Design, synthesis and activity against Staphylococcus epidermidis of 5-chloro-2- or 5-chloro-4-methyl-9H-xanthen-9-one and some of its derivatives

Article abstract of DOI:10.1111/cbdd.13803

Ten new xanthone derivatives have been designed and synthesized for their potential antibacterial activity. All compounds have been screened against Staphylococcus epidermidis strains ATCC 12228 and clinical K/12/8915. The highest antibacterial activity was observed for compound 3: 5-chloro-2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride, exhibiting MIC of 0.8?μg/ml against ATCC 12228 strain, compared to linezolid (0.8?μg/ml), ciprofloxacin (0.2?μg/ml) or trimethoprim and sulfamethoxazole (0.8?μg/ml). For the most active compound 3, genotoxicity assay with use of Salmonella enterica serovar Typhimurium revealed safety in terms of genotoxicity at concentration 75?μg/ml and antibacterial activity against Salmonella at all higher concentrations. A final in silico prediction of skin metabolism of compound 3 seems promising, indicating stability of the xanthone moiety in the metabolism process.

Full text of DOI:10.1111/cbdd.13803

MRS. IWONA SKIBA-KUREK (Orcid ID : 0000-0002-0533-2130)
Article type : Research Article
Design, synthesis and activity against Staphylococcus epidermidis of 5-chloro-2- or 5-chloro-
4-methyl-9H-xanthen-9-one and some of its derivatives
Running title: Antibacterial activity of new xanthone derivatives
Gabriela Mazur a, Iwona Skiba-Kurek b, Elżbieta Karczewska b, Katarzyna Pańczyk-Straszak a,
Joanna Jaworska a, Anna M. Waszkielewicz a*
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Krakow, Poland
b Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Jagiellonian University
Medical College, ul. Medyczna 9, 30-688 Krakow, Poland
* anna.waszkielewicz@uj.edu.pl, tel. +4812-6205576
Acknowledgements
This work was financed from Jagiellonian University Medical College statutory funds N42/
DBS/000032 and K/ZDS/007776. This article is also a result of the research project No.
2018/31/N/NZ6/03339 financed by the National Science Centre, Poland.
Ten new xanthone derivatives have been designed and synthesized for their potential antibacterial
activity. All compounds have been screened against Staphylococcus epidermidis strains ATCC
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through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13803
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12228 and clinical K/12/8915. The highest antibacterial activity was observed for compound 3: 5-
chloro-2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride,
exhibiting MIC of 0.8 µg/mL against ATCC 12228 strain, compared to linezolid (0.8 µg/mL),
ciprofloxacin (0.2 µg/mL) or trimethoprim and sulfamethoxazole (0.8 µg/mL). For the most active
compound 3, genotoxicity assay with use of Salmonella enterica serovar Typhimurium revealed
safety in terms of genotoxicity at concentration 75 µg/mL and antibacterial activity against
Salmonella at all higher concentrations. A final in silico prediction of skin metabolism of
compound 3 seems promising, indicating stability of the xanthone moiety in the metabolism
process.
Key words: antibacterial, xanthone, synthesis, Staphylococcus epidermidis, Salmonella enterica,
UMU-test
1. Introduction
Staphylococcus epidermidis is Gram-positive bacteria belonging to coagulase negative
staphylococci (CNS) group. It is an important element of the physiological flora, colonizing the
skin around the armpits, scalp and nasal atrium (Namvar et al., 2014). Staphylococcus epidermidis
is also an important opportunistic pathogen causing hospital infections particularly in neonatal
intensive care units (NICU) (Eftekhar & Speert, 2009). Blood infections with such pathogens are
especially dangerous for newborns whose immune systems are extremely immature and in whom
invasive treatments are very frequently applied during hospitalization (Cheung & Otto, 2010;
Nash et al., 2013). Rapid and accurate diagnosis of neonatal sepsis is of key importance due to
high morbidity and mortality affecting approximately 40% of patients (Zea-Vera & Ochoa, 2015;
Fell et al., 2017).
Increasing resistance to antimicrobial agents among Staphylococcus epidermidis clinical strains, in
particular lack of susceptibility to β-lactam antibiotics or macrolides, lincosamides and
streptogramins B is currently a serious therapeutic and epidemiological problem (Bizzarro et al.,
2015). A high percentage of Staphylococcus epidermidis isolates acquired from neonates are
resistant to antibiotics: β-lactams (86% – 100%), erythromycin (65% – 100%) and clindamycin
(80% – 100%) (Brzychczy-Włoch et al., 2013). In addition, this species has the ability to produce
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biofilm that increases the pathogenicity of Staphylococcus epidermidis and resistance to
antibacterial drugs used in therapy (Büttner, Mack & Rohde, 2015; Wojtyczka et al., 2014). The
biofilm constitutes extracellular polysaccharide which is termed by polysaccharide intercellular
adhesin (PIA) in S. epidermidis (Fitzpatrick, Humphreys & O’Gara, 2005).
The xanthone moiety (9H-xanthen-9-one, dibenzo-γ-pyrone) is a naturally occurring heterocyclic
structure, present in medicinal plants such as Garcinia mangostana (α-mangostin, Fig. 1) (Jung et
al., 2006; Dharmaratne et al., 2013), Mangifera indica (Pinto, Sousa & Nascimento, 2005),
Cudrania cochinchinensis (Fukai et al., 2005), etc. Therefore, there have been many attempts to
search for biological activity within the group of derivatives of xanthone, where significant
biological activities have been found, such as: anticancer (Szkaradek et al., 2016), anticonvulsant
(Szkaradek et al., 2013; Waszkielewicz et al., 2013), antioxidant (Jung et al., 2006), antifungal
(Klesiewicz et al., 2018), antimicrobial (Fukai et al., 2005), etc. – some amount of research has
been performed by our team.
NH2
CH3
O
O
N
O
F
O
N
N
OH
O
O
CH3
CH3
S
HN
H2N
N
O
N
N
H
O
CH3
H2N
Sulfamethoxazole
Trimethoprim
Ciprofloxacin
H3C
CH3
O
O
O
OH
CH3
N
Cl
O
N
H3C
CH3
N
HO
O
OH
Compound I
-Mangostin
Fig. 1 Popular chemotherapeutics - control and reference compounds: α-mangostin – a natural
antibacterial xanthone derivative and synthetic antifungal compound I (Klesiewicz et al., 2018).
