molecules

Article
Antimicrobial Activity of Some Steroidal Hydrazones
Maia Merlani 1 , Nanuli Nadaraia 1 , Lela Amiranashvili 1 , Anthi Petrou 2 , Athina Geronikaki 2, * , Ana Ciric 3 ,
Jasmina Glamoclija 3 , Tamara Carevic 3 and Marina Sokovic 3

1 TSMU I. Kutateladze Institute of Pharmacochemistry, Tbilisi 0159, Georgia

2 School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
3 Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Siniša Stankovi’c”,
University of Belgrade, 11060 Beograd, Serbia
* Correspondence: geronik@pharm.auth.gr; Tel.: +30-230-1997616

Abstract: Twelve steroid based hydrazones were in silico evaluated using computer program PASS
as antimicrobial agents. The experimental evaluation revealed that all compounds have low to
moderate antibacterial activity against all bacteria tested, except for B. cereus with MIC at a range
of 0.37–3.00 mg/mL and MBC at 0.75–6.00 mg/mL. The most potent appeared to be compound 11
with MIC/MBC of 0.75/1.5 mg/mL, respectively. The evaluation of antibacterial activity against
three resistant strains MRSA, E. coli and P. aeruginosa demonstrated superior activity of compounds
against MRSA compared with ampicillin, which did not show bacteriostatic or bactericidal activities.
All compounds exhibited good antifungal activity with MIC of 0.37–1.50 mg/mL and MFC of
1.50–3.00 mg/mL, but with different sensitivity against fungi tested. According to docking studies,
14-alpha demethylase inhibition may be responsible for antifungal activity. Two compounds were
evaluated for their antibiofilm activity. Finally, drug-likeness and docking prediction were performed.

Keywords: hydrazones; ketosteroids; antimicrobial activity

Citation: Merlani, M.; Nadaraia, N.; 1. Introduction

Amiranashvili, L.; Petrou, A.;
Geronikaki, A.; Ciric, A.; Glamoclija,
The development of new antimicrobial agents is still attracting the interest of medicinal
J.; Carevic, T.; Sokovic, M.
chemists since the resistance of bacterial pathogen strains is a major problem.
Antimicrobial Activity of Some
One of the reasons for the fast multiplication of bacteria is their ability to exchange
Steroidal Hydrazones. Molecules 2023, genes with each other, leading to the development of resistance. On the other hand, the
28, 1167. https://doi.org/10.3390/ interest in the discovery of new antimicrobial agents is because during the past 30+ years,
molecules28031167 the FDA has approved only two new antimicrobial drugs: linezolid and daptomycin.
Despite the fact that many compounds have been synthesized and tested, their clinical
Academic Editors: Danuta
use has been restricted due to the high risk of toxicity and pharmacokinetic deficiencies.
Drozdowska and Robert Bucki
Thus, the scientists have directed their efforts at developing novel approaches to antimi-
Received: 28 December 2022 crobial therapy, aiming to overcome the resistance problem [1–4]. Another big problem
Revised: 15 January 2023 is biofilm formation, which plays a crucial role in bacterial infection and antimicrobial
Accepted: 17 January 2023 resistance. There is increasing proof that cells in biofilms, on a biotic or abiotic surface, are
Published: 24 January 2023 1000-fold more resistant to conventional drugs than planktonic cells [5,6]. The problem is
that upon being established, biofilms become difficult to eliminate and as a result, chronic
and persistent infections [7] appear. As reported in the literature [8,9], one of the main
Gram-positive pathogens causing biofilm-associated infections is Staphylococcus aureus.
Copyright: © 2023 by the authors.
Thus, another need is for the development of new agents that are able to inhibit S. aureus
Licensee MDPI, Basel, Switzerland.
biofilm formation.
This article is an open access article
Hydrazones of different chemical classes possess diverse biological and pharmaco-
distributed under the terms and
conditions of the Creative Commons
logical properties such as antimicrobial, anti-inflammatory, analgesic, antifungal, anti-
Attribution (CC BY) license (https://
tubercular, antiviral, anticancer, antiplatelet, antimalarial, anticonvulsant, cardio protective,
creativecommons.org/licenses/by/
anthelmintic, antiprotozoal, anti-trypanosomal, anti-schistosomiasis etc. [10–12]. Hydra-
4.0/). zones contain two connected nitrogen atoms of different nature and a C-N double bond

Molecules 2023, 28, 1167. https://doi.org/10.3390/molecules28031167 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 1167 2 of 16

that is conjugated with a lone electron pair of the terminal nitrogen atom. These structural
fragments are mainly responsible for the physical and chemical properties of hydrazones.
The combination of thehydrazono group with other functional groups leads to compounds
with a unique physical and chemical character [13]. It is noteworthy that there is an ap-
proved FDA drug with a hydrazone scaffold, namely levosimendan, a calcium sensitizer
used in the management of acutely decompensated congestive heart failure (Figure 1).

Figure 1. Approved by FDA drug.

On the other hand, steroidal compounds are a class of bioactive substances play-
ing a major role in living organisms with a wide representation in the natural world.
Steroidal derivatives attracted the interests of scientists, especially medicinal chemists, due
to their wide range of biological activities [10,13–15]. They are known to possess antimi-
crobial [16,17], antioxidant [17] and anticancer [17] activities. In the last few decades, the
efforts have concentrated on rational modification of steroid molecules due to their lower
toxicity, vulnerability to multi-drug resistance and high bioavailability to penetrate the cell
wall and to be linked to nuclear and membrane receptors. Vollaro et al. [18] reported the
investigation of the in vitro effect of pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1
(PYED-1) on biofilm formation.
Nowadays, a number of steroidal hydrazone derivatives have been developed and
evaluated for their antimicrobial activity [19–24]. Among these hydrazones are some
5α-steroidal derivatives of the androstane and pregnane series with different functional
groups.
Encouraged by these observations, and based on our previous work [25–27], herein
we report the synthesis of two novel 5α-steroidal hydrazones and the evaluation of antimi-
crobial activity of newly and earlier synthesized compounds.
Thus, the purpose of our study was in silico and biological evaluation of the antimi-
crobial potential of twelve steroidal hydrazino derivatives, including action on the resistant
strains.

