Carvacrol codrugs: A new approach in the antimicrobial plan

Article abstract of DOI:10.1371/journal.pone.0120937

Objective: The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs - obtained linking the carvacrol hydroxyl group

Full text of DOI:10.1371/journal.pone.0120937

RESEARCH ARTICLE
Carvacrol Codrugs: A New Approach in the
Antimicrobial Plan
Ivana Cacciatore1☯*, Mara Di Giulio1☯, Erika Fornasari1, Antonio Di Stefano1, Laura
Serafina Cerasa1, Lisa Marinelli1, Hasan Turkez1,2, Emanuela Di Campli1, Soraya Di
Bartolomeo1, Iole Robuffo3, Luigina Cellini1
1 Department of Pharmacy, University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy, 2 Department of
Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum, Turkey,
3 Institute of Molecular Genetics, National Research Council (CNR) section of Chieti, Chieti, Italy
☯ These authors contributed equally to this work.
* cacciatore@unich.it
Abstract
a11111
Objective
The increasing prevalence of antibiotic-resistant bacterial infections led to identify alterna-
tive strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol
codrugs – obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-
containing amino acids via an ester bond – to develop novel compounds with improved anti-
microbial and antibiofilm activities and reduced toxicity respect to carvacrol alone.
OPEN ACCESS
Citation: Cacciatore I, Di Giulio M, Fornasari E, Di
Stefano A, Cerasa LS, Marinelli L, et al. (2015)
Carvacrol Codrugs: A New Approach in the
Antimicrobial Plan. PLoS ONE 10(4): e0120937.
doi:10.1371/journal.pone.0120937
Method
Academic Editor: Muna Anjum, Animal Health and
Veterinary Laboratories Agency, UNITED KINGDOM
All carvacrol codrugs were screened against a representative panel of Gram positive (S. au-
reus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C.
albicans, using broth microdilution assays.
Received: September 9, 2014
Accepted: February 9, 2015
Published: April 10, 2015
Findings
Copyright: © 2015 Cacciatore et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Results showed that carvacrol codrug 4 possesses the most notable enhancement in the
anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial
strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and
10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity
against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol
codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8
and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an
interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, re-
spect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also ana-
lyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans.
Data Availability Statement: The data are contained
within the paper.
Funding: Support was provided by Foundation
Carichieti, Abruzzo-Italy.
Competing Interests: The authors have declared
that no competing interests exist.
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Antimicrobial Effect of Carvacrol Codrugs
Conclusion
The current study presents an insight into the use of codrug strategy for developing carva-
crol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity.
Introduction
The recent increase of bacterial resistance/tolerance to antimicrobial agents—due to genotypi-
cal bacterial modifications—encourages the scientific community towards the research of
novel therapeutic approaches to treat microbial infections [1–5].
The antibacterial properties of natural products, such as essential oils and their components,
are widely explored by both industrial and academic fields [6–7]. Notably, essential oils are
known for their antibacterial, antifungal, and insecticidal activities. Data reported that Gram
positive bacteria are more susceptible to the phenolic components of essential oils than Gram
negative bacteria since they act as membrane permeabilisers [8].
Carvacrol (2-methyl-5-[1-methylethyl]phenol), a phenolic monoterpenoid, is a constituent
of essential oils produced by numerous aromatic plants and spices [9]. To date, several reports
indicate that this monoterpenoid exhibits antimicrobial, fungicidal, anticarcinogenic, and anti-
tumor activities [10–13]. Regarding its antimicrobial activity, carvacrol causes destabilization
of bacterial membrane, decrease in the membrane potential, dissipation of pH gradients, and
perturbation of lipid fractions of bacterial cytoplasmatic membranes [14, 15]. The presence of
the hydroxyl group and a delocalized electron system is the structural requirement for the anti-
bacterial activity of carvacrol [16].
Currently, some carvacrol analogs are reported in literature [17] but no example of carva-
crol codrug is described. The codrug approach is commonly used to improve physicochemical,
biopharmaceutical, and drug delivery properties of therapeutic agents [18–20]. In direct-
coupled codrugs both drugs are directly conjugated to each other via an ester/amide bond that,
after enzymatic cleavage, is able to release both drugs individually. This approach has been suc-
cessfully employed to synthesize anti-Alzheimer, anti-Parkinson, anticancer, antiviral, and an-
tibacterial codrugs [21–24].
In the present study, the direct-coupled codrug strategy was used for generating new and
more effective antimicrobial carvacrol codrugs with antibiofilm properties. Hence, we report
the synthesis, antimicrobial, and antibiofilm activities of carvacrol codrugs 1–10 obtained by
linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids
via an ester bond (Fig 1). This approach allows us to obtain carvacrol codrugs endowed with
antimicrobial and antibiofilm properties and, at the same time, minimize the potential toxic ef-
fects of carvacrol [25] by chemically masking the free phenolic group through linkage to sul-
phur derivatives [N-Acetyl-cysteine (NAC) (codrug 1), several alkyl cysteine analogs (codrugs
2–4, 9), methionine (codrug 5), selenomethionine (codrug 6), (R)-α-lipoic acid (codrug 7),
and cyclic cysteine derivatives (codrugs 8, 10)].
The antibacterial and antibiofilm activities of some sulphur-containing amino acids are
broadly discussed in literature. The well-known anti-biofilm properties of NAC—a mucolytic
agent with antioxidant properties—could improve the antimicrobial profile of carvacrol. In
fact, NAC is able to influence the formation of biofilm by Staphylococcus epidermidis,
inhibit biofilms produced by Pseudomonas aeruginosa, and reduce the damage caused during
Helicobacter pylori infections [26–28]. Moreover, sulphurated compounds extracted from
garlic, such as allyl-cysteine, have been shown to have antibacterial, antifungal, antiviral, and
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Antimicrobial Effect of Carvacrol Codrugs
Fig 1. Chemical structures of carvacrol codrugs 1–10.
doi:10.1371/journal.pone.0120937.g001
antiprotoazoal activities [29]. Also, as demonstrated by Fujisawa et al. [30], sulphurated com-
pounds containing the S-allyl moiety (such as allicin) might be an offensive tool against bacte-
ria. Furthermore, some sulphur-containing aminoacids, such as methionine, are able to
neutralize toxins produced by Candida albicans, such as the gliotoxin which causes extensive
damage to the immune system by killing various kinds of white blood cells [31]. Starting from
these considerations, the effect of all carvacrol codrugs 1–10 containing sulphur moieties was
assayed against Gram positive, Gram negative, and C. albicans reference microorganisms.
Materials and Methods
Chemistry
Carvacrol, compounds 11–15, 22, 24–26, and 28 were purchased from Sigma Chemical Co. (St
Louis Mo, USA). All other chemicals used were of the highest purity commercially available.
1H- and 13C-NMR spectra were recorded on a Varian VXR 300-MHz spectrometer. Chemi-
cal shifts are reported in parts per million (δ) downfield from the internal standard tetra-
methylsilane (Me4Si). The LC-MS/MS system used consisted of an LCQ (Thermo Finnigan)
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Antimicrobial Effect of Carvacrol Codrugs
ion trap mass spectrometer (San Jose, CA) equipped with an electrospray ionization (ESI)
source. The capillary temperature was set at 300°C and the spray voltage at 4.25 kV. The fluid
was nebulized by use of nitrogen (N2) as both the sheath gas and the auxiliary gas. The identity
of all new compounds was confirmed by NMR data and LC-MS/MS system; homogeneity was
confirmed by thin-layer chromatography (TLC) on Merck 60 F254 silica gel. Solutions were
routinely dried over anhydrous sodium sulfate prior to evaporation. Chromatographic purifi-
cations were performed on a Merck 60 70–230 mesh ASTM silica gel column.
