From clove oil to bioactive agents: synthetic routes, antimicrobial and antiparasitic activities of eugenol derivatives

Abstract

Eugenol, a natural compound found in essential oils such as clove oil, has been extensively studied for its diverse biological activities including the therapeutic potential against microbial and parasitic infections. This review provides an overview of the synthetic strategies (shown in Supplementary Material) employed to develop bioactive derivatives and analogues derived from eugenol and related compounds (e.g., dihydroeugenol and isoeugenol), focusing on biological activity of more than 100 bioactive eugenol derivatives against bacterial, fungal, viral and protozoal pathogens. Through a comprehensive survey of literature, this paper shows the impact of structural modifications of these phenylpropanoids on antimicrobial and antiparasitic activity. Key findings highlight promising candidates for further development in antimicrobial drug discovery, suggesting directions for future research in the pursuit of effective therapeutic agents.

Graphical Abstract

Plain language summary

Article highlights.

Biological properties & synthetical versatility of eugenol

• Eugenol exhibits a wide range of biological activities, offering potential for drug development across various therapeutic areas.

• Eugenol's structure includes key functional groups that contribute to its biological activity and allow functionalization.

• Eugenol derivatives present significant potential against a wide range of pathogens, emphasizing the role of chemical structure in improving biological activity.

Biological activities of eugenol derivatives

• Numerous eugenol derivatives have shown antifungal activity, highlighting the potential for discovering new drug candidates for fungal infections.

• Eugenol derivatives may provide new candidates for treating bacterial infections, especially those caused by resistant strains, enhancing the current drug repertoire.

• Some eugenol derivatives have demonstrated potential as antiviral agents, offering hope for developing treatments for viral infections that currently lack effective therapies.

• Various eugenol derivatives show promise in combating parasitic infections, with further studies needed to identify clinically viable candidates as alternatives to current outdated and limited treatments.

1. Introduction

Since the beginning of human history, infectious diseases continue to pose a major global health threat, accounting for millions of deaths annually [1]. Tuberculosis remains a leading cause of death among infectious diseases, with 1.4 million fatalities and 10 million new infections reported in 2019, while malaria continues to threaten half of the world’s population [1,2]; Pottie e Girard, 2021). Emerging of new and resistant pathogens further complicate the treatment scenario, aggravated by a shift in transmission dynamics caused by climate change and other human environmental interventions, such as deforestation, urbanization and increased global travel [3–7].

For instance, a study revealed that out of 7.7 million deaths globally in 2019 caused by bacterial pathogens, 1.3 million were directly attributed to antibiotic resistance [8]. Similarly, multidrug-resistant fungal infections pose a severe threat to public health, with Candida auris as a prominent example. Considered a “superbug”, C. auris exhibits high levels of resistance to commonly used antifungals like azoles, polyenes, and to a lesser extent, echinocandins, leading to frequent therapeutic failures and high mortality rates [9,10]. Moreover, protozoans of clinical relevance continue to pose significant health challenges, with many involved in the so-called neglected tropical diseases, such as Chagas disease, caused by Trypanosoma cruzi, and the parasites responsible for leishmaniasis. Drug treatments against these parasites are often outdated, have limited availability and efficacy, struggle to address the emergence of drug-resistant parasites, and are frequently associated with significant toxicity [11].

In addition, viral resistance and new emerging viruses remain significant challenges, as demonstrated by HIV and, most recently, the COVID-19 pandemics, urging the necessity for new antiviral drug scaffolds [11–13]. Mosquito-borne arboviruses also present a significant healthcare concern, such as dengue virus, whose global incidence has increased more than 30-fold in the past five decades and chikungunya virus and Zika virus, associated with long lasting chronic symptoms and development of neonatal microcephaly, respectively [14]. Unfortunately, despite all efforts, no clinically effective drug has been approved to treat these diseases [15–17].

In the context of natural bioactive compounds that could be explored as hits for the development of new drug candidates, eugenol (Figure 1) is a phenolic representative substance first isolated by the French chemist Eugène Soubeiran in 1834. Soubeiran was a pioneer in the study and extraction of natural substances, including eugenol, which was isolated from the essential oil of clove (Syzygium aromaticum [L.] Merr. & L.M.Perry), a substance long used for its medicinal properties and aroma [18,19].

Figure 1.

Eugenol structure and its chemical versatility.

The initial method for the separation and identification of eugenol involved the direct extraction from an alcoholic solution of clove and this process was followed by its characterization using liquid chromatography. The identification of eugenol was based on its retention time (5.54 min) [20]. The identification of eugenol was later confirmed by gas chromatography/mass spectrometry analysis [21].

The earliest applications of isolated eugenol were closely linked to its medicinal and aromatic properties. It was widely used in dental treatments as an analgesic and anti-inflammatory agent [22] and employed as a flavoring and preservative compound in food [23].

The discovery of eugenol and the elucidation of its chemical structure were significant milestones in the history of organic chemistry, contributing to the understanding of phenolic compounds and their derivatives. Eugenol remains a compound of great interest due to its medicinal properties, including antibacterial [24], antifungal [25], antioxidant [26] and anti-inflammatory activities [27] in addition to use in the cosmetics and food industries [28].

