A review on coumarin backbone: An attractive scaffold for promising bioactive compounds

Background: Coumarin moiety is found in many naturally occurring products that have been used for many decades in traditional medicine around the world. Coumarin has distinctive physicochemical properties and can be easily transformed into a wide range of functionalized coumarins. As a result, a large number of coumarin derivatives have been designed, synthesized, and evaluated to attack a variety of pharmacological targets selectively. These targets may include various selective enzyme inhibitors and the targets, tagged as multitarget-directed ligands, found in diseases like Parkinson's and Alzheimer's, which are considered as multifactorial diseases. Objectives: The most widely used synthetic methods leading to coumarins, besides the major biological routes for their metabolic transformations and biosynthesis, are highlighted and reviewed. Also, the focus was concentrated on some pharmacological activities of coumarin derivatives involving those related to the selective inhibition of cholinesterase and monoamine oxidase enzymes and targeting specific ligands of neurodegenerative diseases. Conclusion: The impacts of substituents pattern/type on the selectivity and potency of the studied coumarins were explained to determine the main structural and molecular factors that may affect the activity and performance of the directed targets.


INTRODUCTION
ogel was discovered coumarin S1 from the seeds of the Dipteryx odorata tree, which has called tonka beans, in 1820, for the first time. It is also known as Coumarou, a French term 1 . Since then, many coumarins derived from plants, bacteria, fungi have been isolated, structurally characterized, synthetically modified, and evaluated for their various biological activities 2, 3 . S1, whose structure is shown in Figure 1, is an oxa-heterocycle with a 2H-chromen-2one (2H-1-benzopyran-2-one or 1,2benzopyrone) that has been extensively investigated due to the presence of its skeleton in many physiologically active products and compounds 4 . Coumarin and some of its derivatives have been developed into medications, as displayed in Figure 2, including the anticoagulants acenocoumarin S2, phenprocoumon S3, and warfarin S4, which all act as vitamin K antagonists. Also, the antibiotic novobiocin S5 that is a potent bacterial DNA gyrase inhibitor, and the choleretics hymecromone (4-methyl umbelliferone) S6, and armillarisin A S7 5 .

Biosynthesis of Coumarins
Coumarins are generated naturally via the main biosynthetic route, which leads to phenylpropanoids.
The enzyme phenylalanine ammonia-lyase (PAL) converts phenylalanine (from the shikimate pathway) into trans-cinnamic acid, which is then converted into the core metabolite 4′coumaroyl-S-CoA. The latter is then subsequently converted into a variety of phenylpropanoids 6 . Coumarin is biosynthesized from the core metabolite via ortho-hydroxylation, followed by trans-cis isomerization of the side chain, and finally cyclization process. The orthohydroxylation, mediated via the enzyme Feruloyl-CoA 6'-hydroxylase 1 (F6′H1), is the first and most important step in this natural synthesis. Kai et al. (2008) suggested the radical mechanism for the biosynthesis of coumarins 7 , which is displayed in Scheme 1.

Metabolism of Coumarins
There are two principal routes for metabolizing coumarin-based compounds, as displayed in Scheme 2, including 7hydroxylation and lactone ring-opening with oxidative decarboxylation. The latter occurs on the intermediate produced in the metabolic pathway's first step, which is coumarin 3,4-epoxide. Other probable coumarin metabolites, which are formed to a considerably lesser degree than at position 7, include hydroxylation of coumarins at positions 8, 6, 5, and 4, and 3,4dihydrocoumarin (DHC) 8 .

Scheme 2: Summarization of the principal coumarin metabolic pathways.
Some cytochrome P-450 isozymes (CYPs) can play a critical role in the metabolic transformation of coumarin-based compounds. The primary families of cytochrome P-450 that are important for most medication's oxidative biotransformation are CYP1, CYP2, and CYP3 9 . In human liver microsomes, the main enzyme responsible for the metabolism of coumarin-based compounds to their corresponding 7-hydroxycoumarin metabolites is CYP2A6 10 . Reduced CYP2A6 activity, which may be due to the CYP2A6's polymorphism and/or high gene multiplicity, may favor alternate coumarin metabolic routes, like the route that leads to 3-hydroxycoumarin via CYP3A4 catalysis. It was proposed that a higher amount of 3hydroxycoumarin may stimulate the production of 2-hydroxyphenyl acetaldehyde (2-HPA), which is a cytotoxic product that might be involved in the coumarin-related toxicity 10 .
Even though coumarin itself can be represented as an effective pharmacological therapy for lymphedema, several medical protocols have prohibited the utilization of this inexpensive and effective medication. The probable hepatotoxic consequences, as previously stated, that demonstrated in mice, rats, and even less frequently in humans may result in this precaution 11 . However, considering the prior results, patients with lymphedema who have a lower activity of CYP2A6 isozyme can be recognized and avoided coumarin treatment. The reason for that is the high possibility for metabolizing coumarin via the cytotoxic route, resulting in o-HPA 11 . The early phenotyping of these individuals can promote a safer application of this effective and inexpensive compound, coumarin, to all other lymphedema-suffering patients with normal CYP2A6 enzyme function 11 .

