FLAVONOIDS: BIOCHEMISTRY, BIOPHYSICS, MEDICINE - Tarakhovsky Yu. S. - 2013

PART 1. MAJOR FLAVONOIDS

1.8. Chalcones

The term "chalcone" was proposed by the Polish chemist Stanisław Kostanecki. It originates from the Greek word "chalkos", meaning "copper". Chemically, chalcones, or 1,3-diaryl-2-propen-1-ones, are open-chain Flavonoids (Fig. 16) in which two aromatic rings are joined by a three-carbon $\alpha,\beta$-unsaturated carbonyl system. In most cases, the aryl rings are hydroxylated. Chalcones can exist in cis- and trans-forms, with the trans-form being thermodynamically more stable. Furthermore, the $\alpha,\beta$-unsaturated ketone groups are likely responsible for most of the observed biological properties of chalcones, as these groups are present in all biologically active molecules, and their removal results in a loss of activity.

Class="center">Fig. 16. Carbon atom numbering in chalcone molecules.

These substances exhibit pronounced anticancer activity by targeting multiple distinct sites within The Cell signaling system. In addition, they are of considerable interest as antioxidants, antihistamines, and anti-inflammatory agents, while also displaying antibacterial and anti-protozoan activities. A key feature of chalcones is their relatively simple chemical Structure, which allows for the synthesis of A wide variety of molecules with biological activity and, in some cases, medicinal properties. Nevertheless, plant-derived chalcones also include numerous substances of significant medical interest (Fig. 17).

Fig. 17. Selected plant-derived chalcones and dihydrochalcones.

Among the most thoroughly studied chalcones are phloretin and its glycoside phloridzin, which are found in apple leaves. These flavonoids have long been recognized as inhibitors of Glucose Absorption by intestinal and renal epithelial Cells. Phloretin is also capable of inhibiting urea transport in various cells, including renal epithelial cells, hepatocytes, and erythrocytes.

A substantial body of research has also been dedicated to butein, named after the leguminous tree *Butea monosperma*, native to Pakistan, Vietnam, and Malaysia. Butein has also been detected in the Tissues of the lacquer tree (*Toxicodendron vernicifluum* or *Rhus verniciflua*, a taxonomic synonym) and rosewood (*Dalbergia odorifera*), which are indigenous to China, Korea, and Japan. The sap of the lacquer tree causes dermatitis due to its urushiol content; however, upon air-drying, it forms a smooth, glossy coating, making it a traditional varnish for artisanal crafts.

Rosewood timber is utilized in furniture making and in traditional medicine. Butein, present in the sap of these plants, is a potent antioxidant and anti-inflammatory agent of medical significance. Concurrently, butein can trigger oxidative stress in malignant tumor cells through the Generation of reactive oxygen species, thereby inducing Cancer cell apoptosis.

Isoliquiritigenin is found in the roots and rhizomes of licorice (*Glycyrrhiza glabra*), which is used to produce licorice confectionery. Isoliquiritigenin has been the subject of intensive research in recent years. This chalcone is known for its action on the Central Nervous system (specifically on gamma-aminobutyric acid receptors), through which it has recently been found to exert not only a calming effect but also hypnotic properties. It also exhibits anticancer activity, as will be discussed in greater detail below. In recent years, considerable research has likewise focused on xanthohumol, a prenylated chalcone present in hop cones and beer.

