Part2: Tailored Functionalization Of Natural Phenols To Improve Biological Activity

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3. Diphenols

Natural diphenols, including catechol, resorcinol, and hydroquinone derivatives, are widespread in nature, being commonly found in several vegetables and fruits. Such natural compounds are usually characterized by peculiar anti-oxidant and anti-inflammatory activity. Some of them have immunomodulatory and anticancer active ingredients. Therefore, natural diphenols are often used as scaffolds to prepare new efficient biologically active drugs. Although there is a widespread presence of bioactive diphenols in nature, in this re-

view, the attention is dedicated to the tailored functionalization of resveratrol, hispolon, and hydroxytyrosol, which constitute abundant and highly active natural phenolic compounds.

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3.1.Resveratrol

Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol) is a natural phenolic stilbene, commonly found in several plants and fruits such as apples, berries, pomegranates, pistachios, as well as in the seed and skin of grapes. Thus, red wine is one of the highest sources of resveratrol [214]. Several studies are currently ongoing on resveratrol and its antibacterial [215], antiviral [216], and antitumoral [217-219] activities. Moreover, its applications in the treatment of diabetic nephropathy [220] and skin disorders [221] have been reviewed. The benefits associated with resveratrol, along with some adverse effects, mainly related to its potential cytotoxicity, have been recently highlighted [222]. Moreover, resveratrol's rapid metabolism leads to low bioavailability of the active compound. Such issues, together with the low water solubility, constitute an important limitation to be considered. Therefore, to improve bioavailability, several resveratrol delivery systems have been proposed, in order to extend the potential biomedical applications of such an interesting natural product [223].

Due to the significant attention on this active compound, several papers on its functionalization have been recently published; additionally, different reviews on the pharmacological and biological activity of resveratrol analogs and a dedicated Special Issue on its functionalization were published in 2017 [224].

Just to mention some examples (Figure5), natural and synthetic resveratrol derivatives and oligomers are effective antibacterial [225] and antiviral [226] agents; di-, tetra-, and hexahydroxy derivatives, as well as di-, tri-, tetra-, and Penta methoxy analogs exhibited higher bioavailability and biological activity with respect to resveratrol[219,225-227]; modification of resveratrol with carboxy ester, acetal, sulfonate, phosphate, carbonate, carbamate and alkyl groups allowed the modulation of resveratrol water solubility, bioavailability, absorption from the gastrointestinal tract and biological properties [228-230]. Resveratrol modification on the aromatic ring has also been investigated, leading to lipophilic derivatives, with anti-oxidant and neuroprotective activity [229]. To improve resveratrol anticancer activity, methoxy, hydroxyl derivatives, as well as other functional groups or heterocyclic esterification have been examined [231,232].

3.2. Hspolon

His polon (6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-diene-2-one) is a natural phenolic compound, extracted from the medicinal mushroom Phellinus linteus. It is characterized by peculiar antioxidant activity as well as important pharmacological properties, being a promising anticancer, antidiabetic, antiviral, and anti-inflammatory agent [233-235]. Recently, several hispolon derivatives have been submitted to in silico predictions to preliminarily evaluate their anticancer activity. The theoretical analysis confirmed that aromatic ring substitution with methoxy and hydroxy groups delivers new his polon analogs with good antiproliferative activity, sometimes even higher than that of his polo itself[236,237]. In fact, the hispolon derivatives dehydroxyhispolon methyl ether and hispolon methyl ether exhibited higher in vitro cytotoxicity than hispolon against colorectal cancer[238,239]. The latter was up to5 times more effective toward colon and prostate cancer cell lines[239]. Dehydrohispolon is a promising antitubercular agent showing lower MIC against M.tuberculosis with respect to his polon [240]. Hispolon and hispolon methyl ether pyrazole derivatives (Scheme 20)showed improved stability with respect to their precursors as well as antigenotoxic effects against radiation exposure [241].

The pyrazole derivative is also more effective than hispolon for scavenging N· and CClO,·radicals [242]. A family of palladium(II) complexes with hispolon analogs have been recently synthesized and they showed higher in vitro cytotoxicity than the corresponding free ligands against different cancer cell lines. In particular, Pd-complexes with methoxy-substituted hispolon analogs (Scheme 21)showed improved activity compared to the corresponding hydroxy compounds [243].

3.3. HydroxcytyroOsol

Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol) is a secondary plant metabolite and it is present in many olive products, being responsible for most of their benefits for human health. The promising pharmacological activities of hydroxytyrosol (as cardioprotective, anticancer, neuroprotective, and antimicrobial agent) have been recently reviewed [79.244]. Analogously, hydroxytyrosol acetate (2-(3,4-dihydroxyphenyl)ethyl acetate), a phenolic constituent of olive oil, is characterized by peculiar biological activities [245-248]. Several hydroxytyrosol derivatives have been explored over the past decades, to extend their biological applications [249-252]. However, the most common functionalization strategies involve esterification or etherification at the alcoholic-OH group or a substitution on the aromatic ring [253].

