Prenylated flavonoids with potential antimicrobial activity: Synthesis, biological activity, and in silico study

International Journal of

Molecular Sciences

Article

Prenylated Flavonoids with Potential Antimicrobial Activity:

Synthesis, Biological Activity, and In Silico Study

Mauricio Osorio ^1,*, Marcela Carvajal ², Alejandra Vergara ², Estefania Butassi ³, Susana Zacchino ³,

Carolina Mascayano ⁴, Margarita Montoya ⁵, Sophia Mejías ⁵, Marcelo Cortez-San Martín ⁶

and Yesseny Vásquez-Martínez ^7,

*

1

2

3

4

5

6

Laboratorio de Productos Naturales, Departamento de Química, Universidad Técnica Federico Santa María,

Valparaíso 2390123, Chile

Centro de Biotecnología CB-DAL, Universidad Técnica Federico Santa María, Valparaíso 2390136, Chile;

marcela.carvajal@usm.cl (M.C.); alejandrajj@gmail.com (A.V.)

Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,

Suipacha 531, Rosario 2000, Argentina; fefabutassi@hotmail.com (E.B.); szaabigil@gmail.com (S.Z.)

Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad de Santiago de Chile,

Santiago 9170022, Chile; carolina.mascayano@usach.cl

Laboratorio Bioquímica Celular, Departamento de Biología, Facultad de Química y Biología, Universidad de

Santiago de Chile, Santiago 9170022, Chile; margarita.montoya@usach.cl (M.M.); soﬁa.mejias@usach.cl (S.M.)

Laboratorio de Virología Molecular y Control de Patógenos, Departamento de Biología, Facultad de Química

y Biología, Universidad de Santiago de Chile, Santiago 9170022, Chile; marcelo.cortez@usach.cl

Programa Centro de Investigaciones Biomédicas Aplicadas, Escuela de Medicina, Facultad de

Ciencias Médicas, Universidad de Santiago de Chile, Santiago 9170022, Chile

7

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

*

Correspondence: mauricio.osorio@usm.cl (M.O.); yesseny.vasquez@usach.cl (Y.V.-M.)

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Osorio, M.; Carvajal, M.;

Vergara, A.; Butassi, E.; Zacchino, S.;

Mascayano, C.; Montoya, M.; Mejías,

S.; Cortez-San Martín, M.;

Abstract: Prenylated ﬂavonoids are an important class of naturally occurring ﬂavonoids with im-

portant biological activity, but their low abundance in nature limits their application in medicines.

Here, we showed the hemisynthesis and the determination of various biological activities of seven

prenylated ﬂavonoids, named 7–13, with an emphasis on antimicrobial ones. Compounds

9, 11, and

Vásquez-Martínez, Y. Prenylated

Flavonoids with Potential

12 showed inhibitory activity against human pathogenic fungi. Compounds 11 12 (ﬂavanones) and

,

13 (isoﬂavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing

that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and

4⁰are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics

synergistically enhanced the antibacterial activity of vancomycin, ciproﬂoxacin, and methicillin in a

factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciproﬂoxacin

was able to decrease the levels of ROS generated by ciproﬂoxacin. According to docking results of S

enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy;

however, more studies are needed to support this result.

Antimicrobial Activity: Synthesis,

Biological Activity, and In Silico

Study. Int. J. Mol. Sci. 2021, 22, 5472.

https://doi.org/10.3390/ijms22115472

Academic Editor: Maurizio Battino

Received: 27 April 2021

Accepted: 18 May 2021

Published: 22 May 2021

Keywords: prenylated ﬂavonoids; synthesis; antibacterial; MRSA; synergism; antifungal

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

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iations.

1. Introduction

Prenylated ﬂavonoids are a subclass of natural ﬂavonoids characterized by the pres-

ence of a prenylated side chain (i.e., prenyl, geranyl and lavandulyl) attached to the

ﬂavonoid skeleton. Since prenylated ﬂavonoids are generally more bioactive than their

non-prenylated precursors, they are of interest as lead compounds for producing new

drugs [1–3]. However, since prenylated ﬂavonoids often exist at trace levels in their nat-

ural sources that make further biological studies difﬁcult, it may be useful to synthesize

prenylated ﬂavonoids in appreciable quantities and at low costs to allow future biological

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distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

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4.0/).

studies [

tion [ ] as well as antifungal [

and anticancer [11] activities and anti-lipid properties, both in vitro and in vivo

prenylation of ﬂavonoids seems to be crucial to the biological activities of these compounds,

4

]. Compounds of this type exhibit signiﬁcant effects in the insect–plant interac-

], antimicrobial [ ], antiviral [ ], anti-inﬂammatory [10],

12]. The C-

[

Int. J. Mol. Sci. 2021, 22, 5472. https://doi.org/10.3390/ijms22115472

https://ww w .mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5472

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which may lead to enhanced cell membrane targeting and thus increased intracellular

activity [13]. From a chemistry point of view, the C-prenylation is complicated, inefﬁ-

cient, and time-consuming, so other methods must be explored; i.e., biocatalysis may be

the solution to these problems [14,15]. The prenylation of the ﬂavonoid core increases

lipophilicity and membrane permeability, which is one of the proposed reasons for the

enhanced biological activities of prenylated ﬂavonoids [16]. Efﬁcient chemistry methods

to obtain 8-prenylnaringenin with reasonable yields (20–33%) have been reported via

Claisen and Claisen–Cope rearrangement [17] in many steps. A good approximation to

obtain these kinds of compounds may be the direct prenylation of commercially available

ﬂavonoids with 3-methyl-2-buten-1-ol using ZnCl₂as Lewis acid by coupling via an aro-

matic electrophilic substitution reaction (ArES), whose conditions have been used to obtain

prenylated phenols in our laboratory [18].

To determine the structural requirements of the prenylated ﬂavonoids to exert any

remarkable biological activity, it is necessary to obtain numerous and varied series of these

compounds and to test them against several types of biological tests. These results will

provide enough data to obtain a reliable structure–activity relationship [17].

Multi-drug-resistant organisms are one of the major causes for the alarming level of

infectious diseases worldwide [19]. The discovery of new drugs with potent antimicrobial

activity, particularly against the resistant strains, is therefore highly desirable. Since

many of the currently available drugs have undesirable side effects, are ineffective against

new or reemerging fungi, or develop a rapid resistance [20], there is an urgent need for

the next generation of new antimicrobial agents that overcome these disadvantages. In

this sense, prenylated ﬂavonoids have been demonstrated to have potent antimicrobial

activity [21]. Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus are the three

human pathogens of greatest concern, associated with hospital- and community-acquired

infections, according to the latest global estimates of antibiotic resistance worldwide [22].

Synergism between natural compounds and traditional antibiotics is a promising

strategy that could solve the problem of antibiotic resistance that some bacterial strains

have been able to develop [23].

As endogenous free radicals and human 5-lipoxygenase (5-hLOX) have been shown

to have major roles in the pathogenesis of various health problems including inﬂammatory

disorders [24,25], the compounds obtained here were tested as 5-hLOX inhibitors and

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavengers. Since inﬂammation is related

to bacterial-mediated infections, a dual anti-inﬂammatory, antibacterial agent with an

improved safety proﬁle is a goal for improved therapeutic beneﬁts and better patient

compliance [26].

In this work, we have synthesized seven prenylated ﬂavonoids and studied their

antimicrobial, anti-inﬂammatory, and antioxidant activities to evaluate the relevance of

adding prenyl groups on the structure of non-prenylated ﬂavonoids and to propose the

use of naturally occurring organic compounds as possible therapeutic agents.

2. Results and Discussion

2.1. Synthesis

The synthesis of prenylated phenols was carried out in one step via the EAS mech-

anism as shown in Scheme 1, using a prenylation methodology applied to hydroxylated

acetophenones and benzaldehydes by us [18], with some modiﬁcations. The one-step

reaction of different ﬂavonoids (

ZnCl₂in ethyl acetate produced prenylated ﬂavonoids (

and side products, mainly. Only 5⁰-prenyl ﬁsetin (10) is new. When the ﬂavonoids chrysin

) and naringenin ( ) were alkylated, we isolated two C-alkylated compounds at positions

8 and 6 of the A-ring. Complex mixtures were found with quercetin ( ) and ﬁsetin ( ), prob-

1

–

5) with 3-methyl-2-buten-1-ol (6) in the presence of

7–

13) in moderate yields (23–36%)

(1

4

2

3

ably because their electron-donating substituents are more susceptible to oxidation [27].

Electron-rich aromatic rings can induce polyalkylations and decompositions, reducing

the yield.