Eugenol acts through distinct mechanisms against bacteria, fungi, viruses and parasites. However, in fungi and bacteria, most evidence points to an involvement with the structure and functioning of the cell membrane. According to studies carried out by Gupta and co-workers [29], the antifungal effect of eugenol against Candida glabrata may be associated with its ability to increase the generation of reactive oxygen species, as well as alter the ergosterol content in the fungal plasma membrane, in addition to reducing virulence factors of the microorganism. The work carried out by Ahmad and co-workers [30] also indicated that eugenol reduces the biosynthesis of fungal ergosterol, directly impacting the structure of the cell membrane. Naz and her team [31] reported that eugenol is an important enzymatic inhibitor of fungal lipase enzymes, which would ultimately result in a reduction of microorganism’s ability to invade infected tissues. In the work developed by Sen and collaborators [32], it was shown that eugenol suppresses the expression of efflux pumps of Aspergillus fumigatus, which reverses the fungistatic effect of azoles in resistant strains when used in combination with this phenylpropanoid. On the other hand, Ahmad and co-workers [33] demonstrated that eugenol is a good inhibitor of fungal membrane ATPase, which results in intracellular acidification and fungicidal effect. This effect may also be the mechanism by which eugenol exerts an antibacterial effect against Escherichia coli and Listeria monocytogenes [34]. Eugenol appears to influence a wide range of other bacterial cellular functions, as shown in the work developed by Hemaiswarya and Doble [35], which demonstrated that it induces structural damage to the bacterial plasma membrane, in addition to activating lysozyme-mediated autolysis of the microorganism. The prospective in silico investigation developed by Vimal and collaborators [36] has shown that eugenol can inhibit L-asparaginase, an enzyme involved in virulence of Salmonella typhimurium. The experimental antimalarial effect of eugenol may be explained by its action in disrupting the development of the protozoan Plasmodium falciparum during the erythrocytic cycle [37]. Its action on another protozoan, T. cruzi, may be related to its inhibitory effect on galactonolactone oxidase, an enzyme involved in the final stage of vitamin C biosynthesis in the parasite, an essential antioxidant for its survival and multiplication [38].

Eugenol contains important functional groups, each contributing to its chemical and biological properties. The hydroxyl group assists in its antioxidant and antimicrobial properties, mainly by contributing to chemical interactions such as hydrogen bonding; the methoxy group modulates the polarity of this compound in addition to its chemical stability. The allylic chain presents chemical reactivity, besides giving the compound analgesic and anti-inflammatory properties [39]. Eugenol’s structural scaffold allows several strategies for functionalization, coupling and chemical modifications. In view of that many researchers around the world have been employing eugenol and its analogues (e.g., dihydroeugenol and isoeugenol) as attractive starting materials in the synthesis of new compounds aimed at treating fungal, bacterial, viral and parasitic infections, which will be addressed in this review. The synthetic routes for obtaining the described eugenol derivatives are detailed in the Supplementary Material (Supplementary Figures S1–S45), while the structures of the bioactive derivatives with MIC/IC₅₀ values and essential groups important for their bioactivity are illustrated in Figures 2–8.

Figure 2.

Chemical structures of eugenol derivatives 1–18.

Figure 3.

Chemical structures of eugenol derivatives 19–33.

Figure 4.

Chemical structures of eugenol derivatives 34 and 35.

Figure 5.

Chemical structures of eugenol derivatives 36–51.

Figure 6.

Chemical structures of eugenol derivatives 52–66.

Figure 7.

Chemical structures of eugenol derivatives 67–84.

Figure 8.

Chemical structures of eugenol derivatives 85–98.

2. Eugenol derivatives with antifungal activity

Fungal infections are still considered neglected diseases by health agencies. There are more than 1 billion infected people globally with approximately 1.6 million deaths annually and among the 6 million fungi species already known, nearly 600 strains are involved in human diseases [9].

In an attempt to discover new eugenol derivatives with antifungal activity, Carrasco and collaborators synthesized 13 derivatives of eugenol and safrole (Figure 2 & Supplementary Figure S1) and tested them as antifungal against Candida albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes [40]. Derivative 4-allyl-2-methoxy-5-nitrophenol (1), obtained by nitration of eugenol, was the most active compound against all strains evaluated, acting as a fungicidal agent. However, its molecular target is yet to be discovered since it does not act by inhibiting the synthesis or assembly of the cell wall of the fungus.

In another study, Koeduka and collaborators reported the extraction of two major phenylpropene compounds 2 and 3 from Illicium anisatum leaves. They conducted a comprehensive study on their antifungal activity and on mite oviposition, along with other natural phenylpropenes as well as derivatives obtained by semi-synthesis (Figure 2 & Supplementary Figure S2). Although guaiacol and allylbenzene showed no activity, eugenol derivatives and analogues exhibited relevant antifungal activity, with very low toxicity, and O-dimethylallyleugenol (2) exhibited a specific and potent activity against mite oviposition [41].

Souza and collaborators employed the glycosylation strategy to synthesize glycosides from eugenol (glucoside, galactoside and lactoside derivatives) as shown in Figure 2 & Supplementary Figure S3 and evaluated their antifungal activity against five Candida strains. The peracetyl glucoside (4) showed activity against all species with a IC₅₀ value of 3.8 μM against Candida glabrata and selectivity index (SI) of 45.3 considering peripheral human blood mononuclear cells [42].

In an effort to increase the antifungal potential of the glucoside 4, chemical modifications were carried out in its saccharide unit with groups other than acetyl, as shown in Figure 2 & Supplementary Figure S4. Although these modifications did not increase the antifungal potency compared with the precursor 4, the benzyl derivative 5 showed both fungistatic (IC₅₀ 18.1 μM) and fungicidal (IC₅₀ 36.2 μM) activities against C. glabrata. This modified glucoside showed a SI of 32 considering the same cells used for evaluating 4 [43].

Considering these first findings with glucosides derived from eugenol, the authors designed a glucosyl-1,2,3-triazol pattern and synthesized twelve derivatives using other aglycones besides eugenol, as dihydroeugenol, isoeugenol, guaiacol, vanillin and phenol, as shown in Figure 2 & Supplementary Figure S5 [44]. Six nonglycosylated analogues were also synthesized to evaluate the importance of the carbohydrate unit for anti-Candida activity. The peracetylated derivatives 6 and 7 were the most promising ones, being active against four of the five Candida spp. evaluated. Eugenol triazole 6 was more potent against Candida krusei than fluconazole with a IC₅₀ value of 26.1 μM. It was less cytotoxic than the control drug, with a SI of 3.3 considering healthy human lung fibroblast cells. Molecular docking and dynamics studies suggested the fungal 14-lanosterol demethylase (CYP-51) as their possible molecular target. It was observed that the carbohydrate moiety is placed near the entrance of the binding site while the aromatic substituent interacts with the hydrophobic pocket adjacent to the heme group.

