Prenylated trans-cinnamic esters and ethers against clinical fusarium spp.: Repositioning of natural compounds in antimicrobial discovery

molecules

Article

Prenylated Trans-Cinnamic Esters and Ethers against Clinical

Fusarium spp.: Repositioning of Natural Compounds in

Antimicrobial Discovery

Safa Oufensou ¹, Stefano Casalini ¹, Virgilio Balmas ¹, Paola Carta ², Wiem Chtioui ¹, Maria A. Dettori ^2,

Davide Fabbri ², Quirico Migheli ^1,3and Giovanna Delogu ²

* ,

1

Dipartimento di Agraria, Università degli Studi di Sassari, Via E. De Nicola 9, 07100 Sassari, Italy;

soufensou@uniss.it (S.O.); stefanocasalini66@yahoo.it (S.C.); balmas@uniss.it (V.B.);

w.chtioui@studenti.uniss.it (W.C.); qmigheli@uniss.it (Q.M.)

2

Istituto CNR di Chimica Biomolecolare, Traversa La Crucca 3, 07100 Sassari, Italy; paola.carta@cnr.it (P.C.);

davidegaetano.fabbri@cnr.it (D.F.); giovanna.delogu@icb.cnr.it (G.D.)

Nucleo di Ricerca sulla Desertiﬁcazione, Università degli Studi di Sassari, Via E. De Nicola 9,

07100 Sassari, Italy

3

*

Correspondence: mariaantonietta.dettori@cnr.it; Tel.: +39-079-284-1224

Abstract: Onychomycosis is a common nail infection mainly caused by species belonging to the

F. oxysporum, F. solani, and F. fujikuroi species complexes. The aim of this study was to evaluate

the in vitro susceptibility of six representative strains of clinically relevant Fusarium spp. toward a

set of natural-occurring hydroxycinnamic acids and their derivatives with the purpose to develop

naturally occurring products in order to cope with emerging resistance phenomena. By introducing a

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

prenylated chain at one of the hydroxy groups of trans-cinnamic acids

1–3, ten prenylated derivatives

Citation: Oufensou, S.; Casalini, S.;

Balmas, V.; Carta, P.; Chtioui, W.;

Dettori, M.A.; Fabbri, D.; Migheli, Q.;

Delogu, G. Prenylated Trans-

(coded 13) were preliminarily investigated in solid Fusarium minimal medium (FMM). Minimal

4–

inhibitory concentration (MIC) and lethal dose 50 (LD₅₀) values were then determined in liquid FMM

0

for the most active selected antifungal p-coumaric acid 3,3 -dimethyl allyl ester 13, in comparison with

Cinnamic Esters and Ethers against

Clinical Fusarium spp.: Repositioning

of Natural Compounds in

the conventional fungicides terbinaﬁne (TRB) and amphotericin B (AmB), through the quantiﬁcation

of the fungal growth. Signiﬁcant growth inhibition was observed for prenylated derivatives

evidencing ester 13 as the most active. This compound presented MIC and LD₅₀values (62–250

and 7.8–125 M, respectively) comparable to those determined for TRB and AmB in the majority

4–13,

µM

Antimicrobial Discovery. Molecules

2021, 26, 658. https://doi.org/

10.3390/molecules26030658

µ

of the tested pathogenic strains. The position and size of the prenylated chain and the presence

of a free phenol OH group appear crucial for the antifungal activity. This work represents the

ﬁrst report on the activity of prenylated cinnamic esters and ethers against clinical Fusarium spp.

and opens new avenues in the development of alternative antifungal compounds based on a drug

repositioning strategy.

Academic Editor: Giulio Rastelli

Received: 28 December 2020

Accepted: 24 January 2021

Published: 27 January 2021

Keywords: onychomycosis; mycoses; Fusarium spp.; drug development; antifungal activity; phenolic

0

inhibitors; hydroxycinnamic acid derivates; p-coumaric acid 3,3 -dimethyl allyl ester

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1. Introduction

Phenolics are among the most desirable food components because of their excellent

antioxidant activity and nutraceutical properties. They also ﬁnd wide-ranging applica-

tion in medicine and agriculture in virtue of their antimicrobial, anti-inﬂammatory, and

antitumoral activities [1,2]. Among the phenolic compounds, cinnamic acids are a group

of aromatic carboxylic acids formed through the biochemical route of shikimate pathway,

leading to the synthesis of lignin, the polymeric material that provides mechanical support

Copyright:

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

to the plant cell wall [

most common cinnamic acids, consisting of a trans-

bonded to a phenol, catechol, and guaiacyl unit, respectively. They possess three distinctive

3

,4

]. p-Coumaric acid

1

, caffeic acid

2, and ferulic acid 3 are the

α, β-unsaturated carboxylic chain

Molecules 2021, 26, 658. https://doi.org/10.3390/molecules26030658

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

Molecules 2021, 26, 658

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structural motifs that may possibly contribute to the free radical scavenging capability of

these compounds (Figure 1).

Figure 1. Chemical structures of p-coumaric acid 1, caffeic acid 2, and ferulic acid 3.

The presence of an electron donating group on the benzene rings provides the addi-

tional property of terminating free radical chain reaction. The carboxylic acid group with a

conjugated C-C double bond provides additional quenching sites for free radicals. These

features of cinnamic acids are reﬂected in many biological processes. Cinnamic acids, the

main constituents of plant defense, prevent the effects of reactive oxygen species (ROS)

formed during fungal infection [5]. The carboxylic acid group of cinnamic acid can act as

an anchor by which the compound binds to the lipid bilayer, providing some protection

against lipid peroxidation, a process spread out across mammal and human cells [6].

Cinnamic acids

tomato, and berries), vegetables (e.g., bean, potato, and onion), and cereals (e.g., maize,

oat, and wheat bran) [ ]. They occupy a key place as intermediates in the synthesis of

1–3 are widely distributed in fruits (e.g., apple, pear, grape, orange,

7

pharmaceuticals, dyes, ﬂavorings, cosmetics, thermoplastics, and materials [8,9]. Because

of their high-promoting health capacities and commercial value and given the availability

of cinnamic acids 1–3 in different plants, extracting processes from biomass are also well

studied even though the main accessibility of these compounds comes from synthetic or

microbial processes [10,11].

Drug repositioning implies the identiﬁcation of novel biological targets for natural-

occurring compounds that may ﬁnd application in ﬁelds where safety and efﬁciency

are still lacking. One approach relies on slight structural modiﬁcation of the natural

compound, aiming to enhance its biological activity toward a speciﬁc target. When the

structural modiﬁcation of the compound concerns the introduction of a natural unit to the

molecule framework, the ﬁnal compound acquires a natural-like feature. Often, such slight

structural modiﬁcations are devoted to improving bioavailability and selectivity of the

parent compound [12–16].

Onychomycosis is a chronic fungal nail infection mainly caused by Fusarium spp.,

particularly those belonging to three specie complexes: F. oxysporum (FOSC), F. solani (FSSC),

and F. fujikuroi species complex (FFSC) [17 –21]. F. oxysporum Schlechtend. emend. Snyder

& Hansen; F. solani (Mart.) Sacc.; F. petroliphilum (Q.T. Chen & X.H. Fu) Geiser, O’Donnell,

D.P.G. Short, & N. Zhang; F. keratoplasticum Geiser, O’Donnell, D.P.G. Short, & Ning Zhang;

F. falciforme (Carrión); and F. verticillioides (Saccardo) Nirenberg are the most representative

species responsible for onychomycosis; moreover, they also infect skin and hair and are

considered as emerging pathogens from superﬁcial mycoses as dermatomycoses. Fusarium

spp. are increasingly reported among the world population. Besides dermatomycoses or

onycomycoses, they are responsible for disseminated infections, particularly in patients

undergoing cancer therapy or those affected by immunological deﬁciency [22–24].

