Potential of 2-Chloro-N-(4-fluoro-3-nitrophenyl)acetamide against Klebsiella pneumoniae and in Vitro Toxicity Analysis

molecules

Article

Potential of 2-Chloro-N-(4-ﬂuoro-3-

nitrophenyl)acetamide Against Klebsiella pneumoniae

and In Vitro Toxicity Analysis

Laísa Cordeiro * , Hermes Diniz-Neto

, Pedro Figueiredo , Helivaldo Souza

,

Aleson Sousa ¹ , Francisco Andrade-Júnior , Thamara Melo , Elba Ferreira

Petrônio Athayde-Filho , José Barbosa-Filho ¹ , Abrahão Oliveira-Filho ³and Edeltrudes Lima

1

, Rafael Oliveira ,

2

1

Department of Pharmaceutical Science, Health Sciences Center, Federal University of Paraíba,

5

8033-455 João Pessoa, Paraíba, Brazil; hermes.dn@hotmail.com (H.D.-N.);

pedrotrﬁgueiredo@gmail.com (P.F.); aleson_155@hotmail.com (A.S.);

juniorfarmacia.ufcg@outlook.com (F.A.-J.); th.rmelo@outlook.com (T.M.); elbaferreira99@gmail.com (E.F.);

jbarbosa@ltf.ufpb.br (J.B.-F.); edelolima@yahoo.com.br (E.L.)

Chemistry Department, Exact and Natural Sciences Center, Federal University of Paraíba,

2

3

5

8033-455 João Pessoa, Brazil; helivaldog3@gmail.com (H.S.); rfarias.quimica@gmail.com (R.O.);

athayde-ﬁlho@quimica.ufpb.br (P.A.-F.)

Rural Health and Technology Center, Federal University of Campina Grande, 58708-110 Patos, Brazil;

abrahao.farm@gmail.com

*

Correspondence: laisavilar@gmail.com; Tel.: +55-83-3216-7347

Received: 9 July 2020; Accepted: 21 July 2020; Published: 31 August 2020

Abstract: Klebsiella pneumoniae causes a wide range of community and nosocomial infections.

The high capacity of this pathogen to acquire resistance drugs makes it necessary to develop

therapeutic alternatives, discovering new antibacterial molecules. Acetamides are molecules

that have several biological activities. However, there are no reports on the activity of

2-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide. Based on this, this study aimed to investigate the

in vitro antibacterial activity of this molecule on K. pneumoniae, evaluating whether the presence of

the chloro atom improves this eﬀect. Then, analyzing its antibacterial action more thoroughly, as well

as its cytotoxic and pharmacokinetic proﬁle, in order to contribute to future studies for the viability of

a new antibacterial drug. It was shown that the substance has good potential against K. pneumoniae

and the chloro atom is responsible for improving this activity, stabilizing the molecule in the target

enzyme at the site. The substance possibly acts on penicillin-binding protein, promoting cell lysis.

The analysis of cytotoxicity and mutagenicity shows favorable results for future in vivo toxicological

tests to be carried out, with the aim of investigating the potential of this molecule. In addition,

the substance showed an excellent pharmacokinetic proﬁle, indicating good parameters for oral use.

Keywords: 2-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide; Klebsiella pneumoniae; antibacterial; toxicity;

minimum inhibitory concentration

1

. Introduction

The Klebsiella pneumoniae bacteria, belonging to the Enterobacteriaceae family, are Gram-negative,

facultative, immobile, and encapsulated anaerobes. It is one of the opportunistic pathogens of greatest

clinical relevance, known to be associated with nosocomial infections. It is estimated that this species,

alone, is responsible for approximately one third of all infections caused by Gram-negative bacteria in

general, causing, for example, pneumonia, urinary tract infections, bacteremia, endocarditis, and liver

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abscesses. This pathogen is also involved in serious community infections, such as necrotizing

pneumonia, pyogenic liver abscesses, and endogenous endophthalmitis [1,2].

In addition to being a highly prevalent species, the appearance of hypervirulent strains of

K. pneumoniae has further expanded the number of people susceptible to infections by this species,

increasing, for example, the number of cases in immunocompetent individuals. The species is also

an important source of resistance to antibiotics, presenting a great capacity to acquire plasmids with

genes that give them resistance to multiple antibacterials [2]. As explained by Paczosa and Mecsas [1],

this pathogen is gaining more notoriety, since the increase in the number of serious infections and the

growing scarcity of eﬀective medications bring a great challenge in the treatment of these diseases.

In 2017, the World Health Organization (WHO) [3] released for the ﬁrst time a list of resistant

microorganisms that pose a threat to human health and for which there is a priority need for the

development of new antibiotics. The experts used as a basis for the construction of this document

criteria such as mortality, prevalence of resistance, and transmissibility. The list was divided into

three levels of need for antibiotic development: critical, high, and medium. Compounding the critical

group are Gram-negative bacteria, more speciﬁcally Acinetobacter baumannii, Pseudomonas aeruginosa

and Enterobacteriaceae—which include K. pneumoniae—resistant to third-generation carbapenemas

and cephalosporins [3].

One of the objectives of the list released by the WHO is to guide research strategies for

new antibacterials. In recent years, experts have already warned of the diﬃculty in combating

Gram-negatives, especially [4]. Quinolones, discovered in 1962, were the last new class of antibiotic

with activity against these bacteria. Of the 44 antibacterials under study for development, currently

only 15 have some type of action against Gram-negative and only ﬁve have progressed to a phase 3

clinical study, but all of these are substances resulting from molecular modiﬁcations of drugs already

on the market [

emphasize that we urgently need to develop new alternatives for the treatment of these infections [

The amide group is widely found and is of fundamental importance for the biological and material

properties of a vast number of compounds created by man and present in nature [ ]. As Aschale [10

5,

6]. Health authorities around the world are looking for solutions to this crisis and

7,8

].

9

]

explains, there has been considerable interest in making substitutions for acetanilide derivatives due

to this group being present in several synthetic drugs related to a wide range of biological activities,

such as antibacterial, antiviral, antifungal, antihelmintic, analgesic, and anti- inﬂammatory activities.

