Synthesis and biological evolution of hydrazones derived from 4-(trifluoromethyl)benzohydrazide

Article abstract of DOI:10.1016/j.bmcl.2017.10.050

Reflecting the known biological activity of isoniazid-based hydrazones, seventeen hydrazones of 4-(trifluoromethyl)benzohydrazide as their bioisosters were synthesized from various benzaldehydes and aliphatic ketones. The compounds were screened for their in vitro activity against Mycobacterium tuberculosis, nontuberculous mycobacteria (M. avium, M. kansasii), bacterial and fungal strains. The most antimicrobial potent derivatives were also investigated for their cytostatic and cytotoxic properties against three cell lines. Camphor-based molecule, 4-(trifluoromethyl)-N′-(1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide, exhibited the highest and selective inhibition of M. tuberculosis with the minimum inhibitory concentration (MIC) of 4 μM, while N′-(4-chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide was found to be superior against M. kansasii (MIC = 16 μM). N′-(5-Chloro-2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide showed the lowest MIC values for gram-positive bacteria including methicillin-resistant Staphylococcus aureus as well as against two fungal strains of Candida glabrata and Trichophyton mentagrophytes within the range of ≤0.49–3.9 μM. The convenient substitution of benzylidene moiety at the position 4 or the presence of 5-chloro-2-hydroxybenzylidene scaffold concomitantly with a sufficient lipophilicity are essential for the noticeable antimicrobial activity. This 5-chlorosalicylidene derivative avoided any cytotoxicity on two mammalian cell cultures (HepG2, BMMΦ) up to the concentration of 100 μM, but it affected the growth of MonoMac6 cells.

Full text of DOI:10.1016/j.bmcl.2017.10.050

Accepted Manuscript
Synthesis and biological evolution of hydrazones derived from 4-(trifluoro-
methyl)benzohydrazide
Martin Krátký, Szilvia Bősze, Zsuzsa Baranyai, Jiřina Stolař íková, Jarmila
Vinšová
PII:
S0960-894X(17)31044-2
https://doi.org/10.1016/j.bmcl.2017.10.050
BMCL 25379
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
30 August 2017
17 October 2017
20 October 2017
Please cite this article as: Krátký, M., Bősze, S., Baranyai, Z., Stolař íková, J., Vinšová, J., Synthesis and biological
evolution of hydrazones derived from 4-(trifluoromethyl)benzohydrazide, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.050
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Synthesis and biological evolution of hydrazones derived from 4-
(trifluoromethyl)benzohydrazide
Martin Krátký a,*, Szilvia Bősze b, Zsuzsa Baranyai b, Jiřina Stolaříková c, and Jarmila Vinšová a
a
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové,
Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd University, Pázmány Péter
b
Sétány 1/A, Budapest, H-1117, P.O. Box 32, 1518 Budapest 112, Hungary
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health
c
in Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic
* Corresponding author. Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
E-mail address: martin.kratky@faf.cuni.cz, tel.: +420-495067343, fax: +420-495067166.
Abstract
Reflecting the known biological activity of isoniazid-based hydrazones, seventeen hydrazones of 4-
(trifluoromethyl)benzohydrazide as their bioisosters were synthesized from various benzaldehydes
and aliphatic ketones. The compounds were screened for their in vitro activity against
Mycobacterium tuberculosis, nontuberculous mycobacteria (M. avium, M. kansasii), bacterial and
fungal strains. The most antimicrobial potent derivatives were also investigated for their cytostatic
and cytotoxic properties against three cell lines. Camphor-based molecule, 4-(trifluoromethyl)-N'-
(1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide, exhibited the highest and selective
inhibition of M. tuberculosis with the minimum inhibitory concentration (MIC) of 4 µM, while N'-
(4-chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide was found to be superior against M.
kansasii (MIC = 16 µM). N'-(5-Chloro-2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide
showed the lowest MIC values for gram-positive bacteria including methicillin-resistant
Staphylococcus aureus as well as against two fungal strains of Candida glabrata and Trichophyton
mentagrophytes within the range of ≤0.49-3.9 µM. The convenient substitution of benzylidene
moiety at the position 4 or the presence of 5-chloro-2-hydroxybenzylidene scaffold concomitantly
with a sufficient lipophilicity are essential for the noticeable antimicrobial activity. This 5-
chlorosalicylidene derivative avoided any cytotoxicity on two mammalian cell cultures (HepG2,
BMMΦ) up to the concentration of 100 µM, but it affected the growth of MonoMac6 cells.
Keywords
Antibacterial activity; antifungal activity; cytostasis; cytotoxicity; hydrazone; Mycobacterium
tuberculosis;
nontuberculous
mycobacteria;
4-(trifluoromethyl)benzohydrazide*
*Abbreviations: BAC: bacitracin; BMMΦ: murine bone marrow culture-derived macrophages; FLU: fluconazole; INH: isoniazid; MRSA:
methicillin-resistant Staphylococcus aureus; Mtb.: Mycobacterium tuberculosis; NTM: non-tuberculous (atypical) mycobacteria.