Systemic antifungals are the most effective treatment, with meta-analyses showing

mycotic cure rates of 76% for terbinaﬁne, 63% for itraconazole with pulse dosing, 59% for

itraconazole with continuous dosing, and 48% for ﬂuconazole [25,26]. The use of these

agents is discouraged in patients suffering from liver, renal, or heart disease, and in those

receiving medications with which there may be signiﬁcant drug–drug interactions [27].

Terbinaﬁne (TRB) belongs to the allylamine class of synthetic antimycotic agents, and

inhibits the squalene epoxidase, a key enzyme involved in the early phase of the ergosterol

biosynthetic pathway [28]. Amphotericin B (AmB) is a broad-spectrum antifungal agent

belonging to the polyene class; its mechanism of action targets membrane function by

Molecules 2021, 26, 658

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forming channels in the fungal cell membrane, hence allowing ions and organic compounds

of the cytoplasm to escape [29].

Susceptibility to fungicides among different Fusarium spp. may vary greatly, and

clinical Fusaria showing multiple resistance to most applied antifungal drugs are increas-

ingly being reported [30–32]. Numerous factors have been cited to explain the lack of

response to therapy, such as nonadherence to treatment, incorrect diagnosis, or advanced

disease [25]. Additionally, antimycotic prophylaxis in high-risk patients may enhance

selective pressure, which favors multidrug-resistant fungi, including Fusaria [33]. This

urges a massive investment in the development of novel agents to treat emerging and

resistant fungi [34].

Considering the antimicrobial activity of cinnamic acids 1–3 [35], their low toxicity, and

large market availability, the aim of this work was to investigate these compounds against

a selection of representative strains belonging to the F. oxysporum (FOSC), F. solani (FSSC),

and F. fujikuroi (FFSC) species complexes; namely, F. oxysporum, F. solani F. keratoplasticum,

and F. verticillioides.

Aiming to improve the bioavailability and to enhance antimicrobial activity, we trans-

formed cinnamic acids 1–3 in esters and ethers using short, medium, and long prenylated

chains as alcoholic unit. The efﬁciency of prenylated phenols in crossing bacterial and fun-

gal membranes [36], as well as their role in exerting antimicrobial activity [37], are generally

acknowledged. O-prenylated phenols are secondary metabolites of plants. Even though

they have been considered for years as biosynthetic intermediates of the most widespread

C-prenylated derivatives, recently, O-prenylated chains are assuming a key role in the

bioactivity of molecules into which they are embedded [38

,39]. We thus predicted that the

preparation of prenylated ethers and prenylated esters of cinnamic acids

1–3

(Figure 2)

could offer an alternative to the conventional antifungal drugs used against clinical Fusaria,

enhancing the antimycotic effect of the parent acid.

Figure 2. Chemical structure of the tested compounds numbered from 1 to 13.

An in vitro assay of clinical isolates of Fusarium spp. grown on solid Fusarium minimal

medium (FMM) amended with compounds 1–13 was carried out and a structure–activity

relationship was described. Minimal inhibitory concentration (MIC) and lethal dose 50

(LD₅₀) values were then determined in liquid FMM for the most active selected antifungal

compound, in comparison with the conventional fungicides terbinaﬁne (TRB) and ampho-

tericin B (AmB). The presence of a O-prenylated chain in natural occurring cinnamic acids

Molecules 2021, 26, 658

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may share a different mode of action compared to conventional antifungal drugs and offers

a successful example of drug repositioning.

2. Results

2.1. Chemistry

Prenylated esters

the corresponding cinnamic acid

4

–

7

and

9

and prenylated ethers 10

1–3

–13 were prepared, starting from

and, according to the reaction, allyl, 3,3⁰-dimethyl

allyl (prenyl), geranyl, and citronellyl bromide or alcohol. Because of the contemporary

presence of two hydroxyl groups in the starting hydroxy cinnamic acid, different synthetic

approaches were applied when ether or ester was the ﬁnal product.

In order to functionalize selectively the phenolic-OH group with a prenylated chain,

esteriﬁcation of the carboxylic group was mandatory (Scheme 1).

Scheme 1. Synthesis of hydroxyl cinnamic ethers 4–7 and 9 and esters 10–13 and 14–16.

Ethers 4–7 and 9 were obtained by the Williamson reaction, starting from the corre-

sponding methyl ester and the appropriate alkenyl bromide under basic conditions and

further ester hydrolysis. Yields varied in the range of 53 and 85%. Methyl esteriﬁcation of

acids 1–3 was carried out under acid conditions under microwave treatment.

For compound 5, the selectivity of etheriﬁcation reaction at the p-phenolic-OH was

conﬁrmed by Nuclear Overhauser Effect Spectroscopy-NMR (NOESY) experiment and by

comparison of NMR spectra with the corresponding methyl ester reported in the literature

(see Materials and Methods for references).

Esters 10

the corresponding parent acid (i.e., 4-hydroxy cinnamic acid

acid ) via activation of the carboxylic group with ethyl chloroformate and triethylamine

–

13 were prepared with yields ranging between 37 and 47%, starting from

1

, caffeic acid , and ferulic

2

3

and further addition of the appropriate prenylated alcohol (Scheme 1) and hydrolysis of

the phenyl ethyl carbonate.

Compound

8 was obtained via Fischer esteriﬁcation of acid 2 using allyl alcohol

as solvent under acidic conditions as reported in the literature. Compound 13 was also

obtained in 87% yield by enzymatic transesteriﬁcation of the corresponding ethyl ester, in

turn achieved by the microwave method, as well as 3,3⁰-dimethyl allyl alcohol, in mild

conditions.

The purity of all new compounds was judged to be >98% by ¹H-NMR spectral deter-

mination.

The remarkable different lipophilicity estimated, as the value of the logarithm of the

partition coefﬁcient of compounds

1–13 for n-octanol/water (LogP), allowed us to evaluate

the possible inﬂuence of this property in the antifungal activity of each compound (Table 1).

Molecules 2021, 26, 658

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Table 1. Values of the logarithm of LogP of compounds

1–

13 for n-octanol/water, estimated by

ChemBioDraw Ultra 13.

Compound

LogP

Compounds

LogP

Compounds

LogP

1

2

3

4

5

1.15

1.42

1.54

2.50

2.11

6

7

8

9

10

2.37

3.04

2.11

4.44

4.90

11

12

13

4.51

4.78

3.04

As expected, in all compounds studied, the lipophilicity increases as the number of

carbon atoms in the prenylated chain increases. Among the three natural occurring acids,

the lipophilicity changes in the following order: ferulic acid

acid . The trend is also retained both in the series of geranyl esters 10

the series of allyl ethers . No difference in LogP value resulted in caffeic acid

derivatives when the p-phenolic-OH or the carboxylic functionalities were protected by an

3

> caffeic acid

2

> p-coumaric

1

>

12 11 and in

>

4

> 6 > 5

allyl group (i.e., compounds 5 and 8 7

) or by a 3,3⁰-dimethyl allyl chain (i.e., compounds

and 13).

2.2. Antifungal Activity of the Parent Compounds 1–3 and Derivatives

The three naturally occurring hydroxycinnamic acids and their ethers and esters

derivatives 13 (Figure 2) were used in a preliminary in vitro screening to compare their

1–3

4–

antifungal activity against six Fusarium spp. clinical isolates associated to onychomycosis

in a hospital in Milan (Italy) (Table 2).