New synthetic xanthone derivatives as well as other groups of compounds and their antibacterial
activity have been already studied by our research team. So far, some significant activity against
the bacteria Helicobacter pylori have been found (Klesiewicz et al., 2016). We noticed interesting
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influence in antibacterial activity of chlorine substituent in xanthone derivatives in positions 6 or 7
as well as in 4-chlorophenoxyalkylamines (Klesiewicz et al., 2018), therefore, the substituent
remains interesting for us when attempting drug design for new antibacterial compounds. This
time we aimed to try a new position – C5. Moreover, we have noticed significant input of
piperazine moiety into antimicrobial activity and this moiety also remains important in our drug
discovery work (Klesiewicz et al., 2016).
The purpose of this research is design, synthesis and evaluation of antibacterial activity of some
new xanthone derivatives, against Staphylococcus epidermidis bacteria. Since the xanthone moiety
by itself exhibits antimicrobial activity, we attempted to enhance it with possible additional
substituents. The general attempt was to achieve xanthone derivatives with chlorine in position 5,
methylene linker in positions 2 or 4, linking with a moiety built of ethylene alkyl groups
connected by proton acceptors (O) or proton donors (NH). Such structure was hoped for creating
hydrogen bonds with any water or polysaccharides in bacterial biofilm. Such interactions would
enable the lipophilic antibacterial chloroxanthone penetrate the biofilm.
2. Results and discussion
2.1. Chemistry
Ten new xanthone derivatives have been designed and synthesized for their potential antibacterial
activity, in terms of chlorine and piperazine containing substituents. Chlorine in position C5 has
been introduced for enhancement of antibacterial activity due to our former observations.
Hydrophilic substituents have been introduced in position C2 or C4, for affinity to the
aforementioned polysaccharide biofilm. The first obtained compounds were hydroxyethyl or
hydroxyethoxyethyl derivatives of 5-chloro-2- or 5-chloro-4-(piperazinemethyl)xanthone. As our
research advanced, we designed pyridine analog, on the basis of formerly beneficial properties of
N-(2-pyridine)piperazine derivatives in our antimicrobial activity research and due to the structure
of reference compound I (Fig. 1) (Klesiewicz et al., 2018), as well as two more lipophilic
structures. We also synthesized three compounds lacking piperazine ring, in order to evaluate the
influence of the presence of piperazine moiety on antibacterial activity. Since we achieved
favorable results for lipophilic substituted derivatives which were poorly soluble in water, it was
necessary to achieve the compounds in the form of hydrochlorides for the significant issue of
affinity towards bacterial biofilm. The form of hydrochloride was possible to achieve in case of 2
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amine groups in the piperazine moiety. Additionally, the chemical characteristics of the
aforementioned biofilm (also produced by S. epidermidis K/12/8915 strain) could require ionized
form of the designed compounds for being mixed and absorbed into proximity of the bacterial cell
membrane. The obtained structures are presented in Table 1.
Additionally, part of our research is analysis how molecular properties such as lipophilicity,
molecular volume, or topological polar surface area (TPSA) influence the biological properties.
Especially that these properties can influence cell membrane permeation and further absorption of
the compound in the bacteria cell. So far, literature concerning properties of antibacterial
compounds against Staphylococcus aureus has described logP range varying between -0.75-4.88
(Wadapurkar et al., 2018). Therefore, our findings are interesting in order to plan further research
of antibacterial compounds.
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Table 1 Structures, calculated physical-chemical data and antibacterial activity of the title compounds 1-10.
O
8
1
4
2
3
7
6
R
O
5
Cl
mV a TPSA a
MIC (µg/mL) Staphylococcus epidermidis
Compound
Position
R
logP a
(Å3)
(Å2)
ATCC 12228 strain
50
K/12/8915 strain
50
1
2
C2
-H
4.62 202.68
3.40 240.14
30.21
H
N
OH
x HCl
62.47
50
50
N
OH
x 2HCl
3
4
3.24 326.47
3.13 369.06
56.91
66.15
0.8
50
50
50
N
N
OH
x 2HCl
O
N
N
N
5
6
5.67 389.86
5.16 396.99
36.69
55.16
6.25
50
50
50
x 2HCl
O
N
O
N
x 2HCl
N
N
N
7
4.67
352.1
49.58
50
50
x 3HCl
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8
9
C4
-H
4.60 202.68
3.7 227.74
30.21
50.44
50
50
50
50
N
OH
N
x 2HCl
N
OH
x 2HCl
O
10
N
3.59 270.32
59.67
50
50
Linezolid b
Ciprofloxacin b
0.8
0.2
1.6
0.4
Trimethoprim/Sulfamethoxazole
in a ratio of 1:19 b
0.8
1.6
a data calculated from www.molinspiration.com; mV – molecular volume; TPSA – topological polar surface area
b quality control (Staphylococcus aureus ATCC 29213, EUCAST QC Tables): Linezolid MIC = 1.6 μg/mL, Ciprofloxacin MIC = 0.4 μg/mL,
Trimethoprim/Sulfamethoxazole in a ratio of 1:19 MIC = 0.4 μg/mL
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The title compounds 1-10 were synthesized similarly to previously published procedures
(Waszkielewicz et al., 2013) and the synthesis is presented in Scheme 1. The first step constituted
the Ullmann’s condensation resulting in formation of the 5-chloro-2- or 5-chloro-4-
methylxanthone skeleton – with use of 2,3-dichlorobenzoic acid and appropriate cresol (para for
1-7 or orto for 8-10). The reaction was performed for a few hours, in paraffin oil, at 180 °C, in the
presence of Cu/Cu2O catalyst. After condensation, intermediate product underwent cyclization in
the presence of H2SO4 (98%) resulting in 5-chloro-2-methyl-9H-xanthen-9-one (Eckstein &
Marona, 1980) (for 1-7) or 5-chloro-4-methyl-9H-xanthen-9-one (Rewcastle et al., 1989) (for 8-
10). For compounds 1 and 8 this step was the final one. For all other compounds free-radical
bromination of the methyl residue with N-bromosuccinimide (NBS) was performed in CCl4, in the
presence of benzoyl peroxide under UV-light, resulting in 5-chloro-2-bromomethyl-9H-xanthen-9-
one (Eckstein & Marona, 1980) or 5-chloro-4-bromomethyl-9H-xanthen-9-one (Rewcastle et al.,
1989), respectively. The final step for compounds 2-7, 9 and 10 was aminolysis of the appropriate
bromo-substituted derivative by means of commercially available amines: colamine for 2,
N-(hydroxyethyl)piperazine for 3 and 9, N-(2-hydroxyethoxyethyl)piperazine for 4 and 10, N-
(phenylethyl)piperazine for 5, N-piperonylpiperazine for 6 and N-(2-pyridyl)piperazine for 7. The
reaction was carried out in toluene in the presence of anhydrous K2CO3 as a proton acceptor. The
obtained amines were washed out from ethanol : water (96 : 4 v/v) mixture. Some portions of
products solutions were converted into hydrochlorides using addition of 37% HCl solution in
water. The salts were purified by means of recrystallization from methanol. Compounds lacking
amine moiety (1 and 8) were crystallized from ethanol.