In another work, Abrão and collaborators synthesized a morpholine Mannich base from eugenol and its O-acylated derivatives. For evaluating the importance of morpholinyl moiety, they also prepared O-acylated derivatives without the amine unit. The products were obtained according to the synthesis route shown in Figure 2 & Supplementary Figure S6 [45]. When evaluated against species of Candida sp. (C. albicans, C. tropicalis, C. krusei, C. parapsilosis and C. glabrata), the products 8, 10, 13 and 14 showed significant activity in the range of 0.86–13.1 μM. Furthermore, most of the synthesized derivatives showed irrelevant cytotoxicity when tested against mononuclear blood cells. They highlighted the good results obtained with these compounds since Candida infections are of great importance in the epidemiology of fungal infections, including infections caused by non-albicans Candida spp. [46].

Carvalho and colleagues described the synthesis and antifungal evaluation of molecular hybrids formed by merging benzoxazoles and eugenol or dihydroeugenol. The synthesis of the derivatives was designed as shown in Figure 2 & Supplementary Figure S7 [47]. Their antifungal activity was verified by the microdilution method against five species of Candida. Cytotoxicity studies were performed in human peripheral blood mononuclear cells using the MTT method. Compounds 15, 16, 17, 18 showed promising fungistatic activity with IC₅₀ values ranging from 321 to 380 μM. The activity of compounds 16 and 18 against C. krusei stands out, since this species is resistant to several azole antifungals available in clinical medicine. In the cytotoxicity assays, an increase in the SI of the synthesized compounds was observed in comparison to eugenol.

Hipólito and coworkers reported the synthesis of 17 new peracetylated/deacetylated D-glucosides derived from eugenol as new anti-Candida agents, as shown in Figure 3 & Supplementary Figure S8 [48]. The new synthesized derivatives were tested against five Candida sp. and nine compounds were active against at least one strain. The nitro sulfonamide 20 showed activity against all species with MIC (minimum inhibitory concentration) values between 11.4 and 151.84 μM. This peracetylated derivative was active against C. tropicalis with a MIC value of 11.4 μM while against C. krusei, it was almost five-times more potent than fluconazole, with a MIC value of 22.8 μM. The non nitrated analogue 19 showed a MIC value of 12.22 μM against C. krusei, being eight-times more active than fluconazole, and this is an interesting result considering the known fluconazole-resistance by C. krusei [49]. The cytotoxicity of these glucosides was investigated against human mononuclear blood cells and derivative 20 showed selectivity indices of 30 (C. tropicalis) and 15 (C. krusei). Finally, molecular docking studies were conducted with different Candida spp. enzymes involved in ergosterol biosynthesis, pointing squalene oxidase as a potential target for 20 due to the affinity of glucosyl and 4-nitrobenzamide groups for the enzyme’s binding site.

The objective of the work by Pinto and collaborators [50] was to evaluate the antifungal and cytotoxic activity of eugenol, some semi-synthetic derivatives and analogues (Figure 3 & Supplementary Figure S9). All the evaluated compounds exhibited antifungal properties against three dermatophyte species: Trichophyton rubrum, T. mentagrophytes and T. tonsurans. Among these derivatives, methyl isoeugenol (21) completely inhibited the radial growth of the mycelium of the three species after 20 days of treatment at 53.43 and 17.81 μM. O-ethyl (22) and O-butyl isoeugenol (23) also demonstrated interesting results.

In another work, Péret and coworkers described the design of miconazole-based eugenol and dihydroeugenol azoles (imidazoles and 1,2,4-triazoles) as new antifungal agents, which were synthesized as shown in Figure 3 & Supplementary Figure S10 [51]. The antifungal activity of these compounds was evaluated against six Candida and Cryptococcus gattii strains and the cytotoxicity was studied using healthy neonatal rat cardiomyoblasts cells (H9c2 cells). Among the most active azoles, the benzyl dihydroeugenol derivative 24 was active against all species evaluated in the range of 18.8–75.3 μM. The eugenol-imidazole 25 showed a MIC value of 4.6 μM against C. albicans being 32-times more potent than miconazole with no relevant cytotoxicity and a SI greater than 28. Furthermore, di-hydroeugenol-imidazole 26 was twice as potent (MIC 36.4 μM) as miconazole and more than five-times more active than fluconazole against the alarming multi-resistant Candida auris. Additionally, the imidazole derivative 25 decreased ergosterol levels in the C. albicans fungal membrane. Docking studies with fungal 14-lanosterol demethylase (CYP51) showed that the imidazole ring of these active azoles interacts with the heme group and that the chlorinated ring inserts into a hydrophobic cavity at the binding site. This behavior is consistent with the interaction patterns observed with the control drugs miconazole and fluconazole.

Given the antifungal potential of various eugenol derivatives, more specific studies could reveal new hits for development as antifungal drug candidates.

3. Eugenol derivatives with antibacterial activity

A study found that of the 7.7 million deaths worldwide in 2019 due to bacterial pathogens, 1.3 million were directly attributable to antibiotic resistance [8]. Considering these data, the search for new antibacterial compounds is of great urgency in the scientific community.

In order to discover new eugenol derivatives with antibacterial potential, Gaonkar and co-workers synthesized oxazolines from a substituted pyridine-containing oxime and various alkenes, including eugenol and its methoxy and methylenedioxy derivatives (Figure 3 & Supplementary Figure S11) [52]. These compounds were tested against different strains of fungi and bacteria and the eugenol derivative 27 stood out, exhibiting activity in the range of 23–42 μM against the evaluated strains (B. subtilis, E. coli, Pseudomonas fluorescens, Xanthomonas campestris pvs., Xanthomonas oryzae, Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Trichoderma sp., Fusarium moniliforme and Penicillium sp.), demonstrating the biological versatility of the isoxazoline–eugenol scaffold.

