Iodinated 1,2-diacylhydrazines, benzohydrazide-hydrazones and their analogues as dual antimicrobial and cytotoxic agents

Article abstract of DOI:10.1016/j.bmc.2021.116209

Hydrazide-hydrazones have been described as a scaffold with antimicrobial and cytotoxic activities as well as iodinated compounds. A resistance rate of bacterial and fungal pathogens has increased considerably. That is why we synthesized and screened twenty-two iodinated hydrazide-hydrazones 1 and 2, ten 1,2-diacylhydrazines 3 and their three reduced analogues 4 for their antibacterial, antifungal, and cytotoxic properties. Hydrazide-hydrazones were prepared by condensation of 4-substituted benzohydrazides with 2-/4-hydroxy-3,5-diiodobenzaldehydes, diacylhydrazines from identical benzohydrazides and 3,5-diiodosalicylic acid via its chloride. These compounds were investigated in vitro against eight bacterial and eight fungal strains. The derivatives were found potent antibacterial agents against Gram-positive cocci including methicillin-resistant Staphylococcus aureus with the lowest values of minimum inhibitory concentrations (MIC) of 7.81 μM. Four compounds inhibited also human pathogenic fungi (MIC of ≥1.95 μM). The derivatives had different degrees of cytotoxicity for HepG2 and HK-2 cell lines (IC₅₀ values from 11.72 and 26.80 μM, respectively). Importantly, normal human cells exhibited lower sensitivity. The apoptotic effect was also investigated. In general, the presence of 3,5-diiodosalicylidene scaffold (compounds 1) is translated into enhanced both antimicrobial and cytotoxic properties whereas its 4-hydroxy isomers 2 share a low biological activity. N′-Benzoyl-2-hydroxy-3,5-diiodobenzohydrazides 3 have a non-homogeneous activity profile. Focusing on 4-substituted benzohydrazide part, the presence of an electron-withdrawing group (F, Cl, CF₃, NO₂) was found to be beneficial.

Full text of DOI:10.1016/j.bmc.2021.116209

Bioorg. Med. Chem. 41 (2021) 116209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Iodinated 1,2-diacylhydrazines, benzohydrazide-hydrazones and their
analogues as dual antimicrobial and cytotoxic agents
a
,
*
b
a
a
´
´
ˇ ´ a
´
ˇ
ˇ
´
´
Martin Kratký , Klara Konecna , Michaela Brablíkova , Jirí Janousek , Vaclav Pflegr ,
a
a
a
´
ˇ
ˇ
´
Jana Maixnerova , Frantisek Trejtnar , Jarmila Vinsova
a
´
´
´
´
´
Faculty of Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b
´
ˇ
Unipetrol Centre of Research and Education, 436 70 Litvínov-Zaluzí 1, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydrazide-hydrazones have been described as a scaffold with antimicrobial and cytotoxic activities as well as
iodinated compounds. A resistance rate of bacterial and fungal pathogens has increased considerably. That is
why we synthesized and screened twenty-two iodinated hydrazide-hydrazones 1 and 2, ten 1,2-diacylhydrazines
3 and their three reduced analogues 4 for their antibacterial, antifungal, and cytotoxic properties. Hydrazide-
hydrazones were prepared by condensation of 4-substituted benzohydrazides with 2-/4-hydroxy-3,5-diiodo-
benzaldehydes, diacylhydrazines from identical benzohydrazides and 3,5-diiodosalicylic acid via its chloride.
These compounds were investigated in vitro against eight bacterial and eight fungal strains. The derivatives were
found potent antibacterial agents against Gram-positive cocci including methicillin-resistant Staphylococcus
aureus with the lowest values of minimum inhibitory concentrations (MIC) of 7.81 µM. Four compounds inhibited
also human pathogenic fungi (MIC of ≥1.95 µM). The derivatives had different degrees of cytotoxicity for HepG2
and HK-2 cell lines (IC50 values from 11.72 and 26.80 µM, respectively). Importantly, normal human cells
exhibited lower sensitivity. The apoptotic effect was also investigated. In general, the presence of 3,5-diiodosa-
licylidene scaffold (compounds 1) is translated into enhanced both antimicrobial and cytotoxic properties
whereas its 4-hydroxy isomers 2 share a low biological activity. N′-Benzoyl-2-hydroxy-3,5-diiodobenzohy-
drazides 3 have a non-homogeneous activity profile. Focusing on 4-substituted benzohydrazide part, the pres-
ence of an electron-withdrawing group (F, Cl, CF3, NO2) was found to be beneficial.
Apoptosis
1,2-diacylhydrazines
Antibacterial activity
Antifungal activity
Cytotoxicity
Hydrazides
Hydrazones
Iodinated compounds
Methicillin-resistant Staphylococcus aureus
1. Introduction
Candida activity with MIC values of 3.9 mg/L.7 Hydrazide-hydrazone
pharmacophore approach for synthesis of new antibacterial agents
Hydrazide-hydrazones containing azomethine group (–NH–N=CH–)
connected with carbonyl group (i.e., with the following moiety, –C
(=O)–NH–N=CH–) have gained a great importance due to their diverse
biological properties and wide spectrum of pharmaceutical applica-
tions.1 Among the biological properties of this class of compounds,
antimicrobial activity is the most frequently encountered in scientific
literature.2–5 Hydrazide-hydrazones of 3-methoxybenzoic acid and 4-
tert-butylbenzoic acid possessed high bactericidal activity against Ba-
cillus subtilis (minimum inhibitory concentrations, MIC, of 1.95–31.25
mg/L).6 Conjugates containing hybrids of bis-pyrazole scaffolds joined
through a hydrazone-hydrazide linker were synthesized and evaluated
for their antimicrobial activity against a panel of Gram-positive and
Gram-negative bacteria. Some of them showed also excellent anti-
was also used in designing of quinoxaline hybrids having incorporated
N-propionic and O-propionic hydrazide moieties.8
The hydrazide-hydrazones have attracted large number of re-
searchers also because of their anticancer activity.9–12 Pyridine-based
bis(hydrazide-hydrazones) were screened for their antibacterial activ-
ities as well as anticancer activities. Some of them showed significant
activity against human colon cancer as well Ishikawa human endome-
trial cancer cell lines (IC50 of 6.78–8.88
μ
M).13
1,2-(or N,N′-)Diacylhydrazine scaffold (–CO–NH–NH–CO–) have
also been employed in medicinal chemistry, although less frequently
than the hydrazide-hydrazones. 1,2-Diacylhydrazines are often used for
the construction of 1,3,4-oxadiazole ring. This approach includes
dehydration followed by simultaneous cyclization of diacylhydrazines
´
´
´
* Corresponding author at: Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho
´
´
1203, 500 05 Hradec Kralove, Czech Republic.
´
E-mail address: martin.kratky@faf.cuni.cz (M. Kratký).
https://doi.org/10.1016/j.bmc.2021.116209
Received 8 March 2021; Received in revised form 3 May 2021; Accepted 4 May 2021
Available online 13 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
under the action of various dehydrating reagents – thionyl chloride,
polyphosphoric acid, phosphorus pentoxide, trichloride or oxychloride,
tosyl chloride, acetic anhydride, sulfuric acid etc. Another approach is
oxidative cyclization of hydrazide-hydrazones.14 Diacylhydrazines are
present in naturally occurring bioactive compounds and they have been
investigated as antioxidant, cytotoxic, antifungal, antimycobacterial
and antiviral agents.15–17
2. Material and methods
2.1. Chemistry
2.1.1. General
All the reagents and solvents were purchased from Sigma-Aldrich
(Darmstadt, Germany), Penta Chemicals (Prague, Czech Republic),
and Lach-Ner (Neratovice, Czech Republic). They were used as received
without any purification. The reactions and the purity of the products
were monitored by thin-layer chromatography (TLC) using a mixture
with a ratio of dichloromethane to methanol of 4:1 (v/v) as the eluent.
Plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck Mil-
lipore, Darmstadt, Germany) and were visualized by UV irradiation
(254 nm). The melting points were determined on a Melting Point B-540
apparatus (BÜCHI, Flawil, Switzerland) using open capillaries. The re-
ported values are uncorrected. Infrared spectra were recorded on a FT-IR
spectrometer using ATR-Ge method (Nicolet 6700 FT-IR, Thermo Fisher
Scientific, Waltham, MA, USA) in the range 600–4000 cmꢀ 1. The NMR
spectra were measured in dimethyl sulfoxide (DMSO)-d6, or tetrahy-
drofuran (THF)-d8 at ambient temperature using a Varian V NMR S500
instrument (500 MHz for 1H and 126 MHz for 13C; Varian Corp., Palo
Alto, CA, USA). The chemical shifts δ are given in ppm and were referred
indirectly to tetramethylsilane via signals of DMSO‑d6 (2.50 for 1H and
39.51 for 13C spectra) or THF-d8 (1.72 and 3.58 for 1H, 25.31 and 67.21
for 13C spectra). The coupling constants (J) are reported in Hz.
Elemental analysis (C, H, N) was performed on an automatic micro-
analyser CHNS-O CE instrument (FISONS EA 1110, Milano, Italy).
Halogenation represents an important approach in optimization for
drug development and about half of the molecules used in high-
throughput screening are halogenated.18 Iodine (I2) is known anti-
septic and disinfectant that acts against bacteria, fungi, and viruses at
millimolar concentrations.19 Its aqueous solutions with potassium io-
dide, called Lugol’s solution was recently tested on long-term preser-
vation of marine plankton.20 The biological significance of iodine in the
group of 2-(aminomethyl)-4-ethynyl-6-iodophenols has been described
for anticancer agents developed as p53-Y220C stabilizers.21 Iodinated
phenolic products were prepared from, e.g., iodovanillin, acetovanillone
or methyl vanillate by laccase catalysis at an excess of potassium iodide.
The iodinated derivatives had significant growth inhibitory effects on
several wood degrading fungal species.22 Iodobenzoboroxoles were
screened in vitro for antimicrobial activity against Gram-positive bac-
teria, Micrococcus luteus and Bacillus cereus, and two Gram-negative
strains, Escherichia coli and Serratia marcescens. The results indicated that
4-iodo-6-methoxybenzo[c][1,2]oxaborol-1(3H)-ol possesses antibacte-
rial activity against Gram-positive strains with MIC of 16 and 13 mg/L
for M. luteus and B. cereus, respectively.23 Hydrazide-hydrazones of 5-
bromo-2-iodobenzoic acid were synthesized and identified as mild
antimicrobial agents together with a significant cytotoxicity.24 Iodin-
ated 4-aryloxymethylcoumarins were evaluated against two cancer cell
lines (MDA-MB human adenocarcinoma mammary gland and A-549
human lung carcinoma) and two mycobacterial strains; they were found
to exhibited a dual anticancer-antimycobacterial action.18
2.1.2. Synthesis
The synthesis and full characterization of 4-iodobenzohydrazide,
3,5-diiodosalicylidenehydrazides 1, 4-hydroxy-3,5-diiodobenzylidene-
hydrazides
2,
N′-(4-fluorobenzoyl)-2-hydroxy-3,5-diiodobenzohy-
In our previous search for antimicrobial agents, we have identified
Schiff bases derived from 2-hydroxy-3,5-diiodobenzaldehyde as prom-
ising derivatives to combat Gram-positive cocci including methicillin-
resistant Staphylococcus aureus (MRSA). Some of them also exhibited a
substantial cytotoxicity for human cell lines (Fig. 1).25–27
drazide 3f and reduced analogues 4a and 4b were published previously
by our group.29
2.1.2.1. Synthesis of hydrazide-hydrazones
1
and 2. Briefly, 4-
substituted benzohydrazides (1 mmol) were treated with 2- or 4-hy-
droxy-3,5-diiodobenzaldehyde (for hydrazones 1 and 2, respectively;
1.1 mmol; 411.3 mg) in boiling methanol for 2 h. The hydrazide-
hydrazones spontaneously precipitated were crystallized from MeOH
if necessary.29
According to the World Health Organization, antimicrobial resis-
tance is a global health threat requiring an urgent multidisciplinary
action. New antimicrobials are urgently needed unequivocally. An
inappropriate use of antimicrobial agents is a major driver in the
development of acquired drug resistance. For common infections, high
rates of resistance against antibiotics frequently used to treat them have
been observed worldwide. Especially alarming is the rapid global spread
of multi- and pan-resistant bacteria causing untreatable infections.
Focusing on Staphylococcus aureus, a common cause of both community
and nosocomial infections, people infected with methicillin-resistant
Staphylococcus aureus (MRSA) infections are 64% more likely to die
than people with methicillin-susceptible infections. Moreover, the
antibacterial and antifungal development pipeline is almost empty.28
2.1.2.2. Synthesis of 1,2-diacylhydrazines 3. 1,2-Diacylhydrazines 3
were prepared by a two-step procedure from appropriate 4-substituted
benzohydrazides and 3,5-diiodosalicylic acid.29 3,5-Diiodosalicylic
acid (389.9 mg, 0.001 mol) was dissolved in thionyl chloride (7 mL)
and after cooling to 0 ◦C, 3 drops of N,N-dimethylformamide (DMF)
were added. The mixture was to let stir without external cooling and
then heated to 60 ◦C under nitrogen atmosphere. After six hours, it was
evaporated to dryness under reduced pressure. Crude 3,5-diiodosalicy-
loyl chloride was used further without any purification. It was added
into dry tetrahydrofuran (THF; 7 mL) under nitrogen atmosphere, and
under vigorous stirring, a solution composed of 1 mmol of 4-substituted
benzohydrazide, 4 mmol of triethylamine (558 µL) and 7 mL of THF was
added dropwise. Alternatively (3j), the hydrazide was added as a solid
together with 2 equivalents of potassium carbonate. The mixture was
stirred for 24 h at room temperature. Then, it was evaporated to dryness
and using ethyl acetate, water and 0.1 M hydrochloric acid, it was
transferred into a separation funnel. The organic phase was washed with
0.1 M aqueous hydrochloric acid, 5% aqueous sodium bicarbonate, 0.1
M hydrochloric acid again, followed by saturated brine. The organic
phase was dried over anhydrous sodium sulfate. The filtrate was
concentrated under reduced pressure and n-hexane was added to start
precipitation. After 24 h at +4 ◦C, the suspension was filtered off to give
required derivatives 3 that were purified using crystallization from ethyl
′
́
Giving together here mentioned findings, we evaluated N -(2-/4-
hydroxy-3,5-diiodobenzylidene)
4-substituted
hydrazides
(i.e.,
hydrazide-hydrazones) and 1,2-diacylhydrazines as potential antimi-
crobial and cytotoxic agents.
Figure 1. Antibacterial 3,5-diiodosalicylidene-imines identified by our
group.25,26,27
2

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
acetate, a mixture of THF/acetonitrile or column chromatography with
2.1.2.2.8. 2-Hydroxy-3,5-diiodo-N′-[4-(trifluoromethyl)benzoyl]ben-
the mixture of toluene with ethyl acetate 4:0.3 (v/v).
zohydrazide 3j. White solid; yield 72%; mp 251–254 ◦C (decomp.). IR
2.1.2.2.1. N′-(Benzoyl)-2-hydroxy-3,5-diiodobenzohydrazide
3a.
(ATR): 3566 (O H), 3361 (N H), 1598 (CO-NH) cmꢀ 1. 1H NMR (500
–
–
◦
–
White solid; yield 48%; mp 219–222 C. IR (ATR): 3225 (N H), 1658
MHz, DMSO‑d6): δ 14.10 (1H, s, OH), 10.85 (1H, s, NH), 8.10 (2H, d, J =
(CO-NH) cmꢀ 1
.
1H NMR (500 MHz, DMSO‑d6): δ 13.16 (1H, s, OH),
8.0 Hz, H3, H5), 7.90–7.85 (3H, m, H2, H6, H4), 7.72 (1H, d, J = 2.1 Hz,
́
́
11.15 (1H, s, NH), 10.73 (1H, s, NH), 8.33 (1H, d, J = 2.0 Hz, H6), 8.26
H6). 13C NMR (126 MHz, DMSO): δ 165.25, 163.16, 148.15, 146.70,
́
́
́
́
́
́
(2H, d, J = 1.9 Hz, H4), 7.94–7.90 (2H, m, H2, H6), 7.64–7.60 (1H, m,
138.12, 137.29, 131.96 (q, J = 31.8 Hz), 128.88, 125.82 (q, J = 3.8 Hz),
123.64 (q, J = 272.3 Hz), 119.26, 88.06, 84.67. Anal. Calcd for
H4), 7.56–7.51 (2H, m, H3, H5). 13C NMR (126 MHz, THF): δ 168.89,
166.65, 161.51, 151.63, 136.10, 133.78, 132.61, 129.16, 128.43,
116.11, 88.55, 80.60. Anal. Calcd for C14H10I2N2O3 (508.05): C 33.10, H
1.98, N 5.51, found: C 33.23, H 1.98, N 5.80.
