**Preface to "Coumarin and Its Derivatives"**

Coumarins are widely distributed in nature and can be found in a large number of naturally occurring and synthetic bioactive molecules. Their unique and versatile oxygen-containing heterocyclic structure makes them a privileged scaffold in medicinal chemistry. The large conjugated system, with electron-rich and charge-transport properties, is important for the interaction of this scaffold with other molecules and ions. Therefore, a great number of coumarin derivatives have been designed, synthetized, and evaluated on different pharmacological targets. In addition, coumarin-based ion receptors, fluorescent probes, and biological stains are growing quickly and have extensive applications to monitor timely enzyme activity, complex biological events, as well as accurate pharmacological and pharmacokinetic properties in living cells. The extraction, synthesis, and biological evaluation of coumarins have become extremely attractive and rapidly developing topics. Research articles, reviews, communications, and concept papers focused on the multidisciplinary profile of coumarins, highlighting natural sources and the most recent synthetic pathways, along with the main biological applications and theoretical studies, constitute this book.

> **Maria Jo˜ao Matos** *Editor*

### *Editorial* **Coumarin and Its Derivatives—Editorial**

**Maria João Matos 1,2**


Coumarins are widely distributed in nature and can be found in a large number of naturally occurring and synthetic bioactive molecules [1]. The unique and versatile oxygen-containing heterocyclic structure makes them a privileged scaffold in Medicinal Chemistry [1]. The large-conjugated system, with electron-rich and charge-transport properties, is important for the interaction of this scaffold with other molecules and ions [1]. Therefore, many coumarin derivatives have been extracted from natural sources, designed, synthetized, and evaluated on different pharmacological targets [2]. In addition, coumarinbased ion receptors, fluorescent probes, and biological stains are growing quickly and have extensive applications to monitor timely enzyme activity, complex biological events, as well as accurate pharmacological and pharmacokinetic properties in living cells [3]. The extraction, synthesis, and biological evaluation of coumarins have become extremely attractive and rapidly developing topics. A large number of research and review papers compile information on this important family of compounds in 2020 [3]. Research articles, reviews, communications, and concept papers focused on the multidisciplinary profile of coumarins, highlighting natural sources, most recent synthetic pathways, along with the main biological applications and theoretical studies, were the main focus of this Special Issue.

The anticoagulatory activity of coumarins is one of the most classic applications of this family of compounds, acenocoumarol and warfarin being the most important approved drugs. The use of one or another depends on different factors. However, the real evidence on their different results is not completely clear. Therefore, the clinical results for both molecules were studied on 2111 MPHV patients included in the nationwide PLECTRUM registry [4]. In addition, the antiplatelet aggregation profile of coumarin, esculetin and esculin, were determined by studying cyclooxygenase I (COX-I) inhibition [5].

Inflammation is another area of constant interest. Hydroxycoumarins are on the top of the list, 4-hydroxy-7-methoxycoumarin being described as an inhibitor of inflammation in LPS-activated RAW264.7 macrophages by suppressing the nuclear factor kappa B (NFκB) and MAPK activation [6]. This simple coumarin reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), being non-cytotoxic for different cell lines. Moreover, this molecule decreased phosphorylation of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK), but not that of p38 MAPK [6]. In addition, coumarins have been described as anti-inflammatory and antioxidant compounds with a potential action in inflammatory bowel disease [7]. These molecules display a protective action in intestinal inflammation by modulating different mechanisms and signaling pathways, mainly modulating immune and inflammatory responses, and protecting against oxidative stress.

Neurodegenerative diseases are another classical application of coumarins in drug discovery. The design of new hybrids, especially looking for a multitarget function, is a trend strategy. Coumarin-chalcone hybrids have been described as potent and selective

**Citation:** Matos, M.J. Coumarin and Its Derivatives—Editorial. *Molecules* **2021**, *26*, 6320. https://doi.org/ 10.3390/molecules26206320

Academic Editor: Thomas J. Schmidt

Received: 13 September 2021 Accepted: 15 October 2021 Published: 19 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

