Pharmacotherapeutics Applications and Chemistry of Chalcone Derivatives

Chalcones have been well examined in the extant literature and demonstrated antibacterial, antifungal, anti-inflammatory, and anticancer properties. A detailed evaluation of the purported health benefits of chalcone and its derivatives, including molecular mechanisms of pharmacological activities, can be further explored. Therefore, this review aimed to describe the main characteristics of chalcone and its derivatives, including their method synthesis and pharmacotherapeutics applications with molecular mechanisms. The presence of the reactive α,β-unsaturated system in the chalcone’s rings showed different potential pharmacological properties, including inhibitory activity on enzymes, anticancer, anti-inflammatory, antibacterial, antifungal, antimalarial, antiprotozoal, and anti-filarial activity. Changing the structure by adding substituent groups to the aromatic ring can increase potency, reduce toxicity, and broaden pharmacological action. This report also summarized the potential health benefits of chalcone derivatives, particularly antimicrobial activity. We found that several chalcone compounds can inhibit diverse targets of antibiotic-resistance development pathways; therefore, they overcome resistance, and bacteria become susceptible to antibacterial compounds. A few chalcone compounds were more active than conventional antibiotics, like vancomycin and tetracycline. On another note, a series of pyran-fused chalcones and trichalcones can block the NF-B signaling complement system implicated in inflammation, and several compounds demonstrated more potent lipoxygenase inhibition than NSAIDs, such as indomethacin. This report integrated discussion from the domains of medicinal chemistry, organic synthesis, and diverse pharmacological applications, particularly for the development of new anti-infective agents that could be a useful reference for pharmaceutical scientists.


Introduction
Chalcone is a collective group of ketones (flavonoids) that has a three-carbon α,β-unsaturated carbonyl group attached to two aromatic rings, namely rings A and B (Figure 1). The numbering system of chalcone shown in Figure 1 is followed throughout this article. Other chemical names of chalcone include benzyl acetophenone or benzylideneacetophenone. They are produced by certain plant species such as Angelica, Glycyrrhiza, Humulus, and Scutellaria as precursors to the biosynthesis of flavonoids and isoflavonoids 1). The numbering system of chalcone shown in Figure 1 is followed throughout t cle. Other chemical names of chalcone include benzyl acetophenone or benzyliden phenone. They are produced by certain plant species such as Angelica, Glycyrrhiza, lus, and Scutellaria as precursors to the biosynthesis of flavonoids and isoflavono intermediates to the synthesis of heterocyclic compounds with biologically inte properties such as pyrazolines, isoxazoles, cyanopyridines and pyrimidines. Chalcone is part of plants' most prominent class of secondary metabolites. used in plant defense mechanisms to combat reactive oxygen species for the plant vive and prevent molecular damage as well as damage caused by microorganis sects, and animals [1]. Chalcone can also be chemically synthesized in the laborator the Claisen Schmidt or aldol condensation reaction [2]. Chalcone has been repo exert multiple beneficial properties, such as anti-inflammatory, antibacterial, ant antidiabetic properties, and anticancer activities. It also improves vision, memor and muscle discomfort, liver and kidney function, sleep, prevents cancer, strength immune system, and beautifies skin and hair. [3][4][5].
Chalcone and its derivatives have shown an inhibitory effect against methic sistant Staphylococcus aureus (MRSA) [6] due to the presence of −OH groups in the and the lipophilicity of the A ring ( Figure 1) [7,8]. A combination of Chalcones an biotics (i.e., oxacillin) has also shown a synergistic effect in treating MRSA infecti 12].
Additionally, some chalcone derivatives exhibited antifungal activities, part against Microsporum gypseum [13][14][15][16]. They inhibit the β (1, 3)-glucan and chitin sy responsible for the formation and normal functioning of the fungal cell wall [17,18 chalcone compounds have shown superior antifungal effects compared to ketoco a broad-spectrum oral antifungal agent [13]. Chalcone compounds are also effec hibitors of inflammatory enzymes, such as cyclooxygenase (COX), lipooxygenase interleukins (IL), and prostaglandins (PGs) [19]. The most researched novel series cones revealed their potent inhibitory effects on nitric oxide (NO) formation and lease of glucuronidase and lysozyme, which are responsible for inflammatory res [20]. Heterocyclic rings and methoxy substitutions on the attached rings of chalcon tribute to the anticancer properties of such chalcone compounds [21][22][23]. The inves chalcone compounds: isoliquiritigenin, flavokawain, and xanthohumol, showed cy and apoptosis induction that promotes antitumor activities [23]. However, the nisms of the compound are unclear, causing uncertainty about chalcones' astoundi ities. It is a compound that has attracted the attention of many scientists due to its p of therapeutic efficacy. Chalcone is part of plants' most prominent class of secondary metabolites. This is used in plant defense mechanisms to combat reactive oxygen species for the plant to survive and prevent molecular damage as well as damage caused by microorganisms, insects, and animals [1]. Chalcone can also be chemically synthesized in the laboratory using the Claisen Schmidt or aldol condensation reaction [2]. Chalcone has been reported to exert multiple beneficial properties, such as anti-inflammatory, antibacterial, antifungal, antidiabetic properties, and anticancer activities. It also improves vision, memory, joint and muscle discomfort, liver and kidney function, sleep, prevents cancer, strengthens the immune system, and beautifies skin and hair. [3][4][5].
Chalcone and its derivatives have shown an inhibitory effect against methicillin-resistant Staphylococcus aureus (MRSA) [6] due to the presence of −OH groups in the B ring and the lipophilicity of the A ring ( Figure 1) [7,8]. A combination of Chalcones and antibiotics (i.e., oxacillin) has also shown a synergistic effect in treating MRSA infections [9][10][11][12].
Additionally, some chalcone derivatives exhibited antifungal activities, particularly against Microsporum gypseum [13][14][15][16]. They inhibit the β (1, 3)-glucan and chitin synthases responsible for the formation and normal functioning of the fungal cell wall [17,18]. Some chalcone compounds have shown superior antifungal effects compared to ketoconazole, a broad-spectrum oral antifungal agent [13]. Chalcone compounds are also effective inhibitors of inflammatory enzymes, such as cyclooxygenase (COX), lipooxygenase (LOX), interleukins (IL), and prostaglandins (PGs) [19]. The most researched novel series of chalcones revealed their potent inhibitory effects on nitric oxide (NO) formation and the release of glucuronidase and lysozyme, which are responsible for inflammatory responses [20]. Heterocyclic rings and methoxy substitutions on the attached rings of chalcones contribute to the anticancer properties of such chalcone compounds [21][22][23]. The investigated chalcone compounds: isoliquiritigenin, flavokawain, and xanthohumol, showed cytotoxic and apoptosis induction that promotes antitumor activities [23]. However, the mechanisms of the compound are unclear, causing uncertainty about chalcones' astounding abilities. It is a compound that has attracted the attention of many scientists due to its plethora of therapeutic efficacy.
Numerous studies have been conducted to explore the pharmacological properties of chalcone and its derivatives in recent years. Even though there are a few reviews available, such as antiviral [24,25], preclinical studies [4], synthesis chalcone [26], and antidiabetic [27], comprehensive reviews focusing on the overall purported health benefits, including molecular mechanisms of pharmacological actions of chalcone and its derivatives evaluating the Molecules 2022, 27, 7062 3 of 24 recent progress of therapeutical applications is insufficient. Therefore, herein, we reviewed the research findings to discuss the synthesis of chalcone and its derivatives and assess their pharmacotherapeutic efficacy, focusing on the recent experimental studies. This report integrated discussion from the domains of medicinal chemistry, organic synthesis, and diverse pharmacological applications, particularly for the development of new anti-infective agents that could be a useful reference for pharmaceutical scientists.

