Synthesis and antifungal activity of carvacrol and thymol esters with heteroaromatic carboxylic acids

Article abstract of DOI:10.1080/14786419.2018.1480618

Aiming to obtain the more effective pathogen inhibitive ingredients and explore the influence of introducing different heterocyclic units to carvacrol and thymol esters, twenty ester derivatives with different heterocyclic units were synthesized. And the

Full text of DOI:10.1080/14786419.2018.1480618

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis and antifungal activity of carvacrol and
thymol esters with heteroaromatic carboxylic
acids
Kaibo Wang, Shanshan Jiang, Yunhai Yang, Liming Fan, Fawu Su & Min Ye
To cite this article: Kaibo Wang, Shanshan Jiang, Yunhai Yang, Liming Fan, Fawu Su & Min
Ye (2018): Synthesis and antifungal activity of carvacrol and thymol esters with heteroaromatic
carboxylic acids, Natural Product Research, DOI: 10.1080/14786419.2018.1480618
To link to this article: https://doi.org/10.1080/14786419.2018.1480618
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1480618
Synthesis and antifungal activity of carvacrol and thymol
esters with heteroaromatic carboxylic acids
Kaibo Wang1, Shanshan Jiang1, Yunhai Yang, Liming Fan, Fawu Su and Min Ye
state Key laboratory for conservation and utilization of Bio-resources in Yunnan, Yunnan agricultural
university, Kunming, china
ABSTRACT
ARTICLE HISTORY
received 13 February 2018
accepted 21 May 2018
Aiming to obtain the more effective pathogen inhibitive ingredients
and explore the influence of introducing different heterocyclic units
to carvacrol and thymol esters, twenty ester derivatives with different
heterocyclic units were synthesized. And the in vitro antifungal activity
of title compounds against five plant pathogenic fungi was evaluated
by mycelium growth rate method. The results showed that some
carvacrol and thymol esters showed good to excellent antifungal
activity, and compound 9d (4-bromo-5-isopropyl-2-methylphenyl
picolinate) exhibited a broad antifungal spectrum. Preliminary study
indicated that the introduction of furan, thiophene and pyridine unit
could enhance the antifungal activity of carvacrol and thymol esters
against Botrytis cinerea and a bromine atom on the para position of
benzene moiety could enhance their antifungal activity.
KEYWORDS
carvacrol; thymol; ester
derivatives; heterocyclic
compound; antifungal
activity
1. Introduction
Plant disease cause major losses in agriculture, which are primarily controlled by application
of synthetic fungicides. But the overuse of synthetic pesticides can lead to fungicides resist-
ance, food and environmental pollution (Aliferis and Jabaji 2011), which encourages the
scientists and researchers to develop novel and improved compounds (Ehler 2006). Natural
products are attracting more attention in research and development of pesticides because
of their different structures, various bioactivities, less side effects, biodegradable properties
and rich source (Dayan et al. 2009). Now, a considerable amount of natural resources have
been applicated in plant disease control (Copping and Duke 2007). Generally, natural
CONTACT Fawu su
su_faw@126.com; Min Ye
yeminpc@126.com
1these authors contributed equally to this work.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1480618.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
K. WANG ET AL.
compounds are not commercially available for non-drug-like physicochemical properties
such as volatility, stability and water solubility (Dayan et al. 2009). An alternate strategy to
combat this problem is combining two or more single active agents or compounds in one
molecule. Hybrid molecules with dual mode of action have been used for generating new
and more active compounds and drugs (Borate et al. 2011; Pete et al. 2012; Shaveta and
Singh 2016).
Carvacrol and thymol are constituent of essential oils produced by numerous aromatic
plants such as the genus Origanum (Stefanakis et al. 2013), Thymus (Porte and Godoy 2008),
Lippia (Bueno-Durán et al. 2014) and Satureja (Yousefzadi et al. 2012; Wesolowska et al. 2015).
The two natural smaller molecules have exhibited potential biological activity, such as anti-
bacterial and antifungal activity (Taha and Azeiz 2010; Friedman 2014; Suntres et al. 2015),
and can potentially be used as antifungal agents against plant pathogenic fungi (Vázquez
et al. 2001; Soković et al. 2002; Numpaque et al. 2011). And in our previous studies, carvacrol
and thymol had showed a significant inhibitory effect on the mycelial growth of several plant
pathogenic fungi, and their ester derivatives were more effective. Thus, we suggested more
ester derivatives of them should be synthesized to obtain the more effective compounds
(Wang et al. 2018). Heterocyclic compounds had played an important role in molecular
design of pesticides due to their diversity of chemical structure, broad-spectrum of biological
activity, low toxicity and high activity (Liu et al. 2007). Therefore, it attracted our interests to
design carvacrol and thymol esters with heterocyclic units such as furan, thiophene, meth-
ylpyrazole and pyridine. Meanwhile, it was reported that the antifungal activity of halogen-
ated thymol derivatives is different from thymol (Kaur et al. 2013). Considering the electronic
and space effects of halogen atom may greatly affect the conjugation system and coplanarity
of the compounds, brominated carvacrol and thymol esters were also synthesized.
In this study, twenty carvacrol and thymol esters were synthesized by linking the carvacrol
or thymol hydroxyl moiety to the carboxyl moiety of heteroaromatic carboxylic acids. And
their antifungal activity against five kinds of important plant pathogenic fungi (Alternaria
solani, Botrytis cinerea, Fusariu oxysporum, Pyricularia oryzae, and Rhizoctonia solani) were
also evaluated.
2. Results and discussion
2.1. Synthesis and characterization
The synthetic routes to carvacrol and thymol ester derivatives are outlined in Scheme 1.
Bromo-carvacrol and bromo-thymol (compounds 3, 4) were respectively prepared via bro-
mination of 1 and 2, according to the procedure described by Soderberg (Soderberg and
Fields 1996). Compounds 6a-f were prepared by the reaction of heterocyclic carboxylic acids
with SOCl2, according to the reported procedure (Cui et al. 2014). Furthermore, carvacrol,
thymol and their brominated derivatives were respectively reacted with compounds 6a-f to
afforded ester derivatives (compounds 7a-d, 8a-d, 9a-f, 10a-f) in a yield of 53–86%. The
chemical structure of compound 3, 4 and all the ester derivatives were elucidated on the
basis of 1H NMR, 13C NMR, and HR-ESI-MS analyses.