Toxicity of Coumarins
The exposure of humans to various natural coumarin-based products is high since they are abundant in fruits, vegetables, nuts, seeds, tea, and coffee 12 . Metabolism-, carcinogenicity-, toxicity-, and safetyrelated studies concerning the coumarin itself found in foods and perfumes for cosmetic application have been reviewed. The authors of this review found that coumarin exposure from foods and cosmetics items has no risk on human health 13 . On the other hand, other publications showed that coumarin and certain coumarin derivatives are significantly hazardous. Indeed, in the hepatocytes from various species, which include humans, hepatotoxic effects have been observed [14][15][16] . Another important article stated that coumarin's cytotoxic effects depend on species and metabolism, so rat models can't be utilized to estimate coumarin toxicity in humans 17 . Recent human investigations have found that 0.1 mg/kg body weight is the tolerated dosage intake for coumarin. So, to avoid hazardous consequences, this dose should not be exceeded 18 .

Strategies for Coumarin Synthesis
The coumarin skeleton is future in a wide range of physiologically active natural entities, medications, agrochemicals, optoelectronic, and polymeric materials 19,20 . As a result, enormous and ongoing efforts were made to develop novel synthetic routes and protocols, which make the crucial cyclization process to the heterocyclic ring and subsequent regioselective derivatization easier to perform. To create properly designed coumarin derivatives, a lot of effort has gone into developing more efficient and environmentally friendly synthetic methods 21 . In recent years, the increased usage of enabling and new technologies, like novel catalysts, ultrasounds, microwaves, greener solvents, and solvent-free reactions, has made access to coumarin derivatives considerably easier 21, 22 . The most common and classical methods for obtaining coumarin derivatives: the Pechmann 23 and Knoevenagel 24 reactions, which are the subjects of several coumarin-synthetic studies, concentrated primarily to improve yields, develop simple work-up procedures, and use green/recyclable catalysts and solvents. Only selected synthetic techniques that were released recently are being discussed briefly here.

Pechmann Reaction for the synthesis of coumarins
One of the most investigated synthetic methods for the synthesis of coumarin and its derived compounds is the Pechmann condensation reaction. The classical version of this coupling reaction was promoted by strong inorganic acids like HCl and H2SO4. The recent advances concerning this reaction type investigated various catalysts to facilitate and initiate this condensation 23 . The incoming are some examples:

A-FeCl3 catalyzed the synthesis of coumarins
The condensing of various invigorated phenols and β-ketoesters was proceeded, utilizing 10% mol FeCl3.6H2O as an initiator, produced moderate-to-good yields 25 .

B-Tin tetrachloride-grafted on silica gel catalyzed the synthesis of coumarins
Under a solvent-free environment heated to 120°C, the titled heterogeneous catalyst (SnCl4-grafted on silica gel) can stimulate the synthesis of substituted coumarins in good yields 26 .

C-γ-Fe2O3@HAp-Ag nanoparticles catalyzed the synthesis of coumarins
The Pechmann reaction was effectively catalyzed by an easily produced promoter, which was silver functionalized on hydroxyapatite-core-shell magnetic gamma-Fe2O3 nanoparticles. The reusable magnetic promoter forms the required coumarin derivatives, under environmentally friendly experimental settings, in high yields, and with a simple work-up procedure 27 .

D-Sulfonated carbon@titania composite loaded with Lewis acid catalyzed the synthesis of coumarins
At 60°C, carbon@titania composite loaded with Lewis acid was used as an efficient initiator in the Pechmann reaction. It gives coumarin derivatives in high yields under a solvent-free environment 28 .

E-Molybdate sulfuric acid (MSA) catalyzed the synthesis of coumarins
At 80°C, the Pechmann reaction proceeded in the water-dioxane blend employing molybdate sulfuric acid as a novel and effective promoter, producing the required coumarins in good yields 29 .