The acetamides are widely used as herbicides in agriculture, showing potent activity against

pests [11]. There are some reports of acetamide derivatives as antimicrobial agents. Katke, Amrutkar,

and Khairnar [12] demonstrated excellent antibacterial and antifungal activity for these compounds,

Patel et al. [13] synthesized an acetamide from coumarin and obtained a product with good activity

against several strains, including Mycobacterium tuberculosis. Jetti, Chidurala, and Meshram [14

]

demonstrated that the combination of an acetamide with monobactamic antibiotic showed good

activity against Gram-positive and negative.

In the acetamide group, the presence of the chloro atom seems to improve the antimicrobial activity

of these molecules. In a study by Bravo et al. [15] the antimicrobial activity of N-(2-hydroxyphenyl)

acetamide against Candida albicans was evaluated. As a result, the absence of activity against this

microorganism was observed. On the other hand, the same substance with the addition of a chloro

atom (2-chloro-N-(2-hydroxyphenyl) acetamide) was able to inhibit 96.6% of C. albicans strains. Thus,

the addition of chlorine to the alpha carbon caused the molecule to show activity against these yeasts.

The N-(4-ﬂuoro-3-nitrophenyl)acetamide (A1) and 2-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide

(A2) are substances commonly used as reaction intermediates [16,17], about which there are still no

reports in the scientiﬁc literature on their biological activities. In view of the antimicrobial potential of

acetamides and chloroacetamides, and the urgent need to develop new therapeutic alternatives for

the treatment of infections caused by K. pneumoniae, this study aimed to investigate the antibacterial

activity of these molecules on K. pneumoniae, evaluating whether the presence of the chloro atom

improves this eﬀect. Then, the most promising molecule was investigated, analyzing its antibacterial

Molecules 2020, 25, 3959

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action more thoroughly, as well as its cytotoxic and pharmacokinetic proﬁle, in order to contribute to

future studies for the viability of a new antibacterial drug.

2

. Results and Discussion

2

.1. Chemistry

The target molecules A1 and A2 were synthesized using speciﬁc acylating agents for each one

(Figure 1). The compound N-(4-ﬂuoro-3-nitrophenyl)acetamide (A1) was obtained with 85% yield

from the acetylation reaction of 4-ﬂuoro-3-nitroaniline with acetic anhydride at room temperature [16].

The compound 2-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide (A2) was obtained from the reaction

between 2-chloroacetyl chloride and 4-ﬂuoro-3-nitroaniline in the presence of triethyamine (Et N),

3

with a yield of 80% [17]. All compounds were puriﬁed using the recrystallization method and their

purities were conﬁrmed by the melting point.

Figure 1. Synthetic route for the synthesis of the target molecules. (A1): N-(4-ﬂuoro-3-nitrophenyl)

acetamide, (A2): 2-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide. Reagents and conditions: (i) Ac O, rt,

2

◦

4 h, 85%; (ii) 2-chloroacetyl chloride, Et N, DCM, 0 C to rt, 20 h, 80%.

3

2

The structures of the compounds A1 and A2 were characterized by infrared (IR), H and ¹³C

nuclear magnetic resonance (NMR) spectroscopy (Supplementary Materials). In the hydrogen spectra

of the compounds, the proton signal attributed to N-H can be observed in the region between 8.47

and 10.37 ppm. For compound A1, a signal assigned to three methyl protons in the form of a singlet

referring to the acetyl group is observed at 2.07 ppm. In the spectrum of compound A2 was observed a

1

referring singlet for two methylenic protons (CH ) at 4.22 ppm. In all compounds, aromatic protons

2

are in the range of 7.29–8.48 ppm. In the carbon spectra of the synthesized compounds, we observed

the characteristic signal of amide carbonyl in the region of 164.53–168.97 ppm. In the spectrum of

compound A1 the signal at 23.89 ppm refers to the methyl carbon of the acetyl group. In compound A2

a signal referring to methylene carbon (CH ) at 42.81 ppm was observed. In all compounds, the signals

2

attributed to aromatic carbons were found at 115.27–152.44 ppm.

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−1

In the infrared (IR) spectra of all compounds, we can observe stretches of approximately 1678 cm

referring to the secondary amide carbonyl. For all compounds, the N-H stretching occurred around

−

1

−1

H for all compounds showed absorption values close to 1610 cm .

3

300 cm . Folding bands of N

−

The asymmetric and symmetrical stretches related to the NO group of all molecules showed absorption

values in the region of 1529 cm and 1332–1338 cm , respectively. The C-F stretches of aromatic

compounds occurred around 1238 cm

2

−

1

−1

.

2

.2. Minimum Inhibitory Concentration (MIC) Determination

Two acetamide derivatives were synthesized: N-(4-ﬂuoro-3-nitrophenyl) acetamide (A1) and

-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide (A2) (Figure 1), in order to assess which molecule, among

2

these, has the best antibacterial activity on K. pneumoniae and whether the presence of the chloro atom

in the molecule is capable of interfering with biological activity. The only structural diﬀerence between

these molecules is at carbon 2 of the amide portion, where in A2 there is a chloro atom, and in A1 this

atom is absent.

The previous screening for the antibacterial activity evaluation of the molecules was done by

determining the minimum inhibitory concentration (MIC). It was possible to observe that A1 presented

MIC of 1024 µg/mL for 100% of the K. pneumoniae strains used in this study, while A2 resulted in a MIC

of 512 µg/mL for the same strains (Table 1).

Table 1. Minimum inhibitory concentration (MIC) of A1 and A2 against K. pneumoniae strains.

MIC (µg/mL)

K. pneumoniae Strains

A1

A2

ATCC-700603

KP-25

1024

512

KP-26

KP-56

KP-83

KP-87

KP-138

KP-143

KP-166

KP-173

KP-176

KP-260

KP-326

The results of the antimicrobial activity suggest that the chloro atom is directly related to the

potency of the antibacterial activity observed, since the addition of this atom (A2 substance) made its

antibacterial activity twice as potent as that of its precursor (A1 substance).