NATURAL PRODUCT RESEARCH
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Scheme 1. synthetic route to carvacrol and thymol esters with heteroaromatic carboxylic acids.
2.2. Antifungal assay
The in vitro antifungal activity of all the synthetic compounds against five plant pathogenic
fungi was evaluated by mycelium growth rate method at 10 μg∙mL−1 and 50 μg∙mL−1, and
the results are listed in Table S1 in the supplementary materials. Preliminary antifungal assay
indicated that all the synthetic compounds exhibited antifungal activity to a certain extent
and significant antifungal activity at the higher concentration (50 μg∙mL−1). At first, all syn-
thetic compounds exhibited low antifungal activity against A. Solani and F. oxysporum, and
their inhibition ratios were below 80 percent at test concentrations. Then, some compounds
exhibited higher antifungal activity than chlorothalonil, such as compounds 8a, 9d, 10a,
10b and 10d against B. Cinerea, and compounds 7a, 7b and 9d against R. Solani. Carvacrol
and thymol exhibited low active against P. oryzae, but their esters compounds 9d, 9e and
10b exhibited higher antifungal activity than them. Interestingly, compound 10b exhibited

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K. WANG ET AL.
100% inhibition ratio against P. oryzae at 50 μg∙mL−1, but it exhibited low inhibition ratio
(5.48 0.27) at 10 μg∙mL−1.
The antifungal activity of the synthetic compounds against B. cinerea were related to their
chemical structure. At first, esters with furan, thiophene and pyridine unit exhibited high
potential antifungal activity, and esters with methylpyrazole unit exhibited low antifungal
activity. Moreover, most of the thymol esters exhibited higher antifungal activity than car-
vacrol esters did. For example, compound 10b, a thymol ester with thiophene unit, was more
effective than its isomer compound 9b. The exactly similar results were also observed in
esters with furan, methylpyrazole or pyridine unit. On the other hand, brominated esters
showed higher inhibition ratios. For example, compound 8b, a thymol ester without bromine
substituent, was less effective than its brominated compound 10b. However, the chlorine
atom in pyridine unit of synthetic compounds could not enhance their antifungal activity.
The antifungal activity of compounds 9e and 10e were not higher than compounds 9d and
10d. Besides, the antifungal activity of synthetic compounds was affected by the position
of ester bond at the heterocyclic moiety. The compounds connected with ester bond at
ortho position in pyridine unit (compounds 9d and 10d) were more effective than at meta
position (compounds 9e and 10e).
In the antifungal assay of synthetic compounds against R. solani, compounds 7a, 7b and
9d were found to be the most effective antifungal compounds, which showed similar or
better antifungal activity than their parent compounds and chlorothalonil. The compounds
with different heterocyclic unit, terpene structure and substituent showed different antifun-
gal activity, but there was no significant regular change. Compound 9d and 9e were the
only two compounds which exhibited some antifungal activity against P. oryzae in this study.
All the other compounds, however, were less effective. Thus, we cannot yet draw a clear
conclusion about the structure-activity relationships of test compounds against R. solani
and P. oryzae according to the current test results.
It is not the first time the hybrid molecules of carvacrol and thymol were synthesized.
Recently, some hybrid molecules were designed through introducing different active struc-
tures to carvacrol and thymol molecules. For example, hybrid molecules of carvacrol and
sulphur-containing amino acids revealed good antifungal activity against Candida albicans
(Cacciatore et al. 2015). Bendre and co-workers (Pete et al. 2012) synthesized benzoyl phenyl
urea derivatives with the structure of carvacrol, and several compounds have exhibited
application possibility in agriculture and medicine. Srinivas and coworkers (James Bound