Chemistry of Chalcone and Its Derivatives
Chalcone and its derivatives have been long used in various traditional medicine systems, including homeopathic and Chinese medicine. They are traditionally prepared by the reaction of benzaldehydes and active methylene ketones under homogeneous conditions using the Claisen-Schmidt condensation and a more recent invention known as the aldol condensation [28]. However, recent discoveries of methods for producing chalcones provide different advantages depending on the type of catalyst, solvent, base, and reaction conditions [29].

Claisen Schmidt Condensation
The Claisen Schmidt condensation reaction involves an aldehyde with the carbonyl group without hydrogen atoms in the α-position, and a ketone, using a heterogeneous acid catalyst to produce the desired α,β-unsaturated ketone. This is one of the methods of synthesizing chalcone in the laboratory ( Figure 2) due to the equimolar quantities of acetophenone and benzaldehyde. Claisen Schmidt condensation uses an aqueous-alcoholic alkali with a (concentration of 10 to 60%) to catalyze the reaction between acetophenone and benzaldehyde by dehydration [30]. The reaction can take place either for 12-15 h at a temperature of 50 • C or for one week at room temperature (20-25 • C).
able, such as antiviral [24,25], preclinical studies [4], synthesis chalcone [26], and antidia betic [27], comprehensive reviews focusing on the overall purported health benefits, in cluding molecular mechanisms of pharmacological actions of chalcone and its derivative evaluating the recent progress of therapeutical applications is insufficient. Therefor herein, we reviewed the research findings to discuss the synthesis of chalcone and its de rivatives and assess their pharmacotherapeutic efficacy, focusing on the recent exper mental studies. This report integrated discussion from the domains of medicinal chemi try, organic synthesis, and diverse pharmacological applications, particularly for the de velopment of new anti-infective agents that could be a useful reference for pharmaceutic scientists.

Chemistry of Chalcone and Its Derivatives
Chalcone and its derivatives have been long used in various traditional medicin systems, including homeopathic and Chinese medicine. They are traditionally prepare by the reaction of benzaldehydes and active methylene ketones under homogeneous con ditions using the Claisen-Schmidt condensation and a more recent invention known a the aldol condensation [28]. However, recent discoveries of methods for producing cha cones provide different advantages depending on the type of catalyst, solvent, base, an reaction conditions [29].

