Part1: Tailored Functionalization Of Natural Phenols To Improve Biological Activity

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Abstract: Phenols are widespread in nature, being the major components of several plants and essential oils. Natural phenols' anti-microbial, anti-bacterial, anti-oxidant, pharmacological, and nutritional properties are, nowadays, well established. Hence, given their peculiar biological role, numerous studies are currently ongoing to overcome their limitations, as well as to enhance their activity. In this review, the functionalization of selected natural phenols is critically examined, mainly highlighting their improved bioactivity after the proper chemical transformations. In particular, functionalization of the most abundant naturally occurring monophenols, diphenols, lipidic phenols, phenolic acids, polyphenols, and curcumin derivatives is explored.

Keywords: carvacrol; thymol; eugenol; resveratrol; hispolon; hydroxytyrosol; lipidic phenols;phenolic acids; polyphenols; curcumin

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1. Introduction

Natural phenols, mainly of vegetable origin, are receiving increasing attention, as insight into their biological activity increases.

In recent years, many reviews appeared about phenolic profiles of plants and/or essential oils, evidencing anti-microbial, anti-bacterial [1-4], antioxidant [5-10], as well as pharmacological [11-18] and nutritional [19-21] properties, together with a very in-forming book [22]. In view of their importance, studies were aimed at breeding plants able to increase the content of bioactive phenols [23]. The research in the field continues, and more and more plants are investigated for their phenolic content and related bioactivity [24-41]. The antioxidant activity of natural phenols has been related to their scavenger ability towards free radicals [42]. Particularly interesting is the possibility to encapsulate phenols—as well as other natural compounds—in chitosan biopolymers [43], or in β-cyclodextrin [44].

It must be noticed that the application of modern extractive techniques [45-52] makes the determination of phenolic compounds in plant matrices more accessible and complete.

New applications of natural phenols in different fields are reported in fish aquaculture [53], sports performances [54], fish gelatin, and gelatin from bovine skin modification by cross-linking with natural phenolic acids[55,56]. Advanced extraction technologies allowed the use of phenolic extracts from some plants for food preservation [57-60]. Moreover, technological applications are becoming available, such as anti-bacterial films based on cellulose/phenolic species |61l, antimicrobials packaging films based on nano-encapsulation of bioactive oils through emulsion polymerization [62], fire-resistant phenolic foams [63], and natural fiber-reinforced composites with lignin phenol binder [64].

Though outside the scope of the present review, it is worth signaling the use of natural phenolic compounds as building blocks to obtain functional materials [65] or as antioxidants for biodiesel [66].

With so much information collected and available, the next step was the effort to understand the structural factors responsible for bioactivity, examining the structure-activity relationship of antioxidant phenolic compounds [67,68].

From the chemical point of view, it may be interesting to look for chemical derivatization of natural phenols leading to eventually enhanced biological activity. As a matter of fact, treatment with diazomethane of phenolic extracts led to derivatives more suitable as antioxidants for lipophilic foods [69]. Considering the importance for human health, representative methods to chemically modify the natural phenols were discussed [70, as well as reviews of enzymatic modification [71] and of metabolic engineering for microbial biosynthesis of natural compounds, among which phenols, were reported [72].

In the present review, we aim to give a general picture of the situation, reporting chemically modified natural phenols and comparing their performances with those of the parent compounds. The number of isolated and bioactive natural phenols is huge and ever-increasing, so our attention is mainly focused on the most abundant ones in nature Moreover, phenolic polymers are not discussed since they deserve a separate review, considering their growing importance. Literature published from 2000 to the beginning of 2021 is considered.

2. Monophenols

Monophenol functionalization is attracting the interest of a growing number of researchers since the synthesis of new biologically active derivatives starting from natural compounds is a proficient tool to improve their properties. In fact, tailored functionalization is a valuable strategy to overcome natural phenol weaknesses such as toxicity, low water solubility, as well as to mild their strong fragrances, which often limit their application [73-78].

As an example, the antioxidant activity of tyrosol (2-(4-hydroxyphenyl)-ethanol), which is an abundant phenol in olive oil, responsible for oil beneficial properties [79], can be sensibly enhanced through the esterification of the alcoholic hydroxyl group with different phenolic acids (Scheme 1)[80]. Analogously, hydroarylation with cinnamic esters improves the antioxidant properties of tyrosol, especially in the presence of additional hydroxyl groups in the aromatic ring of the acidic moiety (Scheme 1) [81].