monoamine oxidase B (MAO-B) inhibitors [8]. A series of fourteen new derivatives were described, an IC<sup>50</sup> in the nanomolar range presenting the best compound. Theoretical approaches corroborated the interaction and selectivity of these compounds for the B isoform. Coumarin-chalcone hybrids also attracted the attention by being adenosine receptor modulators [9]. This family of G-protein-coupled receptors (GPCRs) is especially important in neurological and psychiatric disorders such as Parkinson's and Alzheimer's diseases, epilepsy, and schizophrenia. The studied series proved to be interesting for the design of potent and selective human A<sup>1</sup> or A<sup>3</sup> ligands. In general, molecules bearing hydroxy groups showed more A<sup>1</sup> affinity, while the methoxy counterparts showed A<sup>3</sup> selectivity. On the other hand, extracts from plants and their isolated compounds are also being used as inhibitors of enzymes involved on neurodegenerative diseases. Coumarin glycyrol and liquiritigenin, isolated from *Glycyrrhiza uralensis*, were the most promising molecules [10]. The first one proved to inhibit butyrylcholinesterase (BuChE), acetylcholinesterase (AChE) and MAO-B in the micromolar range, being reversible and noncompetitive inhibitors of BuChE. The second one proved to be reversible and competitive with MAO-B inhibitor in the nanomolar range. Finally, curcumin–coumarin hybrids have been also described as multitarget agents against neurodegenerative disorders [11]. From the studied series, most of the 3-(7-phenyl-3,5-dioxohepta-1,6-dien-1-yl)coumarins proved to be moderate inhibitors of *h*MAO, AChE, and BuChE, also displaying antioxidant activity (scavenging DPPH free radical). Two compounds out of this series also showed neuroprotective activity against hydrogen peroxide (H2O2) in the SH-SY5Y cell line. The formulation of these derivatives in nanoparticles improved this last property.

Anticancer activities for coumarins have been also reported. Coumarin-3-carboxamide derivatives have been reported, and 4-fluoro and 2,5-difluoro benzamides presented activities against HepG2 and HeLa cancer cell lines comparable to doxorubicin, exhibiting low cytotoxicity against LLC-MK2 normal cell line [12]. From the combination of simple coumarins (osthole, umbelliferone, esculin or 4-hydroxycoumarin) with sorafenib, an antiglioma compound was also reported by studying human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells lines [13].

Psoralen derivatives with electrophilic warhead variations at position 3 have been described for their immunoproteasome inhibitory activity [14]. The studied compounds proved to be slightly less active inhibiting the β5i subunit of immunoproteasome than the previously reported 7*H*-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead. These results allowed to establish important structure–activity relationships that will guide the design of potent and selective immunoproteasome inhibitors.

As said before, several coumarins are naturally occurring molecules. Therefore, there is intensive research on plants and extracts analysis. Sixty coumarin derivatives from *Artemisia capillaris* were studied for their constitutive androstane receptor (CAR) activation [15]. Amongst all the molecules studied in the in vitro CAR activation screening, 6,7-diprenoxycoumarin proved to be the most interesting for further studies. A review paper on the natural occurrence, biosynthesis, and biological properties of two 3-prenylated coumarins has been described [16]. A dihydrofuranocoumarin (chalepin) and furanocoumarin (chalepensin) are in the focus of this overview. They were isolated from the first time from the medicinal plant *Ruta chalepensis* L. (Fam: Rutaceae) but are also present in species of the genera Boenminghausenia, Clausena, and Ruta. These two natural products have been described for their anticancer, antidiabetic, antifertility, antimicrobial, antiplatelet aggregation, antiprotozoal, antiviral, and calcium antagonistic properties. The same group focused a second review on the natural origin, biosynthesis, and pharmacological activities of tetracyclic 4-substituted dipyranocoumarins, the calanolides [17]. Ultra-high-performance liquid chromatography coupled with a mass spectrometry (UHPLC-MS) methodology has been used for identifying and quantifying coumarins from a group of twenty-eight plants (roots and leaves) from Arabidopsis natural populations [18]. Simple coumarins such as scopoletin, umbelliferone and esculetin, along

with their glycosides scopolin, skimmin and esculin, respectively, have been identified. Finally, the ability of different coumarins to inhibit quorum sensing when combined with small plant-derived molecules identified in various plants extracts has been described [19].

The development of new chemical tools and strategies to obtain different coumarins, and the update of the traditional ones, are a continuous field of research. Chiral tertiary amine catalyzed asymmetric [4 + 2] cyclization of 3-aroylcoumarins with 2,3-butadienoate has been described [20]. Two reviews on the synthetic strategies to obtain coumarin(benzopyrone) fused five-membered aromatic heterocycles built on the α-pyrone moiety, one centered on five-membered aromatic rings with a single heteroatom and the other one with multiple heteroatoms, have also been published [21,22]. New 3-ethynylaryl coumarin-based dyes for DSSC applications were included in this monographic issue [23]. The synthetic pathways, spectroscopic properties and theoretical calculations were included. The structural characterization (UV-Visible spectroscopy, thermal analysis by differential scanning calorimetry and TGA, <sup>1</sup>H NMR and X-ray diffraction) of mono and dihydroxylated umbelliferone derivatives has been also described [24]. 3-Carboxylic acid and formyl-derived coumarins have been proposed as photoinitiators in the photo-oxidation or photo-reduction processes for photopolymerization upon visible light [25]. These characteristics are related to the potential of these molecules in the photocomposite synthesis and 3D printing applications [25]. Finally, *in silico* tools (i.e. MetFrag, SIRIUS version 4.8.2, CSI:FingerID and CANOPUS) have been used for the structural elucidation of ferulenol, synthetized by engineered *Escherichia coli* [26]. This study highlights the importance of 4-hydroxycoumarins as lead molecules for the chemical synthesis of several bioactive compounds and drugs.