Claisen Schmidt Condensation
The Claisen Schmidt condensation reaction involves an aldehyde with the carbony group without hydrogen atoms in the α-position, and a ketone, using a heterogeneou acid catalyst to produce the desired α,β-unsaturated ketone. This is one of the methods o synthesizing chalcone in the laboratory ( Figure 2) due to the equimolar quantities of ace tophenone and benzaldehyde. Claisen Schmidt condensation uses an aqueous-alcohol alkali with a (concentration of 10 to 60%) to catalyze the reaction between acetophenon and benzaldehyde by dehydration [30]. The reaction can take place either for 12-15 h at temperature of 50 °C or for one week at room temperature (20-25 °C). The heterogeneous acid catalyst is useful in producing chalcones because of the in creased purity of end-products, decreased amounts of undesired products, reduced rea tion time, and cost-effective procedure. Ionic liquids (ILs) are prepared using this conden sation reaction but use a multi-sulfonic acid group ion liquid as the catalyst. ILs hav gained interest due to their high catalytic activity, small catalyst usage, easy filtration recyclable catalyst, and constant catalytic activity ( Figure 2). However, the product yiel will be decreased due to the reaction conditions, which promotes the Cannizzaro reaction This redox reaction produces primary alcohol and carboxylic acid from two aldehyd molecules. The heterogeneous acid catalyst is useful in producing chalcones because of the increased purity of end-products, decreased amounts of undesired products, reduced reaction time, and cost-effective procedure. Ionic liquids (ILs) are prepared using this condensation reaction but use a multi-sulfonic acid group ion liquid as the catalyst. ILs have gained interest due to their high catalytic activity, small catalyst usage, easy filtration, recyclable catalyst, and constant catalytic activity ( Figure 2). However, the product yield will be decreased due to the reaction conditions, which promotes the Cannizzaro reaction. This redox reaction produces primary alcohol and carboxylic acid from two aldehyde molecules.

Aldol Condensation
Aldol condensation is another synthetic method commonly used after the Claisen-Schmidt condensation. The aldol condensation reaction (Figure 3), or the solid-state reaction, replaces aldehydes with benzylidene-diacetate and uses heat (200-350 • C) and a base such as potassium hydroxide as a catalyst for the reaction between the two compounds. It uses calcium, barium or strontium hydroxides or carbonates as catalysts in a liquid mixture containing water with a low boiling point that can perform distillation at a constant temperature. Other synthetic reactions can increase the reaction rate using microwave radiation without solvents; it also provides fluorescence emission profiles that can be used as biological markers.
It uses calcium, barium or strontium hydroxides or carbonates as catalysts in a liquid ture containing water with a low boiling point that can perform distillation at a con temperature. Other synthetic reactions can increase the reaction rate using microwa diation without solvents; it also provides fluorescence emission profiles that can be as biological markers. Reacting a ketone with an aldehyde (with the carbonyl group-containing no h gen atoms in the α position with an acidic heterogeneous catalyst-activated carbon reduce the reaction time, cost, and impurities in the final products. Another tech involves the phase transfer method for synthesizing heterocyclic ring-containing cones, which incorporates a third ring into the chalcone's skeleton chain. The startin stituents used are 1-phenyl-3-aryl-4-formyl pyrazole and acetophenone, with the ca being tetrabutylammonium bromide in the presence of an inorganic alkaline solutio reaction is performed under microwave radiation ( Figure 3).

Synthesisand Chemistry
Another method uses plants such as Sedum jinianum, S. plumbizincicola, S. alfre Potentilla griffithii with a high concentration of metals such as zinc, copper, cadmium magnesium, and at least one of the metal elements is used as a catalyst. They ar heated and treated with acid, then filtered and purified to be attached to a carrier, b ing a metal catalyst in synthesizing chalcones. Although this method promotes less tion, it is unconventional to extract metal from these plants to be used as mere catal A different method uses a fluorine-containing biphasic catalyst as a result of re 4-dimethylamino pyridine with fluorinated alkyl iodide to react benzaldehyde wit tophenone at 50 to 100 °C for 1 to 3 h. The reacted compounds were cooled, filtered tilled, and recrystallized with more than 99% purity. This invention requires an eas cessing method, nature-friendly, low synthesis cost, and ease of re-obtaining the f catalyst.
There are methods to synthesize hydroxyflavones that use soluble resin, speci polyethylene glycol (PEG) that reacted with benzyloxy-2-hydroxy-acetophenone used in a reaction with benzaldehyde with a base as a catalyst. The advantages o method are high accessibility to the reactant compounds and high purity percentag Another popular method is the one-pot synthesis. In this process, a direct react one step is utilized to prepare inorganic components, while the organic component ates as a surface capping material or template. This method shortens the time to sep and purify the products and increases the product yield. Another technique of on Reacting a ketone with an aldehyde (with the carbonyl group-containing no hydrogen atoms in the α position with an acidic heterogeneous catalyst-activated carbon) will reduce the reaction time, cost, and impurities in the final products. Another technique involves the phase transfer method for synthesizing heterocyclic ring-containing chalcones, which incorporates a third ring into the chalcone's skeleton chain. The starting constituents used are 1-phenyl-3-aryl-4-formyl pyrazole and acetophenone, with the catalyst being tetrabutylammonium bromide in the presence of an inorganic alkaline solution; the reaction is performed under microwave radiation ( Figure 3).