Nevertheless, considering their abundance in nature, we examine in detail the functionalization of carvacrol, thymol, and eugenol, since they are amongst the most widespread phenols in nature, usually responsible for beneficial plant properties.

2.1. Car~acrol

Carvacrol(5-isopropyl-2-methyl phenol)is a phenolic monoterpenoid compound, and it is a major component of oregano and thyme essential oils. Together with its isomer, thy-mol (2-isopropyl-5-methyl phenol), it is the main active ingredient responsible for essential oils' biological activity [82-84]. In fact, carvacrol's peculiar antibacterial, anti-fungal, anti-inflammatory, anxiolytic and anticancer activities are currently well established, and the FDA (Food and Drug Administration) has approved its use as an additive in food products.

Nonetheless, the research of new carvacrol analogs is currently inspiring several research groups, with the aim to extend the potential application of the compound [85]. Carvacrol functionalization usually occurs at the -OH moiety; indeed, a wide variety of synthetic carvacrol esters can be found in the literature. Obviously, through phenol esterification, variegated functionalized products can be accessed [86], to be explored in several areas. As an example, carvacrol acetate showed significant anti-inflammatory 87 anti-nociceptive [87], anti-oxidant [88] and anti-fungal[89] effects. It can also be used in the treatment of anxiety disorders [90 and as an acaricidal agent against Rhipicephalus micro plus, a dangerous cattle tick that is causing important economic losses in the cattle industry [91,92]. Similarly, carvacrol propionate, obtained by carvacrol esterification with propionyl chloride in the presence of triethylamine (TEA), showed higher analgesic, anti-inflammatory, and anti-hyperalgesic effects compared to pure carvacrol [93]. Interestingly， esterification with the Boc-protected γ-amino butanoic acid（GABA）， which is the primary inhibitory neurotransmitter of the central nervous system, has been performed with N, N'-dicyclohexylcarbodiimide (DCC), and 4-dimethyl aminopyridine (DMAP) in dichloromethane (DCM)[94,95]. The corresponding ester, obtained upon Boc removal in acid conditions, is a suitable drug for different pharmacological applications. In fact, it can modulate the transient receptor potential (TRP) channels and bind GABA receptors, thus exerting high analgesic and anti-inflammatory effects. In addition, carvacrol esters having hydroxy-substituted cinnamic acids are efficient tyrosinase inhibitors [96].

However, it is worth mentioning that esterification is not always a successful strategy to obtain highly effective derivatives. In fact, the antibacterial activity of carvacrol against S.mutans, S.aureus, B.subtilis, S.epidermidis, and E.coli was reduced upon esterification with different alkyl- or aryl-based acyl chlorides [97]. Similarly, several attempts have been performed to further improve carvacrol activity against Mycobacterium tuberculosis chorismate mutase enzyme: acetylation or etherification of a the-OH group, or introduction of different substituents (-Cl,-Br,-NO2)on carvacrol aromatic rings led to unsatisfactory antitubercular activity [98].

On the contrary, a number of carvacrol and 4-bromocarvacrol esters with furan, thiophene, and pyridine have been synthesized and screened as antifungal agents(Scheme 2)[991.

The different heterocyclic units sensibly influence carvacrol activity: esters with acids of furan and thiophene are more active than carvacrol against R.solani, while pyridine esters of 4-bromocarvacrol exhibited an enhanced antifungal activity vs. P.oryzae.

Carvacrol sulfonate esters, obtained by treating carvacrol with trichloromethyl hypo chlorothionite(ClSCCl3)in the presence of TEA, are remarkable antibacterial agents, being 40 times more effective than carvacrol toward S.epidermidis, and 8 times more active toward P. aeruginosa [100]. Moreover,4-chlorocarvacrol, obtained through the oxychlorination of carvacrol in acetic acid, with LiCl and CuCl2 catalyst, under O2 atmosphere, showed good activity against several bacterial strains. In particular, it is much more effective than its precursor against P. aeruginOSa [101].