2. Results and Discussion

2.1. Chemistry
In the continuation of our research on new bioactive N-containing 5α-steroids, ten
steroidal hydrazine derivatives, that we synthesized earlier on the basis of steroidal ke-
tones [25–29], and two new compounds were prepared and evaluated for their antibacterial
and antifungal actions.
Isonicotinoylhydrazones 1–4 and 7 were synthesized from corresponding ketones
androsta-1,4-diene-3,17-dione, and rosterone, epiandrosterone, allopregnanolone, 3α-hydroxy-
5α-androst-9(11)-en-17-one, by refluxing with hydrazide of isonicotinic acid in ethanol,
respectively [25]. Thiosemicarbazones 5, 6 and 8 were obtained from allopregnanolone, 3α-
hydroxy-5α-androst-9(11)-en-17-oneandandrosta-1,4-diene-3,17-dione by refluxing with
thiosemicarbazide in ethanol, respectively [25]. m-Bromobenzoylhydrazones 10 [26] and 12
were synthesized from acetate epiandrosterone and androsterone, respectively, by refluxing
with m-bromobenzohydrazide. m-Nitro hydrazones 9 [26] and 11 [29] were synthesized
Molecules 2023, 28, 1167 3 of 16

similarly by refluxing the corresponding ketone with m-nitrobenzohydrazide. The synthesis

of all these compounds is presented in Scheme 1.

Scheme 1. Synthetic route of substances (1–12): (a) isoniazide, EtOH, CH3 COOH, reflux 2 h;
(b) thiosemicarbazide, EtOH, CH3 COOH, 2 h; (c) m-bromobenzohydrazide, EtOH, CH3 COOH;
(d) m-nitrobenzohydrazide, EtOH, CH3 COOH, reflux, 5 h; (e) TsCl, pyridine, 0 ◦ C, 20 h; (f) NaN3 ,
DMF, 100 ◦ C, 5 h; (g) NaOH, MeOH, reflux 30 min; (h) ClCOCH2 C6 H5 , pyridine, benzene, reflux 6 h.
Molecules 2023, 28, 1167 4 of 16

2.2. PASS Predictions

PASS prediction of antimicrobial activities was performed for previously synthesized
compounds (1, 3–11), as well as for two new designed ones. The antibacterial activity was
predicted only for two compounds with Pa values in the range 0.164–0.313, and antifungal
activity for almost all compounds with Pa values in the range 0.143–0.470. The calculated Pa
values for all compounds were less than 0.5, indicating their relative novelty compared to
the structures of the compounds from the PASS training set [30]. This may be proof that the
studied compounds have some features dissimilar from those of well-known antimicrobial
agents, which may indicate their innovative potential.

2.3. Biological Evaluation

2.3.1. Antibacterial Activity
Synthesized compounds were tested for their antibacterial activity against a panel of
nine bacteria species, using the microdilution method for the determination of minimal
inhibitory and minimal bactericidal concentrations (MIC and MBC, respectively). As
reference drugs, ampicillin and streptomycin were used. The antibacterial activity of tested
compounds (Table 1) in general was low to moderate, except in some cases where it was
good, with MIC ranging from 0.37 to 3.00 mg/mL and MBC at 0.75–9.00 mg/mL, presented
in Table 1. The order of activity can be presented as follows: 11 = 12 > 4 = 5 > 3 > 1 > 6 > 7 >
10 > 8 > 9 > 2. Compound 11 appeared to be the most potent among those tested, with MIC
and MBC of 0.75/1.5 mg/mL, respectively, but less than for both reference drugs. The most
sensitive bacterium was found to be B. cereus, whereas S. aureus was the most resistant one.

Table 1. Antibacterial activity of compounds 1–12 (MIC/MBC in mg/mL).

Compounds S.a. MRSA B.c L.m. E. coli Rez E. coli P.a. Rez P.a. S.Thy
MIC 1.50 1.50 0.37 0.75 1.00 4.50 1.50 1.00 0.75
1
MBC 3.00 3.00 0.75 1.50 3.00 6.00 3.00 1.50 1.50
MIC 3.00 4.50 0.75 3.00 3.00 6.00 6.00 6.00 6.00
2
MBC 6.00 6.00 1.50 6.00 6.00 9.00 9.00 9.00 9.00
MIC 1.00 0.75 0.37 1.00 1.00 1.50 1.50 0.75 1.00
3
MBC 1.50 1.50 0.75 1.50 1.50 3.00 3.00 1.50 1.50
MIC 0.75 3.00 0.37 0.75 1.50 1.50 0.75 0.75 0.75
4
MBC 1.50 6.00 0.75 1.50 3.00 3.00 1.50 1.50 1.50
MIC 0.75 1.50 0.37 0.75 1.50 1.50 0.75 1.00 0.75
5
MBC 1.50 3.00 0.75 1.50 3.00 3.00 1.50 1.50 1.50
MIC 0.75 1.50 0.37 1.50 1.50 0.75 3.00 1.50 1.50
6
MBC 1.50 3.00 0.75 3.00 3.00 1.50 6.00 3.00 3.00
MIC 0.75 1.50 0.50 1.50 1.50 0.75 3.00 1.50 1.50
7
MBC 1.50 3.00 0.75 3.00 3.00 1.50 6.00 3.00 3.00
MIC 1.50 1.50 0.37 1.50 1.50 1.50 3.00 1.50 1.50
8
MBC 3.00 3.00 0.75 3.00 3.00 3.00 6.00 3.00 3.00
MIC 1.50 3.00 1.50 0.75 3.00 6.00 1.50 3.00 3.00
9
MBC 3.00 6.00 3.00 1.50 6.00 9.00 3.00 6.00 6.00
MIC 1.50 3.00 1.50 0.75 3.00 6.00 1.50 1.50 0.75
10
MBC 3.00 6.00 3.00 1.50 6.00 9.00 3.00 3.00 1.50
MIC 0.75 1.50 0.75 0.75 0.75 1.50 0.75 0.75 0.75
11
MBC 1.50 3.00 1.50 1.50 1.50 3.00 1.50 1.50 1.50
MIC 0.75 1.50 0.75 0.75 0.75 1.50 0.75 0.75 0.75
12
MBC 1.50 3.00 1.50 1.50 1.50 3.00 1.50 1.50 1.50
MIC 0.10 - 0.10 0.15 0.15 0.20 0.30 0.20 0.10
Ampicillin
MBC 0.15 - 0.15 0.30 0.20 - 0.50 - 0.20
MIC 0.10 0.10 0.025 0.15 0.10 0.05 0.10 0.10 0.10
Streptomycin
MBC 0.20 - 0.050 0.30 0.20 0.10 0.20 0.20 0.20
Molecules 2023, 28, 1167 5 of 16