A study by Katke, Amrutkar, and Khairnar [12] investigated the antibacterial potential of thirteen

diﬀerent acetamides using the agar diﬀusion methodology. As results, they observed zones of inhibition

ranging between 7 and 30 mm against Escherichia coli, 16–35 mm against Pseudomonas aeruginosa and

10–36 mm on Staphylococcus aureus. Only two of the thirteen substances evaluated did not show

antibacterial activity for any strain tested, showing the excellent biological potential of acetamides.

In addition, a study by Bravo et al. [15] showed that the presence of the chloro atom seems to

improve the antimicrobial activity of these molecules. The authors evaluated the antimicrobial activity

of N-(2-hydroxyphenyl) acetamide against Candida albicans was evaluated and the substance did not

have the ability to inhibit fungal growth. However, the same substance with the addition of a chloro

atom (2-chloro-N-(2-hydroxyphenyl)acetamide) was able to inhibit 96.6% of C. albicans strains. Thus,

the addition of chloro to the alpha carbon was essential for the biological activity of the molecule,

similarly to the results obtained in this study.

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At the present work, the A1 and A2 MICs against K. pneumoniae show a moderate to weak

antibacterial activity of these acetamides. However, it is important to note that there are no reports

of antimicrobial activity of these substances and the results indicate their potential applicability for

use against bacterial strains, being a starting point for new structural changes to be made, aiming

to improve this biological activity. It is interesting that more analyzes are carried out on the action

of these substances against other bacterial species, since the results may vary and even be more

favorable. Given the current need to discover new antibiotic pharmacological classes, it is interesting

that microbiological studies with this group of molecules are further explored.

Due to the A2 molecule demonstrated greater potential against K. pneumoniae, it was selected as

the object of study in the present work, to better evaluate its antibacterial and cytotoxic eﬀect.

2

.3. Molecular Docking Analysis

To understand what is the possible molecular target on which the A1 and A2 molecules act

to exert the observed antibacterial eﬀect, as well as to better investigate the inﬂuence of the chloro

atom to anchor the molecule in the active site of the target enzyme, molecular docking analyzes

were performed.

The accuracy of molecular docking was validated through re-docking, where it was observed that

the RMSD value for the analyzed enzymes remained within the acceptable range 0–2 Å [18]. Table 2

shows the binding energies of the two molecules with the chosen enzymes. The A2 substance showed

better binding energies compared to A1, for all tested enzymes, showing that the chloro helps the

molecule to have a more eﬀective anchoring in diﬀerent active sites.

Table 2. Binding energies (Moldock score) of PDB enzymes and tested compounds.

Moldock Score

RMSD

(Å)

−1

)

Enzyme

Classiﬁcation

(kcal·mol

kcal·mol⁻

1

)

(

A1

A2

Glucosamine-6-Phosphate Synthase (2VF5)

FosA (5V3D)

Transferase

Hydrolase

−89.1

−62.3

0,18

0,19

0,35

0.33

0.34

0.24

0.47

0.40

−52.9

−42.6

−52.2

−35.7

−55.6

−47.5

−44.0

−54.8

−58.8

−46.5

−59.0

−37.3

−59.0

−55.2

−50.8

−56.8

PBP1b (5HLA)

β-lactamase (2ZD8)

PBP3 (3PBS)

FosAKP (6C3U)

DNA gyrase (1AJ6)

Topoisomerase IV (1S14)

−123.4

−82.5

Hydrolase

−165.5

−95.2

Transferase

Topoisomerase

Isomerase

−121.3

−154.9

Among all tested enzymes, A2 showed better binding energies for the enzymes penicillin binding

−

1

proteins (PBPs) 1 and 3, with

−

59.0 kcal mol for both enzymes. In addition to the values of binding

·

energy, another parameter of extreme importance is the interactions carried out with the active site of

the enzyme. With PBP1, the A2 and A1 molecules performed hydrogen bonding interactions with

the Thr701, Thr 699, Asn574, and Ser510 residues, together with several Van der Walls interactions

with other residues from the active site, as can be seen in Figure 2. However, the presence of chloro

in A2 is fundamental to better stabilize this molecule in the active site of the enzyme, due to Van der

Walls interactions with the Ser510 and Thr702 residues that are important for a good anchorage in this

active site [19]. These diﬀerences in anchoring reﬂect the potency of the antibacterial activity that was

observed previously, conﬁrming that the A2 molecule has a more pronounced antibacterial eﬀect.

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Figure 2. Two and three-dimensional representation of interactions performed by (

A) A1 and (B) A2 at

the PBP1 (5HLA) active site.

Thus, the results suggest that the A2 substance acts on the PBP enzyme. This enzyme is responsible

for maintaining the peptidoglycan layer of the bacterial cell wall, it acts by cross-linking the adjacent

peptide chains of immature peptidoglycan units. Since it plays a fundamental role in the survival of the

bacterial cell, this enzyme is the target of many antibacterial drugs, especially β-lactams. Molecules that

act by inhibiting cell wall synthesis can lead to changes in cell shape and size, induction of responses

to cell stress and, ultimately, cell lysis [20].

2

.4. Minimum Bactericidal Concentration (MBC) Determination

In order to better identify the mode of action of the A2 substance, the minimum bactericidal

concentration (MBC) was determined. As a result, it was observed that the MBC was 512 µg/mL for all

K. pneumoniae strains used in this study, as described in Table 3.

Table 3. Minimum bactericidal concentration (MBC) of A2 against K. pneumoniae.

A2

K. pneumoniae Strains

MBC (µg/mL)

Eﬀect

ATCC-700603

KP-25

512

Bactericide

KP-26

KP-56

KP-83

KP-87

KP-138

KP-143

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Table 3. Cont.

A2

K. pneumoniae Strains

MBC (µg/mL)

Eﬀect

KP-166

KP-173

KP-176

KP-260

KP-326

512

Bactericide

A bactericidal substance is capable of killing the bacterial cell, while bacteriostatic substances

inhibit or slow its growth without causing death. A drug is considered to exhibit bactericidal activity

when the ratio between MBC and MIC is

≤

4 [21,22]. Thus, since the MIC of A2 was 512 µg/mL and the

MBC was also 512 g/mL for all K. pneumoniae strains, there is a 1:1 proportion. Therefore, the substance

µ

A2 acted in a bactericidal manner, from the MIC.