Due to high solubility of compounds required for the antibacterial assays, all achieved bases were
converted to hydrochlorides as described above, and evaluated in this form. However, the mass
spectrometry allows only to show the mass of the base form, and protons in the salt form were not
1
visible in H NMR (expected broad singlet at chemical shift ca. 9-11 ppm) for most compounds.
The achievement of the proper salts of the title compounds was confirmed by means of elemental
analysis. For compounds 1-4 and 8 additional 13C NMR spectra have been obtained.
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O
O
O
1
4
8
1) Ullmann's condensation
2
CH3
7
6
+
OH
CH3
2) cyclization
conc. H2SO4
3
Cl
HO
5
Cl
Compounds
Cl
1 7
para for
orto for
-
1 8
,
8 10
-
1 7
8 10
C2 for
C4 for
-
-
NBS
CCl4, h
O
O
O
O
O
aminolysis
HCl aq
etOH
CH2Br
R
R
toluene, K2CO3
O
x mHCl
Cl
R: amine moiety (see Table 1)
Cl
Cl
2 7, 9 10
Compounds
-
,
2
m: 1 for compound
3 6 9 10
2 for compounds - ,
,
7
3 for compound
Scheme 1 Synthesis of the title compounds 1-10.
2.2. Microbiology
Compounds 3 and 5 were the most active with MIC values equal to 0.8 and 6.25 μg/mL,
respectively – comparable with those obtained for reference chemotherapeutic agents
(http://www.Eucast.Org/Ast_of_bacteria/Media_preparation/).
Interesting observations can be made in terms of position isomerism. Within this group of
compounds, compound 8 is a positional isomer of compound 1, compound 9 is a C4 positional
isomer of compound 3, and similarly compound 10 is the C4 isomer of compound 4. In case of
amines (compounds 3, 4, 9, 10) the molecular volume of the C2 derivatives is larger than that of
C4 derivatives – which could be predicted based on position C5 already taken by Cl atom and the
shape of the compound. Nevertheless, both two most active compounds 3 and 5 are C2 substituted
xanthone derivatives and are more active than e.g. compound 9 – C4 analogue of 3. Therefore, it
can be concluded in this small group that the C2 position seems favourable for the antibacterial
activity. This is unexpected because position C2 brings larger volume of the compound (Table 1),
while smaller compounds are usually expected to be absorbed easier than big ones into the
bacteria membrane.
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LogP values of the most active compounds 3 and 5 are 3.24 and 5.67, respectively, while the range
of logP in this group is 3.13-5.67 – therefore it can be concluded that in this group of compounds
lipophilicity is not determinant for their activity, and this is contrary to the assumptions taken at
the design of the structures.
Considering our assumptions regarding incorporating proton acceptors or donors in the designed
structures for the enhancement of antibacterial activity of xanthone, compounds 1 and 8 lacking
amine/hydroxy group were inactive. On the other hand, compound 4 should be more active than 3
– and this assumption did not prove correct. Compound 4 as hydroxyethoxyethylpiperazine
derivative can produce more hydrogen bonds than 3 as hydroxyethylpiperazine derivative and this
feature did not prove beneficial. The most active are compounds 3 – hydroxyethylpiperazine
derivative and compound 5 – phenethylpiperazine derivative. Therefore, incorporation of
piperazine moiety for possibility to achieve dihydrochlorides as a common feature of the two
compounds, as well as position C2 of the substitution of the 5-chloroxanthone moiety, seem
favourable.
The molecular mechanism of antimicrobial activity observed for compounds 3 and 5 remains
unclear. However, literature data provide some hypotheses. Within xanthone derivatives
exhibiting antimicrobial activity against S. epidermidis, α-mangostin is the most studied one.
Combination of transcriptome and proteome analyses with a use of S. epidermidis RP62A showed
that α-mangostin perturbs cytoplasmic membrane by hydrogen bonds formation with
transmembrane precursor proteins. This process leads to downregulation of genes important for
multiple metabolic pathways (Sivaranjani et al., 2019). Taken the above into account, cytoplasmic
membrane is likely to be a principal target for compounds 3 and 5, especially that both compounds
possess hydrogen bond donors and acceptors in their structure both within the xanthonic system
and the side chain. This hypothesis needs to be tested experimentally.
The difference of activity of compounds 3 and 5 between the strain Staphylococcus epidermidis
ATCC 12228 and clinical strain Staphylococcus epidermidis K/12/8915 can result from presence
of biofilm in K/12/8915 and its absence in the reference ATCC 12228. Moreover, the K/12/8915
is characterized by multidrug resistance. Drug resistance of bacterial strains is a major challenge in
the design of new antibiotics and acts as a driving force for this branch of medical chemistry. The
drug resistance mechanisms developed by bacteria include the expression of enzymes involved in
the degradation of antibiotic molecules, decreased membrane permeability, mutations within
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antibiotic molecular targets or efflux pumps activation. Common strategies of antibiotic resistance
mitigation target particular drug classes or single strains of resistant microbe (Riduan, Armugam
& Zhang, 2020). Modern strategies include among others search for new molecular targets for
antibiotics, design of multitarget drugs, consideration of adjuvant therapies disrupting cell
membrane and deactivating efflux pumps, as well as utilization of reactive oxygen species in
antimicrobial therapy (Riduan, Armugam & Zhang, 2020; Perdih et al., 2015; Perdih et al., 2014;
Baym, Stone & Kishony, 2016; Monserrat-Martinez, Gambin & Sierecki, 2019; Dersch et al.,
2017; Wright, 2016). Within xanthone derivatives, α-mangostin is known to be active against
many drug-resistant strains, probably due to its mechanism of action involving cell membrane
perturbation and – as a consequence – influence on multiple metabolic pathways (Sakagami et al.,
2005; Koh et al., 2013). The xanthon and α-mangostin derivatives therefore represent a promising
group of compounds in the context of combating antibiotic resistance.
