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RESEARCH ARTICLE

A Mild catalyzed imino Diels-Alder reaction for the synthesis of N-(2-

(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)formamide derivatives as
regulators of Quorum Sensing in Pseudomona Aeruginosa

Leidy J. García Maza1, Dayanna F. Orosco Flórez1, Arturo René Mendoza Salgado1, Wendy Rosales2, Evelyn Mendoza-Torres3
and Carlos Mario Meléndez*1
1
Faculty of Basic Science, Molecular Biology Research Laboratory, Organic and BiomedicalChemistry Research Group, Universidad del Atlántico,
Barranquilla, Colombia.
2
Faculty of Exact and Natural Sciences, Biomedicine Advanced Research Group, Universidad Libre, Barranquilla, Colombia.
3
Faculty of Health Sciences, Biomedicine Advanced Research Group, Universidad Libre, Barranquilla, Colombia.

Bacterial resistance is one of the major global public health problems. In addition, virulence factors promoted multiple drug resistance
through cell-to-cell communications by molecules within microbial communities, denominated Quorum Sensing (QS). The regulation of
QS by small molecules appears as a therapeutic alternative through the use of synthetic analogs of agonists. The present work developed the
construction of a series of derivatives of N-(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl) formamide (1a-h) via Povarov-type sequential
reactions to deliver the tetrahydroquinoline (THQ) derivatives in moderate to good yields (24-78%). To evaluate the potential in molecular
diversity of THQ-type autoinducers, a study of these agents as potential inhibitors of bacterial growth and virulence factors involved in QS
systems on P. aeruginosa was carried out both as antibacterial evaluation on E. coli strain. THQs showed significant activity values for
molecules with halogen substituents at the C-6 position (-F, -Cl, and I), ), revealing at a concentration of 75 µg/mL a percentage inhibition
between 27-40%. Furthermore, autoinducers with halogen substitutes in phenotypic studies on P. aeruginosa showed good results. Also,
they exhibited weak formation of both biofilm and low pyocyanin formation, which are indicators of virulence in P. aeruginosa. Overall,
the results suggest that compound N-(6-fluor-2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)formamide (1a) stands out as the most
representative within this series of THQ derivatives.

Keywords: Antibacterial resistance, Quorum Sensing, Synthetic inhibitors, Tetrahydroquinolines.

1. Introduction promising approach for the treatment of bacterial infections.

[8]. Significant efforts have focused on the development of
Resistance of bacterial pathogens to antimicrobials is a
new strategies for the treatment of biofilm infections,
global problem [1],[2]. Conventional therapies and numerous
particularly targeting the processes of cell dispersion in
currently available antibiotics have proven ineffective
biofilm [9]. Other approaches include the use of
against clinically relevant strains of both Gram-negative and
antimicrobial peptides [10], bacteriophage systems [9], and
Gram-positive bacteria, leading to the emergence of several
inhibitors of Quorum Sensing [14], among others.
mechanisms of multi-drug resistance [3], [4]. Gram-negative
pathogens such as Pseudomonas aeruginosa and Escherichia
coli concerning due to their recurrent resistance processes,
which are often more severe compared to Gram-positive
pathogens [5]–[10]. The main challenge in combating
antibiotic resistance lies in the ineffectiveness of current
antibiotic therapy against biofilms established by bacteria
[11]–[15]. Virulence factors such as the formation of
biofilms protect the microbial communities ensuring the
communication and development of the bacterial population,
these factors are promoted through a cell-to-cell
communication system called Quorum Sensing (QS) [16]– Fig. 1. Native autoinducers in Quorum sensing in P. aeruginosa: 3-
[18], a mechanism responsible for the regulation of virulence o-C12-HSL, C4-HSL, PQS, and HHQ. The 2-alkyl-4-quinolones
genes depending on the bacterial density [15], [19], [20]. As molecules share an analogous nucleus with the
tetrahydroquinolines.
a result, targeting biofilm growth has emerged as a