1.8.1. Antioxidant Activity

3,4-Dihydroxychalcones, such as butein, sappan-chalcone, and okanin, display the most pronounced antioxidant activity at concentrations of 0.025–0.1%. The presence of hydroxyl groups at the C-2' carbon atom in ring A or a catechol group in ring B favorably influences antioxidant properties, whereas the absence of the $\alpha,\beta$-double bond diminishes this activity. Broussonin A (or broussonchalcone A) from the paper mulberry tree (*Broussonetia papyrifera*) is capable of inhibiting Lipid Peroxidation in rat Brain homogenates with an IC₅₀ = 0.63 ± 0.03 μM, an activity comparable to that of butylated hydroxytoluene (BHT). Its radical-scavenging capacity can exceed that of $\alpha$-tocopherol. Studies on macrophages have likewise demonstrated that broussonchalcone A suppresses NO production with an IC₅₀ = 11.3 μM. Another chalcone, 3-hydroxyxanthoangelol, derived from the stems of the endemic Japanese plant *Angelica keiskei*, exhibits even greater radical-scavenging activity (IC₅₀ = 0.5 ± 1.1 μM), which is tenfold higher than that of resveratrol used as a control (IC₅₀ = 5.3 μM). Xanthohumol and several of its methylated derivatives isolated from hop cones and present in beer showed higher antioxidant activity than ferulic and p-coumaric acids at concentrations of 0.1–2.0 μM. Isosalipurposide, obtained from the flowers of the blue Water lily (*Nymphaea caerulea*) native to East Africa, India, and Thailand, possesses antioxidant activity (IC₅₀ = 1.7 μg/mL) superior to that of Vitamin C (IC₅₀ = 1.95 μg/mL). Certain chalcones are able to influence cellular systems responsible for regulating cytoplasmic reactive oxygen species levels. For instance, butein inhibits TNF-$\alpha$-dependent generation of reactive oxygen species, contributing to its anti-inflammatory activity.

1.8.2. Anticancer Effects

The capacity of chalcones to exhibit anticancer activity has driven a substantial surge in research on these compounds over the past few years. Remarkably, while acting as potent antioxidants in normal cells, chalcones demonstrate pronounced pro-oxidant activity in tumor cells, leading to the inhibition of cell proliferation and the induction of apoptosis. Thus, butein—which, as mentioned above, normally exerts an anti-inflammatory effect via antioxidant mechanisms—displays pro-oxidant properties in tumor cells by interfering with cellular regulatory systems. The accumulation of reactive oxygen species (ROS) in the Cytoplasm has been observed as a consequence of tumor necrosis factor (TNF-$\alpha$) inhibition, activation of extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase. A similar ROS-mediated mechanism of cell apoptosis was observed upon Treatment of neuroblastoma and Liver cells with butein. ROS activation has also been detected following the treatment of prostate cancer cells with xanthoangelol and Ovarian cancer cells with isoliquiritigenin. Furthermore, ROS activation can enhance tumor radiosensitivity during radiotherapy, as demonstrated in Hep2 hepatocellular carcinoma cells treated with isoliquiritigenin. Hydroxychalcones exert cytotoxic effects on melanoma cells by inducing mitochondrial Glutathione depletion and ATP Cleavage.

Nevertheless, other mechanisms of tumor growth suppression involving chalcones have also been identified. For example, in melanoma cells, butein can induce apoptosis by increasing mitochondrial membrane permeability, which results in the release of cytochrome c into the cytoplasm and the activation of caspases 3 and 9. The anticancer chalcone naringenin, found in citrus fruits and tomatoes, as well as its glycoside isosalipurposide derived from the Japanese plant *Angelica keiskei*, exhibits toxicity against neuroblastoma cells by targeting Mitochondria while remaining non-toxic to normal cells. A comparable phenomenon of increased mitochondrial permeability was discovered when xanthoangelol acted on uterine cancer cells.

In studies using human pleural mesothelioma cells arising from chronic inflammatory processes (such as inhalation of asbestos dust), butein was found to exert an inhibitory effect on the METABOLISM/31.html">Transcription factors NF-$\kappa$B and STAT3, the latter participating in the expression of STAT family Proteins responsible for activating various Kinases. This suppresses the ability of cells to migrate and form clones. The capacity of butein to impede prostate tumor growth has been demonstrated not only *in vitro* but also *in vivo*. In this context, the chalcone was found to affect numerous regulatory systems, including phosphatidylinositol 3-kinases (p85, p110), the phosphorylation of Akt and I$\kappa$B$\alpha$ protein kinases, transcription factor NF-$\kappa$B regulation, and other metabolic regulators.

Xanthoangelol suppressed breast cancer cell proliferation by targeting the H2A histone protein. The cytotoxic effect of xanthoangelol is markedly more potent specifically in tumor cells compared to normal cells, as demonstrated in glioblastoma cell lines. Here, tumor cell apoptosis is triggered by the chalcone's impact on a caspase-dependent regulatory pathway. Another molecular target of xanthoangelol is the chemokine receptor CXCR4, the expression of which is upregulated via modulation of the transcription factor NF-$\kappa$B. It is hypothesized that xanthoangelol may serve as a potent therapeutic agent against acute leukemia. Its action—accompanied by alterations in the levels of FAK and AKT protein kinases and the transcription factor NF-$\kappa$B—is so efficacious that clinical trials of this agent are anticipated.