C
15H9F3I2N2O3 (576.05): C 31.28, H 1.57, N 4.86, found: C 31.21, H
1.59, N 4.83.
2.1.2.2.9. 2-Hydroxy-3,5-diiodo-N′-(4-nitrobenzoyl)benzohydrazide
2.1.2.2.2. 2-Hydroxy-3,5-diiodo-N′-(4-methylbenzoyl)benzohydrazide
3k. Yellow solid; yield 47%; mp 221–224 C. IR (ATR): 3559 (O H),
◦
–
ꢀ 1
3b. White solid; yield 43%; mp 193–196 C. IR (ATR): 3287 (N H),
3361 (N H), 1661 (CO-NH) cm
.
1H NMR (500 MHz, DMSO‑d6): δ
◦
–
–
1659 (CO-NH) cmꢀ 1
.
1H NMR (500 MHz, DMSO‑d6): δ 13.20 (1H, s,
13.23 (1H, s, OH), 10.92 (1H, s, NH), 10.50 (1H, s, NH), 8.31 (2H, d, J =
8.6 Hz, H3, H5), 8.12 (2H, d, J = 8.7 Hz, H2, H6), 7.85 (1H, d, J = 2.6
́
́
OH), 10.87 (1H, s, NH), 10.38 (1H, s, NH), 8.32 (1H, d, J = 2.1 Hz, H6),
8.02 (1H, d, J = 2.1 Hz, H4), 7.84–7.80 (2H, m, H2, H6), 7.38–7.35 (2H,
Hz, H6), 7.69 (1H, d, J = 2.6 Hz, H4). 13C NMR (126 MHz, DMSO): δ
167.46, 164.73, 162.20, 149.21, 146.29, 139.19, 137.89, 129.16,
123.70, 119.16, 87.88, 84.53. Anal. Calcd for C14H9I2N3O5 (553.05): C
30.40, H 1.64, N 7.60, found: C 30.61, H 1.45, N 7.73.
́
́
́
́
́
́
m, H3, H5), 2.46 (3H, s, CH3). 13C NMR (126 MHz, DMSO): δ 165.45,
155.23, 150.65, 142.59, 135.94, 129.25, 128.99, 127.91, 127.36,
118.15, 87.54, 86.09, 21.25. Anal. Calcd for C15H12I2N2O3 (522.08): C
34.51, H 2.32, N 5.37, found: C 34.39, H 2.10, N 5.44.
2.1.2.2.3. 2-Hydroxy-3,5-diiodo-N′-(4-methoxybenzoyl)benzohy-
drazide 3c. Greyish solid; yield 53%; mp 264–267 ◦C. IR (ATR): 3232
2.1.3. Synthesis of reduced analogues 4
The reduced analogue 4a was prepared from the hydrazide-
hydrazone 1f by treatment with lithium aluminium hydride in THF
under nitrogen atmosphere.29
(N H), 1652 (CO-NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 13.17 (1H,
–
s, OH), 11.05 (1H, s, NH), 10.55 (1H, s, NH), 8.30 (1H, d, J = 2.0 Hz,
́
́
́
́
H6), 8.23 (1H, d, J = 1.9 Hz, H4), 7.88 (2H, d, J = 8.7 Hz, H2, H6), 7.03
(2H, d, J = 8.8 Hz, H3, H5), 3.80 (3H, s, CH3). 13C NMR (126 MHz, THF):
δ 168.02, 165.38, 162.43, 159.71, 150.23, 135.49, 129.63, 124.26,
115.18, 114.02, 89.19, 81.97, 55.63. Anal. Calcd for C15H12I2N2O4
(538.08): C 33.48, H 2.25, N 5.21, found: C 33.36, H 2.31, N 5.59.
2.1.2.2.4. N′-[(4-Tert-butyl)benzoyl]-2-hydroxy-3,5-diiodobenzohy-
drazide 3d. Greyish solid; yield 61%; mp 173–176 ◦C. IR (ATR): 3283
2.1.4. Reductive amination29
An appropriate 4-halogenobenzohydrazide (1 mmol) was dissolved
in 7 mL of a mixture of THF with MeOH (1:1, v/v), followed by addition
of 3,5-diiodosalicylaldehyde (411.3 mg, 1.1 mmol). After a complete
dissolution, sodium cyanoborohydride (100 mg; 1.6 mmol) and glacial
acetic acid (100 µL) were added. The mixture was heated under reflux
for 4 h and then let stir for 48 h at room temperature. After this time, the
reaction mixture was diluted with distilled water and let stir for 30 min.
Then, the mixture was evaporated to dryness and suspended in a small
volume of MeOH. After additional 2 h, the resulted precipitate was
filtered off and dried to provide pure product.
(N H), 1644 (CO-NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 10.84 (1H,
–
́
́
s, OH), 9.97 (1H, s, NH), 9.74 (1H, s, NH), 8.27 (1H, d, J = 2.1 Hz, H6),
́
́
8.15 (1H, d, J = 2.2 Hz, H4), 7.87 (2H, d, J = 8.3 Hz, H2, H6), 7.50 (2H,
d, J = 8.3 Hz, H3, H5), 1.36 (9H, s, CH3). 13C NMR (126 MHz, THF): δ
165.42, 155.46, 155.24, 150.65, 146.40, 135.98, 129.03, 127.76,
125.52, 118.15, 87.57, 86.11, 34.93, 31.05. Anal. Calcd for
4-Chloro-N′-(2-hydroxy-3,5-diiodobenzyl)benzohydrazide 4c. White
◦
–
C
18H18I2N2O3 (564.16): C 38.32, H 3.22, N 4.97, found: C 37.49, H 2.96,
solid; yield 68%; mp 256.5–259 C. IR (ATR): 3227 (N H), 1656 (CO-
N 5.14.
NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 11.41 (1H, s, OH), 8.33 (1H, s,
́
2.1.2.2.5. N′-(4-Chlorobenzoyl)-2-hydroxy-3,5-diiodobenzohydrazide
CONH), 8.04 (1H, d, J = 2.0 Hz, H4), 7.91 (2H, d, J = 8.1 Hz, H2, H6),
́
́
◦
́
–
3g. Yellowish solid; yield 46%; mp 246–249 C. IR (ATR): 3233 (N H),
7.63 (1H, d, J = 2.1 Hz, H6), 7.52 (2H, d, J = 8.2 Hz, H3, H5), 4.12 (1H,
1658 (CO-NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 12.11 (1H, s, OH),
s, NH), 2.59 (2H, s, CH2). 13C NMR (126 MHz, THF): δ 162.79, 158.66,
148.33, 148.01, 139.91, 130.14, 129.81, 129.55, 121.12, 87.51, 80.69,
51.93. Anal. Calcd for C14H11ClI2N2O2 (528.51): C 31.82, H 2.10, N
5.30, found: C 31.88, H 2.12, N 5.25.
́
́
10.11 (1H, s, NH), 9.94 (1H, s, NH), 8.28 (1H, d, J = 2.0 Hz, H6), 8.14
́
́
(1H, d, J = 2.0 Hz, H4), 7.93–7.85 (2H, m, H2, H6), 7.52–7.46 (2H, m,
H3, H5). 13C NMR (126 MHz, THF): δ 165.77, 161.00, 156.59, 151.80,
137.81, 130.23, 130.07, 129.40, 129.35, 119.39, 86.40, 84.95. Anal.
Calcd for C14H9ClI2N2O3 (542.50): C 31.00, H 1.67, N 5.16, found: C
30.79, H 1.81, N 5.22.
2.2. Biology
2.1.2.2.6. N′-(4-Bromobenzoyl)-2-hydroxy-3,5-diiodobenzohydrazide
2.2.1. Antibacterial activity
◦
–
3h. Beige solid; yield 55%; mp 235–238 C. IR (ATR): 3545 (O H),
Antibacterial activity was evaluated against four Gram-positive and
four Gram-negative bacterial strains of a clinical importance, namely:
Staphylococcus aureus ATCC (American Type Culture Collection) 29213,
CCM (Czech Collection of Microorganisms) 4223 (methicillin-suscepti-
ble strain, MSSA), methicillin-resistant Staphylococcus aureus ATCC
43300, CCM 4750 (MRSA strain), Staphylococcus epidermidis, ATCC
12228, CCM 4418; Enterococcus faecalis ATCC 29212, CCM 4224;
Escherichia coli ATCC 25922, CCM 3954, Klebsiella pneumoniae ATCC
10031, CCM 4415, Serratia marcescens, clinical isolate, laboratory ID No.