Table 2. List of Fusarium spp. isolates tested in this study.

NRRL

n.^a

PVS-Fu

n.^b

Species/Species Complex/Sequence Type (ST)

Diagnosis

Isolate Source

Date

F. oxysporum/FOSC/ST33

F. keratoplasticum/FSSC/ST2bb

F. solani/FSSC/ST5aa

F. verticillioides/FFSC

a

46603

46606

46443

44903

46599

46442

89

91

93

96

87

Onychomycosis

Dermatomycoses

Onychomycosis

Toe

Foot

Toe

2004

2005

2004

2006

2007

2005

115

NRRL n. Collection number of Agricultural Research Service (ARS); ^bPVS-Fu n. Collection number of Dipartimento di Agraria, Sezione

Patologia Vegetale ed Entomologia, Sassari, Italy.

In the ﬁrst screening, carried out on solid FMM, exposure to cinnamic acids 1–3 did

not determine any signiﬁcant reduction of mycelium fungal growth compared with the

untreated control, with the exception of caffeic acid 2, which induced a slight inhibition on

F. oxysporum 89 colony diameter (Figure S1).

In the case of FOSC and FFSC, we noted a signiﬁcant inhibition of vegetative growth

upon exposure to all the compounds derived from cinnamic acids compared with the

untreated control, except for compound , which did not induce any signiﬁcant reduction

1–3

5

of colony diameter in the two FOSC isolates. FOSC isolates were particularly sensitive

(>53% and >56% of inhibition for F. oxysporum 89 and F. oxysporum 91, respectively) to

compounds , and 13, whereas in the case of FFSC, the most effective compounds

4,

7,

8,

9

were 5, 8, 9, 10, 11, and 13. In both species complexes, compound 13 was by far the most

effective inhibitor of fungal growth, leading to complete inhibition of the two F. verticillioides

isolates (Figures S1–S3).

The two representative isolates of the FSSC displayed a different level of sensitivity

to compounds 4–13: compounds 4, 7, 10, 11, and 13 determined >25% inhibition of radial

growth on F. keratoplasticum 93, whose vegetative growth on solid FMM was completely

inhibited by compound 13, whereas F. solani 96 was only partially inhibited by compounds

10, 11 (36–30% inhibition, respectively), and 13 (63% inhibition; Figure S2).

Molecules 2021, 26, 658

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These preliminary data demonstrate unequivocally that compound 13 has the highest

antifungal activity towards all Fusarium strains investigated (Figures S1–S3). Citronellyl

p-coumaric ester 10 was the second most effective inhibitor towards FSSC and FFSC strains,

while FOSC representative isolates proved more sensitive to compound

was more effective on FOSC and FFSC representatives compared with FSSC ones. The

inhibitory activity of compound towards FOSC isolates was comparable to that displayed

by the ethers and , presenting a shorter prenylated chain. Overall, the ester functionality

8. Compound 9

9

4

7

in the cinnamic acid derivatives appears more efﬁcient in conferring inhibitory properties

to the tested compounds compared with the ether one.

2.3. Determination of the Minimal Inhibitory Concentration (MIC) and Lethal Dose 50 (LD₅₀) for

p-Coumaric acid 3,3⁰-Dimethyl Allyl Ester 13

In a further screening performed on FMM liquid medium, the minimal inhibitory

concentration (MIC) and the lethal dose 50 (LD₅₀) of compound 13 and of two conventional

fungicides were determined (Table 3). p-Coumaric acid 3,3⁰-dimethyl allyl ester 13 con-

ﬁrmed its remarkable good antifungal activity already shown in solid FMM compared with

TRB and AmB, with an MIC range comprised between 125 and 250

and S5), 62 and 125 M in FSSC (Figures S6 and S7), and 125 and 500

S8 and S9) representative isolates. With respect to the LD₅₀, the most effective compound

was TRB (LD₅₀ranging from 2.0 to 64 M), followed by AmB (LD₅₀ranging from 1.0 to 67.5

M) and ester 13 (LD₅₀ranging from <7.8 to 125 M), respectively, for almost all strains. In

the case of F. verticillioides 115, which was less sensitive to AmB (LD₅₀8.40–16.8 M) than

to TRB (LD₅₀2.0–4.0 M) and 13, the latter was able to reduce 50% of the fungal growth at

a concentration <7.8 µM (Table 3, Figure S9).

µM in FOSC (Figures S4

µ

µM in FFSC (Figures

µ

Table 3. In vitro susceptibility of strains belonging to the three Fusarium species complexes, isolated from onychomycosis

0

and dermatomycosis against the selected compound p-coumaric acid 3,3 -dimethyl allyl ester 13 and the two conventional

drugs TRB ^band AmB ^c.

c

b

Ester 13

AmB

LD₅₀(µM)

TRB

LD₅₀(µM)

Species/Species Complex/Sequence

Type (ST)

a

PVS-Fu n.

e

d

LD₅₀(µM)

MIC (µM)

F. oxysporum/FOSC/(ST33)

F. keratoplasticum/FSSC/(ST2bb)

F. solani/FSSC/(ST5aa)

89

91

93

96

87

>125–250

62

62–125

500

31–62

62–125

<7.8

62–125

<7.8

>256

128–256

64–128

>256

8–16

16–64

2.0–4.0

>135

33.7–67.5

4.2–2.1

>135

16.8–33.7

33.7–67.5

>2.1–4.2

1.0

2.1–4.2

8.4–16.8

F. verticillioides/FFSC

115

125–250

>256

>135

a

PVS-Fu n. Collection number of Dipartimento di Agraria, Sezione Patologia Vegetale ed Entomologia, Sassari, Italy; ^bTRB: terbinaﬁne;

AmB: amphotericin B; ^dminimal inhibitory concentration (MIC) range determined visually according to mOD absorbance values (nm)

c

detected after 48 h of incubation at 25 ^◦C; ^elethal dose 50 (LD₅₀) range determined visually according to mOD absorbance values (nm)

detected after 48 h of incubation at 25 ^◦C.

The effects of compounds 13, TRB, and AmB applied at the same concentration

as the MIC determined in liquid FMM on the morphology of representative isolates of

F. solani, F. keratoplasticum, F. oxysporum, and F. verticillioides were examined by optical

microscopy after 72 h (Table 4). While in the untreated controls, a regular morphology of

the mycelium with abundant presence of microconidia as well as normal spore germination

were observed, in the presence of 250 µM of compound 13, degradation of the hyphae and

vacuolisation of the cytoplasm were evident, along with an alteration of the rarely formed

conidia, showing a compromised germination. The release of cell constituents was also

noticeable, which can explain the progressive increase in the absorbance signal over time

in liquid FMM (Figures S4–S9).

After 72 h of exposure to 256 µM TRB, a marked reduction on mycelium formation was

observed, and fungal hyphae and spores showed a distorted morphology with tortuous

growth (Table 4). This concentration of TRB could not totally inhibit the mycelium growth

or the spore production but caused an evident swelling of conidia. The effects of TRB were

Molecules 2021, 26, 658

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more noticeable compared with those induced by AmB at the MIC of 135

µM, causing only

some mycelial distortion and fewer microconidia compared with the control (Table 4).

Table 4. Optical microscopy (100

medium (FMM) amended with 250, 256, or 135

amphotericin B (AmB), respectively, in comparison with the untreated control.