Recently, twenty different amino acid ester prodrugs of carvacrol were synthesized, with the aim to improve carvacrol's solubility in the water while preserving its antimicrobial properties[102]. CAR-1 is highly effective to inhibit C.albicans growth, while C. tropicalis and C.glabrata were successfully inhibited by CAR-2(Scheme 3). Importantly, CAR-1 and CAR-2 did not prove to be cytotoxic at the adopted concentrations.

Similarly, ten carvacrol codrugs, obtained through carvacrol esterification with sulfur-containing amino acids, have been synthesized [103]. Even though such compounds showed reduced toxicity with respect to carvacrol, their antimicrobial activity was poorer. However, CAR-3 (Scheme 4)is more effective than the corresponding free phenol in affecting E.coli mature biofilm. In fact, carvacrol conjugation with Ac-Cys(Allyl)-OH is crucial to promote the permeabilization and the destabilization of the bacterial membrane, thus ensuring reduced biofilm formation. Pharmacokinetic studies also revealed good stability of CAR-3 at stomach pH, in the presence of pepsin and pancreatin, suggesting that after oral administration, CAR-3 can cross the stomach, and it can be absorbed from the intestine, releasing carvacrol after enzymatic hydrolysis.

A more advanced approach was related to the carvacrol anchoring on a gold surface, to develop antimicrobial coats[104]. Indeed, carvacrol functionalization at the phenolic group was performed to obtain a carvacrol ester and an ether, with a-NH, terminal group (Scheme 5). The latter could be covalently attached to a properly modified gold surface. Thus, the antifungal activity of carvacrol functionalized Au surfaces was evaluated against C.albicans and more than 75% of inhibition was observed for the ester derivative, while 65% inhibition was reached with the ether one. Noteworthy, the fungicidal activity was maintained after being stored for one month at 4°C.

Next to carvacrol ester derivatives, ethers have also been extensively studied over the years to implement carvacrol applications [105,106]. In particular, several carvacrol ethers have been explored in the treatment of H.pylori bacterial infection and as antiproliferative agents against human gastric adenocarcinoma cell lines, with promising results [107]. Similarly, a metronidazole carvacrol ether derivative has shown remarkable activity against two strains of H.pylori and one strain of Clostridium perfringens(Scheme 6)[108].

Carvacrol propyl, butyl, octyl, and benzyl ethers demonstrated the ability to reduce fertility and viability of the fruit fly Drosophila melanogaster after oral administration or inhalation exposure [109]. Moreover, different alkyl 4-oxobutanoate p-substituted carvacrol ethyl ethers have been synthesized (Scheme 7) and screened as tyrosinase inhibitors, which are valuable molecules in medicine, agriculture, and cosmetics because of their ability to control melanin overproduction [110]. Data showed that synthetic ethers were more effective in inhibiting tyrosinase with respect to the parent compound.

Docking studies demonstrated that carvacrol derivative CAR-4(Scheme8) is a promising anti-malaria agent [111]. In particular, CAR-4 interacts with amino acid residues in the binding pocket of the protease of P.falciparum parasite, a common target for anti-malarian drugs. Therefore, CAR-4 was synthesized starting from carvacrol and propargyl bromide in the presence of K2CO3. The resulting alkyne was reacted with p-methoxyphenylazide in the presence of a Cu(I)-salt and sodium ascorbate in THF/H2O2, to form the [3+2]cycloaddition product (Scheme 8). CAR-4 showed high anti-malarian activity, with an IC50 value of8.8 μM.In vivo tests showed significant parasite reduction up to 8 days， making CAR-4 a potential lead against the target protease.

Oxypropanolamine derivatives of carvacrol were tested to evaluate their application in different diseases (Scheme 9)[113].In particular, their inhibitory effects towards different types of carbonic anhydrase, α-glycosidase, and acetylcholinesterase enzymes have been evaluated and results showed a very good inhibition effect, even higher than that of reference compounds. Therefore, such synthetic carvacrol derivatives can be further exploited as diuretics, antiepileptics, anti-glaucoma, anti-diabetic and anti-inflammatory agents in the treatment of gastric and duodenal ulcers, and in neurological disorders, such as Alzheimer's disease.