Formation of biofilm by bacterial strains constitutes another problem. Insufficient drug penetration
through the biofilm may lead to reduced or ineffective therapy (Singh et al., 2010). S. epidermidis
biofilm formed on medical devices causes a real threat. Literature data suggest potential use of α-
mangostin and its derivatives also against biofilm-forming strains of S. epidermidis (Sivaranjani et
al., 2017). As compounds 3 and 5 turned out to be active against a strain that does not form
biofilm, it is not possible to make conclusions regarding their biofilm penetration. Further studies
on this issue would be worthy. Due to low MIC values, both compounds are promising for
treatment of infections with S. epidermidis. There exist premises for further research both in this
group of compounds for other strains (including presence and absence of biofilm), as well as in
design and synthesis of new antibacterial compounds.
2.3. Genotoxicity
The most active compound 3 was chosen for additional safety testing, for the purpose of
elimination of genotoxic compounds at an early stage. UMU-Chromotest (commercially available
kit) was used for this purpose. The assay utilizes an engineered Salmonella enterica serovar
Typhimurium strain which possesses a gene coding for the β-galactosidase enzyme tethered to the
umuC gene (one of damage response genes, a part of SOS genetic material repair system). In case
of DNA damage, the SOS system is activated, β-galactosidase gene gets transcribed proportionally
to the level of SOS induction and enzyme activity can be detected calorimetrically (Reifferscheid
et al., 1991).
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The compound was tested in five concentrations ranging 75-1200 µg/mL (Table 2). In order to
receive reliable results, the protocol includes calculation of growth factor G (G < 0.5 indicates
toxicity of tested sample towards the used bacterial strains and makes the experiment unreliable).
Due to low G values, evaluation of genotoxic properties was feasible only at concentration 75
µg/mL, at which the compound proved safe (induction ratio IR < 1.5). Additionally, the low G
values obtained may be a premise for search for antibacterial activity against Salmonella enterica
serovar Typhimurium strain in a group of xanthone derivatives, which is surprising, but very
promising observation.
Table 2 Results of UMU test for compound 3
c
Compound
Concentration
Ga
Usb
IR
[µg/mL]
3
75
0.799
0.473
0.250
0.062
-0.052
0.880
0.508
0.639
NT
0.800
150
300
600
1200
-
NT
NT
NT
NT
NT
NT
NT
10% DMSO in saline
4-NQO
0.941
5.143
1.069
10.126
-
a G (growth factor) = (mean A600 of tested sample) – (mean A600 of blank) / (mean A600 of negative
control) – (mean A600 of blank), G > 0.5 – necessary condition for the reliability of the test results,
G < 0.5 – toxicity towards the used bacterial strains; b Us (β-galactosidase activity, relative units) =
(mean A420 of tested sample) – (mean A420 of blank) / (mean A420 of negative control) – (mean
A420 of blank); c IR (induction ratio) = 1/G*Us, IR > 1.5 – genotoxicity in a particular concentration;
4-NQO – 4-nitroquinoline N-oxide; NT – not tested; - – not applicable.
2.4. In silico metabolism prediction
Additional assays related to safety of active compounds include metabolism prediction. The
MetaSite program (Cruciani et al., 2005; Boyer et al., 2009) allows in silico prediction of possible
routes of metabolism, including skin metabolism. It is particularly important for antibacterial
compounds, which are frequently applied topically. The performed in silico prediction revealed
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that the most probable sites of metabolism are methylene group of hydroxyethyl substituent at the
piperazine ring together with the nitrogen atom, to which the hydroxyethyl is attached. According
to performed simulations, the most probable metabolites (Scheme 2) are products of oxidation: (1)
aldehyde or carboxylic acid resulting from opening of the piperazine ring, (2) amide resulting
from oxidation of methylene group into carbonyl moiety and (3) N-oxide, which have been
previously subject of our research in a group of piperazine derivatives (Żesławska et al., 2020).
Another product is a result of disconnection of the hydroxyethyl substituent at the piperazine ring.
Concluding the observed predictions, together with observations of data provided by the program,
the xanthone moiety remains stable in the in silico prediction, which is also consistent with our
former in silico and in vitro findings for alkoxy derivatives of xanthone with aminoalkanol
substituents (Waszkielewicz et al., 2016). Moreover, the program has indicated that the
hydroxyethyl substituent is oxidized into glycolic acid in the process of oxidative hydrolysis from
piperazine, which may – consistently with the data on beneficial activity of α-hydroxyacids (Yu &
Van Scott, 2004) – contribute to further activity after absorption and metabolism in the skin.
Moreover, from the perspective of our further research objectives, it will be interesting to
synthesize some of the metabolites and study their properties. Metabolites containing aldehyde
group seem too reactive to plan administration in skin (potential reaction with amine groups of
amino acids and resulting colouring properties). Synthesis of other metabolites or research
regarding analysis of in vivo metabolites are interesting for our further research.
O
H
N
N
OH
O
O
O
H
Cl
H
N
N
OH
O
O
HO
Cl
O
O
NH
N
Cl
O-
N+
O
O
3
Compound
N
OH
Cl
O
O
N
N
OH
O
Cl
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Scheme 2 The most probable sites of metabolism of Compound 3 marked as coloured rings on the
structure as well as its five most probable metabolites according to in silico prediction of skin
metabolism.
3. Conclusions
As a conclusion, this research resulted in achievement of a very potent antibacterial compound 5-
chloro-2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride (3),
exhibiting MIC of 0.8 µg/mL against Staphylococcus epidermidis ATCC 12228 strain.
Another conclusion is high importance of position C2 for piperazinemethyl substituents in the
xanthone moiety for the antibacterial activity when Cl is present at C5, in spite of enlargement of
molecular volume of the compound compared to C4 analogs. Nevertheless, calculation of the
physical-chemical properties still remains important part in chemical drug design.