The structure–activity relationship studies revealed that the presence of 3-nitrobenzohy

drazide at position 17 of 3α-hydroxy-5α-androstan-17-one 11 is beneficial for antibacterial
activity. The replacement of the nitro group in the benzene ring by Br led to compound 12
havingthe same good influence as the previous one, on activity. The replacement of the
substituted benzene ring by isonicotinoylhydrazide, and 3α-hydroxy-5α-androstan-17-one
by 3α-hydroxy-5α-pregnan-20-one, resulted in compound 4 with slightly lower activity. It
is interesting to notice that the presence of thethiosemicarbazide substituent at position 20
(5) of the steroid ring, in place of isonicotinoylhydrazide (4) as the substituent in position
20, exhibited the same activity as compound 4. Replacement of hydroxy group in position
3 by phenylacetoxy (3) decreased the antibacterial activity more, while replacement by
theazide group (2) was detrimental.
The evaluation of antibacterial activity of these compounds against three resistant
strains, MRSA, E. coli and P. aeruginosa revealed that compounds were more potent against
MRSA than ampicillin, which did not show bacteriostatic or bactericidal activity, while
against the two other resistant strains, it did not show bactericidal activity. The order of
activity of the tested compounds against resistant strains can be presented as 6 = 7 > 1 > 3 >
4 > 11 = 12 > 5 > 8 > 10 > 9 > 2, with compounds 6 and 7 being the most potent (MIC/MBC
at 0.75–1.50 mg/mL and 1.50–3.00 mg/mL, respectively). It should be mentioned that
compounds 3–5, 8–12 did not show any activity against the resistant E. coli strain. It is
interesting to notice that compounds 6 and 7 were more potent against resistant E. coli than
E. coli strains, while the opposite was observed for compounds 1 and 2. On the other hand,
compounds 1,3,6,7 and 8 exhibited better activity against P. aeruginosa and against resistant
P. aeruginosa, while compounds 2,4,10–12 demonstrated the same activity against both of
these strains. In general, our compounds showed better activity against the resistant P.
aeruginosa strain than against two other resistant strains, being less potent than the reference
drug streptomycin.
In the case of structure–activity relationship studies against resistant strains, it was
found that the presence of athiosemicarbazide substituent (6) as well as anisonicotinoylhy-
drazide one (7) in position 17 of 3α-hydroxy-5α-androst-9(11)-en-17-one was favorable for
the activity against resistant strains.
Finally, it should be mentioned that compounds tested have different behavior against
ATCC and resistant strains. The only common behavior against both strains was observed
for compound 2, which demonstrated a negative effect on antibacterial activity in both cases.

2.3.2. Antifungal Activity

Compounds were also tested for their antifungal activity against six fungal strains
using the microdilution method, and ketoconazole as well as bifonazole were used as
reference drugs. The results are presented in Table 2. In general, compounds showed
moderate to good activity with MIC and MFC in the range of 0.37–3.00 mg/mL and
0.50–6.00 mg/mL, respectively. The order of activity of tested compounds can be presented
as follows: 7 = 8 > 3 > 1 = 9 > 12 > 11 > 6 > 10 > 5 > 2 > 4. The best activity was achieved
for 3α-hydroxy-5α-androst-9(11)-en-17-one isonicotinoylhydrazone (7), with MIC/MFC of
0.37/0/75 mg/mL, respectively, as well as for compound 8. The lowest antifungal effect
was observed for compound 4, with an MIC ranging from 0.75 to 3.00 mg/mL and MFC
from 1.5 to 6.0 mg/mL. It should be noticed that compounds 7 and 8 showed the best
potential against all fungi tested (MIC at 0.37 mg/mL), while compound 3 demonstrated
the same effect against all fungi except for C. albicans. On the other hand, compounds 9 and
12 also showed the same good activity with previous ones against A. fumigatus, T. viride, C.
albicans and A. fumigatus, A. niger, T. viride, respectively. Ketoconazole exhibited antifungal
potential at MIC in the range 0.2–1.0 mg/mL and MFC of 0.3–2.0 mg/mL, while bifonazole
at MIC 0.10–0.20 mg/mL and MFC at 0.2–0.3 mg/mL, respectively. All compounds showed
higher activity than ketoconazole (MIC/MFC of 1.0/1.5 mg/mL) against T. viride, the most
sensitive fungal. However, it is more important that almost all compounds, except for
11 and 12, were more potent than ketoconazole against C. albicans—the most resistant to
Molecules 2023, 28, 1167 6 of 16

our compounds and the deathliest fungal, responsible together with filaments fungal A.
fumigatus for 85–90% of deaths.

Table 2. Antifungal activity of compounds 1–12 (MIC/MBC in mg/mL).

Compounds A.fu A.n. T.v. P.f. P.v.c. C.a.

MIC 0.75 0.75 0.37 0.75 0.37 0.37
1
MFC 1.50 1.50 0.75 1.50 0.75 0.75
MIC 1.50 1.50 1.50 1.50 3.00 0.37
2
MFC 3.00 3.00 3.00 3.00 6.00 0.75
MIC 0.37 0.37 0.37 0.37 0.37 0.75
3
MFC 0.75 0.75 0.75 0.75 0.75 1.50
MIC 1.50 3.00 3.00 3.00 3.00 0.75
4
MFC 3.00 6.00 6.00 6.00 6.00 1.50
MIC 1.50 1.50 1.50 1.50 0.75 0.75
5
MFC 3.00 3.00 3.00 3.00 1.50 1.50
MIC 1.50 1.50 1.50 0.75 0.75 0.75
6
MFC 3.00 3.00 3.00 1.50 1.50 1.50
MIC 0.37 0.37 0.37 0.37 0.37 0.37
7
MFC 0.75 0.75 0.75 0.75 0.75 0.75
MIC 0.37 0.37 0.37 0.37 0.37 0.37
8
MFC 0.75 0.75 0.75 0.75 0.75 0.75
MIC 0.37 0.75 0.37 0.75 0.75 0.37
9
MFC 0.75 1.50 0.75 1.50 1.50 0.75
MIC 0.37 1.50 0.75 1.50 3.00 0.37
10
MFC 0.50 3.00 1.50 3.00 6.00 0.75
MIC 0.75 1.50 0.75 0.75 1.50 1.50
11
MFC 1.50 3.00 1.50 1.50 3.00 3.00
MIC 0.37 0.37 0.37 0.75 1.50 1.50
12
MFC 0.50 0.50 0.75 1.50 3.00 3.00
MIC 0.20 0.20 1.00 0.20 0.20 1.00
Ketoconazole
MFC 0.50 0.50 1.50 0.50 0.30 2.00
MIC 0.15 0.15 0.15 0.20 0.10 0.20
Bifonazole
MFC 0.20 0.20 0.20 0.25 0.20 0.30

According to the structure–activity relationship studies, the presence of isonicotinoyl-

hydrazide (7) in position 17 of 3α-hydroxy-5α-androst-9(11)-en-17-one core and dithiosemi-
carbazide of androst-1,4-dien-3,17-dione moiety (8) have a positive influence on antifungal
activity. Replacement of 3α-hydroxy-5α-androst-9(11)-en-17-one core by 3β-phenylacetoxy-
5α-androstan-17-one and introduction to position 17 isonicotinoylhydrazide substituent
led to compound 3 havingdecreased activity, which decreased more by introduction of
a 3β-azido-5α-androstan-17-one moiety (2) instead of 3β-phenylacetoxy-5α-androstan-
17-one (3). The same influence on antifungal activity resulted from the presence of m-
nitrobenzoylhydrazide in 3β-acetoxy-5α-androstan-17-one core. The replacement of the
3α-hydroxy-5α-androst-9(11)-en-17-one core of compound 7 by 3α-hydroxy-5α-pregnan-
20-one (4) and thiosemicarbazide substituent at position 20 by isonicotinoylhydrazide (4)
weredetrimental for antifungal activity. In general, 3α-hydroxy-5α-pregnan-20-one isoni-
cotynoylhydrazone 4 and thiosemicarbazone 5 as well the 3β-azido-5α-androstan-17-one
isonicotinoylhydrazone 2 were not favorable for antifungal activity.