Development of novel antimicrobial agents represents an up-to-date research topic to achieve a
global control of drug-resistant microbial strains. Modification of compounds with a known activity
to improve their properties belongs to the powerful trends in medicinal chemistry.
The concept of (bio)isosters could be considered as one of the successful approaches for the rational
drug design. It is based on the assumption that single atoms, groups or molecules that exhibit
similar volume, shape, and/or physicochemical properties can produce broadly similar
pharmacologic effects. However, the outcomes of isosteric replacement cannot be predicted
absolutely, since conversion of the action was also observed occasionally. In the molecules of
bioisosters, a different substituent or a group exchanges one moiety from the parent lead compound
to impart similar biological effects. This change can modify both pharmacokinetics and
pharmacodynamics to fine tune of them. Examples of broadly similar isosteric substitution
comprise, e.g., replacement of hydrogen by fluorine, ester bond by amide, phenyl ring by thiophene,
carboxylic acid by tetrazole etc., that are based on chemical and/or physical similarities.1,2
Hydrazones obtained from isoniazid (INH), a first-line antimycobacterial agent, have been reported
as potent antimycobacterial,3,4,5,6,7,8 antibacterial5,7,9 and antifungal5,7,9 agents. An analogue of INH
where heterocyclic nitrogen is replaced by a carbon substituted with a strong electron-withdrawing
group, 4-(trifluoromethyl)benzohydrazide 1 inhibits the growth of Mycobacterium tuberculosis10
(Mtb.). Similarly,
4-(trifluoromethyl)benzohydrazide-derived
INH isosters
preserved
antimycobacterial properties.11,12 Illustratively, hydrazones of 1 with various aldehydes and ketones
have exhibited antimycobacterial,10,11 antibacterial,13,14 antifungal,14 antiparasitic15 and cytotoxic16,17
properties.
Based on here summarized facts, we designed and synthesized 4-(trifluoromethyl)benzohydrazide
hydrazones 2, bioisosters of INH hydrazones (Scheme 1), as potential antimicrobial agents.
Scheme 1. Bioisosteric design of 4-(trifluoromethyl)benzohydrazide hydrazones 2
The synthesis of N'-alkyl/cycloalkyl/benzylidene-4-(trifluoromethyl)benzohydrazides 2 is depicted
in Scheme 2. It involves the reaction of the hydrazide 1 (1.0 equivalent) with commercially
available aldehydes and ketones (1.1 of eq.). If any ketone (acetone, cyclopentanone,
cyclohexanone, camphor) was one of the reactants, a catalytic amount of concentrated sulphuric
acid was added into the reaction mixture. The refluxing in boiling methanol for 2 hours provided
targeted compounds 2 in 85-99% yields (aldehydes) and 68-87% for ketones (Scheme 2). The
lowest yield was observed when camphor was used as a carbonyl compound.