62–2016, and Pseudomonas aeruginosa ATCC 27853, CCM 3955. These
strains were obtained from the Czech Collection of Microorganisms
(CCM, Brno, Czech Republic), one strain (Serratia marcescens) is a clin-
ical isolate kindly provided from the Department of Clinical Microbi-
3241 (N H), 1656 (CO-NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 12.99
–
(1H, s, OH), 10.41 (1H, s, NH), 10.04 (1H, s, NH), 8.24 (1H, d, J = 2.0
́
́
́
́
Hz, H6), 8.11 (1H, d, J = 2.0 Hz, H4), 7.84 (2H, d, J = 8.1 Hz, H2, H6),
7.68 (2H, d, J = 8.1 Hz, H3, H5). 13C NMR (126 MHz, THF): δ 168.85,
165.81, 161.47, 151.69, 136.08, 133.47, 132.49, 132.37, 130.26,
127.19, 88.57, 80.63. Anal. Calcd for C14H9BrI2N2O3 (586.94): C 28.65,
H 1.55, N 4.77, found: C 28.47, H 1.70, N 5.09.
2.1.2.2.7. 2-Hydroxy-3,5-diiodo-N′-(4-iodobenzoyl)benzohydrazide
◦
–
3i. Greyish solid; yield 64%; mp 214–217 C. IR (ATR): 3582 (O H),
3298 (N H), 1647 (CO-NH) cmꢀ 1. 1H NMR (500 MHz, THF-d8): δ 12.86
–
(1H, s, OH), 10.39 (1H, s, NH), 10.08 (1H, s, NH), 8.20 (1H, d, J = 2.0
́
́
́
́
Hz, H6), 8.07 (2H, d, J = 2.1 Hz, H4), 8.02–7.91 (4H, m, H2, H3, H5,
H6). 13C NMR (126 MHz, DMSO): δ 165.68, 163.67, 148.64, 146.51,
138.23, 137.67, 132.97, 129.88, 119.45, 99.16, 87.61, 86.11. Anal.
Calcd for C14H9I3N2O3 (633.94): C 26.52, H 1.43, N 4.42, found: C
26.19, H 1.30, N 4.87.
´
´
ology, University Hospital in Hradec Kralove, Czech Republic.
The microdilution broth method was performed according to
EUCAST (The European Committee on Antimicrobial Susceptibility
Testing) instructions30 with slight modifications starting from the
3

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
concentration of 0.49 µM. Briefly, the cultivation was done in Cation-
adjusted Mueller-Hinton broth (CAMHB, Mꢀ H 2 Broth, Sigma-Aldrich,
St. Louis, MO, USA) at 35 ± 2 ◦C without agitation. Tested compounds
were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) to produce
stock solutions. The final concentration of DMSO in the testing medium
did not exceed 1% (v/v) of the total solution composition and did not
affect the growth of bacteria. Positive (microbe solely), negative
(cultivation medium and DMSO) controls and internal quality standards
were involved in each assay. Antibacterial activity is expressed as
minimum inhibitory concentration (MIC, reported in µM) after 24 and
48 h of static incubation in dark and humidified atmosphere at 35 ±
2 ◦C. The experiments were performed in duplicates. For the results to be
valid, the difference in MIC determined from two parallel measurements
must not be greater than one step on the dilution scale. MIC was
determined by naked eye in the well with the lowest drug concentration,
where no visible growth of microbial agent was detected. MIC deter-
mination scale started from 0.49 µM.
glutamine solution and non-essential amino acid solution in a humidi-
fied atmosphere containing 5% CO2 at 37 ◦C. For subculturing, the cells
were harvested after trypsin/EDTA treatment at 37 ◦C. The human
kidney cells HK-2 (passage 12–14; American Type Culture Collection;
USA) were cultured in DMEM (Dulbecco’s and Eagle’s Modified Me-
dium) under the same conditions. To determine cytotoxicity of the
compounds, the cells treated with the tested substances were used as
experimental groups whereas untreated cells served as control groups.
All used chemicals were obtained from Sigma-Aldrich (St. Louis, MO,
USA).
The cells were seeded in a density of 10 000 cells per well in a 96-well
plate. The next day the cells were treated with each of the tested sub-
stances at a broad range of concentrations (based on their solubility) in
triplicates. The compounds were dissolved in DMSO (maximal incuba-
tion concentration of DMSO was 1% v/v). The controls representing
100% cell viability, 0% cell viability (treated with 10% DMSO), no cell
control and vehiculum control were incubated in parallel also in tripli-
cates. After 24 h of incubation in a humidified atmosphere containing
Broad-spectrum beta-lactam antibiotic piperacillin was used as
reference compound; its results were read after 24 h. Standard antibi-
otics (ciprofloxacin, gentamicin; data not shown) and internal quality
control/reference strains are routinely included in parallel testing-basic
screening of antibacterial activity.
◦
5% CO2 at 37 C, the reagent from the kit CellTiter 96 AQueous One
Solution Cell Proliferation Assay (CellTiter 96; PROMEGA, Fitchburg,
◦
WI, USA) was added. After 2 h of incubation at 37 C, absorbance of
¨
samples was recorded at 490 nm (TECAN, Infinita M200, Grodig,
Austria). A standard toxicological parameter IC50 was calculated by
nonlinear regression from a semilogarithmic plot of incubation con-
centration versus percentage of viability relative to untreated controls
using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla,
CA, USA).
2.2.2. Antifungal activity
Antifungal activity was evaluated against four yeast strains, namely:
Candida albicans ATCC 24443, CCM 8320, Candida krusei ATCC 6258,
CCM 8271, Candida parapsilosis ATCC 22019, CCM 8260, Candida tro-
picalis ATCC 750, CCM 8264; and four strains of filamentous fungi,
namely: Aspergillus fumigatus ATCC 204305, Aspergillus flavus CCM 8363,
Lichtheimia corymbifera CCM 8077, and Trichophyton interdigitale ATCC
9533, CCM 8377. A microdilution broth method was performed ac-
cording to EUCAST instructions (EUCAST 7.3.1. and 9.3.1)31,32 with
slight modifications. Briefly, tested compounds were dissolved in DMSO
and diluted in a twofold manner with RPMI-1640 medium with L-
glutamine, supplemented with 2% glucose (w/v) and buffered to pH 7.0
with 3-(N-morpholino)propane-1-sulfonic acid (MOPS). All these com-
ponents were purchased from Sigma-Aldrich, St. Louis, MO, USA. The
final concentration of DMSO in the tested medium did not exceed 1% (v/
v) of the total solution composition, and it was confirmed that this
concentration did not inhibit the fungal growth. Static incubation was
performed in dark and humidified atmosphere, at 35 ± 2 ◦C for 24 and
48 h (72 and 120 h for Trichophyton interdigitale). Positive controls
consisted of tested fungus solely, while negative controls consisted of
medium and DMSO. The experiments were conducted in duplicates. For
the results to be valid, the difference in MIC determined from two par-
allel measurements must not be greater than one step on the dilution
scale. MIC was determined by naked eye in the well with the lowest drug
concentration, where no visible growth of microbial agent was detected.
MIC determination scale started from 0.49 µM.
Results of the experiments are presented as inhibitory concentrations
that reduce viability of the cell population to 50% from the maximal
viability (IC50). Known cytotoxic compounds tamoxifen (as tamoxifen
citrate) and cisplatin were involved as reference compounds.
2.2.4. Measurement of caspase activities
To determine effect of the tested compounds on cellular apoptosis,
activation of caspase 3/7 in HepG2 cells was evaluated for five the most
interesting compounds using a standard luminescent kit (Caspase-Glo 3/
7® Assay, PROMEGA, Fitchburg, USA). The cells were seeded in 96-well
plates at a concentration of 15 000 cells per well. The compounds were
dissolved in complete MEEM and 0.1 mL of the diluted toxicant in me-
dium per well was added to cells cultured in a 96-well plate. Control
untreated cells were incubated in the vehicle in parallel and only com-
plete medium was added. Following incubation for 24 h in a humidified
atmosphere containing 5% CO2 at 37 ◦C, the caspase activity was
determined according to the manufacturer’s instructions. Reagent from
the Caspase-Glo® 3/7 Assay was added. The plate was incubated in a
shaker at 350 rpm and room temperature for 60 min in the dark. The
addition of the Caspase-Glo reagent resulted in cell lysis, followed by
caspase cleavage of the substrate and generation of a luminescent signal
produced by luciferase. Luminescence is proportional to the amount of
caspase present. Luminescence was measured and recorded using the
plate reader Synergy 2 (BioTek Instruments, Winooski, Inc., VT, USA).
