) δ (ppm): 7.38-
7.44 (m, 1H, ArH), 7.47-7.52 (m, 1H, ArH), 7.59-7.60 (d, 1H, ArH, J = 3 Hz), 7.71-7.74 (m, 1H,
ArH), 7.82-7.83 (d, 1H, ArH, J = 3 Hz), 8.00-8.03 (m, 1H, ArH), 8.86 (s, 1H, =CH), 12.51 (s, 1H,
NH); 13C NMR (DMSO-d6) δ (ppm): 113.9, 117.5, 124.3, 127.9, 128.7, 132.7, 133.1, 133.7, 147.4,
148.3 (10Car), 152.3 (=CH), 153.3 (C=O); For C12H8ClN3O4 (293.66) Calculated: C%: 49.08, H%:
2.75, N%: 14.31; Found: C%: 49.11, H%: 2.75, N%: 14.28.
N-[(4-chlorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (16)
CAS Number: 29448-95-1; Yield: 87%; M.p.: 204°C; 1H NMR (DMSO-d6) δ (ppm): 7.54-7.56 (d,
2H, ArH, J = 6 Hz), 7.56-7.58 (d, 1H, ArH, J = 6 Hz), 7.77-7.80 (d, 2H, ArH, J = 9 Hz), 7.81-7.82
13
(d, 1H, ArH, J = 3 Hz), 8.49 (s, 1H, =CH), 12.32 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm):
115.3, 123.6, 129.1, 129.4, 134.1, 135.1, 150.2, 154.8 (10Car), 156.3 (=CH), 161.3 (C=O); For
C12H8ClN3O4 (293.66) Calculated: C%: 49.08, H%: 2.75, N%: 14.31; Found: C%: 49.01, H%:
2.74, N%: 14.27.
N-[(2-fluorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (17)
1
CAS Number: 125273-98-5; Yield: 83%; M.p.: 208°C; H NMR (DMSO-d6) δ (ppm): 7.28-7.35
(m, 2H, ArH), 7.52-7.54 (d, 1H, ArH, J = 6 Hz), 7.55-7.59 (m, 2H, ArH), 7.81-7.82 (d, 1H, ArH, J
= 3Hz), 8.50 (s, 1H, =CH), 12.36 (s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 115.4, 117.3, 123.5,
124.5, 125.3, 129.4, 130.8, 150.1, 154.8 (9Car), 156.2 (=CH), 160.4 (Car), 167.2 (C=O); For
C12H8FN3O4 (277.21) Calculated: C%: 51.99, H%: 2.91, N%: 15.16; Found: C%: 52.05, H%:
2.90, N%: 15.10.
N-[(3-fluorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (18)
1
CAS Number: 125273-99-6; Yield: 93%; M.p.: 192-194°C; H NMR (DMSO-d6) δ (ppm): 7.30-
7.35 (t, 2H, ArH, J = 6 Hz, J = 9 Hz), 7.56-7.57 (d, 1H, ArH, J = 3 Hz), 7.80-7.81 (d, 1H, ArH, J
13
= 3 Hz), 7.82-7.85 (m, 2H, ArH), 8.50 (s, 1H, =CH), 12.27 (s, 1H, NH); C NMR (DMSO-d6) δ
(ppm): 114.2, 115.4, 116.9, 121.9, 123.6, 130.0, 137.0, 150.2, 154.8 (9Car), 156.1 (=CH), 162.1
This article is protected by copyright. All rights reserved

(Car), 162.7 (C=O); For C12H8FN3O4 (277.21) Calculated: C%: 51.99, H%: 2.91, N%: 15.16;
Found: C%: 52.12, H%: 2.91, N%: 15.12.
N-[(4-fluorophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (19)
1
CAS Number: 125274-00-2; Yield: 71%; M.p.: 222-223°C; H NMR (DMSO-d6) δ (ppm): 7.19-
7.24 (m, 2H, ArH), 7.46-7.52 (m, 1H, ArH), 7.58-7.60 (d, 1H, ArH, J = 6 Hz), 7.82-7.84 (d, 1H,
ArH, J = 6 Hz), 7.93-7.97 (m, 1H, ArH), 8.73 (s, 1H, =CH), 12.54 (s, 1H, NH); 13C NMR
(DMSO-d6) δ (ppm): 113.9, 117.4, 127.4, 129.1, 132.7, 135.9, 140.2, 147.5 (10Car), 152.7 (=CH),
153.3 (C=O); For C12H8FN3O4 (277.21) Calculated: C%: 51.99, H%: 2.91, N%: 15.16; Found:
C%: 51.89, H%: 2.90, N%: 15.19.
N-[(2-iodophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (20)
Yield: 71%; M.p.: 206-208°C; 1H NMR (DMSO-d6) δ (ppm): 7.26-7.31 (t, 2H, ArH, J = 6 Hz, J =
9 Hz), 7.58-7.59 (d, 1H, ArH, J = 3 Hz), 7.74-7.77 (d, 1H, ArH, J = 9 Hz), 7.81-7.84 (m, 2H,
13
ArH) 8.42 (s, 1H, =CH), 12.37 (s, 1H, NH); C NMR (DMSO-d6) δ (ppm): 103.3, 115.4, 123.5,
127.1, 128.2, 129.2, 130.3, 140.0, 150.2, 154.8 (10Car), 156.1 (=CH), 166.3 (C=O); For
C12H8IN3O4 (385.11) Calculated: C%: 37.42, H%: 2.09, N%: 10.91; Found: C%: 37.39, H%: 2.08,
N%: 10.93.