Scheme 2. Synthesis of N´-substituted 4-(trifluoromethyl)benzohydrazides 2.
We chose substituents with different electronic effects as a substitution pattern for benzaldehyde
ring: no substituent (R = H), those with electron-donating properties – methyl (+I effect), hydroxy
and methoxy groups (-I and +M effects), as well as strong electron-withdrawing groups of NO2 (-I
and -M effects) and CF3 (-I effect). Halogens (Cl, Br) as weak deactivating substituents with -I and
+M electronic effects were involved too. Based on the known antimicrobial activity of 5-chloro-2-
hydroxybenzylidene derivatives,18,19 5-chlorosalicylaldehyde was used. We also investigated
various positional isomers (ortho, meta, para; R = Cl, OH) and the activity of hydrazones obtained
from four ketones. In spite of different electronic effects, these derivatives differ also in lipophilic
behaviour and steric parameters.
1
13
All of the compounds 2 were characterized by spectroscopic data involving H, C and IR spectra
(see Supplementary information), and the purity was checked additionally by TLC and elemental
1
analysis. In the H NMR spectra, hydrazone (CONHN=) protons appeared as singlets at 12.31-
11.83 ppm for benzylidene scaffold-based compounds 2a-m concomitantly with azomethine
singlets (N=CH) observed at 8.88-8.36 ppm. Ketone-derived hydrazones 2n-q share CONH proton
signals shifted to upfield within the range of 10.84-10.41 ppm. The 13C NMR spectra contain C=O
and C=N peaks at 162.43-161.83 ppm and 149.14-144.65 ppm, respectively. In the spectra of
ketone-based hydrazones 2n-q, the C=N singlet is shifted to 174.92-161.53 ppm.
The hydrazide 1 and all of the hydrazones 2a-q were screened in vitro for their antimicrobial
properties (see Supplementary information). The panel of pathogens involved Mycobacterium
tuberculosis 331/88 (i.e., H37Rv), Mycobacterium avium 330/88 (resistant to INH, rifamycines,
ethambutol and ofloxacin), Mycobacterium kansasii 235/80 and 6509/96 (a clinical isolate; Table
1); gram-positive bacteria: Staphylococcus aureus CCM 4516/08, methicillin-resistant
Staphylococcus aureus H 5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus
faecalis J 14365/08; Escherichia coli CCM 4517, Klebsiella pneumoniae D 11750/08, extended
spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae J 14368/08, and Pseudomonas
aeruginosa CCM 1961 (gram-negative strains),20 and fungal species of Candida albicans ATCC
44859, Candida tropicalis 156, Candida krusei E28, Candida glabrata 20/I, Trichosporon asahii
1188, Aspergillus fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445
(Table 2).21 This panel of twenty microbial species covers a wide range of important human
pathogens including those with an acquired resistance. It is a useful tool for an initial identification
of potential antimicrobial activity of novel compounds. Bacitracin (BAC), fluconazole (FLU) and
isoniazid were employed as the comparative drugs for antibacterial, antifungal and
antimycobacterial activity, respectively.
The results from antimycobacterial evaluation expressed as minimum inhibitory concentrations
(MICs) are overviewed in Table 1; only active molecules are involved.
Table 1. Antimycobacterial activity of hydrazones 2
MIC [µM]
M. avium
R or X
Mtb. 331/88
M. kansasii 235/80
M. kansasii 6509/96
ClogP
Code
2b
330/88
14 d 21 d
125 125
14 d
21 d
7 d
16
14 d
16
21 d
16
7 d
14 d
21 d
4-Cl
4.84
16
16
16
16
16