F-Ionic-liquid catalyzed the synthesis of coumarins
At 70°C and in a solvent-free environment, 1,3-disulfonic acid imidazolium-hydrogen sulfate (DSIMHS) was found to be an efficient and recyclable ionic-liquid promoter. High yields were obtained in a short period (less than 30 min) 30 .

G-Sawdust-sulfonic acid catalyzed the synthesis of coumarins
The Pechmann reaction proceeded at 110°C and was initiated by a reusable solid catalyst named sawdust-sulfonic acid can form coumarins in high yields. This effort offered several benefits, including a simple work-up procedure, without solvent, and a shorter interaction period (less than 60 min) 31 .

Knoevenagel Reaction for the synthesis of coumarins
The Knoevenagel reaction is among the most frequently examined synthetic techniques for coumarin and its derivatives. An organic base like tertiary amine aided the conventional version of this coupling process that involves the interaction between benzaldehyde functionalized at position-2 with hydroxyl moiety and invigorated methylene. Recent research into this reaction type has looked at a variety of catalysts to promote and trigger this condensation phenotype. Here are a few samples from the newcomers 32 :

A-MgFe2O4 nanopromoter catalyzed the synthesis of 3-functionalized coumarins
Under ultrasound irradiation and in a solvent-free environment, MgFe2O4 is an effective nanopromoter that can initiate the condensation reaction between numerous 1,3-dicarbonyl and salicylaldehydes compounds utilizing Knoevenagel reaction. This strategy has brief reaction times, a simple work-up procedure, and high yields 33 .

B-Potassium phthalimide catalyzed the synthesis of 3-functionalized coumarins
An efficient, fast, and environmentally friendly procedure for high-yielding, potassium phthalimide can promote the synthesis of 3-carboxy-and 3-cyanocoumarin phenotypes S8, as displayed in Scheme 3.
Salicylaldehyde-based derivatives were reacted with invigorated methylene compounds in an aqueous media and at room temperature for several hours using the aforementioned catalyst and under mild reaction settings 34 . Scheme 3: Synthesis of 3-functionalized coumarins using potassium phthalimide as a catalyst.

Pharmacological Activities of Coumarins
Coumarins have impressive pharmacological effects depending on their basic backbone (for example, simple coumarin, bis-coumarin, fused polycyclic coumarin) and substitution pattern 35  activities. The coumarin ring has structural and physicochemical characteristics that making it easy for binding to many target sites.
Because the coumarin ring is aromatic, lipophilic, and planar, it can combine with physiological counterparts, primarily the lipophilic binding sites in proteins, by forming -stacking (non-covalent formedforces generated among aromatic rings) and hydrophobic interactions with aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. Also, coumarins may attach to positively-charged amino acids via strong ion-dipole interaction 50 .
Besides, the coumarin's lactone group can offer the capacity to form strong-polar connections, such as dipole-dipole interactions and hydrogen bonds.
In some cases, coumarins can acylate the protein targets, as suggested for a particular enzyme's covalent inhibition mechanism. The lactone ring can also be opened by esterase-enzyme phenotypes, and biological activity may be caused by molecules that arise from that hydrolysis. In this situation, coumarins are bio-activated to release the actual active metabolites, acting as prodrugs 50 . Several natural coumarins have been reported to have this mode of action for inhibiting carbonic anhydrase, which possesses the catalytic activity of esterase 51 . Pharmacological activities of coumarins on selected targets are discussed below.

Coumarins as ChE Inhibitors
Acetyl-and butyryl-cholinesterase (ACh-E and BCh-E), mainly ACh-E, catalyze the breakdown of acetylcholine (ACh) neurotransmitters at the synaptic gap of cholinergic neurons. Their inhibition can restore ACh levels in the nervous system, so ACh-E inhibitors were used in the treatment of Alzheimer's disease (AD), which led to rising interest in the development of new ACh-E/BCh-E inhibitors 52 . Coumarin derivatives, both synthetic and natural, are a well-studied group of compounds that function as ACh-E/BCh-E inhibitors, with several reviews published on the subject recently 47 . To achieve the maximum inhibition of these enzyme phenotypes, a highly trendy step in the designing of ChE inhibitors was used, which led to conjugated compounds having the moiety of coumarin coupled to a well-known ChEs inhibitor, usually tacrine or donepezil. Although there is no complex for co-crystallization of human ChEs (hChEs) with these kinds of coumarin hybrids yet, inhibition kinetics and docking studies propose that such coumarinbased molecules having a binding posture, typically extending from the peripheral (PAS) to the catalytic active site (CAS) of the enzyme, the term "dual binding site (DBS) inhibitor" was coined to describe these compounds 53 . Also, the crystal structure of the combination of Torpedo californica A-ChE with aflatoxin confirmed that the coumarin ring preferentially binds to a peripheral active site (PAS) of the tested enzyme 54 . As shown in Table 1, many coumarin-based compounds were investigated and identified as ChE inhibitors.
electric eel-(eeAChE) inhibitors Quaternary benzylammonium salt, IC50s in nM-pM range, introducing substituent in position 3 for effective binding with the peripheral active site.
bAChEselective inhibitor IC50= 7.6 nM, a mixed-type inhibitory activity was found, proving the binding with both the PAS and CAS of bAChE.