General method for acetylation. Acetic anhydride (6.6 mmol) was added to a stirred solu-
tion of alkylated cysteine derivatives (5.5 mmol) in acetic acid (11.1 mL) and the reaction
mixture was left under stirring at room temperature for 1 h. The solvent was removed
under vacuum giving the corresponding N-acetylated aminoacids which were used without
further purification.
N-Ac-Cys(Trt)-OH (16). Yield: 59%; Rf = 0.11, CH2Cl2:MeOH (9:1); 1H-NMR (300 MHz,
298.2 K, DMSO-d6): δ = 1.80 (s, 3H, Ac), 2.32–2.46 (m, 2H, Cys β-CH2), 4.05–4.18 (m, 1H,
Cys α-CH), 7.20–7.34 (m, 15H, Ar), 8.22–8.24 (d, 1H, Cys NH); 13C-NMR (300 MHz, 298.2 K,
DMSO-d6): δ = 23.3 (Ac), 33.91 (Cys β-CH2), 52.34 (Cys α-CH), 66.43 (C Trt), 126.12–143.98
(Ar), 178.6 and 178.8 (2 x CO).
N-Ac-Cys(Methyl)-OH (17). Yield: 99%; Rf = 0.11, AcOEt:MeOH (9:1); 1H-NMR (300
MHz, 298.2 K, DMSO-d6): δ = 1.83 (s, 3H, Ac), 2.04 (s, 3H, Cys S-CH3), 2.66–2.84 (m, 2H, Cys
β-CH2), 4.36–4.40 (m, 1H, Cys α-CH), 8.21–8.24 (d, 1H, Cys NH); 13C-NMR (300 MHz, 298.2
K, DMSO-d6): δ = 15.88 (Cys S-CH3), 23.04 (Ac), 35.74 (Cys β-CH2), 52.20 (Cys α-CH),
173.05 and 178.31 (2 x CO).
N-Ac-Cys(Ethyl)-OH (18). Yield: 97%; Rf = 0.24, CH2Cl2:MeOH (9:1); 1H-NMR (300
MHz, 298.2 K, DMSO-d6): δ = 1.11–1.19 (t, 3H, Cys S-CH2CH3), 1.82 (s, 3H, Ac), 2.51–2.55
(q, 2H, Cys S-CH2CH3), 2.67–2.90 (m, 2H, Cys β-CH2), 4.32–4.36 (m, 1H, Cys α-CH), 8.22 (d,
1H, Cys NH); 13C-NMR (300 MHz, 298.2 K, DMSO-d6): δ = 15.22 (Cys S-CH2CH3), 23.02
(Ac), 26.06 (Cys S-CH2CH3), 33.10 (Cys β-CH2), 52.70 (Cys α-CH), 169.99 and 172.96 (2 x
CO).
N-Ac-Cys(Allyl)-OH (19). Yield: 97%; Rf = 0.62, CH2Cl2:MeOH (9:1); 1H-NMR (300
MHz, 298.2 K, DMSO-d6): δ = 1.81 (s, 3H, Ac), 2.59–2.80 (m, 2H, Cys β-CH2), 3.13–3.15 (d,
2H, Cys S-CH2CHCH2), 4.35–4.39 (m, 1H, Cys α-CH), 5.07–5.12 (m, 2H, Cys S-CH2CHCH2),
5.63–5.80 (m, 1H, Cys S-CH2CHCH2), 8.20–8.23 (d, 1H, Cys NH), 12.60 (br s, 1H, COOH);
13C-NMR (300 MHz, 298.2 K, DMSO-d6): δ = 23.03 (Ac), 32.18 (Cys β-CH2), 34.66 (Cys
S-CH2CHCH2), 52.35 (Cys α-CH), 118.09 (Cys S-CH2CHCH2), 134.88 (Cys S-CH2CHCH2),
169.94 and 172.94 (2 x CO).
N-Ac-Met(Se)-OH (23). Yield: 99%; Rf = 0.11, AcOEt:MeOH (9:1); 1H-NMR (300 MHz,
298.2 K, DMSO-d6): δ = 0.92 (s, 3H, Met Se-CH3), 1.33 (m, 2H, Met γ-CH2), 1.84 (s, 3H, Ac),
1.95–2.15 (m, 2H, Met β-CH2), 4.48–4.55 (m, 1H, Met α-CH), 8.05–8.08 (d, 1H, Met NH);
13C-NMR (300 MHz, 298.2 K, DMSO-d6): δ = 11.91 (Se-CH3), 22.74 (Met β-CH2), 23.35 (Ac),
25.42 (Met γ-CH2), 57.18 (Met α-CH), 174.57 and 179.11 (2 x CO).
General method for alkylation. A solution of commercially available NAC (15) (6.13
mmol) and KOH (13.48 mmol) in MeOH (1 mL) was added with propargylic bromide (7.36
mmol) and left for 1 h under reflux. After evaporation of the solvent, the residue was taken up
with water, acidified with HCl 1N (pH = 2), then extracted with AcOEt. The organic layer was
dried over anhydrous Na2SO4 and evaporated under vacuum to give product as oil, which were
used without further purification.
N-Ac-Cys(Propargyl)-OH (20). Yield: 99%. Rf = 0.1, CH2Cl2:MeOH (9:1); 1H-NMR
(300 MHz, 298.2 K, DMSO-d6): δ = 1.85 (s, 3H, Ac), 2.78 (d, 2H, Cys β-CH2), 3.18 (s, 1H,
S-CH2CCH), 3.33 (s, 2H, S-CH2CCH), 4.39 (m, 1H, Cys α-CH), 8.24–8.26 (d, 1H, NH).
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Antimicrobial Effect of Carvacrol Codrugs
13C-NMR (300 MHz, 298.2 K, DMSO-d6): δ = 22.52 (Cys S-CH2CCH), 23.18 (Ac), 32.04 (Cys
β-CH2), 52.45 (Cys α-CH), 73.91 (Cys S-CH2CCH), 77.03 (Cys S-CH2CCH), 178.66 and
178.81 (2 x CO).
General method for coupling reaction. Suitably protected compounds (16–20, 23–26,
and 28) (5.1 mmol) were dissolved in DMF (2 mL) and DCM (10 mL), and then added with
DCC (5.1 mmol). After 1 h under stirring at room temperature, the reaction mixture was
added with DMAP (0.16 mmol) and carvacrol (5.1 mmol) and stirred overnight at room tem-
perature. After reduced pressure filtration, the solvent was evaporated and the residue was ex-
tracted with AcOEt/NaCl ss. The organic layer was dried over anhydrous Na2SO4 and
evaporated under vacuum. Chromatographic purification with DCM/AcOEt 1:1 as eluant pro-
vided the desired compounds in good yields (2–7, 9–10, 21, and 27).