Luminescence data were expressed as fold from untreated controls.
Tamoxifen was involved a positive control. In all experiments, cells were
incubated in triplicates.
Triazole antimycotic drug fluconazole was involved as reference
compound for a comparison. MIC values of fluconazole mean MIC50
values, i.e., the lowest drug concentration giving growth inhibition of
50% of that of the drug-free control. Results were read after 24 h (yeasts)
or 48 h (moulds) microdilution plates cultivation without agitation at
35 ± 2 ◦C in humidified atmosphere. The results were read with a
microdilution plate reader (SynergyTM HTX, BioTek Instruments, Inc.,
VT, USA) at wavelength 530 nm. Internal quality control was included
too. Standard antimycotics (voriconazole, amphotericin B; data not
shown) are routinely included in parallel testing-basic screening of
antibacterial activity.
3. Results and discussion
3.1. Chemistry
Based on the biological activity of hydrazide-hydrazones and iodin-
ated compounds, we used 4-substituted hydrazides and 2-/4-hydroxy-
3,5-diiodobenzaldehydes in drug design to investigate their mutual
hydrazide-hydrazones as potential antimicrobial and cytotoxic agents.
Remaining derivatives, i.e., 1,2-diacylhydrazines 3 and reduced forms of
the most active hydrazones, compounds 4, were designed as their ana-
logues based on the biological activity identified initially in this study.
2.2.3. Cytotoxicity determination
The human hepatocellular liver carcinoma cell line HepG2 (passage
18–19) purchased from Health Protection Agency Culture Collections
(ECACC, Salisbury, UK) was cultured in Minimum Essentials Eagle
Medium (MEEM) supplemented with 10% foetal bovine serum, 1% L-
4

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
Regarding parent hydrazides, we have involved various substitution
I-benzohydrazides were inactive, thus disfavouring lipophilic electron-
donating substituents and the heaviest iodine atom. Contrarily, 4-fluo-
rine, 4-chlorine and 4-CF3-benzohydrazides provided highly active
hydrazones (1f, 1g, and 1j, respectively; MIC of 15.62–62.5 µM), fol-
lowed closely by nitro and hydroxyl groups (1k and 1e). Unsubstituted
hydrazide and 4-brominated analogue (1a and 1h) were moderately
active with slightly higher MIC for E. faecalis. Interestingly, MIC values
for MSSA and MRSA are identical, i.e., the mechanism of action is not
related to beta-lactam antibiotics.
at position 4 of the phenyl. The substituents were representatives of
functional groups with different electron and steric effects: hydrogen
(neutral), alkyls (+I, small methyl and bulky tert-butyl), OH and CH3O
(-I and stronger + M), as well as electron-withdrawing moieties: halo-
gens from fluorine to iodine (negative inductive and weak positive
mesomeric effect), NO2 (strong -I and -M effects) and CF3 (strong -I
effect).
Among eleven hydrazides, only 4-iodobenzohydrazide, precursor of
the derivatives i, was synthesized in-house by a two-step procedure from
Having these promising results of 1 in hands, we prepared and
screened also their analogues: isomeric 2, diacylhydrazines 3 and
reduced analogues 4. However, the series 2 and 3 share only negligible
antibacterial activity generally when compared to the hydrazide-
hydrazones 1. MIC values of the hydrazide-hydrazones 2 started from
125 µM against Gram-positives. In contrast to 1, when 4-halogen, hy-
droxy, nitro or 4-CF3-benzohydrazide was used as a parent compound,
the resulting hydrazones 2 share no or limited activity. Contrarily, 4-hy-
droxy-3,5-diiodobenzylidenehydrazones obtained from 4-alkyl/alkoxyl
hydrazides (2b-2d) exhibited better activity than their isomers 1b-1d,
although with a comparatively high MIC values (≥125 µM) with the tert-
butyl derivative 2d superiority.
4
to iodobenzoic acid via Fischer esterification followed by
hydrazinolysis.29
The hydrazides (1 eq.) were treated with 3,5-diiodosalicylaldehyde
(compounds 1) or its isomeric 4-hydroxy-3,5-diiodobenzaldehyde (de-
rivatives 2) in a mild excess (1.1 of equivalents) in boiling methanol for
2 h (Fig. 2). The yields of hydrazide-hydrazones 1 and 2 were 61–94%
and 44–88%, respectively.29
Based on promising antimicrobial and cytotoxic assessment of these
two series, we decided to prepare also similar 1,2-diacylhydrazines 3
and reduced analogues of the most potent halogenohydrazides 1f and
1g. The derivatives 3 were prepared in two steps from 3,5-diiodosali-
cylic acid that was converted to 3,5-diiodosalicyloyl chloride using an
excess of thionyl chloride and DMF as a catalyst. After evaporation of
volatile components, solution of the hydrazide (1 eq.) and triethylamine
(4 of equivalents) in THF was added to this crude chloride (Fig. 3). The
moderate yields were consistent within the range of 43–72 %. 4-Hydrox-
ybenzohydrazide-based derivative 3e was not obtained due to formation
Focusing on the hydrazides 3, we were not able to determine MIC of
several of them (3a, 3e, 3h, 3j, and 3k) due to their insufficient solu-
bility in the testing medium or a massive precipitation during process-
ing. Three of them exhibited poor inhibition of bacteria (MIC of ≥500
µM; 3c, 3d, 3f), the remaining ones showed an improved activity,
especially those derived from 4-chlorobenzohydrazide and 4-iodobenzo-
hydrazide 3g and 3i with MIC values from 31.25 and 62.5 µM, respec-
tively. N′-(4-Chlorobenzoyl)-2-hydroxy-3,5-diiodobenzohydrazide 3g
was found generally similarly active as the salicylidene derivative 1g.
In fact, the last modification consisting in a reduction of one or two
double bond(s) present in the most active halogenated hydrazones 1f
and 1g was the most successful approach. Among 4-fluorinated com-
pounds, the reduction of both imine and carboxamide bonds provided
the secondary alcohol 4a with a retained activity against MSSA, MRSA
and even improved in the case of S. epidermidis and E. faecalis (up to four
of a complex mixture of particular products.
–
To parallel CH=N and C O bonds reduction, we used lithium
–
aluminium hydride to obtain racemic secondary alcohol 4a (83%;
′
́
Fig. 4). The reduced analogues, i.e., N -substituted hydrazides 4b and 4c
with reduced imine bond were obtained by reductive amination using
sodium cyanoborohydride (Fig. 5) with yields of 64 and 68%,
respectively.
3.2. Biology
–
times). Reduction of only C N bond (4b) led to the molecule with
–
identical inhibition (i.e., ±one dilution) of E. faecalis and S. epidermidis
but higher MIC for S. aureus. For the chlorinated hydrazide-hydrazone
1g is the reduction of imine bond also beneficial since it retains activ-
ity against both S. aureus strains but boosts suppression of E. faecalis and
S. epidermidis (up to four times). In sum, these reduced analogues 4a and
4c belong to the most effective agents to combat MSSA, S. epidermidis
and E. faecalis with MIC values from 7.81 µM (i.e., 4.01 mg/L).
3.2.1. Antibacterial activity
Initially, we investigated antimicrobial activity of the synthesized
compounds 1–4. MIC values were determined by the microdilution
broth method against four Gram-positive strains (Staphylococcus aureus
ATCC 29213; MRSA, i.e., methicillin-resistant Staphylococcus aureus
ATCC 43300; Staphylococcus epidermidis ATCC 12228; Enterococcus fae-
calis ATCC 29212) and four Gram-negative strains (Escherichia coli ATCC
25922; Klebsiella pneumoniae ATCC 10031; Serratia marcescens, clinical
isolate laboratory ID No 62–2016; Pseudomonas aeruginosa ATCC
27853). A broad-spectrum beta-lactam antibiotic piperacillin (PIP) was
used for comparison of MIC (Table 1). From chemical point of view, it is
an ureidopenicillin, from pharmacological perspective, it is highly
active against Gram-negative bacteria including Pseudomonas aeruginosa
and Gram-positive pathogens that do not produce penicillinase and are
susceptible to methicillin.
