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

PART 1. MAJOR FLAVONOIDS

1.10. Aurones

The name "aurones" comes from the Latin word aurum, meaning gold. Aurones impart a golden-yellow color to plants, which is responsible for the floral hues of several well-known garden plants (Fig. 22).

Class="center">Fig. 22. Examples of some of the best-known aurones. The general Chemical Structure of aurones is also shown as two stereoisomers, along with the atomic numbering in the molecule.

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For instance, leptosidin, found in the flowers of large-flowered coreopsis (Coreopsis grandiflora), was the first aurone discovered in 1943. Another prominent example is aureusidin, present in the flowers of snapdragons (Antirrhinum). Currently, attempts are underway to create Transgenic Plants containing aureusidin to enhance their nutritional properties. Specifically, a transgenic lettuce (Lactuca sativa) containing aureusidin in its leaves has been successfully developed. It has been shown that the altered foliage color of plants producing aureusidin is accompanied by an enhanced capacity to scavenge superoxide radicals.

Hispidol and its Glycosides have been found in soybeans (Glycine max). Sulfuretin is present in the ornamental plant bastard teak (Butea monosperma), as well as in various species of the genera Coreopsis, Dahlia, and Simsia. 4,6,4'-trihydroxyaurone is found in the Indian kino tree (Pterocarpus marsupium). Although the majority of aurones occur in higher dicotyledonous plants, these compounds can also be found in brown Algae (Spatoglossum variabile).

In nature, aurones are less widely distributed than other flavonoids and have been less extensively studied. An aurone molecule consists of a benzofuran ring joined to a benzylidene group at position 2. Notably, the five-membered ring distinguishes aurones from most other flavonoids, which possess six-membered rings. Aurone molecules can form two isomers, designated as the E-configuration and the Z-configuration (Fig. 22). Plant Tissues predominantly contain Z-aurones because this configuration is thermodynamically more stable.

In plants, aurones serve as a defense against fungal and bacterial infections and herbivorous insects, while the bright coloration of aurone-containing flowers acts as an attractant for pollinators. Their potential biomedical Applications have driven a surge in research dedicated to aurone study. The possibility of synthesizing novel aurones with enhanced therapeutic activity has provided further impetus for research in this direction. Advances in synthetic techniques, such as The Use of ultrasound, can substantially reduce reaction times to 5–30 min.

1.10.1. Anticancer activity

In the Treatment of numerous pathologies, particularly cancers, The phenomenon of multidrug resistance is of critical importance. It has been discovered that 4-hydroxy-6-methoxy-aurones and 4,6-dimethoxy-aurones (Fig. 23) exhibit a high affinity for the C-terminal nucleotide-binding domain of P-glycoprotein (also known as ABCB1), an ABC transporter class protein responsible for the efflux of pharmacological agents from Cells.

Fig. 23. Comparative binding affinity for P-glycoprotein among synthetic aurones, Chalcones, and flavones.

It has been experimentally demonstrated that, by disrupting drug efflux mechanisms, aurones stimulate the intracellular accumulation of the chemotherapeutic agent Paclitaxel significantly more effectively than chalcones or flavones. For example, synthetically derived 4,6,3',4',6'-pentamethoxyaurone reduced The rate of drug efflux from cells by a factor of ten. Aurones can also exert an inhibitory effect on the breast Cancer resistance protein (BCRP). It was shown that in the presence of micromolar concentrations of synthetic 4,6-dimethoxyaurone, the accumulation of the drug mitoxantrone in a tumor Cell culture increased more than twofold (Fig. 24). This aurone also exhibited antiproliferative activity, resulting in a 20-fold reduction in the IC₅₀ value of mitoxantrone. Furthermore, 4,6,3',4'- and 4,6,3',5'-tetramethoxylated aurones were shown to suppress the expression of the ABCG2 drug-resistance protein. In addition, methoxylated aurones can directly interact with the substrate-binding site of this protein, enhancing ATPase activity and inhibiting the ability of ABCG2 to pump drugs out of The Cell.