N-Ac-Cys(Methyl)-CAR (2). Yield: 30%. Rf = 0.62, DCM:AcOEt (1:1); 1H-NMR (300
MHz, 298.2 K, CDCl3): δ = 1.19–1.23 (d, 6H, 2 x CH3, i-Pr), 2.04 (s, 3H, SCH3), 2.16 (s, 3H,
CH3), 2.22 (s, 3H, Ac), 2.78–2.91 (m, 1H, i-Pr), 3.08–3.21 (m, 2H, Cys β-CH2), 5.08–5.15 (m,
1H, Cys α-CH), 6.55–6.57 (d, 1H, NH), 6.68–7.16 (m, 3H, Ar). 13C-NMR (300 MHz, 298.2 K,
CDCl3): δ = 15.75 (SCH3), 16.69 (CH3), 23.29 (Ac), 24.13–24.29 (2x CH3, i-Pr), 33.91 (CH i-
Pr), 36.58 (Cys β-CH2), 52.25 (Cys α-CH), 113.26–148.50 (Ar), 169.83 and 170.66 (2 x CO).
N-Ac-Cys(Ethyl)-CAR (3). Yield: 32%; Rf = 0.67, DCM:AcOEt (1:1); 1H-NMR (300 MHz,
298.2 K, CDCl3): δ = 1.21 (d, 6H, 2 x CH3, i-Pr), 1,29 (t, 3H, SCH2CH3), 2.08 (s, 3H, CH3), 2.15
(s, 3H, Ac), 2.60–2.68 (m, 2H, SCH2CH3), 2.82–3.07 (m, 2H, Cys β-CH2), 5.08–5.12 (m, 1H,
Cys α-CH), 6.38–6.41 (d, 1H, NH), 6.87–7.16 (m, 3H, Ar). 13C-NMR (300 MHz, 298.2 K,
CDCl3): δ = 14.97 (SCH2CH3), 16.13 (CH3), 23.40 (Ac), 24.13 (CAR, 2 x CH3, i-Pr), 27.15
(SCH2CH3), 33.78 (CH i-Pr), 34.05 (Cys β-CH2), 52.31 (Cys α-CH), 119.67–148.46 (Ar),
162.81 and 170.63 (2 x CO).
N-Ac-Cys(Allyl)-CAR (4). Yield: 35%; Rf = 0.79, DCM:AcOEt (1:1); 1H-NMR (300 MHz,
298.2 K, CDCl3): δ = 1.19–1.21 (d, 6H, i-Pr), 2.09 (s, 3H, Ac), 2.18 (s, 3H, CH3), 2.78–2.83 (m,
1H, i-Pr), 2.85–3.08 (m, 2H, Cys β-CH2), 3.17 (d, 2H, SCH2CHCH2), 5.02–5.09 (m, 1H, Cys α-
CH), 5.10–5.19 (m, 2H, SCH2CHCH2), 5.71–5.82 (m, 1H, SCH2CHCH2), 6.24–6.27 (d, 1H,
NH), 6.83–7.18 (m, 3H, Ar). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 15.76 (CH3), 23.30
(Ac), 24.14–24.29 (2 x CH3, i-Pr), 32.25 (CH i-Pr), 32.93 (Cys β-CH2), 35.59 (S-CH2CHCH2),
58.62 (Cys α-CH), 118.52 (SCH2CHCH2), 121.44–131.33 (Ar), 133.59 (SCH2CHCH2), 170.95
and 199.09 (2 x CO).
N-Ac-Met-CAR (5). Yield: 47%; Rf = 0.74, DCM:AcOEt (1:1); 1H-NMR (300 MHz, 298.2
K, CDCl3): δ = 1.20–1.25 (d, 6H, 2 x CH3, i-Pr), 2.04 (s, 3H, Ac), 2.08 (s, 3H, CH3), 2.11 (s, 3H,
SCH3), 2.11–2.44 (m, 2H, Met β-CH2), 2.63–2.68 (m, 2H, Met γ-CH2), 2.82–2.91 (m, 1H, CH
i-Pr), 4.97–5.04 (m, 1H, Met α-CH), 6.25–6.27 (d, 1H, NH), 6.85–7.16 (m, 3H, Ar). 13C-NMR
(300 MHz, 298.2 K, CDCl3): δ = 15.81 (SCH3), 16.10 (CH3), 23.39 (Ac), 24.13 (2 x CH3, i-Pr),
30.39 (CH i-Pr), 32.06 (Met γ-CH2), 33.79 (Met β-CH2), 51.99 (Met α-CH), 119.64–149.02
(Ar), 170.52 and 170.90 (2 x CO).
N-Ac-Met(Se)-CAR (6). Yield: 35%; Rf = 0.65, DCM:AcOEt (1:1); 1H-NMR (300 MHz,
298.2 K, CDCl3): δ = 1.20–1.23 (d, 6H, 2 x CH3, i-Pr), 2.04 (s, 3H, Ac), 2.08 (s, 3H, CH3), 2.13
(s, 3H, SeCH3), 2.17–2.46 (m, 2H, Met β-CH2), 2.64–2.69 (m, 2H, Met γ-CH2), 2.85–2.90 (m,
1H, CH i-Pr), 4.97–5.04 (m, 1H, Met α-CH), 6.25–6.27 (d, 1H, NH), 6.85–7.16 (m, 3H, Ar).
13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 4.64 (SeCH3), 16.12 (CH3), 20.59 (Met β-CH2),
23.45 (Ac), 24.12 (2 x CH3, i-Pr), 33.34 (Met γ-CH2), 33.79 (CH i-Pr), 52.79 (Met α-CH),
119.65–149.0 (Ar), 170.34 and 170.79 (2 x CO).
LA-CAR (7). Yield: 38%; Rf = 0.81, DCM; 1H-NMR (300 MHz, 298.2 K, CDCl3): δ = 1.21–
1.24 (d, 6H, 2 x CH3, i-Pr), 1.54–1.97 (m, 8H, 4 x CH2), 2.13 (s, 3H, CH3), 2.45–2.52 (m, 1H,
CH i-Pr), 2.57–2.62 (t, 2H, CH2), 2.85–2.89 (m, 1H, SCH), 3.11–3.20 (m, 2H, SCH2), 6.84–7.15
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Antimicrobial Effect of Carvacrol Codrugs
(m, 3H, Ar). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.12 (CAR CH3), 24.16 (2 x CH3, i-
Pr), 25.03 (CH2), 29.07 (CH2), 33.79 (CH i-Pr), 34.27 (CH2), 34.87 (CH2), 38.76 (SCH2), 40.48
(CH2), 56.56 (SCH), 119.99–149.43 (Ar), 171.99 (CO).
N-Ac-Cys(Propargyl)-CAR (9). Yield: 42%; Rf = 0.23, DCM:AcOEt (1:1); 1H-NMR (300
MHz, 298.2 K, CDCl3): δ = 1.21–1.23 (d, 6H, 2 x CH3, i-Pr), 1.63–1.88 (m, 2H, Cys β-CH2),
2.08 (s, 3H, Ac), 2.15 (s, 3H, CH3), 2.31–2.33 (s, 1H, SCH2CCH), 2.84–2.89 (m, 1H, i-Pr),
3.13–3.38 (2H, m, SCH2CCH), 5.14 (m, 1H, Cys α-CH), 6.49–6.51 (d, 1H, NH), 6.89–7.15 (m,
3H, Ar). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.15 (CH3), 20.31 (SCH2CCH), 23.36
(Ac), 24.13 (2 x CH3, i-Pr), 33.78 (Cys β-CH2), 34.14 (CH i-Pr), 52.11 (Cys α-CH), 72.50
(SCH2CCH), 79.45 (SCH2CCH), 119.68–149.04 (Ar), 169.58 and 170.41 (2 x CO).