Synthesisand Chemistry
Another method uses plants such as Sedum jinianum, S. plumbizincicola, S. alfredi and Potentilla griffithii with a high concentration of metals such as zinc, copper, cadmium, and magnesium, and at least one of the metal elements is used as a catalyst. They are first heated and treated with acid, then filtered and purified to be attached to a carrier, becoming a metal catalyst in synthesizing chalcones. Although this method promotes less pollution, it is unconventional to extract metal from these plants to be used as mere catalysts.
A different method uses a fluorine-containing biphasic catalyst as a result of reacting 4-dimethylamino pyridine with fluorinated alkyl iodide to react benzaldehyde with acetophenone at 50 to 100 • C for 1 to 3 h. The reacted compounds were cooled, filtered, distilled, and recrystallized with more than 99% purity. This invention requires an easy processing method, nature-friendly, low synthesis cost, and ease of re-obtaining the fluoric catalyst.
There are methods to synthesize hydroxyflavones that use soluble resin, specifically polyethylene glycol (PEG) that reacted with benzyloxy-2-hydroxy-acetophenone to be used in a reaction with benzaldehyde with a base as a catalyst. The advantages of this method are high accessibility to the reactant compounds and high purity percentage.
Another popular method is the one-pot synthesis. In this process, a direct reaction in one step is utilized to prepare inorganic components, while the organic component operates as a surface capping material or template. This method shortens the time to separate and purify the products and increases the product yield. Another technique of one-pot synthesis is slowly adding primary alcohol with chromium oxide (CrO 3 ) to produce furochalcones, or the addition of cheap catalysts, which are copper salt, 2,2 -bipyridine, and 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) kept at a temperature of −10 to 100 • C for 10 to 96 h to produce a milder reaction.
A different synthesis method to produce the α,β-unsaturated carbonyl system that does not require condensation reaction offers more direct response with fewer vigor conditions, a cheaper cost of reactants, a more straightforward operating system, and a higher yield of products. The raw materials are the halogenated aromatic hydrocarbons with a ketone in the carbon skeleton and aromatic alkynes, using an alkali, a phosphine ligand, and palladium as catalysts, at a temperature of 60 to 150 • C [31,32].

Approach to Design of Chalcone Derivatives from the Natural Sources
Different approaches have been used to design chalcone derivatives, as outlined below:

Isoliquiritigenin
It can be extracted from the plant Radix Glycyrrhizae. Isoliquiritigenin is used in cosmetics due to its beneficial effects on the skin, including treatment of skin conditions such as acne, eczema, and irritation, as well as desirable effects, such as skin whitening, antiaging, and eye drop preparations. There have been claims that isoliquiritigenin can activate the GABA A receptor, bind to the γ-subunit and act as a positive allosteric modulator that elicits similar effects as a benzodiazepine. It also prevents and treats cardio-cerebrovascular diseases [33] (Figure 4).
does not require condensation reaction offers more direct response with fewer vigor conditions, a cheaper cost of reactants, a more straightforward operating system, and a higher yield of products. The raw materials are the halogenated aromatic hydrocarbons with a ketone in the carbon skeleton and aromatic alkynes, using an alkali, a phosphine ligand, and palladium as catalysts, at a temperature of 60 to 150 °C [31,32].

Approach to Design of Chalcone Derivatives from the Natural Sources
Different approaches have been used to design chalcone derivatives, as outlined below:

Isoliquiritigenin
It can be extracted from the plant Radix Glycyrrhizae. Isoliquiritigenin is used in cosmetics due to its beneficial effects on the skin, including treatment of skin conditions such as acne, eczema, and irritation, as well as desirable effects, such as skin whitening, antiaging, and eye drop preparations. There have been claims that isoliquiritigenin can activate the GABAA receptor, bind to the γ-subunit and act as a positive allosteric modulator that elicits similar effects as a benzodiazepine. It also prevents and treats cardio-cerebrovascular diseases [33] (Figure 4).

Licochalcone A
It is present in high concentrations in the plant Glycyrrhiza inflate [34]. Licochalcone A and isoliquiritigenin compounds are used in the cosmetic industry; they are both used in acne treatment and skin whitening. Licochalcone A is also used to prepare skin toner and hair cosmetics. The compound in essential oils produces bath salts to clean pores in the skin, control sebum production and retain skin moisture. Licochalcone A is also used for the treatment, improvement, and prevention of adenosine 5 monophosphate-activated protein kinase (AMPK)-related diseases, an enzyme involved in metabolism, specifically for lipids [34]. Influenza virus infection-related diseases were treated for other conditions in which licochalcone A was used [35][36][37] (Figure 4).

Xanthoangelol
It is a major component of the plant Angelica keiskei. Xanthoangelol is an isoprenylchalcone compound with antioxidant properties that are used to prevent diseases involving lipid metabolism or inflammation [38] (Figure 4).

Isobavachalcone
It can be isolated from Psoralea corylifolia or Piper longum fruits. It belongs to the same chalcone family as xanthoangelol but has different properties [39]. It inhibits melanin formation causing skin whitening [40]. Isobavachalcone is also used to reduce nerve inflammation and inhibit cholesterol absorption [41]. Other uses of isobavachalcone prevent and control diseases [42] (Figure 4).

Xanthohumol
It is present in the Humulus lupulus and belongs to the prenylated chalcone family. Its antioxidant property is a 'broad spectrum' cancer chemo-preventive agent. Along with hydroxytyrosol, it is used as a nasal spray for viral infection, allergic reactions, or vasomotor rhinitis of the nasal mucosa [43]. Xanthohumol has demonstrated the inhibitory activity of the enzyme α-glucosidase, an enzyme responsible for carbohydrate metabolism. Therefore, it is used in metabolic diseases such as diabetes and other diseases such as AIDS, osteoporosis, and malignant tumors [44,45] (Figure 4).

Nardoaristolone A
It can be extracted from Nardostachys chinensis and classified as terpenoid chalcones [46]. Like any other chalcones, it has been used to treat different skin cancers. It has systemic effects such as increasing the red blood cell count and aids in small bowel movements. Nardoaristolone A has been used in medications for tuberculosis and endometrium cancers [1,45] (Figure 4).