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 2

The reaction between anilines (2), o-tolualdehyde (3), and N-

Therefore, comprehending, controlling, and/or regulating vinyl formamide (4) in the presence of p-TsOH 10 mol% p-
these bacterial chemical communication systems hold the toluenesulfonic acid, and solvents such as ethanol and
potential to establish new objectives for developing acetonitrile at room temperature provides ready access to (2-
alternative or complementary treatments to conventional (o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)formamide
antibiotic agents [8], [18], [21], the development of these derivatives (1a-h) (Scheme 1). While striving to extend the
strategies prevents or limits the formation of biofilms scope of this transformation, we have discovered that the use
through the use of synthetic analogs of natural autoinducers at high temperatures or carrying out the reaction in one pot
[9], [16], [22]–[24]. (Tetrahydro)quinoline (THQ) would cause the reaction to deviate to a different course,
derivatives represent a significant strategy in the molecular forming several degradation products.
design of synthetic analogs of natural autoinducers [25]–
[29]. The structural diversity of these heterocyclic Table 1. Catalyst and solvent screening for imino Diels-Alder
compounds [30], [31] along with their shared nucleus reaction of substituted anilines (2), o-tolualdehyde (3), and N-vinyl
analogous to the native autoinducers PQS corresponding to formamide (4).
the HHQ signals [23], [24], [32]–[34] (Fig. 1), make them as
Entry Catalyst Solvent (b/c) Temp Yield
a promising antimicrobial agent against resistant pathogens,
(°C) (%)
in addition to the exploration of new systems that can
modulate QS circuits [14], [21], [35]–[37]. On the other 1 ZnCl2d EtOHb,c r.t 30
hand, although THQ derivatives have been widely reported 2 BiCl3e,f CH2Cl2/MeCN r.t 42
for their broad spectrum of antibacterial activities [38]–[41], 3 ZnCl2e EtOH/MeCN 80-120g nr
their potential as QS inhibitors has not been fully developed 4 BiCl3d,f CH2Cl2/MeCN r.t 42
yet [32]. Based on the above, searching for new Micellar
5 MeOHi r.t 37
antimicrobials and using effective synthetic routes to obtain catalysis
THQ derivatives with potent biological activities is 6 BiCl3d EtOH/MeCN r.t 50
imperative. The imino-Diels-Alder reaction (iDA) emerge as 7 Ce(SO4)2d EtOH/MeCN r.t nr
an effective methodology [31], [42]–[44] for HOAc 1
tetrahydroquinoline compounds. [30], [42] The iDA reaction 8 EtOH/MeCN r.t 18
mmol
offer several advantages as readily available reagents [45], 9 TsOHe EtOH/MeCN r.t 71
high regioselectivity and stereospecificity [46], and high Amberlyst
yields, among others. EtOH/PEG
10 15 (40% r.t 27
600
w/w)
a
This work aims to synthesize new non-native autoinducers Catalyst in second step; b Solvent in first step; c Solvent in second step; d 5
% mol; e 10 % mol; f Na2SO4 (2.0 equiv, 4.0 mmol); g refluxing EtOH; h 15
using the iDA reaction, via a simple and versatile protocol, mL SDS-H2O/HCl 0,1 M; i one-pot reaction; r.t, room temperature; nr, no
with readily available arylamines, o-tolualdehyde, and N- reaction.
vinyl formamide. Additionally, the effectiveness of the
synthesized compounds will be evaluated in the regulated Overall, moderate yields (24-78%) were obtained during
QS by observing phenotypic changes in E. coli and P. Diels-Alder imino cycloaddition, the compounds with
aeruginosa strains. This research offers a new perspective on electron-donating groups such as -CH3 (71%) and -OMe
the significance of exploring the molecular diversity of (78%) showed the best global yields. Moreover, compounds
synthetic analogs of autoinducers such as with electron-withdrawing groups e.g., -F (56%), -Cl (44%),
tetrahydroquinoline compounds, as a starting point for and -I (56%) exhibited moderate yields and do not reveal a
further investigations on QS systems and the potential marked trend as atomic radius increases and
application as therapeutic agents against drug-resistant
electronegativity decreases. The results obtained from the
bacterial strains.
reaction yields are consistent with those reported in studies
2. Chemistry: synthesis of the 2-(o-tolyl)-1,2,3,4- related to the synthesis of tetrahydroquinolines by the imino
tetrahydroquinoline Diels-Alder reaction with the use of arylamines and aromatic
aldehydes, with moderate to good yields [51].
The iDA sequential methodology has been extensively
employed to explore and optimize reaction conditions using
The yield of the product in the reaction can be attributed to
readily available and commercially accessible substrates
the easy formation and stability of the imine intermediate
(Table 1). Diverse catalysts [31], [47], [48] and solvents
(5). Additionally, aromatic amines with electron-donating
have been analyzed to identify the most efficient reaction
groups have demonstrated similar reactivity, contributing to
conditions, various reaction media as micellar catalysis with
the yield of the reaction. These groups donate some of their
SDS-H2O/HCl 0. 1 M used in green chemistry, as well as
electron density to the aromatic ring of aniline, which allows
solvents such as PEG-400 and glycerol [49], and catalysts
most of the electron density to be concentrated in the
such as Amberlyst 15 [50] have also been used. The optimal
nitrogen atom leading to imine formation more easily than
reaction conditions for obtaining the desired product were
with a group that reduces electron density in the molecule
identified to be achieved using the catalyst p-TsOH and
around the nitrogen atom. Moreover, arylamines with
EtOH/MeCN as solvent (entry 9).
electron-donating bulky groups (1b, 1h) do not react
completely with the aromatic aldehyde to give the desired

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intermediate possibly due to the steric hindrance. This causes
the electron density to be partially concentrated on the
nitrogen atom and the complete formation of the imine does
not occur, although the reaction time is prolonged. The series
Scheme 1. Synthesis of N-(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)formamide (1a-h) under optimal conditions.
3. Biology
3.1 Antibacterial activity
The inhibitory capacity of tetrahydroquinoline (1a-h) was
assessed against the Gram-negative strains of clinical
importance E. coli and P. aeruginosa [13]. Growth control
experiments were conducted to observe the initial phases of
growth for each concentration (10, 20, 35, 50, 75 µg/mL) of
THQ derivatives Figure 2. The latency phase corresponds to
the adaptation period (first two hours), followed by the
exponential phase characterized by cell division (last six
hours) [52]. The results demonstrated a direct relationship
between the P. aeruginosa and E. coli growth and the
concentration of THQ derivatives, showing consistent trends
in growth control for both bacterial strains. All compounds
exhibited growth inhibition effects as early as 4 hours,
particularly at concentrations of 50 and 75 µg/mL.
Compared those results with the positive control
(Gentamycin 10 µg/mL), we notice the remarkable
effectiveness and potency of this drug [40], [53], [54].
Regarding cytotoxicity control (yellow line), it is
corroborated that the solvent used (DMSO) for the
preparation of stock solutions of the derivatives did not
influence the bacteria growth.
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of N-(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)formamide
1a-h were structurally characterized using spectroscopy
techniques such as IR, NMR one-dimensional (1H, 13C) and
two-dimensional (COSY, HMBC, HSQC, DEPT 135).
Bacterial growth curves show the maximum absorbance
recorded at 8 hours. Therefore, the mean absorbances of the
culture medium obtained from 3 independent experiments
were calculated and as a result the inhibition percentages of
the THQ compounds (1a-h) for each bacterial strain were
acquired. The substituted 6-fluor compound 1a, followed by
the halogenated compounds 1d and 1f showed the greatest
decrease in absorbance at a concentration of 75 µg/mL for
both P.aeruginosa and E. coli, in an inhibition range of 20-
40 %. The absorbance values obtained determine the
inhibition percentages of compounds evaluated (Figure 3)
which evidence higher values for the compounds mentioned
above. Whereas compound 1e does not induce a decrease in
the growth of both microorganisms.
Figure 3 illustrates a progressive increase in the inhibition
percentage as the concentration of the compounds increases.
However, it is important to identify the minimum
concentration of tetrahydroquinolines compounds at which
growth inhibition initiates. To determine it, the average
absorbance of the cultures at 8 hours of incubation was
calculated for each concentration in µg/mL. Subsequently, a
post hoc Tukey test was performed to identify significant
statistical differences between the group of concentrations
and growth control. This analysis allows the determination
 4

of minimum inhibitory concentration (MIC), finding that the tetrahydroquinoline derivatives with halogenated
MIC inhibits bacterial growth for most compounds starting substituents at the C-6 position [55][39] exhibited higher
at 10 and 20 µg/mL with a range of p < 0,05-0,0001. inhibition percentages compared to compounds with
electron-donating groups such as
The obtained results are consistent with the trend observed in
previous structure-activity relationship studies, where

a b

P. aeruginosa E. coli P. aeruginosa E. coli

1.2 1.2 1.2 1.2
Abs690 (AU)