Data on the classiﬁcation of a given substance as bacteriostatic or bactericidal can provide valuable

information on the potential action of antibacterial agents in vitro. However, these concepts are

applicable to experiments in standardized conditions in vitro and can vary according to the type of

bacteria, amount of inoculum and duration of the test. These variations are, therefore, observed in

clinical practice, where the conditions found are as varied as possible [22]. Thus, it is necessary to

combine this information with the pharmacokinetic and pharmacodynamic data of this acetamide,

to provide a more signiﬁcant prediction of in vivo eﬃcacy.

2

.5. Time-Kill Kinetics Against K. pneumoniae

To assess the time of action of A2 on K. pneumoniae, time-kill kinetics was performed, evaluating

the number of colony-forming units (CFU) over 24 h of cell exposure to the substance. As shown in

Figure 3, after 10 h of treatment with A2 it is possible to observe the total reduction of viable cells.

The same eﬀect is observed after 6 h in the presence of A2 in the concentration corresponding to

2

×

MIC. Bactericidal activity was deﬁned as a 3-log reduction in bacterial counts (log₁₀CFU/mL) [23],

≥

therefore, these results conﬁrm the data observed in the determination of the minimum bactericidal

concentration, proving that A2 acts as a bactericide agent, from the MIC.

Figure 3. Time-kill curve for K. pneumoniae (

and 2× MIC concentrations.

A) ATCC-700603 and (B) KP-56 treated with A2 at MIC

Although A2 MIC and 2 MIC are bactericidal, the time-kill kinetics is reduced with increasing

×

concentration, as observed in the dead time curve (Figure 3). Thus, the higher the concentrations of A2

substance, the shorter the time required to obtain the bactericidal eﬀect.

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2

.6. Eﬀect on Bacterial Cell Integrity

The analysis of molecular docking suggests that A2 acts by inhibiting the PBP enzyme, responsible

for maintaining the bacterial cell wall. Agents acting on this target cause a series of destabilizations,

which result in cell lysis [20] and consequent release of cytoplasmic content. In these cases, cytoplasmic

ions such as potassium and phosphate are ﬁrst released to the external environment, followed by

larger molecules such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), in addition to

other materials. The nucleotides have a strong absorption at 260 nm and, therefore, it is possible to

measure cell lysis through UV absorption of the supernatant of cells treated with the antibacterial

substance [24,25].

The Figure 4 shows the results when K. pneumoniae is treated with diﬀerent concentrations of A2

(

MIC and 2 MIC) compared to untreated cells (negative control). After 10 h of treatment with A2 at

×

MIC, there is a signiﬁcant release of cytoplasmic contents. Cell lysis is also evident after six hours of

A2 action at a concentration corresponding to 2× MIC.

Figure 4. The eﬀect of A2 on bacterial cell integrity. Release of 260 nm absorbing material from K.

pneumoniae suspensions treated with MIC and 2 MIC of A2 for 10 h and 6 h, respectively. Statistical

×

analysis compared to negative controls (NC): **** p ≤ 0.0001.

The results indicated that irreversible damage to K. pneumoniae cells can occur, leading to the

loss of cellular constituents, such as nucleic acid and some essential molecules, causing bacterial lysis.

These results reinforce the previous data, conﬁrming the bactericidal eﬀect of A2 and its possible action

on PBP enzyme.

2

.7. Hemolytic Activity

The toxicity of bioactive substances must be assessed to ensure less toxic eﬀect on the system to

which it is intended for therapeutic application. In this context, tests with in vitro cytotoxicity screenings

are a good alternative to assess the toxic potential of substances. In a study by Rangel et al. 26],

[

red blood cells were used to determine the toxicity of promising drugs. Its eﬀect was classiﬁed by

percentage considering: low (0% and 40%), moderate (40% and 80%), and high above (80%).

The analysis of the hemolytic potential of the A2 molecule showed a low hemolysis, being

approximately 4–20% at 50–500

µg/mL concentrations, in all tested blood types. The highest

concentration of the tested molecule (1000

µg/mL) against the erythrocytes of the ABO system

demonstrated a percentage of lysis that varied according to blood type. On types A and B, moderate

hemolysis was obtained, ranging from 37–47%. However, for type O, a low percentage of hemolysis

was observed, with an average of 8% of erythrocyte lysis, as can be seen in Figure 5.

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Figure 5. Cytotoxic eﬀect of A2 on red blood cells (RBC) of the diﬀerent blood types of the ABO system:

) type A, ( ) type B, and ( ) type O. NC: negative control. PC: positive control. Statistical analysis

compared to positive control: *** p ≤ 0.001.

(

A

B

C

The results suggest that A2 molecule has less potential for hemolysis in the absence of surface

carbohydrates from red cell membranes, such as N-acetylgalactosamine (serotype A) and d-galactose

(

serotype B), since the assay revealed a better interaction of the molecule in order O < B < A blood types.

Other acetamide derivatives with signiﬁcant biological activity also demonstrate low cytotoxic

potential. In studies carried out by Gentile and Calabrese [27] evaluating the in vitro toxicity of

acetamide-derived molecules with potential herbicide, the production of oxidative stress against the

erythrocytes of rats, sheep, and dogs was not observed. The test describes, in addition to low levels of

hemolysis, the reduction in oxidative stress of the test on the formation of MetHb. Likewise, in the

present study, the results indicate that the A2 molecule has a low toxicological eﬀect when tested in

human red blood cells.

2

.8. Antihemolytic Activity

The anti-hemolytic evaluation of the A2 molecule showed a percentage of hemolysis that is

classiﬁed as moderate (>40% and <80%) [26], for all concentrations evaluated in the three blood types

of the ABO system, except for the concentration of (1000 g/mL), which presented hemolysis (>80%) in

µ

type B red blood cells. The osmotic protection performance was also perceived with an increase in the

concentration of the tested substance, where, the higher the concentration tested, the better the red cell

protection eﬀect, as can be seen in Figure 6.

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Figure 6. Anti-hemolytic eﬀect of A2 on red blood cells (RBC) of the diﬀerent blood types of the ABO

system: ( ) type A, ( ) type B, and ( ) type O. NC: negative control. PC: positive control. Statistical

analysis compared to positive control: *** p ≤ 0.001; ** p ≤ 0.01.

