Synthesis and Antimicrobial Resistant Modulatory Activity of 2,4-Dinitrophenylhydrazone Derivatives as Agents against Some ESKAPE Human Pathogens

Article abstract of DOI:10.1155/2020/2720697

A library of six novel phenylhydrazones were synthesized and evaluated for their in vitro antimicrobial and resistance modulating activity against a panel of Gram-positive, Gram-negative, and fungal species. The compounds were produced in good yields of 6

Full text of DOI:10.1155/2020/2720697

Hindawi
Journal of Chemistry
Volume 2020, Article ID 2720697, 9 pages
https://doi.org/10.1155/2020/2720697
Research Article
Synthesis and Antimicrobial Resistant Modulatory Activity of
2,4-Dinitrophenylhydrazone Derivatives as Agents against Some
ESKAPE Human Pathogens
Alberta Ade,1 Cedric D. K. Amengor ,2 Abena Brobbey ,1 Isaac Ayensu,1
Benjamin K. Harley,3 and Yaw Duah Boakye4
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
2Department of Pharmaceutical Chemistry, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana
3Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana
4Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
Correspondence should be addressed to Abena Brobbey; abena23@yahoo.com
Received 27 July 2020; Accepted 21 August 2020; Published 7 October 2020
Academic Editor: Pedro M. Mancini
Copyright © 2020 Alberta Ade et al. &is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A library of six novel phenylhydrazones were synthesized and evaluated for their in vitro antimicrobial and resistance modulating
activity against a panel of Gram-positive, Gram-negative, and fungal species. &e compounds were produced in good yields of
60–92% w/w and characterized using melting point, UV-visible spectroscopy, infrared, and nuclear magnetic resonance (1H, 13C,
and DEPT-Q) techniques. Mass spectroscopy was used to confirm the identity of one of the most active compounds, 5 [SA5]. &e
phenylhydrazones showed activity against all the six selected microorganisms with minimum inhibitory concentration (MIC)
values of the most active compounds, 1 [BP1] and 5 [SA5], at 138 µM (Klebsiella pneumoniae) and 165 µM (Streptococcus
pneumoniae), respectively. Compound 1 [BP1] further demonstrated a high resistance modulatory activity at 1.078 µM against
Streptococcus pneumoniae and Klebsiella pneumoniae.
17 million people annually, with the emergence of at least
thirty new diseases. &e world is currently battling the
pandemic COVID-19 caused by corona virus that has al-
ready claimed over 25,000 lives globally within three months
(WHO, 2020). &ese diseases threaten the health of millions
of people especially as no cure or vaccine exists. &e in-
creasing trend of microbial infections is largely due to the
perennial issue of antimicrobial resistance which has been
on the ascendency. Key myriad causes of antimicrobial
resistance include prolonged chemotherapy and noncom-
pliance to dosage regimen [4].
1. Introduction
&e world in recent decades is running out of effective
antibiotics due to the increased prevalence of multidrug
resistant organisms [1]. &is has led to an increase in re-
sistant infections requiring scientists to relentlessly explore
the possibility of synthesizing analogues of active or lead
compounds as novel antimicrobials to curb these infections
and resultant diseases. Infectious diseases over the centuries
have become a major threat to the existence of mankind as
they continue to negatively impact society significantly [2].
&e pathogenic agents such as bacteria, viruses, fungi, and
parasites continue to emerge and are on the upsurge es-
pecially in the 21st century despite several attempts to curtail
them [3]. Infectious diseases account for the death of about
&e growing resistance to antibiotics has led to the in-
creasing trend of pathogens like methicillin resistant
Staphylococcus aureus (MRSA), multidrug resistant Myco-
bacterium tuberculosis (MDR-TB), and multidrug resistant

2
Journal of Chemistry
Escherichia coli (MDR-Escherichia coli) [5, 6]. Moreover, of
most concern in the treatment of nosocomial infections are
the ESKAPE pathogens (Enterococcus faecium, Staphylo-
coccus aureus, Klebsiella pneumoniae, Acinetobacter bau-
mannii, Pseudomonas aeruginosa, and Enterobacter species)
[7]. &eir presence in infections has been a worrying situ-
ation in the health sector since most of them are resistant to
many antibiotics, and understanding the mechanism of
resistance of these strains is useful in the development of
novel antimicrobial agents [8, 9].
ml. A new agent, 3, 5-dibenzoylvanillin hydrazone, and
transition metal complexes were found to exhibit impressive
antibacterial activity [18, 19].
Hence in this study, six novel phenylhydrazone deriv-
atives have been successfully synthesized by nucleophilic
condensation reaction, with their antimicrobial activities
and resistance modulation effects investigated.
2. Materials and Methods
2.1. Synthesis: General Materials and Methods. A round
bottom flask (100 mL) equipped with a magnetic stirring bar
was charged with 2,4-dinitrophenylhydrazine (1 eq.) in
methanol (10 mL) maintained in an ice bath. &e resulting
suspension was stirred and cooled to 0°C before adding
concentrated H2SO4 (98% v/v, 2 mL) dropwise which gave a
pale yellow solution. After cooling to room temperature, an
aldehyde or ketone derivative (1.04 eq.) in methanol (5 mL)
was added and the mixture stirred until there was a gradual
formation of precipitate which was left for 24 hours. &e
progress of the chemical reactions was monitored by thin
layer chromatography (TLC) intermittently using alumin-
ium plates coated with silica gel (60 GF254). &e plates were
visualized under UV light at 254 nm and 366 nm, which was
followed by spraying with anisaldehyde for spot identity
confirmation. &e crude product was filtered by suction
filtration and recrystallized from hot absolute ethanol (96%
v/v). &e solid product was obtained by suction filtration,
dried, and stored at room temperature.