2.3.3. Inhibition of Biofilm Formation

After the observation of the antifungal activities of compounds, antibiofilm activities
were assessed. We observed that compounds 1 and 8 possessed higher antifungal activity
against C. albicans and all tested micro fungi than other used compounds. The strain used
for the antibiofilm assay was C. albicans. Incubation with compounds 1 and 8 hasreduced
the ability of C. albicans (Figures 2 and 3) to attach to the surface and begin the process of
biofilm formation. A concentration equal to the previously determined MIC has reduced
Molecules 2023, 28, 1167 7 of 16

the biofilm biomass by 33% and 15% for compounds 1 and 8, respectively. When applied in
0.5 and 0.25 MIC concentrations of compound 1, inhibition percentages were almost the
same, about 18% (Figure 3). The reference drug, Ketoconazole, possessed better biofilm
activity than the compounds, reducing the biofilm biomass by 50%, 47% and 25% for MIC
concentrations 0.5 MIC and 0.25 MIC, respectively (Figure 2).

Figure 2. Percentages of inhibition of C. albicans ATCC 10231 biofilm formation by compound 1 and
Ketoconazole.

Figure 3. Percentages of inhibition of C. albicans ATCC 10231 biofilm formation by compound 8 and
Ketoconazole.

Even twice as low concentrations (0.5 MIC) of compound 8 limited the biofilm forming
ability and induced more than 16% inhibition in C. albicans. The impact on the fungal
biofilm was less profound and the 0.25 MIC concentration of 8 was able to reduce the
biofilm formation byless than 5% (Figure 3).

2.4. Docking to Antifungal Targets

In order to investigate the possible mechanism of antifungal activity of compounds,
all of them along with the reference drug ketoconazole were docked to lanosterol 14α-
demethylase of C. albicans and DNA topoisomerase IV. The results are presented in Table 3.
Molecules 2023, 28, 1167 8 of 16

Table 3. Molecular docking free binding energies (kcal/mol) to antifungal targets.

Est. Binding Energy

Residues Residues Residues
(kcal/mol)
Involved in Involved in Involved in Interactions
DNA CYP51 of C. H-bond Hydrophobic Aromatic with HEM601
Comp. TopoIV albicans Formation Interactions Interactions
1S16 5V5Z
Tyr118, Thr122,
Hydrophobic,
1 −4.11 −8.52 Tyr132 Thr311, Phe380, Hem601
aromatic
Met508, Hem601
Tyr118, Thr311,
2 −3.31 −6.38 - - Hydrophobic
Leu376, Hem601
Tyr118, Thr122,
Phe228, Phe233,
Hydrophobic,
3 −3.58 −9.04 Met508 Thr311, Leu376, Hem601
aromatic
Phe380, Met508,
Hem601
Tyr118, Leu376,
4 −1.77 −6.01 - - Hydrophobic
Met508, Hem601
Tyr118, Tyr122,
5 −2.47 −6.80 - Leu376, Met508, Tyr118 Hydrophobic
Hem601
Tyr118, Tyr122,
6 −4.20 −7.32 - Thr311, Leu37, Tyr118 Hydrophobic
Hem601
Tyr118, Leu121,
Hydrophobic,
7 −3.18 −9.86 Tyr132 Thr311, Met508, Hem601
aromatic
Hem601
Tyr118, Thr122,
Ile131, Tyr132, Hydrophobic,
8 −2.54 −10.23 Tyr64 -
Leu376, Met508, Fe binding
Hem601
Tyr118, Leu121,
Hydrophobic,
9 −3.61 −8.48 Tyr132 Thr122, Thr311, Hem601
aromatic
Met508, Hem601
Tyr118, Thr311,
10 −1.38 −7.11 - Tyr118 Hydrophobic
Met508, Hem601
Tyr118, Leu121,
11 −2.47 −7.55 Tyr118 - Hydrophobic
Met508, Hem601
Tyr118, Tyr122,
Hydrophobic,
12 −1.28 −8.02 Tyr118 Thr311, Met508, Hem601
aromatic
Hem601
Tyr118, Ile131,
Tyr132, Leu300, Hydrophobic,
ketoconazole - −8.93 Tyr64 Hem601
Ile304, Leu376, aromatic
Met508, Hem601

Based on docking studies, all compounds bind to the CYP51 Ca enzyme similarly to
the reference drug ketoconazole (Figure 4). The most active compound 8 binds to the Fe
of the heme and interacts hydrophobically and aromatically with the heme. Additionally,
compound 8 forms a hydrogen bond between the oxygen atom of the C=O group and
the side-chain hydrogen of Tyr64. Hydrophobic interactions were also detected between
residues I Tyr118, Thr122, Ile131, Tyr132, Leu376, Met508 and the compound (Figure 5).
Ketoconazole also forms aromatic and hydrophobic interactions with the heme group. It
has been shown, however, that compound 8 forms a more stable complex with the enzyme,
possibly due to its interaction with heme’s iron. It is likely that this is the reason why this
compound has a better antifungal effect than ketoconazole.
Molecules 2023, 28, 1167 9 of 16

Figure 4. Docked conformation of ketoconazole in lanosterol 14α-demethylase of C. albicans

(CYP51ca ).

Figure 5. (A) Superposition of compound 8 (magenta) and ketoconazole (blue) in lanosterol 14α-
demethylase of C. albicans (CYP51ca ). (B) Docked conformation of the most active compound 8 in
lanosterol 14α-demethylase of C. albicans (CYP51ca ). Red dotted arrows indicate H-bond, blue arrows
aromatic interactions and yellow spheres hydrophobic interactions.

The superposition of compound 3 and ketoconazole (Figure 6) explains its good

antifungal activity. Similar to ketoconazole, compound 3 inserts the binding site of the
enzyme, forming an additional hydrogen bond with residue Met508. In addition, it exhibits
the same hydrophobic and aromatic interactions with the heme group as ketoconazole,
which explains its good inhibition profile.