N-[(3-iodophenyl)methylidene]-5-nitrofuran-2-carbohydrazide (21)
1
Yield: 76%; M.p.: 222°C; H NMR (DMSO-d6) δ (ppm): 7.05-7.09 (t, 1H, ArH, J = 6 Hz), 7.44-
7.46 (d, 1H, ArH, J = 6 Hz), 7.56-7.57 (d, 1H, ArH, J = 3 Hz), 7.69-7.71 (d, 1H, ArH, J = 6 Hz),
7.84-7.85 (d, 1H, ArH, J = 3 Hz), 8.00-8.03 (m, 1H, ArH), 8.45 (s, 1H, =CH), 10.94 (s, 1H, NH);
13C NMR (DMSO-d6) δ (ppm): 99.3, 115.4, 123.5, 126.4, 130.3, 134.3, 134.9, 137.9, 150.2, 154.7
(10Car), 156.1 (=CH), 161.4 (C=O); For C12H8IN3O4 (385.11) Calculated: C%: 37.42, H%: 2.09,
N%: 10.91; Found: C%: 37.50, H%: 2.09, N%: 10.89.
2.4 In vitro antimicrobial assay
The examined compounds 2 – 21 were screened in vitro for antibacterial and antifungal
activities using the broth microdilution method according to European Committee on
Antimicrobial Susceptibility Testing (EUCAST) (EUCAST discussion document E. Dis 5.1) and
Clinical and Laboratory Standards Institute (CLSI M27-S4) guidelines against a panel of reference
This article is protected by copyright. All rights reserved

and clinical or saprophytic strains of microorganisms including Gram-positive bacteria, Gram-
negative bacteria and fungi belonging to yeasts. Detailed antimicrobial assays procedures were
described by our group earlier (Popiołek, 2015).
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 a MIC from 126 to 500 µg/ml, good
bioactivity as a MIC in the range 26 – 125 µg/ml, strong bioactivity with a 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) effect of the tested compounds
(Wiegand, 2008; O'Donnell, 2010).
3. RESULTS AND DISCUSSION
3.1 Chemistry
The main aim of this research was the synthesis, chemical structure identification and in vitro
antimicrobial activity evaluation of new hydrazide-hydrazones. As a starting material for the
synthesis of new compounds we used methyl ester of 5-nitrofuran-2-carboxylic acid (1), which
was converted to an appropriate hydrazide (2) in the reaction with 100% hydrazine hydrate. New
compounds were synthesized on the basis of the condensation reaction of hydrazide of 5-
nitrofuran-2-carboxylic acid (2) with diverse aliphatic and aromatic aldehydes (Scheme 1.).
Among nineteen hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid seven compounds are
registered in CAS database (12, 13, 15 - 19). The rest of the compounds (3-11, 14, 20, 21) are new
in the literature and their chemical structures, physio-chemical properties and biological activity
have not been reported so far. Chemical structures of all synthesized compounds were confirmed
by spectral methods (1H NMR and 13C NMR).
1
In the case of the synthesis of hydrazide of 5-nitrofuran-2-carboxylic acid (2) on the H NMR
spectrum we observed characteristic signals which correspond to hydrazide moiety [-(C=O)-
NHNH2]. For the NH group we found singlet peak at δ 10.21 ppm and for the NH2 group singlet
13
peak appeared at δ 4.71 ppm. On C NMR spectrum signal for carbonyl group was found at δ
156.1 ppm.
Hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid (3-21) possessed the following
1
characteristic signals on the H NMR spectra, which confirmed the successful conduction of
condensation reactions: singlet for the NH group in the range of 10.94 – 12.54 ppm and signal for
This article is protected by copyright. All rights reserved

the =CH group at δ 7.74 – 8.91 ppm. Signals for other aliphatic and aromatic fragments of
13
synthesized molecules were observed at expected values of chemical shift. In case of C NMR
spectra signals for =CH and C=O groups appeared in the range of δ 152.3 – 156.6 ppm and 153.5
– 166.3 ppm, respectively.
This article is protected by copyright. All rights reserved

3.2 In vitro antimicrobial assays
Our data presented in Table 1. indicated that newly synthesized compounds 2 – 21 showed
interesting antimicrobial activity. Those substances exhibited comparable antibacterial effect
against all reference Gram-positive bacteria. Their minimal inhibitory concentrations (MICs) were
0.24 – 500 µg/ml and minimal bactericidal concentrations (MBCs) ranged from 0.48 µg/ml to
>1000 µg/ml. Compounds 12 – 20 exhibited the highest activity towards them (MIC = 0.48 – 500
µg/ml and MBC = 0.98 µg/ml – >1000 µg/ml). The most sensitive were staphylococci to 12 – 20.
Those were Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 43300,
Staphylococcus aureus ATCC 6538, and from other bacteria – Bacillus subtilis ATCC 6633 and
Bacillus cereus ATCC 10876. Compounds 12 – 20 showed very strong or strong bactericidal
activity against the above bacteria (MIC = 0.48 – 15.62 µg/ml and MBC = 0.98 – 62.5 µg/ml,
MBC/MIC = 1 – 4). Additionally, S. epidermidis ATCC 12228 was also very susceptible to
hydrazide of 5-nitrofuran-2-carboxylic acid 2 and hydrazide-hydrazones 3 – 9 and 11 with (MIC =
0.24 – 7.81 µg/ml, MBC = 0.48 – 31.25 µg/ml). These compounds showed also bactericidal effect
(MBC/MIC = 1 – 4) towards reference S. epidermidis. Among Gram-positive bacteria,
Micrococcus luteus ATCC 10240 (MIC = 125 – 500 µg/ml, MBC = 250 – > 1000 µg/ml) was the
least sensitive to substances 2 – 21.