&e World Health Organization has made it clear that
the trend of increasing infectious diseases would continue
due to a number of factors including rural-urban migration,
increase in global population, microbial adaptation, and
climate change [10]. &ese would favor the emergence and
spread of pathogens, so stakeholders including medicinal
chemists are continuously urged to design strategies to
discover new chemotherapeutic agents to overcome the
antimicrobial resistance menace [11]. Resistant modulatory
activity of a compound is the ability of the compound to
have controlling influence on the already known standards
especially in a positive way. Due to the increasing resistance
of microorganisms to antibiotics, agents from natural or
synthetic sources seem to modulate the activity of some
standard antimicrobial agents such as amoxicillin (a),
ciprofloxacin (b), and fluconazole (c) (Figure 1).
Resistance modulatory activities of products (both
natural and synthetic) on standard antibiotics have gained
scientific interest in recent years. &is aims at maximizing
antimicrobial potency with major strides in curbing mi-
crobial resistance and hence leading to potential drug dis-
covery. An important class of such synthetic agents with
promising resistance modulation effect is hydrazones [11].
Hydrazones and their analogues possess the azomethine
functionality, an important group of compounds with broad
spectrum of biological activities [11, 12]. Hydrazones were
chosen due to their wide array of pharmacological activities
such as anticonvulsant, anti-inflammatory, antimicrobial,
antiprotozoal, and anticancer activities. &ey play crucial
roles not only in biology but also in the fields of photo-
chemistry, analytical chemistry, and inorganic chemistry.
Hydrazones are related to ketones and aldehydes by sub-
&e structures of the synthesized compounds were
established with melting point determinations, mass spec-
troscopy, 1D NMR (proton and carbon-13), and 2D NMR
(DEPT-Q) spectroscopy with the support of infrared (IR)
and ultraviolet-visible (UV-Vis) spectroscopy techniques
[20].
2.1.1. 1-(2,4-Dinitrophenyl)-2-(diphenylmethylene) Hydra-
zine [C19H14N4O4, 1]. 2, 4-Dinitrophenylhydrazine (0.50 g,
2.74 mmol, 1 eq.) in the presence of benzophenone (0.53 g,
1.04 eq., 2.64 mmol) yielded the crude product which was
purified by recrystallization from hot ethanol to obtain the
product [1] (0.84 g, 85%) as a light orange solid. Rf (Pet.
ether 70%: EtOAc 30%): 0.90. Mpt: 141–143°C; UV-V is
(MeOH) λmax: 382 nm. Infrared (neat) υmax cm−1: 3382
(OH), 3286 (NH), 1586 (C � CH), 848, 614 (ArH). 1H NMR
(400 MHz, CDCl3): δH 11.24 (1H, H-C1, s, NH), 9.09–9.10
(1H, H-C3, s, ArH), 8.41 (1H, H-C5, d, J � 2.4, ArH),
8.37–8.38 (1H, H-C6, m, ArH), 7.66–7.72 (5H, H-C4′,
H-C5′, H-C6′, H-C5′, H-C3′, m, ArH), 7.35 (3H, H-C6″,
H-C5″, H-C4″, m, ArH), 7.57 (2H, H-C5′, H-C3′, m, ArH)
ppm. 13C NMR (400 MHz; CDCl3) δc 155.7, 145.1, 136.5,
131.9, 130.5, 130.4, 130.1, 129.9, 128.6, 128.2, 127.9, 123.4,
116.6 ppm.
stitution of oxygen with −NNH
2 functional moiety [13, 14].
Due to varying synthetic protocols and detailed structure-
activity relationship (SAR) studies, different hydrazone
derivatives have been developed and discovered to be
pharmacologically active at various targets [15]. Some
hydrazone derivatives of isonicotinoyl were found to possess
antitubercular activity. Furthermore, benzoic acid hydra-
zone derivative 4-hydroxybenzoic acid [(5-nitro-2-furyl)
methylene]-hydrazide (nifuroxazide) was found to be active
against intestinal worms, and derivative 4-fluorobenzoic
acid [(5-nitro-2-furyl) methylene]-hydrazide showed anti-
bacterial activity against Staphylococcus aureus ATCC 29213
at 3.13 μg/mL and susceptible Mycobacterium tuberculosis
strain H37RV also at 3.13 μg/mL [16, 17]. Recently syn-
thesized hydrazones including nifuroxazide were found to
be active against Mycobacterium tuberculosis strain H37RV
at minimum inhibitory concentration range of 0.78–6.25 μg/
2.1.2.