×

) images of the fungal growth of Fusarium spp. after 72 h on liquid Fusarium minimal

µ

M of p-coumaric acid 3,3⁰-dimethyl allyl ester 13, terbinaﬁne (TRB), or

Species

Control

0 (µM)

Ester 13

MIC (250 µM)

TRB

MIC (256 µM)

AmB

MIC (135 µM)

F. keratoplasticum

F. solani

F. oxysporum

F. verticillioides

Molecules 2021, 26, 658

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3. Discussion

By introduction of a short, medium, or long prenylated chain at one of the hydroxyl

functionalities of trans-cinnamic acids , a series of prenylated cinnamic derivatives

13 was prepared to test the hypothesis that differences in the chemical and physical

1–3

4–

proprieties would inﬂuence the antifungal activity of the compounds against clinical

Fusarium spp. The presence of a conjugated double bond confers particular conformational

and electronic characteristic to these compounds strongly inﬂuenced by the phenol-OH

group in para position. In order to observe the effect of the hydroxylated aromatic ring

on the fungicide activity, a set of cinnamic esters 10

of the corresponding carboxylic acid with citronellol, while a set of allyl cinnamic ethers

was prepared with the aim to evaluate the inﬂuence of the phenolic-OH group in para

–12 was prepared by functionalisation

4–6

position to the alpha, beta-unsaturated carboxylic chain. No signiﬁcant differences were

observed in the synthesis and yields of each set of compounds with different aromatic

rings, whereas lower yields in esters in comparison with ethers were achieved, evidencing

the higher reactivity of the phenolic-OH group.

The Food and Drug Administration (FDA) considers trans-cinnamic acids

“generally recognized as safe” (GRAS), enabling their use in the ﬁeld of food additives [40].

Compounds are commercially available at an affordable price and can been obtained by

direct extraction from plants biomass where the compounds are the main components [41

or by chemical and biotechnological processes [42]. Besides cinnamic acids , prenylated

cinnamic ester 13 and ethers and are plant components, whose extracts were studied for

their remarkable biological properties [43]. In particular, compounds and 13 are present in

propolis, a source of valuable compounds with antioxidant and antimicrobial activity [44].

Compound is not toxic to human cells and presents antitumoral and anti-inﬂammatory

activities, in addition to acting as an inhibitor of bioﬁlm formation by oral pathogenic

bacteria [45]. While 4⁰-geranyloxy ferulic acid

is generally extracted from citrus fruit,

1–3 as

1–3

]

1–3

7

9

quinoa seeds, and several vegetable oils, compound 13 was only detected in propolis

extract. Propolis, produced by honeybees, is a very complex mixture composed of 50%

resin, 30% wax, 10% essential oil, and 5% of polyphenols as ﬂavonoids, terpenes, fatty acids,

stilbenes, β-steroids, cinnamic acids, and their prenylated derivatives [46,47]. Change in

chemical composition of propolis is frequently observed [44]. A few studies have been

conducted on the antifungal and antibioﬁlm activity of propolis against onychomycosis

caused by Fusarium spp. [48,49], but no studies aimed to identify the active component of

the propolis extract against these fungi.

In a previous article reporting the activity of natural phenols against clinical Fusarium

spp., we observed that the percentage of growth inhibition measured in liquid medium

(Vogel’s) and solid (PDA) was comparable [50]. To achieve full solubilisation of compounds

1

–

13 at 0.5 mM, the preliminary screening was carried out with a sustainable solid medium

based on gellan/water, where each compound was solubilized in a 0.1% water/gellan

solution. In the preliminary in vitro screening of compounds 13 against Fusarium spp.,

cinnamic acids were generally ineffective, whereas signiﬁcant growth inhibition was

achieved by prenylated derivatives 13, evidencing ester 13 as the most active. Among

the Fusarium spp. investigated, F. solani was the most resistant to compounds 13, whereas

1–

1–3

4–

1–

F. verticillioides was the most sensitive, in accordance with data present in the literature

for these species. In fact, Fusarium spp. are increasingly reported as resistant to many

antifungal compounds in vitro; among them, F. solani is considered as the most resistant

taxon, albeit some reports pointed out that the resistance may be species- and even isolate-

dependent [51].

The antifungal activity of compounds

1

–13 may be explained by the key role played

by some moieties of their structure. The prenylated chain present in compounds

4–

13

has the ability to penetrate and to accumulate within the fungal cell membrane, resulting,

according to the size, in the disruption of its integrity as generally acknowledged for

prenylated phenols [36

–38]. The position and size of the prenylated chain in the studied

compounds appear crucial for their antifungal activity. Although we did not perform a

Molecules 2021, 26, 658

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proper bioavailability assay, we observed a detrimental effect on the fungal membrane

when treated with compound 13 (Table 4).

Esters were more active than ethers as inhibitors of all tested Fusarium spp. Ester 13

0

was deﬁnitely more active than the corresponding ether

7

containing the same 3,3 -dimethyl

allyl moiety, even though an identical lipophilicity was measured for both compounds

0

(LogP 3.04). In the esters series, the alcoholic unit represented by a 3,3 -dimethyl allyl chain

(compound 13) was more active than the citronellyl one (compound 10), a substituent that

signiﬁcantly increases the lipophilicity of the molecule (LogP 4.9).

We suppose that different prenylated chains may change the bioavailability of the

compound inﬂuencing the hydrolytic degradation of the prenylated esters within the

fungal cell. In fact, hydrolytic degradation, mediated by fungal enzymes, of the esters in

the parent cinnamic acid and the corresponding alcohol cannot be ruled out. In esters 11

and 10, a too long prenylated chain could be partially metabolized by the fungus at the ﬁrst

stages of contact, whereas in ester 13, the hydrolysis would take a longer time, allowing

it to reach sensitive compartments of the fungal cell where the prenyl alcohol may exert

its antifungal activity. A similar effect has been reported by farnesol on F. keratoplasticum,

which is associated with bioﬁlm formation in hospital water systems and internal pipelines:

this prenylated alcohol has a remarkable anti-bioﬁlm activity; causes the destruction of

hyphae and of the extracellular matrix; and prevents the adhesion of conidia, ﬁlamentation,

and the formation of bioﬁlm [52].

Compounds 4–13 contain an α,β-unsaturated Michael acceptor pharmacophore effec-

tive in interacting with nucleophiles present in the fungal cell; nevertheless, this feature

is not exhaustive for the antifungal activity. The presence of a free phenol-OH in para

position would play a key role in the radical scavenging and stabilisation of the radical

by electronic delocalisation along the structure. In general, we observed that compounds

with a catechol and guaiacyl ring favouring an intramolecular hydrogen bond and ham-

pering the availability of the H donor to scavenge radicals were less active as antimycotic

(compounds 5, 8, and 12).

The antifungal activity of compound 13 was compared with that of TRB and AmB,

two of the most effective conventional fungicides for clinical use [53]. TRB and AmB were

applied at clinical dosage ranging between 2–256

µM and 1–135

µM, respectively, whereas

compounds 13 was amended at concentrations comprised between 7.8 and 500

µM. Both

TRB and AmB interact at the level of fungal cell membrane, the ﬁrst one by inhibiting

squalene epoxidase, a key step along the ergosterol biosynthesis pathway, and the second

one by a complex interaction with phospholipid bilayers [54]. Our results demonstrate that

compound 13 presents MIC and LD₅₀values against F. verticillioides 115 and F. oxysporum

89 that are consistent with those reported for AmB. Similarly, while AmB was indeed

the most effective compound in terms of MIC and LD₅₀against F. keratoplasticum 93 and

F. solani 96, the antifungal efﬁcacy of compound 13 against these members of the FSSC

was comparable to that of terbinaﬁne. Besides its remarkable biological activity, ester

13 presents some attractive advantages; that is, it is as natural compound with a simple

structure, a straightforward synthesis, low production cost with easy recovery of the

starting materials. Considering the increasing frequency of multi-drug resistance patterns

in opportunistic Fusarium spp. [55], the development of compound 13 as an effective

antifungal drug represents a valuable alternative to the conventional therapeutic agents in

onychomycosis treatment.