The huge and growing range of applications of coumarins described in this Special Issue is a demonstration of the potential of this family of compounds in Organic Chemistry, Medicinal Chemistry, and different sciences related to the study of natural products. This Special Issue includes 24 articles: 18 original papers and 6 review papers. The versatility of this scaffold is also being demonstrated by the number of manuscripts revealing and highlighting its potential. Based on the current results, it may be expected that the utility of coumarins as scaffolds for drug design, as structures for chemical synthesis and as fluorescent probes, may grow in the next years. Finally, it seems that simple coumarins are still more explored than complex derivatives.

**Author Contributions:** Writing, review, and editing, M.J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.J.M. would like to thank Fundação para a Ciência e Tecnologia (FCT, CEECIND/02423/2018 and UIDB/00081/2020).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


### *Review* **Trending Topics on Coumarin and Its Derivatives in 2020**

**Aitor Carneiro <sup>1</sup> , Maria João Matos 1,2, Eugenio Uriarte 1,3 and Lourdes Santana 1,\***


**Abstract:** Coumarins are naturally occurring molecules with a versatile range of activities. Their structural and physicochemical characteristics make them a privileged scaffold in medicinal chemistry and chemical biology. Many research articles and reviews compile information on this important family of compounds. In this overview, the most recent research papers and reviews from 2020 are organized and analyzed, and a discussion on these data is included. Multiple electronic databases were scanned, including SciFinder, Mendeley, and PubMed, the latter being the main source of information. Particular attention was paid to the potential of coumarins as an important scaffold in drug design, as well as fluorescent probes for decaging of prodrugs, metal detection, and diagnostic purposes. Herein we do an analysis of the trending topics related to coumarin and its derivatives in the broad field of drug discovery.

**Keywords:** coumarins; biological applications; drug discovery; fluorescent probes

**Citation:** Carneiro, A.; Matos, M.J.; Uriarte, E.; Santana, L. Trending Topics on Coumarin and Its Derivatives in 2020. *Molecules* **2021**, *26*, 501. https://doi.org/10.3390/ molecules26020501

Academic Editor: Emerson F. Queiroz Received: 10 December 2020 Accepted: 15 January 2021 Published: 19 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Coumarins are molecules that belong to a very special family. Their conjugated double ring system makes them interesting molecules for different fields of research. Coumarins can be found in industry as cosmetics and perfume ingredients, as food additives, and especially in the pharmaceutical industry in the synthesis of a large number of synthetic pharmaceutical products [1]. This last application is the main focus of our overview.

Coumarin (Figure 1) is found in nature in a wide variety of plants, particularly in high concentration in the tonka bean (*Dipteryx odorata*). It can also be found in sweet woodruff (*Galium odoratum*), vanilla grass (*Anthoxanthum odoratum*), and sweet grass (*Hierochloe odorata*), among others. This explains the great interest in the extraction and characterization techniques of natural coumarins, and in the synthesis of their derivatives. In addition, the simplicity of its chemical backbone is very attractive, as well as the reactivity of the benzene and pyrone rings. Conjugated double bonds are responsible for an electronic environment that plays a very important role in this family of compounds.

**Figure 1.** Basic classification of coumarins: chemical structures of the three main classes.

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This review is based on the most relevant literature that comprises new data from recent research articles and overviews on the development of new therapeutic solutions and fluorescent probes based on the coumarin scaffold. The research articles and reviews organized to prepare this manuscript have been compiled from various electronic databases, including SciFinder, Mendeley, and PubMed. The latter was the main source of information, due to its specificity in the biomedical field.

#### **2. Discussion**

Searching for the word "coumarin" in Mendeley, PubMed, and SciFinder in early November 2020, and filtering by year "2020", more than a thousand references appeared. Another search was conducted in late December to include as much information as possible in this manuscript. A diversity of journals from different fields publishes research articles and reviews related to the biological interest of both plant extracts containing coumarins and/or synthetic molecules based on this scaffold. Hybrid molecules containing different pharmacophores [2,3] like piperazines or pyrazolines [4] are at the top of the list. For simplicity, in the current review the information is organized taking into account the potential pharmacological/biological applications of coumarin derivatives.