Int. J. Mol. Sci. 2021, 22, 5472

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a

Flavonoids

Prenylated flavonoids

7 - 13

1 - 5

R₉

R₁₀

R₁₁

R₁₀

R₁₁

R₈

R₅

R₇

R₆

O

R₇

R₆

O

R₃

R₅

O

1. R₃= R₆= R₈= R₉= R₁₀= R₁₁= H; R₅= R₇= OH

2. R₆= R₈= R₁₁= H; R₃= R₅= R₇= R₉= R₁₀= OH

3. R₅= R₆= R₈= R₁₁= H; R₃= R₇= R₉= R₁₀= OH

7. R₃= R₆= R₉= R₁₀= R₁₁=H; R₈= Prenyl; R₅= R₇= OH

8. R₃= R₈= R₉= R₁₀= R₁₁=H; R₆= Prenyl; R₅= R₇= OH

9. R₆= R₈= H; R₁₁= Prenyl; R₃= R₅= R₇= R₉= R₁₀= OH

10. R₅= R₆= R₈= H; R₁₁= Prenyl; R₃= R₇= R₉= R₁₀= OH

4. R₆= R₈= R₉= R₁₁= H; R₅= R₇= R₁₀= OH

11. R₈= R₉= R₁₁= H; R₆= Prenyl; R₅= R₇= R₁₀= OH

12. R₆= R₉= R₁₁= H; R₈= Prenyl; R₅= R₇= R₁₀= OH

R₈

R₅

R₇

R₆

O

R₁₀

5. R₆= R₈= H; R₅= R₇= R₁₀= OH

13. R₆= H; R₈= Prenyl; R₅= R₇= R₁₀= OH

Conditions and reagents: (a) 3-methyl-2-buten-1-ol (6), ZnCl2/AcOEt, 40 °C–reflux, 4 h → 7 -13 (23–36%).

Scheme 1. Representative synthesis of prenylated ﬂavonoids in one step.

Structure Determination

The place of C-alkylation was determined by 2D ¹H- ¹³C HMBC NMR spectra. The

main J₃and J₂interactions between the methylene proton CH₂of the prenyl chain and

aromatic peaks are schematically shown in Figure 1. Only the interaction between the

methylene protons with the quaternary 8a carbon in the structure occurs when the C-

prenylation is produced in C-8. Correlations J₃and J₂between the methylene protons

CH₂and quaternary 8a or 4a carbon are impossible when the prenylation is bounded at

Position 6. For the rest of the molecules, the same reasoning was used to determine the

place of C-alkylation.

The presence of the carbonyl and hydroxyl groups was conﬁrmed by observing the

typical signals in IR spectra. Stretching absorption values between 3550 and 3200 cm⁻¹as

a broad signal indicated the presence of phenol groups and stretching absorption values

between 1750 and 1680 cm⁻¹as one of the strongest singlet IR absorptions conﬁrmed the

presence of carbonyl groups.

The hydrogen bond to carbons was determined by J₁interactions in the 2D ¹H-¹³C

HSQC NMR spectra. 2D NMR spectra (HSQC and HMBC) for all prenylated compounds

were included in Supplementary Materials.

Int. J. Mol. Sci. 2021, 22, 5472

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3'

2'

1'

4'

5'

2'

1'

4'

5'

8

HO

O

1

HO

O

1

7

8a

4a

8a

4a

8

2

6'

7

2

6'

6

4

6

4

3

5

OH

O

OH

O

8

7

Figure 1. 2D ¹H-¹³C HMBC spectrum obtained for compounds

7 and 8

. Here, the projections of the full spectrum ¹H NMR

and projections between 100 and 158 ppm for ¹³C NMR spectrum are represented. Only the main correlations between

aromatic peaks and prenyl chain are shown.

2.2. In Vitro Antibacterial Effects on Human Pathogen Bacteria

From the results of bacterial growth inhibitory activity on sensitive and resistant

S. aureus (Table 1), we can observe that not all prenylated ﬂavonoids showed a good

antibacterial effect, being prenylated ﬂavanones 7 to 10, weak to moderate inhibitors. In

Table 2, compounds 11 and 12, corresponding to prenylated naringenin, together with

compound 13 (prenylated genistein), showed strong activity both on the sensitive strain

and on the resistant strains, with MIC values ranging from 5 to 50 µg/mL. Compound 11

is the one that shows the best antibacterial action, especially on the resistant strain MRSA

97-7 with an MIC lower than that of the commercial agents tested.

Table 1. In vitro antibacterial assay. The expressed values are the percent inhibition of bacterial

growth achieved by prenylated ﬂavonoids at a concentration of 50 µg/mL.

Compound

MRSA 97-7

MRSA 622-4

ATCC 6538

7

8

9

10

11

12

13

21%

5%

61%

13%

95%

98%

91%

0%

3%

18%

93%

98%

96%

6%

26%

52%

17%

100%

99%

Table 2. Effect of prenyl substituent in the ﬂavonoids on the antibacterial activity expressed as

minimum inhibitory concentration (MIC) (µg/mL).

Compound

MRSA 97-7

MRSA 622-4

ATCC6538

4

11

12

>50

5

15

10

25

100

50

>50

25

50

5

10

50

>50

10

15

13

10

Vancomycin

Ciproﬂoxacin

Methicillin

3.5

2.5

10

The results show that the saturation of the double bond at the C2–C3 position appears

to be important for the antibacterial activity of ﬂavonoids (compounds 11 and 12). This

coincides with those reported in the literature, where prenylated ﬂavanones show better

activity than ﬂavones [28,29]. The position of the hydroxyl substituents, as well as that of

Int. J. Mol. Sci. 2021, 22, 5472

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the prenyl group, appears to be important in the inhibitory activity of bacterial growth,

observing that derivatives with OH at positions C-5, 7, and 4⁰show better inhibitory

activity, as well as the presence of prenyl groups in the C6 and C8 positions of Ring A, as

previously reported [30–32]. Araya–Coultier et al. (2018) showed that the position of the

prenyl group affects the antimicrobial activity of ﬂavonoids, when comparing the effect

of lupiwighteone (5,7,4⁰-trihydroxy-8-prenylisoﬂavone) and wighteone (5,7,4⁰-trihydroxy-

6-prenylisoﬂavone), on the Gram-positive strain L. monocytogenes. Lupiwighteone, chain

prenylated at C-8, showed no inhibition, whereas wighteone, chain prenylated at position

C-6, had high antibacterial activity [33].

When comparing compound 4 with compounds 11 and 12, where the base structure is

maintained and only one prenyl substituent has been added in different positions, we can

observe that the introduction of a prenyl group has an important effect on the inhibitory

activity, reducing by up to at least one order of magnitude the MIC in MRSA 97-7 (Table 2)

It has been reported that prenylated ﬂavonoids are more hydrophobic than common

ﬂavonoids, facilitating this characteristic the ability to penetrate the cell membrane, thus

improving their action at the active site [29].

.

Mun et al. (2014) proposed that prenylated ﬂavonoids act through interaction with

the bacterial membrane, binding directly with peptidoglycan, observing morphological

changes when MRSA strains were treated with the prenylated ﬂavonoid sophora-ﬂavanone

B [34].

Tables 3 and 4 show that a synergistic effect is produced between compounds 11, 12,

and 13 when acting in conjunction with commercial antibiotics, showing that the molecules

11 and 12 reduce the MIC of ciproﬂoxacin over MRSA 97-7 by 10 times (FICI values 0.6

and 0.16, respectively) and 11, 12, and 13 reduce 100 times its MIC over MRSA 622-4 with

FICI values from 0.1 to 0.2 (results shown in Table 4). They also acted synergistically with

Vancomycin (FICI values from 0.15 to 0.43), decreasing its MIC 10 times against MRSA

97-7. Compound 11 also acted synergistically with Methicillin with an FICI value of 0.01,

decreasing its MIC 100 times against MRSA 97-7.

Table 3. Synergistic effects of prenylated ﬂavonoids with commercial antibiotics on isolated MRSA 97-7.

MIC Alone

(µg/mL)

MIC Combined

Fold Reduction in Commercial

Antibiotic MIC

Agents

FICI

Outcome

(µg/mL)

11

Vancomycin

12

Vancomycin

13

Vancomycin

11

Ciproﬂoxacin

12

5

25

15

25

10

25

5

100

15

100

5

0.5

2.5

5

2.5

0.5

2.5

10

1

10

0.02

0.5

0.2

0.43

0.15

0.6

Synergy

10

Synergy

10

Partial Synergy

Synergy

10

0.16

0.01

10

Ciproﬂoxacin

11

Methicillin

Synergy

100

50

This potentiating effect of the antimicrobial agent action exerted by prenylated molecules

is not observed when combining them with each other, as observed in Table 5, where, when

combining compounds 11 and 12, an FICI value between 1.1 and 2 was achieved, given an

account of an indifferent effect. This difference in the behavior of prenylated molecules,

when compared to the action they exert when combined with antimicrobial agents, may be

due to the fact that prenylated ﬂavonoids act by a different mechanism than the commercial

antibiotics evaluated, e.g., through the inhibition of pumps of expulsion such as ABC

transporters, thus being able to enhance the action of the commercial antibiotic, as proposed

by Wagner et al. (2009), who pointed out that the synergistic effect of phytodrugs in

conjunction with antimicrobial agents can contribute to effects on the permeability of

Int. J. Mol. Sci. 2021, 22, 5472

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the cell wall of bacteria, the inhibition of the efﬂux pump, and the inhibition of bacterial

enzymes, and that all these mechanisms would help the antimicrobial agent to reach its

site of action [35].

Table 4. Synergistic effects of prenylated ﬂavonoids with commercial antibiotics on isolated MRSA 622-4.