Abs660 (AU)

Abs690 (AU)

Abs660 (AU)
0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (hours) Time (hours) Time (hours) Time (hours)

c d
P. aeruginosa E. coli P. aeruginosa E. coli
1.2 1.2 1.2 1.2
Abs690 (AU)

Abs660 (AU)

Abs690 (AU)

Abs660 (AU)
0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (hours) Time (hours) Time (hours) Time (hours)

e f
P. aeruginosa E. coli P. aeruginosa E. coli
1.2 1.2 1.2 1.2
Abs690 (UA)

Abs660 (AU)

Abs690 (AU)

0.8 0.8 0.8 Abs660 (AU) 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (hours) Time (hours) Time (hours) Time (hours)

g h
P. aeruginosa E. coli P. aeruginosa E. coli
1.2 1.2 1.2 1.2
Abs690 (UA)

Abs660 (UA)

Abs690 (UA)

Abs660 (UA)

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Time (hours) Time (hours) Time (hours) Time (hours)

10 µg/mL 20 µg/mL 35 µg/mL 50 µg/mL 75 µg/mL C+ gentamicin 10 µg/mL

Cytotoxicity (DMSO) Growth control

Figure 2. Growth curves of P. aeruginosa and E. coli when exposed to different concentrations of N-(2-(o-tolyl)-(1,2,3,4-
tetrahydroquinoline-4-yl)formamide derivatives using the absorbance variable as a growth indicator. (n=3, independent experiments). a)
compound 1a, b) compound 1b, c) compound 1c, d) compound 1d, e) compound 1e, f) compound 1f, g) compound 1g y h) compound 1h.

-Me and -OMe (F > I > Cl > Me > H > C4H10 (isopropyl)). breakage of bacterial chromosomes [56]. Therefore, it is
This is possibly due to atoms such as fluorine enhancing the noteworthy that the compound N-(6-fluor-2-(o-tolyl)-1,2,3,4-
cellular barrier penetration of the compounds and might tetrahydroquinoline-4-yl)formamide 1a is the most
effectively inhibit the DNA gyrase complex, resulting in the representative of the series of THQ derivatives. It exhibited

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the highest inhibition percentages on the studied strains E. 10 µg/mL with p < 0,0001). Other compounds show
coli (35,9%) and P. aeruginosa (39,1%), being the MIC = 10 moderate antibacterial activity such as 1c and 1g on P.
µg/mL where the inhibition of bacterial growth on both aeruginosa likewise compound 1f when was evaluated on
pathogens begins with con p < 0,0001. In addition, the both pathogens. Overall, the analyzed compounds exhibited
compound N-(6-chloro-2-(o-tolyl)-1,2,3,4- lower antibacterial activity compared to the reference drug
tetrahydroquinoline-4-yl) formamide 1d showed a similar (positive control), as indicated by the yellow bars in Figure
effect but in this case only on P. aeruginosa (36,8%, MIC = 3.

68,7
80

70,1

70,3

68,3

67,4

68,3

68,5
65,3
60
% Inhibition

39,1

36,8
35,4

35,6
34,7

34,5
32,8
40

26,2
26,2

25,9
22,1
22,1

21,7
20,9

19,1
19,0

18,2

17,6
16,7

17,6
17,4
15,8

15,0

16,7
14,6

14,8

14,5
13,6

13,3

12,8
11,9
12,5

11,2
20

9,0

9,4
8,7

7,5
6,8
4,9
3,3

0
1a 1b 1c 1d 1e 1f 1g 1h
Compound

80,7
80,7

b
78,8

71,7

71,5

71,8

68,49
68,49
80

60
% Inhibition

35,9

32,8

40
26,9
26,4

21,56

20,32
20,76
18,12
20,9

21,5

20,2

20,7
19,3

16,78

18,5
13,9
16,5

15,7

14,2

13,03
17,2

10,85
12,7

12,8

11,2
12,1

20
11,2
8,50
9,50

8,80

8,10

6,86

7,34
6,12

7,02
6,34

5,53
5,60

5,72
4,25

0
1a 1b 1c 1d 1e 1f 1g 1h
Compound
10 µg/mL 20 µg/mL 35 µg/mL 50 µg/mL 75 µg/mL
C+ gentamicin 10 µg/mL

Figure 3. Growth inhibition of a) P. aeruginosa and a) E. coli, after exposure to different concentrations of THQ derivatives (1a-f).