et al. 2016) synthesized a series of novel 2,3-unsaturated and 2,3-dideoxy 1-O-glucosides of
carvacrol, thymol, and perillyl alcohol, the 2,3-dideoxyglucosides of carvacrol and thymol
showed high antifungal activity and could potentially be used as antifungal agents. And in
this study, we synthesized some carvacrol and thymol esters and the antifungal activity
in vitro of the most effective compound (compound 9d) was close to chlorothalonil against
both B. cinerea, P. oryzae and R. solani. In previous studies, carvacrol and thymol ester deriv-
atives with structure of 5-phenyl-2-furan were synthesized and their antifungal activity was
evaluated, and the results showed that most of the title compounds had a considerable
effect on the selected plant pathogenic fungi (Cui et al. 2014). In our study, our primary
concern was that the influence of introducing different heterocyclic units in carvacrol and
thymol esters, and heteroaromatic carboxylic acids without substitute was chemically com-
bined with the carvacrol and thymol molecules. Fortunately, some hybrid molecules also
exhibited excellent antifungal activity, which were higher than previously reported carvacrol

NATURAL PRODUCT RESEARCH
5
or thymol esters. Nevertheless, more picolinate derivatives should be synthesized to get
more effective antifungal ingredient for our further research.
3. Experimental section
3.1. General experimental
All of the reagents and solvents for reaction were purchased from Aladdin (China) or
Sinopharm Chemical Reagent Co., Ltd (China) were of analytical grades and used without
further treatments. Solvents for extraction and chromatography were of technical grade and
distilled prior to use. Reactions were monitored by thin layer chromatography (TLC) by using
silica gel coated glass slides (silica gel 60 GF 254, Qingdao Haiyang Chemical, China).
Detections were conducted under UV (254 nm). 1H and 13C NMR spectra were recorded on
a Bruker Avance III 500 NMR spectrometer. The chemical shifts (δ) were reported in ppm
with reference to internal TMS, and coupling constants (J) were given in Hz. ESI-MS spectra
were recorded on a Thermo Fisher Scientific TSQ Endura MS.
3.2. Chemistry
3.2.1. 4-Bromocarvacrol (3)
To a solution of carvacrol (1.5 g) in glacial acetic acid (10 mL) cooled to 0 °C was added Br2
(0.51 mL) over 20 min, then the reaction mixture was stirred for 3 h at room temperature.
The mixture was poured out on ice water (20 mL), and the aqueous solution was extracted
with dichloromethane (3 × 10 mL). The combined organic phase was dried over anhydrous
Na2SO4, filtered, and the solvent was removed in vacuo to give a yellow oil. The oil was purified
by column chromatography using silica gel with PE/EA (40:1, v/v) as eluent to afford com-
pound 3.
Compound 4 was prepared as described for compound 3.
3.2.2. 5-isopropyl-2-methylphenyl furan-2-carboxylate (7a)
A solution of furan-2-carboxylic acid (1.2 mmol, 1.2 equiv) in thionyl chloride (1 mL) was
refluxed for 3 h, then thionyl chloride was removed in vacuo. The residues were dissolved in
anhydrous THF (1 mL) and was added to a solution of carvacrol (1 mmol, 1.0 equiv) and
triethylamine (1.5 mmol, 1.5 equiv) in anhydrous THF (4 mL). The mixture was stirred for 1 h
at room temperature. After the reaction was completed, 5 mL of water was added to the
reaction mixture, then the reaction mixture was extracted with EtOAC (2 × 10 mL). The
organic layer was dried over anhydrous Na2SO4. The solvent was removed in vacuo, and the
oil was purified by silica gel colunm chromatography (Petroleum ether : EtOAC = 20:1) to
give compound 7a.
Compounds 7b-d, 8a-d, 9a-f and 10a-f were prepared as described for compound 7a.
3.3. Antifungal assays
The in vitro antifungal activity of target compounds was determined by the mycelium growth
rate method. Five kinds of important plant pathogenic fungi (Alternaria solani, Botrytis
cinerea, Fusariu oxysporum, Pyricularia oryzae, and Rhizoctonia solani) were chosen for