OTC-CAR (10). Yield: 33%; Rf = 0.78, DCM:AcOEt (9:1); 1H-NMR (300 MHz, 298.2 K,
CDCl3): δ = 1.21–1.23 (d, 6H, 2 x CH3, i-Pr), 2.11 (s, 3H, CH3), 2.85–2.89 (m, 1H, i-Pr), 3.77–
3.87 (m, 2H, OTC β-CH2), 4.66–4.71 (m, 1H, OTC α-CH), 6.86–7.17 (m, 3H, Ar), 6.91 (s, 1H,
NH). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.00 (CH3), 24.12 (2 x CH3, i-Pr), 32.28
(CH, i-Pr), 33.80 (OTC β-CH2), 56.44 (OTC α-CH), 119.47–148.82 (Ar), 168.83 and 175.08 (2
x CO).
N-Ac-Cys(Trt)-CAR (21). Yield: 33%; Rf = 0.18, DCM:AcOEt (1:1); 1H-NMR (300 MHz,
298.2 K, CDCl3): δ = 1.20 (d, 6H, i-Pr), 1.99 (s, 3H, Ac), 2.08 (s, 3H, CH3), 2.67–2.95 (m, 2H,
Cys β-CH2), 4.81–4.88 (m, 1H, Cys α-CH), 6.57 (d, 1H, NH), 6.88–7.46 (m, 18H, Ar).
13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.15 (CH3), 23.34 (Ac), 24.13 (2x CH3, i-Pr), 34.16
Cys (β-CH2), 51.48 (Cys α-CH), 67.34 (C Trt), 119.76–149.11 (Ar), 168.79 and 170.34 (2 x
CO).
Boc-TCA-CAR (27). Yield: 40%; Rf = 0.64, DCM; 1H-NMR (300 MHz, 298.2 K, CDCl3):
δ = 1.21–1.25 (d, 6H, 2 x CH3, i-Pr), 1.48 (s, 9H, Boc), 2.16 (s, 3H, CH3), 2.85–2.90 (m, 1H, i-
Pr), 3.43–3.49 (m, 2H, TCA β-CH2), 4.50–4.75 (m, 2H, TCA δ-CH2), 4.96–5.16 (m, 1H, TCA
α-CH), 6.85–7.15 (m, 3H, Ar). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.07 (CH3), 24.16
(2 x CH3, i-Pr), 28.55 (Boc), 33.67 (CH i-Pr), 35.01 (TCA β-CH2), 48.39 (TCA δ-CH2), 62.02
(TCA α-CH), 81.53 (Boc), 119.44–148.39 (Ar), 153.56 (OCONH), 169.23 (CO).
Removal of protecting groups
NAC-CAR (1). A solution of 21 (100 mg, 0.17 mmol) in DCM (0.3 mL) was added with
TIPS (0.27 mL, 0.21 mmoL) and TFA (0.52 mL, 6.8 mmol) under nitrogen atmosphere. The re-
action mixture was stirred for 48 h at room temperature and then dried under vacuum. The
crude was purified by chromatographic column using DCM/MeOH 9:1 as eluant to afford the
final deprotected compound 1. Yield: 88%; 1H-NMR (300 MHz, 298.2 K, CDCl3): δ = 1.21–
1.23 (d, 6H, i-Pr), 2.07 (s, 3H, Ac), 2.15 (s, 3H, CH3), 2.87 (m, 1H, i-Pr), 3.17–3.23 (m, 2H, Cys
β-CH2), 5.16–5.19 (m, 1H, Cys α-CH), 6.57–6.60 (d, 1H, NH), 6.87–7.17 (m, 3H, Ar).
13C-NMR (300 MHz, 298.2 K, CDCl3): δ = 16.19 (CH3), 23.35 (Ac), 24.12 (2 x CH3, i-Pr),
27.21 (CH i-Pr), 33.79 (Cys β-CH2), 53.91 (Cys α-CH), 119.70–148.97 (Ar), 169.08 and 170.32
(2 x CO).
TFAꢀTCA-CAR (8). A solution of 27 (327 mg, 0.9 mmol) in TFA (1.35 mL) was left for 2
h under stirring at room temperature [32]. Yield: 100%; Rf = 0.21, DCM; 1H-NMR (300 MHz,
298.2 K, CDCl3): δ = 1.21–1.25 (d, 6H, 2 x CH3, i-Pr), 2.09 (s, 3H, CH3), 2.84–2.90 (m, 1H, i-
Pr), 3.54–3.64 (m, 2H, TCA β-CH2), 4.35–4.52 (m, 2H, TCA δ-CH2), 5.04–5.08 (m, 1H, TCA
α-CH), 6.85–7.16 (m, 3H, Ar), 9.48 (s, 2H, NH2). 13C-NMR (300 MHz, 298.2 K, CDCl3): δ =
15.69 (CH3), 24.00 (2 x CH3, i-Pr), 33.45 (CH i-Pr), 33.77 (TCA β-CH2), 49.09 (TCA δ-CH2),
62.82 (TCA α-CH), 119.44–148.99 (Ar), 166.24 (CO).
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Antimicrobial Effect of Carvacrol Codrugs
Antimicrobial activity
Two Gram-positive (S. aureus ATCC 29213 and S. epidermidis ATCC 35984) and two Gram-
negative (E. coli ATCC 8739 and P. aeruginosa ATCC 9027) strains, and one fungal strain (C.
albicans ATCC 10231) were used for the detection of antibacterial activity of carvacrol, NAC,
Ac-Cys(Allyl)-OH, and carvacrol codrugs 1–10. All tested compounds were dissolved in 20%
DMSO to prepare the stock solution (100 mg/mL). The final concentration of DMSO in each
well appeared to be not toxic for all studied microorganisms, as confirmed by the solvent con-
trol test included in the study.
The effects of carvacrol codrugs 1–10 and reference compounds on planktonic cells were
evaluated by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concen-
tration (MBC) determination using the broth microdilution method according to EUCAST
guidelines [33]. Bacterial suspensions, grown in Mueller–Hinton Broth (MHB) at logarithmic
phase, were incubated on microtiter plates at a concentration of 5 x 105 CFU/mL, with several
dilutions (0.07–40 mg/mL) for 24 h at 37°C. The MIC was defined as the lowest concentration
of substances giving a complete inhibition of visible growth in comparison with a control well,
and the MBC was determined as the lowest concentration at which no bacterial growth oc-
curred on Mueller–Hinton Agar plates.
The MIC detection of C. albicans ATCC 10231 was performed using the broth microdilu-
tion method according to EUCAST guidelines [34] in RPMI 1640 plus 2% glucose with a final
inoculum of 1–5 x 105 CFU/mL for 24–48 h at 37°C. The Minimum Fungicidal Concentration
(MFC) was determined as the lowest concentration of substances at which no fungal growth
occurred on Sabouraud agar plates.
Data were obtained from at least three independent experiments performed in duplicate.
The susceptibility of the bacterial strains against ciprofloxacin was used as an internal standard
during MIC determinations.