Indeed, certain studies propose that antibacterial agents can

interfere with the bacterial transcription process, preventing 3.2 Biofilm Formation Index (BFI)
RNA or DNA transcription [57]. Moreover, DNA gyrase is P. aeruginosa is associated with biofilm-forming bacteria,
the primary target for most Gram-negative bacteria, which often cause resistance to antibiotics used to treat
including the opportunistic pathogens P. aeruginosa and E. acquired clinical infections. The mechanisms of biofilm
coli [56]. Therefore, the antibacterial activity exhibited by formation in P. aeruginosa are regulated by Quorum
THQ compounds with electron-withdrawing groups is Sensing, mainly by 3 systems las, rhl, and pqs, the latter
attributed to the interaction with the DNA polymerase. These being mediated by signaling molecules 2-heptyl-3-hydroxy-
molecules facilitate cellular penetration and contribute to the 4(1H)-quinolone (PQS) [59][60]. However, the mechanism
inhibition of the DNA gyrase complex [56]. The ability of of action of PQS in promoting biofilm formation remains
bacteria agents to pass through the bacterial envelope and unclear [59], [61], [62]. There is potential for PQS analogs to
reach their biological activity is crucial for their efficacy. serve as biofilm inhibitors. Despite recent studies on the
The moderate activity exhibited by the synthesized effect of tetrahydroquinolines compounds on P. aeruginosa
compounds may be due to their ability to penetrate the cell biofilm formation are lacking, it is possible that analogous of
barrier and, by the efficiency of efflux pumps that decrease the native autoinducer PQS as N-(2-(o-tolyl-(1,2,3,4-
intracellular concentration of bacterial agents in the tetrahydroquinoline-4-yl)formamide (1a-h), could act as
cytoplasm [58]. inhibitors of biofilm formation. First, absorbance values

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 6

were obtained at the different concentrations evaluated after
24 hours of incubation with the bacterial strain for
subsequent analysis of the biofilm inhibition capacity of
possible THQ autoinducers (Figure 4).
The results showed a strong tendency for the reduction of
biofilm production (decrease in absorbance) with the
progressive increase of the concentration of synthetic
autoinducers (Figure 4) [33]. It observes the negative
control (bacterial strain without autoinducers) with a
progressive increase in absorbance measurements and then
remaining constant but with a significant difference with the
assays that contained the autoinducers analogous to the
different concentrations.
0.8
0.7
Abs570 (AU)
35, and 75 μg/mL, i.e., it has the greatest decrease in biofilm
formation in P. aeruginosa. This result is consistent with the
observed inhibition of bacterial growth. Compounds with
more than one or bulky substituents in the aromatic ring (1b
and 1h) increase biofilm formation; including compound 1e
without electron-withdrawing or -donating groups in its
structure.
The effect observed by the synthetic analogs (THQ) at the
lowest concentration (10 μg/mL) is particularly intriguing. It
is noteworthy that some of the studied molecules exhibit a
biofilm formation rate very similar to the negative control,
indicating no significant influence on the inhibition of
biofilm formation. However, as the concentration of the
compounds increases, a decrease in bacterial biofilm
formation becomes evident, suggesting a potential alteration
1a in bacterial communication circuits mainly targeting
1b compound 1a. Recent research has found that the amount of
substituents in the phenyl ring influences the inhibitory

1c
0.6 activity of biofilm formation [33]. This explains the low
1d
0.5 1e activity (high rates of biofilm formation) of the compound
1f 1h because the aromatic ring is replaced more than once. In
0.4 1g addition, increases in the length of the substituted group
1h chains in the benzene ring drastically increase the biofilm
0.3
0 10 20 30 40 50 60 70 Control (-) formation, as in the case of the synthesized antagonist 1b.
Concentration (g/mL)
It is observed in Table 2 that more than 98% of the
concentrations of the autoinducers present BFI ≥ 1.0 being
Figure 4. Recorded absorbance after incubation for 24 hours of o- classified as moderate to strong biofilm formers and, BFI <
tolyl (1,2,3,4-tetrahydroquinoline-4-yl)formamide derivatives (1a-f) 1.0 is considered as weak formers, according to studies
on P. aeruginosa strain. Control (-), growth control of the bacterial performed on biofilm formation [63]. Therefore, it is
strain without autoinducer.
evidenced in this study that the evaluated molecules induce
The optical density results expressed in terms of the Biofilm the P. aeruginosa bacterial strain to develop moderate to
Formation Index (BFI) allow us to classify the ability of the weak biofilm indices at 75 µg/mL with p < 0.0001, i.e., these
compounds (1a-h) to modify the capacity of biofilm analogs can decrease biofilm formation by up to 50% if we
formation on P. aeruginosa (Table 2). Moreover, compound compare with the BFI data of C- (100% biofilm formed by
1a with a halogen substituent at the C-6 (-F) position has the the bacterial strain). These results open the possibility of
lowest biofilm formation index compared to the other future molecular studies to determine the specific genes that
synthetic autoinducers evaluated at concentrations of 10, 20, are expressed for this type of virulence mechanism.

Table 2. Biofilm formation Index (BFI) of P.aeruginosa of each of the autoinducers of N-(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl)
formamide at the concentration evaluated. (N = 3, number of independent essays).

Synthetic Concentration (µg/mL)

autoinducer
10 20 35 50 75 (C-)
1a 1,50 1,24 1,19 1,08 0,81 1,95
1b 1,93 1,54 1,45 1,19 1,09 1,95
1c 1,64 1,50 1,20 1,04 0,97 1,95
1d 1,62 1,40 1,33 1,15 0,89 1,95
1e 1,94 1,52 1,32 1,24 1,09 1,95
1f 1,64 1,24 1,24 1,20 0,96 1,95
1g 1,63 1,39 1,43 1,04 0,95 2,01
1h 1,77 1,55 1,36 1,08 1,05 1,83