Among newly synthesized hydrazide-hydrazones, compounds 3 – 7 showed also good, strong
and even very strong bactericidal activity against Gram-negative bacteria from Enterobacteriaceae
family, with MIC = 7.81 – 62.5 µg/ml, MBC = 15.62 – 125 µg/ml and MBC/MIC ≤ 4. The
activity of other substances towards Enterobacteriaceae, namely 8 – 10, 14 – 16 and 19 – 20 was
lower (MIC = 125 – 500 µg/ml, MBC = 125 – > 1000 µg/ml). Moreover, some of them: 11 – 13,
17 – 18 and 21 were not active against these bacteria. In turn, the compounds (except substances
11 with 2-bromophenyl and 19 with 4-fluorophenyl substituent) showed especially mild or
moderate effects towards Bordetella bronchiseptica ATCC 4617. Furthermore, hydrazide 2 and
hydrazide-hydrazone with pentylidene substituent 6 exhibited some activity towards Pseudomonas
aeruginosa ATCC 9027 (MIC = 500 – 1000 µg/ml, MBC = 500 – > 1000 µg/ml) and these
microorganisms were the least sensitive to tested compounds.
New hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid 3 – 21 indicated also some
anticandidal activity. The values of MICs of compound 20, which contains the 2-iodophenyl
substituent, were the highest (from 7.81 µg/ml to 15.62 µg/ml), indicating its strong or very strong
activity against all reference Candida spp. Additionally, minimal fungicidal concentrations
This article is protected by copyright. All rights reserved

(MFCs) of compound 20 ranged from 15.62 µg/ml to 31.25 µg/ml and this compound had
fungicidal effect (MFC/MIC = 1 – 2). In the case of remaining compounds, antifungal activity was
varied (MIC = 15.62 – 1000 µg/ml, MFC = 15.62 – >1000 µg/ml). Moreover, some of the
compounds, had no any activity. This applies in particular to compounds 11, 12 and 21 with 2-
bromophenyl, 3-bromophenyl and 3-iodophenyl substituents, respectively. All reference strains
were insensitive to them.
In comparison to the ampicillin, which was used as the reference substance the activity against
Bacillus subtilis ATCC 6633 was the same as that of ampicillin (MIC = 62.5 µg/ml) for
compounds 3, 7 and 9 (MIC = 62.5 µg/ml). Hydrazide 2 and hydrazide-hydrazones 4 and 11
displayed two times higher activity (MIC = 31.25 µg/ml), hydrazide-hydrazones 6 and 19 were
sixteen times more active (MIC = 3.91 µg/ml), whereas hydrazide-hydrazones with
halogensubstitued phenyl 12 – 18 were thirty two times more active (MIC = 1.95 µg/ml) than
ampicillin against this bacterium (MIC = 62.5 µg/ml). Compound 20 with 2-iodophenyl
substituent was the most active. Its MIC value against Bacillus subtilis ATCC 6633 (MIC = 0.48
µg/ml) was 130 times lower than for ampicillin.
Antibacterial activity of hydrazide-hydrazone 7 against Staphylococcus epidermidis ATCC
12228 was the same as the activity of cefuroxime (MIC = 0.24 µg/ml). Towards Bacillus subtilis
ATCC 6633 compounds 6 and 19 (MIC = 3.91 µg/ml), which contain pentylidene and
4-fluorophenyl substituents were four times more active than cefuroxime (MIC = 15.62 µg/ml),
compounds 12 – 18 (MIC = 1.95 µg/ml) were eight times more active, whereas hydrazide-
hydrazone 20 showed the highest activity and was thirty two times more active (MIC = 0.48
µg/ml). When comparing the activity towards Bacillus cereus ATCC 10876, substances with
pentylidene 6, 4-bromophenyl 13 and 4-fluorophenyl 19 substituents showed the activity (MIC =
31.25 µg/ml) equal to the activity of cefuroxime against this bacterium (MIC = 31.25 µg/ml). The
MIC values of hydrazide-hydrazones with phenyl substituent substituted with chlorine at meta-
position 15 and para- position 16 (MIC = 15.62 µg/ml) were two times lower than the MIC of
cefuroxime and for hydrazide-hydrazone with 2-iodophenyl 20 it was four times lower (MIC =
7.81 µg/ml).