3-Cl
4-OH
3-OH
125
62.5
62.5
250
62.5
32
125
500
500
4.84
3.9
3.9
2c
2e
2f
2g
2h
2m
2n
2o
2p
125
125
250
125
62.5
125
125
500
250
125 125 125
125
125
125
250
125
62.5
125
1000
1000
125
125
250
250
125
62.5
125
1000
1000
125
250
250
250
62.5
250
125
500
500
125
500
500
250
125
250
125
1000
1000
125
500
500
250
125
250
125
1000
1000
125
250
250
62.5
>250
250
250
125
62.5
500
250
250
125
2-OH
3.9
2-OH-5-Cl
4-NO2
4.45
3.49
2.71
3.52
3.93
250 250
propan-2-ylidene
cyclopentylidene
cyclohexylidene
1,7,7-trimethyl-
bicyclo[2.2.1]hept
an-2-ylidene
BH (1)
125 125 125
500
500
125 125
125 125
5.42
2q
4
4
250 250 250
250
250
250
250
250
250
0.5
500
1
1.61
-
500 500 250
250 250 250
250
250
250
250
250
8
250
8
250
8
INH
INH: isoniazid; BH: 4-(trifluoromethyl)benzohydrazide 1; Mtb.: Mycobacterium tuberculosis. The lipophilicity (ClogP)
was computed using the program CS ChemOffice Ultra version 16.0.
One or two of the best MIC value(s) for each strain are shown in bold.
Eleven hydrazones (2b-c, 2e-h, 2m-q) exhibited an antimycobacterial activity, remaining
derivatives (2a, 2d, 2i-l) were inactive. Hydrazide 1 itself showed only a mild activity against Mtb.
(250/500 µM). M. avium inhibited only by phenolic derivatives was the most resistant strain (MICs
≥ 62.5 µM). The lowest MIC values for Mtb. were produced by terpenic hydrazone obtained from
camphor 2q (4 µM concomitantly with an evident selectivity for this strain) followed by 4-
chlorobenzylidene derivative 2a. This molecule was also found to be the most active against both
strains of M. kansasii (16 µM). N'-(4-Nitrobenzylidene)-4-(trifluoromethyl)benzohydrazide 2m
inhibited significantly only the growth of the collection strain of M. kansasii 235/80 with MIC
values starting from 32 µM.
In sum, for the antimycobacterial activity it is essential the presence of one chlorine atom at the
position of 4 (2b) since its structural isomers are virtually (2c) or totally inactive (2d). The phenolic
group represents another favourable substituent preferably at the position 4 again (2e), but in this
case isomeric hydroxybenzaldehydes provided derivatives (2e-g) with a similar biological response.
The halogenation of salicylaldehyde improves antimycobacterial properties up to four times (2g vs.
2h). Generally, an increased lipophilicity is translated into an enhanced antimycobacterial action
(2q, 2b vs. 2e and 2m, 2g vs. 2h). On the other hand, the most lipophilic benzaldehyde-based
hydrazones (i.e., 4-Br 2k and 4-CF3 2l with C logP >5) did not display any growth inhibition.
Although essential, the lipophilicity is not the only factor determining this property and similarly,
the occupation of the 4-position does not ensure bioactivity automatically.
The chemical modification of the parent hydrazide 1 by carbonyl compounds led mainly to more
potent antimycobacterial agents. Four derivatives (2e-h) exceeded the in vitro activity of INH for
M. avium and M. kansasii 235/80, additional four hydrazones (2b-c, 2m-n) produced lower MIC
value(s)
against
the
last
strain
demonstrably.
N'-(4-Chlorobenzylidene)-4-
(trifluoromethyl)benzohydrazide 2b was comparable to INH against the clinical isolate of M.
kansasii 6509/96 (± one dilution). Our study did not confirm previous finding about the activity of
camphor-based hydrazones against NTM.22
The results of antibacterial and antifungal evaluation are reported in Table 2. Unfortunately, two
compounds (2k, 2q) are not sufficiently soluble in the media for both antibacterial and antifungal
testing (Mueller-Hinton broth, RPMI-1640), thus making the MIC values determination unreliable.
The majority of the hydrazones 2 as well as the parent hydrazide 1 avoided any growth inhibition of
eight bacterial strains investigated. Klebsiella pneumoniae and Pseudomonas aeruginosa showed a
complete resistance, E. coli was the only one gram-negative species susceptible to 2
(salicylaldehyde-based hydrazones 2g-h, 250-500 µM). The MIC values of 5-chloro-2-