3.3 Pyocyanin Production

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Pyocyanin is an important virulence factor of P. aeruginosa
[33]. This is one of the phenazine compounds with redox
activity produced and secreted by this pathogen. Although
this exotoxin has a wide range of toxic effects on different
host cells, its different toxicity originates from the
production of reactive oxygen species (ROS) capable of
causing significant oxidative stress [64], [65]. Moreover, the
exoproduct pyocyanin (PCN) is a zwitterion that easily
penetrates biological membranes [66]. Therefore, pyocyanin
is an essential component for complete virulence in a variety
of Pseudomonas aeruginosa infection models [67], [68].
The formation of PCN in the culture of P. aeruginosa was
determined by evaluating the molecules that presented the
lowest rates of biofilm formation which correspond to 1a,
1c, 1d, 1f, and 1g. The absorbance of the cultures at 600 nm
was recorded after 20 hours of incubation for calculations of
pyocyanin production. As phenotypic identification, the
production of pyocyanin is responsible for the blue-green
color characteristic of P. aeruginosa, observing a decrease as
Table 3. Mean absorbances of the culture medium (an indicator of pyocyanin formation) after exposure of the P. aeruginosa inoculum to
each concentration at 20 hours of incubation (n=3). The means ± Standard Deviation (SD) of each of the recorded absorbances are shown.
Growth control as bacterial strain without autoinducer.
Compound 1a
Control 1,16(±0,03)
10 µg/mL 1,04(±0,004)
20 µg/mL 1,02(±0,024)
35 µg/mL 0,946(±0,04)
50 µg/mL 0,843(±0,039)
75 µg/mL 0,732(±0,031)
4 Materials and Methods
4.3 General Information
All reagents and solvents used were reactive grades
purchased from (Sigma Aldrich and Merck). Column
chromatography was performed using spherical silica gel 70
Å, 40–75 μm. One-dimensional (1H, 13C) and two-
dimensional (COSY, HMBC, HSQC, and DEPT 135)
Nuclear Magnetic Resonance (NMR) were recorded on a
Bruker Avance-400 (400 MHz) spectrometer (Universidad
Industrial de Santander, Bucaramanga, Colombia) with the
solvent resonance as the internal standard (CDCl3: δ = 7.26;).
Infrared spectra (FT-IR) were measured on a
Spectrophotometer FTIR-4700 + ATR PRO ONE
(Universidad del Atlántico, Barranquilla, Colombia).
Melting points were measured on a Differential Scanning
Calorimeter (DSC) (823) (Universidad del Atlántico,
Barranquilla, Colombia). The reactions were carried out for
the synthesis of 1a-h.
To determine the biological activity of the derivatives of N-
(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-4-yl) formamide
(1a-h), three types of assays were performed to analyze the
regulation of these systems using the synthesized molecules.
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the concentration of the evaluated autoinducers increases.
Table 3 shows the average absorbances for each of the
concentrations of the molecules tested, showing a decrease in
pyocyanin formation of 44% and 43% at 75 µg/mL for
compounds 1a and 1d, respectively. Hence, the effectiveness
of halogenated substituents is observed in regulating some
virulence factors such as pyocyanin production and biofilm
formation. Statistical analysis by Tuckey's test indicated a
significant difference between the means of the data versus
growth control, starting at 10 µg/mL with p < 0.05 for 1a
and p < 0.01 for 1g. Furthermore, a significant statistical
difference with p < 0.0001 was observed at 35 µg/mL for all
synthetic autoinducers studied in this assay.
As the compounds (1a-h) show an effect on bacterial growth
inhibition < 40%, it is suggested that the synthetic
autoinducers do not cause the death of the evaluated P.
aeruginosa pathogen. However, they regulate some
virulence factors that generate resistance to standard
antibiotics.
1c 1d 1f 1g
1,10 (±0,03) 1,11 (±0,03) 1,14 (±0,03) 1,18(±0,03)
1,06(±0,014) 1,05(±0,02) 1,074(±0,03) 1,04(±0,04)
1,02(±0,021) 1,02(±0,02) 1,03(±0,013) 1,01(±0,03)
0,826(±0,043) 0,991(±0,01) 0,951(±0,04) 0,943(±0,002)
0,801(±0,033) 0,93(±0,04) 0,876(±0,04) 0,812(±0,04)
0,797(±0,011) 0,749(±0,04) 0,801(±0,03) 0,791(±0,01)
The reagents and other materials were acquired from
certified chemical companies. Dimethyl sulfoxide (DMSO)
was purchased from Merck. Müeller Hinton broth (MH
broth) and Müeller Hinton agar (MH agar) were obtained
from Merck KGaA (Darmstadt, Germany). Gentamicin
reactive grade Sigma Aldrich. The bacterial strains were
from the American Type Culture Collection (ATCC):
Pseudomonas aeruginosa (ATCC 27853) by Microbiologics
and Escherichia coli (ATCC 25922) by Sigma Aldrich.
ALTA ELISA Plate Analyzer (405 – 630 nm) was used for