In the study conducted by Suresh and co-workers a diverse library of 52 novel aryl ether compounds, including triphenyl ethers and heteroaromatic analogues, were designed in silico using eugenol and dihydroeugenol as the base molecules. They aimed to obtain potent inhibitors of enoyl-acyl carrier protein reductase (ENR), an enzyme encoded by the fatty acid biosynthesis long-chain reductase (FabL) gene of Bacillus anthracis (BaENR). This enzyme has emerged as a significant target due to the increasing virulence and antibiotic resistance exhibited by this pathogen [53]. After obtaining these compounds by synthesis (Figure 3 & Supplementary Figure S12) and evaluating them, they pointed out the dihydroeugenol derivatives 28 and 29 as interesting candidates with MIC values of 0.8 and 0.5 μM, respectively, against the cited enzyme.

Martins and coworkers described the synthesis of twenty-one esters derived from eugenol as shown in Figure 3 & Supplementary Figure S13 and evaluated their activities against Candida sp., Gram-positive and Gram-negative bacteria [54]. Among the active compounds, derivatives 30 and 31 showed IC₅₀ values between 86.5 and 292 μM for the evaluated microorganisms, except against E. coli. The compounds exhibited activity comparable or superior to eugenol.

Rodrigues and collaborators reported an intriguing dimeric structural pattern of eugenol, achieved through oxidative coupling and partial methoxylation (Figure 3 & Supplementary Figure S14). This process resulted in the monomethyl ether and its dimethyl ether derivative. The leishmanicidal potential of these compounds against the promastigote evolutive form of Leishmania amazonensis was evaluated, and derivative 32 was active with IC₅₀ value of 19.13 μM. There was a significant increase in leishmanicidal activity for derivative 33, which showed a IC₅₀ value of 4.85 μM against the parasite [55].

Eugenol was also explored in the context of polymerization, representing an inexpensive biosource for such materials. Based on this application, Modjinou and colleagues prepared a UV-cured network (34) using allyl eugenol combined with eugenol or linalool, an antibacterial monoterpene [56], expecting to enhance their antibacterial activity [57] as shown in Figure 4 & Supplementary Figure S15. For this purpose, they employed a thiol–ene reaction between synthesized allyl eugenol, eugenol (or linalool) with trimethylolpropane tris(3-mercaptopropionate), a trithiol compound, and 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator. The network containing 30% w/w of eugenol exhibited improved anti-adhesion activity against Staphylococcus aureus compared with the pure allyl eugenol network, evidenced by a 57% reduction in the CFU/cm². Additionally, the inclusion of eugenol in the allyl eugenol network improved its antioxidant property from 23% to 90%, as demonstrated in the discoloration assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical. The authors attribute these encouraging results to the presence of free phenol moieties in the network, which serve as hydrogen donors to neutralize free radicals.

A subsequent study further explored the use of eugenol derivatives to produce UV-cured networks with biological activities but now using a different synthetic approach [58]. Networks of resorcinol diglycidyl ether containing 0 to 70% of epoxy eugenol were prepared (35). Epoxy eugenol derivative was previously prepared using 3-chloroperbenzoic acid (MCPBA) and bis(4-dodecylphenyl) iodonium hexafluroantimonate (Deuteron UV 1242) was used as photoinitiator (Figure 4 & Supplementary Figure S16). Similarly, the presence of the eugenol moiety increased the antioxidant activity from 11 to 94% in the co-network prepared from 70% epoxy eugenol. The antibacterial properties were also increased, with a reduction of the bacterial adhesion of 91% for S. aureus with the same co-network. Together, these studies offer significant insights into the development of eugenol-based materials capable of inhibiting bacterial growth and biofilm formation on surfaces, which is of critical importance in environments such as hospitals [59].

In the study conducted by Glaive and colleagues involving sustainable antibacterial and antioxidant networks based on natural products, bio-based release networks incorporating active phenols were prepared by photoinduced thiol–ene reaction, as shown in Figure 5 & Supplementary Figure S17 [60]. The incorporation of 10% by tannic acid into the eugenol-based polymer network (36) resulted in a substantial reduction of 77.9% in the adhesion of Gram-negative bacteria E. coli. The system based on 20% of carvacrol (37) worked as an effective and diffusion-controlled system, as it allowed a complete inhibition of bacterial growth and a reduction in bacterial adhesion of 100 and 98.4% for E. coli and S. aureus, respectively.

In another study carried out by Bansode, pyridine esters of eugenol were prepared as shown in Figure 5 & Supplementary Figure S18 and the products were evaluated against B. subtilis, E. coli, C. albicans and A. niger by disk diffusion method. Three compounds (38–40) showed inhibition zones between 14 and 22 mm at 11.9 μM, an activity superior to the reference drugs ampicillin and fluconazole used as positive controls [61].

Employing the molecular hybridization strategy, Bkhaitan and coworkers prepared two eugenol derivatives linked with metronidazole for biological evaluation against the species of Helicobacter pylori and Clostridium perfringens. The hybrids 41 and 42, ester and ether derivatives respectively, were obtained as shown in Figure 5 & Supplementary Figure S19 [62]. Derivative 41 showed a IC₅₀ value of 21.6 μM against H. pylori P12 and a MIC value of 3.8 μM against C. perfringens. For derivative 42 the IC₅₀ value against H. pylori 26695 was 31.2 μM and against C. perfringens, 1.9 μM.

In the next study [63], the authors explored the biological potential of eugenol and their esters as antibacterial and antifungal agents by modifying the phenolic group to obtain a series of compounds (Figure 5 & Supplementary Figure S20). The derivatives were obtained by classical esterification reaction of eugenol with acyl chlorides and they were evaluated against five bacteria and two fungi strains. The most active derivatives were 43–46, with MIC values in the range of 1.2–1.38 μM. In addition, derivatives 43–46 were active against B. subtilis, and 46 against C. albicans.

Silva and coworkers proposed the synthesis of new eugenol derivatives as potential antibacterial agents, modifying its allyl group, as shown in Figure 5 & Supplementary Figure S21, and evaluated them against Pseudomonas aeruginosa, E. coli, S. aureus, Streptococcus, Klebsiella pneumoniae and Bacillus cereus [24]. As a result, some compounds were active, with inhibition zones between 6 and 20 mm, at 55 μM. The hydroxylated derivative 47 presented an inhibition zone in the range of 10–20 mm for the tested pathogens. In addition, the acyl derivative 48 also stands out as the only derivative in the study to be active against P. aeruginosa, with inhibition zones in the range of 6–12 mm.