Antibiofilm assay. The efficacy on established biofilm was evaluated by determining Bio-
film Inhibitory Concentration (BIC) and Biofilm Eradication Concentration (BEC) according
to the method described by Johnson et al. [35] with some modifications. Bacterial suspensions,
grown in Tryptic soy broth supplemented with 0.5% (v/v) glucose at logarithmic phase, as well
as C. albicans grown in RPMI 1640 plus 2% glucose, were incubated on flat-bottomed microti-
ter plates at a concentration of 5 x 105 CFU/mL. After 24 h of incubation at 37°C, the plankton-
ic cells were gently removed and wells were washed with sterile phosphate-buffered saline
solution (PBS) pH 7.3 and filled with carvacrol, carvacrol codrug 4, Ac-Cys(Allyl)-OH with di-
lutions ranging from the MIC values to a maximum concentration of 50 mg/mL. The OD600
was measured at time 0 and after incubation for 24 h at 37°C. The BIC values were determined
as the lowest concentrations where no growth occurred in the supernatant fluid, confirmed by
no increase in optical density compared with the initial reading. The BEC values were deter-
mined as the lowest concentrations at which no bacterial growth occurred on Tryptic soy agar
for bacteria and on Sabouraud agar for C. albicans. Data were obtained from at least three inde-
pendent experiments performed in duplicate.
Hemolytic Activity. Experimental protocols to study hemolysis on human red cells were
approved by Ethics Committee of the University “G. d’Annunzio” Chieti-Pescara. Human red
cells were from two volunteers, who signed the informed consent for this study.
Hemolytic activity of carvacrol codrugs 1–10 was tested against human red blood cells (h-
RBC). Fresh human blood, collected with EDTA, was centrifuged at 3000 rpm for 5 min then
washed three times with PBS pH 7.3. Red blood cells were diluted to 4% in PBS and incubated
with different codrugs concentrations ranging from 25 to 0.05 mg/mL. After 1 h of incubation
at 37°C, the suspensions were sedimented by centrifugation and the release of hemoglobin was
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Antimicrobial Effect of Carvacrol Codrugs
determined by absorbance measurement at 405 nm and compared with a 0% hemolysis control
(PBS) and a 100% hemolysis control (PBS with 1% v/v Triton X-100). The percentage of hemo-
lysis was calculated using the following equation:
Hemolysis ð%Þ ¼ ½ðOD405 sample ꢁ OD4050% lysis controlÞ=ðOD405100% lysis control
ꢁ OD4050% lysis controÞꢂ X 100
Transmission electron microscopy (TEM). For transmission electron microscopy, micro-
rganisms were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed with 1%
OsO4, dehydrated in graded ethanol and embedded in Epon 812. Ultrathin sections (60 to 80
nm) were then mounted on 200-mesh copper grids and stained with uranyl acetate and lead
citrate for ultrastructural observation. Sections were photographed using a Philips 268 D elec-
tron microscope (FEI).
Pharmacokinetic studies
HPLC–UV assays. The HPLC system consisted of a Waters 600 HPLC pump (Waters
Corporation, Milford, MA, USA), equipped with a Waters 2996 photodiode array detector.
The column was a Kinetex RP-C8 column (3.0 x 150 mm, 5 μm). The mobile phase consisted
of 0.1% TFA in acetonitrile/0.1% TFA in 95% H2O and 5% acetonitrile 50:50, under isocratic
conditions at a flow rate of 1 mL/min. The UV detector was set at a length of 220 nm.
Solubility. The water solubility was determined by stirring an excess of the compound in 1
mL of water for 15 min at room temperature. Then the mixture was filtered using a Millipore
filter (0.45 μm) and the concentration of the compound in its saturated solution was deter-
mined by HPLC analysis [36].
Lipophilicity. The calculated clogP values were determined using ACD LogP software
package, version 4.55 (Advanced Chemistry Development Inc., Toronto, Canada).
Kinetics of chemical hydrolysis. The chemical hydrolysis rate of compound 4 was studied
in solution of 0.02 M hydrochloric acid buffer of pH 1.3, as non-enzymatic simulated gastric
fluid (SGF), and a 0.02 M phosphate buffer of pH 7.4 at 37°C, as simulated intestinal fluid
(SIF). Reactions were initiated by adding 1 mL of 10-4 M stock solution (in acetonitrile) of the
compound to 10 mL of the appropriate aqueous buffer solution, containing 20% acetonitrile.
At appropriate time intervals, samples of 20 μL were withdrawn and analyzed by HPLC. Pseu-
do-first-order rate constants (kobs) for the hydrolysis of the compounds were then calculated
from the slopes of the linear plots of log (% residual compound) against time. The experiments
were run in triplicate and the mean values of the rate constants were calculated [37].
Kinetics of enzymatic hydrolysis. The enzymatic hydrolysis was evaluated in human and
rat plasma. Stock solution of drug was prepared in methanol (1 mg/mL) which did not have ef-
fects on the enzymatic degradation (data not shown). A volume of 200 μL of this solution was
added with pre-heated (37°C) plasma fractions (4 mL) previously diluted with 0.02 M phos-
phate buffer (pH 7.4) to give a final volume of 5 mL (80% plasma). Aliquots (100 μL) were
taken at various times and deproteinized by mixing with 200 μL of 0.01 M HCl in methanol.
After centrifugation for 5 min at 5000 × g, 10 μL of the supernatant layer were analysed by
chromatography as described above. The amounts of remaining intact compound were plotted
as a function of incubation time [38].
Drug stability testing in intestinal fluids. To assess enzymatic stability, hydrochloric
buffer with pepsin (10 or 40 mg/mL) and phosphate buffer with pancreatin (10 or 40 mg/mL)
were used. Buffer solution (250 μL) was preincubated at 37°C and 50 μL of drug stock solution
(50 μM in ethanol and FASSIF 1:9) were added and shaken at 37°C and 650 rpm. Samples of
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Antimicrobial Effect of Carvacrol Codrugs
100 μL were withdrawn at various times and 100 µL of ice-cold acetonitrile containing 0.5 v/v
% formic acid were added to stop enzymatic activity [39]. Samples were vortexed and centri-
fuged at 2°C and 10000 rpm for 10 min. The drug content in the supernatant was analyzed by
HPLC.
PAMPA Method. Parallel Artificial Membrane Permeability Assay (PAMPA) was used to
measure the permeability coefficient (Pe) of codrug 4 through the artificial membrane to pre-
dict oral absorption. The carvacrol codrug 4 permeability was determined using PAMPA-GI
protocols already developed [37]. Briefly, donor and acceptor plates were assembled to form a
“sandwich” and incubated at room temperature for 18 h.
Pe can be calculated from the equation below:
(
!)
½drugꢂacceptor
½drugꢂequilibrium
VD ꢀ VA
Pe ¼
ꢀ ln 1 ꢁ
ð1Þ
ðVD þ VAÞ ꢀ A ꢀ t
where Pe is the effective permeability coefficient (cm × s-1), VD is the volume of the donor
compartment (0.15 cm3) and VA is the volume of the acceptor compartment (0.30 cm3), A is
the effective filter area (0.28 cm2), t is the incubation time for the assay (s), [drug]acceptor is
the concentration of the compound in the acceptor compartment at assay completion, and
[drug]equilibrium is the concentration of the compound at theoretical equilibrium.
Results and Discussion
Synthesis of carvacrol codrugs 1–10
Carvacrol codrugs 1–10 were synthesized as outlined in Figs 2–4 employing solution phase
procedures by elongation of the suitably protected Cys aminoacid chain in the C direction.
Acetylation of aminic groups of cysteine derivatives 11–14, and 22 was performed as previous-
ly described [40] to obtain compounds 16–19, and 23 in good yields (Figs 2–3). Alkylation of
commercial NAC (15) was performed using propargyl bromide in dry MeOH for 1 h at 60°C
in basic conditions to afford the derivative 20 in good yield (Fig 1).