2.5. Drug-Likeness
All tested compounds were evaluated for their drug-likeness and bioavailability score-
sand the results are presented in Table 4. According to the prediction, the bioavailability
score for most of the compounds was about 0.55, except for compounds 3, 9, 10 and 12
with 0.17 values. Despite these compounds exhibiting two violations of Lipinski’s rule of
Molecules 2023, 28, 1167 10 of 16

five, they have excellent drug-likeness scores ranging from 0.74 to 1.54. Thus, it can be
concluded that they have good oral bioavailability and drug-likeness profile (Figure 7).

Figure 6. (A) Superposition of compound 3 (yellow) and ketoconazole (blue) in lanosterol 14α-
demethylase of C. albicans (CYP51ca ). (B) Docked conformation of compound 3 in lanosterol 14α-
demethylase of C. albicans (CYP51ca ). Red dotted arrows indicate H-bond, blue arrows aromatic
interactions and yellow spheres hydrophobic interactions.

Table 4. Drug-likeness predictions of tested compounds.

Drug-
Number Number Log Po/w d e Bioavailability Likeness
No MW Log S TPSA Lipinski
of HBA a of HBD b (WLOGP) c Score Model
Score
Moderately
1 522.64 6 2 5.09 108.70 0 0.55 1.09
soluble
Moderately
2 448.60 6 1 6.28 104.10 0 0.55 1.30
soluble
2
violations:
Moderately MW >
3 541.72 5 1 6.75 80.65 0.17 1.54
soluble 500,
MLOGP >
4.15
Moderately
4 423.59 4 2 3.75 74.58 0 0.55 1.48
soluble
Moderately
5 405.64 2 3 4.61 102.73 0 0.55 0.71
soluble
Moderately
6 375.57 3 2 3.44 102.73 0 0.55 0.74
soluble
Moderately
7 407.55 4 2 4.49 74.78 0 0.55 1.29
soluble
8 430.66 2 4 3.10 Soluble 165.00 0 0.55 0.78
Molecules 2023, 28, 1167 11 of 16

Table 4. Cont.

Drug-
Number Number Log Po/w Bioavailability Likeness
No MW Log S d TPSA e Lipinski
of HBA a of HBD b (WLOGP) c Score Model
Score
2
violations:
Poorly MW >
9 509.64 6 1 6.05 113.58 0.17 0.98
soluble 500,
MLOGP >
4.15
2
violations:
Poorly MW >
10 543.54 4 1 6.90 67.76 0.17 1.18
soluble 500,
MLOGP >
4.15
Moderately
11 467.60 5 2 5.47 107.51 0 0.55 0.85
soluble
2
violations:
Moderately MW >
12 501.50 3 2 6.33 61.69 0.17 1.07
soluble 500,
MLOGP >
4.15
a number of hydrogen bond acceptors; b number of hydrogen bond donors; c lipophilicity; d Water solubility

(SILICOS-IT [S = Soluble]); e topological polar surface area (Å2 ).

Figure 7. Drug-likeness model and bioavailability radar of the compounds 3 and 4. The pink area
represents the optimal range for each property for oral bioavailability, (Lipophilicity (LIPO): XLOGP3
between −0.7 and +5.0, Molecular weight (SIZE): MW between 150 and 500 g/mol, Polarity (POLAR)
TPSA between 20 and 130 Å2 , Solubility (INSOLU): log S not higher than 6, Saturation (INSATU):
fraction of carbons in the sp3 hybridization not less than 0.25 and Flexibility (FLEX): no more than
9 rotatable bonds.
Molecules 2023, 28, 1167 12 of 16

3. Materials and Methods

3.1. Chemistry—General Information
All commercially available reagents, isonicotinic acid hydrazide, m-bromobenzoic
acid hydrazide, m-nitrobenzoic acid hydrazide and androsta-1,4-diene-3,17-dione were of
analytical grade and used without further purification (all from Sigma Aldrich, Schnelldorf,
Germany).
1 H NMR and 13 C NMR spectra were recorded using DMSO-d or CDCl as a solvent
6 3
at 22 ◦ C with a Bruker AC 400 instrument. IR spectra were recorded using a JASCO
FT/IR-4600 spectrometer. Mass spectra were obtained on an HPLC-APCIMS (positive
mode)-Agilent 1100 Series with an Inertsil PREP-ODS column (6.0 × 250 mm) and elution
of steroids by H2 O–MeCN (20:80). The melting points were recorded on a Gallenkamp
apparatus and are uncorrected. The IR, 1 HNMR and 13 C NMR spectra can be found in
Supplementary Materials.
3β-Phenylacetoxy-5α-androstan-17-one isonicotinoylhydrazone (3)
A mixture of 3β-phenylacetoxy-5α-androstan-17-one (1 g, 2.44 mmol), isonicotinic
acid hydrazide (0.39 g, 2.92 mmol) and acetic acid (1 mL) in ethanol (20 mL) was boiled for
2 h and cooled to room temperature. The resulting precipitate was filtered off, washed with
water and crystallized from ethanol. Yield 75%, m.p. 180–182 ◦ C. IR(KBr): 3169, 1729, 1639,
1598, 1548, 1494, 1452, 1132, 1010, 928, 841 cm−1 . 1 H NMR (400 MHz, CDCl3 , δ, ppm, J/Hz):
δ, 0.89 and 1.00 (6H, s, 18-CH3 , 19-CH3 ), 3.62 (2H, s, CH2 C6 H5 ), 4.75 (1H, m, H-3), 7.28–7.37
(5H, m, C6 H5 ), 7.65–7.80 (2H, H-Pr), 8.50 (1H, s, NHCO), 8.75–8.79 (2H, dd, J = 5.8, J = 2.2,
H-Pr). 13 C NMR spectrum (100 MHz, CDCl3 , δ, ppm): 12.3, 16.9, 20.7, 23.5, 25.3, 26.2, 27, 4,
28.3, 31.4, 33.9, 34.9, 35.7, 36.7, 41.8, 44.8, 44.9, 45.5, 53.4, 54.5, 74.0, 121.0, 124.0, 126.9, 128.5,
129.2, 134.4, 140.7, 141.1, 149.6, 150.7, 167.6, 168.1, 171.2, 174.0.
3α-Hydroxy-5α-androstan-17-one m-bromobenzoylhydrazone (12)
A mixture of 3α-hydroxy-5α-androstan-17-one (0.05 g, 0.14 mmol), m-bromobenzoic
acid hydrazide (0.04 g, 0.2 mmol) and 0.1 mL acetic acid was refluxed in ethanol for 5 h.
The resulting precipitate was filtered off, washed with water and crystallized from ethanol.
Yield 80%, m.p. 266–268 ◦ C. IR(KBr): 3465, 3360, 1675, 1643, 1609, 1565, 1518, 1363, 1259,
1003, 930, 890, 606 cm−1 . 1 H NMR (400 MHz, DMSO-d6 , δ, ppm, J/Hz): δ, 0.78 (3H, s,
18-CH3 ), 0.86 (3H, s, 19-CH3 ), 2.36–2.60 (2H, m, H-16), 4.47 (1H, s, H-3), 7.44 (1H, m, H-Ar),
7.75 (2H, m, H-Ar), 7.95 (1H, s, H-Ar), 10.34 (1H, s, NHCO). 13 C NMR spectrum (100 MHz,
DMSO-d6 , δ, ppm J/Hz): 12.0, 16.7, 20.2, 22.7, 26.7, 28.1, 30.8, 31.2, 34.5, 35.0, 36.4, 37.9, 44.3,
44.5, 52.7, 53.9, 69.1, 121.4, 126.6, 130.2, 133.8, 136.4, 150.4, 161.8, 175.1.