#### *2.1. Anticancer Activity*

The activity of coumarins as anticancer agents is at the top of reviews published in 2020 [5–8], as well as research papers. Potent inhibitors (Figure 2, general structure **I**) of aldo–keto reductase (AKR) presenting an iminocoumarin scaffold, with activities between 25 and 56 nM, have been described for the treatment of prostatic cancer [9]. The design of sulfamide 3-benzylcoumarin hybrids bearing an oxadiazole ring at position 7 (Figure 2, general structure **II**) has allowed the preparation of new multitarget mitogen-activated protein kinase (MEK) inhibitors and nitric oxide (NO) donors, both with antiproliferative properties [10]. In other cases, the anticancer profile has been directed to other targets. Such is the case of new inhibitors of cyclin-dependent kinases, specifically CDK9, designing hybrids that incorporate an aminopyrimidine fragment to coumarin, both pharmacophores of known activity on these therapeutic targets [11]. We highlight here compound **III** (Figure 2), with high activity and selectivity for these receptors in comparison with other kinases.

Other important targets for cancer treatment, especially lymphomas, are histone deacetylases (HDACs). A series of coumarins (Figure 2, general structure **IV**) exhibiting a hydroxamate structure similar to HDACi vorinostat (SAHA) has been published [12]. The compounds show inhibitory activity in the nanomolar range, being higher in the case of propyl or methoxypropyl derivatives.

In addition, it is worth highlighting the design of hybrids in which one part of the molecule provides fluorescent properties, and another provides therapeutic action (theranostic). Such is the case of the fusion of a 7-aminocoumarin fluorescent ring with a chalcone fragment (Figure 2, compound **V**). This molecule is an inhibitor of thioredoxin reductases (TrxRs), presenting high antitumor activity (IC<sup>50</sup> = 3.6 µM), and is also used as a diagnostic agent [13]. Coumarin scaffold fluorescence is being explored extensively in biomedicine, as described at the end of this review.

The preparation of photo-triggered drug delivery systems (PTDDSs, Figure 2, general structure **VI**) has also been described, in which the chlorambucil pharmacophore is incorporated into more complex carbazole–coumarins (electron donor and electron acceptor fragments, respectively), carriers of a mitochondrial triphenylphosphonium ligand. This system allows, by irradiation, the controlled release of the chemotherapeutic agent [14].

Finally, coumarins are widely used as ligands in the formation of metal complexes, as described in a very recent review [15] focusing on their application as anticancer agents. Such is the case of complexes with platinum, palladium, gold, copper, or ruthenium, many of which are also used as described below, in the design of antimicrobial agents.

**Figure 2.** Structures of coumarin derivatives as anticancer agents.

Within the group of compounds with anticancer activity, coumarins exhibiting an antiglioma profile may be highlighted. Simple coumarins such as osthole, umbelliferone, esculin, and 4-hydroxycoumarin, combined with sorafenib (a kinase inhibitor drug approved for the treatment of primary kidney cancer, advanced primary liver cancer, FLT3- ITD positive acute myeloid leukemia (AML), and radioactive iodine-resistant advanced thyroid carcinoma) were studied [16]. The same group also studied a combination of the same simple coumarins with temozolomide (used in the treatment of brain tumors such as glioblastoma multiforme or anaplastic astrocytoma) [17].

#### *2.2. Antimicrobial Activity*

There is also an abundant bibliography related to the interest of coumarins as antimicrobials. Most of the projects are still inspired by the classic antibiotic novobiocin. There are several works in which antibacterial activity is found due to the presence of an azole ring introduced in different positions of the coumarin system. Articles have been published recently on the antibacterial activity of azole–coumarins, as well as 3/4/7 substituted arylcoumarins (Figure 3, general structures **VII** and **VIII**), especially active on Gram-positive and negative bacteria according to substitution patterns [15,18–20]. In other cases, thiazolidinedione–coumarin hybrids have been described (Figure 3, general structure **IX**) that show activity on methicillin-resistant *Staphylococcus aureus* (MRSA) [21].

Interestingly, coumarin metal complexes also show antibacterial activity. Such is the case of 3-arylcoumarins that present general structures **X** (Figure 3), coordinated with Re(**I**), active against MRSA in nanomolar concentrations [22]; or the complexes of general structure **XI**, a coordination of coumarin–quinoline hybrids with Cu(**I**), with activity against *Flavobacterium psychrophilum*, a Gram-negative bacterium that causes significant septicemia in fish, causing devastating economic problems in aquaculture [23].