6
K. WANG ET AL.
antifungal assay. The experimental details and computing methods are the same as we
described in our recent paper (Wang et al. 2018).
4. Conclusions
In conclusion, twenty carvacrol and thymol esters with heteroaromatic carboxylic acids were
synthesized, and their antifungal activity was evaluated against five phytopathogens. The
results showed that some synthetic compounds exhibited higher antifungal activity than
their precursor compounds and commercial chlorothalonil, and compound 9d (4-bro-
mo-5-isopropyl-2-methylphenyl picolinate) was the most potential antifungal agent in this
study, which exhibited a broad antifungal spectrum against B. cinerea, P. oryzae and R. Solani.
The introduction of different heterocyclic units in the carvacrol and thymol molecules could
influence their antifungal activity, carvacrol and thymol esters with furan, thiophene and
pyridine unit exhibited good antifungal activity and a bromine atom on the para position
of pyridine unit can help to enhance their antifungal effect. We are in the process of additional
antifungal assay to investigate the bioactivity of compound 9d. And for our further work,
more picolinate derivatives should be synthesized.
Supplementary material
Spectral data of all synthetic compounds and experimental data of antifungal assay relating to this
article can be found in the online versions as Appendix.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by Project of the National Natural Science Foundation of China [grant num-
ber 31460487]; Project of the Yunnan Provincial Natural Science Foundation of China [grant number
2016FB065].
References
Aliferis KA, Jabaji S. 2011. Metabolomics – a robust bioanalytical approach for the discovery of the
modes-of-action of pesticides: a review. Pestic Biochem Physiol. 100:105–117.
Borate HB, Sawargave SP, Chavan SP, Chandavarkar MA, Iyer R, Tawte A, Rao D, Deore JV, Kudale AS,
Mahajan PS, et al. 2011. Novel hybrids of fluconazole and furanones: Design, synthesis and antifungal
activity. Bioorg Med Chem Lett. 21:4873–4878.
Bueno-Durán AY, Cervantes-Martínez J, Obledo-Vázquez EN. 2014. Composition of essential oil from
Lippia graveolens. Relationship between spectral light quality and thymol and carvacrol content.
J Essent Oil Res. 26:153–160.
Cacciatore I, Di Giulio M, Fornasari E, Di Stefano A, Cerasa LS, Marinelli L, Turkez H, Di Campli E, Di
Bartolomeo S, Robuffo I, et al. 2015. Carvacrol codrugs: a new approach in the antimicrobial plan.
Plos One. doi:10.1371/journal.pone.0120937.
Copping LG, Duke SO. 2007. Natural products that have been used commercially as crop protection
agents. Pest Manag Sci. 63:524–554.