3-Fluorophenyl carbamate derivative of carvacrol(CAR-5, synthesized from the reaction between carvacrol with3-fluorophenyl isocyanate in DCM, Scheme 10)is 130-fold more active compared to carvacrol in acetylcholinesterase inhibition and 400-fold more efficient in inhibiting butyrylcholinesterase, with negligible cell death [114]. More interestingly, a series of carvacrol amide derivatives have been screened against acetylcholinesterase and butyrylcholinesterase enzymes [115]. The carvacrol derivative modified with a quinoline moiety (CAR-6, Scheme 10)is 149-fold more effective than carvacrol in inhibiting acetyl-cholinesterase and more than 8000-fold more efficient for butyrylcholinesterase inhibition. The higher activity compared to carvacrol was related to the presence of the heterocyclic aromatic quinoline core that can interact with the amino acid residues in the active site of the enzyme through T-7T interactions.

Sulfonic acid-functionalized carvacrol, synthesized through the electrophilic aromatic sulfonation reaction with concentrated H2SO, and the corresponding potassium salt, are less effective anti-bacterial agents with respect to carvacrol, but their remark-able water solubility and reduced odor allow their use in the food industry for preservation of foodstuffs and for a shelf-life increase [116]. A series of interesting differently substituted carvacrol analogs, such as sulfonate esters [117](obtained by reaction with ethane sulfonyl chloride or aryl sulfonyl chloride in dichloromethane, in the presence of TEA), dihydroxy-[118], acetohvdrazone-[119], hydrazone-, sulfonyl hydrazone-[120], and hydrazide-based sulfonamide-[121] carvacrol derivatives have been synthesized and screened for their antimicrobial, antioxidant, and anti-cancer activities. Results suggest that the synthesized compounds exhibit very promising biological properties in the studied fields, even though their efficiency was not directly compared with that of carvacrol. In a recent paper, carvacrol has been successfully coupled with phthalocyanines[122]:3-nitrobenzene-1,2-dicarbonitrile was initially reacted with carvacrol and then the resulting compound was subjected to macrocyclization under MW irradiation, to obtain the corresponding phthalocyanine, CAR-7 (Scheme 11).

The photodynamic antibacterial activity of the synthesized phthalocyanine was evaluated: upon excitation with light, the carvacrol-substituted phthalocyanine showed an increased photoinactivation at 100 uM with respect to the sole zinc(I)phthalocyanine. Lower dark toxicity was observed in comparison to pure carvacrol, likely because of the lower bacterial membrane penetration of bulk phthalocyanine with respect to carvacrol. Nonetheless, the lower photostability of the conjugate was observed [122].

2.2.Tlymol

Next to carvacrol, its isomer, thymol, is widely used as an antibacterial, antifungal, antioxidant, and anti-inflammatory active ingredient in several products, as well as a food preservative [84,123].

Indeed, several natural and synthetic thymol derivatives have been proposed over the years to further broaden their application at the industrial level[124-126].

Quite a few thymol derivatives have been synthesized and evaluated for different biological purposes [86,127-130]. Thymol functionalization through esterification or etherification reactions constitutes one of the most useful approaches to access a wide library of different bioactive molecules. Thymol esterification usually occurs in classical conditions, reacting thymol with the appropriate anhydride or acyl chloride in the presence of a base. MW-assisted procedures in an aqueous medium have also been proposed, to perform reactions in reduced times and with improved yields [131].

Thymol acetylation has been widely studied, since the product, i.e., thymol acetate, is more effective than thymol against plant pathogenic fungi, such as A. solani, B.cinerea, P. grisea, and R. solami[89]and Gram-positive bacterial strains such as S.mutans, B.subtilis and S.epidermidis[97]. Higher or equal activity with respect to thymol was assessed for Gram-negative E.coli, S. Typhimurium, P. aeruginosa, and K.pneumonia [132]. Similar enhancements in anti-bacterial action have been achieved with thymol propanoate and methyl propanoate derivatives [97], while thymol esterification with heteroaromatic carboxylic acids led to efficient anti-fungal compounds[99]. Moreover, in a screening of different synthetic thymol esters and ethers, thymol benzoateexhibited the highest larvicidal potency on Aedes aegypti, which is a dangerous mosquito and vector of dengue fever and other diseases in the world [133]. Importantly, thymol protection through esterification proved to be effective to reduce thymol toxicity. In fact, thymol acetate and benzoate are promising candidates as antileishmanial drugs, being less toxic and more active than thymol against the parasite Leishmania infant umchagasi [134]. Thymol acetylation has been considered effective also in the treatment of gastrointestinal nematode infection of small ruminants because of the reduced toxicity of the ester compared to the parent compound, even though thymol acetate was actually less effective than thymol in vitro studies [135].