The results of this study provide useful insights to the optimal design of the structure

of cinnamic esters with improved antifungal properties. Although cinnamic acids and their

derivatives have been studied on some plant pathogenic fungi [56], to the best of our knowl-

edge, no investigation was conducted on prenylated cinnamic esters and ethers on clinical

Fusarium spp., thereby offering an intriguing opportunity in drug repositioning strategy.

Molecules 2021, 26, 658

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4. Materials and Methods

4.1. Chemical Synthesis

4.1.1. General

Unless otherwise noted, starting materials and reagents were obtained from commer-

cial suppliers and were used without further puriﬁcation. Melting points were determined

on a Büchi 530 apparatus and are uncorrected. All ¹H-NMR and ¹³C-NMR spectra were

recorded in CDCl₃(if not otherwise indicated) solution at 399.94 MHz and 75.42 MHz,

respectively, with a Varian VXR 5000 spectrometer (Varian, Palo Alto, CA USA). Chem-

ical shifts are given in ppm (δ); multiplicities are indicated by s (singlet), d (doublet), t

(triplet), q (quartet), m (multiplet), or dd (doublet of doublets). Elemental analyses were

performed using an elemental analyser model 240 C (Perkin-Elmer, Walthan, MA USA).

Acetone was freshly distilled from CaCl₂. Flash chromatography was carried out with

silica gel 60 (230–400 mesh; VWR; Radnor, AF, USA) eluting with appropriate solution in

the stated v:v proportions. Analytical thin-layer chromatography (TLC) was performed

with 0.25 mm thick silica gel plates (Sigma Aldrich, Munich, Germany). All reactions were

monitored by TLC performed on 0.2 mm thick silica gel plates (60 F254 Merck). Microwave

reactions were carried out on a MW instrument (CEM-Discover SP MW, Matthews, NC,

USA). Melting points were determined on a 530 apparatus (Büchi, Flawil, Switzerland) and

are uncorrected. The purity of all new compounds was judged to be >98% by ¹H-NMR

spectral determination. Lipase from Candida antarctica (Novozym 435 CAL-B) is immo-

bilized on a macroporous polyacrylic resin beads (recombinant, expressed in Aspergillus

niger, activity

≥

5000 PLU/g (propyl laurate units/g) and purchased from Merck (Milan,

Italy). Compound 8 was prepared according to the literature [57].

Lipophilicity of the compounds was estimated using the logarithm of the partition

coefﬁcient for n-octanol/water (log P), which was calculated using 403 ChemBioDraw

Ultra 13.0.

4.1.2. General Procedure for the Synthesis of Compounds 10–13

Ethyl chloroformate (2 eq for 10

for 10 12, and 13 or 3 eq for 11) were added to a suspension of appropriate cinnamic

acid (1 eq) in dichloromethane (10 mL) and stirred for 1 h at

, 12, and 13 or 3 eq for 11) and triethylamine (2 eq

,

◦

30 C until all of the

−

starting material disappeared, as determined by TLC. Appropriate alcohol (1 eq) and

4-dimethylaminopyridine (0.2 eq) were then added, and the mixture was stirred at room

temperature for 6 h. The reaction mixture was acidiﬁed with hydrochloridric acid (10%

solution) and extracted with dichloromethane (3

×

50 mL), and the organic phases were

combined and dried over anhydrous sodium sulphate. The product was then concentrated

under reduced pressure and ﬁltered on a pad of silica gel using dichloromethane as eluent

to give a yellow oil. The oil was diluted in dichloromethane (15 mL) and piperidine

(30 eq) was added at 0 ^◦C. The reaction mixture was stirred at room temperature for 3 h,

acidiﬁed with hydrochloridric acid (10% solution), and extracted with dichloromethane

(3

×

50 mL), and the organic phases were combined and dried over anhydrous sodium

sulphate. The crude product was concentrated under reduced pressure and puriﬁed by

ﬂash chromatography using a 1:1 mixture of petroleum ether/ethyl acetate as eluent to

give the pure ester.

20

(E)-3,7-Dimethyloct-6-en-1-yl 3-(4-hydroxyphenyl)acrylate 10: oil; 44%; [

α

]

0.5 (c = 0.9,

D

CHCl₃); ¹H-NMR

δ

0.94 (d, J = 6.4 Hz, 3H), 1.22 (m, 1H), 1.38 (m, 1H), 1.51 (m, 1H), 1.60

(m, 1H), 1.61 (s, 3H), 1.67 (s, 3H), 1.76 (m, 1H), 1.99 (m, 2H), 4.26 (m, 2H), 5.11 (m, 1H), 6.28

(d, J = 16.0 Hz, 1H), 6.81 (m, Ar, 2H), 7.42 (m, Ar, 2H), 7.63 (d, J = 16.0 Hz, 1H); ¹³C-NMR

δ

17.66, 19.43, 25.40, 25.73, 29.54, 35.48, 36.99, 63.40, 114.97, 115.99, 124.57, 126.63, 130.07,

131.37, 145.12, 158.51, 168.44; Anal. Calcd. for C₁₉H₂₆O₃: C, 75.46; H, 8.67; Found: C, 75.44;

H, 8.60.

(E)-3,7-Dimethyloct-6-en-1-yl 3-(3,4-dihydrophenyl)acrylate 11: brown solid; 47%; mp

20

100–101 ^◦C; [

α

]

2.9 (c = 0.4, CHCl₃); ¹H-NMR

δ 0.93 (d, J = 6.4 Hz, 3H), 1.21 (m, 1H),

D

1.36 (m, 1H), 1.52 (m, 1H), 1.59 (m, 1H), 1.60 (s, 3H), 1.67 (s, 3H), 1.74 (m, 1H), 1.99 (m, 2H),

Molecules 2021, 26, 658

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4.30 (m, 2H), 5.09 (m, 1H), 6.28 (d, J = 15.6 Hz, 1H), 6.88 (d, J = 8.4 Hz, Ar, 1H), 6.99 (dd,

J = 2.1, 8.4 Hz, Ar, 1H), 7.12 (d, J = 2.1 Hz, Ar, 1H), 7.59 (d, J = 15.6 Hz, 1H); ¹³C-NMR

δ

17.66, 19.42, 25.37, 25.72, 29.51, 35.42, 36.97, 63.61, 114.44, 115.14, 115.46, 122.46, 124.52,

127.17, 131.42, 144.03, 145.56, 146.78, 168.69; Anal. Calcd. for C₁₉H₂₆O₄: C, 71.67; H, 8.23;

Found: C, 71.60; H, 8.26.