The syntheses of the carvacrol codrugs 1–10 were successfully carried out by using N,N’-
dicyclohexylcarbodiimide (DCC), which proves to be an effective catalyst for the conversion of
carboxylic acids to esters and amides (Figs 2–4). Particularly, compounds 16–20, 23–26 and 28
were treated with DCC in DMF/DCM for 1 h at room temperature; after the addition of carva-
crol and DMAP, the mixture was left under stirring for 15 h at room temperature. The carva-
crol codrugs 2–7 and 9–10 were obtained and purified on silica gel using DCM/AcOEt as
eluant (yields 30–47%).
A further step was necessary to obtain the final compounds 1 and 8; in case of carvacrol
codrug 1 the deprotection of the cysteine-SH group was reached when the corresponding pro-
tected precursor 21 was treated with TIPS, TFA in DCM for 48 h under nitrogen atmosphere
at room temperature (yield 88%) (Fig 2); the removal of the Boc protecting group on 27 by
treatment with TFA for 2 h at room temperature afforded the corresponding trifluoracetate 8
in quantitative yield (Fig 4). Before performing biological studies, all carvacrol codrugs 1–10
were fully characterized by 1H-NMR, 13C-NMR spectra and the purity was checked by HPLC
analysis. A grade of purity higher than 98% after purification was obtained for all the
final compounds.
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Antimicrobial Effect of Carvacrol Codrugs
Fig 2. Synthesis of carvacrol codrugs 1–4 and 9. Reagents and conditions: a) Ac2O, AcOH, 4 h, rt for compounds 11–14; KOH, Propargyl bromide, in dry
MeOH, 1 h, 60°C (reflux) for compound 15; b) DCC in DMF/DCM, 1 h, rt, then carvacrol, DMAP, 15 h, rt; c) TIPS, TFA in DCM, 48 h, rt under
nitrogen atmosphere.
doi:10.1371/journal.pone.0120937.g002
Antimicrobial activity of carvacrol codrugs 1–10
In this study, the antimicrobial activity of carvacrol codrugs 1–10, carvacrol, NAC, and Ac-Cys
(Allyl)-OH (the last three as reference compounds) was evaluated against pathogenic microor-
ganisms and the results are outlined in Table 1.
When considering antimicrobial potency against Gram positive and Gram negative, carva-
crol codrug 4 resulted the most active with bacteriostatic and bactericidal values equal to 2.5
mg/mL for all bacterial strains except for P. aeruginosa ATCC 9027, for which MIC and MBC
values were 5 mg/mL and 10 mg/mL, respectively. We observed a lower antibacterial potency
for all the other carvacrol codrugs (MIC values ranging from 2.5 mg/mL to 10 mg/mL)
(Table 1). We also tested the reference compounds: a) the antimicrobial effect exerted by NAC
—MIC and MBC values ranged from 5 to 20 mg/mL for Gram positive and Gram negative test-
ed bacteria—was similar to those reported by Aslam and Darouiche [41], suggesting that this
amino acid may competitively inhibit bacterial utilization of cysteine or react via its sulfhydryl
group with bacterial membranes; b) Ac-Cys(Allyl)-OH showed a better antimicrobial activity
(MIC values from 2.5 to 10 mg/mL) respect to NAC probably due to the presence of the allyl
moiety that might be a more offensive tool against bacteria, as previously reported [30]. In fact,
the better antibacterial activity of Ac-Cys(Allyl)-OH compared to NAC is reflected in the cor-
responding carvacrol codrugs 4 and 1, respectively, suggesting that the presence of the allyl-
cysteine moiety in 4 improves the antibacterial properties. However, compound 4 showed a
weaker potency against P. aeruginosa confirming the much lower outer membrane
Fig 3. Synthesis of carvacrol codrugs 5–6. Reagents and conditions: a) Ac2O, AcOH, 4 h, rt for compound
22; b) DCC in DMF/DCM, 1 h, rt, then carvacrol, DMAP, 15 h, rt.
doi:10.1371/journal.pone.0120937.g003
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Antimicrobial Effect of Carvacrol Codrugs
Fig 4. Synthesis of carvacrol codrugs 7–8 and 10. Reagents and conditions: a) DCC in DMF/DCM, 1 h, rt,
then carvacrol, DMAP, 15 h, rt for compounds 25–28; b) TFA, 2 h, rt for compound 27.
doi:10.1371/journal.pone.0120937.g004
Table 1. MICa and MBCa values of carvacrol codrugs 1–10 against bacterial strains and C. albicans.
Compounds (mg/mL)
S. aureus
ATCC
29213
S.
E. coli
ATCC 8739
P.
C. albicans
ATCC 10231
epidermidis
aeruginosa
ATCC 9027
ATCC
35984
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MFCa
Carvacrol
0.6
5
1.25
20
5
0.3
5
1.25
5
0.6
5
1.25
5
5
10
5
0.3
20
0.6
80
2.5
0.3
5
NAC
5
Ac-Cys(Allyl)-OH
5
5
5
2.5
10
10
10
2.5
10
5
2.5
10
10
10
2.5
10
10
10
5
10
10
10
10
5
10
10
10
10
10
10
10
10
10
10
10
2.5
0.3
5
1
2
3
4
5
6
7
8
9
10
10
10
10
2.5
10
10
10
5
10
10
10
2.5
10
10
10
10
10
10
5
20
10
10
2.5
10
10
10
5
2.5
10
2.5
10
5
2.5
1.25
5
2.5
1.25
5
5
5
0.15
2.5
0.6
1.25
2.5
0.6
5
10
2.5
5
5
5
5
10
10
10
0.6
2.5
2.5
10
10
10
10
10
10
10
10
10
a Abbreviations: MIC, Minimum Inhibitory Concentration; MBC, Minimum Bactericidal Concentration; MFC,
Minimum Fungicidal Concentration.
doi:10.1371/journal.pone.0120937.t001
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Antimicrobial Effect of Carvacrol Codrugs
permeability than E. coli [42] probably due to the presence of efflux pumps that can transport
anti-infective agents out of the periplasmatic space and to the reduced number and alteration
of porins.
In this study, the same set of ten carvacrol codrugs was also tested against C. albicans ATCC
10231. Most of them, including carvacrol, showed antifungal activity with MIC values ranging
from 0.15 mg/mL to 5 mg/mL (Table 1). In particular, carvacrol codrugs 6, 1, 8 displayed fun-
gicidal activity with MIC values equal to 0.15, 0.3, and 0.6 mg/mL, respectively. On the other
hand, NAC was inactive against this fungal strain (MIC 20 mg/mL) while Ac-Cy(Allyl)-OH
displayed antifungal activity with MIC and MFC equal to 2.5 mg/mL. However, the carvacrol
codrug 1, containing the NAC moiety, showed a good antifungal activity while carvacrol
codrug 4, containing the allyl-cysteine, was less active. This antifungal behavior is opposite to
the antibacterial one for these carvacrol codrug 1 and 4. The antifungal activity is related to the
presence and hydrophobicity of the carvacrol structure that is able to distribute into lipids of
fungal membrane and interfere with its integrity causing fungal death. However, the overall
lipophilicity of carvacrol codrugs 1 and 4 does not correlate with the results since codrug 1 is
less hydrophobic (calculated LogP 2.66) than codrug 4 (calculated LogP 3.77) suggesting a po-
tential involvement of the free thiol group in the antifungal activity of the carvacrol codrug.
This consideration is in accordance with Bertling et al. [31] that support the hypothesis the
thiol group is able to inactivate the gliotoxin produced by C. albicans by oxidation reactions.