As an example, to broaden usage in foods and cosmetics, antioxidant lipophilic hydroxytyrosol esters have been synthesized through esterification at the alcoholic-OH, with different fatty acids [254-257], or through the chemoselective transesterification procedure [258]. Interestingly, decanoate and dodecanoic hydroxytyrosol esters resulted in good anti-trypanosomal and anti-leishmanial agents, active against T. brucei and L. donovani, respectively [259]. Hydroxytyrosol antioxidant activity was enhanced upon esterification to the corresponding butyrate [260], fenofibrate [261], and nicotinate [262] esters(HT-1 and HT-2 respectively, Scheme 22), the latter also being a potent α-glucosidase inhibitor.

Similarly, a polyacrylate polymer bearing hydroxytyrosol in its side-chain exhibited antioxidant and antimicrobial activity against Staphylococcus epidermidis [263].In addition, hydroxytyrosol phosphodiesters (Scheme 23) are promising antioxidant agents, suitable for the prevention or therapy of Alzheimer's disease [264].

Likewise, hydroxytyrosol alkyl ethers are characterized by interesting biomedical properties. In fact, hydroxytyrosol hexyl ether showed antiangiogenic [265] and anti-platelet [266] effects, while the ethyl ether exhibited intestinal anti-carcinogenic activity [267], as well as high anti-oxidant properties when added to commercial olive oils [268]. Moreover, the pharmacological potential activity of hydroxytyrosol glycosides has been recently explored; hence, neuroprotective hydroxytyrosol glycosides have been synthesized through a sustainable enzymatic reaction, using a fungal β-xylosidase as the catalyst to perform the regioselective trans-xylosylation reaction with xylobiose [269].

Nitrohydroxytyrosol, synthesized by the reaction of hydroxytyrosol with sodium nitrite in acetate buffer (pH 3.8), and nitro hydroxy tyrosyl ester derivatives(namely acetate, butyrate, hexanoate, octanoate, decanoate, laurate, myristate, and palmitate)have been successfully obtained in good yields [270]. NO,-hydroxytyrosol showed improved antioxidant activity with respect to the parent compound, while the activity of the ester derivatives was highly dependent on acyl chain length, but good effects were detected with acetate and butyrate. Similarly, the antioxidant activity of alkyl nitro hydroxy tyrosyl ethers were maintained or even improved for ethyl and butylethers, while, with longer side chains, it was reduced with respect to hydroxytyrosol [271].

The synthesis of alkyl carbonate derivatives of hydroxytyrosol is strategic to increase hydroxytyrosol antioxidant activity [272]. Carbonate derivatives have been achieved by a multi-step process in which the chloroformate hydroxytyrosol derivative (having protected phenolic groups)was reacted with alkyl alcohols or diols of different chain lengths [272,273](Scheme24). Hydroxytyrosol carbonates also exhibited higher anti-trypanosome activity against Trypanosoma Brunei with respect to the precursor [273].

4. Phenolic Acids

Phenolic acids are hydroxy or methoxy derivatives of benzoic acid or of cinnamic acid(3-phenylpropanoid acid). They are diffused in many plants(for example, they are among the most abundant natural antioxidants of virgin olive oil [274]). The more frequent phenolic acids are summarized in Figure 6.

Occurrence, biological and pharmacological functions of monocyclic phenolic acids were reviewed, evidencing their wide distribution and variety of functions [275-277]together with their promising therapeutic applications [278-282]. As an example, the development of gallic acid derivatives as pharmacological agents [283,284]was recently discussed. Similarly, features and potential application of ferulic acid derivatives[285-289] and natural and synthetic products derived from caffeic acid [290] were reviewed.

Therefore, the appealing properties of phenolic acid derivatives are unambiguously affirmed. Nevertheless, in the following, the most interesting derivatives of natural phenolic acids are discussed.

4.1.Caffeic Acid

Caffeic acid (3,4-dihydroxybenzoic acid) can be found in coffee, wine, and tea. It is characterized by an antioxidant, anti-inflammatory, and anticarcinogenic activity. Esters of phenolic acids have been of significant interest, and the most interesting biological properties and synthetic methods of caffeic acid phenethyl ester and derivatives have been reviewed [291]. Indeed, caffeic acid phenethyl esters are among the small molecules with anti-inflammatory activity for the treatment of acute lung injury [292]. Non-natural biosynthetic pathways were tailored incorporating caffeic acid into various aromatic alcohol or amine. The new platform permitted the bacterial production of a library of caffeic acid-derived phenethyl esters or amides in Escherichia coli[293](Scheme 25).