MIC Alone

(µg/mL)

MIC Combined

Fold Reduction in Commercial

Antibiotic MIC

Agents

FICI

Outcome

(µg/mL)

11

25

10

25

10

50

10

2.5

0.1

5

0.1

5

0.1

0.2

0.1

Synergy

100

Ciproﬂoxacin

12

Ciproﬂoxacin

13

Ciproﬂoxacin

0.1

Table 5. Synergistic effects between prenylated ﬂavonoids on an isolated MRSA strain.

Strain

Agents

MIC Alone

MIC Combined

FICI

Outcome

11

12

5

15

5

15

MRSA 97-7

2

Indifferent

11

12

25

2.5

25

MRSA 622-4

1.1

Indifferent

To corroborate the synergistic action of compound 11 with vancomycin, a time kill

assay experiment was carried out, the result of which is shown in Figure 2. We can see that

compound 11 alone manages to slightly lower the bacterial growth curve, decreasing by less

than 2 log the CFU/mL, showing a moderate bacteriostatic action. Vancomycin achieved

a greater effect, lowering CFU/mL by more than 3 log10, but this effect is enhanced by

acting in conjunction with compound 11, lowering CFU/mL by more than 5 log10, thus

verifying the synergistic effect that compound 11 would exert on ciproﬂoxacin on MRSA

97-7. It has been described in the literature that prenylated ﬂavonoids isolated from Morus

alba L. show a broad spectrum of synergistic action towards Staphylococcus aureus, acting in

conjunction with antibacterial agents such as aminoglycosides,

β-lactams, glycopeptides,

and ﬂuoroquinolones [28].

Figure 2. Time kill assay vancomycin with compound 11 on MRSA 97-7.

Int. J. Mol. Sci. 2021, 22, 5472

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The results of the bacterial ROS level are shown in Figure 3. We can see that there

is a signiﬁcant decrease in ROS levels inside the bacteria as compounds 11, 12, and 13

are present, showing a protective effect by reducing ROS, with an effect as important as

that shown by the agent Trolox, a known ROS species trapper. It has been described in

the literature that polyhydroxylated ﬂavonoids can act as antioxidant agents, trapping

ROS [36], and this antioxidant property can be related to the ROS-reducing effect observed

in this assay.

Figure 3. Effect of prenylated compounds in intrabacterial ROS levels in Staphylococcus aureus

ATCC6538. Control: DMSO. Trolox: ROS scavenger control. 1,4-NQ (1,4-Naphtoquinone): ROS

generator control. Ciproﬂoxacin (0.05 µg/mL): commercial antibiotic control. 11–13: prenylated

compounds in evaluation (1/2 MIC concentration). Statistical analysis: Mann–Whitney U test (*)

p < 0.05, between each treatment with the negative control.

It has been previously reported that ciproﬂoxacin generates oxidative stress in S. aureus,

generating an increase in ROS levels within the bacteria [37]. This result was corroborated in

this study, where the levels of ROS generated in the bacteria in the presence of ciproﬂoxacin

were statistically higher than those of the control. As a way to explain the synergistic action

observed in the previous experiments, the effect on intrabacterial ROS levels was evaluated

by combining ciproﬂoxacin with compound 11. As we can see in Figure 4, this combination

markedly decreases the levels of ROS when compared to those generated by ciproﬂoxacin

alone. From these results, it can be deduced that the prenylated compounds may be acting

as trappers for the ROS generated by ciproﬂoxacin.

Figure 4. Effect of prenylated Compound 11 on intrabacterial ROS levels produced by ciproﬂoxacin in

Staphylococcus aureus ATCC6538. Control: DMSO. Ciproﬂoxacin (0.05

µg/mL): commercial antibiotic

control. Cipro + 11 (ciproﬂoxacin 0.05

Mann–Whitney U test (*) p < 0.05.

µg/mL + 11 (1/2 MIC concentration). Statistical Analysis:

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Docking of 11 (S and R Enantiomers) and 8 with ATP-Binding Cassette (Multi-Drug ABC

Transporter Sav1866 from S. aureus in Complex with AMP-PNP)

We used a fast and predictive tool as the molecular docking, which allowed us to

obtain by the genetic algorithm the most suitable location of ligand on macromolecule

without using molecular dynamic simulations. Thus, we carried out the analysis according

to the most favorable binding energy, by grouping the results in a cluster. To analyze

the good result of 11 and their correlation with the antibacterial activity, we used the in

silico tool with a known and essential pharmacological target linked to this property. For

the above, the ABC transporter was selected, which is crucial for balancing nutrients in

bacteria and humans. This transporter is also responsible for several processes, such as

drug-resistant bacteria [38]. Based on these reasons, we used docking tools to observe which

interactions were relevant, especially with the most promising molecule 11, although it is

worth taking into account that the biological tests were carried out with a racemic mixture.

In this study, we decided to study both enantiomers in order to be able to discriminate

them and determine whether the binding was stereoselective. The results showed a priority

for the S enantiomer (

−

5.06 Kcal/mol

±

0.012) versus the R enantiomer ( 3.37 Kcal/mol

−

±

0.016) (a difference of approximately 2 Kcal/mol between them) both binding in the

similar place of the transporter. These analyses of results effectively indicated a different

binding mode: The S enantiomer showed two hydrogen bonds with Glu320 and Asp321 of

the ABC transporter; on the other hand, the R enantiomer showed two hydrogen bonds

but with Glu353 and Gly378 (Figure 5), and in both molecules, the hydrophobic interaction

is important to the binding. We carried out a similar analysis by docking studies with

8

which has a bad antibacterial activity. The binding energy (-2.39 Kcal/mol) obtained

was worse than the 11 enantiomers and the molecule only showed one hydrogen bond

with Asp220 for stabilization in the binding area. Finally, we observed that the molecule is

located in the same area as the isomers as we observed in Figure 5.

Figure 5. Docking results: main interactions of the stereoisomers of 11 ((a) (R)-11 and (b) (S)-11) and 8 (c) with ATP

binding cassette.

2.3. In Vitro Antifungal Effects on Human Pathogenic fungi

In order to have a look into the potential usefulness of these compounds as candidates

for the development of new antifungal drugs, we investigated the antifungal properties of

compounds 1–5 and 7–13 against a panel of clinically important fungal species including

two yeasts (Candida albicans and Cryptococcus neoformans), three Aspergillus spp. (A. niger,

A. fumigatus, and A. ﬂavus), and three dermatophytes (Trichophyton rubrum, T. mentagro-

phytes, and Microsporum gypseum) (voucher numbers are included in Table 6 ). The minimum

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inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of all com-

pounds were determined with the microbroth dilution methods M27-A3 and M38-A2 of the

Clinical and Laboratory Standards Institute (CLSI) [39,40]. The evaluation of the minimum

fungicidal concentration (MFC) of the prenylated ﬂavonoids showed whether the com-

pounds kill fungi in addition to inhibiting them. This characteristic is highly appreciated

in an antifungal drug since the recurrence of fungistatic drugs is mostly avoided because

long treatments are no longer necessary.

Table 6. Antifungal activity (MIC/MFC in µg/mL) of prenylated ﬂavonoids 7–13 against pathogenic fungi from American

Type Culture Collection (ATCC) and Centro de Referencia en Micología Culture Collection (CCC). Amph: Amphotericin B;

Terb: Terbinaﬁne.

Trichophyton

mentagro-

phytes

Candida

albicans

ATCC10231

Cryptococcus

neoformans

ATCC32264

Aspergillus

ﬂavus

ATCC 9170

Aspergillus

fumigatus

ATCC26934

Aspergillus

niger

ATCC9029

Microsporum Trichophyton

Comp.

gypseum

CCC115,

rubrum

CCC113

ATCC9972

1

2

3

4

5

7

8

9

11

12

10

13

Amph

Terb

>250

62.5/62.5

>250

125/250

>250

62.5/62.5

125/125

62.5/125

250/>250

>250

1.0/2.0

-

>250

2.0/2.0

-

>250

2.0/2.0

-

>250

2.0/2.0

-

>250

62.5/62.5

15.6/31.25

250/250

>250

62.5/62.5

15.6/31.25

250/250

>250

62.5/62.5

31.25/31.25

250/250

>250

1.0/1.0

-

0.008/0.015

0.004/0.008

0.004/0.015

Compounds with MICs of >250

and 125 g/mL were considered lowly active; those in the range 62.5–31.25

considered moderately active; and MICs of <31.25 µg/mL were considered highly active.

µg/mL were considered inactive; those between 250

µ

µg/mL were

The results showed (Table 6) that the non-prenylated structures (the ﬂavone 1, the

ﬂavonols 2 and 3, the ﬂavanone 4, and the isoﬂavone 5) were all inactive.

When these ﬂavonoids were prenylated, the following results were obtained: (i) The

prenylation of the ﬂavone or the isoﬂavone did not improve the antifungal activity,

since , and 13 were inactive (MIC 250 g/mL). (ii) The prenylation of the ﬂavonols

and led to (which showed activity against the yeasts C. albicans and C. neoformans and

dermatophytes at 62.5 g/mL) and 10, which was inactive. Both compounds possessed

the prenyl substituent at position 5⁰but differed in that

possesses a hydroxyl group

in position 5. This structural difference could play a role in the antifungal activity of

. (iii) The prenylation of the ﬂavanone led to both 11 (MIC = 125 g/mL against C.

neoformans and 62.5 g/mL against dermatophytes) and the most active compound 12

which displayed MIC = 125 g/mL against C. albicans, MIC = 62.5 g/mL against C.

neoformans, MIC = 15.6 µg/mL against the dermatophytes T. rubrum and T. mentagrophytes,

and 31.2 g/mL against M. gypseum). Probably the prenyl substituent at position 8 of the

1

5

7

,

8

≥

µ

2

3

9

µ

9

4

µ

,

µ

trihydroxyﬂavanone naringenin confers high activity to the whole ﬂavanone molecule.