Isoliquiritigenin halts Cell Division and initiates apoptosis in lymphoblastoid cells. This process is accompanied by altered pairing and function of NUCLEOTIDES within the mRNA-microRNA complex. These disruptions are thought to lead to a substantial downregulation of Gene Expression pathways, including the regulatory pathway of the p53 transcription factor, which is involved in Cell Cycle regulation. Isoliquiritigenin inhibits the growth of multiple myeloma cells (a subtype of leukemia) by blocking the interleukin-6 (IL-6) signaling pathway. A significant reduction in ERK kinase phosphorylation and STAT3 transcription activation is observed. Isoliquiritigenin is also capable of suppressing tumor angiogenesis—The Development of capillaries essential for sustaining tumor growth. This action is mediated by the chalcone activating c-Jun kinase while inhibiting ERK kinase, whereas during the suppression of prostate cancer cell growth, conversely, activation of both ERK and AMPK kinases is observed.

Tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL) is a natural anticancer agent that displays no toxicity toward healthy cells because it specifically interacts with the death receptors TRAIL-R1 and TRAIL-R2 On the surface of cancer cells. It has been discovered that chalcones such as licochalcone, isobavachalcone, xanthohumol, butein, and certain dihydrochalcones including phloretin significantly enhance TRAIL cytotoxicity in prostate cancer cells through a sensitization effect.

The diverse alterations in regulatory systems reported by various research groups do not yet yield a unified picture of how chalcones influence carcinogenesis, but they strongly indicate the promising potential of investigating the anticancer properties of these agents.

1.8.3. Anti-inflammatory Activity

Anti-inflammatory activity is directly linked to anticancer defense and is frequently mediated through the Antioxidant Properties of these compounds. Thus, chalcones such as butein, xanthoangelol, 4-hydroxyderricin, cardamonin, 2',4'-dihydroxychalcone, isoliquiritigenin, isosalipurposide (phloridzin), and naringenin chalcone (phloretin) are capable of suppressing The activity of nuclear factor NF-$\kappa$B, which controls Introduction/24.html">DNA Transcription and participates in carcinogenesis. Specifically, the chalcone butein, along with several other polyphenolic compounds from the medicinal plant *Butea monosperma*, suppressed the p65 subunit of the NF-$\kappa$B complex, reduced the levels of tumor necrosis factor TNF-$\alpha$ and interleukins IL-6 and IL-8, promoted Erk1/2 MAPK phosphorylation, and inhibited nitric oxide synthase expression, thereby accounting for the anti-inflammatory action of this plant in traditional medicine. Isoliquiritigenin from licorice exerts a comparable action on osteoclasts. It can be observed how isoliquiritigenin impedes signal Transduction from Toll-like receptors responding to bacterial lipopolysaccharides, leading to the suppression of the inflammatory process, which—as noted above—operates via nuclear factor NF-$\kappa$B and nitric oxide synthase. The action of isoliquiritigenin on Toll-like receptors can interrupt the signaling cascade leading to interferon-$\beta$ (TRIF), which activates immune system T-cells, thereby repressing the expression of the RANTES cytokine involved in leukocyte immune responses. A analogous regulatory process concerning inflammatory mechanisms mediated by THP-1 monocytes can be suppressed by xanthoangelol and its derivatives. The regulatory process

begins with the interaction between bacterial lipopolysaccharides and the Toll-like receptor on The surface of leukocytes, which initiates cytokine production and triggers the inflammatory process. It is hypothesized that these chalcones can penetrate the specialized pocket of the MD-2 protein, which is responsible for recognizing lipopolysaccharides and activating the Toll-like receptor, thereby halting the progression of inflammation.