Acrylate derivatives of thymol have been synthesized according to a multi-step process (Scheme 12) [136].

Thymol esterification with acrylic acid in the presence of DCC and a catalytic amount of DMAP in DCM was performed. The obtained product was reacted with nitro-substituted benzaldehyde in dry acetonitrile, with the nucleophilic catalyst 1,4-diazabicyclo[2.2.2]octane (DABCO). Reactions proceeded with good yields, and a product with higher antileishmanial activity than thymol against Leishmania amazonensis was obtained [136].

Promising results have also been achieved with the conjugation of nonsteroidal anti-inflammatory drugs (NSAIDs)to thymol, to prevent adverse gastrointestinal mucosal reactions, which are typical side effects related to long-term use of NSAIDs[137-140]. The formation of gastric ulceration related to NSAID therapy is usually due to the local generation of reactive oxygen species(ROS); thus, the introduction of antioxidant components in NSAIDs'structure can limit such undesired effects. Accordingly, thymol esterification product with indomethacin, etodolac, and tolfenamic acid showed retention of pharmacological activity with respect to the parent drug and significant reduction in ulcerogenic side effects of the corresponding NSAID [137]. Similarly, thymol has been included in ketoprofen drug (2-(3-benzoylphenyl)propanoic acid), through a glycolic acid spacer(Scheme 13) [139].

Modified ketoprofen showed better analgesic and anti-inflammatory activities and reduced gastrointestinal toxicity, thus demonstrating the great advantage in using such prodrugs for chronic inflammatory disorders treatment.

Thymol conjugation with diacerein (1,8-diacetoxy-3-carboxyanthraquinone), an anthraquinone derivative used as an antiarthritic, moderate anti-inflammatory, antipyretic, and analgesic drug, has been exploited. Diacerein linkage to thymol through esterification with DCCimproved lipophilicity and bioavailability of the drug, while decreasing gastric irritant effect and enhancing anti-inflammatory activity [140].

Because of their already known antioxidant and mushroom tyrosinase inhibitory activity, substituted benzoic acids and cinnamic acids bearing a thymol moiety have been synthesized in order to discover new effective tyrosinase inhibitors [141-144]. To this aim, thymol esterification has been carried out, with properly substituted benzoic or cinnamic acids, in the presence of TEA (Scheme 14).

Among the tested compounds, derivatives possessing 4-hydroxyl substituted cinnamic acid are the most active ones, having the maximum binding affinity with the receptor protein [141,144]. Thus, the synthesized derivatives may serve as lead structures for developing even more effective tyrosinase inhibitors.

2-Isopropoxy-1-isopropyl-4-methylbenzene, obtained by thymol etherification with 2-chloropropane in the presence of TEA in diethyl ether, exhibited an enhanced antibacterial activity with respect to thymol against E.coli, S.typhimurium, S.aureus, P. aeruginosa, and K. pneumonia[132]. Interesting thymoloxypropanolamine derivatives showed powerful antibacterial activity on different Gram-negative and Gram-positive bacteria, as well as good inhibition of some metabolic enzymes, such as human carbonic anhydrases isoenzymes I and I, α-glycosidase, and acetylcholinesterase [145]. Enhancements of the thymol biological activity have also been detected with thymol glucosides.

Glycosylation is a versatile method that allows us to improve the hydrophilicity of organic compounds, also widening their pharmacological applications. Several thymol glucosides derivatives have been synthesized and tested over the years [146-148]. Notably, the in vitro antifungal activity of 2-isopropyl-5-methylphenyl-4,6-di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-2-enepyranoside(THY-1),2-isopropyl-5-methylphenyl-2,3-dideoxy-α-D-erythrohex-2-enepyranoside (THY-2) and 2-isopropyl-5-methylphenyl-23-dideoxy-α-D-erythrohexanopyranoside(THY-3)(Figure 1)has been evaluated, and larger inhibition zones and lower MIC were achieved against A. flavors, A.ochraceus and F.oxysporum compared to thymol. Thus, because of the improved hydrophilicity, next to the biological activity, glucosides thymol derivatives can be suggested as antifungal agents in food systems [149].