Also in the case of the best antifungal carvacrol codrug 6 (MIC 0.15 mg/mL) the hydrophobic-
ity (calculated LogP 2.61) does not seem to be linked to the activity indicating that the presence
of selenium might promote further interactions with the fungal membrane destabilizing it.
Antibiofilm effect of carvacrol codrug 4. BIC and BEC for carvacrol, Ac-Cys(Allyl)-OH,
and the most active carvacrol codrug 4 were determined for all studied microorganisms
(Table 2). BIC and BEC values of the three compounds for S. aureus ATCC 29213 were two-
fold (2MIC) or four-fold (4MIC) greater than the concentration required to inhibit growth in
suspension (MIC value); for S. epidermidis ATCC 35984 BIC and BEC values for carvacrol
codrug 4 were 2MIC and 8MIC, equal to MIC and 2MIC for carvacrol, and 4MIC for Ac-Cys
(Allyl)-OH; for E. coli ATCC 8739, Ac-Cys(allyl)-OH showed BIC and BEC values of 2MIC
and 8MIC, while codrug 4 and carvacrol showed higher values: 8MIC, 16MIC, and 64MIC, re-
spectively. BIC and BEC values of codrug 4, carvacrol, and Ac-Cys(Allyl)-OH coincided with
MIC and 2MIC values for P. aeruginosa ATCC 9027. As shown in Table 2, for C. albicans
ATCC 10231 BIC and BEC values of codrug 4 were 8MIC, for carvacrol these coincided with
MIC and MFC, and for Ac-Cys(Allyl)-OH BIC and BEC were 2MIC.
Our results outlined that carvacrol possesses antimicrobial and antibiofilm properties but,
except for S. epidermidis ATCC 35984 and C. albicans ATCC 10231, did not show an
Table 2. BICa and BECa values of carvacrol codrug 4 against bacterial strains and C. albicans.
Compounds (mg/mL)
S. aureus
ATCC
29213
S.
E. coli
ATCC 8739
P.
C. albicans
ATCC
10231
epidermidis
aeruginosa
ATCC 9027
ATCC
35984
BIC
BEC
BIC
BEC
BIC
BEC
BIC
BEC
BIC
BEC
Carvacrol
1.2
10
5
2.5
20
10
0.3
20
5
0.6
20
20
40
5
40
20
40
5
10
20
10
0.3
5
0.6
5
Ac-Cys(Allyl)-OH
10
5
4
20
10
10
aAbbreviations: BIC, Biofilm Inhibitory Concentration; BEC, Biofilm Eradicating Concentration.
doi:10.1371/journal.pone.0120937.t002
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Antimicrobial Effect of Carvacrol Codrugs
interesting and considerable effect on bacterial sessile phase respect to the planktonic one.
Escherichia coli ATCC 8739 mature biofilm was more affected by Ac-Cys(Allyl)-OH and
codrug 4 than carvacrol, suggesting that carvacrol is able to reduce bacterial biofilm formation
interfering with the quorum sensing signaling mechanism but not with the mature biofilm, as
reported by Burt et al. [43]. Probably, carvacrol alone is unable to penetrate the microbial bio-
film matrix of E. coli while the conjugation to Ac-Cys(Allyl)-OH renders the codrug 4 able to
promote the permeabilization and destabilization of the bacterial membrane.
Hemolytic Activity. In vitro hemolytic assay represents a suitable screening tool to verify
the in vivo toxicity to host cells. In the present study, the hemolytic activities of codrugs 1–10,
carvacrol and NAC, as reference compounds, were evaluated (Fig 5). All analyzed codrugs pro-
duced human blood hemolysis below 50% at their MIC values, except for codrugs 8 and 9
which showed hemolytic activity values over 50%. In particular, carvacrol codrugs 7 and 10 he-
molytic activities were never more than 12% at the tested concentrations; codrug 1 never ex-
pressed hemolytic activity higher than 40% and for carvacrol codrugs 2, 3, 5, and 6 the
hemolytic activities were more than 50% only over 20 mg/mL of concentration. The carvacrol
codrugs 4, 8, and 9 displayed hemolytic activity over 50% at 5 mg/mL, 3 mg/mL, and 10 mg/
mL, respectively. These data correlate with the hydrophobicity parameters: the carvacrol
codrugs 8 and 9, showing the highest LogP values, resulted the most toxic compounds.
Furthermore, carvacrol codrug 8 was obtained as trifluoracetate salt, which is substantially
more toxic than hydrochloride salt even though they are both able to cross the intestinal
membranes [44].
In particular, the hemolytic activity of carvacrol codrug 4 was evaluated in a wider range of
values between 6.02–2.96 mg/mL to verify if the hemolysis occurs around the MIC value (2.5
mg/mL) for S. aureus ATCC 29213, S. epidermidis ATCC 35984, and E. coli ATCC 8739. Re-
sults showed that codrug 4 resulted not toxic at the MIC values for S. aureus ATCC 29213, S.
epidermidis ATCC 35984, E. coli ATCC 8739, and C. albicans ATCC 1023.
Transmission Electron Microscopy of carvacrol codrug 4. To elucidate the physiological
effects of carvacrol codrug 4 against S. aureus, E. coli, and C. albicans transmission electron mi-
croscopy was utilized. As illustrated in Fig 6, the micrographs clearly demonstrated that the
growth of S. aureus, E. coli, and C. albicans in media containing carvacrol codrug 4 at 12.5 mg/
mL for 3 h generated profound changes in cell morphology [45]. We found that carvacrol
codrug 4 had no effect on the envelope in S. aureus, but induced abnormal septum formation
with irregular features (Fig 6B, arrows) probably with the same mechanism of action of some
antibiotics such as penicillin [46], erythromycin [47], and vancomycin [48]. On the contrary,
E. coli treated cells were evidently affected on the cell wall: alteration of outer membrane (Fig
3D), formation of membrane blebbing with numerous electron-dense bubbles protruding from
the cell surface (Fig 6D–6c and 6b) and electron-dense granules internalized into the cytoplasm
were observed (Fig 6D-d). In C. albicans we observed the disintegration of the membranes (Fig
6F) confirming that the carvacrol codrug 4 hydrophobicity allows it to interact with the fungal
cell membrane and to interfere with its integrity. In fact, compound 4 could distribute into lip-
ids of fungal membranes, rendering them more permeable and damaging their integrity, conse-
quently causing mycelia death [49].
Pharmacokinetic studies of carvacrol codrug 4
The pharmacokinetic parameters were studied only for carvacrol codrug 4 since it resulted the
compound with the best antimicrobial activity.
Solubility is one of the most important parameters to reach the desired concentration of
drug in systemic circulation for achieving required pharmacological response. Table 3 shows
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Antimicrobial Effect of Carvacrol Codrugs
Fig 5. Hemolytic activity of carvacrol codrugs 1–10, CAR, and NAC (A) and in a wider range of values
for carvacrol codrug 4 (B).
doi:10.1371/journal.pone.0120937.g005
the water solubility of codrug 4 and its lipophilicity (LogP and cLogP). Our data revealed that
codrug 4 has a low water solubility (0.15 mg/mL) and a cLogP value (3.77) similar to carvacrol
(3.35), suggesting that this hydrophobic character could allow codrug 4 to interact with the
bacterial membrane just like carvacrol.