Fig. 24. Comparison of The activity of a synthetic aurone and a chalcone against ABCG2.

The anticancer effect of aurones is also manifested in their ability to inhibit protein Kinases that regulate Cell Division. For instance, cyclin-dependent kinases (CDKs) represent a promising yet challenging target for pharmacological agents intended for cancer therapy, particularly chronic Lymphocytic Leukemia. Flavonoids have proven to be highly effective agents against these kinases. Clinical trials evaluating the efficacy of flavopiridol in this disease are currently underway. However, the therapeutic utility of flavopiridol is constrained by its low Specificity across various CDK isoforms, whereas certain synthetic aurones (Fig. 25) have demonstrated greater efficacy specifically against CDK1 kinase, which is advantageous for treating this condition.

Fig. 25. Comparison of the Molecular structure and inhibitory activity against various CDK forms for flavopiridol and a synthetic aurone.

The effects of synthetic aurones on sphingosine kinase (SphK)—an enzyme regulating the synthesis of bioactive cell Membrane Lipids, sphingosines, and ceramides—have also been investigated. These lipid mediators participate in cell signaling, apoptosis, and the Pathogenesis of numerous disorders, including cancer. As demonstrated in animal studies, 3',4'-dihydroxyaurone can modulate sphingosine kinase activity and thereby suppress tumor growth. Treatment over an 18-day period successfully achieved a 50% reduction in tumor volume.

The cytotoxicity of compounds toward tumor cells is widely exploited in oncology. Certain synthetic aurones (Fig. 26) can exhibit high toxicity against cancer cells. Minor structural modification of an aurone originally isolated from the tropical plant Uvaria hamiltonii yields an agent highly toxic to rapidly dividing myeloid leukemia cells (K562), which is capable of arresting cell division at the G₂/M phase. This effect stems from the ability of this substance to interact with tubulin at the colchicine-binding site and thereby inhibit its polymerization.

Fig. 26. Cytotoxicity of synthetic aurones against human myeloid leukemia cells (K562), gastric adenocarcinoma cells (SGC-7901), and human umbilical vein endothelial cells (HUVEC).

It should be noted that in this particular context, aurones are inferior to certain other flavonoids. For instance, analogous molecules derived from chalcones exhibit hundreds of times higher tubulin-binding activity than aurones, which is likely attributable to the greater conformational flexibility of the chalcone Skeleton. Conversely, the synthetic molecule 6-alloyloxyl-4'-trifluoromethylaurone emerges as the most potent inhibitor of cell division against gastric adenocarcinoma cells, although its exact MECHANISM OF ACTION remains to be fully elucidated. Furthermore, replacing the B-ring of the aurone with a piperazine moiety yielded highly effective inhibitors of cell division in various carcinoma forms at the G₀/G₁ phase, which are also capable of triggering apoptosis with an IC₅₀ of 4.1–13.1 µM.

One of the strategies in tumor growth therapy is the suppression of their Vascular System development. Specifically, agents that inhibit vascular epithelium growth are employed. It was established that the presence of a diethylamine group at the 4’ position of the aurone molecule is essential for obtaining highly effective inhibitors of HUVEC vascular endothelial cell division with an IC₅₀ = 0.25 μM. These molecules also proved to be inhibitors of lung and breast cancer cell growth while exhibiting low toxicity toward non-cancerous cells.

The use of antioxidants represents one of the mechanisms in carcinogenesis therapy. The flavoprotein NAD(P)H:quinone oxidoreductase 1 (NQO1) is part of the cellular defense system against the action of reactive oxygen species (ROS) and is capable of detoxifying harmful Quinones. The elimination of quinones ensures the Stability of the p53 tumor suppressor factor. Therefore, therapeutic interventions aimed at increasing cytoplasmic NQO1 levels are frequently regarded as effective measures in cancer treatment. It was demonstrated that fluorinated derivatives of 4,6-dimethoxyaurone (Fig. 27) double NQO1 activity at submicromolar concentrations.