1-Benzylidene-2-(2,4-dinitrophenyl)
Hydrazine
[C13H10N4O4, 2]. 2, 4-Dinitrophenylhydrazine (0.90 g,
4.53 mmol, 1 eq.) in the presence of benzaldehyde (0.50 g,
1.04 eq., 4.71 mmol) yielded the crude product which was

Journal of Chemistry
3
NH2
O
O
O
N
N
H
N
F
N
H
HO
N
S
HO
HO
N
N
N
N
O
F
NH
N
O
HO
F
(a)
(b)
(c)
Figure 1: Chemical structures of some standard antibiotics [11]: (a) amoxicillin; (b) ciprofloxacin; (c) fluconazole.
purified by recrystallization from hot ethanol to obtain the
7.97 (1H, H-C2′, d, J � 4.0, ArH), 7.66–7.76 (1H, H-C6′, d,
product [2] (1.01 g, 78%) as a yellow solid. Rf (Pet. ether
70%: EtOAc 30%): 0.83. Mpt: 178–180°C; UV-V is (MeOH)
λmax: 224 nm and 378 nm. Infrared (neat) υmax cm−1: 3337
(OH), 3283 (NH), 3100 (C � CH), 1618, 1584 (ArC � C). 1H
NMR (400 MHz, CDCl3) δ 11.24 (1H, H-C1, s, NH), 9.09
(1H, H-C3, s, ArH), 8.30–8H.31 (1H, H-C1′, s, N � CH), 8.05
(1H, H-C5, m, ArH), 8.02 (1H, H-C6, m, ArH), 7.41–7.69
(2H, H-C2′, H-C6′, m, ArH), 7.40 (1H, H-C4′, m, ArH), 7.39
(2H, H-C3′, H-C5′, m, ArH). 13C NMR (400 MHz, CDCl3)
δc 147.9, 145.8, 144.9, 144.7, 131.0, 130.0, 129.0, 127.6, 123.5,
116.8 ppm.
J � 8.0, ArH), 6.38–6.35 (1H, H-C5′, d, J � 12.0, ArH), (3H,
H-C4′, Ar-OCH3). 13C NMR (400 MHz, CDCl3) δc 150.7,
150.2, 148.6, 144.9, 137.1, 130.2, 125.6, 123.6, 123.1, 117.2,
116.1, 110.3, 56.2 ppm.
2.1.5. (Z)-2-(2, 4-Dinitrophenyl) Hydrazono) Methyl Phenol
[C13H10N4O5, 5]. 2, 4-Dinitrophenylhydrazine (0.78 g,
3.93 mmol, 1 eq.) in the presence of salicylaldehyde (0.50 g,
4.09 mmol, 1.04 eq.) yielded the crude product which was
purified by recrystallization from hot ethanol to obtain the
product [5] (1.09 g, 92%) as a bright orange solid. Rf (Pet.
ether 70%: EtOAc 30%): 0.84. Mpt: 176–180°C; UV-Vis
(MeOH) λmax: 386 nm. Infrared (neat) υmax (cm−1): 3334
(OH), 3267 (NH), 3059 (C � CH), 1583 (ArC � C), 759
(ArH) 1H NMR (400 MHz, CDCl3) δH 11.25 (1H, H-C3′, s,
ArOH), 9.98 (1H (C1), s, NH), 9.11 (1H H-C3, s, ArH),
8.34–8.36 (1H, H-C1′, s, N � CH), 8.33 (1H, H-C5, d, J � 4.0,
ArH), 7.61–7.58 (1H, H-C6, d, J � 4.0, ArH), 7.31 (1H, H-C6′,
m, ArH), 7.25 (1H, H-C4′, m, ArH), 7.24 (1H, H-C7, m,
ArH), 6.95 (1H, H-C5′, m, ArH) ppm. 13C NMR (400 MHz,
CDCl3) δ 157.9, 151.3, 132.9, 131.4, 130.6, 123.7, 120.3,
117.2, 116c.9, 115.3 ppm. HRMS (ESI): m/z calculated for [M-
H]+ C13H10N4O5: 302.2400, found: 301.0000.
2.1.3. 3-(2-2-(4-Dinitrophenyl) Hydrazono) Methylphenol
[C13H10N4O5, 3]. 2, 4-Dinitrophenylhydrazine (0.78 g,
3.93 mmol, 1 eq.) in the presence of m-hydroxybenzaldehyde
(0.50 g, 1.04 eq., 4.09 mmol) yielded the crude product which
was purified by recrystallization from hot ethanol to obtain
the product (0.76 g, 64%) as a dark red solid. Rf (Pet. Ether
70%: EtOAc 30%): 0.74. Mpt: 277–280°C. UV-Vis (MeOH)
λmax: 392 nm. Infrared (neat) υmax cm−1: 3420 (OH), 3257
(NH), 3116 (C � CH), 1607, 1584 (ArC � C). 1H NMR
(400 MHz, CDCl3) δH 11.56 (1H, H-C1, s, NH), 10.04 (1H,
H-C2′, s, ArOH), 8.88 (1H, H-C3, s, ArH), 8.86 (1H, H-C7, s,
N � CH), 8.35–8.37 (1H H-C5, d, J � 8.0, ArH), 8.08–8.34
(1H, H-C6), d, J � 12.0, ArH), 8.05 (1H, H-C5′, m, ArH), 7.66
(1H, H-C1′, s, ArH), 7.14 (1H, H-C4′, m, ArH), 6.87–6.89
(1H (H-C3′, m, ArH). 13C NMR (400 MHz, CDCl3) δ 160.5,
150.5, 144.9, 137.1, 130.2, 129.8, 129.5, 125.2, 123.6c, 117.1,
116.4 ppm.