the reading of absorbances (Universidad Libre, Barranquilla,
Colombia).
4.4 Synthesis of N-(2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1a-h)
In a 50 mL round bottom ball were added 1 mmol of 2a-f
aniline, 1 mmol of 3 o-tolu aldehyde, and 10 mL EtOH, then
it was covered with a septum and subjected to constant
agitation at room temperature. Once the 5a-h N-phenyl-2-(o-
tolyl)methanimine (observed by TLC) was performed, then
the reaction crude was transferred to a 100 mL round bottom
ball, and the solvent was evaporated at reduced pressure. To
the 100 mL round ball which contained 1 mmol of N-phenyl-
2-(o-tolyl)methanimine (without purification) was added 10
mol% of p-toluenesulfonic acid (p-TsOH), 1 mmol of 4 N-
vinyl formamide and 20 mL MeCN, then it was covered with
a septum and subjected to constant agitation at room
 8
temperature. Product formation was confirmed by TLC. The
solvent of the reaction crude was evaporated at reduced
pressure. The reaction mass was washed with brine and the
organic phase was extracted with dichloromethane (3x20
mL) and deposited on anhydrous sodium sulfate (Na2SO4).
The organic phase was concentrated under reduced pressure.
The crude material was purified by column chromatography
(silica gel, hexane: ethyl acetate; in variable proportions) to
provide the desired N-(2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide 1a-h.
4.4.1 N-(6-fluor-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1a)
White crystals; yield: 0.28 g (0.98 mmol, 56%); mp: 148,76
°C. IR (ATR): v max (cm-1) 3367, 3226, 3041, 2962, 2925,
2869, 2755, 1650, 1502, 1454, 1382, 1261, 750. NMR 1H
(400 MHz, CDCl3): δ 8.29 (s, 1H, (CO)H), 7.56 (d, J = 2.6
Hz, 1H, 6’-HAr), 7.26 – 7.20 (m, 3H, 3’,4’,5’-HAr), 6.93 –
6.89 (m, 1H, 5-HAr), 6.82 – 6.81 (m, 1H, 7-HAr), 6.52 (dd, J
= 8.8, 4.7 Hz, 1H, 8-HAr), 5.92 (d, J = 9.3 Hz, 1H, N-
H(CO)), 5.57 (ddd, J = 10.0, 9.8, 6.1 Hz, 1H, 4-Hax), 4.79
(dd, J = 11.0, 2.5 Hz, 1H, 2-Hax), 3.92 (s, 1H, N-H), 2.42
(dd, J = 6.0, 2.4 Hz, 1H, 3b-Hec), 2.40 (s, 3H, 2’-CH3), 1.94
– 1.78 (m, 1H, 3a-Hax) ppm. NMR 13C (101 MHz, CDCl3): δ
161.1, 141.8, 140.5, 135.2, 130.8, 127.6, 126.6, 125.5, 115.7,
115.6, 115.4, 113.7, 113.5, 52.0, 45.1, 36.8, 19.1 ppm.
4.4.2 N-(8-isopropyl-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1b)
White solid; yield: 0.12 g (0.39 mmol, 24%); mp: 162,98
°C. IR (ATR): v max (cm-1) 3324, 3264, 3193, 3035, 2960,
2927, 2869, 1671, 1596, 1544, 1494, 1247, 742. NMR 1H
(400 MHz, CDCl3): δ 8.34 (s, 1H, (CO)H), 7.29 (s, 1H, 6’-
HAr), 7.08 – 7.03 (m, 3H, 3’,4’,5’-HAr), 6.77 – 6.70 (m, 3H,
5, 6, 7-HAr), 5.75 (d, J = 9.1 Hz, 1H, N-H(CO)), 5.51 – 5.43
(m, 1H, 4-Hax), 4.92 – 4.85 (m, 1H, 2-Hax), 3.76 (s, 1H, N-
H), 1.33 (dd, J = 6.3, 3.8 Hz, 1H, 3b-Hec), 2.81 (hept, J = 6.7
Hz, 1H, 8-CH), 1.28 – 1.27 (m, 3H, 8-CH3), 1.27 – 1.26 (m,
3H, 8-CH3), 1.25 – 1.24 (m, 3H, 2’-CH3), 0.93 – 0.85 (m,
1H, 3a-Hax) ppm. NMR 13C (101 MHz, CDCl3): δ 160.7,
142.3, 140.7, 138.5, 137.7, 129.5, 128.6, 127.6, 127.1, 126.6,
125.3, 124.5, 123.8, 50.9, 48.1, 38.7, 29.7, 24.8, 19.5 ppm.
4.4.3 N-(6-methyl-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1c)
Beige solid; yield: 0.36 g (1.28 mmol, 71%); mp: 149.38 °C.
IR (ATR): v max (cm-1) 3366, 3293, 3252, 3024, 2951, 2916,
2851, 1667, 1615, 1506, 1233, 766. NMR 1H (400 MHz,
CDCl3): δ 8.30 (s, 1H, (CO)H), 7.57 (d, J = 2.7 Hz, 1H, 6’-
HAr), 7.25 – 7.18 (m, 3H, 3’,4’,5’-HAr), 7.01 (s, 1H, 5-HAr),
6.93 (d, J = 8.2 Hz, 1H, 7-HAr), 6.53 (d, J = 8.1 Hz, 1H, 8-
HAr), 5.81 (d, J = 9.2 Hz, 1H, N-H(CO)), 5.58 (ddd, J = 10.0,
9.9, 6.2 Hz, 1H, 4-Hax), 4.79 (dd, J = 10.9, 2.5 Hz, 1H, 2-
Hax), 3.90 (s, 1H, N-H), 2.44 (dd, J = 6.1, 2.5 Hz, 1H, 3b-
Hec), 2.40 (s, 3H, 6-CH3), 2.26 (s, 3H, 2’-CH3), 1.91 – 1.80
(m, 1H, 3a-Hax) ppm. NMR 13C (101 MHz, CDCl3): δ 161.1,
143.2, 140.9, 135.2, 130.8, 129.3, 127.7, 127.5, 127.4, 126.6,
125.6, 120.7, 114.9, 51.9, 45.1, 37.2, 20.5, 19.2 ppm.
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4.4.4 N-(6-chloro-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1d)
White solid; yield: 0.22 g (0.73 mmol, 44%); mp: 150,82 °C.
IR (ATR): v max (cm-1) 3382, 3234, 3029, 2958, 2925, 2865,
1652, 1490, 1380, 1301, 1089, 754. NMR 1H (400 MHz,
CDCl3): δ 8.31 (s, 1H, (CO)H), 7.53 (d, J = 2.7 Hz, 1H, 6’-
HAr), 7.26 – 7.20 (m, 3H, 3’,4’,5’-HAr), 7.15 (d, J = 1.6 Hz,
1H, 5-HAr), 7.04 (dd, J = 8.9, 2.0 Hz, 1H, 7-HAr), 6.52 (d, J =
8.5 Hz, 1H, 8-HAr), 5.81 (d, J = 9.3 Hz, 1H, N-H(CO)), 5.56
(ddd, J = 10.3, 10.0, 6.3 Hz, 1H, 4-Hax), 4.83 (dd, J = 10.9,
2.6 Hz, 1H, 2-Hax), 4.03 (s, 1H, N-H), 2.44 – 2.42 (m, 1H,
3b-Hec), 2.40 (s, 3H, 2’-CH3), 1.92 – 1.81 (m, 1H, 3a-Hax)
ppm. NMR 13C (101 MHz, CDCl3): δ 161.1, 144.0, 140.3,
135.2, 130.9, 128.5, 127.7, 127.0, 126.6, 125.5, 122.4, 122.1,