20

(E)-3,7-Dimethyloct-6-en-1-yl 3-(4-hydroxy-3-methoxyphenyl)acrylate 12: oil; 37%; [

α]

D

0.6 (c = 0.4, CHCl₃); ¹H-NMR

δ

0.93 (d, J = 6.8 Hz, 3H), 1.22 (m, 1H), 1.37 (m, 1H), 1.52 (m,

1H), 1.60 (m, 1H), 1.61 (s, 3H), 1.67 (s, 3H), 1.73 (m, 1H), 2.01 (m, 2H), 3.99 (s, 3H), 4.23 (m,

2H), 5.09 (m, 1H), 6.29 (d, J = 16.0 Hz, 1H), 6.89 (d, J = 8.4 Hz, Ar, 1H), 7.02 (d, J = 2.0 Hz,

Ar, 1H), 7.06 (dd, J = 2.0, 8.4 Hz, Ar, 1H), 7.59 (d, J = 16.0 Hz, 1H); ¹³C-NMR

δ 17.64, 19.44,

25.39, 25.71, 29.54, 35.55, 37.01, 55.90, 62.94, 109.32, 114.74, 115.61, 123.03, 124.59, 127.01,

131.31, 144.67, 146.78, 147.30, 168.41; Anal. Calcd. for C₂₀H₂₈O₄: C, 72.26; H, 8.49; Found:

C, 72.34; H, 8.40.

(E)-3-Methylbut-2-en-1-yl 3-(4-hydroxyphenyl) acrylate 13: oil; 45%; ¹H-NMR

δ

1.73 (s,

3H), 1.77 (s, 3H), 4.71 (d, J = 7.2 Hz, 2H), 5.41 (m, 1H), 6.29 (d, J = 16.0 Hz, 1H), 6.87 (m,

Ar, 2H), 7.38 (m, Ar, 2H), 7.64 (d, J = 16.0 Hz, 1H); ¹³C-NMR

18.08, 25.82, 61.73, 114.91,

δ

115.99, 118.42, 126.69, 130.07, 139.50, 145.19, 158.42, 168.36; Anal. Calcd. for C₁₄H₁₆O₃: C,

72.39; H, 6.94; Found: C, 72.45; H, 6.96.

4.1.3. General Procedure for the Synthesis of Compounds 14–17

In a 30 mL glass pressure microwave tube, equipped with a magnetic stirrer bar, a few

drops of concentrated sulphuric acid were added to a solution of hydroxycinnamic acid

(p-coumaric acid or caffeic acid or ferulic acid) (1 eq) in methanol (for 14

(for 17) (10 mL). The mixture was subjected to microwave irradiation (power: 150 W;

temperature: 80 ^◦C for 14 16 and 98 ^◦C for 17) for 15 min, basiﬁed with aqueous sodium

bicarbonate (5% solution), and extracted with dichloromethane (3 5 mL). The collected

–16) or ethanol

–

×

organic phases were dried over anhydrous sodium sulphate and evaporated to dryness to

give the pure ester.

_◦(E)-Methyl 3-(4-hydroxyphenyl)acrylate 14: white solid; 82%; mp 125–127 ^◦C ([58] 132–

134 C); ¹H-NMR

δ 3.79 (s, 3H), 5.37 (bs, 1H), 6.28 (d, J = 16.0 Hz, 1H), 6.85 (m, Ar, 2H), 7.42

(m, Ar, 2H), 7.63 (d, J = 16.0, 1H); ¹³C-NMR

δ 51.75, 114.52, 115.91, 125.98, 129.89, 144.79,

158.12, 167.65. Anal. Calcd. for C₁₀H₁₀O₃C, 67.41; H, 5.66; Found: C, 67.53; H, 5.56.

(E)-Methyl 3-(3,4-dihydroxyphenyl)acrylate 15: brown solid; 88%; mp 155–156 ^◦C ([59

]

160 ^◦C); ¹H-NMR

δ

3.80 (s, 3H), 6.25 (d, J = 16.0 Hz, 1H), 6.87 (d, J = 8.4 Hz, Ar, 1H), 7.01

(dd, J = 2.0, 8.4 Hz, Ar, 1H), 7.07 (d, J = 2.0 Hz, Ar, 1H), 7.58 (d, J = 16.0 Hz, 1H); ¹³C-NMR

51.78, 114.33, 115.27, 115.49, 122.46, 127.52, 143.74, 145.03, 146.28, 168.18. Anal. Calcd. for

δ

C₁₀H₁₀O₄; C, 61.85; H, 5.19; Found: C, 62.05; H, 5.78.

(E)-Methyl 3-(4-hydroxy-3-methoxyphenyl)acrylate 16: brown solid; 92%; mp 62–64 ^◦C

◦

([60] 65 C); ¹H-NMR

δ

3.75 (s, 3H), 3.83 (s, 3H), 6.14 (bs, 1H), 6.25 (d, J = 16.0 Hz, 1H), 6.86

(d, J = 8.0 Hz, Ar, 1H), 6.96 (d, J = 2.0 Hz, Ar, 1H), 6.96 (dd, J = 2.0, 8.0 Hz, Ar, 1H), 7.58

(d, J = 16.0 Hz, 1H); ¹³C-NMR

51.64, 55.84, 109.52, 114.72, 114.81, 122.98, 126.78, 145.12,

146.92, 148.12, 167.92. Anal. Calcd. for C₁₁H₁₂O₄C, 63.45; H, 5.81; Found: C, 63.51; H, 5.76.

δ

◦

(E)-Ethyl 3-(4-hydroxyphenyl)acrylate 17: brown solid; 87%; mp 70–72 C ([61] 73–74 C);

¹H-NMR

1.32 (t, J = 7.1 Hz, 3H), 4.26 (q, J = 7.1 Hz, 2H), 6.39 (d, J = 16.0 Hz, 1H), 7.35 (m,

Ar, 2H), 7.47 (m, Ar, 2H), 7.58 (d, J = 16.0 Hz, 1H); ¹³C-NMR

14.43, 60.74, 119.14, 124.58,

δ

129.54, 132.22, 133.52, 143.29, 166.78. Anal. Calcd. for C₁₁H₁₂O₃: C, 68.74; H, 6.29; Found:

C, 68.81; H, 6.34.

4.1.4. Procedure for the Synthesis of Compound 13 with Lipase

To a solution of 17 (1 eq) in cyclohexane (2.5 mL), 3,3-dimethylallyl alcohol (2 eq) was

added. The reaction mixture was stirred at 60 ^◦C for 15 min at a speed of 300 rpm. The

reaction was initiated by adding a known ﬁxed quantity of lipase (100 mg). The progress

of the reaction was monitored by TLC using a 1:1 mixture of petroleum ether/ethyl acetate

as eluent. After three days, the starting material was still present and another aliquot of

Molecules 2021, 26, 658

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lipase (100 mg) was added, and the mixture was left stirring at 60 ^◦C for two additional

days. The reaction mixture was ﬁltered over Buchner funnel, solvent concentrated under

reduced pressure, and puriﬁed by ﬂash chromatography using a 1:1 mixture of petroleum

ether/ethyl acetate as eluent to obtain compound 13 (0.19 g, 80% yield).

4.1.5. General Procedure for the Synthesis of Compounds 4, 6, 7, and 9

Compound 14 or 15 or 16 (1 eq) was dissolved in dry acetone (15 mL) and then

anhydrous potassium carbonate (1 eq) and appropriated alkenyl bromide (1 eq) were

added. The resulting mixture was stirred at 50 ^◦C for 12 h, then sodium hydroxide 2 N

◦

(15 mL) was added and the reaction mixture was stirred at 90 C for an additional 3 h.

The cooled solution was acidiﬁed to pH 2 with hydrochloridric acid (10% solution) and

extracted with dichloromethane (3

anhydrous sodium sulphate and evaporated to dryness to give the pure ether.