Almeida and collaborators carried out the synthesis of three ester derivatives (49–51, Figure 5 & Supplementary Figure S22) and these compounds were evaluated against mycobacteria, Gram-positive and Gram-negative bacteria [64]. The compounds presented MIC values in the range 24–932 μM against Mycobacterium tuberculosis strains and 14.54–1213 μM against M. massilliense, M. fortuitum, M. smegmatis, M. chelonae, M. avium, M. kansasii, M. szulgai and M. gordonae, while eugenol did not show significant activity (MIC: 1523 μM). The substances did not show significant activity (MIC > 3000 μM) against Gram-positive and Gram-negative bacteria species. Furthermore, another work showed the synthesis of compound 50 [65] and its evaluation against Bacillus megaterium, S. aureus, E. coli and Rhodotorula rubra. They found inhibition zones in the range of 24–77.3 mm at 0.02 μM, while for eugenol this range was 1.0–7.2 mm. The authors believe the better activities were due to an increase in the lipophilicity of esters compared with eugenol.

In the work of Bilgiçli and colleagues [66], five oxypropanolamine derivatives containing eugenol proved to be effective in an in vitro antibacterial assay against Acinetobacter baumannii, P. aeruginosa, E. coli and S. aureus, being even better than the standard antibiotic control drugs: ceftazidime (CAZ), ciprofloxacin (CIP), imipenem (IPM), amikacin (AK), gentamicin (CN), piperacillin/tazobactam (TZP) and ampicillin/sulbactam (SAM). These compounds (52–56) were synthesized as shown in Figure 6 & Supplementary Figure S23. They were also effective as inhibitors of the enzymes α-glucosidase, cytosolic carbonic anhydrase I and II (hCA I and II) and acetylcholinesterase (AChE) with EC50 values being respectively 0.98 μM, 2.04 μM, 3.84 μM, 0.81 μM, and 0.90 μM for hCA I; 1.83 μM, 3.84 μM, 4.08 μM, 1.04 μM, and 1.72 μM for hCA II; 8.73 μM, 9.34 μM, 4.71 μM, 11.73 μM, and 16.20 μM for α-glucosidase and 22.05 μM, 48.20 μM, 31.93 μM, 29.94 μM, and 34.04 μM for AChE.

In another study reported by Lapa and coworkers [67], 16 piperazine-based eugenol/dihydroeugenol Mannich bases were obtained and evaluated as antimicrobial compounds against Mycobacterium abcessus, Mycobacterium fortuitum, S. aureus, Staphylococcus epidermidis, K. pneumoniae and P aeruginosa. As results, all compounds showed greater activity than their phenolic predecessors, with emphasis on compounds 57–59 (synthesized as shown in Figure 6 & Supplementary Figure S24), that showed MIC values between 10.91 and 24.14 μM. Furthermore, most of the derivatives were able to inhibit biofilm formation of resistant M. fortuitum at ¼ of the MIC value, showing better results than sulfamethoxazole, which was not able to do the same even in MIC concentrations.

Santos and coworkers designed a series of eugenol 1,2,3-triazoles as new antimycobacterial agents from modifications in its allyl group, as shown in Figure 6 & Supplementary Figure S25 [68]. The antimicrobial activity of these compounds was evaluated against Mycobacterium abscessus, M. massiliense and M. fortuitum while their cytotoxicity was investigated against African green monkey kidney cells (Vero cells). The benzyl derivative 60 was active against M. abscessus at 48.8 μM (SI of 2.4), the galactosyl 61 triazole showed activity against M. massiliense at 31.7 μM (SI of 7.1) and the sulfonate eugenol derivative 62 was active against M. fortuitum with a MIC value of 88.6 μM (SI of 0.6).

Bashyal and collaborators described the biocatalytic synthesis and antibacterial activity of O-glycosides derived from eugenol and isoeugenol. The molecules were obtained from enzymatic catalysis using the Bacillus glycosyltransferase YjiC from Bacillus licheniformis as shown in Figure 6 & Supplementary Figure S26 [69]. These compounds were evaluated against different Gram-positive and Gram-negative strains [70]. Eugenol 63 and isoeugenol 64 glucosides showed MIC values 2 to four-times higher than the starting phenols, evidencing the importance of the saccharide unit for the observed antibacterial activity.

Azevedo-Barbosa and coworkers developed new potentially antibacterial sulfa derivatives using eugenol or dihydroeugenol as part of the structure [71]. The rationale used is that the new substances could be active on the same target enzyme as sulfa drugs (dihydropteroate synthase), in addition to being able to act through other mechanisms of action resulting from the presence of the phenylpropanoid subunit [72]. Using the synthetic route shown in Figure 6 & Supplementary Figure S27, the authors obtained eight new compounds, which were evaluated against Gram-positive and Gram-negative bacteria, in addition to fast-growing mycobacteria. In general, all new compounds showed antimicrobial activity much higher than that of the starting phenylpropanoids. Highlight is given to the derivatives 65 and 66, with a MIC value of 56 μM against Staphylococcus aureus and Staphylococcus saprophyticus while for eugenol this value was 15.2 mM against both bacteria. Additionally, most of the compounds presented poses and interactions like sulfamethoxazole in molecular modeling studies with dihydropteroate synthase, as the hydrogen bonds between the compounds sulfonyl group and the serine-222 residue in the enzyme active site. This suggests that inhibition of this enzyme may be one of the sulfa derivatives mechanisms of action.

Eugenol was also used by Oliveira and coworkers in the preparation of new agents aimed at combating bacteria and mycobacteria strains [73]. These authors relied on the molecular hybridization strategy and rationalized that merging pharmacophore groups from eugenol and the antibiotic chloramphenicol could lead to new entities with superior antimicrobial potential [74]. The four evaluated products (67–70) were synthesized through the synthetic route shown in Figure 7 & Supplementary Figure S28. The antimicrobial activity of the products increased when moving through the series from compound 67 to compound 70, the latter being the one that bears the greatest structural similarity to chloramphenicol. The hybrid 70 showed significant activity (MIC values in the range of 10.5–41.8 μM) against clinical isolates of P. aeruginosa, S. aureus and fast-growing mycobacteria, being in most cases superior to chloramphenicol itself.