Role of Chalcone Moiety in Synthesis of Derivatives
The chalcone moiety can be used to produce other chalcone derivatives such as cyanopyridines, pyrazolines, isoxazoles, and pyrimidines with different heterocyclic ring systems. These derivatives containing hydroxyl, ether, acid, or amino groups have diverse functionality and can be used to produce more complex chalcones. Aminochalcones, a chalcone moiety, can produce benzothiazole chalcones and other derivatives through alkylation, hydrolysis, and esterification or amide formation. Chalcone derivatives can be produced through the Phase Transfer Catalysis through alkylation, using oxygen or sulfur.
Chalcone derivatives can also be produced using the α,β-unsaturated system by substitution of the functional group at the two positions, and dihydrochalcones can be produced by reducing the double bond in the saturated system; these derivatives will be used in the synthesis of pyrazoles and flavonoids as well as other heterocyclic compounds.
A chalcone derivative which is 3-phenyl-1-(4-methyl) phenyl-2-bromo-propylene-1one, can be produced by reacting ρ-toualdehyde with acetophenone using an inorganic base as a catalyst in an aldol condensation reaction, and the products react with a halogen in an addition reaction followed by an elimination reaction with an inorganic base. Another method also used a heterogeneous catalyst (hexagonal boron nitride h-BN) that is hydrogenated and has a Frustrated Lewis Pair (FLP)-type electronic structure that produces 100% yield.
Another method to obtain dihydrochalcones, specifically phloretin, used bacterial or plant chalcone isomerases and enolate reductase as a catalyst. An electrolytic hydrogenation method using hydrogen, which becomes active by electrolysis of water, is needed, followed by an addition reaction with a ketone without a catalyst to obtain neohesperidin dihydrochalcones. Dihydrochalcones and quinazolinyl derivatives can be produced by substituting carboxylic or nitro groups at the β carbon of the carbon skeleton of chalcones, respectively [47] (Figure 5). tion method using hydrogen, which becomes active by electrolysis of water, is needed, followed by an addition reaction with a ketone without a catalyst to obtain neohesperidin dihydrochalcones. Dihydrochalcones and quinazolinyl derivatives can be produced by substituting carboxylic or nitro groups at the β carbon of the carbon skeleton of chalcones, respectively [47] (Figure 5).

Semi Synthetic Derivatives of Chalcones
Chalcone isocordoin and its semisynthetic derivatives were tested for Anti-inflammatory and anti-hypersensitive effects in mice ( Figure 6).

Semi Synthetic Derivatives of Chalcones
Chalcone isocordoin and its semisynthetic derivatives were tested for Anti-inflammatory and anti-hypersensitive effects in mice ( Figure 6). The structure of the synthesized chalcones can be characterized by IR, NMR and mass spectroscopy.

UV Spectrum
The UV spectrum of chalcones consists of two essential absorption band: band I and relatively a minor band, band II. In chalcones, band I usually appears in 340-390 nm, alt-

Characterization of Chalcones
The structure of the synthesized chalcones can be characterized by IR, NMR and mass spectroscopy. The UV spectrum of chalcones consists of two essential absorption band: band I and relatively a minor band, band II. In chalcones, band I usually appears in 340-390 nm, although a minor inflection or peak often occurs at 300-320 nm. Band II appears in 220-270 nm.

IR Spectrum
In the IR spectra of chalcones asymmetric and symmetric stretching vibrations of the aromatic C-H bonds are seen at 3120-3080 cm −1 and 3060-3040 cm −1 ranges with two low intensity bands. C-H stretching band of the =C-H group is observed at 3030-3010 cm −1 . The bands at 1610-1570 cm −1 are assigned to the vibrations of the aromatic ring. The inplane deformation of the =C-H bond is appeared as broad weak band at 1460-1430 cm −1 . The carbonyl stretching vibrations for the enones (=C-C=O) can be found between 1650 and 1685 cm −1 .

NMR Spectrum
The 1 H-NMR spectrum of double bonds of chalcones were seen at 5.4 and 6.1 ppm. The aromatic regions were observed at 6.9-8.1 ppm.
In 13 C-NMR spectrum of chalcones, the carbonyl carbon usually appears between δ 186.6 and 196.8. The αand βcarbon atoms with respect to the carbonyl group give characteristic signals between δ 116.1-128.1 and δ 136.9-145.4 respectively.

Mass Spectrum
Basic fragmentation pathways of chalcones are obtained by loss of the phenyl group from the A or B ring, and loss of CO.

Basic Fragmentation Pathways of Chalcones Are Obtained by Loss of the Phenyl Group from the A or B Ring, and Loss of CO. Pharmacotherapeutic Activities
Chalcone and its derivatives have shown diverse pharmacological activities. A summary of different pharmacological properties with their salient mechanisms of action is shown in Figure 7. The pharmacophore responsible for various activities changes depending on the activity. The details of these functions have been discussed comprehensively in the following sections.

Anti-Bacterial Agent
Chalcone is an antibacterial agent with moderate to high activity due to the presence of the reactive αβ-unsaturated system. Their flexibility to change their structure by incorporating different types of substituent groups into the aromatic ring can potentially achieve a higher potency, lower toxicity, and a wider spectrum of antibacterial activity [54].
Many chalcone derivatives showed potential antibacterial activities against different