115.8, 51.7, 44.9, 36.5, 19.1 ppm.
4.4.5 N-(2-(o-tolyl)-1,2,3,4-tetrahydroquinoline-
4-yl)formamide (1e)
Beige oil; yield: 0.21 g (0.79 mmol, 42%); mp: 110,95 °C.
IR (ATR): v max (cm-1) 3319, 3269, 3050, 3019, 2952, 2923,
2863, 1663, 1603, 1476, 1381, 1310, 1247, 1038, 745. Peso
molecular: 266.34 g/mol. NMR 1H (400 MHz, CDCl3): δ
8.28 (s, 1H, (CO)H), 7.56 (d, J = 6.3 Hz, 1H, 6’-HAr), 7.23 –
7.20 (m, 4H, 3’,4’,5’,5-HAr), 7.12 – 7.08 (m, 1H, 6-HAr), 6.77
– 6.73 (m, 1H, 7-HAr), 6.59 (dd, J = 8.0, 1.2 Hz, 1H, 8-HAr),
5.88 (d, J = 9.3 Hz, 1H, N-H(CO)), 5.60 (td, J = 10.1, 5.9
Hz, 1H, 4-Hax), 4.84 (dd, J = 10.9, 2.6 Hz, 1H, 2-Hax), 4.02
(s, 1H, N-H), 2.45 (dd, J = 6.0, 2.6 Hz, 1H, 3b-Hec), 2.41 (s,
3H, 2’-CH3), 2.07 (s, 1H, 3a-Hax) ppm. NMR 13C (101 MHz,
CDCl3): δ 161.2, 145.5, 140.8, 135.2, 130.8, 128.7, 127.6,
127.3, 126.6, 125.6, 120.7, 118.0, 114.7, 51.7, 45.1, 37.0,
19.2 ppm.
4.4.6 N-(6-iodo-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1f)
Dark green solid; yield: 0.28 g (0.71 mmol, 56%); mp:
154,63 °C. IR (ATR): v max (cm-1) 3388, 3257, 3035, 2958,
2925, 2861, 1656, 1482, 1382, 1247, 1041, 756. NMR 1H
(400 MHz, CDCl3): δ 8.28 (s, 1H, (CO)H), 7.51 (d, J = 3.2
Hz, 1H, 6’-HAr), 7.43 (s, J = 1.6 Hz, 1H, 5-HAr), 7.33 (dd, J
= 8.5, 2.1 Hz, 1H, 7-HAr), 7.28 – 7.16 (m, 3H, 3’,4’,5’-HAr),
6.37 (dd, J = 8.5, 2.1 Hz, 1H, 8-HAr), 5.88 (d, J = 9.3 Hz, 1H,
N-H(CO)), 5.54 (ddd, J = 10.3, 10.0, 6.3 Hz, 1H, 4-Hax),
4.82 (dd, J = 10.8, 2.6 Hz, 1H, 2-Hax), 4.08 (s, 1H, N-H),
2.39 (s, 3H, 2’-CH3), 2.36 (d, J = 5.3 Hz, 1H, 3b-Hec), 1.90 –
1.82 (m, 1H, 3a-Hax) ppm. NMR 13C (101 MHz, CDCl3): δ
161.2, 145.1, 140.3, 137.2, 135.6, 135.2, 130.9, 127.7, 126.6,
125.5, 123.2, 116.7, 78.2, 51.6, 44.7, 36.4, 19.2 ppm.
4.4.7 N-(6-methoxy-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1g)
Beige solid; yield: 0.39 g (1.32 mmol, 78%); mp: 151.56 °C.
IR (ATR): v max (cm-1) 3365, 3294, 3246, 3030, 2958, 2927,
2843, 1667, 1502, 1462, 1384, 1232, 1159, 1037, 809, 756,
665. NMR 1H (400 MHz, CDCl3): δ 8.29 (s, 1H, (CO)H),
7.58 (d, J = 2.3 Hz, 1H, 6’-HAr), 7.25 – 7.19 (m, 3H, 3’,4’,5’-
 9
HAr), 6.77 (s, 1H, 5-HAr), 6.73 (d, J = 8.6 Hz, 1H, 7-HAr),
6.56 (d, J = 8.6 Hz, 1H, 8-HAr), 5.86 (d, J = 9.3 Hz, 1H, N-
H(CO)), 5.60 (ddd, J = 10.1, 9.1, 6.3 Hz, 1H, 4-Hax), 4.76
(dd, J = 10.9, 2.4 Hz, 1H, 2-Hax), 3.76 (s, 1H, N-H), 3.75 (s,
3H, 6-OCH3), 2.44 (dd, J = 6.2, 2.4 Hz, 1H, 3b-Hec), 2.40 (s,
3H, 2’-CH3), 1.92 – 1.79 (m, 1H, 3a-Hax). NMR 13C (101
MHz, CDCl3): δ 161.12, 152.46, 140.86, 139.73, 135.16,
130.77, 127.48, 126.53, 125.61, 121.85, 116.03, 115.17,
112.51, 77.40, 77.09, 76.77, 55.92, 52.07, 45.35, 37.24,
19.16 ppm.
4.4.8 N-(5,8-dimethoxy-2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide (1h)
White solid; yield: 0.19 g (0.58 mmol, 38%); mp: 126,25 °C.
IR (ATR): v max (cm-1) 3267, 3198, 3135, 3067, 2970, 2873,
1673, 1599, 1542, 1490, 1439, 1399, 1296, 1142, 748, 691.
NMR 1H (400 MHz, CDCl3): δ 7.78 (s, 1H, (CO)H), 7.39 (d,
J = 6.5 Hz, 1H, 6’-HAr), 7.19 – 7.16 (m, 3H, 3’,4’,5’-HAr),
6.20 (d, J = 3.2 Hz, 1H, 6-HAr), 6.17 (d, J = 3.3 Hz, 1H, 7-
HAr), 5.48 (q, J = 5.9 Hz, 1H, 4-Hax), 5.19 (d, J = 8.6 Hz, 1H,
N-H(CO)), 4.81 (d, J = 6.2 Hz, 1H, 2-Hax), 3.86 (s, 3H, 5-
OCH3), 3.77 (s, 1H, N-H), 3.76 (s, 3H, 8-OCH3), 2.50 – 2.45
(m, 1H, 3b-Hec), 2.39 (s, 3H, 2’-CH3), 2.34 – 2.22 (m, 1H,
3a-Hax). NMR 13C (101 MHz, CDCl3): δ 159.90, 152.81,
141.69, 140.61, 136.91, 135.00, 131.16, 127.15, 126.26,
125.05, 109.33, 107.91, 97.17, 56.03, 55.53, 49.90, 40.43,
34.62, 19.09 ppm.
4.5 Biological Assays
4.5.1 General procedure for the determination
of the antibacterial activity
Samples and reference control. The micro broth method
was used according to the protocol of the Clinical &
Laboratory Standards Institute (CLSI) [69]. Stock solutions
were prepared for each compound, dissolving 1000 µg of
each compound in 1 mL dimethyl sulfoxide (DMSO) 1%.
The dilution equation was used to calculate the amount of
compound to be used at each of the concentrations to be
tested in the bacterial culture (10, 20, 35, 50, and 75 μg/mL).
The different concentrations of the compounds were chosen
taking into account previous research where
tetrahydroquinolines were very effective against these
bacterial strains [39]. For the reference control, a stock
solution of Gentamicin at 100 μg/mL was prepared from a
concentrated solution (8 mg/mL). The amount of gentamicin
required for 2 mL culture strain and a final concentration of
10 μg/mL was determined.
Biological media. 0,013 g nutrient broth was added and
dissolved in 100 mL of ultra-pure water, then, it was
sterilized in an autoclave at 120°C with a pressure of 15
Pounds Per Square Inch (PSI) for approximately 45 minutes.
In sterile test tubes, one inoculum of each bacterium was