Caffeic acid enzymatic esterification with phenethyl alcohol in the presence of Novozym 435 in isooctane at 70°C has been exploited [294]. The enzyme maintained more than 90%of its original activity up to the third run. It is worth signaling that the enzymatic synthesis of caffeic acid phenethyl ester was successfully ultrasound-accelerated [295]. Similarly, the transesterification of methyl caffeate to caffeic acid phenethyl ester analogs was performed with Candida Antarctica lipase B, using the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide as the solvent [296].2-Cyclohexylethyl caffeate and 3-cydlohexylpropyl caffeate exhibited strong antiproliferative activities. Alkyl esters of caffeic acid with 2-8 C atoms were obtained from caffeic acid and the proper alcohol in the presence of DCC [297]. A screening for their antifungal activity was carried out against Candida albicans, with the best antifungal performances shown by propyl caffeate. Other alkyl esters of caffeic acid(alkyl =methyl, ethyl, butyl, octyl, benzyl, and phenethyl) were prepared to reflux caffeic acid with alkanol in acetyl chloride [298]. In vitro and in vivo experiments confirmed the anti-inflammatory efficiency of the esters.

Twenty-one caffeic acid phenethyl ester derivatives were synthesized, characterized, and investigated for their cytoprotective effects [299]. Some of them showed stronger cytoprotective activities than the parent phenethylester, so the authors suggested a potentiality as functional food ingredients for the prevention of neurodegenerative diseases.

Caffeic acid and its phenyl propyl ester were investigated for their ability to suppress the proliferation of human colorectal cancer cells both in vitro and in vivo[300], resulting in potent anti-cancer agents.

Twenty ester derivatives of caffeic acid were synthesized to investigate the inhibitory activities against the nitric oxide production induced by lipopolysaccharides [301]. All of them showed inhibitory activity.

Semi-synthetic esters derivatives of caffeic acid with a triazole moiety have been designed as potential 5-lipoxygenase inhibitors [302].5-Lipoxygenase is involved in the biosynthesis of leukotrienes, the well-known inflammatory mediators with implications in different diseases (asthma, allergic rhinitis, cardiovascular diseases, and certain types of cancer). Structure-guided drug design delivered compounds with excellent 5-lipoxygenase inhibition, especially compounds reported in Scheme 26 that showed enhanced activity compared to caffeic acid.

Amides have been chosen as bioactive derivatives of caffeic acid: several alkyl and aryl amines were used to prepare antioxidant caffeic acid derivatives [303]. Anilides of caffeic acids were found to be efficient as lipid peroxidation inhibitors.

Since some plant-derived polyphenols were found to exert antiviral effects against the influenza virus, inhibiting the viral surface protein neuraminidase, a library of caffeic acid-based compounds was built, forming variously substituted amides [304]. The amides presented a moderate activity on neuraminidases and the compound evidenced in Figure7 is the most promising one.

Amides were prepared from caffeic acid and the heteroaromatic amine tacrine with alkyl spacers of different lengths (Scheme 27)[305]. All the tacrine derivatives have much more antioxidant efficiency than caffeic acid. In particular, the compound with a3 Clinker and a Cl atom in the aromatic ring was the more selective one, since it had a potent neuroprotective effect, inhibiting β-amyloid aggregation. These properties make the amide a good candidate for the development of beneficial therapeutic tools in Alzheimer's disease treatment.

A number of acyl hydrazides of caffeic acid were prepared and investigated for anti-inflammatory, analgesic, and ulcerogenic activities [306]. Caffeic acid was tert-butylated in position 5 of the aromatic ring 307]. Both caffeic and t-butylated caffeic acids showed strong antioxidant capacity during squalene peroxidation under direct UVA irradiation, but only the latter could reduce the generation of reactive oxygen species. 

4.2.Ferulic Acid

Ferulic acid (4-hydroxy-3-methoxy cinnamic acid) is an effective antioxidant compound, typically found in seeds, leaves, as well as in plant cell walls. Several ferulic acid derivatives have been recently explored in different areas. Lipases from Candida Antarctica (Novozyme 435), Candida rugosa, Chromobacterium viscosum, and Pseudomonas sp. were used to catalyze transesterifications of vinyl ferulate with hydroxyl steroids and arbutin [308](Scheme 28). Arbutin ferulate possesses a 19% higher antiradical activity than ferulic acid.

Other enzymatic esterification reactions allowed the synthesis of ethyl ferulate from ferulic acid and ethanol, using Novozym 435 as the biocatalyst, which could be reused eight times before significant loss of activity [309]. Lipase-catalyzed preparation of mono-and diesters of ferulic acid was reported 310]. In fact, the exploitation of the antioxidant capacity of ferulic acid is limited by its scarce solubility in hydrophobic media such as fats and oils. A strategy to maximize the therapeutic benefits is to transform the acid into esters [31l](Scheme 29). Once prepared, esters were evaluated for in vitro antioxidant potential. Moreover, molecular docking indicated that ferulic esters inhibit target proteins in breast cancer and in oxidative stress.