3.2. In Vitro Evaluation of Antimicrobial Activity

The following Gram-negative bacteria: Escherichia coli (ATCC 35210), Salmonella Ty-
phimurium (ATCC 13311), Pseudomonas aeruginosa (ATCC 27853) as well as Gram-positive
bacteria: Listeria monocytogenes (NCTC 7973), Bacillus cereus (clinical isolate) and Staphylo-
coccus aureus (ATCC 6538) were used. Resistant strains used were Methicillin resistant S.
aureus (IBRS MRSA 011), resistant E. coli (IBRS E003) and resistant P. aeruginosa (IBRS P001)
obtained as described in the previous paper [31]. The organisms were obtained from the
Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research
“Siniša Stankovic”, National Institute of Republic of Serbia, Belgrade, Serbia.
The antifungal activity of all investigated samples was tested on strains obtained
from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological
Research “Sinisa Stankovic”, National Institute of Republic of Serbia, Belgrade, Serbia.
The following fungi Aspergillus fumigatus (ATCC 1022), Aspergillus niger (ATCC 6275),
Trichoderma viride (IAM 5061), Penicillium funiculosum (ATCC 36839), P. verrucosum var.
cyclopium (food isolates) and Candida albicans (ATCC 10231) were tested. The detailed
explanation is given in our previous papers [32,33].
Commercial antibiotics, ampicillin and streptomycin, and fungicides, bifonazole and
ketoconazole, were used as positive controls. EtOH 30% was used as a negative control.
All experiments were performed in duplicate and repeated three times.
Molecules 2023, 28, 1167 13 of 16

The minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC)

concentrations were determined by the modified microdilution method, as previously
reported [34,35].

Inhibition of Biofilm Formation

The potential of compounds to inhibit biofilm formation was investigated as previously
described, with some modifications [36] C. albicans ATCC 10231 was incubated in 96-well
microtiter plates with an adhesive bottom (Sarstedt, Germany), with MIC and sub-MIC
concentrations of tested compounds/referent drug in YPD medium at 37 ◦ C for 24 h.
Afterwards, wells were washed thrice with sterile PBS (Phosphate buffered saline, pH 7.4)
and biofilms were fixed with methanol for 20 min. Then, methanol was removed and
stained with 0.1% crystal violet (Bio-Merieux, France) for 30 min. The plate was slowly
washed, air dried and 96% ethanol (Zorka, Serbia) was added to dissolve bounded crystal
violet. The absorbance (620 nm) was read on a Multiskan™ FC Microplate Photometer,
Thermo Scientific™. Lastly, the percentage of inhibition of biofilm formation was calculated
by the formula:

Percentage of inhibition = ((A620(control) − A620sample )/A620control )) × 100

3.3. Docking
AutoDock 4.2® software was used for the in silico studies and a detailed procedure is
reported in our previous paper [37].

3.4. Drug-Likeness
Drug-likeness [38] scores of compounds were predicted using the Molsoft software
and SwissADME program (http://swissadme.ch, accessed on 19 October 2022) via the
ChemAxon’s Marvin JS structure drawing tool [39].

4. Conclusions
This work presents the synthesis of two new steroid derivatives and the study of
antibacterial and antifungal activities together with previously synthesized compounds
against a panel of bacterial and fungal pathogens of twelve steroid derivatives, two of
which are new. The antibacterial activity of tested compounds was low to moderate with
minimal inhibitory concentration being 0.37–1.5 mg/mL and minimal bactericidal being
1.5–3.0 mg/mL, except against B. cereus which was good. The antibacterial activity against
resistant strains MRSA, E. coli and P. aeruginosa was superior against MRSA than ampicillin,
which did not show bacteriostatic or bactericidal activity, while against the two other strains,
it did not show bactericidal activity. All compounds exhibited moderate to good antifungal
potency with MIC and MFC in the range of 0.37–3.00 mg/mL and 0.50–6.00 mg/mL,
respectively. Compound 7 demonstrated the best activity among all tested with MIC/MFC
of 0.37/0/75 mg/mL, respectively. The most sensitive fungal to compounds tested was
T. viride, while C. albicans was the most resistant one. Despite this, almost all compounds
except for 11 and 12 were more potent than ketoconazole against C. albicans, the deathiest
fungal. Antibiofilm activity assessed for the two most potent compounds 1 and 8 in
concentrations of MIC, 0.5 MIC and 0.25 MIC revealed that it was lower (33 and 15%,
respectively, in concentration of MIC) than that of ketoconazole. According to docking
results, it seems that the inhibition of CYP51 reductase is responsible for the antifungal
activity of the compounds. All compounds showed good drug-likeness scoresin the range
of 0.71–1.54. Three compounds showed two violations to the Lipinski rule.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28031167/s1; Figure S1: 13 C NMR spectrum of com-
pound 1 in DMSO-d6; Figure S2: 1 H NMR spectrum of 3β-azido-5α-androstan-17-one isonicotinoyl-
hydrazone (compound 2) in CDCl3 ; Figure S3: 13 C NMR spectrum of 3β-azido-5α-androstan-17-one
isonicotinoylhydrazone (compound 2) in CDCl3 ; Figure S4: IR spectrum of 3β -phenylacetoxy-5α-
Molecules 2023, 28, 1167 14 of 16

androstan-17-one isonicotinoylhydrazone (compound 3) in KBr; Figure S5: 1 H NMR spectrum of

3β-phenylacetoxy-5α-androstan-17-one isonicotinoylhydrazone (compound 3) in CDCl3 ; Figure S6:
13 C NMR spectrum of 3β-phenylacetoxy-5α-androstan-17-one isonicotinoylhydrazone (compound