Considering that C. neoformans is the main cause of cryptococcal meningoencephalitis

among HIV patients, which many times has not responded to any antifungal and has led

to death [41], we decided to test compounds 9, 11, and 12 (which showed some activity

against the ATCC strain) against a panel of ﬁve clinical C. neoformans strains. The results

showed (Table 7) that the behavior of the three compounds against clinical isolates is similar

to that against the standard ATCC strain.

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Table 7. MIC/MFC (in

µg/mL) of selected prenylated compounds against clinical isolates of C. neo-

formans from Malbrán Institute (IM, Buenos Aires Argentina). Amph: Amphotericin B.

C. neoformans Clinical Isolates

Compound

IM983040

IM972724

IM042074

IM983036

IM00319

9

11

12

125/250

250/>250

125/125

0.25

125/250

250/>250

125/125

0.25

125/125

250/>250

62.5/62.5

0.12

125/125

250/>250

62.5/125

0.25

125/125

250/>250

125/125

0.5

Amph

In turn, since C. albicans is the fourth leading cause of nosocomial bloodstream infec-

tion (BSI) in intensive care units, causing fatal invasive candidiasis in a high percentage

of patients [42], we decided to test

9 and 12 against ﬁve clinical isolates of C. albicans, in

order to determine whether these two compounds have the potential to be further devel-

oped. The results showed (Table 8) that both compounds behave against C. albicans clinical

isolates similarly to how they behave against the standard ATCC strain.

Table 8. MIC/MFC (in µg/mL) of selected prenylated compounds against clinical isolates of C. albi-

cans from Centro de Referencia en Micología, (CCC, Rosario, Argentina): Amph: Amphotericin B.

C. albicans Clinical Strains

Compound

C. albicans

CCC 126

C. albicans

CCC 127

C. albicans

CCC 128

C. albicans

CCC 129

C. albicans

CCC 130

9

12

Amph

62.5/62.5

125/125

1.56

62.5/62.5

125/125

0.78

62.5/62.5

125/125

1.56

62.5/62.5

125/125

0.78

62.5/62.5

125/125

0.50

2.4. In Vitro Antibacterial Effects on Plant Pathogen bacteria

The results of the antibacterial activity against phytopathogenic bacteria showed that

all the prenylated compounds were able to inhibit the in vitro growth of the three bacteria

tested. Interestingly, most of the compounds signiﬁcantly inhibited the growth of the three

bacterial species in high percentages, obtaining IC₅₀values of less than 3.9

µM for several

of them. Despite the above considerations, the , and molecules showed the highest

3,

4

5

IC₅₀values in the series (Table 9). Statistical analysis revealed that the values obtained

from inhibition are signiﬁcant (p < 0.05) compared to the negative control.

Table 9. IC₅₀and MIC values of compounds towards phytopathogenic bacteria.

IC₅₀/MIC (µM)

Compound

P. syringae

A. tumefaciens

P. carotovorum

1

2

3

4

5

7

8

9

11

12

10

13

>31.3/>250

>62.5/>250

>250/>250

<3.9/>250

<15.6/>250

<3.9/125

>250/>250

<3.9/>250

>250/>250

<3.9/>250

<7.8/>250

<3.9/>250

<3.9/125

Antibacterial activities are shown as IC₅₀and MIC values in

µM concentrations; IC₅₀

values were calculated from the ﬁrst kinetic assay. MIC: minimum inhibitory concentration.

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IC₅₀values for each compound indicated that the bacteria tested had different levels

of sensitivity to the compounds tested. The most sensitive bacteria were P. syringae, and the

most resistant were A. tumefaciens. This behavior could be due to the different mechanisms

of action of the compounds on each tested Gram-negative pathogen.

After the ﬁrst kinetic test, the bactericidal capacity of each compound was evaluated

in a second growth kinetics test, using an inoculum of the ﬁrst kinetic plate [43]. This

new culture was incubated for 7 h, and the absorbance was measured. It was observed

that the bacterial culture was able to recover its growth rate (with respect to the culture

without compound) by 50–85%. This means that the compounds were bacteriostatic but

not bactericide at the concentrations tested. Compound 13 showed selectivity against

P. syringae and P. carotovorum and to a lesser extent against A. tumefaciens (>250, MIC).

2.5. In Vitro 5-hLOX Enzyme Inhibition Assay

The results showed that, of all the evaluated molecules (Table 10), only two (com-

pounds

9 and 10) had a relevant inhibitory effect. The structural characteristic that dif-

ferentiates these compounds from the rest of the structures is the catechol group (1,2-

dihydroxybenzene) in Ring B. Many known inhibitors of 5-LOX possess the catechol group

that is a relevant characteristic for their inhibitory properties [44].

Table 10. Activity of prenylated phenols as 5-hLOX inhibitors.

Compound

% Inhibition

7

8

9

10

11

12

13

4

1

51

38

20

12

17

2.6. DPPH Radical Scavenging Activity

The results obtained from antioxidant activity assays are shown in Table 11. All

compounds were compared with Trolox. All compounds possessing the catechol moiety

were active. It is known that the ﬂavonoids quercetin (

2

) and ﬁsetin (3) have better

antioxidant activity than Trolox [45]; however, their prenylated derivatives (

9

and 10) show

an even greater degree of this activity. This behavior follows the same trend found in some

of our previous work [18].

Table 11. Screening results of DPPH radical scavenging activity of ﬂavonoids and their preny-

lated derivatives.

Compound

IC₅₀µM ± SD

Compound

IC₅₀µM ± SD

1

2

3

4

5

NA

13.22 ± 0.49

12.82 ± 0.48

NA

7

8

9

10

11

12

13

NA

9.49 ± 0.36

10.32 ± 0.03

NA

Trolox

22.54 ± 0.61

Antioxidant activity is shown as IC₅₀values in

µM concentrations; NA = no activ-

ity. All compounds were analyzed in triplicate, and the results expressed as average

standard deviation.

±

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2.7. Cytotoxic Activity of 11 and 12

Anticancer activity for naringenin in diverse tumor cell lines has been previously re-

ported [46 48]. However, in vitro activities were commonly observed using high ﬂavonoid

concentrations often exceeding 100 M, which have limited physiological signiﬁcance

–

µ

and must be interpreted with caution. Given this anticancer activity and that prenylated

ﬂavonoids have enhanced biological activities, we measured cytotoxic activity prenylated

naringenin at positions 6 and 8 (11 and 12) against MDA-MB-231 (the human breast adeno-

carcinoma cell line), B16-F10 (mouse melanoma cells), and MEF (primary mouse embryonic

ﬁbroblasts). Our results in Table 12 show that the cytotoxic effect of both prenylated

naringenins were low for both cancer cell lines and did not show any selectivity com-

pared to the effect on non-cancerous cells (MEFs). When the cytotoxic effect of 11 and 12

against cell lines were compared with commonly used chemotherapeutic drugs, it could

be veriﬁed that both prenylated ﬂavonoids showed from 100- to 250-fold lower activity

than Taxol and from 30- to 50-fold lower activity than etoposide. It is possible that the

anticancer activity described for naringenin in vivo could be related to antiangiogenic and

immunostimulating effects [49,50].

Table 12. Cytotoxic activities of 11 and 12 against cancer and non-cancer cells.

IC₅₀(µM) ± SD

Compound

MDA-MB-231

B16-F10

MEF

11

12

Taxol

53.94 ± 9.66

59.02 ± 3.25

0.21 ± 0.05

1.33 ± 0.28

49.14 ± 3.38

45.53 ± 3.82

0.48 ± 0.08

1.74 ± 0.32

48.45 ± 3.44

54.66 ± 3.35

0.40 ± 0.07

1.03 ± 0.17

Etoposide

SD: standard deviation; MDA-MB-231: human breast adenocarcinoma cell line; B16-F10: mouse melanoma cells;

MEF: primary mouse embryonic ﬁbroblast.

On the other hand, compounds 11 and 12 exhibited the most effective antibacterial

activity, which had a synergistic effect with commercial antibiotics. A signiﬁcant synergic

activity was observed using approximately 7 µM for 11 and 15 µM for 12 (Table 4 ), which

did not generate a cytotoxic effect in MEF cells. This clearly suggests that 11 and 12 could be

useful for in vivo treatment against pathogenic resistant bacteria, although further studies

are needed to conﬁrm this possibility.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Data

All chemical reagents obtained were purchased from Merck (Darmstadt, Germany),

Sigma–Aldrich (St. Louis, MO, USA), or Alfa Aesar (Kandel, Germany), were of the highest

commerciall_◦y available purity, and were used without previous puriﬁcation. Melting

points (mp: C) were measured on a melting point apparatus Stuart-Scientiﬁc SMP3 and

are uncorrected. IR spectra were recorded as a KBr disk in a Thermo Scientiﬁc Nicolet

6700 FT-IR spectrometer (Massachusetts, USA), and frequencies are reported in cm⁻¹

.