hAChEselective inhibitor
One of the donepezil-like compounds, which was early released and advanced to pharmacological and biochemical testing.

hChEsinhibitors
Potent ChE inhibitors designed by using a recurrent approachs.  65 . Such exceptional selectivity and activity were examined further by many researchers, as shown in Table 2.

Coumarins as MAO Inhibitors
The easily functionalize coumarin backbone synthetically enables the investigation of various substitution patterns. The resolution of co-crystallized complexes of human MAOs with many reversible/irreversible inhibitors by X-ray crystallography led to an advance in the design of MAOs inhibitors 66,67 . By using the structural information obtained from the studies of X-yay as a guide, a target-dependent design of expanded sequences of selective inhibitors of MAO-B was created, by retaining the substituent 7-m-chlorobenzyloxy, to ensure selectively and desirable binding to MAO-B and trying to introduce charged or polar substituent at position 4. B/A selectivity and MAO-B affinity were preserved in nearly all of the proposed compounds, which also demonstrated high solubility in water and low lipophilicity, all of which are essential requirements for progressing to pre-clinical and clinical investigations 67 .
Also, to discover and justify the most significant molecular factors for high selectivity of MAO A/B and MAO affinity, as well as to give drug-like characteristics to recently developed inhibitors, different computational studies were carried out (computer-promoted molecular design) 68 . Lately, molecular dynamics (MD) simulations used to check the interaction of hMAO-B and -A with selective coumarinbased inhibitors at the molecular level. A significant finding has been the discovery of water-mediated H-bond between Flavin adenine dinucleotide (FAD) cofactor and the selective MAO-B 7-benzyl oxycoumarin, an interaction that was not anticipated by docking-studies of similar analogs 69 . 7-Benzyloxy and 7arylsulfonyl oxycoumarins 70 .

MAOinhibitors
IC50s in the nM range, selective and potent .

rMAOinhibitors
The ex vivo studies on this group of coumarins were carried out on the rat's brain and liver for the first time. The half-life of esters hydrolysis in a buffer correlated with the R group's steric hindrance.

rMAOinhibitors
Strong selectivity of MAO B/A and high rat MAO-B inhibitory action in low-to the sub-µM range.

hMAOinhibitors
Different selectivity of MAO B/A, high inhibitory potency of human MAO, with IC50s ranging from µM to sub-µM. A decrease in MAO-B affinity occurred due to the addition of the benzyloxy group in position 7.

MAOinhibitors
Higher selectivity of MAO B/A and lower inhibitory potencies in comparison with 7-benzyl oxycoumarins analogs.

hMAOinhibitors
Potent and selective human MAO-B inhibitors and IC50s in the sub-µM to the sub-nM range. The addition of substituents in position 6 of coumarins seemed tolerable, except for the insertion of a bulky group.

hMAOinhibitors
High selectivity of MAO B/A, an affinity for human MAO-B in the µM range, and good predicted pharmacokinetic properties (ADMET).

rMAOinhibitor
High B/A selectivity and rat MAO-B inhibitory potency. Good pharmacokinetic properties, low cytotoxicity, and high blood-brain barrier (BBB) permeation. IC50 of MAO-A= 5.94µM and MAO-B= 13nM.

Potent hMAO-Binhibitors
Excellent selectivity of MAO B/A and the activity on human MAO-B in the nM to the sub-nM range. These compounds developed through target-and ligand-depended screening of well-known selective inhibitors of MAO.

Highly selective hMAO-Ainhibitors
Derived by using a combined QSAR-CN (complex networks) model approach and MARCH-INSIDE approach (computational method).