The last conclusion is the safety in terms of genotoxicity of the active compound 3 at 75 µg/mL
and surprisingly potential antibacterial activity against Salmonella enterica at higher
concentrations, as well as promising in silico predictions of the compound’s metabolism in the
skin. Therefore, there exist premises for further research in this group of compounds in terms of
multiple antibacterial activity.
4. Experimental protocols
4.1. Chemistry
All reagents were manufactured by Alfa Aesar (purchased from Chemat, Gdansk, Poland or
Trimen Chemicals S.A., Łódź, Poland), or Sigma-Aldrich (purchased from Sigma-Aldrich,
Poznan, Poland). Solvents were commercially available materials of reagent grade. Melting points
(mp) were determined by means of a Büchi Melting Point M-560 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and were uncorrected. The purity of the obtained compounds was confirmed
by LCMS at Faculty of Pharmacy, Jagiellonian University Medical College. For mass
spectrometry analysis samples were prepared in acetonitrile/water (10/90 v/v) mixture. The
LC/MS system consisted of a Waters Acquity UPLC, coupled to a Waters TQD mass spectrometer
(electrospray ionization mode ESI-tandem quadrupole). All the analyses were carried out using an
Acquity UPLC BEH C18, 1.7 lm, 2.1 x 100 mm column. A flow rate of 0.3 mL/min and a gradient
of (5 – 95) % B over 10 min and then 100% B over 2 min was used. Eluent A: water / 0.1 %
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HCO2H; eluent B: acetonitrile / 0.1% HCO2H. LC/MS data were obtained by scanning the first
quadrupole in 0.5 s in a mass range from 50 to 1000 Da; eight scans were summed up to produce
the final spectrum.
1
The H NMR spectra were obtained at Faculty of Pharmacy, Jagiellonian University Medical
College, using Bruker AVANCE III 600 (600.2 MHz) spectrometer (Bruker, Karlsruhe,
Germany). Results are presented in the following format: chemical shift δ (ppm), multiplicity, J
values in Hertz (Hz), number of protons, protons’ position. Multiplicities are showed as the
abbreviations: s (singlet), br s (broad singlet), d (doublet), br d (broad doublet), t (triplet), br t
(broad triplet), m (multiplet).
In silico registration and interpretation of LCMS and NMR spectra were facilitated by ACDLabs
2019 Spectrus Processor 1.3.
Elemental analysis was performed at Faculty of Pharmacy, Jagiellonian University Medical
College, with use of Elementar Analysensysteme GmbH VarioEL V2.10 20.Aug. 2001 CHNS
Mode.
4.1.1. General procedure for preparation of the final compounds 1-10.
In the first step, reaction of condensation of 2,3-dichlorobenzoic acid with p-cresol or o-cresol,
respectively, was performed. 23 g of metallic sodium was slowly (in small portions) dissolved in
500 mL of methanol. Next, 500 g of 2,3-dichlorobenzoic acid, 60 mL of p-kresol or o-kresol
(respectively) and catalysts: 0,1 g Cu2O and 0,3 g Cu were added. Reaction was heated on
o
electromagnetic stirrer to 120 C for 5 hours. During this time all the solvent (methanol) was
distilled of. Then, 250 mL of paraffin oil was added and the mixture was stirred in temperature
180 oC for next 4 hours. After his time, the precipitate was filtered off, washed with toluene, than
heptane and dried. The precipitate was dissolved in 20% water solution of NaOH, heated to 100 oC
and stirred for 30 minutes. After this time undissolved precipitate was filtered off on a Büchner
funnel. The filtrate was transferred to a separating funnel, benzene was added and the phases were
separated. The water phase was collected, active carbon was added. After night the carbon and
impurities were filtered of. Obtained filtrate was acidified with 10% HCl in order to obtain
product of condensation (3-chloro-2-(2-methylphenoxy) or 3-chloro-2-(4-methylphenoxy) benzoic
acid, respectively). The resulting precipitate was filtered off and dried.
The next step was cyclization. The crude product of condensation was dissolved in 500 mL of
o
concentrated sulfuric acid (H2SO4 conc.), heated to 100 C and stirred for 4 hours. The mixture
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was then poured onto ice and filtered on a Büchner funnel. The precipitate was washed with water,
transferred to a beaker and 20% NaOH was added, in order to wash away the 2,3-dichlorobenzoic
acid that has not reacted. Undissolved precipitate was filtered off on a Büchner funnel and washed
with water to give 5-chloro-4-methylxanthone. The product of cyclization was then recrystallized
from ethanol, in order to obtain pure compound 1 or 8 respectively. These compounds were also
used in the next step as a substrates for bromination.
For bromination of 5-chloro-4-methylxanthone or 5-chloro-2-methylxanthone, respectively,
appropriate substrate, together with N-bromosuccinimide and benzoyl peroxide were dissolved in
o
CCl4, stirred in temperature of 100 C on electromagnetic stirrer with reflux condenser and with
additional light from UV-VIS lamp for 20 hours. After this time evolved succinimide was filtered
off. The filtrate was placed in the refrigerator overnight. Obtained precipitate was filtered off,
dried and recrystallized from toluene, to obtain 5-chloro-4-methylxanthone or 5-chloro-4-
methylxanthone bromide, respectively.
0.02 mole of appropriate bromide was refluxed with 0.02 mole of appropriate N-substituted
piperazine in toluene with presence of anhydrous K2CO3 at 110 °C for 72 h. Then the solvent was
distilled off and the remaining solid, containing substrates and the product were boiled in water
acidified with 10% HCl for acidic pH in order to dissolve KCl, K2CO3, unreacted amine and the
product. The mixture was added carbon for binding the unreacted bromide and then filtered off. In
order to achieve basic pH, 30% NaOH was added to the filtrate so that the product was
precipitated and obtained in the form of base (the unreacted piperazine substrate was liquid). The
mixture was left for several h and then filtered off. After drying the product was dissolved in
boiling 96% ethanol and carbon was added to the solution for purification. After filtering of the
solution, a few milliliters of 37% HClaq was added in order to precipitate the hydrochloride
product, which was then left to cool down and the precipitate was then filtered off and
recrystallized from methanol. The purity was verified by LC-MS. The structure was verified with
1
13
LC-MS (mass), H NMR (and C NMR in cases of 1-4, 8) and – since protons at N were not
1
visible in H NMR – elemental analysis for determination if the salts were mono-, di- or
trihydrochlorides.