An important contribution to the anti-inflammatory effect of lacquer tree flavonoids, including butein, is also associated with reduced ROS levels, as well as the expression of nitric oxide synthase and cyclooxygenase (COX-2). A similar effect on cytoplasmic ROS levels and the expression of pro-inflammatory genes IL-1a, IL-6, MCP-1, and ICAM-1 was observed when xanthoangelol acted on liver cells under ischemic conditions or toxic liver injury, as well as during wound healing and the suppression of surface Skin inflammation. In this process, not only did oxidative stress decrease in damaged tissues, but angiogenesis—essential for wound healing—was also activated.

Another target of anti-inflammatory agents is histone deacetylase (HDAC), which removes the acetyl group from Lysine residues on histone proteins, thereby influencing DNA packaging and the expression of specific genes. It has been discovered that certain polyphenols, including butein, can regulate HDAC activity, thereby suppressing inflammatory processes.

Another key mechanism in the development of inflammatory processes involves the leucine zipper bZIP domain of the NRF2 protein, a transcription factor that participates in the cell's anti-inflammatory and antioxidant pathways. It is well established that bZIP zipper domains mediate protein-DNA interactions during transcription. The chalcone xanthoangelol can interact with the bZIP domain of the NRF2 protein, modulating the expression of inflammatory mediators such as nitric oxide synthase, certain interleukins, and the transcription factor TNF-α. Furthermore, it significantly upregulates the transcription of NADPH:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), and the levels of glutathione—the primary cellular antioxidant.

1.8.4. Chalcones against Diabetes and Obesity

Chalcones derived from the ashitaba plant (Angelica keiskei), used in traditional Japanese medicine, exhibit Insulin-like activity and enhance glucose uptake by adipocytes, thereby displaying antihyperglycemic effects. These compounds hold promise as potential agents for diabetes treatment. Furthermore, these chalcones can help prevent metabolic syndrome (abdominal obesity), thereby lowering the risk of type 2 diabetes. This effect is achieved by activating the expression of adiponectin—a hormone involved in regulating glucose and fatty acid metabolism, as well as governing adipose tissue development. Chalcones isolated from licorice (Glycyrrhíza glabra) were found to inhibit pancreatic lipase activity and reduce Blood levels of Cholesterol, Fatty acids, and triglycerides, thereby countering the onset of metabolic syndrome.

It was also discovered that sulfonamide derivatives of chalcone represent a novel class of highly active α-glucosidase inhibitors (IC₅₀ 0.4–1.0 μM). A recently investigated series of chalcone derivatives, designated as Chana, exhibits inhibitory activity against α-glucosidase and promotes adipocyte differentiation, which prevents fat accumulation. Methoxy-substituted chalcones are capable of suppressing hyperglycemia on par with the pharmaceutical drug insulin lispro. Naphthylchalcone can lower blood glucose levels by stimulating insulin secretion. Notably, THE POSITION OF the nitro group on the phenyl ring was found to be critical for this function.

1.8.5. Chalcone-Based Antibiotics

Chalcones can be synthesized artificially, making industrial production feasible (Fig. 18). Chemical modifications allow for a wide variety of chalcone derivatives to be produced, facilitating the development of compounds with diverse biological activities and even potent antibiotics.

Fig. 18. One of the potential synthetic pathways for chalcones. R1 and R2 represent various substituents.

Both natural chalcones and their synthetic analogues can exhibit antibacterial activity. For instance, licochalcones A and C, isolated from licorice, demonstrate The ability to protect against Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus with a minimum inhibitory concentration (MIC) of 3.3–12.5 μg/mL. Research on licochalcone A analogues revealed that an OH group at position 4 on ring A is essential for inhibiting S. aureus growth. Introducing a longer hexyl group enhances antibacterial activity; conversely, removing the lipophilic prenyl group or replacing it with a propyl group leads to a

decrease in activity. Thus, it was established that the Hydrophobicity of the molecule plays a crucial role in the antibacterial efficacy of this compound.

The dihydrochalcone asebogenin demonstrated the ability to inhibit the growth of S. aureus and methicillin-resistant S. aureus (MRSA), with IC₅₀ values of 10 μg/mL and 4.5 μg/mL, respectively. Replacing the 4’-hydroxyl group with a carboxyl group yields an active compound with high water solubility. Imparting a cationic charge via the attachment of piperazine at position 2 of ring B (Fig. 19, formula 1) produces a compound highly active against various strains of E. faecium and E. coli.