**Figure 3.** Structures of coumarin derivatives as antimicrobial agents.

In addition to the antibacterial activity, in a recent and comprehensive review on coumarins, activity against protozoa of the genus *Leishmania* was described [24]. The most promising compounds are prenylated, glycosylated, furan/pyranocoumarins, or simple hydroxy- or methoxy-substituted coumarins, along with the natural coumarin mammea A/BB (Figure 3, structure **XII**). Derivatives of this natural product have been prepared and substitutions at positions 6 and 8, as well as the phenyl ring at position 4, turned out to be mandatory for the studied activity. This structure–activity relationship (SAR) study led to the synthesis of the simplest and most lipophilic analogue **XIII** (Figure 3), the most promising member of the group as an antileishmanial agent. Similar structures, some also derived of the *Mammea* genus, have been evaluated against *Mycobacterium tuberculosis*, an activity that also shows simpler synthetic analogues derived from 4-hydroxycoumarin (Figure 3, general structure **XIV**) [25].

Finally, it is worth mentioning two articles reported this year on the design and preparation of coumarin derivatives with potential antiviral activity. This is the case of the dual hybrid inhibitors inspired by the antiviral activity of calanolide, known as reverse transcriptase (RT) inhibitor. With this in mind, dual inhibitors of HIV-1 RT and protease (PR) have been designed, in which the coumarin fragment responsible for RT activity is linked to the fragment of the antiretroviral darunavir, active against PR of the HIV, through different amide, carbamate, or amine linkers (Figure 3, general structure **XV**) [26]. The second case described the introduction of a piperidine ring through a linker in position 7 of the coumarin scaffold, originating compounds with outstanding activity against certain filoviruses such as Marburg virus (MARV) or Ebolavirus (EBOV). From the SAR studied, it is interesting to highlight the role of substitution in *para* position with a trifluoromethoxy group that originated compound **XVI** (Figure 3) with IC<sup>50</sup> = 0.5 µM and 1.2 µM against EBOV and MARV, respectively [27]. μ μ

μ μ

#### *2.3. Antioxidant and Anti-Inflammatory Activities*

Although we have found very few publications related to these activities, in some cases the antioxidant activity of coumarin derivatives of both natural [28] and synthetic [29] origin has been reported. This is the case of NOs inhibitors, an activity described for coumarins that bind through different linkers to phenolic fragments capable of acting as radical scavengers (Figure 4, general structure **XVII**), hybrids that can therefore be used in the treatment of immunomodulatory diseases.

**Figure 4.** Structures of coumarin derivatives as antioxidant and anti-inflammatory agents.

Regarding the anti-inflammatory activity, it is worth mentioning a review on the coumarins of natural origin (simple coumarins, prenylcoumarins, furocoumarins, coumestans, and benzocoumarins) with a detailed anti-inflammatory activity due to the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2 factor) that protects cells against stress oxidative [30]. In other cases, the anti-inflammatory activity found for coumarin esters (Figure 4, general structure **XVIII**) as inhibitors of the Kallikrein-related peptidase 9 (KLK9) involved in inflammatory processes of the skin is reported [31]. Finally, the replacement of the carboxylic group by a sulfone or sulfoxide group (Figure 4, general structure **XIX**) gives rise to new inhibitors of cyclooxygenase-2 (COX-2) with activities comparable, in many cases, to indomethacin [32].

#### *2.4. Adenosine Ligands*

The affinity of the coumarin system for adenosine receptors has also been published recently. The 3-arylcoumarins (Figure 5, compound **XX**) have been described as antagonists of *h*A<sup>3</sup> receptors, showing a high affinity (in the low nanomolar range) and selectivity for this subtype [33], while the 3-aroylcoumarins (Figure 5, general structure **XXI**) have been described as dual *h*A1/*h*A<sup>3</sup> antagonists in the low micromolar range [34]. These works are aligned with the already known potential of these derivatives as modulators of the different adenosine receptors, published in the last decade.

**Figure 5.** Structures of coumarin derivatives as adenosine ligands.

#### *2.5. Enzymatic Inhibitory Activity: α-Glucosidase, Carbonic Anhydrase, Tyrosinase, Sulfatase, and Xanthine Oxidase*

The activity of coumarin derivatives on α-glucosidase was also reviewed in 2020 [35], in a study in which the influence of the substitution pattern was evaluated, and an important SAR was established. In addition to α-glucosidase, aldehyde dehydroge-

nase 1A1 (ALDH1A1) is another target for the treatment of diabetes and obesity, and 3-amidocoumarins have been described as inhibitors of this enzyme, with compound **XXII** (Figure 6) being a very promising derivative (IC<sup>50</sup> = 3.87 mM) [36]. This activity has also recently been found for hybrids of coumarin and cinnamic acid, with compound **XXIII** (Figure 6) being described as a very promising derivative (IC<sup>50</sup> = 12.98 mM) [37]. μ

μ

*α*

α

α

**Figure 6.** Structures of coumarin derivatives as enzymatic inhibitors.

Closely related to these structures, works have been published on coumarin derivatives with inhibitory activity on carbonic anhydrase **IX** and **XII**. These are coumarins that incorporate arylacrylamide substituents at position 3 (Figure 6, general structure **XXIV**) that showed inhibitory activity in the nanomolar range [38]. In other cases, anhydrase inhibitory activity was reported for hybrids connected by a methyleneoxy linker at position 7, oxadiazole heterocycles [39] that the same authors extend to the triazole ring (Figure 6, general structure **XXV**) [40].