4.1.2 Characterization of final products
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5-chloro-2-methyl-9H-xanthen-9-one (1) White solid; yield = 73%; C14H9ClO2; M = 244.03; mp
1
110-112 °C; H NMR (600 MHz, D2O) δ (ppm): 8.15 (dd, J=7.73, 1.43 Hz, 1 H, X-H8), 7.99 (s,
1H, X-H1), 7.68 (dd, J=7.73, 1.43 Hz, 1H, X-H6), 7.44 - 7.49 (m, 1H, X-H3), 7.38 (d, J=8.59 Hz,
1H, X-H4), 7.22 (t, J=7.73 Hz, 1H, X-H7), 2.40 (s, 3H, -CH3); 13C NMR (126 MHz,
CHLOROFORM-d) δ ppm 176.55 (s, 1C, C=O), 154.02 (s, 1C, X-C4a), 151.74 (s, 1C, X-C4b),
136.45 (s, 1C, , X-C3), 134.78 (s, 1C, X-C6), 134.42 (s, 1C, X-C2), 126.00 (s, 1C, X-C1), 125.35
(s, 1C, X-C7), 123.65 (s, 1C, X-C8), 123.08 (s, 1C, X-C5), 122.80 (s, 1C, X-C8a), 121.00 (s, 1C,
X-C8b), 118.00 (s, 1C, X-C4), 20.91 (s, 1C, -CH3); LC-MS [M + H]+ m/z: 245.23, 99.19%.
5-chloro-2-(((2-hydroxyethyl)amino)methyl)-9H-xanthen-9-one hydrochloride (2), Light beige
1
solid; yield = 63%; C14H9ClO2; M = 303.07; mp 281-283 °C; H NMR (600 MHz, DMSO-d6) δ
(ppm): 9.66 (br s, 1H, NH+), 8.36 (d, J = 2.3 Hz, 1H, X-1), 8.16–8.12 (m, 1H, X-8), 8.12–8.09 (m,
1H, X-6), 8.02–7.99 (m, 1H, X-3), 7.74 (d, J = 8.6 Hz, 1H, X-4), 7.43 (t, J = 7.7 Hz, 1H, X-7),
5.31 (br s, 1H, -OH), 4.28 (br s, 2H, -NH), 3.68 (t, J = 5.4 Hz, 2H, X-CH2-NH), 3.34 (br s, 2H,
13
NH-CH2-CH2-OH), 2.95 (br s, 2H, NH-CH2-CH2-OH); C NMR (126 MHz, DMSO-d6) δ ppm
175.89 (s, 1C, C=O), 155.79 (s, 1 C, X-C4a), 151.57 (s, 1C, X-C4b), 138.39 (s, 1C, X-C2), 136.09
(s, 1C, X-C6), 129.47 (s, 1C, X-C3), 128.93 (s, 1C, X-C1), 125.62 (s, 1C, X-C7), 125.38 (s, 1C,
X-C8), 123.16 (s, 1C, X-C5), 122.24 (s, 1C, X-C8a), 121.10 (s, 1C1, X-C8b), 119.12 (s, 1C, X-
C4), 56.86 (s, 1C, NH-CH2-CH2-OH), 49.44 (s, 1C, X-CH2-NH), 49.19 (s, 1C, NH-CH2-CH2-
OH); LC-MS [M + H]+ m/z: 304.18, 99.31%.
5-Chloro-2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride (3).
calc
Yellow solid; yield = 92%; C20H23N2O3Cl3; M = 372.12; mp 290-292 °C; el.an.
/
anal: N6.28
/
;
6.18
C53.89 53.89; H5.20 5.21; 1H NMR (600 MHz, D2O) δ (ppm): 7.71 (d, J = 1.7, 1H, X-1), 7.64-7.60 (m,
/
/
1H, X-6), 7.45-7.41 (m, 1H, X-4), 7.33-7.29 (m, 1H, X-3), 7.09 (d, J = 8.6, 1H, X-8), 6.92 (t, J =
8.0, 1H, X-7), 4.35 (s, 2H, X-CH2-N<), 3.86-3.81 (m, 2H, >N-CH2-CH2-OH), 3.64 (br s, 4H, pip),
13
3.56 (br s, 4H, pip), 3.37-3.33 (m, 2H, >N-CH2-CH2-OH). C NMR (126 MHz, D2O) δ (ppm):
176.8 (X-9), 155.6 (X-4’), 150.4 (X-5’), 138.4 (X-3), 136.2 (X-6), 128.9 (X-2), 124.9 (X-7), 124.8
(X-1), 124.4(X-8), 122.1 (X-5), 120.9 (X-8’), 119.8 (X-9’), 119.5(X-4), 59.2 (X-CH2-N<), 58.2
(>N-CH2-CH2-OH), 54.9 (>N-CH2-CH2-OH), 48.9 (pip: N-CH2-CH2-N-), 48.1(pip: N-CH2-CH2-
N-); LC-MS [M + H]+ m/z: 373.23, 98.03%.
5-chloro-2-((4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one
calc
dihydrochloride (4): White solid; yield = 82%; C22H27N2O4Cl3; M = 453,13; el.an.
/
:
anal
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N5.72 5.63; C53.95 53.74; H5.56 5.55; mp 270-272 °C; 1H NMR (600 MHz, D2O) δ (ppm): 7.70 (s,1H, X-
/ / /
1), 7.65-7.59 (m, 1H, X-8), 7.43-7.37 (m, 1H, X-3), 7.27 (d, J = 8.0, 1H, X-4), 7.06 (d, J = 8.6,
1H, X-6), 6.93-6.85 (m, 1H, X-7), 4.36 (s, 2H, X-CH2-N<), 3.82-3.73 (m, 3H, >N-CH2-CH2-O, -
OH), 3.72-3.61 (m, 6H, pip, O-CH2-CH2-OH), 3.60-3.53 (m, 6H, pip, >N-CH2-CH2-O), 3.46-3.44
ppm (m, 2H, >N-CH2-CH2-O). 13C NMR (126 MHz, D2O) δ (ppm): 176.67 (X-C9, C=O), 155.51
(X-C4a), 150.35 (X-C4b), 138.43 (X-C3), 136.22 (X-C6), 128.96 (X-C2), 124.87 (X-C7),
124.71(X-C1), 124.35 (X-C8), 122.01 (X-C5), 120.83 (X-C8a), 119.73 (X-C9a), 119.47 (X-C4),
71.85 (O-CH2-OH), 63.51 (>N-CH2-CH2-O), 60.44 (X-CH2-N<), 59.1 (O-CH2-CH2-OH), 48.96
(pip: N-CH2-CH2-N), 48.16 (pip: N-CH2-CH2-N); LC-MS [M + H]+ m/z: 417.23, 99.36%.