Chalcones containing two allyloxy groups, particularly 2’,4’-allyloxy-6’-methoxy chalcones (Fig. 19, formula 2), exhibit selective activity against Trypanosoma cruzi at concentrations below 25 μM. Attaching the well-known antibacterial agent oxazolidone to ring B (Fig. 19, formula 3) yields substances with enhanced antibacterial activity against various strains of S. aureus and Enterococcus faecalis, with an MIC of 32 μg/mL. Piperazine-containing chalcones (Fig. 19, formula 4) acted as potent antimicrobial agents with MIC₅₀ values ranging between 2 and 100 μg/mL against S. aureus and E. coli. Chalcone derivatives bearing thiazolidinedione and benzoic acid groups (Fig. 19, formula 5) showed exceptionally high activity against gram-positive S. aureus strains (MIC 1 μg/mL), surpassing the potency of the established antibiotic oxacillin and rivaling norfloxacin. Against certain resistant strains, the activity of these chalcone derivatives exceeded that of the aforementioned antibiotics by tens of times. Replacing one of the chalcone rings with a thiazole ring (Fig. 19, formula 6) yields compounds even more active than ampicillin.

Developing drugs against pathogens of the genus Mycobacterium that cause tuberculosis is one of the most pressing challenges, given that approximately 2 million new cases are registered worldwide each year. Highly active compounds have been derived from chalcones (Fig. 19, formula 7) with an MIC of 3.2 μg/mL against M. tuberculosis, showing no toxicity toward human cells. In another study involving a series of chalcones in which various hydroxyl groups were replaced with N-methylpiperazine, a compound was obtained (Fig. 19, formula 8) with an IC₅₀ of 3.5 μg/mL against M. tuberculosis, whereas an analogous compound with an N,N-dimethylaminopropylamine substituent exhibited even greater potency (IC₅₀ = 0.035 μg/mL) against the malaria parasite. Investigations into a series of aryloxy-azolyl chalcones identified compounds with MIC values of 0.78–3.12 μg/mL against M. tuberculosis and low cytotoxicity toward cells of various Organs. These substances also demonstrated moderate in vivo therapeutic efficacy in animals infected with a virulent strain of M. tuberculosis. A recently synthesized series of naphthylchalcones proved capable of inhibiting Tyrosine phosphatase from M. tuberculosis cells with exceptionally high efficiency, offering promising molecular models for optimizing enzyme-inhibitor interactions.

Fig. 19. Synthetic analogues of chalcones.

Chalcones can also exhibit activity against Other types of infections. For example, a series of chalcone derivatives was synthesized (Fig. 19, formula 9) that display potent activity against a wide spectrum of pathogenic Fungi, including Microsporum canis (MIC 25 μg/mL), Microsporum gypseum (1.5 μg/mL), Trichophyton mentagrophytes (MIC 3 μg/mL), Trichophyton rubrum (MIC 3 μg/mL), and Epidermophyton floccosum (MIC 0.5 μg/mL). Recently, high activity of an oxathiolone-chalcone derivative against pathogenic fungi of the genus Candida was discovered. Additionally, a series of novel guanolinyl chalcones exhibits strong antifungal activity against Candida, Cryptococcus gattii, and Paracoccidioides brasiliensis.

Chalcones are also capable of demonstrating considerable activity against viral infections. Certain successes have been achieved in combating the HUMAN IMMUNODEFICIENCY VIRUS (HIV). For instance, xanthohumol from hop cones acts as a selective inhibitor of the p24 antigen and HIV-1 Reverse Transcriptase, with MIC values of 1.28 μg/mL and 0.5 μg/mL, respectively. Screening over 90,000 compounds from the National Cancer Institute (USA) antiviral compound collection revealed that a chalcone (Fig. 19, formula 10) inhibits HIV integrase with an IC₅₀ of 2 μM in the presence of Ca²⁺ and Mg²⁺ cations, which serve as Cofactors for this process. Given that the integrase enzyme is essential for viral Replication, this discovery represents a significant milestone in antiviral drug development. Subsequently, several other chalcone derivatives exhibiting activity against this enzyme were identified, such as ferrocenyl-chalcone-difluoroborate. Currently, A number of analogous compounds active against HIV-1 integrase are undergoing clinical trials in China.