Highly selective hMAO-Binhibitors
Derived by using a combined QSAR-CN (complex networks) model approach and MARCH-INSIDE approach (computational method).

Coumarins as Multitarget-Directed Ligands
The use of a multi-target drug therapy to treat neurodegenerative and other complicated illnesses has become a "revolution" in drug development. Because of their multiple etiology, multifactorial diseases are usually treated with a medication cocktail, which has a higher risk of drug-drug interactions and toxicity. The multi-target strategy is based on the idea of one drug, several targets, which intends to provide a single-drug therapy with multiple pharmacological actions that may be used to treat the same illness 82 .
The benefits of this monotherapy include primarily the absence of drug-drug interactions, increased compliance of patients who are taking only one therapy for their condition, and the simplicity of ADMET screening and therapeutic and pharmacological characterization. However, such a multi-target profile necessitates a correct balance of potencies to achieve the required pharmacological activity for every target addressed. The simplest method of preparing multi-target ligands is to attach two pharmacophoric moieties, each of which is responsible for different biopharmacological actions, via a linker that can be cleaved metabolically (conjugationtype) or through a covalent bond that is formed directly (fusion-type). A higher advanced multi-target ligand design involves merging (hybridizing-type) two or more functional groups into a novel molecular unit, capable of displaying the original actions of both moieties 83,84 , as shown in Figure 3. The disadvantage of creating conjugated or fused molecules is that it frequently leads to big molecules with low water solubility, high lipophilicity, high molecular weight, and a large number of rotatable bonds. As a result, these compounds are likely to have poor pharmacokinetic characteristics, including limited bioavailability and higher susceptibility to being substrates for detoxification systems. On the other hand, hybrid molecules may maintain more drug-like properties and may be easier to create as hit drugs for pharmacological investigations 85, 86 .
The significant interactions of the coumarin core inside the binding sites of ChEs and MAOs enzymes lead to molecular conjugates having this skeleton, which is also known as dual ChE-MAO inhibitors 87 . Some coumarin derivatives that can act as multitarget-directed ligands are reported in Table 3.

S75-85
Protonatable 7-substituted coumarins, a flexible alkoxy chain was used to connect the basic N-benzyl group to position 7 89 .

Dual ChE-MAO-B inhibition
Good inhibitory activities at MAO-B, AChE, and BChE, but low selectivity.

S86-90
Protonatable 7-substituted coumarins, A more rigid 7benzyloxy moiety was used to connect the basic N-benzyl group to position 7 89 .

Dual ChE-MAO-B inhibition
The presence of CH2OH group in position 4 of the coumarin led to higher selectivity of MAO B/A, better balance of MAO-B and AChE activities, and good ADMET properties. This group of congeners has the potential to be developed into hit structures in the future.
Potency in the nM range for hMAO-B and sub µM for hAChE.

Dual ChE-MAO-B inhibition
High selectivity and potency, good ADMET properties, especially high water solubility and BBB permeability, high cytoprotective effect against oxidative stress, and low cytotoxicity.

Dual ChE-MAO inhibition
Excellent activity profile as S91 and S92 compounds.

S95
7-Substituted coumarins using a linker with second basic nitrogen 92 .

Dual ChE/MAO inhibitors
The multi-target action was retained, but the potency was reduced.

Dual ChE/MAO inhibitors
The multi-target action was retained, but the potency was reduced. The addition of charged residues, in particular, had a negative effect on hMAO-B affinity.

Multitargetdirected ligands
Good activity profiles, although protonatable piperazine is present in a spacer, the length of the linker allows for a more marked increase in the affinity of hMAO-B and hBChE.

Multitargetdirected ligands
The piperazine ring in a spacer connected to tacrine by an amide-bond. Efficient inhibitors of eeAChE/esBChE, with metal-chelating, and antiamyloid properties.

Dual ChE-MAO inhibitors
Act by inhibiting MAO-B through another mechanism while retaining good dual AChE-MAO-B inhibitory action, with cytoprotective effects and high BBB permeability.

CONCLUSION
The main structural and molecular factors that affect the activity and performance in the directed targets. Such knowledge can help in designing new coumarins with more selectivity and better pharmacological action. NMR spectrophotometer and X-ray crystallography are used to determine their three-dimensional structures. The safety of coumarins is another factor to consider. The danger may occur just at extremely high coumarin doses, which is difficult to achieve in typical diets. Despite recent significant discoveries, the development of selective and potent coumarins remains a significant promising goal for pharmaceutical chemists.