With the aim of discovering active compounds as antituberculosis, Ghallab and collaborators relied on the pharmacological potential of Mannich bases to propose a series of new derivatives of this nature from eugenol [75]. In previous works, eugenol proved an interesting compound against M. tuberculosis [64,76]. Here they used different secondary amines and formaldehyde, which resulted in a set of five Mannich bases, synthesized as shown in Figure 7 & Supplementary Figure S29. Although none of the products were more potent than the control drug, isoniazid, two of them (71 and 72) were more active (MIC 45.2 and 40.5 μM, respectively) than eugenol (MIC 121.8 μM) against M. tuberculosis Mtb-H₃₇Rv strain. In molecular docking studies, the substance 72 was shown to be a possible inhibitor of pantothenate kinase enzyme, essential for mycobacteria growth. From the point of view of antioxidant action, Mannich base 73 was the most relevant as a free radical scavenger, although all five were better than eugenol. The authors then point to these substances as interesting candidates to be explored in optimization studies.

Ghallab and colleagues reported the synthesis (Figure 7 & Supplementary Figure S30) and antimycobacterial activity of eugenol and five derivatives as well as their iron chelation capacity [77]. Ether 74 and carbonate 75 showed the best inhibition against M. tuberculosis growth with IC₅₀ values of 3.90 e 2.36 μM, respectively. The propargylic derivative 76 and again carbonate 75 were the best iron chelators, with EC₅₀ values of 0.19 and 0.078 μM, respectively.

Based on these data, it can be concluded that eugenol derivatives may enhance the repertoire of new drug candidates for treating bacterial infections, particularly those caused by resistant species.

4. Eugenol derivatives with antiviral activity

Emerging viral diseases, as COVID-19, and resistance to antiviral drugs, as seen in AIDS therapy, highlight the need for new antiviral drugs. Mosquito-borne arboviruses like dengue, chikungunya and Zika also pose significant health risks, with increasing global incidence and severe long-term effects [11,14,15].

With the aim of discovering new antiviral agents derived from eugenol, Oliveira and collaborators described the synthesis (Figure 7 & Supplementary Figure S31) and antiviral activity of 1,2,3-triazole derivatives from this allylphenol [78]. The antiviral activity of these compounds was initially verified by enzymatic inhibition assays against the NS2B–NS3 protein of West Nile virus. In addition, the antiviral evaluation was performed in vitro against dengue virus and the cytotoxicity of them was investigated against Vero cells [79]. The brominated derivative 77 showed promising antiviral activity, acting as a competitive inhibitor of NS2B–NS3 with an inhibition constant value of 3.06 μM. Furthermore, this triazole was active against the four dengue virus strains in the range of 14.17–70.1 μM assays, showing the antiviral potential of this compound. To perform the molecular docking studies, the crystallographic structure of the NS2B–NS3 protease with code 2JIO obtained from the Protein Data Bank was used. The results showed that the interactions of the compounds with that enzyme possibly occurs through interactions with residues His⁵¹, Thr¹³⁴, Ser¹³⁵ and Tyr¹⁶¹.

Gan and collaborators reported the synthesis of hybrids of the ferulic acid-eugenol type (Figure 7 & Supplementary Figure S32), obtained by aryl ether-type particles and investigation of its antiviral properties against tobacco mosaic virus and cucumber mosaic virus, two common viruses that infect plants [80]. The results of this bioassay were promising, with compounds 78, 79, 80 and 81 showing notable curative, protective and inactivating effects on tobacco mosaic virus and cucumber mosaic virus at 1.39; 1.31 μM; 1.34 μM; 1.21 μM, respectively. The EC50 values for these compounds were higher than those of ferulic acid, eugenol, isoeugenol and ningnanmycin for both evaluated viruses.

Despite efforts, no effective clinical treatments are still available for these infections and some eugenol derivatives showed the capacity to represent new hits for development of antiviral agents.

5. Eugenol derivatives with antiparasitic activity

Neglected tropical parasitosis, like Chagas disease and leishmaniasis, remain major health challenges. Current treatments are often outdated, ineffective against drug-resistant parasites, and associated with high toxicity [11].

In the context of the search for new antiparasitic agents derived from eugenol, Xavier and coworkers synthesized various compounds known as Morita–Baylis–Hillman adducts, which are a type of chemical product formed through a specific reaction involving aldehydes and acids. The work investigated their biological potential as antileishmanial agents. It was demonstrated that the α,β-unsaturated carbonyl and the electron-withdrawing groups linked to the aromatic ring were responsible for the activity optimization of MBHAs [81]. They reported the synthesis (Figure 7 & Supplementary Figure S33) and biological activity of new ortho, meta and para-nitro MBHA hybrids based on the synergistic biological properties of eugenol [82]. The synthesized compounds were more active than eugenol with IC₅₀ values seven-times lower against Leishmania amazonensis. Derivatives 82, 83 and 84 presented IC₅₀ values of 8.75, 10.49 and 4.71 μM, respectively, with the most active compound containing the o-nitro group.

Another research involving eugenol esters was carried out by Raja and coworkers [83] who synthesized thirty-six eugenol derivatives, twenty of which were esters (Figure 8 & Supplementary Figure S34). These compounds were evaluated against promastigote and amastigote forms of Leishmania donovani, in addition to their cytotoxicity in vitro. All the esters showed higher activity than eugenol, which showed IC₅₀ values of 441.66 μM (promastigote forms) and 173.26 μM (amastigote forms). The best results were observed for compounds 85–87, which showed IC₅₀ values in the range of 20.13–33.2 μM for promastigote and EC₅₀ values in the range of 4.25–9.28 μM for amastigote forms. Furthermore, these acylated derivatives presented low cytotoxicity against mouse peritoneal macrophages (EC₅₀ >300 μM), proving to be potential as new leishmanicidal drugs.