The antioxidant activity of a series of heterocyclic sulfide derivatives of thymol has been recently exploited [150]; they were prepared following a multi-step procedure (Scheme 15), where the natural phenol was firstly subjected to methylation reaction with cesium carbonate and methyl iodide in DMF. To note, DMC can efficiently replace CH3I in thymol methylation reaction [151]. Next, Friedel-Crafts acylation of the methyl-protected thymol with chloroacetyl chloride led to 2-chloro-1-(5-isopropyl-4-methoxy-2-methylphenyl)Ethan-1-one in 48% yield. Nucleophilic substitution of Cl- was then performed, with the proper heterocyclic aromatic thiols, in the presence of potassium carbonate and potassium iodide in acetonitrile, to obtain the desired thymol derivatives.

Synthesized compounds showed good antioxidant activity, and docking studies on tyrosinase revealed higher affinity with respect to thymol and the reference compound (kojic acid) toward the binding site of the enzyme. In particular, the oxadiazole derivatives presented the largest binding affinities with the enzyme because of favorable H-bond interactions with amino acid residues in the active site [150].

Recently, new thymol sulfonamide derivatives have been prepared. The synthetic pathway firstly requires diazonium salt synthesis from the aromatic amine, and then electrophilic substitution on thymol aromatic ring, in basic solution. The conjugate thymol-sulfadiazine derivative is the most antibacterial active one, showing inhibitory activity against S.aureus and E.faecalis [152]. Differently functionalized thymol derivatives have been obtained, such as thymol-based paracetamol analogs [153] or aryl-azo-substituted thymol [154], as well as N-methylcarbamate derivatives [155,156] and they showed good anti-oxidant and anti-microbial activities. A 1,3,5-triazine piperazines thymol derivative displayed very interesting therapeutic perspectives as a drug against memory and cognitive impairment,i.e., Alzheimer's disease and dementia, having good pharmaceutical and safety profiles in vitro[157]. Increased antibacterial and antifungal activity has been observed for thymol pyridazine [158] and thymol pyridine [159] derivatives, with respect to thymol, while 2-(4H-12,4-triazole-3-yl)thioacetamide thymol derivatives exhibited promising anti-cancer activity [160]. Mannich bases of thymol were investigated as carbonic anhydrase inhibitors, showing moderate activity [161].

Thymol-based substituted pyrazolines and chalcones have been tested against human malaria parasite strain Plasmodium falciparum activity [162]. The proposed synthetic pathway to access such bioactive compounds firstly requires the synthesis of 3-isopropyl-4-methoxy-6-methylbenzaldehyde. Chalcones are then obtained through Claisen-Schmidt condensation of the aldehyde with different acetophenones in methanol, with KOH ex-cess. The reaction of the thymol-based chalcones with diisopropyl azodicarboxylate (DIAD)in the presence of PPh, and toluene afforded the functionalized pyrazolines, under MW irradiation, with good yields (Scheme 16).

The synthesized compounds showed enhanced anti-malarian activity with respect to thymol, and in particular, chalcones THY-4 and THY-5 and pyrazoline THY-6 exhibited the highest activity against human malaria parasite P. falciparum, being much more effective than the parent compound [162].

Different studies also demonstrated that halogenation is a proficient strategy to enhance thymol biological activity. Yet, thymol chlorination affords 4-chloroethyl as the main product [163,164], which is up to six times more active than thymol against S.aureus, S. epidermis, and different C.albicans strains [163]. More interestingly, thymol bromination in mild conditions leads to 4-bromothymol [165-167, which is a very effective antimicrobial active compound [168]. In fact, its activity is up to 15 times stronger than that of the parent compound, against several bacterial and fungal strains pathogenic for humans and animals. Thus, 4-bromothymol sustainable synthesis was the object of several studies[167], and biocompatible drug delivery methods have been also developed to study the potential application of such an interesting antimicrobial compound for topical applications in cosmetics [151]. 