(E)-3-(4-(Allyloxy)phenyl)acrylic acid

: white solid; 85%; mp 161–162 ^◦C ([62] 160 ^◦C);

¹H-NMR

4.56 (d, J = 5.2 Hz, 2H), 5.29–5.44 (series of m, 2H), 6.03 (m, 1H), 6.29 (d,

J = 16.0 Hz, 1H), 6.91 (m, Ar, 2H), 7.49 (m, Ar, 2H), 7.73 (d, J = 16 Hz, 1H); ¹³C-NMR

×

50 mL). The collected organic phases were dried over

4

δ

68.85, 114.72, 115.13, 118.08, 126.91, 130.07, 132.66, 146.67, 160.73, 172.55. Anal. Calcd. for

C₁₂H₁₂O₃C, 70.57; H, 5.92; Found C, 70.78; H, 5.87.

◦

(E)-3-(4-(Allyloxy)-3-methoxyphenyl)acrylic acid

6: white solid; 53%; mp 152–154 C ([63

]

151–153 ^◦C); ¹H-NMR

δ

3.91 (s, 3H), 4.66 (m, 2H), 5.30–5.44 (series of m, 2H), 6.08 (m, 1H),

6.29 (d, J = 16.0 Hz, 1H); 6.86 (d, J = 8.4 Hz, Ar, 1H), 7.08 (dd, J = 2.0, 8.4 Hz, Ar, 1H), 7.09 (d,

J = 2.0 Hz, Ar, 1H), 7.71 (d, J = 16.0 Hz, 1H); ¹³C-NMR

55.92, 69.70, 110.08, 112.71, 114.90,

δ

118.51, 122.92, 127.14, 132.61, 147.01, 149.52, 150.46, 172.72. Anal. Calcd. for C₁₃H₁₄O₄C,

66.66; H, 6.02; Found: C, 66.87; H, 6.12.

_◦(E)-3-(4-((3-Met_◦hylbut-2-en-1-yl)oxy)phenyl)acrylic acid

7: white solid; 69%; mp 146–

δ 1.73 (s, 3H), 1.79 (s, 3H), 4.52 (d, J = 6.4 Hz, 2H), 5.47 (s,

147 C ([16] 148–150 C); ¹H-NMR

1H), 6.28 (d, J = 16 Hz, 1H), 6.89 (d, J = 8.8 Hz, Ar, 2H), 7.47 (d, J = 8.4 Hz, Ar, 2H), 7.73 (d,

J = 16 Hz, 1H); ¹³C-NMR

25.81, 29.19, 64.91, 114.62, 115.04, 119.11, 126.67, 130.04, 138.73,

146.60, 161.04, 171.36. Anal. Calcd. for C₁₄H₁₆O₃C, 72.39; H, 6.94; Found C, 72.59; H, 6.03.

(E)-3-(4-(((E)-3,7-Dimethylocta-2,6-dien-1-yl)oxy)-3-methoxyphenyl)acrylic acid : white

solid; 75%; mp 59–60 ^◦C ([64] 60–61 ^◦C); ¹H-NMR

1.63 (s, 3H), 1.70 (s, 3H), 1.77 (s, 3H),

δ

9

δ

2.01–2.24 (series of m, 4H), 3.95 (s, 3H), 4.62 (m, 2H), 5.11 (m, 1H), 5.44 (m, 1H), 6.33 (d,

J = 16.0 Hz, 1H), 6.86 (d, J = 7.6 Hz, Ar, 1H), 7.01–7.15 (series of m, Ar, 2H), 7.37 (d, J = 16.0

Hz, 1H); ¹³C-NMR

δ 16.70, 17.70, 25.70, 26.20, 39.53, 55.91, 65.82, 109.91, 112.52, 114.71,

119.11, 123.01, 123.71, 126.81, 131.84, 141.22, 147.01, 149.52, 150.83, 172.11. Anal. Calcd. for

C₂₀H₂₆O₄C, 72.70; H, 7.93; Found: C, 72.80; H, 7.92.

4.1.6. Synthesis of Compound 5

(E)-3-(4-(Allyloxy)-3-hydroxyphenyl)acrylic acid 5: Compound 15 (0.5 g, 2.57 mmol)

was dissolved in dry acetone (15 mL) and then anhydrous potassium carbonate (0.35 g,

2.57 mmol) and allyl bromide (0.31 g, 2.57 mmol) were added. The resulting mixture was

stirred at 50 ^◦C for 12 h. The cooled solution was acidiﬁed to pH 2 with hydrochloridric

acid (10% solution) and extracted with dichloromethane (3

×

50 mL). The collected organic

phases were dried over anhydrous sodium sulphate and evaporated. The crude product

was puriﬁed by ﬂash chromatography using a mixture of 3:1 petroleum ether/acetone as

eluent to give compound 18 as a white solid (0.47 g, 78%).

◦

(E)-Methyl 3-(4-(allyloxy)-3-hydroxyphenyl)acrylate 18: mp 95–96 C ([65] 94–95 C);

¹H-NMR

3.78 (s, 3H), 4.65 (d, J = 5.6 Hz, 2H), 5.33–5.43 (series of m, 2H), 5.69 (s, 1H),

6.08 m, 1H), 6.30 (d, J = 15.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, Ar, 1H), 6.99 (dd, J = 2.0 8.4 Hz, Ar,

1H), 7.14 (d, J = 2.0 Hz, Ar, 1H), 7.61 (d, J = 15.6 Hz, 1H); ¹³C-NMR

51.59, 69.80, 11.84,

δ

113.21, 115.94, 118.80, 121.64, 123.13, 128.20, 132.26, 144.60, 146.02, 147.39, 167.68. Anal.

Calcd. for C₁₄H₁₆O₄C, 67.73; H, 6.50; Found: C, 67.52; H, 6.42. To compound 18 (0.47 g,

2.00 mmol) in a 3:1 solution of MeOH:H₂O, sodium hydroxide 2 N (15 mL) was added and

the mixture was stirred at 90 ^◦C for 3 h. The cooled solution was acidiﬁed to pH 2 with

Molecules 2021, 26, 658

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hydrochloridric acid (10% solution) and extracted with dichloromethane (3

×

50 mL). The

collected organic phases were dried over anhydrous sodium sulphate and evaporated to

◦

5 δ (acetone

as white solid (0.33 g, 75%); mp 184–185 C; ¹H-NMR

dryness to give the pure

d₆) 4.66 (m, 2H), 5.26 (m, 1H), 5.46 (m, 1H), 6.07 (m, 1H), 6.32 (d, J = 16 Hz, 1H), 7.01 (d,

J = 8.4 Hz, Ar, 1H), 7.09 (dd, J = 2.4, 8.4 Hz, Ar, 1H), 7.19 (d, J = 2.4, Hz, Ar, 1H), 7.56 (d,

J = 16 Hz, 1H); ¹³C-NMR

δ 64.41, 112.87, 113.95, 115.94, 117.05, 121.08, 128.03, 133.44, 144.61,

147.08, 148.52, 167.09. Anal. Calcd. for C₁₂H₁₂O₄C, 65.45; H, 5.49; Found: C, 65.52; H, 5.32.

4.2. Fungal Strains and Culture

Two monosporic isolates collected from human samples from an Italian hospital

(Table 2 ) were selected as representative of each Fusarium oxysporum, F. solani, and F. fu-

jikuroi species complexes. Conidial suspensions of each strain were pre-cultured in a

carboxymethyl cellulose medium (CMC; [66]) for 5 days on a rotary shaker at 24 ^◦C and

180 rpm. Cultures were ﬁltered through four layers of sterile cheesecloth, and spores were

collected by centrifugation, adjusted to 1

water, and used as inoculum.