2.1.6. 4-(2-(2, 4-Dinitrophenylhydrazono) Methyl) Benzene-
1, 3-diol [C13H10N4O6, 6]. 2, 4-Dinitrophenylhydrazine
(0.69 g, 3.48 mmol, 1 eq.) in the presence of 2, 4-dihydroxy
benzaldehyde (0.50 g, 1.04 eq., 3.62 mmol) yielded the crude
product which was purified by recrystallization from hot
ethanol to obtain the product [6] (0.66 g, 2.07 mmol, 60%) as
a dark red solid. Rf (Pet. ether 70%: EtOAc 30%): 0.51. Mpt:
270–274°C; UV-Vis (MeOH) λmax: 403 nm. Infrared (neat)
υmax (cm−1): 3364 (OH), 3094 (C � CH), 1612, 1584
(ArC � C) 592 (ArH). 1H NMR (400 MHz, DMSO-d6) 11.58
2.1.4. 4-(2-(2,4-Dinitrophenyl) Hydrazono) Methyl)-2-methox-
yphenol [C14H12N4O6, 4]. 2, 4-Dinitrophenylhydrazine
(0.78 g, 3.93 mmol, 1 eq.) in the presence of 4-hydroxy-3-
methoxybenzaldehyde (vanillin) (0.50 g, 1.04 eq.,
4.09 mmol) yielded the crude product which was purified by
recrystallization from hot ethanol to obtain the product [4]
(1H, H-C3′, s, ArOH), 10.11 (1H, H-C1, s, NH), 9.94 (1H,
(0.91 g, 70%) as a bright red solid. Rf (Pet. ether 70%: EtOAc
30%): 0.66. Mpt: 270–273°C. UV-Vis (MeOH) λmax: 218 nm
and 394 nm. Infrared (neat) υmax cm−1: 3363 (OH), 3274
H-C5′, s, ArOH), 8.87 (1H, H-C3, s, ArH), 8.86 (1H, s,
N � CH), 8.33 (1H, H-C4′, s, ArH), 8.36 (1H, H-C5, d,
J � 12.0, ArH), 7.95–7.97 (1H, H-C6, d, J � 8.0, ArH), 7.54
(NH), 3111 (C � CH), 1605 (ArC � C), 699 (ArH). 1H NMR
(400 MHz, CDCl3) δ 11.58 (1H, s, NH), 10.11 (1H, H-C4′, s,
ArOH), 9.94 (1H, HH-C3, s, ArH), 8.87 (1H, H-C5, s, ArH),
8.86 (1H, H-C7, s, N � CH), 8.35 (1H, H-C6, d, J � 4.0, ArH),
(1H, H-C7, m, ArH), 6.38 (1H, H-C6′, d, J � 8.0, ArH) ppm.
13C NMR (400 MHz, DMSO-d6) δc 161.8, 159.1, 148.2, 144.6,
136.7, 130.1, 129.3, 128.8, 123.6, 116.9, 112.0, 108.7,
102.1 ppm.

4
Journal of Chemistry
2.2. Antimicrobial Evaluation of the Compounds
(Scheme 1). A retrosynthetic disconnection approach
allowed us to identify various aldehydes and a ketone as key
intermediates for the synthesis of the desired compounds.
&e process involves a rate-determining aromatic nucleo-
philic attack of 2, 4-dinitrophenylhydrazine on the carbonyl
in an acidified solution. &is was followed by dehydration of
the reactive intermediate to give the final product 2, 4-
dinitrophenylhydrazone derivative.
&e data for the synthetic procedures are included in the
experimental section and interpreted according to the se-
quence: melting point range (Mpt.) in degree Celsius (°C),
wavelength of maximum absorption (λmax) from the ul-
traviolet-visible spectrum, infrared spectrum, 1H NMR
[chemical shift, integral, multiplicity, coupling constant, and
interpretation], 13C NMR, and HRMS as a confirmatory tool
for SA5 (compound 5) (one of the most active compounds).
2.2.1. Source of Test Organisms. Pure cultures of Staphylo-
coccus aureus (SA) (ATCC 25923), Escherichia coli (EC)
(ATCC 25922), and Pseudomonas aeruginosa (PA) (ATCC
27853) were obtained from the Microbiology Division of the
Department of Pharmaceutics, Faculty of Pharmacy and
Pharmaceutical Sciences, Kwame Nkrumah University of
Science and Technology (KNUST), Kumasi. However,
Klebsiella pneumoniae (KP), Candida albicans (CA), and
Streptococcus pneumoniae (SP) were clinical strains obtained
from the Komfo Anokye Teaching Hospital, Kumasi, and
cultured in the Department of Pharmaceutics, KNUST.
2.2.2. Determination of Minimum Inhibitory Concentration
(MIC). &e microbroth dilution method was employed for
determining the minimum inhibitory concentrations
(MICs) of the hydrazone derivatives and the reference drugs
ciprofloxacin and fluconazole. 96-well microtiter plates were
filled with 125 µL of double strength nutrient broth, and
different concentrations of the hydrazone derivatives added.