3) in CDCl3 ; Figure S7: MS spectrum of 3β -phenylacetoxy-5α-androstan-17-one isonicotinoylhy-

drazone (compound 3); Figure S8: 13 C NMR spectrum of compound 4 in d6 -DMSO; Figure S9: 1 H
NMR spectrum of 3α-hydroxy-5α-pregnan-20-one thiosemicarbazone (compound 5) in d6 -DMSO;
Figure S10: 13 C NMR spectrum of 3α-hydroxy-5α-pregnan-20-one thiosemicarbazone (compound
5) in d6 -DMSO; Figure S11: 13 C NMR spectrum of compound 6 in d6 -DMSO; Figure S12: 1 H NMR
spectrum of 3α-hydroxy-5α-androst-9(11)-en-17-one isonicotinoylhydrazone (compound 7) in d6 -
DMSO; Figure S13: 13 C NMR spectrum of 3α-hydroxy-5α-androst-9(11)-en-17-one isonicotinoylhy-
drazone (compound 7) in d6 -DMSO; Figure S14: 13 C NMR spectrum of androsta-1,4-diene-3,17-dione
dithiosemicarbazone (compound 8) in d6 -DMSO; Figure S15: 13 C NMR spectrum of 3β-acetoxy-5α-
androstan-17-one m-nitrobenzoylhydrazone (compound 9) in d6 -DMSO; Figure S16: 13 C NMR spec-
trum of 3β -acetoxy-5α-androstan-17-one m-bromobenzoylhydrazone (compound 10) in d6 -DMSO;
Figure S17: 1 H NMR spectrum of 3α -hydroxy-5α-androstan-17-one m-nitrobenzoylhydrazone
(compound 11) in d6 -DMSO; Figure S18: 13 C NMR spectrum of 3α -hydroxy-5α-androstan-17-one
m-nitrobenzoylhydrazone (compound 11) in d6 -DMSO; Figure S19: IR spectrum of 3α-hydroxy-5α-
androstan-17-one m-bromobenzoylhydrazone (compound 12) in KBr; Figure S20: 1 H NMR spec-
trum of 3α-hydroxy-5α-androstan-17-one m-bromobenzoylhydrazone (compound 12) in d6 -DMSO;
Figure S21: 13 C NMR spectrum of 3α-hydroxy-5α-androstan-17-one m-bromobenzoylhydrazone
(compound 12) in d6 -DMSO; Figure S22: Fragment of 13 C NMR spectrum 3α-hydroxy-5α-androstan-
17-one m-bromobenzoylhydrazone (compound 12) in d6 -DMSO; Figure S23: MS spectrum of 3α-
hydroxy-5α-androstan-17-one m-bromobenzoylhydrazone (compound 12).
Author Contributions: Conceptualization, A.G., M.M.; methodology, L.A., N.N.; software, A.P.;
investigation, M.M., A.C., J.G., T.C.; data curation, A.G., A.C.; writing—original draft preparation,
A.G., M.M.; writing—review and editing, A.G., A.C.; visualization, A.P.; supervision, A.G., M.M.
funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.
Funding: The authors thank the Serbian Ministry of Education, Science and Technological Develop-
ment, for the financial support (grant number 451-03-68/2022-14/200007).
Institutional Review Board Statement: IR, 1 HNMR, 13 C NMR, spectra.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.

References
1. Coates, A.R.M.; Hu, Y. Novel approaches to developing new antibiotics for bacterial infections. Br. J. Pharmacol. 2007, 152,
1147–1154. [CrossRef] [PubMed]
2. Spellberg, B.; Bartlett, J.; Wunderink, R.; Gilbert, D.M. Novel approaches are needed to develop tomorrow’s antibacterial therapies.
Am. J. Respir. Crit. Care Med. 2015, 191, 135–140. [CrossRef] [PubMed]
3. Cataldo, M.A.; Granata, G.; Petrosillo, N. Clostridium difficile infection: New approaches to prevention, non-antimicrobial
treatment, and stewardship. Expert Rev. Anti Infect. Ther. 2017, 15, 1027–1040. [CrossRef] [PubMed]
4. Hughes, G.; Webber, M.A. Novel approaches to the treatment of bacterial biofilm infections. Br. J. Pharmacol. 2017, 174, 2237–2246.
[CrossRef]
5. Parsek, M.R.; Singh, P.K. Bacterial biofilms: An emerging link to disease pathogenesis. Annu. Rev. Microbiol. 2003, 57, 677–701.
[CrossRef]
6. Verderosa, A.D.; Totsika, M.; Fairfull-Smith, K.E. Bacterial Biofilm Eradication Agents: A Current Review. Front. Chem. 2019, 28,
824–841. [CrossRef] [PubMed]
7. Bjarnsholt, T. The role of bacterial biofilms in chronic infections. APMIS Suppl. 2013, 136, 1–51. [CrossRef] [PubMed]
8. Tong, S.Y.; Davis, J.S.; Eichenberger, E.; Holland, T.L.; Fowler, V.G.J. Staphylococcus aureus infections: Epidemiology, pathophysi-
ology, clinical manifestations, and management. Clin. Microbiol. Rev. 2015, 28, 603–661. [CrossRef] [PubMed]
9. Otto, M. Staphylococcal infections: Mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity.
Annu. Rev. Med. 2013, 64, 175–188. [CrossRef]
Molecules 2023, 28, 1167 15 of 16