High-resolution mass spectra were recorded on an Exactive^TMPlus Orbitrap spectrometer

(Thermo Scientiﬁc, Bremen, Germany) by infusion, applying a voltage of 5 or 8 kV in the

positive ionization mode and 4 or 7 kV in the negative ionization mode. The spectra were

recorded using full-scan mode, covering a mass range from m/z 214 to 470. The resolution

was set to 140,000, and the maximum loading time for the ion cyclotron resonance (ICR) cell

1

was set to 200 ms. H-, ¹³C-(DEPT 135), 2D HSQC, and 2D HMBC spectra were recorded

in DMSO-d₆solutions and referenced to the residual peaks of DMSO at

and

39.5 ppm for ¹³C, respectively, on a Bruker Avance 400 Digital NMR spectrometer,

operating at 400.1 MHz for ¹H and 100.6 MHz for ¹³C. Chemical shifts are reported in

δ

2.50 ppm for ¹H

δ

ppm and coupling constants (J) are given in Hz. Silica gel (Merck 200–400 mesh) was used

for C.C. and silica gel plates HF-254 for TLC. TLC spots were detected by both under a UV

Int. J. Mol. Sci. 2021, 22, 5472

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lamp and heating after drenching in 10% H₂SO₄in H₂O. Antioxidant determinations were

performed in a Thermo Scientiﬁc Multiskan GO 96-well plate photometer.

3.1.2. General Experimental Procedure for the Prenylation of Flavonoids

A solution of ﬂavonoid (1 mol equiv) and dry ZnCl₂(4 mol equiv) was placed in a

round bottom ﬂask and dissolved in ethyl acetate (100 mL). Under vigorous stirring, a

solution of 3-methyl-2-buten-1-ol (6) (4 mol equiv) in ethyl acetate (10 mL) was added

dropwise for 1 h at 40 ^◦C. The reaction mixture was then allowed to warm up to reﬂux

temperature and the stirring continued. After 4 h, water at pH 1 (100 mL) was added

to the reaction mixture to decompose the ZnCl₂. The organic layer was then separated,

and a new extraction with ethyl acetate was made. The organic solutions obtained after

extractions were mixed and dried over anhydrous sodium sulphate and ﬁltered, and the

solvent was evaporated under reduced pressure. The mixture was then subjected to silica

gel ﬂash column chromatography (ethyl acetate/hexane or dichloromethane/methanol or

acetone/hexane mixtures were used as mobile phases) to obtain pure products.

All structures were conﬁrmed by IR and NMR spectra as discussed below.

3.1.3. Physical Data of Prenylated Flavonoids

2-phenyl-8-(3-methyl-2-buten-1-yl)-5,7-dihydroxy-4H-chromen-4-one (

6-(3-methyl-2-buten-1-yl)-5,7-dihydroxy-4H-chromen-4-one ( ) were obtained from chrysin

) (3.9 mmol), 3-methyl-2-buten-1-ol ( ) (15.7 mmol), and ZnCl₂(15.7 mmol), as described

above. The crude mixture was puriﬁed using ethyl acetate-hexane in a gradient system

(0 70% of ethyl acetate) as the mobile phase to afford as a yellow powder (329 mg,

26%); mp: 222 ^◦C; HRMS m/z, observed: 321.1131; C₂₀H₁₇O₄[M–H]⁻requires: 321.1127.

IR (KBr):

_maxcm⁻¹: 3431, 2960, 2927, 2858, 1731, 1647, 1615, 1579, 1507, 1384, 1363, 1275.

¹H-NMR (DMSO-d₆)

7) and 2-phenyl-

8

(1

6

→

7

ν

δ

ppm: 12.75 (s, 1H, ArOH-5); 10.6–11.0 (br. s, 1H, ArOH-7); 8.03 (d,

2H, J = 6.8 Hz, 2⁰,6⁰-ArH); 7.59–7.57 (m, 3H, 3⁰,4⁰,5⁰-ArH); 6.95 (s, 1H, ArH-3); 6.30 (s, 1H,

ArH-6); 5.18 (br. t., 1H, CH = C(CH₃)₂); 3.44 (d, 2H, J = 6.7 Hz, CH₂CH = C(CH₃)₂); 1.74

(s, 3H, -CH₂CH = CCH₃CH₃); 1.61 (s, 3H, -CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-d₆)

δ

ppm: 17.8 (-CH = CCH₃CH₃); 21.3 (-CH₂CH = CCH₃CH₃); 25.4 (CH₂CH = CCH₃CH₃);

98.5 (ArC-6); 103.9 (ArC-4a); 105.0 (ArC-3); 106.3 (ArC-8); 122.4 (CH = C(CH₃)₂); 126.3

(ArC-2⁰,6⁰); 129.2 (ArC-3⁰,5⁰); 131.1 (CH = C(CH₃)₂); 132.0 (ArC-4⁰); 154.6 (ArC-8a); 159.1

(ArC-5); 161.9 (ArC-7); 163.1 (ArC-2); 182.2 (4-C = O).

◦

Compound

8

was obtained as a yellow powder (291 mg, 23%) mp: 221–222 C (lit. [51

]

213–214 ^◦C). HRMS m/z, observed: 321.1130; C₂₀H₁₇O₄[M–H]⁻requires: 321.1127. IR

(KBr):

ν

cm⁻¹: 3400, 2969, 2918, 2651, 1639, 1580, 1553, 1483, 1449; ¹H-NMR (DMSO-

max

d₆)

δ

ppm: 13.08 (s, 1H, ArOH-5); 8.05 (d, 2H, J = 7.1 Hz, 2⁰,6⁰-ArH); 7.60–7.56 (m, 3H,

3⁰,4⁰,5⁰-ArH); 6.95 (s, 1H, ArH-3); 6.57 (s, 1H, ArH-8); 5.17 (br. t, 1H, CH = C(CH₃)₂); 3.22

(d, 2H, J = 6.8 Hz, -CH₂CH = C(CH₃)₂); 1.72 (s, 3H, -CH₂CH = CCH₃CH₃); 1.61 (s, 3H,

-CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-d₆)

δ ppm: 17.7 (-CH = CCH₃CH₃); 21.0 (-CH₂CH

= CCH₃CH₃); 25.5 (CH₂CH = CCH₃CH₃); 93.4 (ArC-8); 103.7 (ArC-4a); 105.0 (ArC-3); 111.2

(ArC-6); 122.1 (CH = C(CH₃)₂); 126.4 (ArC-2⁰,6⁰); 129.1 (ArC-3⁰,5⁰); 130.7 (CH = C(CH₃)₂);

130.8 (ArC-1⁰); 131.9 (ArC-4⁰); 155.2 (ArC-8a); 158.3 (ArC-5); 162.2 (ArC-7); 162.9 (ArC-2);

181.9 (4-C = O).

5,7-dihydroxy-6-(3-methyl-2-buten-1-yl)-2-(4-hydroxyphenyl)chroman-4-one (11) and

5,7-dihydroxy-8-(3-methyl-2-buten-1-yl)-2-(4-hydroxyphenyl)chroman-4-one (12) were ob-

tained as racemic mixtures because these were synthesized from (

mmol) commercially available, 3-methyl-2-buten-1-ol ( ) (7.3 mmol), and ZnCl₂(7.3 mmol)

as described above. The crude mixture was puriﬁed using ethyl acetate-hexane in a gradi-

±

) naringenin (4) (3.7

6

ent system (0

→

70% of ethyl acetate) as the mobile_◦phase to afford 11 as a yellow powder

◦

(378 mg, 30%); mp: 206–207 C (lit. [52] 212–214 C); HRMS m/z, observed: 341.1384;

C₂₀H₂₁O₆[M + H]⁺requires: 341.1389. IR (KBr):

ν

_maxcm⁻¹: 3420, 2960, 2926, 2857, 1728,

1

1633, 1586, 1519, 1457, 1384, 1296. H-NMR (DMSO-d₆)

(br. s, 1H, ArOH-7); 9.55 (br. s, 1H, ArOH-4 ) 7.29 (d, 2H, J = 8.4 Hz, ArH-2 ,6 -); 6.78 (d, J =

δ

ppm: 12.40 (s, 1H, Ar-OH-5); 10.74

0

Int. J. Mol. Sci. 2021, 22, 5472

14 of 22

8.4 Hz, 2H, ArH-3⁰,5⁰); 5.94 (s, 1H, ArH-8); 5.39 (dd, 1H, J₁= 12.5 Hz, J₂= 2.6 Hz, CH-2);

5.11 (br. t, 1H, CH = C(CH₃)₂); 3.22 (dd, 1H, J₁= 17.1Hz, J₂= 12.7 Hz, CHH-3); 3.10 (d,

2H, J = 7.0 Hz, CH₂CH = C(CH₃)₂); 2.66 (dd, 1H, J₁= 14.3 Hz, J₂= 2.9 Hz, CHH-3); 1.68 (s,

3H, CH₂CH = CCH₃CH₃); 1.60 (s, 3H, CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-d₆)

δ ppm:

17.6 (-CH = CCH₃CH₃); 20.6 (-CH₂CH = CCH₃CH₃); 25.4 (-CH₂CH = CCH₃CH₃); 42.0

(CH₂-3); 78.3 (ArC-2); 94.3 (ArC-8); 101.5 (ArC-4a); 107.5 (ArC-6); 115.1 (ArC-3⁰,5⁰); 122.6

(CH = C(CH₃)₂); 128.3 (ArC-2⁰,6⁰); 128.98 (ArC-1⁰); 130.2 (CH = C(CH₃)₂); 157.7 (ArC-4⁰);

160.5 (ArC-7,8a); 164.3 (ArC-5); 196.4 (4-C = O).