A

B

C

The concentration of 500 µg/mL of the A2 molecule revealed a moderate potential to promote

the stability and protection of the erythrocytes of the ABO system. However, there was a variation in

lysis according to the blood type and concentration of the substance evaluated, in the following order

O < A < B.

According to a study by Autore et al. [28] acetamide derivatives may have antioxidant and

anti-inﬂammatory activity, revealing low toxicity potential in tests with liver cell lines. In this present

study, the anti-hemolytic test revealed a moderate percentage of lysis, suggesting a higher concentration

of the A2 molecule to achieve good levels of protection for red blood cells. However, more studies

need to be carried out to better investigate this protective activity.

2

.9. Mutagenic Eﬀect on Oral Mucosa Cells

In vitro genotoxicity studies evaluate the cytotoxic capacity of substances on certain cell types

isolated in liquid media. The possible action of toxicity is revealed through the damage caused to the

nucleus by the time of exposure to test substances. Cytotoxic eﬀects can be veriﬁed by suggestive

nuclear alterations, such as: micronucleus, binucleation, karyolysis, and cariorrexe [29].

The A2 genotoxicity test showed the appearance of few cellular alterations compatible with toxic

damage. The toxic ﬁndings were more present in the groups exposed to the H O solution (positive

2

control). The cells treated with diﬀerent concentrations of A2 showed few cellular changes compared

to the positive control, as can be seen in Table 4.

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Table 4. Genotoxic proﬁle of A2.

Group

Karyolysis

Karyorrhexis

Micronucleus

Binucleation

Normal

NC

1.50%

7.00%

5.00%

4.00%

3.00%

1.00%

2.00%

10.75%

7.00%

5.00%

4.00%

1.00%

2.75%

6.50%

2.00%

0.70%

0.50%

0.25%

1.00%

0.50%

0.00%

93.50%

77.75%

83.50%

86.80%

92.50%

97.50%

PC-H O₂

2

A2-1000 µg/mL

A2-500 µg/mL

A2-100 µg/mL

A2-50 µg/mL

NC: negative control. PC: positive control.

The data reveal that A2 showed low toxicity, with the presence of normal cells in amounts

greater than 80% in all tested concentrations. Among the nuclear alterations found, karyolysis,

and karyorrhexis were identiﬁed in greater quantities, although these indices are lower than those

found in the positive control, suggesting a safe threshold of low toxicity of the A2 molecule on the oral

mucosa cells.

Other studies, such as the one carried out by Moore et al. [30], point out that some acetamides

have low cytotoxic, clastogenic, aneugenic, or mutagenic potential, which reinforces the data found in

this work. However, in order to better evaluate the possibility of using A2 and its derivatives as an

antibacterial drug, further studies must be performed to ensure the safety of in vivo use.

2

.10. In Silico Analysis of Pharmacokinetic Parameters

In this work, the physical-chemical parameters of the A2 molecule were obtained through an

in silico approach using the free online software SwissADME to verify its theoretical absorption.

According to Table 5, the physical-chemical parameters analyzed according to the rules of Lipinski [31],

Ghose [32], Veber [33], and Egan [34] it is possible to predict whether the molecule under study has the

potential to be developed as a drug.

0

Table 5. In silico studies of Lipinski s parameters of terpinen-4-ol.

Parameters

Physicochemical Properties

A2

Formula

C8H6ClFN2O3

Molecular Weight

232.60 g/mol

Num. Heavy atoms

Fraction Csp3

15

0.12

4

1

54.33

74.92 Å

Num. Rotatable Bonds

Num. H-bonds acceptors

Num. H-bonds donors

Molar Refractivity

1

2

TPSA

Lipophilicity

Consensus ²Log Po/w³

1.33

Water Solubility

Log S (Ali)

Class

−2.30

4

Soluble

Druglikeness

5

Lipinski

Yes; 0 violation

0.55

6

Ghose

7

Veber

8

Egan

Bioavailability Score

1

TPSA: Topological Polar Surface Area; ²Consensus Log Po/w = Average of all ﬁve predictions; ³Log Po/w = The

4

partition coeﬃcient between n-octanol/water; Class = Ali class: insoluble <

soluble <

−

10 < poor <

−

6 < moderately <

−

4 <

5

−

2 < very < 0 < highly.; Lipinski = MM

≤

500; Log Po/w ≤ 5; H-bond donors

≤

5; H-bond acceptores

6

≤

10;. Ghose = 180

≤

MM

≤

480; 20

≤

No. of atoms

≤

70; 40

≤

Molar Refractivity

≤

130; -0.4

≤

Log Po/w ≤ 5.6;

7

2

8

2

Veber = Num. Rotatable Bonds ≤ 10; TPSA ≤ 140 Å ; Egan = Log Po/w ≤ 5.88; TPSA ≤ 131.6 Å .

Molecules 2020, 25, 3959

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The in silico approach showed that the substance A2 presents values of physicochemical parameters

within the acceptable region based on the rules of Lipinski [31], Ghose [32], Veber [33], and Egan [34].

The hydrogen acceptors calculated was 4 and that of hydrogen donors was 1 and are in accordance

0

with Lipinski s rule. To develop a molecule with the potential for a particular drug, the molecule must

be able to cross plasma membranes. Thus, the partition coeﬃcient of a substance in n-octanol and

water (Log P), is an important parameter. According to the rules of Lipinski (Log P_o_/_w

Log P_o_/_w5.6), and Egan (Log P_o_/_w5.8) the A2 molecule ﬁts the required conditions imposed, as its

Log P is 1.33.

≤

5), Ghose

(

≤

Another important factor for the development of a compound is the molar mass, since it is

related to the transport of the drug within the body. The molecule under study has a molar mass of

2

32.60 g/mol, which meets the criteria of Lipinski and Ghose. Other important criteria are the number

of rotatable connections and the value of the TPSA. According to Veber, molecules that have a number

of rotatable bonds 10 and TPSA values 140 Å, indicate that there is a high probability of being

≤

administered orally. According to Table 5, the molecule showed values of 4 for the number of rotatable

bonds and a TPSA of 74.92 Å2. As for solubility (Log S), it considers an important quality of a drug

for the absorption and distribution of the molecule in the body. According to the class presented in

Table 5, substance A2 is soluble, as it presented the Log S (coeﬃcient of solubility determined by the

ESOL method) with a value of −2.30.