The activity of synthesized hydrazide 2 and hydrazide-hydrazones 3 – 21 in comparison with
the activity of nitrofurantoin is as follows: against Staphylococcus aureus ATCC 6538 hydrazide 2
and hydrazide-hydrazones 6 and 12 – 18 showed the same MIC values as nitrofurantoin (MIC =
15.62 µg/ml) against this bacterium. Compound with 4-chlorophenyl substituent 16 was two times
This article is protected by copyright. All rights reserved

more active (MIC = 7.81 µg/ml), hydrazide-hydrazone with 4-fluorophenyl substituent 19 was
four time more active (MIC = 3.91 µg/ml) and the compound substituted with 2-iodophenyl 20
was eight times more active (MIC = 1.95 µg/ml) than nitrofurantoin. The MIC values of
synthesized derivatives of 5-nitrofuran-2-carboxylic acid for hydrazide-hydrazones 11, 13, 15, 16,
17 and 18 (MIC = 7.81 µg/ml) against Staphylococcus aureus ATCC 43300 were equal to the
MIC value of nitrofurantoin (MIC = 7.81 µg/ml). The compounds substituted with halogen at
ortho- position 14 (chlorine) and 20 (iodine) (MIC = 3.91 µg/ml) were two times more potent than
nitrofurantoin towards this bacterium. Towards Staphylococcus epidermidis ATCC 12228
hydrazide of 5-nitrofuran-2-carboxylic acid 2 and hydrazide-hydrazones 3, 4, 5, 8 and 17
displayed activity equal to nitrofurantoin (MIC = 3.91 µg/ml), whereas compounds 11, 15, 18 and
21 (MIC = 1.95 µg/ml) were two times more potent than nitrofurantoin (MIC = 3.91 µg/ml)
against this bacterium. The MIC values for compounds 6, 13, 14, 16 (MIC = 0.98 µg/ml) and for
compounds 12, 19, 20 (MIC = 0.48 µg/ml) were four and eight times lower, respectively, than the
MIC values for nitrofurantoin (MIC = 3.91 µg/ml). Hydrazide-hydrazone with hexylidene moiety
7 (MIC = 0.24 µg/ml) proved to be the most active and showed the activity sixteen times higher
than nitrofurantoin. In the case of the activity against Bacillus subtilis ATCC 6633 compounds 6
(substituted with pentylidene substituent) and 19 (substituted with 4-fluorophenyl) were active at
the same level as nitrofurantoin (MIC = 3.91 µg/ml). Hydrazide-hydrazones with
halogensubstituted phenyl 12 – 18 (MIC = 1.95 µg/ml) were two times more active, and
compound 20 substituted with 2-iodophenyl (MIC = 0.48 µg/ml) was almost eight times more
active than nitrofurantoin (MIC = 3.91 µg/ml) on the basis of their MIC values. Compound 20
showed also activity at the same level in comparison to the nitrofurantoin towards Bacillus cereus
ATCC 10876 (MIC = 7.81 µg/ml). Hydrazide 2 showed activity equal to nitrofurantoin (MIC =
125 µg/ml) against Gram-negative strain Bordetella bronchiseptica ATCC 4617, whereas
hydrazide-hydrazones with alkyl chain 6 (pentylidene) and 7 (hexylidene) (MIC = 7.81 µg/ml)
were two times more active towards Klebsiella pneumoniae ATCC 13883 than nitrofurantoin
(MIC = 15.62 µg/ml). Towards Proteus mirabilis ATCC 12453 compounds with short substituents
3, 4, 5 were as active as nitrofurantoin (MIC = 62.5 µg/ml), hydrazide 2 had its MIC values two
times lower (MIC = 31.25 µg/ml) and compounds 6 and 7 had their MIC values four times lower
(MIC = 15.62 µg/ml) than nitrofurantoin (MIC = 62.5 µg/ml). In comparison to nitrofurantoin
compounds 3, 4, 5 against Salmonella typhimurium ATCC 14028 showed activity equal to
nitrofurantoin (MIC = 31.25 µg/ml), hydrazide 2 and hydrazide-hydrazone 7 were two times more
This article is protected by copyright. All rights reserved

active (MIC = 15.62 µg/ml) and compound 6 was four times more active (MIC = 7.81 µg/ml). The
activity against Escherichia coli ATCC 25922 for compounds 2 and 6 was equal to the activity of
nitrofurantoin (MIC = 7.81 µg/ml).
4. CONCLUSIONS
In this research we synthesized and tested a new series of hydrazide-hydrazones of
5-nitrofuran-2-carboxylic acid for in vitro bioactivity. Our antimicrobial activity assay results
indicated that the newly synthesized compounds 3 - 21 showed high antibacterial activity,
especially against Gram-positive bacteria. In the case of Gram-negative bacteria, the most active
were compounds 3 – 7. In turn, 20 was particularly effective against fungi belonging to yeasts
from Candida spp. The hydrazide-hydrazones which contain alkyl chains as substituents (3 – 10)
showed higher activity against Gram-negative bacteria than hydrazones with aryl substituents (11
– 21), with the exception of heptylidene (8), octylidene (9) and nonylidene (10) substituents.
Whereas hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid with halogensubstituted phenyl
as substituents (11 – 21) inhibited the growth of Gram-positive bacterial strains and fungal
Candida spp. species more efficiently than hydrazones with alkyl chains substituents (3 – 10).
Performed structure-activity analysis suggests that synthesized hydrazide-hydrazones may be
regarded as promising antimicrobial agents what specifically applies to hydrazide-hydrazones of
5-nitrofuran-2-carboxylic acid with short alkyl chains and halogensubstituted phenyl at ortho-
position, especially with iodine atom.
CONFILCT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
REFERENCES
Ahmad, P., Woob, H., Jun, K.-Y., Kadi, A. A., Abdel-Aziz H. A., Kwon, Y., Rahman, A. F. M.
M. (2016). Design, synthesis, topoisomerase I & II inhibitory activity, antiproliferative activity,
This article is protected by copyright. All rights reserved

and structure–activity relationship study of pyrazoline derivatives: An ATP-competitive human
topoisomerase IIa catalytic inhibitor. Bioorganic & Medicinal Chemistry, 24, 1898–1908.