&e reference drugs in the range 12.5 µL/mL to 40 µL/mL
were similarly treated. An aliquot of 1 × 105 cfu/mL of test
organisms was added to each well. &e control plate was
filled with nutrient broth and test organisms only. &e test
and control plates were incubated at 37°C (24 hours for
bacteria and 48 hours for fungus), after which 20 µL of
1.25 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphe-
nyltetrazolium bromide (MTT) was introduced into each
well. Observations were made after 20 minutes for a purple
coloration, which indicates growth. &e minimum con-
centrations of the hydrazone derivatives and reference drugs
that did not show color change in the wells were deemed to
be the MIC values [21, 22]. &e determinations were carried
out in replicates.
3.1.1. UV-Vis Spectroscopy. &e spectra for all the six
compounds [refer to high resolution electrospray ionization
mass spectrum (HRESIMS)] were determined in both the
ultraviolet and visible wavelengths (200–800 nm) [23, 24] as
a confirmatory tool for BP1 in the supplementary infor-
mation using methanol as the blank. &e absorption bands at
203 nm and 403 nm, which were obtained in the electronic
spectra of the synthesized phenylhydrazones, can be credited
to (π − π∗) transitions, which are contributed by the various
substituent auxochromes on the phenylhydrazones. Phe-
nylhydrazones are known to be absorbed at various wave-
lengths within the UV-visible range [25, 26].
For instance, compound BP1, when dissolved in
methanol and scanned within the UV-visible region, showed
absorption at 203 nm, 314 nm, and 382 nm which could be
attributed to extensive conjugation in the phenylhydrazones
after the coupling of benzophenone and 2,4-dini-
trophenylhydrazine. However, compound BP1 showed
maximum absorption at 382 nm, and this could be attributed
to an increase in conjugation. Also, compounds BA2,
MHB3, VL4, SA5, and DHB6 showed maximum absorption
at 378 nm, 392 nm, 394 nm, 386 nm, and 403 nm, respec-
tively, as observed in the supporting information.
2.2.3. Resistant Modulation Studies. For the modulation
studies, the 96-well plates were filled with 125 µL of double
strength nutrient broth, and the same volume of 40 µL
phenylhydrazones was added. Different concentrations in
the ranges 50 µL/ml to 7.812 µL/ml and 15.625 µL/ml to
7.812 µL/ml of ciprofloxacin and fluconazole were, respec-
tively, added. 25 µL containing 1 × 105 cfu of test organisms
was added to each well, and the plates were incubated at 37°C
(for 24 and 48 hours for bacteria and fungus, respectively),
after which 20 µL of MTT was added to each well. &e
appearance of a change in color from cloudy wells to purple
was recorded and compared with MICs of the reference
drugs only [22, 23]. &e determinations were carried out in
replicates.
&e extensive conjugation can be attributed to the
presence of chromophores in the phenylhydrazones which
include BP1 (two aromatic ring chromophores), MHB3
(meta hydroxy auxochrome), VL4 (a hydroxy and methoxy
auxochrome), SA5 (ortho hydroxy auxochrome), and DHB6
(two hydroxy auxochromes). &is suggests that the hydra-
zones with auxochromes and extra chromophore had ex-
tended conjugation leading to a shift in λmax to the right
(bathochromic shift).
3.1.2. Infrared Spectroscopy. &e infrared spectrum gives an
idea of the functional groups in a compound. From the
infrared spectrum of compound 5 as a sample, the presence
of broad band stretching from around 3300 cm−1 and
sloping into the aliphatic CH region of around 3000 cm−1
indicates the presence of a hydroxy functional group, in this
case that of a phenol as observed in the supporting
information.
3. Results and Discussion
3.1. Structural Analysis of the Phenylhydrazones. &e syn-
thetic route employed for the synthesis of the compounds
was followed by the standard condensation reaction between
aldehydes or ketones and hydrazines, taking into consid-
eration the commercial availability of the building blocks

Journal of Chemistry
5
NO2
R
NO2
NHN=C
NHNH2
Methanol/H+
0–25°C
R
R′
+
C=O
+
H2O
O2N
O2N
R′
2, 4-DNP hydrazine
Carbonyl
2, 4-DNP hydrazone
Compound
R
R′
–C6H5
–H
–C6H5
–C6H5
1
2
3
C6H5 (m-OH)
–H
C6H5OH (m-OCH3)
C6H5 (o-OH)
4
5
6
–H
–H
–H
C6H5OH (m-OH)
Scheme 1: Synthetic procedures and reagents for compounds 1–6. Reagents and conditions: methanol/concentrated H2SO4 (98% v/v, 2 mL),
0–25°C, 24 h.
It was also observed from the IR spectrum (supple-
mentary information) that compounds like MHB3, VL4,
SA5, and DHB6 showed sharp strong peak of their C � C
stretching band in the region of 1138–1620 cm−1 compared
to BP1 and BA2 which do not have the hydroxy function as
shown in Table 1. &e presence of the C � N, which is formed
from the condensation reaction between the hydrazine and
the carbonyls, was overlapped by aromatic sp2 hybridized
carbons (C � C) in the benzene which vibrate at
1138–1640 cm−1 depending on the substituent attachments.