2.3. Eugenol

Eugenol (4-allyl-2-methoxyphenyl) is the major component of clove essential oils, but it can be also found in minor amounts in cinnamon, clover pepper, and other plants. It is used in perfumeries for its pleasant fragrance, as a flavoring agent in foods, as antiseptic and disinfectant in dental products, and in many other fields [169]. Eugenol can be readily functionalized through the chemical transformation of the phenolic-OH group (mainly via the classical etherification and esterification reactions)[170-176], on the aromatic ring (through nitration reaction or Mannich bases formation)[177-180], as well as on the allylic functionality, through epoxidation [175] (Figure 2).

Thanks to its highly versatile structure, several eugenol derivatives have been synthesized for different biological purposes in the past ten years [181,182]. Moreover, eugenol can be used as a scaffold to synthesize biologically active natural products [183,184]. The possibility to introduce eugenol skeleton in complex structures such as phthalocyanines [185], platinum(II) complexes [186], and organic antimicrobial polymers [187I has been explored, obtaining new interesting biologically active species (Figure 3).

Alkyl and aryl eugenol esters have promising anti-inflammatory agents for skin inflammation [188], anti-oxidants[189], as well as effective antibacterial and anti-fungal compounds[190]. In particular, different eugenolesters with high anti-oxidant activity have been synthesized for application in cosmetics. Results showed that after esterification, skin penetration of the active compounds was increased; therefore, eugenol ester derivatives could explicate their anti-oxidant activity in the deeper layers of the skin [191]. Eugenol tosylate derivatives have also been synthesized by reaction with different sulfonyl chlorides in the presence of pyridine. The obtained tosylates are effective inhibitors for Candida albicans [192-194].

Eugenol esterification has been also performed with aspirin (acetylsalicylic acid, previously activated with SOCl, to form the corresponding acyl chloride). The obtained ester is a very promising compound, having fewer toxic effects than aspirin and eugenol [195]and showing interesting therapeutic effects [196-198]. In fact, it is an anti-inflammatory and antipyretic drug, with stronger and longer effects than its precursors, likely indicating a synergistic effect between the two moieties [195]. Moreover, eugenol esterification with ibuprofen led to a prodrug with retention of anti-inflammatory activity and minimized gastrointestinal toxicity [199].

Eugenol epoxidation at the allylic position, followed by ring-opening with different nucleophiles, gives access to a wide library of eugenol derivatives that have been tested as carbonic anhydrase, acetylcholinesterase, and α-glycosidase inhibitors, with good results (Scheme 17)[200,201. Oxypropanolamine derivatives, obtained by ring-opening with amines, showed antibacterial activity on Gram-negative (A.baumani, P. aeruginosa, and E. coli) and Gram-positive(S.aureus)bacteria [202].

Numerous eugenol derivatives bearing triazole functionalities have been successfully accessed through the"click chemistry" approach (Figure 4, Scheme 18). Several examples are present in the literature about () O-alkylation of eugenol with terminal alkynes, followed by reaction with different benzyl azides [203-205];(ii) eugenol conversion in epoxide and ring-opening to obtain the corresponding alkyl azides, followed by reaction with different alkynes [206];(ii) hydroboration oxidation at the allylic position of eugenol, followed by methylation and oxidation reactions, to achieve the eugenol azide; then, reaction with phenylacetylene to afford the triazole. Importantly, the first step of this latter process requires-OH protection through silylation, thus allowing the synthesis of variously substituted products (Scheme 18) [207].

Synthesized eugenol triazole derivatives showed leishmanicidal [205], antimycobacterial [207], trypanocidal [206],anticancer [203] as well as protease inhibitory [204] activity. Triazole eugenol glucosides also showed significant bactericidal activity and low toxicity to normal cells [208].

A series of hydrazones of eugenol has been recently synthesized by condensation of a eugenol hydrazide with various aromatic aldehydes or ketones (Scheme 19) [209]. All the obtained hydrazones showed a promising antitubercular activity, measured by in vitro antimycobacterial activity test against M. tuberculosis. Docking studies revealed that the hydrazone eugenol derivative EUG-5 interacts with the active site amino acid residues of the target enzyme, through the amino and phenyl functionalities.

Great attention has been recently devoted to new eugenol glucoside derivatives. Here, the synthesis is generally performed through a nucleophilic substitution reaction between the phenol group of eugenol and α-D-tetra-O-acetylglucopyranosyl bromide [210-212]. Some of the obtained derivatives showed strong anti-bacterial[211]and anti-fungal activity, mainly against different Candida species [210,212,213].