Based on the above, in silico results show a favorable pharmacokinetic proﬁle for A2 substance.

Inadequate penetration of the infection site is one of the main factors related to the failure of

antibacterial therapies. The active drug needs to reach the bacteria in appropriate body ﬂuids and

tissues at concentrations necessary to kill or suppress pathogens’ growth. Even for substances with

good in vitro activity, the pharmacokinetic parameters are decisive for its clinical use [22]. Thus, A2

presents characteristics that suggest a good drug candidate and can display a high prospect of being

used orally, with excellent theoretical oral bioavailability and good solubility, which can guarantee

adequate absorption and distribution in vivo.

3

. Materials and Methods

3

.1. Chemistry

The reagents 4-ﬂuro-3-nitroaniline, triethylamine (Et N), acetic anhydride, chloroacetyl chloride,

3

®

chloroform, and ethanol were purchased from Merck/Sigma-Aldrich (Darmstadt, Germany).

The recrystallization technique was used to purify the compound and its purity was conﬁrmed from

the melting point measurement using the MQAPF-302 (Microqu

A Shimadzu spectrometer model IRPrestige-21 FT-IR (Barueri, S

í

mica, S

ão Paulo, Brazil) apparatus.

ão Paulo, Brazil) with an attenuated

total reﬂection (ATR) accessory produced the infrared (IR) spectrum. The hydrogen and carbon nuclear

magnetic resonance (NMR) spectra were obtained from a Bruker Avance 400 apparatus. The hydrogen

1

spectra ( H) were obtained at the frequency of 400 and 500 MHz using deuterated solvents chloroform

13

(

CDCl ) and dimethylsulfoxide (DMSO). The carbon spectra ( C) were obtained at the frequency

3

of 101 and 126 MHz using deuterated solvents chloroform (CDCl ) and dimethylsulfoxide (DMSO).

3

The chemical distances (

δ) were generated in parts per million (ppm) and in Hertz (Hz) the coupling

constants (J) were determined.

3

.2. Preparation of N-(4-ﬂuoro-3-nitrophenyl)acetamide (A1)

The 4-ﬂuro-3-nitroaniline (3.12 g, 20 mmol) is added, in small portions, in a 50 mL ﬂask containing

acetic anhydride (11 mL, 116 mmol) under constant stirring at room temperature. The reaction mixture

is stirred for 12 h. At the end of the reaction, ice water is added and the reaction mixture was stirred

for 30 min. The precipitate formed was ﬁltered, washed with water and dried. The product was

◦

recrystallized from an ethanol/water mixture (8:2). Yield: 85%, brown solid. m.p.: 142–144 C (Lit. [16]:

◦

−1

140–141 C). IV (ATR):

ν

/cm 3356 (N-H), 3124, 3064 (C-HAr), 1678 (C=O), 1610 (N-H), 1529 and

Molecules 2020, 25, 3959

13 of 19

1

332 (NO ), 1482 (C=C), 1238 (CAr-F), 1155, 1141 (C-HAr), 833 (C-N for ArNO2). 1H NMR (400 MHz,

2

DMSO-d₆)

δ

10.37 (s, 1H, NH), 8.48 (dd, J = 6.9, 2.7 Hz, 1H, CHAr), 7.81 (ddd, J = 9.1, 3.9, 2.8 Hz, 1H,

168.97,

50.19 (J = 257.8 Hz), 136.31 (J = 7.8 Hz), 136.05 (J = 3.3 Hz), 126.23 (J = 8.1 Hz), 118.75 (J = 21.8 Hz),

15.27 (J = 3.3 Hz), 23.89 [16].

CHAr), 7.50 (dd, J = 11.2, 9.1 Hz, 1H, CHAr), 2.07 (s, 3H, CH3). 13C NMR (101 MHz, DMSO)

1

δ

3

.3. Preparation of 2-Chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide (A2)

The chloroacetyl chloride (2.71 g, 24 mmol) is added slowly in a 50 mL ﬂask containing a mixture

of 4-ﬂuoro-3-nitroaniline (1) (3.12 g, 20 mmol) and Et N (3, 3 mL, 0.024 moles) solubilized in 20 mL

3

◦

of CHCl at 0 C in an ice bath. At the end of the addition of chloroacetyl chloride, the ice bath is

3

removed and the reaction is stirred at room temperature. After 20 h of reaction, the reaction mixture

was subjected to extraction. The organic phase is washed with water (3

dried over anhydrous sodium sulfate and then concentrated under reduced pressure, providing the

×

30 mL), brine (3

×

30 mL),

precipitate. The material was puriﬁed using a mixture of ethanol/water (9:1) in a recrystallization

◦

−1

process. Yield: 80%, brown solid, m.p.: 91–93 C (Lit. [17]: 90–92 C). IV (ATR):

ν

/cm 3332 (N-H),

3

132, 3074 (C-H_A_r), 2943 (C-H

), 1678 (C=O), 1606 (N-H), 1529 and 1338 (NO ), 1492 (C=C), 1228

Aliph

1

2

(

C_A_r-F), 831 (C-N for ArNO ). H-NMR (500 MHz, CDCl ) δ 8.47 (s, 1H, NH), 8.32 (dd, J = 6.4, 2.8 Hz,

2

3

1

H, CH_A_r), 7.89–7.85 (m, 1H, CH_A_r), 7.29 (dd, J = 10.1, 9.1 Hz, 1H, CH_A_r), 4.22 (s, 2H, CH₂). ¹³C-NMR

164.53, 152.43 (J = 263.6 Hz), 133.44, 133.41, 127.13 (J = 8.1 Hz), 119.13 (J = 22.1 Hz),

17.55 (J = 2.9 Hz), 42.81 [17].