Since C � N vibrational frequencies fall within
1580–1600 cm−1, their bands cannot be clearly distinguished
in the presence of their aromatic C � C counterparts as
shown in the experimental data.
deshielding effect of the electronegative oxygen [27]. &is
chemical shift was slightly higher than that of the hydroxyl
group (δ 9.94 ppm) because the former is closer to the
chemical environment of the electronegative nitrogen atom
in the imine bond. Hence, the first hydroxy singlet signal at δ
11.54 ppm was followed by a singlet signal secondary amino
proton at δ 10.11 due to electronegativity of the nitrogen
group before the other hydroxy signal. &e aromatic proton
sandwiched between two electronegative nitro groups was
the next signal resonating downfield at δ 8.86 ppm. &is was
followed by the proton close to the tertiary amino group. &e
tertiary amino group attached to the imine proton caused
deshielding and downfield resonating of imine protons at δ
8.86 ppm with a singlet signal. &e lone proton closest to the
nitro group at the para position was the next signal with a
doublet peak at δ 7.95–7.97 ppm. &e other lone proton
closest to the less electronegative secondary amino group
also gave a doublet signal at δ 7.64–7.66 ppm. &e two other
lone protons (one in between the diol and the other ortho to
the imine bond) gave a multiplet signal at δ 7.52–7.54 ppm,
while the proton closest to the para hydroxy group and two
carbon bonds away from the imine group gave a doublet
signal at δ 6.38 ppm.
3.1.3. Nuclear Magnetic Resonance (NMR) Spectroscopy.
NMR determinations were performed for all the six syn-
thesized phenylhydrazones using a one-dimensional (1D)
NMR technique (1H and 13C) and two-dimensional NMR
technique, distortionless enhancement by polarization
transfer for quaternary carbons (DEPT-Q). &ese data are
assigned to each compound in the experimental section. &e
DEPT-Q is used for the differentiation of the primary,
secondary, tertiary, and quaternary carbons. In DEPT-Q, the
1
structures were confirmed with H NMR, 13C NMR, and
DEPT-Q spectral data as shown in the supplementary in-
formation for compounds BP1, BA2, MHB3, VL4, SA5, and
DHB6, respectively. Considering DHB6 (compound 6), [4-
(2-(2, 4-dinitrophenylhydrazono) methyl) benzene-1, 3-
diol] (Figure 2, Table 1), and from the proton NMR (sup-
plementary information), a chemical shift showing reso-
nance far downfield signified the presence of the hydroxy
proton with a singlet signal. It resonated further downfield
than the secondary amine proton because of the increased
methyl (CH
3) and methine (CH) signals appear up in the
positive phase, while methylene (CH2) and quaternary
signals appear down in the negative phase. &e DEPT-Q
spectrum of DHB6 (supplementary information) reveals the
presence of upward carbon signals representing only six
methine carbons in this case occurring at δ 161.8, 159.1,
148.2, 136.7, 130.1, and 112.0 confirming the aromatic
methine carbons and seven quaternary carbons occurring at
δ 148.2, 130.1, 129.2, 123.6, 116.9, 108.7, and 102.9. &e

6
Journal of Chemistry
Table 1: Chemical structures and physical data of the synthesized phenylhydrazones.
Compounds
Code
Color
Mol. For.
Mol. Wt. (g/mol)
Yield (%)
85
NO2
NH–N=C
BP1
Light orange
C19H14N4O4
362.34
286.24
302.24
O2N
NO2
NH–N=C
H
BA2
Dark yellow
Dark red
C13H10N4O4
78
92
O2N
OH
NO2
NH–N=C
H
MHB3
C13H10N4O5
O2N
OCH3
OH
NO2
NH–N=C
H
VL4
SA5
Bright red
Bright orange
Dark red
C14H12N4O6
C13H10N4O5
C13H10N4O6
332.27
302.24
318.24
70
64
60
O2N
OH
NO2
CH
NH–N
O2N
HO
NO2
OH
NH–N=C
H
DHB6
O2N
peak in negative mode was 301.0000 (M−H+) which is the
actual mass having a tolerance range of 0.24 (0.41%) from
the theoretical mass of 302.2400. &is confirmed the mo-
lecular weight of compound SA5.
&e mass spectroscopy was used to confirm the mo-
lecular weight of one of the most active compounds and
hence was not applied to the others.
HO
NO2
OH
N
H
N=C
H
O2N
Figure 2: Chemical structure of DBH6 (compound 6) [4-(2-(2, 4-
dinitrophenylhydrazono) methyl) benzene-1, 3-diol].
4. Antimicrobial Evaluation
4.1. Minimum Inhibitory Concentrations (MICs).
Minimum inhibitory concentration (MIC) is the least
concentration of an antimicrobial agent that would inhibit
the visible growth of the pathogen after 24 h (bacteria) and
48 h (fungi) incubation [28]. &e MICs of the phenyl-
hydrazones and two standard antibiotics (ciprofloxacin and
fluconazole) were evaluated with the microbroth dilution
test against a panel of six pathogenic microorganisms:
Gram-negatives [P. aeruginosa (ATCC 27853), E. coli
(ATCC 25922), K. pneumoniae (clinical)], Gram-positives
DEPT-Q for the other compounds can be found in the
supplementary information.
3.1.4. Mass Spectroscopy. Mass spectroscopy is used for the
determination of accurate molecular weights and frag-
mentation of compounds using ionization techniques [28].
Compound SA5 phenylhydrazone was one of the most active
compounds after antimicrobial evaluation. From the elec-
trospray ionization (ESI) mass spectrum, the molecular ion

Journal of Chemistry
Test organisms
7
Table 2: Antimicrobial activity of the compounds against the panel of organisms.