Compound 12 was obtained as a yellow powder (453 mg, 36%) mp: 193 ^◦C (lit. [53

]

183–184 ^◦C,

requires: 339.1232. IR (KBr):

¹H-NMR (DMSO-d₆)

±

-8-prenylnaringenin); HRMS m/z, observed: 339.1236; C₂₀H₁₉O₅[M–H]⁻

ν

cm⁻¹: 3169, 2966, 2912, 1635, 1519, 1439, 1383, 1347.

max

δ

ppm: 12.09 (s, 1H, Ar-OH-5); 10.75 (s, 1H, ArOH-7); 9.56 (s, 1H,

ArOH-4⁰) 7.30 (d, 2H, J = 8.4 Hz, ArH-2⁰,6⁰); 6.78 (d, J = 8.4 Hz, 2H, ArH-3⁰,5⁰); 5.95 (s, 1H,

ArH-6); 5.40 (dd, 1H, J₁= 12.4 Hz, J₂= 2.7 Hz, CH-2); 5.07 (br. t, 1H, CH = C(CH₃)₂); 3.19

(dd, 1H, J₁= 17.1 Hz, J₂= 12.6 Hz, CHH-3); 3.06 (d, 2H, J = 7.1 Hz, CH₂CH = C(CH₃)₂); 2.70

(dd, 1H, J₁= 17.2 Hz, J₂= 2.7 Hz, CHH-3); 1.57 (s, 3H, CH₂CH = CCH₃CH₃); 1.52 (s, 3H,

CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-d₆)

δ ppm: 17.6 (-CH = CCH₃CH₃); 21.2 (-CH₂CH

= CCH₃CH₃); 25.5 (-CH₂CH = CCH₃CH₃); 41.9 (CH₂-3); 78.2 (ArC-2); 95.3 (ArC-6); 101.7

(ArC-4a); 106.9 (ArC-8); 115.1 (ArC-3⁰,5⁰); 122.6 (CH = C(CH₃)₂); 128.0 (ArC-2⁰,6⁰); 129.2

0

(ArC-1 ); 130.1 (CH = C(CH₃)₂); 157.5 (ArC-4 ); 159.7 (ArC-8a); 161.1 (ArC-5); 164.3 (ArC-7);

196.6 (4-C = O).

2-(3,4-dihydroxy-5-(3⁰-methyl-2⁰-buten-1⁰-yl)phenyl)-3,5,7-trihydroxy-4H-chromen-4-

one (

(13.2 mmol), and ZnCl₂(13.2 mmol) as described above. The crude mixture was puriﬁed

using ethyl acetate-hexane in a gradient system (0 80% of ethyl acetate) as the mobile

9; Uralenol) was obtained from quercetin (2) (3.3 mmol), 3-methyl-2-buten-1-ol (6)

→

phase to afford the ti_◦tle compound as a pale green powder (417 mg, 34%); mp: 205–206

^◦C (lit. [54] 176–178 C); HRMS m/z, observed: 369.0974; C₂₀H₁₇O₇[M–H]⁻requires:

369.0972. IR (KBr):

1363, 1313, 1283, 1247, 1167. H-NMR (DMSO-d₆)

ν

cm⁻¹: 3599, 3390, 2969, 2914, 1655, 1635, 1601, 1566, 1518, 1462,

max

1

δ

ppm: 12.52 (s, 1H, ArOH-5); 10.75 (br.

s, 1H, ArOH-7); 9.31 (s, 1H, ArOH-3⁰); 9.03 (s, 1H, ArOH-4⁰); 8.93 (s, 1H, ArOH-3); 6.83 (s,

1H, ArH-2⁰); 6.66 (s, 1H, ArH-6⁰); 6.29 (d, 1H, J = 1.6 Hz, ArH-8); 6.18 (d, 1H, J = 1.5 Hz,

ArH-6); 5.10 (br. t, 1H, CH = C(CH₃)₂); 3.12 (d, 2H, J = 7.0 Hz, CH₂CH = C(CH₃)₂); 1.56

(s, 3H, CH₂CH = CCH₃CH₃); 1.47 (s, 3H, CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-d₆)

δ

ppm: 17.5 (CH = CCH₃CH₃); 25.4 (CH₂CH = CCH₃CH₃); 31.1 (CH₂CH = CCH₃CH₃); 93.3

(ArC-8); 98.2 (ArC-6); 103.5 (ArC-4a); 116.5 (ArC-6⁰); 117.1 (ArC-2⁰); 120.3 (ArC-5⁰); 123.5

(CH = C(CH₃)₂); 131.0 (CH = C(CH₃)₂); 132.2 (ArC-3⁰); 136.3 (ArC-2); 142.9 (ArC-1⁰); 147.1

(ArC-4⁰); 150.0 (ArC-3); 156.7 (ArC-8a); 160.9 ₀(ArC-5); 163.8 (ArC-7); 176.2 (4-C = O).

0

2-(3,4-dihydroxy-5-(3 -methyl-2 -buten-1 -yl)phenyl)-3,7-dihydroxy-4H-chromen-4-one

(

10) was obtained from ﬁsetin (

ZnCl₂(7 mmol) as described above. The crude mixture was puriﬁed using ethyl acetate-

hexane in a gradient system (0 80% of ethyl acetate) as the mobile phase to afford the title

3) (1.75 mmol), 3-methyl-2-buten-1-ol (6) (7 mmol), and

→

compound as a dark green powder (155 mg, 25%); mp: 229–230 ^◦C; HRMS m/z, observed:

355.1176; C₁₈H₁₉O₆[M + H]⁺requires: 355.1182. IR (KBr):

ν

cm⁻¹: 3350, 2971, 2926,

δ ppm: 10.68 (br. s, 1H,

max

1

2855, 1698, 1612, 1596, 1508, 1458, 1417, 1272. H-NMR (DMSO- d₆)

ArOH-7); 9.25 (s, 1H, ArOH-3 ); 9.01 (s, 1H, ArOH-4 ); 8.62 (s, 1H, ArOH-3); 7.93 (d, 1H, J =

0

8.6 Hz, ArH-5); 6.89 (d, 1H, J = 8.2 Hz, ArH-6); 6.83 (s, 1H, ArH-2 ); 6.78 (s, 1H, ArH-8); 6.66

(s, 1H, ArH-6⁰); 5.11 (br. t, 1H, CH = C(CH₃)₂); 3.12 (d, 2H, J = 6.8 Hz, CH₂CH = C(CH₃)₂);

1.54 (s, 3H, CH₂CH = CCH₃CH₃); 1.44 (s, 3H, CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO-

d₆)

δ ppm: 17.5 (CH = CCH₃CH₃); 25.4 (CH₂CH = CCH₃CH₃); 31.2 (CH₂CH = CCH₃CH₃);

101.8 (ArC-8);114,6 (ArC-6); 114.8 (ArC-4a); 116.4 (ArC-6⁰); 117.1 (ArC-2⁰); 120.9 (ArC-5⁰);

123.6 (CH = C(CH₃)₂); 126.5 (ArC-5); 130.8 (CH = C(CH₃)₂); 132.0 (ArC-3⁰); 137.8 (ArC-1⁰);

142.8 (ArC-2); 146.8 (ArC-4⁰); 148.1 (ArC-3); 156.7 (ArC-8a); 162.1 (ArC-7); 172.1 (4-C = O).