In another study, Teixeira and coworkers synthesized twenty-six new 1,2,3-triazole derivatives from eugenol, according to the synthetic route described in Figure 8 & Supplementary Figure S35 [84]. The compounds were designed as anti-Leishmania agents and evaluated against L. amazonensis. Among the active compounds, derivative 88 was active against promastigote (IC₅₀ 7.4 μM) and intracellular amastigote (IC₅₀ 1.6 μM) forms of parasite. The cytotoxicity of this triazole was investigated against healthy peritoneal macrophages (CC₅₀ = 211.9 μM), which resulted in a SI of 132. This compound was more active than both reference drugs employed as positive control: glucantime and pentamidine [85,86]. The authors also reported that the predicted physicochemical and pharmacokinetic properties of this derivative are promising for an effective oral use.

The need to discover potential leishmanicidal agents also led Coelho and coworkers to evaluate the antiparasitic potential of eight esters obtained from the morpholine Mannich base of eugenol, as shown in Figure 8 & Supplementary Figure S36 [87]. In addition to in vitro studies with the cysteine protease rCPB 2.8, an important enzyme active in different stages related to Leishmania sp. [88] infection, molecular modeling studies were carried out targeting this molecular target. Derivative 10 (already synthesized in another study and previously shown in Figure 7) presented the highest inhibitory activity in in vitro studies with the recombinant cysteine protease type B (rCPB 2.8) (88.2% inhibition at 100 μM), besides being the one with the greatest affinity for the active site of this enzyme in in silico studies.

Julianto and colleagues hypothesized that an amino alcohol derived from O-methyleugenol could be an antimalarial candidate agent. They relied on reports of new 1-aryl-3-substituted propanol derivatives as promising antimalarial agents [89,90]. To this find they subjected O-methyleugenol to epoxidation of its side chain with peracetic acid, followed by opening the epoxide with aniline, as shown in Figure 8 & Supplementary Figure S37 [91]. The obtained product was evaluated in the heme polymerization inhibition test, frequently used as an indication of potential antimalarial action. The amino alcohol 89 was able to promote inhibition with an IC₅₀ value of 672 μM, compared with 14.5 mM for the control drug chloroquine.

Functionalization adjacent to the hydroxyl group in eugenol opens the possibility for the synthesis of numerous heterocycles. For instance, Sharma and colleagues synthesized a series of 1,3-benzoxazines derived from eugenol and isoeugenol, which exhibited activity against P. falciparum [92]. The cyclization reaction occurred via a Mannich condensation, adhering to the principles of green chemistry [93]: a one-pot reaction with atom economy, solvent-free, producing only water as a byproduct, and utilizing starting materials derived from natural sources, including furfurylamine, derived from agricultural byproducts such as corn cobs. The scheme for the synthesis of the 1,3-benzoxazines is illustrated in Figure 8 & Supplementary Figure S38 [94,95]. Benzoxazines 90 and 91 were active against trophozoites of P. falciparum strain 3D7, with EC₅₀ values of 22 and 17.54 μM, respectively. Furthermore, they showed no cytotoxicity against human cells (HepG2) when tested at 50 μM. An increase in Na⁺ levels accompanied by swelling of the parasites was observed in treated parasites, suggesting disruption of sodium homeostasis as the mechanism of action. Docking studies on a homology model of P falciparum ATPase 4 (PfATP4), a sodium homeostasis regulator, reinforces this hypothesis as the most active compound showed stronger binding affinity then eugenol (-8.4 vs -6.2 kcal/mol).

Coupling of eugenol via Povarov reaction with aryl imines derived from isatin could afford spiroindolones with antiplasmodial activity [96]. The Povarov reaction is a multicomponent reaction involving the condensation of an aldehyde/ketone with an aniline, followed by coupling with an activated olefin to yield a tetrahydroquinoline [97]. Mathebula and colleagues employed this aza-Diels-Alder transformation with various alkenes, including isoeugenol, to synthesize a series of spiroindolones with antiplasmodial activity (Figure 8 & Supplementary Figure S39). Among these, only the isoeugenol derivatives (93, 95, and 96) showed antiplasmodial activity against the drug-sensitive strain 3D7, with IC₅₀ values ranging from 1.48 to 1.80 μM. The spiroindolone 94 was only active against the drug-resistant strain FCR-3 (IC₅₀ 4.20 μM). Remarkably, compounds 92, 95 and 96 showed broad-spectrum efficacy, being active against both drug-sensitive and drug-resistant strains, with IC₅₀ values of 2.28, 1.67 and 1.52 μM, respectively (values for FCR-3 strain). Despite the fact that none of them was more active than positive control chloroquine (FCR-3 IC₅₀ 0.11 μM), these findings highlight the potential of the isoeugenol-derived spiroindolone promising antiplasmodial scaffold.

In another work, Souza e coworkers synthesized new 1,2,3-triazoles derived from eugenol and dihydroeugenol as shown in Figure 8 & Supplementary Figure S40 [98]. The phenyl derivatives 97 and 98 were active against epimastigote forms of T. cruzi in preliminary in vitro assays, showing IC₅₀ values of 88.4 and 42.8 μM, respectively. In in vivo studies using infected rats with Y strain of T. cruzi, the triazole 98 (100 mg/Kg) reduced more than 50% of parasitemia. The cytotoxicity of these compounds was evaluated against healthy cardiac cells from neonatal rats (H9c2 cells) and the SI observed were 1.6 (for 97) and 3.6 (for 98). Furthermore, docking studies suggested these compounds could inhibit cruzain, an essential enzyme for T. cruzi metabolism. The main interactions observed between these triazoles and cruzain active site involve the residues GLY 23, CYS 25, TRP 26, SER 64, GLY 65, GLY 66, LEU 67, MET 68, LEU 160, HIS 162 and GLY 163.