Minimum inhibitory concentrations (µM) of the phenylhydrazones
BP1
BA2
699
699
699
>699
MHB3
VL4
602
300.96
602
602
SA5
662
165
662
662
DHB6
CPR
4.72
2.36
4.72
2.36
FLZ
Nd
Nd
Nd
Staphylococcus aureus (+)
Streptococcus pneumoniae (+)
Escherichia coli (−)
>552
>662
>628
552
>662
314
>552
>662
628
Pseudomonas aeruginosa (−)
>552
>662
628
Nd
Klebsiella pneumoniae (−)
138
552
699
699
>662
>562
602
602
331
662
314
314
2.36
Nd
Nd
Candida albicans
327
BP1 [benzophenone derivative], BA2 [benzaldehyde derivative], MHB3 [m-hydroxy benzaldehyde], VL4 [vanillin derivative], SA5 [salicylaldehyde de-
rivative], DHB6 [2, 4-dihydroxybenzaldehyde derivative]; standard antibiotics: CPR [ciprofloxacin] and FLZ [fluconazole]. Nd [not determined]. All tests
were carried out in triplicate (n � 3).
Table 3: Resistant modulatory activity of the compounds against the panel of organisms.
Minimum inhibitory concentrations (µM) of the phenylhydrazones
Test organisms
BP1
BA2
MHB3
VL4
SA5
DHB6
CPR
FLZ
Staphylococcus aureus (+)
>138
87.3
1.36
165
>150
>165
>157
4.72
2.36
4.72
2.36
2.36
—
—
—
—
—
—
327
Streptococcus pneumoniae (+)
1.078
1.29
1.176
1.29
1.23
Escherichia coli (−)
>138
>17.2
1.078
2.156
>175
87.3
5.46
273
>165
>150
>165
2.58
1.29
1.29
>157
39.3
1.23
1.23
Pseudomonas aeruginosa (−)
Klebsiella pneumoniae (−)
Candida albicans
82.7
75.2
1.29
1.176
1.176
5.109
[S. aureus (ATCC 25923), S. pneumoniae (clinical strain)],
and C. albicans (fungus, clinical strain).
against S. pneumoniae lowered the MIC of ciprofloxacin
from 2.36 to 1.078 µM representing 54.32% reduction in the
MIC of the ciprofloxacin. BP1 also improved the perfor-
mance of the ciprofloxacin at sub-MIC of 50 µM against
K. pneumoniae from 2.36 µM to 1.078 µM representing
54.32% reduction in the MIC of the antibiotics. Again, BP1
modulated the activity of fluconazole against C. albicans
from 327 to 2.156 µM representing 99.34% reduction in the
MIC of the fluconazole. Similarly, BA2 improved the activity
of ciprofloxacin against S. pneumoniae and reduced the MIC
of ciprofloxacin from 2.36 to 1.36 µM showing 42.37% re-
duction. In addition, BA2 also modulated the activity of
fluconazole at sub-MIC of 50 µM against C. albicans and
caused a reduction in its MIC from 327 to 1.23 µM which is
indicative of 99.6% reduction in the MIC.
MHB3 modulated the activity of the ciprofloxacin against
S. pneumoniae and Klebsiella pneumoniae causing a reduction
in the MIC of antibiotics from 2.36 to 1.29 µM. &is decrease
in the MIC indicated a reduction of 45.33%. MHB3 also
showed the modulation of the activity of fluconazole at sub-
MIC of 50 µM against Candida albicans and lowered the MIC
to 5.109 from 327 µM indicating a maximum reduction of
98.43%. VL4 enhanced the performance of ciprofloxacin at
sub-MIC of 50 µM against S. pneumoniae and K. pneumoniae.
&e MIC of the antibiotic decreased from 2.36 to 1.176 µM. in
both instances. &is decrease in MIC depicted 50.17% re-
duction. VL4 again modulated the performance of flucona-
zole at sub-MIC of 50 µM against C. albicans and lowered the
MIC to 1.176 µM from 327 µM, which showed a maximum
reduction of 99.64%.
All the compounds showed weak antimicrobial activity
against test organisms at test concentrations as observed in
Table 2. BP1 showed the highest antimicrobial activity among
the six compounds against the selected test organisms. BA2,
however, had the least antimicrobial activity with MIC of
699 µM against the entire test organisms. MHB3 had MIC of
562 µM against C. albicans and 662 µM and above against the
rest of the test organisms. VL4 recorded an MIC of 602 µM
against all test organisms except S. pneumoniae
(MIC � 300 µM). &e MICs of SA5 against S. pneumoniae and
K. pneumoniae were 165 µM and 331 µM, respectively, while
for S. aureus, E. coli, P. aeruginosa, and C. albicans it had a
higher MIC of 662 µM. DHB6 showed activity against
S. pneumoniae, K. pneumoniae, and C. albicans with MIC of
314 µM, while for S. aureus, E. coli, and P. aeruginosa it had
MIC of 628 µM and above. &e reference antibacterial drug
used was ciprofloxacin, and the MICs recorded were 2.36 µM
against P.aeruginosa, S. pneumoniae, and K. pneumoniae,
while 4.72 µM was recorded against S. aureus and E. coli. &e
reference antifungal drug used was fluconazole, and the MIC
obtained was 327 µM against C. albicans. &e MIC of the
phenylhydrazones against the two Gram-negative organisms
ranged from 552 µM to 699 µM, while the standard cipro-
floxacin was at 4.72 and 2.36 µM, respectively (Table 2).