Anti-Bacterial Agent
Chalcone is an antibacterial agent with moderate to high activity due to the presence of the reactive αβ-unsaturated system. Their flexibility to change their structure by incorporating different types of substituent groups into the aromatic ring can potentially achieve a higher potency, lower toxicity, and a wider spectrum of antibacterial activity [54].
Many chalcone derivatives showed potential antibacterial activities against different pathogenic strains, including antibiotic-resistant bacteria. A summary list is shown in Table 1. Several chalcones were significantly potential than the standard antibiotics (Table 1). For example, compounds 1-4, 6-8, 11-13, 15-19, 21-29 (Table 1) have shown strong antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis, and against Gram-negative bacteria Escherichia coli and Salmonella enterica [55]. Grampositive bacteria were more susceptible to cationic molecules than Gram-negative bacteria. For example, compounds 1-4 showed the highest activity (MIC ranged 1-2 µg/mL) against Gram-positive bacteria, whereas MIC ranged 2-8 1-2 µg/mL for Gram-negative bacteria. The hydrophobicity of the alkyl chain was responsible for varying strength of antibacterial potentiality. Compounds 7 with medium hydrophobicity exhibited the highest activity against the tested bacteria; however, compounds 6-10 with different alkyl chain lengths showed different antibacterial sensitivity. The results reported in Table 1 demonstrated that increasing alkyl chain length (i.e., compounds 8-10) causes decreasing antibacterial activity. Since the long hydrophobic chain has an aggregation tendency, this might cause this decreasing antibacterial activity [56]. Another study reported that 31 compounds, among them, compounds 47, 50, and 51 (Table 1), were more active against the tested bacteria, B. cereus, E. coli, P. aeruginosa, and S. aureus [57]. Another study tested both sulfones and bisulfones chalcones (11 compounds) for their antibacterial activity against Gram-positive strains B. subtilis and S. aureus and Gram-negative strains P. aeruginosa and S. typhimurium [58]. In this study, in comparison to standard antibiotics Ampicillin and Kanamycin, compound 61 was slightly better against B. subtilis and compounds 65, 66, and 67 were significantly potential against S. typhimurium. In another study using monomeric chalcone compounds, there was higher antibacterial activity in Gram-positive bacteria than the Gram-negative bacteria [59]. A screening method of chalcone derivatives against S. aureus, B. subtilis, E. coli, and P. aeruginosa showed increased lipophilic area, and the smaller molecular size of chalcones increased their antimicrobial activity [60].         Chu et al. [55] reported the antibacterial activity of several cationic Chalcones against antibiotic resistance strains, including nine clinical isolates of MRSA, 12 clinical isolated of KPC-2-producing Klebsiella pneumoniae (KPC), and 12 clinical isolated of New Delhi metallo-β-lactamase 1 (NDM-1)-producing Enterobacteriaceae. A few selected molecules (compounds 1-4, 7, 12, 16-19, 26, and 28) were tested against MRSA and found MIC values of 0.25-32 µg/mL (Table 1). Among these, compound 7 showed the lowest MIC 0.25 µg/mL, and exhibited the highest activity against clinical isolates 6 of MRSA. The MICs of the other compounds were primarily 0.5-8 µg/mL [55].
For KPC, compounds 17, 26, and 28 exhibited the best antibacterial activity (MIC 2 µg/mL), which was equivalent to vancomycin. However, compound 3 showed average activity MIC ranging from 4-8 µg/mL. The MIC for NDM ranged mainly from 1-16 µg/mL; however, among them, compound 28 exhibited the highest activity (MIC: 2-8 µg/mL) [55]. Overall, compound 28 was highly effective against KPC and NDM, whereas compound 7 was for MRSA. Furthermore, the antibacterial activity of the tested molecules against NDM was better than the KPC.

Antibacterial Mechanisms
Chalcone and its derivatives have the potential to target diverse receptors. This diverse inhibition process includes (i) efflux pump inhibitor (EPI), (ii) type II fatty acid biosynthetic pathway (FAS-II), (iii) DNA replication, (iv) filamentous temperature-sensitive mutant Z (FtsZ), (v) virulence factor, and (vi) protein tyrosine phosphatases (PTPs). A schematic representation of the antibacterial mechanisms of chalcone and its derivatives is shown in Figure 8. The details mechanisms can be found elsewhere (see review [5]).