Drawing a comparison to piperacillin, the derivatives 1–4 were able
to supress PIP-resistant S. epidermidis and MRSA strains. The most active
hydrazine derivatives 4a and 4c exhibited similar in vitro efficacy for
E. faecalis in both molar and weight concentration scales (±one dilu-
tion). On the other hand, our derivatives were less active against beta-
lactams-susceptible S. aureus and Gram-negative bacteria that are
main therapeutical areas of PIP, predominantly used in a fixed combi-
nation with beta-lactamase inhibitor tazobactam.33
In general, the derivatives 1–4 have antibacterial properties only
against all Gram-positive cocci at comparable concentrations whereas
they are de facto inactive towards Gram-negative species. Only the 4-
hydroxybenzohydrazide derivative 1e inhibited E. coli, remaining
pathogens showed a complete resistance (P. aeruginosa, K. pneumoniae,
S. marcescens). Importantly, there are no differences of MIC values for
MSSA and MRSA that means no cross-resistance to beta-lactams, fortu-
nately. The optimal structural requirements are as follows: (1) benzo-
hydrazide substituted by fluorine, chlorine, trifluoromethyl or nitro
group at the position 4; (2) substituted by 2-hydroxy-3,5-diiodobenzyli-
At the beginning, we evaluated hydrazones 1 prepared from 3,5-diio-
dosalicylaldehyde (Table 1). They abolished growth of all Gram-positive
bacteria predominantly with MIC values from 15.62 µM (7.97 mg/L).
The hydrazones 1b-1d and 1i derived from 4-(O)CH3, 4-tert-butyl and 4-
′
́
dene at N , this imine double bond can be reduced individually or
together with carboxamide moiety.
Among other salicylidene hydrazides, 3,5-diiodosalicylidene hydra-
zides have also been screened for their inhibition activity on type III
Figure 2. Synthesis of the hydrazide-hydrazones 1 and 2 (R = H a, CH3 b,
OCH3 c, t-Bu d, OH e, F f, Cl g, Br h, I i, CF3 j, NO2 k).
5

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
Figure 3. Synthesis of 1,2-diacylhydrazines 3 (DMF: N,N-dimethylformamide, THF: tetrahydrofuran; R = H a, CH3 b, OCH3 c, t-Bu d, F f, Cl g, Br h, I i, CF3 j, NO2 k).
Figure 4. Reduction of 1f (THF: tetrahydrofuran).
vs. 1f and 4b).
T. interdigitale was the most susceptible strain. Contrarily, C. tropicalis
and both Aspergillus strains tolerated all the derivatives 1–4. Impor-
tantly, our derivatives exhibited comparable or superior activity than
clinically used drug fluconazole against C. albicans (1k), C. krusei (1f,
1k), L. corymbifera (1f, 1k, 4b) and T. interdigitale (all four active
compounds).
Previously, iodinated salicylidene benzohydrazides (free or in a
Figure 5. Reductive amination (THF: tetrahydrofuran).
combination with copper salts) have been reported as effective agents
against phytopathogenic fungi,35 but here presented derivatives are
secretion in Gram-negative pathogen Yersinia pseudotuberculosis. Type III
active against human fungal pathogens at low concentrations.
secretion is a virulence system constituting a potential drug target
discovered in a wide range of clinically important Gram-negative
3.2.3. Cytotoxicity
pathogens. However, three investigated derivatives showed only a
Cytotoxicity of the tested compounds 1–4 was measured using two
mild inhibition.34 To the best of our knowledge, this is the first report of
types of cells, a cancer cell line HepG2 and a HK-2 cells derived from
iodinated salicylidene benzohydrazide-hydrazones as potent antibacte-
normal kidney tissue. The used CellTiter 96 assay is based on the
rial agents inhibiting Gram-positive cocci so far.
reduction of tetrazolium of MTS dye ([3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)–2H-tetrazolium]) in living
3.2.2. Antifungal activity
cells to formazan, which is then determined colorimetrically. The
We screened 1,2-disubstituted hydrazine derivatives 1–4 also against
reduction of the reagent is related to availability of NADH or NADPH.
eight strains of human fungal pathogens. The panel of microorganisms
Their drop causes lowered production of formazan indicating decreased
covers four yeasts (Candida albicans ATCC 24443, Candida krusei ATCC
cell viability.
6258, Candida parapsilosis ATCC 22019 and Candida tropicalis ATCC
Results of the experiments are presented as inhibitory concentration
750) and four moulds (Aspergillus fumigatus ATCC 204305, Aspergillus
which reduces viability of the cell population to 50% from the maximal
flavus CCM 8363, Lichtheimia (Absidia) corymbifera CCM 8077, and Tri-
viability (IC50). This IC50 parameter was used as a quantitative measure
chophyton interdigitale ATCC 9533). Triazole fungistatic drug fluconazole
of cytotoxicity, which allows the quantitative comparison of the toxicity
(FLU) was used for comparison of MIC values (Table 2). MIC of the
among tested compounds through three series of hydrazine derivatives.
investigated compounds means a complete inhibition of fungal growth
We were able to determine IC50 of the majority of the investigated
determined by naked eye, whereas MIC of FLU mean IC50 values
substances. However, IC50 of seven hydrazide-hydrazones (1a, 1b, 2b,
determined spectrophotometrically.
2c, 2g-2i) couldn’t be determined due to the precipitation in the culture
Among the iodinated derivatives, only four of them exhibited sig-
medium at higher concentrations than reported in Table 3. However,
nificant antifungal properties (Table 2; 1f, 1k, 4a, and 4b), MIC of
they have exhibited no signs of cytotoxicity among soluble concentra-
remaining ones were greater than 125 µM. Obviously, for antifungal
tions. The effective cytotoxic concentrations of the compounds 1–4 are
action, 4-fluoro- and 4-nitrobenzohydrazide scaffold are optimal choice
listed in Table 3. Considerable differences in cytotoxicity of the tested
in combination with 2-hydroxy-3,5-diiodobenzylidene substitution (1f,
compounds were found also in HK-2 cells as treatment led to different
1k). 4-Hydroxy-3,5-diiodobenzylidenes 2 and 1,2-diacylhydrazines 3
rate of damage. However, the found IC50 values of the five selected
did not offer any antifungal properties. 4-Nitrobenzohydrazide-based
compounds (based on activity against HepG2) did not have so broad
hydrazone 1k exhibited the most potent antimycotic action with MIC
dispersion (Table 4). For this cell line, the toxic concentrations were
from 1.95 µM (1.05 mg/L) inhibiting four strains from total eight
possible only to be assessed in three agents due to limited solubility of 2d
(C. albicans, C. krusei, L. corymbifera and T. interdigitale). 4-Fluorinated
and 3i.
hydrazide-hydrazone 1f showed inhibition of five fungi, but at higher
Focusing on cancer HepG2 cells, the IC50 values among the hydrazine
concentrations than 1k. Focusing on fluorinated analogues, the reduc-
derivatives 1–4 compounds differ in two orders of magnitude (from
tion of imine bond is partly tolerated (4b, that has lower MIC for
11.72 to 190.90 µM). We were able to identify relatively non-toxic
L. corymbifera but higher for yeasts); on the other hand, an additional
compounds for HepG2 cells (IC50 greater than 100 µM: 2a-2c, 2e-2g,
reduction of carboxamide double bond brought a drop in the activity (4a
3c, 3j, and 3k). On the other hand, cytotoxicity of the most toxic
6

´
M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
Table 1
Structures and antibacterial activity of hydrazines 1–4.