×

10⁶colony-forming units (CFU)/mL in sterile

4.3. Evaluation of the Antifungal Activity of Compounds 1–13 in FMM Solid Medium

A total of 13 phenolic compounds (Table 1) were tested for their antifungal activity

against the six Fusarium spp. isolates (Table 2) in Fusarium minimal medium (FMM) [67].

Each phenolic compound was resuspended in H₂O/gellan 0.1 % solution and sonicated

at room temperature for 1 h at 80 Hz (Elmasonic P 180 H, Elma Schmidbauer GmbH,

Germany). Solid FMM with nitrate sodium NaNO₃as nitrogen source was distributed

into Ø90 mm Petri dishes (15 mL/Petri dish) and amended with each compound at a

◦

ﬁnal concentration of 0.5 mM at a temperature of 45 C. Ten microliters of the conidial

suspension of each strain were spotted onto the center of the Petri dish amended FMM.

◦

Antifungal activity of each compound was measured after 5 d of growth at 25 C in the dark

and expressed as the colony diameter (percentage relative to control). Three replicates were

prepared for each isolate/inhibitor combination and the experiment was repeated once.

4.4. Evaluation of the Antifungal Activity of p-Coumaric Acid 3,3⁰-Dimethyl Allyl Ester (13) in

FMM Liquid Medium

The conventional antifungal drugs used in the study were AmB and TRB. AmB was

purchased by Sigma Aldrich (A2942, Germany) as a standard solution. TRB was extracted

from Terbinaﬁne Hexal 250 mg tablets by ﬁne crushing and dissolution in a solution 1:2

(v:v) dichloromethane and water. The emulsion was stirred at room temperature until two

phases clearly appeared. The organic phase was extracted and dried on Na₂SO₄and the

TRB was recovered in neat form after evaporation of the solvent under vacuum. NMR

∼

spectra of the solid extract conﬁrmed the presence of TRB with a purity 98%. TRB was

=

dissolved in 60% ethanol/H₂O (v/v), while AmB was diluted with water to reach the

desired concentration and was frozen in aliquots at

were selected according to clinical dosage and standard experimental procedures with

some modiﬁcations [68].

−

20 ^◦C. AmB and TRB concentrations

The minimal inhibitory concentration (MIC) and the lethal dose 50 (LD₅₀) of each

strain were assayed by a standardized micro-dilution method in the 96-well plate. Two-

fold serial dilutions of each antifungal agent in liquid FMM in a total volume of 200

µL

were tested. Further, 10 L of fungal spore suspension (4

µ

×

10⁶CFU/mL) was added. A

blank control with water was used for each treatment. The optical density mOD of each

microplate well was measured at 2 h intervals during 72 h of incubation with a microplate

spectrophotometer SpectrostarNano (Euroclone, Germany) at a 595 nm wavelength. The

inhibitory activity of each compound was expressed as MIC, representing the lowest

concentration of active ingredient (

µM) that is sufﬁcient to inhibit the absorbance signal,

whereas the LD₅₀of each compound was calculated as the concentration of active ingredient

Molecules 2021, 26, 658

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(

µM) able to reduce by 50% the mOD₅₉₅signal. The experiments were repeated at least

two times in quadruplicate.

4.5. Optical Microscopy Examination

A drop (15 µL) of the total volume present on the wells corresponding to the MIC of

each isolate/compound combination was pipetted after 72 h of incubation onto a glass

slide. A clean glass cover slip was placed on the sample prepared with emulsion oil. Each

slide was examined at 100

microconidia, and germinating spores, using an optical microscope (LEICA ICC50) at a

×

for the presence of mycelium, branched hyphae, ﬁalides,

scale of 20 µm.

4.6. Data Acquisition and Analysis

In the ﬁrst screening, an analysis of variance (one-way ANOVA) followed by multiple

comparisons by Tukey HSD test at the signiﬁcance level p < 0.05 using Minitab for Windows,

release 17 was performed.

In the second screening, data were recorded and analysed with Mars Data Analysis

Software, BMG Labtech, and exported to Microsoft Excel for generation of the graphs.

Graphs for the determination of the MIC and LD₅₀(Figures S4–S9) were generated for an

incubation time between 0 h and 48 h because, after this interval time, the drug free-test

(control) curve started to reach the stationary phase for almost all strains investigated.

Optical microscopy images were captured and treated with LAS V4.13 Leica applica-

tion software.

5. Conclusions

The design of cinnamic derivatives

modiﬁcation of the parent compounds

with bioactive prenylated chains, with the aim to enhance the antifungal activity of the

ﬁnal compound. Compounds are commercially available at a reasonable price and

4–

13 was focused on both electronic and steric

1–3 by esteriﬁcation and etheriﬁcation reaction

1–3

offer a successful example of repositioning of natural compounds. In this study, we

provided data that may contribute to increasing the knowledge about the promotion of

the importance of antifungal susceptibility testing. p-Coumaric acid 3,3⁰-dimethyl allyl

ester 13, a component of propolis, showed good antifungal activities against Fusarium spp.,

causing onychomycosis, and identiﬁes prenylated hydroxy cinnamic acids as interesting

pharmacophore for developing new drugs effective against this pathology. The activity of

this compound will be investigated over a larger number of isolates belonging to different

species complexes and haplotypes. Furthermore, the mechanism of action of compound

13 needs to be fully characterized and possibly tested in combination with other bioactive

molecules that may be enabled to reach their target within the fungal cell.

Noteworthy, this study cannot be adopted as a clinical guideline, and the MIC values

obtained must be tested in an appropriately designed clinical study.

Supplementary Materials: The following are available online. Figures S1–S3: Antifungal activity of

compounds 1–13 against FOSC, FFSC, and FFSC, respectively. Figures S4–S9: MIC and LD₅₀ranges

expressed as absorbance (milliOD) at 595 nm at 48 h of ester 13 (A), TRB (B), and AmB (C) against

FOSC, FSSC, and FFSC strains, respectively.

Author Contributions: Conceptualization, G.D. and Q.M.; methodology, S.O., V.B., M.A.D., and D.F.;

software, S.O.; validation, S.O., P.C., and D.F.; formal analysis, S.O., V.B., S.C., W.C., P.C., and D.F.;

resources, Q.M. and D.F.; data curation, S.O., S.C., and P.C.; writing—original draft preparation, G.D.,

M.A.D., S.O., and S.C.; writing—review and editing, G.D. and Q.M.; funding acquisition, Q.M. and

D.F. All authors have read and agreed to the published version of the manuscript.

Funding: This study has been supported by the following projects: CNR-CONICET Bilateral project

2016–2018; RASSR73282: “IDEASS: Approcci innovativi nella difesa delle colture agrarie: studi

in silico, selezione e sintesi di composti a ridotta tossicità per il contenimento di funghi”; CUP:

Molecules 2021, 26, 658

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J86C16000070009 SSPIT: Selection of sustainable phenolic inhibitors of trichothecene biosynthesis:

in vitro studies and computational insight into the structure- activity relationship”.

Conﬂicts of Interest: The authors declare no competing ﬁnancial interest. The funders had no role

in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the

manuscript; or in the decision to publish the results.

Ethics Statement: This article does not contain any studies with human participants or animals

performed by any of the authors.

Sample Availability: Samples of the compounds 1–13 are available from the authors.

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Article Doi

Prenylated trans-cinnamic esters and ethers against clinical fusarium spp.: Repositioning of natural compounds in antimicrobial discovery

DOI: 10.3390/molecules26030658

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