Backes, G. L., Neumann, D. M., Jursic B. S. (2014). Synthesis and antifungal activity of
substituted salicylaldehyde hydrazones, hydrazides and sulfohydrazides. Bioorganic &
Medicinal Chemistry, 22, 4629–4636.
Bala, S., Uppal, G., Kajal, A., Kamboj, S., Sharma, V. (2013). Hydrazones as promising lead with
diversity in bioactivity-therapeutic potential in present scenario. International Journal of
Pharmaceutical Sciences Review and Research, 18(1), 65–74.
CLSI M27-S4, Clinical and Laboratory Standards Institute (2012).Reference method for broth
dilution antifungal susceptibility testing of yeasts, Wayne, PA, USA.
Coates, A., Hu, Y., Bax, R., Page, C. (2002). The future challenges facing the development of new
antimicrobial drugs. Nature Reviews Drug Discovery, 1, 895–910.
Dehestani L., Ahangar, N., Hashemi, S. M., Irannejad, H., Masihi, P. H., Shakiba, A., Emami, S.
(2018). Design, synthesis, in vivo and in silico evaluation of phenacyl triazole hydrazones as
new anticonvulsant agents. Bioorganic Chemistry, 78, 119–129.
EUCAST discussion document E. Dis 5.1, European Committee for Antimicrobial Susceptibility
Testing (EUCAST). (2003). Determination of minimum inhibitory concentrations (MICs) of
antibacterial agents by broth dilution. Clinical Microbiology Infections, 9, 1–7.
Zha, G.-F., Leng, J., Darshini, N., Shubhavathi, T., Vivek, H. K., Asiri, A. M., Marwani, H. M.,
Rakesh, K. P., Mallesha, N., Qin, H.-L. (2017). Synthesis, SAR and molecular docking studies
of benzo[d]thiazolehydrazones as potential antibacterial and antifungal agents. Bioorganic &
Medicinal Chemistry Letters, 27, 3148–3155.
Gökçe, M., Utku, S., Küpeli, E. (2009). Synthesis and analgesic and anti-inflammatory activities
6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted
derivatives. European Journal of Medicinal Chemistry, 44, 3760–3764.
benzal)hydrazone
Kumar, D., Kumar, N. M., Ghosh, S., Shah, K. (2012). Novel bis(indolyl)hydrazide–hydrazones
as potent cytotoxic agents. Bioorganic & Medicinal Chemistry Letters, 22, 212–215.
Manikandan, V., Balaji, S., Senbagam, R., Vijayakumar, R., Rajarajan, M., Vanangamudi, G.,
Arulkumaran, R., Sundararajan, R., Thirunarayanan, G. (2017). Synthesis and antimicrobial
activities of some (E)-N’-1-(substituted benzylidene)benzohydrazides. International Journal of
Advanced Chemistry, 5(1), 17–24.
This article is protected by copyright. All rights reserved

Metwally, K. A., Abdel-Aziz, L. M., Lashine, E.-S. M., Husseiny, M. I., Badawy, R. H. (2006).
Hydrazones of 2-aryl-quinoline-4-carboxylic acid hydrazides: Synthesis and preliminary
evaluation as antimicrobial agents. Bioorganic & Medicinal Chemistry, 14, 8675–8682.
Moellering Jr, R. C. (2011). Discovering new antimicrobial agents. International Journal of
Antimicrobial Agents, 37, 2–9.
Moldovan, C. M., Oniga, O., Pârvu, A., Tiperciuc, B., Verite, P., Pîrnau, A., Crişane, O., Bojiţa,
M., Pop, R. (2011). Synthesis and anti-inflammatory evaluation of some new acyl-hydrazones
bearing 2-aryl-thiazole. European Journal of Medicinal Chemistry, 46, 526–534.
Nasr T., Bondock, S., Rashed, H. M., Fayad, W., Youns, M., Sakr, T. M. (2018). Novel hydrazide-
hydrazone and amide substituted coumarin derivatives: Synthesis, cytotoxicity screening,
microarray, radiolabeling and in vivo pharmacokinetic studies. European Journal of Medicinal
Chemistry, 151, 723–739.
Nasr, T., Bondock, S., Youns, M. (2014). Anticancer activity of new coumarin substituted
hydrazide-hydrazone derivatives. European Journal of Medicinal Chemistry, 76, 539–548.
Navidpour L., Shafaroodi H., Saeedi-Motahar, G., Shafiee, A. (2014). Synthesis, anti-
inflammatory and analgesic activities of arylidene-2-(3-chloroanilino)nicotinic acid hydrazides.
Medicinal Chemistry Research, 23, 2793–2802.
Nikolova-Mladenova B., Momekov G., Ivanov, D., Bakalova, A. (2017). Design and drug-like
properties of new 5-methoxysalicylaldehyde based hydrazones with anti-breast cancer activity.
Journal of Applied Biomedicine, 15, 233–240.
O'Donnell, F., Smyth, T. J., Ramachandran, V. N., Smyth, W. F. (2010). A study of the
antimicrobial activity of selected synthetic and naturally occurring quinolines. International
Journal of Antimicrobial Agents, 35, 30–38.
Popiołek, Ł., Biernasiuk, A., Malm, A. (2015). Synthesis and antimicrobial activity of new 1,3-
thiazolidin-4-one derivatives obtained from carboxylic acid hydrazides. Phosphorus Sulfur,
Silicon and the Related Elements, 190(2), 251–260.