5-chloro-2-((4-phenethylpiperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride (5). Light grey
calc
solid; yield = 78%; C26H27N2O2Cl3; M = 504.11; mp 291-293 °C; el.an.
/
anal: N5.54
/
;
5.42
C61.73 61.67; H5.38 5.48;1H NMR (600 MHz, acetone-d6) δ (ppm): 8.24-8.14 (m, 2H, X-6, X-7); 7.96-
/
/
7.93 (m, 1H, X-8), 7.87 (dd, J = 8.59, 2.29, 1H, X-3), 7.65 (d, J = 8.59, 1H, X-4), 7.46-7.43 (m,
1H, X-1), 7.25 - 7.19 (m, 4H, Fen-2, Fen-3, Fen-5, Fen-6), 7.15-7.12 (m, 1H, Fen-4), 3.62 (s, 2H
CH2-N<), 2.79-2.71 (m, 4H, >N-CH2-CH2-Fen), 2.54- 2.42 (m, 8H, pip). LC-MS [M + H]+ m/z:
433.24, 100.00%.
2-((4-(benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)methyl)-5-chloro-9H-xanthen-9-one
dihydrochloride (6). Light beige solid; yield = 65%; C26H25N2O4Cl3; M=535.85; mp 296-298 °C;
calc
1
el.an.
/ / / /4.60; H NMR (600 MHz, DMSO-d6) δ (ppm) 10.90 (br s,
anal: N5.23 5.13; C58.28 57.91; H4.70
1H, HCl), 8.14 – 8.04 (m, 2H, X-1, X-8), 8.01 (br d, J = 7.4, 1H, X-6), 7.86 – 7.73 (m, 1H, X-4),
7.67 (br d, J = 8.0, 1H, X-3), 7.43 (br t, J = 7.9, 1H, X-7), 7.23 (br s, 1H, PIPER-4 ), 7.00 (br s,
1H, PIPER-7), 6.92 (br d, J = 7.2, 1H, PIPER-6), 6.01 (br s, 2H, PIPER-2, (O-CH2-O)), 3.66 (br s,
2H, >N-CH2-PIP), 3.31 (s, 6H, X-CH2-N<, N<CH2, (piperazine)), 3.07–2.75 (m, 4H, CH2>N
(piperazine)); LC-MS [M + H]+ m/z: 463.24, 100.00%.
5-chloro-2-((4-(pyridin-2-yl)piperazin-1-yl)methyl)-9H-xanthen-9-one trihydrochloride (7). Light
calc
beige solid; yield: 61%; C23H23N3O2Cl4; M = 513.05; mp 281-283 °C; el.an.
/
anal: N8.16
/
;
8.22
C53.61 54.61; H4.50 4.71;1H NMR (600 MHz, CDCl3) δ (ppm): 8.27-8.22 (m, 2H, X-1, pyr-6); 8.18-
/
/
8.14 (m, 1H, X-8); 7.86-7.82 (m, 1H, X-6); 7.77 (d, J = 8.02, 1H, X-4); 7.59 (d, J = 8.59, 1H, X-
3); 7.48-7.41 (m, 1H, pyr-4); 7.30 (t, J = 7.73, 1H, X-7); 6.64-6.57 (m, 2H, pyr-3,5); 3.66 (s, 2H,
2X-CH2); 3.58-3.50 (m, 4H, Pip-N1-(CH2-CH2)2-N4); 2.58 (br t, J = 4.87, 4H, Pip-N1-(CH2-
CH2)2-N4). LC-MS [M + H]+ m/z: 406.128, 100.00%.
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4-chloro-5-methyl-9H-xanthen-9-one (8) Light yellow solid; yield: 71%; C14H9ClO2; M= 244.03;
mp 116.8-117.7°C 1H NMR (500 MHz, DMSO-d6) δ ppm 8.00 (dd, J=8.02, 1.43 Hz, 1H, X-H8),
7.90 (dd, J=7.73, 1.72 Hz, 1H, X-H1), 7.80 (d, J=1.15 Hz, 1H, X-H2), 7.57 (dd, J=8.73, 2.15 Hz,
1H, X-H6), 7.44 (d, J=8.59 Hz, 1H, X-H3), 7.34 (t, J=7.88 Hz, 1H, X-H7), 2.34 (s, 3H, -CH3); 13C
NMR (126 MHz, DMSO-d6) δ ppm 175.80 (s, 1C, C=O), 153.84 (s, 1C, X-C4a), 151.39 (br s, 1C,
X-C4b), 137.38 (br d, J=3.62 Hz, 1C, X-C3), 135.64 (br s, 1C, X-C6), 134.90 (br s, 1C, X-C7),
125.69 (s, 1C, X-C4), 125.47 (s, 1C, X-C2), 124.87 (s, 1C, X-C8), 122.95 (d, J=3.02 Hz, 1C, X-
C8a), 122.11 (s, 1C, X-C5), 120.81 (br s, 1C, X-C8b), 118.48 (br s, 1C, X-C1), 20.83 (s, 1C, -
CH3); LC-MS [M + H]+ m/z: 245.04.128, 100.00%.
4-chloro-5-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride (9).
calc
White solid; yield: 76%; C20H23N2O3Cl3; M=445.77; el.an.
/
anal: N6.28
/
6.22; C53.89
/
54.71; H5.20
/
;
5.60
1
mp 291-293 °C; H NMR (600 MHz, D2O) δ (ppm): 7.89 (d, J = 8.0, 1H, X-1), 7.81 (d, J = 7.4,
1H, X-8), 7.67 (d, J = 8.0, 1H, X-3), 7.65-7.61 (m, 1H, X-6), 7.31 (t, J = 7.7, 1H, X-7), 7.12 (t, J =
7.7, 1H, X-2), 4.45 (s, 2H, X-CH2-N<), 3.81-3.77 (m, 2H, >N-CH2-CH2-OH), 3.68 -3.45 (br s, 8H,
-CH2- (pip)), 3.31-3.27 (m, 2H, >N-CH2-CH2-OH). LC-MS [M + H]+ m/z: 373.15, 99.21%.