These last structures are closely related to others that present tyrosinase inhibitory activity in the sub-micromolar range. This is the case of coumarins (umbelliferone and other phenolic analogues) that incorporate a kojic acid fragment through a triazole linker at position 4 (Figure 6, compound **XXVI**), both fragments with demonstrated tyrosinase inhibitory activity [41].

Likewise, the introduction of a sulfamate group in the coumarin scaffold originates a hybrid prototype (Figure 6, general structure **XXVII**) that presents a high inhibitory activity of the steroid sulfatase (best compound of the series with IC<sup>50</sup> = 0.13 µM), which is of interest in the treatment of hormone-dependent breast cancers [42].

During 2020, 3-phenylcoumarins were also studied as xanthine oxidase inhibitors [43]. Methoxy and nitro substituents were introduced into the framework. The best compound in the series proved to be 3-(4-methoxyphenyl)-6-nitrocoumarin, with an IC<sup>50</sup> = 8.4 µM, being also non-cytotoxic in B16F10 cells.

#### *2.6. Anti-Neurodegenerative Diseases Activity: MAO and AChE/BChE Inhibitors*

The role played by coumarin derivatives as agents that exhibit biological activities associated with neurodegenerative diseases, such as Alzheimer's disease, is very important. Throughout this year, a large number of manuscripts related to this field have been found. Due to the multidirectional nature of these diseases, there are also many works on hybrid coumarins directed at different pharmacological targets, such as monoamine oxidase B

(MAO-B) or acetylcholinesterase (AChE), amyloid aggregation, or oxidative stress, among others. Hybrids of general structure **XXVIII** (Figure 7) have been described, in which the rasagiline fragment with MAO-B inhibitory activity and neuroprotection properties is incorporated into the coumarin scaffold also with demonstrated MAO-B inhibitory activity, antioxidant, and neuroprotective properties [44]. The incorporation at position 3 of a pyridazine ring (Figure 7, general structure **XXIX**) is another case of hybrid structures as selective MAO-B inhibitors [45]. The incorporation of isoxazole-type heterocycles in carboxamide–coumarins (Figure 7, general structure **XXX**) allowed obtaining derivatives with significant inhibitory activities of AChE/BuChE and beta-secretase 1 (BACE1) [46]. In other cases, taking into account the importance of metals in the pathogenesis of Alzheimer's disease, a pyridinone fragment (Figure 7, general structure **XXXI**) with iron-chelating properties was incorporated [47].

**Figure 7.** Structures of coumarin derivatives as anti-neurodegenerative diseases agents.

Other multitarget structures are the benzotriazole–coumarin (Figure 7, general structure **XXXII**) and carbazole–coumarin (Figure 7, general structure **XXXIII**) hybrids [48,49]. In both cases, the molecules show antioxidant activity, as well as AChE and β-amyloid aggregation inhibitory properties. Finally, it is worth mentioning another type of hybrid, this time a furocoumarin that incorporates two fragments of resveratrol (Figure 7, general structure **XXXIV**) [50]. Compounds containing this scaffold present AChE and BACE1 inhibitory properties related to the furocoumarin fragment, and antioxidant (radical scavenging) and COX-2 inhibition, related to the resveratrol [50].

The 7-amidocoumarins (Figure 7, general structure **XXXV**) have recently been published for their potential against monoamine oxidase A (*h*MAO-A), *h*MAO-B, *h*BACE1, *h*AChE, and butyrylcholinesterase (*h*BuChE) [51]. The research project is based on a screening of compounds with potential activity against Alzheimer's and Parkinson's diseases, since these multifactorial pathologies share some of their pharmacological targets. Five derivatives of the studied series were described as potent and selective *h*MAO-B inhibitors in the nanomolar range; six turned out to be *h*MAO-A inhibitors in the low micromolar range; one showed inhibitory activity of *h*BACE1, and another one *h*AChE inhibitory activity, both in the micromolar range. In addition to the enzymatic inhibition, all of the studied molecules proved to be non-cytotoxic to neurons in the motor cortex. As a main conclusion, results suggest that by modulating the substitution pattern at position 7 of the scaffold, selective or multitarget molecules can be achieved.