Popiołek, Ł., Biernasiuk, A. (2016A). Design, synthesis and in vitro antimicrobial activity of
hydrazide-hydrazones of 2-substituted acetic acid. Chemical Biology & Drug Design, 88(6),
873–883.
Popiołek, Ł., Biernasiuk, A. (2016B). Hydrazide-hydrazones of 3-methoxybenzoic acid and 4-tert-
butylbenzoic acid with promising antibacterial activity against Bacillus spp. Journal of Enzyme
Inhibition and Medicinal Chemistry, 31(S1), 62–69.
This article is protected by copyright. All rights reserved

Popiołek, Ł. (2017A). Hydrazide-hydrazones as potential antimicrobial agents: overview of the
literature since 2010. Medicinal Chemistry Research, 26(2), 287–301.
Popiołek, Ł., Biernasiuk, A. (2017B). New hydrazides and hydrazide-hydrazones of 2,3-dihalogen
substituted propionic acids: synthesis and in vitro antimicrobial activity evaluation. Chemistry
& Biodiversity, 14(8), e1700075, 1–11.
Popiołek, Ł., Biernasiuk, A. (2017C). Synthesis and investigation of antimicrobial activities of
nitrofurazone analogues containing hydrazide-hydrazone moiety. Saudi Pharmaceutical
Journal, 25(7), 1097–1102.
Popiołek, Ł., Biernasiuk, A., Berecka, A., Gumieniczek, A., Malm, A., Wujec, M. (2018A). New
hydrazide-hydrazones of isonicotinic acid: synthesis, lipophilicity and in vitro antimicrobial
screening. Chemical Biology & Drug Design, 91(4), 915–923.
Popiołek, Ł., Piątkowska-Chmiel, I., Gawrońska-Grzywacz, M., Biernasiuk, A., Izdebska, M.,
Herbet, M., Sysa, M., Malm, A., Dudka, J., Wujec, M. (2018B). New hydrazide-hydrazones
and 1,3-thiazolidin-4-ones with 3-hydroxy-2-naphthoic moiety: Synthesis, in vitro and in vivo
studies. Biomedicine & Pharmacotherapy, 103, 1337–1347.
Rollas, S., Gulerman, N., Erdeniz, H. (2002). Synthesis and antimicrobial activity of some new
hydrazones of 4-fluorobenzoic acid hydrazide and 3-acetyl-2,5-disubstituted-1,3,4-
oxadiazolines. Il Farmaco, 57, 171–174.
Rollas, S., Küçükgüzel, Ş. G. (2007). Biological activities of hydrazone derivatives. Molecules,
12, 1910–1939.
Sondhi, S. M., Dinodia, M., Kumar, A. (2006). Synthesis, anti-inflammatory and analgesic activity
evaluation of some amidine and hydrazone derivatives. Bioorganic & Medicinal Chemistry, 14,
4657–4663.
Sreenivasulu, R., Reddy, K. T., Sujitha, P., Kumar, C. G., Raju, R. R. (2019). Synthesis,
antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-
hydrazone derivatives. Bioorganic & Medicinal Chemistry, 27, 1043–1055.
Tantak, M. P., Klingler, L., Arun, V., Kuma, A., Sadana, R., Kumar, D. (2017). Design and
synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel
tubulin inhibitors. European Journal of Medicinal Chemistry, 136, 184–194.
Wiegand, I., Hilpert, K., Hancock, R. E. W. (2008). Agar and broth dilution methods to determine
the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols,
3(2), 163–175.
This article is protected by copyright. All rights reserved

Compound
number
Yield
(%)
Compound
number
Yield
(%)
R
R
Br
CH
3
3
3
10
13
68
CH
Cl
CH
CH
3
3
CH
3
4
5
72
47
14
15
79
67
Cl
S
CH3
Cl
CH3
6
14
16
87
F
F
CH
3
7
8
37
45
17
18
83
93
CH3
F
CH
3
9
19
19
71
I
I
CH
10
11
24
81
20
21
71
76
3
Br
This article is protected by copyright. All rights reserved

Br
12
91
Scheme 1. Synthesis of new hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid
This article is protected by copyright. All rights reserved