The precondition of codrug strategy is that the codrug can be converted to the parent drugs
to exert the pharmacological effect. For this purpose, the chemical and enzymatic stabilities of
carvacrol codrug 4 were evaluated in three buffers (pH 1.3, 5.0, and 7.4), in simulated fluids
(SGF and SIF) with different concentrations (10 mg/mL and 40 mg/mL) of enzymes (pepsin
and pancreatin), and in human and rat plasma. As reported in Table 4, codrug 4 resulted more
stable at pH 1.3 (t = 75.38 h) and pH 5.0 (t = 62.08 h), respect to pH 7.4 (t = 22.9 h). These
½
½
½
values suggest that codrug 4 is able to pass unhydrolyzed the g.i. tract. In the presence of in-
creasing concentrations of enzymes, the rate of hydrolysis of codrug 4 was significantly faster
at pH 7.4 than 1.3; in fact, in the presence of pepsin (10 mg/mL) at pH 1.3, codrug 4 resulted
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Antimicrobial Effect of Carvacrol Codrugs
Fig 6. Transmission electron microscopy demonstrating the effects of carvacrol codrug 4 on S. aureus ATCC 29213 (B), E. coli ATCC 8739 (D), C.
albicans ATCC 10231 (F), and untreated cultures (control), respectively (A, C, and E). Microorganisms incubated for 3 h in media containing 12.5 mg/mL
[45] of carvacrol codrug 4 (B, D, and F). Irregular features of septa in S. aureus ATCC 29213 (B: arrows); numerous electron-dense bubbles protrude from the
cell surface (higher magnification b 140000x and c 110000x) in E. coli ATCC 8739 treated (D) and electron-dense granules of the substance internalized into
the cytoplasm (d 110000x); integrity of the membrane (a 110000x) in the controls (C); disintegration of membrane in C. albicans ATCC 10231 (F).
doi:10.1371/journal.pone.0120937.g006
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Antimicrobial Effect of Carvacrol Codrugs
Table 3. Physicochemical properties of carvacrol codrug 4.
Lipophilicity
Water Solubilitya (mg/mL)
LogPa
cLogP
1.21 ( 0.04)
3.35
0.15 ( 0.01)
aValues are means of three experiments, standard deviation is given in parentheses.
doi:10.1371/journal.pone.0120937.t003
stable with a t higher than 74 h (Table 4); however, with the increase in pepsin concentration
½
the half-life of codrug 4 lowered (t = 5.79 h). On the other hand, in physiological buffer
½
codrug 4 degraded with a half-life of 5.5 h and about 1 h in the presence of 10 and 40 mg/mL of
pancreatin, respectively (Table 4).
These data evidenced that codrug 4 resulted 13-fold more stable at pH 1.3 respect to pH 7.4
in the presence of pepsin and pancreatin (10 mg/mL), suggesting that the hydrolysis of ester
linkage is preferred at physiological pH (Table 4). Codrug 4 showed the same rate of hydrolysis
in 80% human and rat plasma (t = 0.50 h) suggesting that both plasma may have more selec-
½
tive enzymes for the hydrolysis of ester bond (Table 4). Taken together, these results point out
that codrug 4 is quite stable to potentially cross unmodified the acidic environment of the
stomach, to be absorbed intact from the intestine after oral administration, and able to release
carvacrol and Ac-Cys(Allyl)-OH after enzymatic hydrolysis.
Depending on the phospholipid type, PAMPA can mimic different adsorption/permeation
environments. For predicting the ability of carvacrol codrug 4 to diffuse through the gastroin-
testinal tract, lecithin in dodecane has been used for g.i. permeation studies (PAMPA-GI)
(Table 5). To carefully reproduce the g.i. tract, the permeability of codrug 4 was determined at
pH 5.0, 6.5, and 7.4. Since our carvacrol codrug 4 has a low solubility in the conventional solu-
tion used for the PAMPA, permeability tests were performed in the presence of cosolvents (2%
of Tween 80 and 10% of MeOH). The employed cosolvents did not change the permeability of
the phospholipid layer at the investigated concentrations, as previously reported [37]. After 18
h of incubation, codrug 4 showed good permeability through PAMPA-GI membrane (Pe > 1 x
10-6 cm/s) at each value of pH, thus suggesting a good absorption along g.i. tract.
Table 4. Kinetic data for hydrolysis of carvacrol codrug 4 at 37°C.a
Chemical hydrolysis
t
(h)
kobs (h-1)
½
pH 1.3
75.38 ( 2.94)
62.08 ( 2.11)
22.9 ( 0.6)
0.009 ( 0.001)
0.11 ( 0.01)
pH 5.0
pH 7.4
0.030 ( 0.001)
Enzymatic hydrolysis
SGFb
pepsin 10 mg/mL
74.09 ( 3.19)
5.79 ( 0.16)
5.51 ( 0.13)
0.84 ( 0.03)
0.50 ( 0.01)
0.50 ( 0.02)
0.009 ( 0.001)
0.119 ( 0.003)
0.126 ( 0.003)
0.83 ( 0.02)
1.38 ( 0.03)
1.38 ( 0.05)
pepsin 40 mg/mL
SIFb
pancreatin 10 mg/mL
pancreatin 40 mg/mL
Human plasma
Rat plasma
aValues are means of three experiments, standard deviation is given in parentheses.
bAbbreviations: SGF, simulated gastric fluid; SIF, simulated intestinal fluid.
doi:10.1371/journal.pone.0120937.t004
PLOS ONE | DOI:10.1371/journal.pone.0120937 April 10, 2015
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Antimicrobial Effect of Carvacrol Codrugs
Table 5. PAMPA-GI permeability of carvacrol codrug 4 after 16 h of incubation.
PAMPA-GI permeability Pe (10-6 cm s-1)
pH 7.4a
pH 6.5a
pH 5.0a
2.34
17
8.6
a pH of both donor and acceptor compartment.
doi:10.1371/journal.pone.0120937.t005
Conclusion
In summary, we synthesized ten carvacrol codrugs with the aim of developing innovative anti-
microbial compounds. However, Ac-Cys(Allyl)-CAR (4) resulted the most active compound
against S. aureus, S. epidermidis, and E. coli with MIC values of 2.5 mg/mL and endowed with
low toxicity. Even though antibacterial activity of Ac-Cys(Allyl)-CAR is not particularly good,
pharmacokinetic data evidenced a good stability at pH 1.3 respect to pH 7.4 in the presence of
pepsin and pancreatin, suggesting that, after absorption it might be able to release carvacrol
and Ac-Cys(Allyl)-OH due to the enzymatic activity.
Further experiments will be necessary to evaluate the in vivo antimicrobial activity of Ac-
Cys(Allyl)-CAR and assess the real hydrolysis of the ester bond that would guarantee the re-
lease of the free hydroxyl group of carvacrol able to form hydrogen bonds and act as a trans-
membrane carrier of monovalent cations through the bacterial cytoplasmatic membrane.
In conclusion, new approaches like codrug strategy applied to natural products (essential
oils), could be useful in the antimicrobial plan to overcome the issue of the antibiotic resistance.
Acknowledgments
Authors wish to thank the “Foundation Carichieti”, Abruzzo-Italy, for the fellowship attributed
to Laura Serafina Cerasa and MIUR for the financial support.
Author Contributions
Conceived and designed the experiments: IC ADS LC. Performed the experiments: EF LSC HT
LM EDC SDB IR. Analyzed the data: IC MDG EF ADS LSC LM HT EDC SDB IR LC. Contrib-
uted reagents/materials/analysis tools: LC ADS IC. Wrote the paper: IC LC MDG ADS.
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