(

126 MHz, CDCl₃) δ

1

3

.4. Strains

The clinical isolates strains of K. pneumoniae were used in this study belong to the MICOTECA of

the Antibacterial and Antifungal Activity Research Laboratory of the Federal University of Para ba,

í

Brazil, which are: KP-25, KP-26, KP-56, KP-83, KP-87, KP-138, KP-143, KP-166, KP-173, KP-176,

KP-260, and KP-326. The American Type Culture Collection strain ATCC-700603 was used as controls.

For use in the assays, bacterial suspensions were prepared in 0.9% saline solution, from fresh cultures,

and adjusted to the McFarland standard 0.5 scale.

3

.5. Minimum Inhibitory Concentration (MIC)

To assess the antibacterial activity of substances A1 and A2, the minimum inhibitory concentration

against K. pneumoniae strains was determined. The substances were solubilized in dimethyl sulfoxide

DMSO) at 5% and Tween-80 at 2%, to obtain emulsions in the concentrations necessary for use in the

tests. The MIC determination was performed using the broth microdilution technique in a 96-well

plate to obtain diﬀerent concentrations of A1 and A2 35]. In parallel, controls for sterility, cell viability,

(

[

and interference of the vehicles used in the preparation of substance emulsions (DMSO and Tween-80)

were also performed. MIC is deﬁned as the lowest concentration capable of causing complete inhibition

◦

of bacterial growth after 24 h at 35 ± 2 C.

3

.6. Molecular Docking

The chemical structures of the compounds were designed using the software MarvinSketch 18.5,

their energies minimized in the program Hyperchem v. 8.0.3, using the molecular mechanics method

MM +) and the semi-empirical method AM1 (Austin Model 1) [36]. The enzymes were obtained from

(

the Protein Data Bank (https://www.rcsb.org /) together with their co-crystallized inhibitors, with PDB

ID: 2VF52 (2.9 Å) [37], 5V3D3 (1.54 Å) [38], 5HLA4 (1.7 Å) [19], 2ZD85 (1.05 Å) [39], 3PBS6 (2.00 Å) [40],

6C3U7 (1.85 Å) [41], 1AJ68 (2.30 Å) [42], 1S149 (2.00 Å) [43]. Molecular docking was performed at

Molegro Virtual Docker (MVD) software (v 6.0.1, Molegro ApS, Aarhus, Denmark), using the standard

parameters of the software, the water molecules were removed and a template was generated in the

co-crystallized inhibitor of the PDB.

Molecules 2020, 25, 3959

14 of 19

3

.7. Minimum Bactericidal Concentration (MBC)

After MIC reading, the A2 minimum bactericidal concentration was determined by removing

aliquots from the microdilution plates in the wells corresponding to concentrations equivalent to MIC,

MIC, 4 MIC, and 8 MIC and inoculating in new plates containing only culture broth. All controls

2

×

were performed in parallel. MBC is deﬁned as the lowest concentration capable of causing complete

◦

inhibition of bacterial growth after 24 h at 35 ± 2 C [21,44].

3

.8. Time-Kill Kinetics

The determination of the action time of A2 substance against K. pneumoniae (ATCC-700603 and

KP-56) was carried out exposing the microorganisms to concentrations equivalent to MIC and 2

during times 0, 2, 4, 6, 8, 10, and 24 h. After incubation on Mueller–Hinton agar, bacterial colonies were

counted. Bactericidal activity was deﬁned as a 3-log reduction in bacterial counts (log₁₀CFU/mL) [45].

×

MIC

≥

3

.9. Eﬀect on Bacterial Cell Integrity

The cell integrity can be assessed by releasing intracellular material, mainly nucleotides, which

absorb 260 nm wavelengths. The K pneumoniae strains ATCC-700603, KP-26, KP-56, and KP-143 were

7

used to perform this test. First, the bacterial inoculum was incubated in BHI broth (1

×

10 CFU/mL)

◦

overnight at 35

±

2 C. Then, centrifugation was performed at 400

×

g for 10 min. The cell pellet was

washed three times with sterile PBS (phosphate-buﬀered saline) and resuspended in PBS containing the

A2 substance at MIC and 2 MIC concentrations. As a negative control, suspensions that underwent

×

the same procedures, but without treatment with the substance, were used. The tubes corresponding

◦

to the MIC were incubated for 10 h at 35

±

2 C, and the tubes corresponding to the 2

×

MIC, for 6 h.

g for 10 min and the supernatants were

read on a spectrophotometer at 260 nm [46]. All analyses were performed in triplicate.

After incubation, the suspensions were centrifuged at 400

×

3

.10. Human Erythrocytes and Oral Mucosa Cells Collection

The tests were performed according to the recommendations established by the Code of Ethics of

the World Medical Association, after approval by the ethics committee of the Centro Universitário de

Patos (number: 3621284). Blood samples (types A, B, and O) and smears of oral mucosa were donated

by young, healthy adults.

3

.11. Hemolytic Activity

The red blood cell (RBC) samples from blood donors were diluted in 0.9% NaCl at a 1:30 ratio

and centrifuged at 2500 rpm for ﬁve minutes to obtain the 0.5% suspension free of white blood

cells and platelets. Then, the A2 substance was added to the red blood cell suspensions, at 50,

100, 500, and 1000 µg/mL concentrations. As controls, a suspension of red cells without treatment

(

0% hemolysis) were used as a negative control and a suspension of red cells treated with 1% Triton

◦

X-100 (100% hemolysis) as a positive control. The assay was incubated for one hour at 22

After that time, the samples were centrifuged at 700

spectrophotometry at a wavelength of 540 nm [26].

±

2 C.

×

g for 5 min and hemolysis was quantiﬁed by

3

.12. Antihemolytic Activity

The analysis of the osmotic fragility of the red cells was performed using previously prepared

red blood cell suspensions at 0.5%. Diﬀerent concentrations (50, 100, 500, and 1000

µ

g/mL) of the

◦

A2 substance were used for the treatment of 0.5% RBC suspensions for 1h at 22

were centrifuged at 2500 rpm for 5 min and the supernatant was discarded. The erythrocytes were

±

2 C. The samples

◦

resuspended in a hypotonic solution of 0.24% sodium chloride for one hour at 22

period, the samples were centrifuged at 700

quantiﬁed by spectrophotometry at 540 nm [47]. As controls, a suspension of untreated red blood

±

2 C. After this

g for 5 min and the hemolysis of the supernatant was

×

Molecules 2020, 25, 3959

15 of 19

cells was used as a negative control (0% hemolysis) and in a hypotonic solution as a positive control

100% hemolysis).