hydroxybenzylidene derivative 2h against four gram-positive strains (≤3.9 µM) qualify this
molecule for further investigation. Importantly, the MIC values of 2h were uniformly clearly
superior to BAC, an established antibiotic drug used for the topical treatment of gram-positive
infection. The hydrazone 2b obtained from 4-chlorobenzaldehyde exhibited MICs starting from
31.25 µM that was comparable to BAC in the case of Staphylococcus epidermidis. Interestingly,
there is no cross-resistance to methicillin as could be observed from the comparison of identical
MIC values against a drug-susceptible Staphylococcus aureus and a methicillin-resistant (MRSA)
strain. The antibacterial activity of remaining compounds reported in the Table 2 (2e, 2g, and 2n) is
rather detrimental (≥125 µM). The structure-activity relationships are similar to those obtained for
the action against mycobacteria: the presence of chlorine atom (2b) or hydroxyl group (2e vs. 2g) at
the position 4 of the benzylidene moiety as well as chlorination of salicylic ring (2g vs. 2h)
modulate antibacterial properties positively.
Three positive hits from antibacterial screening, 2b, 2g, and 2h, produced also an inhibition of four
fungal strains: Candida tropicalis, Candida krusei, Candida glabrata and a filamentous fungus,
Trichophyton mentagrophytes. Four species showed a complete resistance to all of the hydrazones 2
(Candida albicans, Trichosporon asahii, Aspergillus fumigatus, Absidia corymbifera). N'-(4-
Chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide 2b exhibited a mild antifungal activity with
MICs within the range of 62.5-125 µM. Salicylidene derivatives 2g-h were able to affect C.
glabrata and T. mentagrophytes at the concentration of ≤0.49-15.62 µM with the superiority of the
halogenated counterpart 2h again (up to 16 times). Almost all of these values are comparatively
lower than those obtained for fluconazole, a clinically used antimycotic drug. Especially the
inhibition of C. glabrata at low micromolar or submicromolar concentrations is worth. This
pathogen is one of the most common causative agents of invasive candidoses with a high mortality
rate of 40-50 % concomitantly with an increasing incidence and drug-resistance.23
4-(Trifluoromethyl)benzohydrazide 1 and its hydrazones 2 were also screened for their in vitro
cytotoxic and cytostatic properties on two human cell cultures: hepatocellular carcinoma cells
(HepG2)24 and on monocytic cell line MonoMac625 using MTT-assay. The in vitro cytotoxic
activity of a representative compound set was also determined on murine bone marrow culture-
derived macrophages (BMMΦ)26,27 using MTT assay.
Importantly, none of the derivatives 1-2 exhibited cytotoxicity (i.e., direct killing of cells) for
HepG2 cells up to the concentration of 100 µM and majority of them were non-cytostatic. The most
antimicrobial active hydrazones (2b, 2g-h, 2q) were subjected to additional tests on MonoMac6 and
BMMΦ cells (Table 3). The HepG2 cells represent an in vitro model for the hepatotoxicity, the
MonoMac6 cells and BMMΦ are models of the macrophages.28 The selectivity indexes (SI) were
defined as a ratio of the MIC and IC50 value for cytotoxic action on HepG2 cells. Its values higher
than 10 indicate an acceptable toxicity (based on the analogy of the therapeutic index).
The natural pharmacophore-based hydrazone 2q exhibited neither cytotoxicity nor cytostasis for all
three cell lines at a single concentration of 100 µM, thus targeting Mtb. selectively (SI>25) without
any undesired effects on mammalian cells. 4-Chlorobenzylidene derivative 2b did not affected both
HepG2 and MonoMac6 at 100 µM, but it was cytotoxic on the BMMΦ cells (IC50 = 7.05 µM).
These cells can be also considered as a sensitive macrophage model from normal primary cell
culture, therefore a suitable choice to estimate selectivity of promising compounds.26,27,28,29
Regarding hepatotoxicity, it is the most promising agent against NTM (SI>6.25 µM). In contrast to
these two compounds, salicylaldehyde derivatives 2g-h share a strong cytostatic behaviour (IC50
within the range of 1.70-13.55 µM), i.e., they arrest cell growth, multiplication and proliferation.
Simultaneously, they were cytotoxic on MonoMac6 model (IC50 values for 2g and 2h of 2.28 and
5.8 µM, respectively), but not for HepG2 and BMMΦ (IC50 >100 µM). Interestingly, the

concomitant cytostatic action together with no cytotoxicity was described for salicylanilide
derivatives.30