The 3-arylcoumarins are a family of compounds with proven activity on different targets related to neurodegenerative diseases, especially Alzheimer's and Parkinson's diseases, the two most prevalent. In the last decade, several manuscripts described very promising activities of this scaffold, both as selective and multitarget compounds. SAR studies were performed, and important conclusions were drawn based on the substitution patterns in the main scaffold. Due to the large number of molecules based on this scaffold currently synthetized and studied as *h*MAO inhibitors, in 2020 a theoretical work was published comparing different QSAR models and docking calculations, in order to predict the *h*MAO-B activity of the 3-arylcoumarins [52]. Based on the predictions, a small series of compounds was synthetized and evaluated against both *h*MAO-A and *h*MAO-B, and the most promising models were validated. Selective activities were found in the low nanomolar range against this isoenzyme for 6 and 8 methyl-substituted 3-arylcoumarins, also presenting methoxy groups or bromine atoms in different positions of the 3-phenyl ring (Figure 7, general structure **XXXVI**). These advancements may represent robust tools in the design of potent and selective derivatives.

Analogues of 3-phenylcoumarins were also published during 2020. The discovery and optimization of 3-thiophenylcoumarins (Figure 7, general structure **XXXVII**) as novel and promising agents against Parkinson's disease have been described [53]. This study explores, for the first time, the potential of these structures as in vitro and in vivo agents against this disease. The inhibitory activities of *h*MAO-A and *h*MAO-B, antioxidant profile, neurotoxicity in neurons of the motor cortex, and neuroprotection against hydrogen peroxide production were studied. The in vivo effect on locomotor activity was also evaluated by an open field test (OFT) for the most potent, selective and reversible *h*MAO-B inhibitor of the series: 3-(4′ -bromothiophen-2′ -yl)-7-hydroxycoumarin (IC<sup>50</sup> = 140 nM). In reserpinized mice pre-treated with levodopa and benserazide, this molecule exhibited a slightly better in vivo profile than selegiline, currently a therapeutic option for Parkinson's disease. The results suggested that the 7-position substitution of the coumarin scaffold is interesting for enzyme inhibition. Furthermore, the presence of a catechol at positions 7 and 8 exponentially increases the antioxidant potential and the neuroprotective properties.

The neuroprotective effects of xanthotoxin and umbelliferone on streptozotocin (STZ) induced cognitive dysfunction in rats were evaluated [54]. Alzheimer's disease was induced in these animals and both compounds were administrated, proving to prevent cognitive deficits in the Morris water maze and object recognition tests. In addition, both compounds reduced the activity of hippocampal AChE and the level of malondialdehyde,

increasing the glutathione content. These coumarins also modulated neuronal cell death by reducing the level of proinflammatory cytokines, inhibiting the overexpression of inflammatory markers (nuclear factor κB and cyclooxygenase **II**), and upregulating the expression of NF-κB inhibitor (IκB-α). An attenuation of cognitive dysfunction by these compounds was observed. This effect can at least be attributed to the inhibition of AChE and the reduction of oxidative stress, neuroinflammation, and neuronal loss, opening a new door for these classic coumarins.

#### *2.7. Anticoagulant Activity*

The classic anticoagulant effect of specific coumarin derivatives, based on acenocoumarol and warfarin, also remains one of the classic applications for this family. During 2020, a review on this topic was published [55].

#### *2.8. Fluorescent Probes*

In addition to the interest of coumarins as a versatile scaffold in drug design, the important role that this scaffold plays as fluorescent probes to detect metals, enzymes, and biomaterials, among others, should be highlighted [56–59]. These fluorescent probes have a great imaging potential for the diagnosis of several pathologies.

Coumarins are being used in the selective detection of metals such as copper (Figure 8, general structure **XXXVIII**) [60] or its determination in drinking water (Figure 8, compound **XXXIX**) [61,62]. A recent review focuses on the detection of iron in water and its applications [63]. Other works study the fluorescence determination of the presence of silver in aqueous medium (Figure 8, compound **XL**) [64]. In the case of mercury, there are also published works in which the selective determination in water is studied (Figure 8, general structure **XLI**) [65]. In some cases, this determination is selective, but in this case, the innovative methodology can be applied over a wide pH range (Figure 8, compound **XLII**) [66].

In some cases, these metal complexes serve as probes for the detection of biothiols, as in the case of copper complexes with benzothiazoles (Figure 8, compound **XLIII**) used in the determination of cysteines [67], or coumarin–quinoline complexes used in the detection of glutathione (Figure 8, compound **XLIV**) [68]. In other cases, aromatization to form the coumarin ring is used as a fluorescence test to detect the superoxide anion (Figure 8, compound **XLV**) [69].