Alkyl esters and amides of ferulic acid were prepared and evaluated for their anti-cancer activity [312]. According to the authors, all the synthesized amides showed good cytotoxic activity, a characteristic attributed to lipophilicity. Several amides of substituted ferulic acids were also prepared from O-acetyl feruloyl chloride with amines [313]. Most of the amides exhibited significant promotion of insulin release from rat pancreatic cells.

A number of amides were prepared from ferulic acid and other phenolic acids (Scheme 30), with the aim to evaluate their antioxidant activity [314]. The results of four different in vitro tests indicated a very high antioxidant activity, especially when-OH or -OMe groups were present in the amide.

Several ferulic acid amides and amides of the corresponding 3-(4-ethoxy-3-hydroxy)phenylpropanoid acid were synthesized and tested for antiviral and insecticidal activity, against the tobacco mosaic virus[315]. Most of the examined amides presented not only good antiviral activity, inhibiting the plant viral infection, but also insecticidal efficiency against the insect vectors, thus helping in the prevention of virus spreading in the crops (Figure 8).

Amides of hydrogenated ferulic acid,3-(4-hydroxy-3-ethoxy)phenylpropanoid acid, were synthesized(Scheme 31)[316]. Some of them displayed excellent protection and curative activity against the tobacco mosaic virus.

The feruloyl and phenethyl groups were combined to synthesize the ester phenethyl trans-3-(4-hydroxy-3-methoxyphenyl)acrylate and the amide trans-3-(4-hydroxy-3-methoxyphenyl)-N-phenethylacrylamide [317]. Phenethyl ester and amides were studied for bioactivity as anticancer agents, proving more effective than the parent compound [318].

Many other amides of ferulic acid were prepared under solvent-free conditions by MW-assisted condensation reaction [319]. Twenty-one amides showed noticeable in vitro anticancer activity, nine presented in vitro free radical scavenging activity higher than that of ferulic acid.

Hexyl caffeate and ferulate, as well as caffeovlhexylamide and ferulovlhexylamide, were tested in human breast cancer cell lines [320]. Differently from parent compounds, the new compounds inhibited cell proliferation and induced cell cycle alterations and cell death.

Twenty-seven ferulic acid derivatives, with halogens-acetanilide as novel surface recognition moiety, were prepared with the aim to obtain compounds able to act as histone deacetylase inhibitors. The latter class of compounds was important for cancer therapy [321]. Synthesized compounds evidenced at the bottom of Scheme 32 exhibited significant enzymatic inhibitory activities.

Notably, the geranylated derivative of ferulic acid, 3-(4O-geranyloxy-3-methoxyphenyl)-2-propenoate resulted in more activity than the parent ferulic acid as an inhibitory agent on aberrant crypt foci, reducing the incidence of adenocarcinomas in the colon [322].

4.3. Miscellanea

Methyl, ethyl, propyl, and butyl esters of sinapic acid(4-hydroxy-3,5-dimethoxycinnamic acid, a bioactive natural compound found in fruits, vegetables, and medicinal plants), pre-pared by acid-catalyzed esterification of alcohols, presented almost the same antioxidant activity, which is slightly lower than that of sinapic acid [323]. However, they have the advantage of higher lipophilicity, which is valuable for antioxidants designed to act as membrane protectors in vivo.

Alkyl esters of protocatechuic and gallic acids (3,4-dihydroxybenzoic acid and 3,4,5-trihydroxy benzoic acid, respectively), prepared reacting the acid with different alkanols in presence of DCC, were found to inhibit HIV-1 protease dimerization [324], with efficiency depending on the length of the alkyl chain. In fact, no inhibition was observed with alkyl chains with less than eight carbon atoms. Protocatechuic acid and its ethyl and heptyl esters were investigated for photoprotective activity [325]. The photoprotection and antiaging activity were higher with the esters than with the parent acid, probably because of their higher lipophilicity. Unfortunately, cytotoxicity also increased.

Alkyl esters of gallic acid were prepared by the classical Fisher esterification, from acid and alcohols in an acid medium [326]. Besides anti-cancer, antiviral and antimicrobial properties, they are able to scavenge and reduce reactive oxygen species formation. Additionally, gallic acids esters are potential inhibitors of metastasis [327]. The propyl ester of gallic acid was transformed into the acyl hydrazide that, upon reaction with the appropriate chalcone, was transformed into pyrazole derivatives of gallic acid [328]. The compounds were screened for their anti-inflammatory activity, with good results in some cases.

New syringic hydrazones from the aldehyde related to syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) and substituted hydrazines (with electron-withdrawing or electron attracting groups)[329] were efficient against the oxidative stress.