Antibacterial Mechanisms
Chalcone and its derivatives have the potential to target diverse receptors. This diverse inhibition process includes (i) efflux pump inhibitor (EPI), (ii) type II fatty acid biosynthetic pathway (FAS-II), (iii) DNA replication, (iv) filamentous temperature-sensitive mutant Z (FtsZ), (v) virulence factor, and (vi) protein tyrosine phosphatases (PTPs). A schematic representation of the antibacterial mechanisms of chalcone and its derivatives is shown in Figure 8. The details mechanisms can be found elsewhere (see review [5]). Figure 8. Antibacterial mechanisms of chalcone and its derivatives. Antibiotic resistance bacteria inhibit the function of antibiotic or antimicrobial agents by activating the efflux pump, modifying the active site of the enzyme, and degrading the enzyme. However, chalcone and its derivatives inhibit or block the functions of resistance plasmids through different mechanisms. Additionally, they inhibit DNA gyrase, DNA replication, and FtsZ. Therefore, bacteria cannot cell divide, and ultimately the bacteria die (Information was sourced from reference [61]. Figure 8. Antibacterial mechanisms of chalcone and its derivatives. Antibiotic resistance bacteria inhibit the function of antibiotic or antimicrobial agents by activating the efflux pump, modifying the active site of the enzyme, and degrading the enzyme. However, chalcone and its derivatives inhibit or block the functions of resistance plasmids through different mechanisms. Additionally, they inhibit DNA gyrase, DNA replication, and FtsZ. Therefore, bacteria cannot cell divide, and ultimately the bacteria die (Information was sourced from reference [61].
Chalcones were the potential to exhibit cytoplasmic membrane depolarization and inner membrane permeabilization. Compounds 1-4, 7, 11, 12, 16-19 were used to evaluate the ability of the cytoplasmic membrane depolarization and the inner membrane permeabilization against S. aureus and E. coli to understand their mode of action [55]. The highest cytoplasmic membrane depolarization activity of S. aureus was exhibited by compounds  3, 7, and 18, whereas compounds 11, 1, and 7 showed for E. coli. Both S. aureus and E. coli cytoplasmic membrane depolarization was at least level by the other compounds.
The ability of bacterial efflux pumps to rapidly export antibacterial drugs makes them sustainable against drugs. Even though the origin of antibiotic resistance is numerous and complex, efflux pumps are one of the critical class of resistance determinants [62][63][64][65][66][67][68][69].
About 117 Chalcone molecule was tested against the NorA efflux pump of S. aureus and found at least 20 effective compounds that were able to inhibit this efflux pump. However, five compounds were active, and among them, two compounds, 4-phenoxy-4 -dimethylaminoethoxychalcone and 4-dimethylamino-4 -dimethylaminoethoxychalcone were highly active, which were equipotent to reserpine with IC 50 -values of 9.0 and 7.7 lM, respectively. Additionally, three compounds synergistically increased the effect of ciprofloxacin on S. aureus, of which 4-phenoxy-4 -dimethylaminoethoxychalcone exhibited a fourfold higher activity at 3.13 µg/mL, being twice as potent as reserpine [70]. Additionally, the ability to inhibition of efflux pump was concentration-dependent. Higher molecule concentration increases many-fold inhibition of efflux pump.
Condensed pyrimidine derivatives, ring-fused chalcones, and flavanones were also reported to have antibacterial activity. In treating certain oral infections caused by bacteria, chalcones were included along with the antibiotics, which were found to enhance the effects of the antibiotics. Rhodanine derivatives have strong antimicrobial activity against methicillin-resistant and non-methicillin-resistant S. aureus strains. Quinoxalines derivatives inhibited bacterial activity against the gram-positive bacteria, Bacillus subtilis, and Staphylococcus aureus [72].

Anti-Fungal Agent
Chalcones act on glutathione, cysteine molecules, and proteins involved in the cell separation of yeast cells. A hybrid molecule of pharmacophore of fluconazole and chalcone was tested for their antifungal activity and showed inhibitory activity against Candida albicans. Chalcone with unsubstituted thiophene ring B and thiomethyl substitution at the p-position of ring A (compared to oand m-) produced credible antifungal activity on fluconazole-sensitive and fluconazole-resistant strains of yeast [73].
The highest antifungal efficacy against standardized strains of Trichophyton rubrum was demonstrated by the chalcone 3-(2-chlorophenyl)-1-(2 -hydroxy-4 ,6 -dimethoxyphenyl) prop-2-en-1-one (MIC = 12.5 µg/mL), which also inhibited all ten clinical isolates of T. rubrum (IC 50 12.5 and IC 90 25 µg/mL). The Neurospora crassa assay revealed a blotchy look in the inhibitory halo formed by this chalcone, strongly indicating that it may work by inhibiting the fungal cell wall [74]. This study also demonstrated that noticeable hyphal curling was observed in the hazy zone at a magnification of 400 or less; therefore, this chalcone appears to be a hyphal deformity inducer.
The highest antifungal activity (MIC: 1.95 µg/mL) was shown by the compounds 62, 63, 68, and 69 against C. albicans (Table 2) Other observations included significant antifungal activity by fluoro-substitution at the pposition on ring A and chalcone compounds with smaller halogen sizes [73]. Several studies on the antifungal activity of chalcones have results that contradict one another. However, they have a common finding: placing the hydroxyl group at the m-position in ring A produces a significant antifungal effect [54]. Table 2. Antifungal activity of chalcone and its derivatives [66].

No. Materials Tested Drug Test Assay
Antifungal Strains (MIC µg/mL) Standard Antifungal
A study reported that the C2-C3 double bond is responsible for the high inhibitory activity of chalcones because it links the A and B rings as well as stabilizes the molecular conformation, which allows the drug molecule to bind to the active site more efficiently; there is decreased inhibitory activity by steric interactions due to the substitutions on the bridge portion of chalcone derivatives; increased inhibitory activity was seen in electrondonating substitution on ring A and chloro-or fluoro-substitution on ring B; an increase in antimalarial activity in placing quinolinyl group to ring B [90]. A small hydrophobic nitrogen heterocyclic group at ring B and a small lipophilic functional group at ring A can increase antimalarial activity [90].

Anti-Protozoal and Anti-Filarial Agent
Even though chalcone and its derivatives have diverse biological applications; however, limited applications have been found for antiprotozoal activity [91][92][93]. Since structural modifications of the main pharmacophore of chalcone is the synthesis process of new derivatives, expecting high biological activity of novel compounds is normal. However, the antiparasitic potencies of the α,β-double bond modified chalcones only differ marginally from the potencies of the parent chalcones [93]. This may be limited the further evaluation of chalcone for antiprotozoal activity.
TIe chalcIne of interest in having anti-leishmanial activities is licochalcone A; it modifies the structure and function of the Leishm'nia braziliensis's mitochondria as well as the activity of mitochondrial dehydrogenases. In addition, the aforementioned chalcone prevents the promastigotes and amastigotes, which are produced by the parasite, from developing. There is also a decrease in parasite load inside the liver and spleen [94]. Another protozoon called Trypanosoma cruzi was used to study the effects of chalcone. The chalcones with no substitution groups exhibit anti-trypanosomal activity [95].
Chalcones were tested on Setaria cervi extracted from female adults for anti-filarial effects by inhibiting glutathione-S-transferase (GST) enzyme. There was a significant inhibitory activity of GST, which led to irreversible inhibition of the viability and motility of the parasites, as well as reduced glutathione levels. Results indicated that lipophilic groups of the ring in chalcone containing oxygen or nitrogen were behind the anti-filarial effects, where methoxy groups exhibited the most significant activity [96].