3-(4-hydroxyphenyl)-8-(3-methyl-2-buten-1-yl)-5,7-dihydroxy-4H-chromen-4-one (13

;

lupiwighteone) was obtained from genistein ( ) (1.9 mmol), 3-methyl-2-buten-1-ol (6)

5

Int. J. Mol. Sci. 2021, 22, 5472

15 of 22

(7.4 mmol), and ZnCl₂(7.4 mmol) as described above. The crude mixture was puriﬁed

using ethyl acetate-hexane in a gradient system (0

→

80% of ethyl acetate) as the mobile

phase to afford the title compound as a white powder (231 mg, 36 %); mp: 140 ^◦C (lit. [55

]

133–135 ^◦C); HRMS m/z, observed: 339.1228; C₂₀H₁₉O₅[M + H]⁺requires: 339.1232. IR

(KBr):

ν

cm⁻¹: 3350, 3174, 2921, 1705, 1651, 1613, 1570, 1513, 1428, 1364, 1301, 1257,

max

1

0

1198. H-NMR (DMSO-d₆):

δ

ppm: 12.98 (s, 1H, ArOH-5); 9.58 (br. s, 1H, ArOH-4 ); 8.38 (s,

0

1H, ArH-2); 7.37 (d, 2H, J = 8.6 Hz, ArH-2 ,6 ); 6.80 (d, 2H, J = 8.6 Hz, ArH-3 ,5 ); 6.30 (s, 1H,

ArH-6); 5.14 (br. t, 1H, CH = C(CH₃)₂); 3,34 (overload with H₂O, CH₂CH = C(CH₃)₂); 1.74

(s, 3H, -CH₂CH = CCH₃CH₃); 1.62 (s, 3H, -CH₂CH = CCH₃CH₃). ¹³C-NMR (DMSO- d₆)

δ

ppm: 17.7 (-CH = CCH₃CH₃); 21.0 (-CH₂CH = CCH₃CH₃); 25.4 (CH₂CH = CCH₃CH₃); 98.5

0

(ArC-6); 104.4 (ArC-4a); 105.8 (ArC-8); 115.0 (ArC-3 ,5 ); 121.3 (ArC-1 ); 121.9 (ArC-3); 122.1

(CH = C(CH₃)₂); 130.1 (ArC-2⁰,6⁰); 131.0 (CH = C(CH₃)₂); 154.0 (ArC-2); 154.8 (ArC-8a);

157.3 (ArC-4⁰); 159.5 (ArC-5); 161.7 (ArC-7); 180.5 (4-C = O).

3.2. Biological Assays

3.2.1. In Vitro Antibacterial Activity Assays: Human Pathogens

Minimum Inhibitory Concentration Assay

Two clinical isolates of methicillin-resistant S. aureus (622-4 and 97-7) and one clinical

isolate of E. coli 33.1 were kindly donated by Dr. Marcela Wilkens from Universidad de

Santiago de Chile. S. aureus (NCTC8325-4) and E. coli (ATCC25922) were used as the control

strains. The antimicrobial activities of the isolated compounds against E. coli (ATCC25922),

multi-resistant E. coli (33.1), S. aureus (NCTC8325-4), and methicillin-resistant S. aureus

(MRSA) 97-7 strains were assessed using the Clinical and Laboratory Standards Institute

(CLSI) microdilution method [56]. Brieﬂy, stock solutions (5 mg/mL) of compounds in

DMSO were two-fold diluted in Mueller–Hinton broth (MHB). The ﬁnal concentration

of DMSO was

≤

2.5% and does not affect the microbial growth. The obtained solution

was added to MHB and serially two-fold diluted in a 96-well microplate. A sample of

100 mL of inoculum 1.5

×

10⁶colony-forming units (CFU) ml⁻¹in MHB was added and

then incubated at 37 ^◦C for 18 h. The assay was repeated three times. Wells containing

MHB, 100 mL of inoculum, and DMSO served as negative controls. The MIC was deﬁned

as the lowest concentration of compounds resulting in the complete inhibition of visible

growth [57].

Checkerboard Dilution Test

The assessments of synergy of prenylated ﬂavonoids 11 and 12 combined with methi-

cillin were investigated with the checkerboard method [57]. Brieﬂy, the uppermost row (A)

of a 96-well microtiter plate contained substance

X in a concentration of about four times

the expected MIC of the microorganism examined. Each following row (B–H) contained

half the concentration of the previous one. The same procedure was carried out along the

columns (1–12) with substance Y, but not necessarily with the same starting concentration.

Therefore, each well contained a unique combination of the two substances (X and Y).

Lastly, 100 µ

L of Mueller–Hinton broth containing about 10⁵(CFU/mL) were added to the

wells and incubated at 37 ^◦C for 24 h. The concentrations of the ﬁrst wells without visible

growth along the stepwise boundary between inhibition and growth were used to calculate

the FICI values. The fractional inhibitory concentration index (FICI) was calculated as

follows: FICI = fractional inhibitory concentration of A (FIC_A) + (FIC_B) = (MIC of drug

A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug

B alone). FIC index was interpreted as follows: synergy, <0.5; partial synergy, 0.5–0.75;

additive effect, 0.76–1.0; indifference, >1.0–4.0; antagonism, >4.0 [58].

Time Kill Assay

Time kill curves were conducted with a ﬁnal concentration of antimicrobial agent at

four times the MIC [59]. Flasks containing 50 mL of MHB with the appropriate antimicrobial

agent were inoculated with 50 mL of the test organism in a logarithmic growth phase

Int. J. Mol. Sci. 2021, 22, 5472

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adjusted to the appropriate density. Aliquots were removed, diluted, and plated at the 0, 2,

4, 6, and 24 h time points. The plates were incubated for 24 h, and the viable counts were

determined. Bactericidal activity was deﬁned as a reduction of 99.9% ( 3 log10) of the

≥

total count of CFU/mL in the original inoculum [56]. Bacteriostatic activity was deﬁned as

the maintenance of or a reduction of less than 99.9% (3 log10) of the total count of CFU/mL

in the original inoculum.

Determination of Reactive Oxygen Species (ROS) Intrabacterial Levels

The production of ROS by S. aureus ATCC 6538 after treatment with the prenylated

0

compounds was evaluated using the peroxynitrite indicator 2 ,7 -dichlorodihydroﬂuorescein

diacetate (DCFH-DA) (Sigma–Aldrich), which can detect a broad range of ROS includ-

ing nitric oxide and hydrogen peroxide. The adjusted bacterial culture (0.5 McFarland

exponential phase bacteria culture) was treated with a concentration of each compound

of 2 µM, using ciproﬂoxacin as an antibiotic control, Trolox as a ROS scavenger control,

1,4-naphtoquinone as a ROS generation control, and the presence of DCFH-DA at a ﬁnal

concentration of 5 mM in 0.85% saline and incubated at 37 ^◦C aerobically at 200 rpm for

24 h. Untreated bacterial culture was served as the negative control. The ﬂuorescence

emission of DCFH-DA was measured at 525 nm using a Tecan microtiter plate reader

with an excitation wavelength of 485 nm. The background ﬂuorescence of 0.85% saline

and autoﬂuorescence of the bacterial cells incubated without the probe was measured to

calculate the net ﬂuorescence emitted from the assay itself. The experiment was conducted

in triplicate [60].

3.2.2. In Vitro Antifungal Activity Assays against Human Pathogens

Microorganisms and Media

For the antifungal evaluation, standardized strains from the American Type Culture

Collection (ATCC, Manassas, VA, USA), Centro de Referencia en Micología (CCC), Facultad

de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-(2000)-Rosario, Argentina and

Malbrán Institute (IM), Av. Velez Sársﬁeld 563, Buenos Aires, Argentina were used. The

voucher specimens of standardized strains are as follows: C. albicans ATCC 10231, C. neo-

formans ATCC 32264, A. ﬂavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029,

T. rubrum CCC 110, T. mentagrophytes ATCC 9972, and M. gypseum CCC 115. Clinical isolates

of C. neoformans (n = 5) were provided by IM. They included ﬁve strains whose voucher

specimens are presented in Table 7. Clinical isolates of C. albicans (n = 5) were provided by

CCC; their voucher specimens are presented in Table 8.

◦

Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 C,

maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and sub-cultured every

15 days to prevent pleomorphic transformations. Inocula were obtained in accordance

with reported procedures [39

units (CFUs)/mL.

,40] and adjusted to 1–5

×

10³cells with colony-forming

Antifungal Susceptibility Testing

The minimum inhibitory concentration (MIC) of each compound was determined by

using broth microdilution techniques according to the guidelines of the CLSI for yeasts

(M27-A3) [39] and for ﬁlamentous fungi (including dermatophytes) M38-A2 [40]. MIC

values were determined in RPMI-1640 (Sigma–Aldrich) and buffered to pH 7.0 with MOPS.

◦

Microtiter trays were incubated at 35 C for yeasts and Aspergillus spp. and at 28–30 C for

dermatophyte strains in a moist, dark chamber. MICs were visually recorded at 48 h for

yeasts, and at a time according to the control fungus growth, for the rest of the fungi. For

the assay, stock solutions of pure compounds were two-fold diluted with RPMI-1640 from

250 to 0.98

volume of 100

µ

g/mL (ﬁnal volume = 100

µ

L) and a ﬁnal DMSO concentration of

≤

1%. A

µL of inoculum suspension was added to each well with the exception of the

sterility control, where sterile water was added to the well instead. Terbinaﬁne (Novartis

Co, Basel, Switzerland) and amphotericin B (Sigma–Aldrich) were used as positive controls.

Int. J. Mol. Sci. 2021, 22, 5472

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Endpoints were deﬁned as the lowest concentration of drug resulting in the total inhibition

of visual growth compared to the growth in the control wells containing no antifungal drug.

The MFC of each compound against an isolate was determined as follows: After

determining the MIC, an aliquot of 5 µL was withdrawn from each clear well of the

microtiter tray and plated onto a 150 mm RPMI-1640 agar plate buffered with MOPS

◦

(Remel Inc., Lenexa, KS, USA). Inoculated plates were incubated at 30 C, and the MFC was

recorded after 48 h. The MFC was deﬁned as the lowest concentration of each compound

that resulted in the total inhibition of visible growth.