Table 1. The activity data of compounds 2 – 21 expressed as MIC (MBC or MFC) [µg/ml] and {MBC/MIC or MFC/MIC} against the reference strains of bacteria and fungi
MIC (MBC or MFC) [µg/ml] and {MBC/MIC or MFC/MIC} of the tested compounds
Species
CIP/
NY*
NIT
CFX
APC
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Staphylococcus
aureus
62.5
(62.5)
{1}
62.5
(125)
{2}
62.5
(125)
{2}
125
(125)
{1}
62.5
(62.5)
{1}
125
(125)
{1}
250
(500)
{2}
250
(250)
{1}
250
(250)
{1}
125
(500)
{4}
250
(1000)
{4}
62.5
(62.5)
{1}
62.5
(125)
{2}
31.25
(62.5)
{2}
31.25
(125)
{4}
62.5
(62.5)
{1}
31.25
(62.5)
{2}
62.5
(62.5)
{1}
31.25
(62.5)
{2}
250
(>1000)
{>4}
125
0.48
15.62
0.49
nd
(0.48)
(15.62)
ATCC 25923
Staphylococcus
aureus
15.62
(31.25)
{2}
62.5
(62.5)
{1}
31.25
(62.5)
{2}
31.25
(62.5)
{2}
15.62
(31.25)
{2}
31.25
(62.5)
{2}
62.5
(250)
{4}
62.5
(125)
{2}
125
31.25
(62.5)
{2}
15.62
(31.25)
{2}
15.62
(15.62)
{1}
15.62
(15.62)
{1}
15.62
(62.5)
{4}
7.81
15.62
(31.25)
{2}
15.62
(15.62)
{1}
3.91
1.95
(3.91)
{2}
0.24
15.62
(250)
{2}
(15.62)
{2}
(15.62)
{4}
(>1000)
{>8}
250
0.98
nd
nd
nd
(0.24)
(15.62)
ATCC 6538
Staphylococcus
aureus
31.25
(31.25)
{1}
62.5
(62.5)
{1}
15.62
(31.25)
{2}
62.5
(62.5)
{1}
15.62
(31.25)
{2}
15.62
(62.5)
{4}
125
125
250
7.81
(7.81)
{1}
15.62
(62.5)
{4}
7.81
3.91
7.81
7.81
7.81
7.81
15.62
(31.25)
{2}
3.91
(7.81)
{2}
0.24
7.81
(250)
{2}
(125)
{1}
(250)
{1}
(7.81)
{1}
(7.81)
{2}
(15.62)
{2}
(7.81)
{1}
(15.62)
{2}
(15.62)
{2}
(1000)
{4}
(0.24)
(15.62)
ATCC 43300
Staphylococcus
epidermidis
ATCC 12228
Micrococcus
luteus
3.91
3.91
(3.91)
{1}
3.91
3.91
(15.62)
{4}
0.98
0.24
3.91
(15.62)
{4}
7.81
(31.25)
{4}
31.25
(31.25)
{1}
1.95
(7.81)
{4}
0.48
0.98
0.98
1.95
0.98
3.91
1.95
0.48
0.48
(1.95)
{4}
1.95
0.12
3.91
(3.91)
{1}
(7.81)
{2}
(1.95)
{2}
(0.48)
{2}
(1.95)
{4}
(3.91)
{4}
(3.91)
{4}
(7.81)
{4}
(3.91)
{4}
(7.81)
{2}
(7.81)
{4}
(1.95)
{4}
(3.91)
{2}
0.24
0.98
15.62
31.25
nd
nd
(0.12)
(7.81)
125
250
250
250
125
250
500
500
500
250
125
125
31.25
500
500
125
500
250
250
500
0.98
62.5
(250)
{>2}
31.25
(62.5)
{2}
(500)
{>2}
62.5
(62.5)
{1}
(500)
{>2}
31.25
(62.5)
{2}
(500)
{>2}
125
(250)
{>2}
3.91
(500)
{>2}
62.5
(500)
{1}
(1000)
{4}
(>1000) (>1000) (>1000) (>1000) (>1000)
(1000)
{2}
(>1000) (>1000) (>1000) (>1000) (>1000) (>1000)
nd
(1.98)
(62.5)
ATCC 10240
Bacillus
{>2}
125
{>4}
31.25
(31.25)
{1}
{>8}
1.95
{>8}
1.95
{>32}
1.95
(3.91)
{2}
{>2}
1.95
{>8}
1.95
(3.91)
{2}
{>2}
1.95
(3.91)
{2}
{>4}
3.91
{>4}
0.48
{>2}
250
125
62.5
(62.5)
{1}
1.95
0.03
3.91
subtilis
(125)
{1}
(7.81)
{2}
(62.5)
{1}
(125)
{1}
(125)
{1}
(3.91)
{2}
(3.91)
{2}
(3.91)
{2}
(7.81)
{4}
(7.81)
{2}
(0.98)
{2}
(>1000)
{>4}
62.5
nd
(0.03)
(3.91)
ATCC 6633
Bacillus
62.5
125
62.5
125
31.25
(62.5)
{2}
125
250
125
62.5
(250)
{4}
125
125
31.25
(250)
{8}
62.5
(62.5)
{1}
15.62
(62.5)
{4}
15.62
(31.25)
{2}
62.5
(125)
{2}
62.5
(125)
{2}
31.25
(125)
{4}
7.81
250
0.06
7.81
cereus
(62.5)
{1}
(250)
{2}
(125)
{2}
(500)
{4}
(125)
{1}
(250)
{1}
(250)
{2}
(250)
{2}
(>1000)
{>8}
1000
(15.62)
{2}
(>1000)
{>4}
(0.12)
(15.62)
ATCC 10876
Bordetella
bronchiseptica
ATCC 4617
Klebsiella
125
250
250
250
250
500
1000
500
1000
1000
250
1000
1000
1000
1000
250
1000
0.98
125
(250)
{2}
(500)
{2}
(500)
{2}
(1000)
{4}
(500)
{2}
(1000)
{2}
(>1000) (>1000) (>1000)
-
-
-
-
(>1000) (>1000) (>1000) (>1000) (>1000) (>1000) (>1000)
-
(>1000) (>1000)
nd
(0.98)
(>1000)
{>1}
250
{>2}
250
{>1}
500
{>1}
-
{>1}
-
{>4}
500
{>1}
500
{>1}
125
{>1}
-
{>1}
-