Employing the triazole 98 as a hit compound, Reis and colleagues synthesized new 1,2,3-triazoles modifying different regions in this compound, such as the propyl, hydroxyl and phenyl groups, as presented in Figure 9 & Supplementary Figure S41 [99]. The dihydroeugenol derivatives 99 and 100 were the most promising in in vitro assays against epimastigote and tripomastigote forms of T. cruzi (IC₅₀ values of 19.7 and 20.7 μM for 99; and 7.3 and 8.4 μM for 100, respectively). These compounds were selected for in vivo studies using infected rats with T. cruzi and nitro derivative 100 showed very interesting results, since it did not present toxicity by the evaluation of behavioral and physiological parameters, mortality, effect in body weight gain and through the measurement of liver biomarkers aspartate transaminase and alanine aminotransferase enzymes. The triazole 100 also reduced 99.4% of parasitemia and myocarditis in infected animals at 100 mg/Kg.

Figure 9.

Chemical structures of eugenol derivatives 99–108.

Pelozo and collaborators proposed the synthesis of new trypanocidal agents through molecular hybridization of metronidazole with eugenol and analogues using two strategies: a coupling using a triazole ring as a connector (Figure 9 & Supplementary Figure S42) and a classical direct etherification (Figure 9 & Supplementary Figure S43). The trypanocidal activity of these compounds was evaluated against epimastigote and trypomastigote forms of Trypanosoma cruzi. Regarding the first strategy (Figure 9 & Supplementary Figure S42) and considering epimastigote forms, the hybrids 101 (IC₅₀ value of 52 μM) and 102 (IC₅₀ 52 μM) presented the most promising results [100]. When evaluated against trypomastigote forms of parasite, the eugenol derivative 101 (IC₅₀ 10 μM) showed a similar trypanocidal activity to the reference benznidazole (IC₅₀ 7 μM). Regarding the direct etherification strategy (Figure 9 & Supplementary Figure S43) and considering epimastigote forms, the analogue 103 (IC₅₀ 24.2 μM) presented a trypanocidal activity similar to the reference benznidazole (IC₅₀ 21.5 μM). Comparing the activity against trypomastigote forms of these hybrids with benznidazole (IC₅₀ 5.1 μM) the dihydroeugenol derivative 103 (IC₅₀ 1.3 μM), 104 (IC₅₀ 7.1 μM) and 105 (IC₅₀ 7.3 μM) showed the most promising results [101,102].

Reis and coworkers used eugenol and dihydroeugenol in the preparation of α,β-unsaturated ketones with potential activity against T. cruzi [103]. The authors relied on previous studies by Braga and coworkers [104] and Alkhaldi and coworkers [105] that showed the antiparasitic potential of other α,β-unsaturated ketones and of phenylpropanoid derivatives [98]. The nine ketone derivatives from these phenylpropanoids were synthesized as shown in Figure 9 & Supplementary Figure S44. Derivative 106, obtained from dihydroeugenol was the most active against amastigote forms of T. cruzi, with an IC₅₀ value in the nanomolar range (5.2 nM), which represents a potency 700-times greater than that of the control drug, benznidazole. The authors highlight the importance of the cyclic ketone subunit in 106, as the acyclic ketone analogue was 20,000-times less active.

The isoxazoline heterocycle was another structure pattern explored in eugenol chemistry which showed different promising biological activities. For instance, in a recent study Rani and colleagues discovered eight antileishmanial compounds with IC₅₀ values lower than eugenol [106]. In order to obtain a series of isoxazolines, they reacted oximes prepared from aldehydes bearing different substituents in the aromatic ring with acetylated or benzylated eugenol, in a 1,3-dipolar cycloaddition with chloramine-T (Figure 9 & Supplementary Figure S45). Derivatives 107 and 108 were the most potent against Leishmania donovani promastigotes, with IC₅₀ values of 13.0 and 7.5 μM and SI of 6.3 and 9.2, respectively, considering J774A.1 macrophages. Additionally, the compounds were equally active against intracellular amastigote forms, with IC₅₀ values of 11.6 and 5.8 μM. Mechanistic studies on treated parasites revealed increased reactive oxygen species production, reduced intracellular ATP levels, disruption of mitochondrial membrane potential, increased formation of acidic vesicular organelles and accumulation of lipid bodies.

Given the antiparasitic potential of various eugenol derivatives, these compounds could undergo more advanced studies to identify new potential candidates for clinically useful drugs.

6. Conclusion

The strategy of exploring eugenol to develop new derivatives has shown significant potential in combating a wide range of microbial and parasitic infections. The synthetic strategies suggested the importance of structural modifications in enhancing their efficacy against bacterial, fungal, viral and protozoal pathogens. As evidenced by the diverse biological activities observed with these compounds, from antimicrobial to antiparasitic properties, there is clear promise for further development in drug discovery. However, most of the studies presented herein still lack robust investigations on the mechanisms of action for the reported eugenol derivatives and analogues.

7. Future perspective

Future research directions should prioritize the refinement of these eugenol derivatives and the development of innovative analogues, particularly focusing on the underexplored eugenol allyl chain. It is crucial to conduct comprehensive and rigorous clinical studies to effectively translate these promising findings into therapeutic agents capable of addressing the persistent challenges posed by infectious diseases. With the growing resistance of various microbial and parasitic species against the existing drugs, there is an urgent need for new substances that can serve as prototypes for potential antimicrobial and antiparasitic treatments. Novel bioactive compounds hold significant value as therapeutic candidates, not only because they have the potential to overcome resistance issues but also because they can target mechanisms that current drugs do not address. By continually advancing research and innovation in this area, we can maximize the therapeutic potential of eugenol and its derivatives, ultimately contributing to improved global health outcomes.

Funding Statement

This work was supported by PROPP/UFOP, FAPEMIG (APQ-00686-18; APQ-00352-18, APQ-00544-23, APQ-01455-24, RED-00110-23, APQ-00490-22 and APQ-00975-21) and CNPq (403194/2023-7; 405032/2021-8). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2419376

Financial disclosure

This work was supported by PROPP/UFOP, FAPEMIG (APQ-00686-18; APQ-00352-18, APQ-00544-23, APQ-01455-24, RED-00110-23, APQ-00490-22 and APQ-00975-21) and CNPq (403194/2023-7; 405032/2021-8). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.