In addition to the determination of metals, in many cases coumarin derivatives are used as fluorescence probes for the detection of hypochlorite, as in the case of coumarin– thiophene complexes (Figure 8, compound **XLVI**) [70] or of 2-thiocoumarins in which the presence of ClO– allows the formation of a fluorescent coumarin (Figure 8, compound **XLVII**) [71].

Coumarins can also be used as photocleavable linkers in the controlled release of drugs or biomaterials. This is the case of the in vivo photolysis of the microtubule inhibitor 4 pyridinomethylcoumarin (Figure 8, compound **XLVIII**) [72]. 7-Hydroxymethyl substituted aminocoumarins are used as iron complexes (Figure 8, general structures **XLIX** and **L**), and can be used to photochemotherapeutically target the mitochondria in the treatment of cancer [60,73].

Other reviews report on the use of 7-hydroxycoumarin and its derivatives in determining the activity of cytochromes P450 (CYP) enzymes [74] as well as 7-aminocoumarin derivatives in determining amino acids from serine or cysteine proteases [58]. In other cases, they are used to detect the metabolism of mitochondrial cysteines, the oxidation of which is a measure of cellular oxidative stress (Figure 8, compound **LI**) [75]. The use of the chiral coumarin-BINOL hybrid allows the enantioselective detection of amino acids (Figure 8, compound **LII**) [76]. Finally, coumarins can be used for easy detection of bacterial carbapenamases in which the coumarin fluorophore binds to the carbapenemic structure via a reactive linker (Figure 8, general structure **LIII**) [77,78].

**Figure 8.** Structures of coumarin derivatives as fluorescent probes.

#### **3. Perspectives**

Coumarins are privileged structures for biological applications. Their conjugated double ring system allows different spots for chemical modifications, and a large number of derivatives can be obtained. Therefore, structure/activity studies appear to be the hottest emerging topic. During the year 2020, more than a thousand research articles and reviews related to coumarins could be found. This highlights the great potential that these molecules can have in different fields of research. For simplicity, our overview focused on the potential of coumarins in medicinal chemistry. The most relevant studies were included. The range of applications described in this document, and some others outside the scope of this general description (i.e., optoelectronic applications [79], polymers [80], etc.), reflect the versatility of this scaffold.

In our opinion, the potential of coumarins as fluorescent probes appears to be the most promising field of research for the next few years, since several coumarin derivatives have shown great potential in prodrug degradation (drug release) and diagnostics.

Due to the length of this general overview, synthetic strategies for obtaining new coumarins have not been discussed in detail. To find information on the most recent synthetic pathways, we recommend the manuscripts by Molnar and co-authors [81] and Kovaˇc and co-authors [82], both from 2020. To find an overview of the wide range of biological activities of coumarins, a 2020 review by Pinto and co-authors [1] is strongly recommended. Finally, to find information on the most recent analytical methods (fundamentals, instrumentation, purification and quantification applications, optimization of experimental conditions, emerging ecological methods, etc.) we recommend the review by Xue-song and co-authors [83].

#### **4. Conclusions**

Coumarins belong to a privileged family for biomedical proposes. Their simplicity, chemical properties, and the efficiency of the synthetic routes to obtain a wide range of substitution patterns make these compounds highly attractive and versatile for medicinal and biological chemists. To date, and during 2020, more than a thousand research articles and reviews containing information on coumarins appear in the PubMed, SciFinder, and Mendeley databases. The number of research groups working on this scaffold, and the impact of the results, highlight the potential of these molecules. Special attention has been paid to the potential of coumarins in drug design, as well as to fluorescent probes. This last application seems to be the most promising field of research for the next few years, since several coumarin derivatives have shown great potential in the decaging of prodrugs (drug release) and for diagnostic purposes.

**Author Contributions:** Conceptualization, M.J.M. and L.S.; methodology, A.C., M.J.M., E.U., and L.S.; formal analysis, A.C., M.J.M., E.U., and L.S.; investigation, A.C., M.J.M., E.U., and L.S.; resources, M.J.M., E.U., and L.S.; writing—original draft preparation and editing, M.J.M. and L.S.; writing review and editing, M.J.M., L.S., and E.U.; visualization, M.J.M., E.U., and L.S.; supervision, L.S.; project administration, M.J.M., E.U., and L.S.; funding acquisition, M.J.M., E.U., and L.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Xunta de Galicia (Galician Plan of Research, Innovation and Growth 2011–2015, Plan I2C, ED481B 2014/027-0, ED481B 2014/086–0 and ED481B 2018/007) and Fundação para a Ciência e Tecnologia (FCT, CEECIND/02423/2018 and UIDB/00081/2020).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data presented is original and not inappropriately selected, manipulated, enhanced, or fabricated.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