{>4}
500
{>1}
-
31.25
(31.25)
{1}
31.25
(31.25)
{1}
31.25
(62.5)
{2}
62.5
(62.5)
{1}
7.81
7.81
250
(250)
{1}
0.12
15.62
pneumoniae
ATCC 13883
Proteus
(15.62)
{2}
(15.62)
{2}
(1000)
{4}
(250)
{1}
(500)
{1}
(1000)
{2}
(500)
{1}
(>1000)
{>8}
(1000)
{2}
nd
nd
(0.12)
(31.25)
31.25
(62.5)
{2}
62.5
(62.5)
{1}
62.5
62.5
(125)
{2}
15.62
(31.25)
{2}
15.62
(31.25)
{2}
250
250
500
500
500
125
250
500
0.03
62.5
mirabilis
(125)
{2}
(1000)
{4}
(500)
{2}
(1000)
{2}
-
-
-
-
(1000)
{2}
(500)
{1}
(>1000)
{>8}
-
-
-
-
(500)
{2}
(500)
{1}
-
-
nd
nd
(0.03)
(125)
ATCC 12453
Salmonella
typhimurium
ATCC 14028
Escherichia
coli
15.62
(31.25)
{2}
31.25
(125)
{4}
31.25
(125)
{4}
31.25
(62.5)
{2}
7.81
15.62
(62.5)
{4}
250
125
500
500
250
250
250
500
0.06
31.25
(62.5)
(31.25
{4}
(500)
{2}
(250)
{2}
(500)
{1}
(1000)
{2}
(1000)
{4}
(>1000)
{>4}
(250)
{1}
(1000)
{2}
nd
nd
(0.06)
7.81
31.25
(125.5)
{4}
31.25
(125)
{4}
31.25
(31.25)
{1}
7.81
31.25
(31.25)
{1}
125
125
500
500
500
500
125
500
0.004
7.81
(15.62)
{2}
(15.62)
{2}
(125)
{1}
(125)
{1}
(500)
{1}
-
-
-
-
-
-
(500)
{1}
(1000)
{2}
(>1000)
{>2}
-
-
-
-
(250)
{2}
(1000)
{2}
-
-
nd
nd
nd
nd
(0.004)
(15.62)
ATCC 25922
Pseudomonas
aeruginosa
500
1000
(>1000)
0.48
-
-
-
-
-
-
-
-
-
-
-
-
-
(500)
(0.98)
This article is protected by copyright. All rights reserved

ATCC 9027
Candida
{1}
62.5
{>1}
62.5
(250)
{4}
1000
(>1000)
{>1}
125
(500)
{2]
250
(500)
{2}
125
(250)
{2}
62.5
(250)
{4}
125
(1000)
{4}
250
(>1000)
{>4}
125
500
15.62
(250)
{16}
31.25
(250)
{8}
15.62
(500)
{32}
500
125
31.25
(250)
{8}
15.62
(15.62)
{1}
0.24*
(0.24)
albicans
(250)
{4}
-
-
-
-
-
-
-
-
-
-
(>1000) (>1000)
(>1000) (>1000)
-
-
-
-
-
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
ATCC 2091
Candida
{>8}
62.5
{>2}
-
{>2}
-
{>8}
250
125
1000
250
250
125
250
250
125
500
15.62
(15.62)
{1}
31.25
(250)
{8}
7.81
0.48*
(0.48)
albicans
(500)
{4}
(>1000)
{>1}
(1000)
{4}
(1000)
{4}
(500)
{4}
(500)
{2}
(250)
{1}
(500)
{4}
(>1000)
{>2}
(250)
{4}
(>1000)
{>4}
(15.62)
{2}
ATCC 10231
Candida
1000
(>1000)
{>1}
1000
(>1000)
{>1}
500
1000
1000
500
500
500
500
500
1000
(>1000)
{>1}
500
250
15.62
(31.25)
{2}
0.24*
(0.48)
parapsilosis
ATCC 22019
Candida
-
(>1000) (>1000) (>1000) (>1000) (>1000) (>1000) (>1000)
-
(>1000)
{>2}
500
-
-
-
-
(>1000)
{>4}
250
{>1}
1000
{>1}
1000
{>2}
250
{>2}
500
{>2}
500
{>2}
500
{2}
250
1000
(>1000)
{>1}
7.81
0.24*
(0.48)
glabrata
-
(>1000) (>1000)
(500)
{2}
(500)
{1}
(>1000) (>1000)
-
-
(>1000) (>1000)
-
(>1000)
{>4}
15.62
(500)
{32}
(15.62)
{2}
ATCC 90030
Candida
{>1}
1000
{>1}
500
{>2}
500
{>2}
250
{>4}
250
{>2}
62.5
1000
250
250
250
15.62
(31.25)
{2}
1000
31.25
15.62
(15.62)
{1}
0.24*
(0.24)
krusei
(1000)
{2}
(>1000) (>1000) (>1000)
{>1} {>1} {>2}
(500)
{2}
(500)
{2}
(>1000) (>1000)
{>2} {>4}
-
(>1000) (>1000)
{>4} {>4}
(1000)
{16}
(>1000) (>1000)
{>1} {>32}
ATCC 14243
“-“ – no activity; nd – not determined; na – not applicable; The standard chemotherapeutics used as positive controls: ciprofloxacin (CIP), nitrofurantoin (NIT), cefuroxime (CFX), ampicillin (APC) for bacteria and nystatin
(NY*) for fungi. Compounds with bactericidal effect (MBC/MIC ≤ 4) or fungicidal effect (MFC/MIC ≤ 4), MBC/MIC or MFC/MIC in these cases is bolded. The standard antibiotics used as positive controls: ciprofloxacin
(CIP) for bacteria and nystatin (NY*) for fungi; “-“ no bioactivity
This article is protected by copyright. All rights reserved