Code
R
MIC [µM] (mg/L)
SA
MRSA
24 h
SE
EF
EC
24 h
48 h
48 h
24 h
24 h
24 h
48 h
24 h
48 h
1a
2a
3a
1b
2b
3b
1c
2c
3c
1d
2d
3d
1e
2e
1f
H
62.5
125
62.5
125
62.5
125
250
500
>500
>500
ND
>500
>500
ND
H
250
250
500
>500
ND
>500
ND
>500
ND
>500
ND
>500
ND
H
ND
ND
ND
CH3
>500
250
>500
500
>500
>500
>125
>500
>500
500
>500
>500
>125
>500
>500
>500
>500
250
>500
>500
125
>500
>500
>125
>500
>500
>500
>500
250
>500
>500
>125
>500
>500
500
>500
>500
>125
>500
>500
>500
>500
500
>500
>500
>125
>500
>500
>500
>500
>500
>500
250
>500
>500
>125
>500
>500
>500
>500
>500
>500
250
CH3
CH3
125
>125
>500
500
OCH3
>500
500
>500
>500
500
OCH3
OCH3
500
>500
>500
125
t-Bu
>500
125
>500
125
>500
125
>500
250
t-Bu
t-Bu
>500
31.25
500
>500
62.5
500
>500
62.5
>500
62.5
>500
31.25
>500
500
>500
62.5
>500
125
>500
250
>500
250
OH
OH
>500
15.62 (7.97)
500
>500
31.25
>500
500
>500
31.25
>500
500
>500
31.25
>500
500
>500
62.5
>500
>500
>500
>500
>500
>500
>500
>250
>500
ND
>500
>500
>500
>500
>500
>500
>500
>250
>500
ND
F
15.62 (7.97)
125
31.25
500
2f
F
>500
500
3f
F
500
500
500
1g
2g
3g
1h
2h
3h
1i
Cl
31.25
>500
62.5
31.25
>500
62.5
62.5
>500
ND
31.25
>500
62.5
62.5
>500
62.5
125
31.25
>500
31.25
62.5
62.5
62.5
62.5
Cl
>500
62.5
>500
125
>500
125
Cl
Br
62.5
62.5
125
250
250
Br
>500
ND
>500
ND
>500
ND
>500
ND
>500
ND
>500
ND
>500
ND
Br
I
>125
>250
250
>125
>250
250
>125
>250
125
>125
>250
250
>125
>250
62.5
>125
>250
125
>125
>250
125
>125
>250
250
>125
>250
>250
>250
>500
ND
>125
>250
>250
>250
>500
ND
2i
I
3i
I
1j
CF3
31.25
125
31.25
500
31.25
500
31.25
500
31.25
500
31.25
500
31.25
500
31.25
500
2j
CF3
3j
CF3
ND
ND
ND
ND
ND
ND
ND
ND
1k
2k
3k
4a
4b
4c
PIP
NO2
31.25
250
62.5
250
31.25
500
62.5
>500
ND
31.25
>500
ND
62.5
62.5
125
>125
>500
ND
>125
>500
ND
NO2
>500
ND
>500
ND
>500
ND
NO2
ND
ND
ND
F; R1: –OH
F; R1: =O
Cl; R1: =O
–
15.62 (8.03)
62.5
15.62 (8.03)
125
31.25
62.5
62.5
125
7.81 (4.01)
62.5
7.81 (4.01)
62.5
15.62 (8.03)
31.25
15.62 (8.26)
7.41 (4)
15.62 (8.03)
62.5
>500
>500
>500
3.71 (2)
>500
>500
>500
31.25
1.85 (1)
62.5
31.25
14.83 (8)
62.5
15.62
>59.31 (>32)
15.62
15.62 (8.26)
PIP = piperacillin sodium salt. ND: not determined due to solubility issues. SA: Staphylococcus aureus ATCC 29213; MRSA: methicillin-resistant Staphylococcus aureus
ATCC 43300; SE: Staphylococcus epidermidis ATCC 12228; EF: Enterococcus faecalis ATCC 29212; EC: Escherichia coli ATCC 25922. ND: not determined due to solubility
issues.
The lowest MIC values for each strain are given in bold. MIC in mg/L were calculated for the lowest MIC identified in µM.
Table 2
Antifungal activity of hydrazine derivatives 1 and 4.
Code
R
MIC [µM]
CA
CK
CP
LC
TI
24 h
48 h
24 h
24 h
24 h
48 h
24 h
48 h
72 h
120 h
1f
F
62.5
1.95
>500
125
125
125
125
62.5
>125
>500
>125
3.3
125
125
125
15.62
3.9
62.5
1k
NO2
7.81
>500
>125
6.5
62.5
>125
>500
>125
>104.5
>125
>500
>125
3.3
31.25
>500
15.62
>104.5
62.5
15.62
62.5
4a
>500
>125
>104.5
>500
31.25
>104.5
62.5
15.62
52.2
4b
31.25
52.2
FLU*
6.5
FLU = fluconazole. *: MIC means IC50 values. CA: Candida albicans ATCC 24443; CK: Candida krusei ATCC 6258; CP: Candida parapsilosis ATCC 22019; AC: Lichtheimia
corymbifera CCM 8077; TI: Trichophyton interdigitale ATCC 9533.
One or two of the best MIC value(s) for each strain are shown in bold.
7

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M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
Table 3
Toxicity of the hydrazine derivatives 1–4 in HepG2 cells.
Code
IC50 [µM]
Range of concentrations tested
Code
IC50 [µM]
Range of concentrations tested
1a
H
>50*
1–1000
1–250
1–100
1–1000
1–100
1–250
1–1000
1–100
1–50
1g
2g
3g
1h
2h
3h
1i
Cl
14.75
>100*
48.65
22.27
>50*
1–500
1–100
1–50
2a
H
190.90
98.03
>25*
Cl
3a
H
Cl
1b
CH3
CH3
CH3
OCH3
OCH3
OCH3
t-Bu
t-Bu
t-Bu
OH
OH
F
Br
Br
Br
I
1–500
1–50
2b
>100*
86.42
45.15
>100*
132.40**
41.59
45.92
87.32
19.17
110.40
12.07
116.70
86.99
19.6
3b
62.79**
31.81
>50*
1–50
1c
1–500
1–50
2c
2i
I
3c
3i
I
25.05
11.72
57.43
167.00
24.65
92.08
158.80**
41.91
52.20
52.22
1–100
1–250
1–250
1–100
1–1000
1–500
1–100
1–1000
1–1000
1–1000
1d
1–1000
1–250
1–100
1–1000
1–500
1–500
1–500
1–100
1–500
1–500
1j
CF3
CF3
CF3
NO2
NO2
NO2
F
2d
2j
3d
3j
1e
1k
2k
3k
4a
4b
4c
2e
1f
2f
F
3f
F
F
tamoxifen
Cl
cisplatin
21.3
*
Higher concentrations precipitated in the culture medium.
Estimated value based on the curve.
**
Also, the most cytotoxic tert-butylated 4-hydroxybenzylidene derivative
2d showed a lesser inhibition of HK-2 (at least 5.4× due to its limited
solubility in DMEM) as well as triiodinated diacylhydrazine 3i (more
than 4 times). These results indicate a higher sensitivity of cancer cells,
fortunately. The found differences in cytotoxicity between HepG2 and
HK-2 cells can be explained by different origin and character of the
employed cell lines.
Table 4
Toxicity of the selected hydrazine derivatives in HK-2 cells.
Code
IC50
[
μM]
Range of concentrations tested [μM]
2d
1f
˃250
1–250
1–500
1–100
1–250
1–1000
55.61
˃100
3i
1j
49.85
26.80
To describe potential mechanism of the antiproliferative effect of the
tested compounds, we evaluated selected ones (hydrazide-hydrazones
1f, 1j, 1k, 2d, and 1,2-diacylhydrazine 3i) for changes in caspase 3/7
activities in HepG2 cells (Fig. 6). Tamoxifen was involved as a
comparator. Caspase activity was demonstrated in cells treated with this
1k
derivatives was comparable or higher in comparison with anticancer
drugs tamoxifen and cisplatin (19.6 and 21.3 µM, respectively): 1e-1k,
and 3i. Focusing on remaining derivatives, they have toxicities between
these values. The small substituents with a moderate rate of lipophilicity
and electron-donating or neutral properties (hydrogen a, methyl b,
methoxy c) are associated with a lower toxicity for eukaryotic cancer
cells.
drug at concentrations of 25 and 50 μM (1.69- and 1.81-fold control,
respectively). The performed standard test did not show a significant
proapoptotic effect in most of the agents. A small increment in caspase
3/7 activity was found in two fluorinated hydrazide-hydrazones 1f and
1j. The CF3-derivative 1j at the concentration of 100 μM led to 1.37-fold
In general, the presence of 2-hydroxy-3,5-diiodobenzylidenehydra-
zide (compounds 1) is associated with an escalated toxicity especially
when combined with halogens and strong electron-withdrawing sub-
stituents (CF3, NO2). The 4-CF3-benzohydrazide-derived hydrazone 1j
(IC50 of 11.72 µM) was the most cytotoxic compound within this study.