seeded in 2 mL of sterile nutrient broth and incubated at 37
°C for 12 hours [69]. The inoculum was standardized with a
0.5 McFarland pattern, which was prepared by adding 0.5
mL BaCl2 0.048 M with 99.5 mL H2SO4 0.18 M and stirring
for 2 minutes. This solution was read in a spectrophotometer
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at a wavelength of 690 nm, obtaining a turbidity
(absorbance) of 0.082 corresponding to 0.5 on the
McFarland scale, comparable to a bacterial suspension
containing 1.5 x 108 CFU/mL [70]. After 12 hours, the
absorbance of the inoculum was measured to the 0.5
McFarland scale (absorbance approximately 0.082).
Antibacterial effect of N-(2-(o-tolyl)-1,2,3,4-
tetrahydroquinoline-4-yl)formamide derivatives (1a-h)
on the bacterial culture of P. aeruginosa ATCC 27853
and E. coli 25922 (in situ). The assay was prepared
according to the indications established by the Clinical &
Laboratory Standards Institute (CLSI) [69]. 10 μL of a 0.5
McFarland culture were seeded in 200 μL Mueller-Hinton
broth (MH) at various concentrations (10, 20, 35, 50, and 75
μg/mL) of each of the synthetic autoinducers (1a-h) from a
stock solution of 1000 μg/mL diluted in DMSO at 1%. This
was done in corning 96-well plates and incubated at 37°C
under agitation. Absorbance was measured at 690 nm for P.
aeruginosa and 660 nm for E. coli every hour for 8 hours
[69]. Each concentration of the compounds was evaluated in
duplicate for an N=3, on the whole with the positive
inhibition control consisting of Gentamicin 10 μg/mL (C+),
growth control (C-) consisting of bacterial culture without
compound, and cytotoxicity control being the bacterial
culture with DMSO at 1%. After the absorbance values were
measured using the culture medium to calibrate, the
percentages of inhibition in each measurement were
calculated using a T-test and one-way ANOVA, with the
GraphPad Prism 8.0 and Excel 2013 statistical package.
4.5.2 General procedure for the identification of
phenotypic switching on the bacterial
culture of P. aeruginosa ATCC 27853
Biofilm Formation Index. The tests were carried out on
corning 96-well plates at the Laboratorio de Biología
Molecular e inmunología de la Universidad Libre seccional
Barranquilla. The 0.5 McFarland strain of P. aeruginosa was
sown in nutrient broth in the presence and absence of
different concentrations (10, 20, 35, 50, and 75 μg/mL) of
tetrahydroquinoline compounds. Inoculated plates were
incubated at 37°C for 24 hours. Then, the plates were gently
shaken in a microplate shaker to mix the planktonic cells.
The supernatant from each well was transferred to the
corresponding well of a new microplate to measure the
absorbance at 600 nm (AbsRemaining). The remaining intact
biofilm in each well was stained with a 1% violet crystal
solution (CV) [71] at 37 °C for 30 min, then, washed with
sterile distilled water and dried at 55 °C for 1 hour. Biofilm
cells were destained with 95% ethanol and absorbance was
measured at 570 nm (AbsBiofilm). The biofilm formation index
was calculated with statistical significance using a T-test and
one-way ANOVA, with the GraphPad Prism 8.0 statistical
package and Excel 2013.
Pyocyanin Production. It was measured from the bacterial
culture supernatant following the protocol reported by
O’Loughlin et al. 2013 [70] with some modifications. 1 mL
of a 1:1000 subculture of P. aeruginosa was seeded in 9 mL
LB broth and incubated at 37°C. Then, a stock solution of
 10

tetrahydroquinoline compounds (1a,c,d,f) was prepared in
1% DMSO, and the corresponding quantities of each
compound were added in 1.5 mL microtubes to obtain
concentrations of 10, 20, 35, 50, and 75 μg/mL. The test
inoculum was prepared by a 1:10 dilution of the 16-hour
culture in fresh medium and 200 μL aliquot of this
subculture was added to each of the microtubes and
incubated at 37°C for approximately 20 hours. Then,
absorbance was determined by spectrophotometric reading at
600 nm. The remaining volume of each of the test tubes was
centrifuged at 2500 RPM at 15 °C for 10 minutes and 200
μL of the supernatant was transferred to new microplates for
absorbance measurement at 695 nm.
Supportive/Supplementary Material and include a brief
caption line for each file describing its contents.
Declaration of Generative AI and AI-assisted
technologies in the writing process
During the preparation of this work, the author(s) used
Grammarly: Free Writing AI Assistance to check grammar,
style, and punctuation in the writing. After using this
tool/service, the author(s) reviewed and edited the content as
needed and take(s) full responsibility for the content of the
publication.

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