3.2.3. In Vitro Antibacterial Activity against Plant Pathogens

Broth microdilution methods were used to evaluate the effects of the compounds on

the growth of P. carotovorum (NCPPB 312), A. tumefaciens (strain C58C1), and P. syringae

(NCPPB 281). Bacteria were grown in sterile tubes with 10 mL of Mueller–Hinton (MH)

medium and incubated at 27 ^◦C for 12 h with shaking to produce an initial culture. The

antimicrobial activity was evaluated by observing the growth response of both microor-

ganisms in samples with different concentrations of the compounds [61

were performed on sterile 96-well microplates with a ﬁnal volume of 200

ing Mueller–Hinton broth (MH) inoculated with 1

L of bacterial suspension (10⁵–10⁶

UFC/mL, initial culture) in the presence of different concentrations of test compounds

(3.9, 7.8, 15.6, 31.3, 62.5, 125, and 250 M). MH was used as the negative control [C( )],

–

63]. All assays

µL contain-

µ

−

and MH with streptomycin [64] was used as the positive control [C(+)]. The plates were

incubated for 7 h at 27 C. Bacterial growth was monitored by measuring the optical density

◦

at 595 nm every hour with a microplate reader. All tests were performed in 10 repetitions

for each microorganism evaluated. Bacterial growth was shown as the arithmetic mean

expressed in terms of the negative control (100% growth). The lowest concentration of

the compound preventing the appearance of turbidity was considered to be the minimal

inhibitory concentration (MIC).

The ﬁrst experiment (ﬁrst kinetic assay) in which the compound was exposed to the

bacterial cultures in MH was carried out over a period of 6 h. Subsequently, to determine

the minimal bactericidal concentration (MBC), a second experiment (second kinetic assay)

was conducted; this experiment involved taking inoculum from the ﬁrst kinetic assay and

adding it to MH in a new 96-well microplate containing culture medium, which was then

cultured for 7 h. The aim of this second culture (second kinetic assay) was to determine

whether the compounds have bactericidal or bacteriostatic properties [65].

3.2.4. Statistics

The Mann–Whitney U test (*) with p < 0.05 was performed to identify signiﬁcant

differences among the treatment and control groups and between the two treatments.

3.3. In Vitro 5-LOX Enzyme Inhibition Assay

The commercially available enzyme by Cayman Chemicals Inc., Ann Arbor, MI, USA,

was diluted (1:500) in the assay buffer (HEPES 50 mM, EDTA 2 mM, ATP 10

CaCl₂10 M at pH 7.5) and mixed with 10 M H₂DCFDA dye in the reaction mixture

and incubated for 15 min in the assay plate. Subsequently, 280 L of buffer was added,

and 10 L of inhibitor with a ﬁnal concentration of 10 M was placed per well in the

reaction mixture and incubated for 30 min. The reaction was started by the addition

of a suitable concentration of arachidonic acid (0.5 M). The ﬂuorescence was read in

a multimode detector Synergy HT Multi-Mode Microplate Reader (Biotek) at 480 nm

µM, and

µ

™

excitation/520 nm emission after 1 h of incubation at room temperature. The inhibition per-

centage was obtained for analysis of oxidation of H₂DCFDA dye to the highly ﬂuorescent

2⁰,7⁰-dichloro-ﬂuorescein (DCF) product.

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3.4. Docking of 11 (S and R Enantiomers) and with ABC Transporter

The prenylated ﬂavonoids structures were built with the Molecular Operating Envi-

ronment software [66]. ChelpG charges were obtained at the B3LYP/6-31G** level theory,

employing the Gaussian 09 package [67]. Docking was done with the crystal structure of the

ABC transporter (PDB code: 2ONJ, 3.40 Å resolution) using the AutoDock4 package [68

and a Lamarckian algorithm, assuming total ﬂexibility of the inhibitors. The grid maps

were made up ﬁrst to 126 126 126 points and later to 60 60 60, with a grid-point

]

×

spacing of 0.375 Å on the grid map. The AutoTors option was used to deﬁne the ligand

torsions, and the docking results were then analyzed by a ranked cluster analysis, resulting

in conformations with the highest overall binding energy (the most negative Gibbs free

energy binding value, –∆G).

3.5. General Procedure to Determine the DPPH Radical Scavenging Activity

The radical scavenging activity of the prenylated compounds and starting materials

towards the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was measured as described [18],

adapted to a screen to 96-well plates. Brieﬂy, stock solutions of each compound were

prepared in methanol at a 1-mM concentration (10 mL). Dilutions (1–200

µM) were prepared

from the stock solutions. Methanol (90 L), dilutions (150 L), and DPPH (60 µ

µ

L, Sigma–

Aldrich) in methanol (0.5 mM), resulting in a ﬁnal concentration of 0.1 mM of DPPH were

added in a 96-well plate. Methanol was used as the blank sample. The mixtures were left

for 30 min at room temperature, and the absorbances were then measured at 517 nm. Trolox

was used as the standard antioxidant. The radical scavenging activity was calculated as

follows: % Inhibition = [(blank absorbance

The mean of three IC₅₀(concentration causing 50% inhibition) values for each compound

was determined graphically.

−

sample absorbance)/blank absorbance]

×

100.

3.6. In Vitro Anticancer Activity of 11 and 12

The cell lines used in this work included MDA-MB-231 human breast adenocarcinoma

cells, B16-F10 mouse metastatic melanoma cells, and MEF primary mouse embryonic ﬁbrob-

lasts. Cells were maintained in a DMEM high glucose medium (Mediatech, Manassas, VA,

USA) supplemented with 10% (MDA-MB-231 and B16-F10) or 15% (MEF) heat-inactivated

fetal bovine serum (HyClone Laboratories), 100 IU/mL penicillin, and 100

mycin and maintained at 37 ^◦C in a 5% CO₂humidiﬁed atmosphere. Cell viability was

measured using CyQuant Direct Cell Proliferation Assay Kit (Life Technologies) following

the manufacturer’s instruction. Brieﬂy, 5.000 cells/well were seeded onto a ﬂat-bottomed

96-well plate in a 200 L ﬁnal volume. Six hours after seeding, the culture medium was

replaced with the medium containing the tested compounds at concentrations ranging from

0 to 100 M dissolved in DMSO (a 0.1% ﬁnal concentration) for 72 h. The concentrations

used to calculate the IC₅₀values were 100, 30, 10, 3, 1, 0.3, 0.1, 0.01, and 0 M. Untreated

cells (medium containing 0.1% DMSO) were used as controls. At the end of the incubation,

100 L of culture medium was removed from each experimental well and replaced by 2

µg/mL strepto-

®

µ

×

detection reagent. Cells were incubated for 1 h, and ﬂuorescence emission was measured

at 535 nm with excitation at 480 nm in a microplate reader (Inﬁnite 200 PRO, Tecan). At

least four independent experiments were performed for each concentration. The results

from each experiment were transformed to a percentage of controls, and the IC₅₀values

were graphically obtained from the dose–response curves. The IC₅₀value was obtained

adjusting the dose–response curve to sigmoidal curves (variable slope), generated using

GraphPad Prisma 6.0 software [69].

4. Conclusions

Our results demonstrated that the 6-prenylated-(

±

)-naringenin (11), 8-prenylated-( )-

±

naringenin (12) and the 8-prenylated genistein (13) are active molecules against Staphy-

lococcus aureus-resistant bacteria with a strong synergistically effect in combination with

commercial antibiotics vancomycin, ciproﬂoxacin, and methicillin, enhancing their effects

Int. J. Mol. Sci. 2021, 22, 5472

19 of 22

in a factor of 10–100 times against drug-resistant bacteria. Compound 12 is the most active

against the dermatophytes T. rubrum, T. mentagrophytes, and M. gypseum fungus. Prenylation

substitution was shown to be fundamental for these biological activities, speciﬁcally at

positions 6 and 8 of the ( )-naringenin (11 and 12, respectively); however, others structural

±

requirements are important as well, such as hydroxylation at positions 5, 7 of ring A, and

4⁰of ring B. According to the docking results of 11 with ATP-binding cassette transporter,

the S isomer showed better interaction energy than the R isomer. It is relevant to continue

this study with enantiomerically pure isomers of prenylated ﬂavanones 11 and 12.

Supplementary Materials: Supplementary materials can be found at https://ww w .mdpi.com/

article/10.3390/ijms22115472/s1.

Author Contributions: Synthesis of prenylated compounds and antioxidant assay, M.O.; phy-

topathogenic bacteria assays, M.C. and A.V.; human-resistant bacteria studies, Y.V.-M. and M.C.-S.M.;

human pathogenic fungi studies, E.B. and S.Z.; 5-hLOX and docking studies, C.M.; cytotoxic studies,

M.M. and S.M.; writing—original draft preparation, M.O. and Y.V.-M. All authors have read and

agreed to the published version of the manuscript.

Funding: This work was supported by CONICYT, grant number 1130924, by the Dirección General

de Investigación, Innovación y Emprendimiento (DGIIE) of the Universidad Técnica Federico Santa

María, and by DICYT-VRIDEI of the Universidad de Santiago de Chile, grant numbers 021901VM

and 021641MC. E.B. acknowledges CONICET.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Acknowledgments: M.O. acknowledges Luis Espinoza for lab facilities (NMR, IR, and other equip-

ment).

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

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Prenylated flavonoids with potential antimicrobial activity: Synthesis, biological activity, and in silico study

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