10. Verma, G.; Marella, A.; Shaquiquzzaman, M.; Akhtar, M.; Ali, M.R.; Alam, M.M. A review exploring biological activities of
hydrazones. J. Pharm. Bioallied Sci. 2014, 6, 69–80.
11. Rollas, S.; Küçükgüzel, Ş.G. Biological Activities of Hydrazone Derivatives. Molecules 2007, 12, 1910–1939. [CrossRef] [PubMed]
12. Belskaya, N.P.; Dehaen, W.; Bakulev, V.A. Synthesis and properties of hydrazones bearing amide, thioamide and amidine
functions. Arch. Org. Chem. 2010, 1, 275–332. [CrossRef]
13. Tantawy, M.A.; Nafie, M.S.; Elmegeed, G.A.; Ali, I.A. Auspicious role of the steroidal heterocyclic derivatives as a platform for
anti-cancer drugs. Bioorg. Chem. 2017, 73, 128–146. [CrossRef] [PubMed]
14. Ericson-Neilsen, W.; Kaye, A.D. Steroids: Pharmacology, complications, and practice delivery issues. Ochsner J. 2014, 14, 203–207.
15. Krstić, N.M.; Bjelaković, M.S.; Pavlović, V.D.; Robeyns, K.; Juranić, Z.D.; Matić, I.; Novakovic, I.; Sladić, D.M. New androst-4-en-
17-spiro-1, 3, 2-oxathiaphospholanes. Synthesis, assignment of absolute configuration and in vitro cytotoxic and antimicrobial
activities. Steroids 2012, 77, 558–565. [CrossRef]
16. Hryniewicka, A.; Malinowska, M.; Hauschild, T.; Pieczul, K.; Morzycki, J.W. Synthesis and antimicrobial properties of steroid-
based imidazolium salts. J. Steroid. Biochem. Mol. Biol. 2019, 189, 65–72. [CrossRef]
17. Farhan, A.M.; Alshamusi, Q.K.; Jebur, M.H. Synthesis of steroid bearing heterocyclic derivatives and biological activity. Review
2014–2020. J. Phys. Conf. Ser. 2021, 1853, 01205.
18. Vollaro, A.; Esposito, A.; Esposito, E.P.; Zarrilli, R.; Guaragna, A.; De Gregorio, E. PYED-1 Inhibits Biofilm Formation and Disrupts
the Preformed Biofilm of Staphylococcus aureus. Antibiotics 2020, 9, 240. [CrossRef]
19. Mistry, S.; Singh, A.K. Synthesis and in vitro antimicrobial activity of new steroidal hydrazone derivatives. FJPS 2022, 8, 7.
[CrossRef]
20. Popiołek, Ł. Hydrazide–hydrazones as potential antimicrobial agents: Overview of the literature since 2010. Med. Chem. Res.
2016, 26, 287–301. [CrossRef]
21. Visbal, G.; San-Blas, G.; Maldonado, A.; Alvarez-Aular, A.; Capparelli, M.V.; Murgich, J. Synthesis, in vitro antifungal activity and
mechanism of action of four sterol hydrazone analogues against the dimorphic fungus Paracoccidioides brasiliensis. Steroids
2011, 76, 1069–1081. [CrossRef] [PubMed]
22. Gan, C.; Cui, J.; Su, S.; Lin, Q.; Jia, L.; Fan, L.; Huang, Y. Synthesis and antiproliferative activity of some steroidal thiosemicar-
bazones, semicarbazones and hydrozones. Steroids 2014, 87, 99–107. [CrossRef] [PubMed]
23. Khan, S.A.; Asiri, A.M. Synthesis and spectroscopic studies of Ru(II) complexes of steroidal thiosemicarbazones by multi step
reaction: As anti-bacterial agents. Steroids 2017, 124, 23–28. [CrossRef] [PubMed]
24. Loncle, C.; Brunel, J.M.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis and antifungal activity of cholesterol-hydrazone
derivatives. Eur. J. Med. Chem. 2004, 39, 1067–1071. [CrossRef] [PubMed]
25. Merlani, M.I.; Amiranashvili, L.S.; Mulkidzhanyan, K.G.; Shelar, A.R.; Manvi, F.V. Synthesis and antituberculosis activity of
certain steroidal derivatives of the 5α –series. Chem. Nat. Compd. 2008, 44, 618–620. [CrossRef]
26. Merlani, M.I.; Amiranashvili, L.S.; Kemertelidze, E.O.; Mulkidzhanyan, K.G. Synthesis and antimicobacterial activity of some
steroidal derivatives of tigogenin. Chem. Nat. Compd. 2009, 45, 389–392. [CrossRef]
27. Shelar, A.R.; Merlani, M.; Shelar, M.; Amiranashvili, L.; Shelar, B. Medicinal Applications of Benzoic Acid Hydrazones Synthesized
on the Basis of Steroidal Tigogenin. U.S. PATENT 8623849B2, 7 January 2014.
28. Nadaraia, N.S.; Amiranashvili, L.S.; Merlani, M.; Kakhabrishvili, M.I.; Barbakadze, N.N.; Geronikaki, A.; Petrou, A.; Poroikov,
V.; Ciric, A.; Glamoclija, J.; et al. Novel antimicrobial agents’ discovery among the steroid derivatives. Steroids 2019, 144, 52–65.
[CrossRef]
29. Amiranashvili, L.; Nadaraia, N.; Merlani, M.; Kamoutsis, C.; Petrou, A.; Geronikaki, A.; Pogodin, P.; Druzhilovskiy, D.; Poroikov,
V.; Ciric, A.; et al. Antimicrobial Activity of Nitrogen-Containing 5-α-Androstane Derivatives: In Silico and Experimental Studies.
Antibiotics 2020, 9, 224. [CrossRef]
30. Lagunin, A.; Stepanchikova, A.; Filimonov, D.; Poroikov, V. PASS: Prediction of activity spectra for biologically active substances.
Bioinformatics 2000, 16, 747–748. [CrossRef]
31. Kartsev, V.; Lichitsky, B.; Geronikaki, A.; Petrou, A.; Smiljkovic, M.; Kostic, M.; Radanovic, O.; Soković, M. Design, synthesis and
antimicrobial activity of usnic acid derivatives. MedChemComm 2018, 9, 870–882. [CrossRef]
32. Haroun, M.; Tratrat, C.; Kositsi, K.; Tsolaki, E.; Petrou, A.; Aldhubiab, B.; Attimarad, M.; Harsha, S.; Geronikaki, A.; Venugopala,
K.N.; et al. New Benzothiazole-based Thiazolidinones as Potent Antimicrobial Agents. Design, synthesis and Biological
Evaluation. Curr. Top. Med. Chem. 2018, 18, 75–87. [CrossRef] [PubMed]
33. Kritsi, E.; Matsoukas, M.T.; Potamitis, C.; Detsi, A.; Ivanov, M.; Sokovic, M.; Zoumpoulakis, P. Novel Hit Compounds as Putative
Antifungals: The Case of Aspergillus fumigatus. Molecules 2019, 24, 3853. [CrossRef] [PubMed]
34. Clinical and Laboratory Standards Institute—CLSI. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow
Aerobically. Approv. Stand. 2009, 29, 1–65.
35. Horishny, V.; Kartsev, V.; Matiychuk, V.; Geronikaki, A.; Anthi, P.; Pogodin, P.; Poroikov, V.; Ivanov, M.; Kostic, M.; Soković, M.D.;
et al. 3-Amino-5-(indol-3-yl)methylene-4-oxo-2-thioxothiazolidine Derivatives as Antimicrobial Agents: Synthesis, Computational
and Biological Evaluation. Pharmaceuticals 2020, 13, 229. [CrossRef] [PubMed]
36. Smiljkovic, M.; Matsoukas, M.T.; Kritsi, E.; Zelenko, U.; Grdadolnik, S.G.; Calhelha, R.C.; Ferreira, I.C.F.R.; Sankovic-Babic, S.;
Glamoclija, J.; Fotopoulou, T.; et al. Nitrate Esters of Heteroaromatic Compounds as Candida albicans CYP51 Enzyme Inhibitors.
ChemMedChem 2018, 13, 251–258. [CrossRef]
Molecules 2023, 28, 1167 16 of 16

37. Fesatidou, M.; Zagaliotis, P.; Camoutsis, C.; Petrou, A.; Eleftheriou, P.; Tratrtat, C.; Haroun, M.; Geronikaki, A.; Ciric, A.; Sokovic,
M. 5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design, synthesis, molecular docking and evaluation.
Bioorg. Med. Chem. 2018, 26, 4664–4676. [CrossRef]
38. Lipinski, C.A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov. Today Technol. 2004, 1, 337–341.
[CrossRef]
39. SwissADME Program. Available online: https://swissadme.ch (accessed on 19 October 2022).

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