Table 2. Antibacterial and antifungal activity of hydrazone derivatives 2
MIC/IC95/IC50 [µM]
SA
MRSA
SE
EF
EC
CT
CK
CG
TM
120 h
125
Code
24 h
62.5
250
500
1.98
48 h
62.5
250
>500
1.98
24 h
48 h
62.5
250
24 h
31.25
125
250
3.9
48 h
62.5
125
500
3.9
24 h
62.5
48 h
24 h
48 h
24 h
125
48 h
125
24 h
62.5
48 h
62.5
24 h
48 h
72 h
125
62.5
250
500
1.98
>125 >125 >125
>125 >125
2b
2e
2g
2h
2n
BH (1)
BAC
FLU
>250 >250 >250 >250
>125
>125
>125
>500
>500
-
>125
>125
>125
>500
>500
-
>125 >125 >125 >125 >125 >125
>125 >125
>125 >125
>500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500
>500
1.98
250
1.98
250
250
3.9
500
500
250
500
250
3.9
15.62
7.81
1.98
15.62
3.9
≤0.49
0.98
>500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500
7.81 15.62 15.62 15.62 15.62 31.25 15.62
500
>500
>500 >500
62.5
-
>500 >500
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
>500
>500
125
250
31.25
500
7.81
125
BAC: bacitracin; BH: 4-(trifluoromethyl)benzohydrazide 1; FLU: fluconazole. Bacteria: SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus
aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; EF: Enterococcus sp. J 14365/08; Escherichia coli CCM 4517. Fungi: CT: Candida tropicalis; CK: Candida krusei
E28; CG: Candida glabrata 20/I; TM: Trichophyton mentagrophytes 445.
One or two of the best MIC value(s) for each strain are shown in bold.

In contrast to antimicrobial activity, the halogenation of salicylidene core decreases both
cytotoxicity and cytostasis (2g vs. 2h) thus being beneficial in term of the selectivity. N'-(5-Chloro-
2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide 2h was identified as the most potent and
selective molecule against gram-positive cocci and several fungal strains (SI on HepG2 of >50.5
and >25.64, respectively).
Table 3. Cytostatic and cytotoxic properties of the most potent hydrazones 2
Cytotoxicity Cytostasis
Cytotoxicity
(MonoMac6)
IC50 [µM]
>100
2.28±0.32
5.8±0.10
>100
Cytostasis
(MonoMac6)
IC50 [µM]
>100
1.70±0.20
10.3±2.40
>100
Cytotoxicity
(BMMΦ)
IC50 [µM]
7.05±0.15
>100
SI for
myco-
bacteria
>6.25
NA
SI for S.
aureus
SI for
fungib
(HepG2)
IC50 [µM]
>100
(HepG2)
IC50 [µM]
>100
Code
>1.6
NA
>50.5
NA
NA
>6.40
>25.64
NA
2b
2g
2h
2q
>100
>100
>100
6.20±1.0
13.55±0.75
>100
>100
>100
>1.6
>25a
SI = IC50 (cytotoxicity for HepG2)/MIC. a = only for Mtb. b = for C. glabrata and T. mentagrophytes. NA = not
applicable.
In conclusion, seventeen hydrazones were synthesized by the treatment of 4-
(trifluoromethyl)benzohydrazide with corresponding substituted benzaldehydes or ketones. These
novel compounds were characterised and they underwent biological evaluation in order to
determine their antibacterial, antifungal, antimycobacterial, cytotoxic and cytostatic action. Four
molecules exhibited an interesting and potentially promising activity profile as potential
antimicrobial agents especially against Mycobacterium tuberculosis, staphylococci including one
MRSA strain, Candida glabrata and Trichophyton mentagrophytes with an acceptable toxicity and
thus selectivity for eukaryotic cells. They represent auspicious hits for further structure
optimization.
Acknowledgments
This work was supported by the Czech Science Foundation [grant number 17-27514Y]; and the
Hungarian Research Fund [grant number OTKA K-104275].
We would like to thank Ida Dufková for the excellent performance of antibiotic and antimycotic
susceptibility tests, the staff of the Department of Organic and Bioorganic Chemistry, Faculty of
Pharmacy in Hradec Králové for the technical assistance and J. Urbanová, M.A., for improving the
grammar of the manuscript. The authors also thank Dr. Rita Szabó for participating in the isolation
and maintenance of murine bone marrow culture-derived cells.
Conflicts of interest: none.
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Highlights
•Hydrazones of 4-(trifluoromethyl)benzohydrazide, an isoniazid isoster, were obtained.
•These hydrazones were investigated as potential antimicrobial agents.
•Hydrazone of camphor is selective inhibitor of Mycobacterium tuberculosis (MIC=4 µM).
•A high activity against gram-positive bacteria including MRSA strain (MIC ≥1.98 µM).
•Antifungal activity from ≤0.49 µM and no cytotoxicity for HepG2 cells.