4-chloro-5-((4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one
dihydrochloride (10). White solid; yield = 75%; C22H27N2O4Cl3; M = 489.82; mp 281-283 °C;
el.an. calc anal: N5.72 5.60; C53.95 54.36; H5.56 5.81; 1H NMR (600 MHz, D2O) δ (ppm): 7.80 (br d, J = 7.4,
/ / / /
1H, X-1), 7.78 (br d, J = 8.6, 1H, X-8), 7.53 (t, J = 7.2, 2H, X-3, X-6), 7.26 (t, J = 7.7, 1H, X-7),
7.02 (t, J = 7.7, 1H, X-2), 4.45 (s, 2H, X-CH2-N<), 3.76–3.71 (m, 2H, -O-CH2-CH2-OH), 3.68–
3.58 (m, 8H, CH2- (pip)), 3.58 (br d, J = 4.6, 2H, >N-CH2-CH2-O-), 3.51–3.48 (m, 2H, -O-CH2-
CH2-OH), 3.41–3.37 (m, 2H, >N-CH2-CH2-O-). LC-MS [M + H]+ m/z: 417.29, 100.00%.
4.2. Microbiology
The bacteria used are Staphylococcus epidermidis ATCC 12228 (Winslow and Winslow) and
clinical strain K/12/8915 obtained from the collection of the Department of Pharmaceutical
Microbiology, Faculty of Pharmacy, Jagiellonian University Medical College. Strains were
exposed to the newly synthetized compounds using dilution method in a 96-well polystyrene flat-
bottomed microtiter plates. Protocols for MIC (Minimum Inhibitory Concentration)
determinations
were
based
on
the
EUCAST
guidelines
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(http://www.eucast.org/ast_of_bacteria/media_preparation/). The MIC value of the newly tested
compounds was determined in CAMHB II broth (Cation-Adjusted Mueller-Hinton Broth, BD
BBLTM). New synthetized compounds were dissolved in DMSO (1% of the final volume, Merck)
and diluted with culture broth to a concentration of 50 μg/mL. Further 1:2 serial dilutions were
performed by addition of culture broth to reach concentrations ranging from 50 to 0.1 μg/mL. In
the assay, control of the Cation-Adjusted Mueller-Hinton Broth (CAMHB II) with the new
compounds was also performed and no increase in the absorbance value was observed compared
to the control of the broth itself. The reference chemotherapeutic agents: linezolid, ciprofloxacin,
sulfamethoxazole in combination with trimethoprim were purchased from Merck and dissolved in
DMSO. The study carried out quality control of sensitivity determination for linezolid,
ciprofloxacin and trimethoprim / sulfometaxazole against the Staphylococcus aureus ATCC 29213
control strain (internal quality control EUCAST). The plates were incubated at 37 °C for 18 to 20
h with shaking and the optical density was read in a microtiter plate reader SunriseTM (Tecan,
Switzerland) at 600 nm. MICs for antimicrobial compound-treated cultures were calculated as the
lowest concentration of compound/drug at which growth was not apparent, as measured by optical
density at 600 nm. All experiments were performed at least in triplicate and the average MIC was
listed as the MIC in data Table 1.
4.3 Genotoxicity
Potential genotoxicity was tested with a use of commercially available UMU-Chromotest kit
(EBPI, environmental bio-detection products inc., Canada, http://www.ebpi.ca, accessed 06
February 2020; ISO 13829: Water quality-determination of the genotoxicity of water and waste
water using the umu-test, 2003-03-15). All reagents and solvents were provided with the kit.
Photometer Eppendorf BioPhotometer plus LabtechLT-4000 Microplate Reader were used for
absorbance measurements. Experiments were carried out according to a procedure attached to the
kit (UMU-Chromotest Procedure, Version 6.6).
Briefly, one day before the experiment dried bacteria (engineered Salmonella typhimurium
strain) were rehydrated and incubated overnight at 37 °C. After 18 h the previously prepared
bacterial culture showed OD600 reading approximately 0.5. After dilution by half with a growth
medium, it was placed in an incubator for additional 1.5 h (37 °C). In the meantime, a test
microplate A has been prepared. Stock solutions of tested compounds were prepared in 10%
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DMSO in saline. Subsequently, they were pipetted on a microplate and diluted with distilled water
in order to obtain final concentrations (each tested in triplicate). 10% DMSO in saline served for a
solvent control, water for a negative control, 4-NQO solution (included in the kit) for a positive
control, while appropriate amounts of distilled water and growth media served for blank. Finally,
each well was supplemented with 20 µL of growth media enriched with glucose. After 1.5 h
incubation, bacterial culture were pipetted to all wells (excluding blank) and the microplate A was
incubated at 37 °C for 2 h. Then, 30 µL from each well of microplate A was pipetted into the
corresponding well of microplate B and diluted tenfold with growth media enriched with glucose.
Subsequently, microplate B was incubated at 37 °C for 2 h and after that time absorbance at 600
nm was measured in order to calculate growth factor (G = mean A600 of tested sample – mean A600
of blank / mean A600 of negative control – mean A600 of blank, final results considered valid if
G>0.5). Then, microplate C was prepared by adding to each well: 270 µL of B-buffer, 30 µL from
a corresponding microplate B well and 30 µL of o-nitrophenyl-ß-galactoside solution in phosphate
buffer. Plate C was immediately placed in an incubator at 37 ℃ for 30 min, after which time
yellow colour developed in a positive control wells and 120 µL of Stop Solution was added to all
wells of microplate C. The absorption of the solution in each well was measured at 420 nm. β-
galactosidase activity (Us = mean A420 of tested sample – mean A420 of blank / mean A420 of
negative control – mean A420 of blank) and induction ratio (IR = 1/G*Us) were calculated for
samples with G > 0.5. If IR > 1.5, a compound was considered genotoxic in a particular
concentration.
4.4. Metabolism prediction
The prediction of metabolism was performed with use of MetaSite 6.0.1 x64 (Molecular
Discovery), version built on March 20th 2018.
Conflicts of interest
There are no conflicts to declare.
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The data that supports the findings of this study are available in the supplementary material of this
article.
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