(

3

.13. Genotoxic Eﬀects

First, the oral mucosa cells were collected from the donors, which was done with a cyto-brush

(

smear collector) in the cheek area, and the material obtained was placed in 5 mL of 0.9% NaCl [48].

As controls, cells treated with hydrogen peroxide 0.0005% (positive control) and untreated cells

negative control) were used. The cells were washed twice with saline and centrifuged for 10 min at

00 g and kept in saline. After the third wash, the cells were exposed (ex vivo) to A2 substance at 50,

00, 500, and 1000 g/mL concentrations for 30 min. The material was centrifuged and the supernatant

−

(

4

1

×

µ

was discarded before smear preparation. The cells were homogenized in a vortex and placed on the

slides at room temperature for drying for 15 min. Then, they were ﬁxed with methanol: acetic acid

(

3:1) for 15 min and stained with 2% Giemsa. The cells were observed under an optical microscope and

about 1000 cells were analyzed on each slide [49,50].

3

.14. Statistical Analysis

The data were analyzed by the One-way Analysis of Variance (ANOVA) and by the Bonferroni

post hoc test, using the GraphPadPrism software (version 6.0 for Windows, San Diego, CA, USA).

The data were considered signiﬁcant when p < 0.05.

3

.15. In Silico Analysis of Pharmacokinetic Parameters

Violations of the rules of Lipinski [31], Ghose [32], Veber [33], and Egan [34] help to evaluate the

pharmacokinetic characteristics of drug candidate substances. The following parameters were evaluated

about A2 substance: Physicochemical properties, lipophilicity, water solubility, and druglikeness,

using the free online software SwissADME (www.swissadme.ch ). Such results were analyzed using

the rules of Lipinski [31], Ghose [32], Veber [33], and Egan [34].

4

. Conclusions

Based on the above, this study showed a good antibacterial potential for acetamides, especially

-chloro-N-(4-ﬂuoro-3-nitrophenyl) acetamide (A2), of which chloro is responsible for improving its

2

antibacterial activity, stabilizing the molecule at the site target enzyme. The substance possibly acts

on PBP, promoting cell lysis. The analysis of cytotoxicity and mutagenicity show favorable results

for future in vivo toxicological tests to be carried out, with the aim of investigating the potential of

this molecule. In addition, the substance showed an excellent pharmacokinetic proﬁle, indicating

good parameters for oral use. Given the need to discover new antibacterial drugs in order to have

alternatives against resistant infections, it is interesting that this substance serves as a starting point for

future molecular changes aimed at improving its biological activity.

Supplementary Materials: The following are available online. Figure S1. FTIR (ATR) spectrum of A1 substance;

Figure S2. 1H NMR spectrum (400 MHz, DMSO-d6) of A1 substance; Figure S3. 13C NMR spectrum (101 MHz,

DMSO-d6) of A1 substance; Figure S4. FTIR (ATR) spectrum of A2 substance; Figure 5. 1H NMR spectrum

(500 MHz, CDCl3) of A2 substance; Figure S6. 13C NMR spectrum (126 MHz, CDCl3) of A2 substance.

Author Contributions: Conceptualization: L.C., H.D.-N., P.F., A.S., and E.L.; methodology: L.C., H.D.-N., P.F.,

H.S., A.S., R.O., and P.A.-F.; software: L.C., H.D.-N., P.F., H.S., A.S., F.A.-J., and E.L.; validation: L.C., A.S., F.A.-J.,

T.M., E.F., R.O., J.B.-F., A.O.-F., and E.L.; formal analysis: L.C., P.F., and H.S.; investigation: L.C., P.F., H.S., A.S.,

F.A.-J., T.M., E.F., J.B.-F., A.O.-F., and E.L; resources: L.C., J.B.-F., A.O.-F., and E.L.; data curation: L.C., J.B.-F.,

A.O.-F., E.M., and E.L.; writing—original draft preparation: L.C., P.F., H.S., and A.S.; writing—review and editing:

L.C., P.F., H.S., A.S., J.B.-F., A.O.-F., and E.L.; visualization: L.C., P.F., H.S., A.S., J.B.-F. and E.L.; supervision: J.B.-F.,

A.O.-F., and E.L.; project administration: J.B.-F., P.A.-F., A.O.-F., and E.L.; funding acquisition: L.C., J.B.-F., A.O.-F.,

and E.L. All authors have read and agreed to the published version of the manuscript.

Molecules 2020, 25, 3959

16 of 19

Funding: “Coordenaç

ã

o de Aperfeiçoamento de Pessoal de N

í

vel Superior” (CAPES), concession number:

8

8882.347018/2019-01; and the “Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico” (CNPq),

concession number: 302519/2012-2.

Acknowledgments: The authors wish to thank for the support provided by the Federal University of Para

(UFPB) and the Brazilian funding agencies: CAPES, CNPq.

íba

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

ATCC

BHI

American Type Culture Collection

Cerebral Heart Infusion (Broth)

Colony Forming Units

CFU

DMSO

MBC

MIC

MVD

MW

Dimethyl Sulfoxide

Minimum Bactericidal Concentration

Minimum Inhibitory Concentration

Molegro Virtual Docker

Molecular Weight

NC

Negative control

PBS

PC

Phosphate Buﬀered Saline

Positive control

PDB

RBC

RMSD

TPSA

Protein Data Bank

Red Blood Cell

Root Mean Standard Deviation

Topological polar surface area

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Sample Availability:

Samples of the compounds N-(4-ﬂuoro-3-nitrophenyl)acetamide and

2

-chloro-N-(4-ﬂuoro-3-nitrophenyl)acetamide are available from the authors.

©

2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

CC BY) license (http://creativecommons.org /licenses /by /4.0 /).

(

Article Doi

Potential of 2-Chloro-N-(4-fluoro-3-nitrophenyl)acetamide against Klebsiella pneumoniae and in Vitro Toxicity Analysis

DOI: 10.3390/molecules25173959

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