4.2. Resistant Modulation Activity. &e tendency of the
phenylhydrazones at sub-MICs to improve the activities of
some commercial antibiotics against known resistant strains
was investigated through the resistant modulation test. From
Tables 2 and 3, BP1 (compound 1) with sub-MIC of 50 µM
In addition, compound 5 (SA5) modulated the activity of
the ciprofloxacin at sub-MIC of 50 µM against S. pneumoniae
and K. pneumoniae. &e MIC was lowered from 2.36 to

8
Journal of Chemistry
1.29 µM in both cases resulting in 45.34% reduction. SA5
again modulated the performance of fluconazole at sub-
MIC of 50 µM against C. albicans and suppressed its MIC
from 327 to 1.29 µM, indicating a good reduction of
99.61%. DHB6 (compound 6) also modulated the activity
of ciprofloxacin at sub-MIC of 50 µM against
S. pneumoniae and K. pneumoniae and lowered its MIC
from 2.36 to 1.23 µM in both instances representing
47.88% reduction in the MIC. DHB6 also modulated the
activity of fluconazole at sub-MIC of 50 µM against
C. albicans and lowered its MIC from 327 µM to 1.23 µM
representing 99.62% reduction in MIC. SA5 and BP1 were
better than the other four phenylhydrazones in resistance
modulatory activity together with ciprofloxacin which
had MIC of 2.58 µM against P.aeruginosa, followed by BP1
(17.2 µM). Hence, the presence of an ortho hydroxy group
in SA5 and aromatic ketone group might be essential for
resistant modulatory activity in combination with
ciprofloxacin against Gram-negative P.aeruginosa and
E. coli. Furthermore, the presence of meta hydroxy group
(MHB3), a hydroxy and methoxy (VL4), and absence of a
substituent on the benzaldehyde portion of BA2 also
improved activity and could be considered for future drug
development. K. pneumoniae, the other Gram-negative
counterpart which causes serious infections, was more
susceptible to the phenylhydrazones at 138 to 699 µM. By
the combination of subinhibitory concentrations of the
phenylhydrazones with ciprofloxacin, the resistance
barrier of K. pneumoniae was reduced allowing for a far
lower MIC of 1.078 from 5.46 µM, a trend that is more
impressive than that of P. aeruginosa and E.coli.
NTCC: National collection of type cultures
MTT:
(4, 5-Dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide
Minimum inhibitory concentration
&in layer chromatography
Colony forming units
MIC:
TLC:
Cfu:
Eq.:
Equivalent
DEPT-Q: Distortionless enhancement of polarization
transfer for quaternary carbons
HRSMS: High resolution mass spectrum.
Data Availability
Data of the research is available in the archives of the De-
partment of Pharmaceutical Chemistry, Faculty of Phar-
macy, Kwame Nkrumah University of Science and
Technology.
Conflicts of Interest
&e authors declare no conflicts of interest regarding the
publication of this paper.
Authors’ Contributions
A.A., A.B., I.A., Y.D.B., C.D.K.A., and B.K.H. conceived the
research work and prepared the manuscript. C.D. K.A., A.B.,
and A.A. designed and carried out the synthesis of the
compounds. C.D.K.A., A.A., and I.A. developed the con-
ceptual framework and prepared the manuscript. C.D.K.A.
and B.K.H. interpreted the spectral results and carried out
the structure elucidation. Y.D.B. and A.A. carried out the in
vitro antimicrobial assay and provided its experimental data
and interpretation of the results.
&e phenylhydrazones had similar activity against the
Gram-positive organisms ranging from 87.3 to 699 µM
which is also evident of the resistant properties of Staphy-
lococcus aureus and Streptococcus pneumoniae.
Acknowledgments
5. Conclusion
&e authors are very grateful to all the staff and technicians
of the Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, KNUST, Kumasi, Ghana, for their support. &e
authors greatly acknowledge Mr. Francis Amankwah of the
Department of Pharmaceutics (Microbiology Section),
KNUST, for the technical support.
A library of six novel phenylhydrazones was successfully
synthesized and characterized: 1-(2, 4-dinitrophenyl)-2-
(diphenylmethylene) hydrazine [BP1], 1-benzylidene-2-(2,
4-dinitrophenyl) hydrazine [BA2], 3-(2–2-(4-dinitrophenyl)
hydrazono) methylphenol, 4-(2-(2, 4-dinitrophenyl)
hydrazono) methyl)-2-methoxyphenol [VL4], (Z)-2-(2, 4-
dinitrophenyl) hydrazono) methylphenol [SA5], and 4-(2-
(2, 4-dinitrophenylhydrazono) methyl) benzene-1, 3-diol.
As part of future drug design and optimization, the
phenylhydrazones can be considered as structural back-
bones due to their synthetic feasibility and good yield. &e
compounds exhibited weak antimicrobial property but
demonstrated strong resistant modulating action when
combined with the standard drugs. &e activities of the
standards improved significantly with a good reduction in
the MIC values.
Supplementary Materials
&e supplementary file contains all the spectra. (Supplementary
Materials)
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MRSA:
ATCC:
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