NATURAL PRODUCT RESEARCH
7
Cui Z, Li X, NishidaY. 2014. Synthesis and bioactivity of novel carvacrol and thymol derivatives containing
5-Phenyl-2-furan. Lett Drug Des Discovery. 11:877–885.
Dayan FE, Cantrell CL, Duke SO. 2009. Natural products in crop protection. Bioorg Med Chem. 17:4022–
4034.
Ehler LE. 2006. Integrated pest management (IPM): definition, historical development and
implementation, and the other IPM. Pest Manage Sci. 62:787–789.
Friedman M. 2014. Chemistry and multibeneficial bioactivities of carvacrol (4-Isopropyl-2-
methylphenol), a component of essential oils produced by aromatic plants and spices. J Agric Food
Chem. 62(31):7652–7670.
James Bound D, Murthy PS, Srinivas P. 2016. 2,3-Dideoxyglucosides of selected terpene phenols and
alcohols as potent antifungal compounds. Food Chem. 210:371–380.
Kaur R, Darokar MP, Chattopadhyay SK, Krishna V, Ahmad A. 2013. Synthesis of halogenated derivatives
of thymol and their antimicrobial activities. Med Chem Res. 23:2212–2217.
LiuY, Cai B, LiY, Song H, Huang R, Wang Q. 2007. Synthesis, crystal structure, and biological activities of
2-cyanoacrylates containing furan or tetrahydrofuran moieties. J Agric Food Chem. 55:3011–3017.
Numpaque Oviedo MA, Gil LA, García JH, Durango CM, Diego L. 2011. Thymol and carvacrol:
biotransformation and antifungal activity against the plant pathogenic fungi Colletotrichum
acutatum and Botryodiplodia theobromae. Trop Plant Pathol. 36:3–13.
Pete UD, Zade CM, Bhosale JD, Tupe SG, Chaudhary PM, Dikundwar AG, Bendre RS. 2012. Hybrid
molecules of carvacrol and benzoyl urea/thiourea with potential applications in agriculture and
medicine. Bioorg Med Chem Lett. 22:5550–5554.
Porte A, Godoy RLO. 2008. Chemical composition of Thymus vulgaris L. (thyme) essential oil from the
Rio de Janeiro State (Brazil). J Serb Chem Soc. 73:307–310.
Shaveta Mishra S, Singh P. 2016. Hybrid molecules: the privileged scaffolds for various pharmaceuticals.
Eur J Med Chem. 124:500–536.
Soderberg BC, Fields SL. 1996. expedient syntheses of espintanol, -methoxycarvacrol and thymoqutnol
dimethyl ether. Org Prep Proced Int. 28:221–225.
Soković M, Tzakou O, Pitarokili D, Couladis M. 2002. Antifungal activities of selected aromatic plants
growing wild in Greece. Mol Nutr Food Res. 46:317–320.
Stefanakis MK, Touloupakis E, Anastasopoulos E, Ghanotakis D, Katerinopoulos HE, Makridis P. 2013.
Antibacterial activity of essential oils from plants of the genus Origanum. Food Control. 34:539–546.
Suntres ZE, Coccimiglio J, Alipour M. 2015. The bioactivity and toxicological actions of carvacrol. Crit
Rev Food Sci Nutr. 55:304–318.
Taha M, Azeiz AZA. 2010. Antifungal effect of thymol, thymoquinone and thymohydroquinone against
yeasts, dermatophytes and non-dermatophyte molds isolated from skin and nails fungal infections.
Egypt J Biochem Mol Biol. 28:109–126.
Vázquez BI, Fente C, Franco CM, Vázquez MJ, Cepeda A. 2001. Inhibitory effects of eugenol and thymol
on Penicillium citrinum strains in culture media and cheese. Int J Food Microbiol. 67:157–163.
Wang K, Jiang S, Pu T, Fan L, Su F, Ye M. 2018. Antifungal activity of phenolic monoterpenes and
structure-related compounds against plant pathogenic fungi. Nat Prod Res. doi:10.1080/14786419.
2017.1419232.
Wesolowska A, Grzeszczuk M, Jadczak D. 2015. Influence of distillation method on the content and
composition of essential oil isolated from summer savory (Satureja hortensis L.). J Essent Oil-Bear
Plants. 18:215–221.
Yousefzadi M, Riahi-Madvar A, Hadian J, Rezaee F, Rafiee R. 2012. In vitro cytotoxic and antimicrobial
activity of essential oil from Satureja sahendica. Toxicol Environ Chem. 94:1735–1745.