Contrarily, their positional isomers 2 are predominantly the least toxic
analogues (up to ten-times less, as demonstrated in the pair 1f and 2f).
Also, the replacement of imine bond by second carboxamide (hydrazide-
hydrazones 1 → 1,2-diacylhydrazines 3) led to a substantial lower
toxicity. The switch of 2-hydroxy-3,5-diiodobenzylidene substituent for
4-fluoro- and 4-chlorobenzohydrazide to 2-hydroxy-3,5-diiodobenzyl
(1f and 1g to 4b and 4c, respectively), i.e., reduction of imine double
bond, resulted in a decreased toxicity (up to 4.3 times), thus improving
selectivity for microbes.
control, while 1f increased caspase 3/7 activity at two concentrations of
1 μM and 100 μM (1.36- and 1.33-fold control, respectively). However,
the apoptotic effect is not a class-effect. This finding means that inhib-
itory effect of the compounds towards the cells is likely based on
impairment of another metabolic process than apoptosis.
The selectivity to particular biological activities can be quantified
using selectivity indexes (SI). In analogy to therapeutic index, SI is
calculated as a ratio of the IC50 and MIC values. Its value above a
threshold of 10 means a selective action. The nitro hydrazide-hydrazone
1k provides selective activity against C. albicans (SI of 13.7), other
compounds share a balanced dual antibacterial (for 1f and 1k; 4a and 4b
antifungal as well) and cytotoxic action, for some compounds with a
more pronounced toxicity, for others with a stronger antimicrobial
action.
Keeping in hands these results, we investigated the most active
compounds also against normal human cell line to compare their inhi-
bition of cancer and healthy cells. We used HK-2 line, epithelial kidney
cortex/proximal tubule-derived cells. Following representatives were
chosen: the most cytotoxic 2-hydroxy-3,5-diiodobenzylidene hydra-
zones 1f, 1j, and 1k; 4-hydroxy-3,5-diiodobenzylidene hydrazone 2d
and 1,2-diacylhydrazine 3i. IC50 data are reported in Table 4.
Importantly, four hydrazines showed lower toxicity for normal than
for cancer cells, only the nitro compound 1k was an exception with
identical IC50 values (26.80 and 24.65 µM, respectively). HK-2 line was
inhibited at at least four times higher concentrations than HepG2 (4.5×
and 4.3× for fluorine substituted molecules 1f and 1j, respectively).
Previously, some iodinated organic compounds have been reported
and investigated as potential cytotoxic/anticancer agents.24,26,36–38
Based on IC50, the most cytotoxic here presented derivatives are at least
comparable to these molecules, thus opening a new field for their
possible application.
The coincidence of cytotoxic and antibacterial/antifungal properties
in one molecular entity can be also beneficial since anticancer drugs
with an adjuvant antimicrobial action are of a special interest.39 For
some compounds clinically used as antimicrobials, a potent and prom-
ising anticancer action has been described later. Although used for
treatment of bacteria and protozoa-caused diseases widely, they may be
useful for oncologic patients not only with a cancer-associated infection
8

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M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
Figure 6. Caspase 3/7 activities in HepG2 cell treated with selected tested compounds (mean ± SD; n = 6). Cytostatic drug tamoxifen was used as a positive control.
The lower value at the highest concentration of 1k indicates cellular necrosis.
due to their direct cytostatic and cytotoxic activities (both in vitro and in
vivo), e.g., salicylic derivative niclosamide,40 fluoroquinolones, 8-
hydroxyquinolines, thiazole antibiotics etc.39,41 In addition to various
anticancer effects, antimicrobial agents can benefit cancer patients by
killing oncogenic-related microorganisms and protecting from
immunosuppression-induced opportunistic infections.39 From another
point of view, also “classical” anticancer drugs have shown an inter-
esting inhibition of various microbes, e.g., anthracycline anti-
biotics,39–41 5-fluorouracil, busulfan, mitomycin C, methotrexate42 or
tamoxifen.42–44 This repurposing represents a viable concept for intro-
ducing “novel” drugs.
compounds are easily synthetically available.
The 4-substituted benzohydrazide modification using 2-/4-hydroxy-
3,5-diiodobenzaldehydes and 3,5-diiodosalicylic acid offers compounds
that are active against Gram-positive bacteria including MRSA strain
and some of them are also antifungal agents with low MIC values from
1.95 µM. The cytotoxicity ranged from low micromolar concentrations
to hundreds of µM. Structure-activity analysis found following re-
lationships: (1) the presence of 3,5-diiodosalicylidene moiety is required
for potent bioactivity; (2) the imine bond can be also reduced; (3) 4-hy-
droxy-3,5-diiodobenzylidene and 3,5-diiodosalicyloyl scaffolds led to
lower antimicrobial action, but they contribute to cytotoxicity; (4) for
substitution of benzohydrazide, electron-withdrawing atoms/groups are
optimal (smaller halogens, CF3, NO2). Several compounds provided
“pure” cytotoxic action, while some members of the series share a dual
antimicrobial-cytotoxic action that can be promising, too. Two fluori-
nated hydrazide-hydrazones induce an increase in caspase-3/7 activity,
i.e., they have proapoptotic effect.
4. Conclusions
In this study, we evaluated twenty-two iodinated hydrazide-
hydrazones and two their reduced analogues as potential antimicro-
bial and cytotoxic agents. Based on their promising results, we designed
and synthesized eleven additional analogues, iodinated 1,2-diacylhydra-
zines and one reduced molecule. Advantageously, the targeted
9

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M. Kratký et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116209
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CRediT authorship contribution statement
´
Martin Kratký: Conceptualization, Investigation, Writing - original
18 Basanagouda M, Jambagi VB, Barigidad NN, Laxmeshwar SS, Devaru V.
Narayanachar Synthesis, structure-activity relationship of iodinated-4-
aryloxymethyl-coumarins as potential anti-cancer and anti-mycobacterial agents. Eur
J Med Chem. 2014;74:225–233.
´
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draft, Methodology, Supervision. Klara Konecna: Investigation,
´
Writing - original draft. Michaela Brablíkova: Investigation, Supervi-
ˇ
ˇ
´
´
sion. Jirí Janousek: Investigation. Vaclav Pflegr: Investigation. Jana
19 McDonnell G, Russell AD. Antiseptics and disinfectants: Activity, action, and
resistance. Clin Microbiol Rev. 1999;12:147–179.
´
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Maixnerova: Investigation. Frantisek Trejtnar: Methodology, Writing
20 Sano M, Makabe R, Kurosawa N, Moteki M, Odate T. Effects of Lugol’s iodine on
long-term preservation of marine plankton samples for molecular and stable carbon
and nitrogen isotope analyses. Limnol Oceanogr Methods. 2020;18:635–643.
21 Wilcken R, Liu X, Zimmermann MO, et al. Halogen-enriched fragment libraries as
leads for drug rescue of mutant p53. J Am Chem Soc. 2012;134:6810–6818.
22 Ihssen J, Schubert M, Thony-Meyer L, Richter M. Laccase Catalyzed Synthesis of
Iodinated Phenolic Compounds with Antifungal Activity. PLoS ONE. 2014;9, e89924.
23 Al-Zoubi RM, Al-Zoubi MS, Jaradat KT, McDonald R. Design, Synthesis and X-ray
Crystal Structure of Iodinated Benzoboroxole Derivatives by Consecutive Metal-
Iodine Exchange of 3,4,5-Triiodoanisole. Eur J Org Chem. 2017;2017:5800–5808.
24 Popiolek L, Patrejko P, Gawronska-Grzywacz M, et al. Synthesis and in vitro
bioactivity study of new hydrazide-hydrazones of 5-bromo-2-iodobenzoic acid.
Biomed Pharmacother. 2020;130, 110526.
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- review & editing, Supervision. Jarmila Vinsova: Writing - review &
editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
´
´
ˇ
Acknowledgements
25 Kratký M, Dzurkova M, Janousek J, et al. Sulfadiazine Salicylaldehyde-Based Schiff
Bases: Synthesis, Antimicrobial Activity and Cytotoxicity. Molecules. 2017;22:1573.
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2020;10:9.
This work was supported by the Czech Science Foundation [grant
number 20-19638Y]; the EFSA-CDN [grant No. CZ.02.1.01/0.0/0.0/
16_019/0000841] co-funded by ERDF; and the Charles University [SVV
260 547].
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