Anti-Inflammatory Agent
The main component of chalcone that contributes to its anti-inflammatory property is the α,β-unsaturated carbon skeletal system. Polymethoxychalcones showed potent inhibitory effects on inflammation, mainly through the production of high IL-1 β-induced PGE 2 , prostaglandin inhibitors, inflammatory cytokines, and normal human epidermal keratinocyte inhibitors. A series of pyran-fused chalcones and trichalcones can inhibit the NF-κB signaling complement system involved in inflammation. At the same time, some compounds showed better inhibitory activity against lipoxygenase than the standard drug indomethacin. Inflammatory cytokines, particularly TNF-α, IL-1β, IL-6, and IL-8 production, were inhibited by chalcones with heterocyclic systems in their moiety. Chalcones converted to Mannich bases also inhibit the release of inflammatory mediators from mast cells, macrophages, neutrophils, and microglial cells. Other chalcone derivatives, such as dihydroxychalcones, also exhibit anti-inflammatory activity [46].
The anti-inflammatory activity also depends on the position of the substituted group on the phenyl ring A and increases in the order: −3, 4-(OH) 2 Chalcones with fluoride or chloride groups also show potent anti-inflammatory activity, with 4-Cl and 3-NO 2 being more powerful than 2, 4-Cl , and 2-NO 2 , respectively [2].

Anti-Cancer Agent
In recent years, numerous studies evaluated and revealed the anticancer potential of chalcone and chalcone-based different types of derivatives through in vitro, in vivo as well as molecular docking studies [102][103][104][105][106][107][108][109][110][111]. Both in vitro and in vivo methods were used to explore the anti-melanoma effects of flavokawain B on human melanoma cells and the processes of cell death that were mediated by the generation of reactive oxygen species (ROS) [108]. The findings show that flavokawain B decreased the viability of human melanoma cells as well as the expression of the B-Raf proto-oncogene, serine/threonine kinase (BRAF), and extracellular signal-regulated kinase. The execution of apoptosis was aided by Caspase-3 activation, the PARP cleavage pathway, and Bcl2-associated X (Bax)/Bcell lymphoma 2 (Bcl-2) dysregulation. Additionally, flavokawain B also inhibited tumor growth in nude mice with xenografts [108]. This study concluded that flavokawain B could potentially be used for managing human melanoma cancer by executing and producing ROS-modulated apoptotic and autophagic cell death.
Chouiter et al. [105] synthesized novel chalcone derivatives and tested for their anticancer activity using human lung (A549) and stomach (AGS) cancer cell lines and evaluated them in the noncancer human lung fibroblast (MRC-5) cell lines. All the cell lines tested showed no toxicity for 2-pyrazolines, although the AGS cell line was hazardous for chalcones containing a benzimidazole moiety. Mechanistic studies revealed that these substances cause loss of cell viability and mitochondrial membrane potential while inducing morphological characteristics consistent with regulated cell death by caspase activation, particularly by caspase-3. Boronic chalcones were investigated for anticancer activity, and results indicated the chalcones, particularly AM114, produced a cytotoxic effect in cancer cells by inhibiting the chymotrypsin-like activity of the 20S proteasome in these cells, which in turn accumulated p53 and p21 proteins, creating a favorable condition for cell apoptosis [112]. In other studies, the double bond was replaced with a thiophene ring, which showed the antiproliferative activity of cancer cells and the growth inhibition activity of cancer cell lines at nanomolar to low micromolar concentrations. Some studies have shown inhibition of tubulin polymerization by binding [3H] colchicine to tubulin and arresting the G2/M phase of K562 cells [113,114]. 2 -hydroxychalcone was studied for their antitumor effects on HepG2 hepatocellular carcinoma cells, and results showed they initiate cell apoptosis and inhibit cell proliferation. This was due to the 'ring A,' which does not contain methoxy groups. Other chalcones promoted cell apoptosis and cytotoxicity in prostate cancer cells through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [114].

Conclusions
This review summarized the recent antimicrobial and other pharmacological activities of chalcone compounds with their mode of action. Moreover, we have discussed different methods of chalcone derivatives synthesis and characterization. Notably, chalcone derivatives demonstrated potential antimicrobial activities, including antibiotic-resistant strains like MRSA, KPC, and NDM. Chalcone derivatives potentially inhibit the different targets pathway of microbial resistance. Several chalcone compounds demonstrated optimum characteristics of an effective antimicrobial agent. However, their potential must be warranted using preclinical and clinical studies. Chalcone derivatives can also be a lead compound with anti-inflammatory and anticancer properties, but further study is required to corroborate its clinical activities. Since structure-activity has a potential relationship, even though some modifications revealed negligible improvement, further study is needed for more extensive evaluation, including molecular mechanisms of actions. Even though a plethora of health benefits are evident from experimental studies, evaluating the pharmacological activities of chalcone and its derivatives is still required, particularly in preclinical and clinical studies. As chalcone and its derivatives can be synthesized in the laboratory and chemical structure can be modified, more effective action against a specific disease pathway can be achieved.