Fig. 27. Induction of NQO1 and scavenging of the superoxide radical by synthetic aurone derivatives. CD is the aurone concentration required to double NQO1 activity.

The induction of NQO1 occurs As a result of the activation of the xenobiotic defense system involving the AhR polyphenol receptor and the Nrf2 METABOLISM/31.html">Transcription factor. Aurones also exhibit high radical-scavenging activity, particularly against the superoxide radical. Under experimental conditions, the antiradical activity of aurones was investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), toward which aurones display an activity 100 times higher than that of ascorbic acid.

1.10.2. Anti-inflammatory Action

Replacing the benzyldiene group of aurones with a 2,2-bis-aminomethyl group yields a compound capable of inhibiting The production of inflammatory cytokines—tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The highest activity was exhibited by 2,2-bis-pyrrolidinomethyl aurone, which completely suppressed cytokine synthesis at a concentration of 10 μM. Furthermore, it was shown that aurones act as inhibitors of nitric oxide production and are capable of scavenging Lipid Peroxidation products.

In traditional Asian medicine, the Histology/2.html">EXTRACT FROM THE heartwood of the lacquer tree (Rhus verniciflua) is used for the treatment of Blood stasis and cancer. It was discovered that sulfuretin, contained in this plant, inhibits the expression of nitric oxide synthase, cyclooxygenase-2, the proinflammatory cytokines TNF-α, IL-1β, and prostaglandin E₂. Among the aurones present in this plant, 6-O-methylsulfuretin exhibited the highest activity in reducing the levels of nitric oxide (IC₅₀ = 9.3 μM) and prostaglandin E₂ (IC₅₀ = 1.6 μM).

1.10.3. Treatment of Brain Function Disorders

Aminomethylaurones and their indanone analogue are acetylcholinesterase inhibitors, which can be utilized for the temporary amelioration of condition in patients suffering from Alzheimer's disease. In in vitro experiments, aurones were more active (IC₅₀ = 0.082–1.54 μM) than the commonly used drug rivastigmine (IC₅₀ = 2.07 μM). Favorable results were also obtained in behavioral tests on animals treated with these aurones. Molecular modeling demonstrates that the indanone core fits well into the Active Site of acetylcholinesterase. The activity of these substances can be substantially enhanced by replacing the B-ring with an N-benzylpyridine group (Fig. 28). The potency of these aurone derivatives (IC₅₀ = 10–22 nM) exceeds that of the reference drug donepezil (IC₅₀ = 28 nM).

Fig. 28. Synthetic aurone analogues containing a pyridine group act as acetylcholinesterase inhibitors.

1.10.4. Hormonal Action

Hyperthyroidism (Graves' disease) is caused by the Excessive production of the THYROID Hormones triiodothyronine and thyroxine. One of the therapeutic targets for this condition is the enzyme iodothyronine deiodinase, whose overexpression leads to the overproduction of the corresponding hormone. Plant extracts with antihormonal activity, lacking aurones, have long been used in the treatment of Graves' disease. However, it was discovered that certain natural aurones—aureusidin, sulfuretin, and 4,6,4’-trihydroxyaurone—are among the most potent natural inhibitors of this enzyme. As early as the mid-1980s, it was demonstrated that introducing an iodine atom at the 3’ position of the 4,6,4’-trihydroxyaurone molecule yields an agent capable of successfully competing with thyroxine, thereby inhibiting the enzyme (IC₅₀ = 0.5 μM). It is hypothesized that an aurone molecule bearing hydroxyl groups at positions 4 and 4’ may be mistakenly recognized by the enzyme as a hormone molecule.

1.10.5. Protection Against Obesity and Diabetes

The aurone sulfuretin from the plant Rhus verniciflua is one of the most active flavonoid-type inhibitors of aldose reductase (ALR2), an enzyme involved in the reduction of glucose to sorbitol, which is a contributing factor in The Development of diabetes. The potency of sulfuretin is comparable to that of the pharmaceutical agent epalrestat. In addition, sulfuretin acts as an inhibitor of advanced glycation end-product (AGE) formation, the accumulation of which causes various health complications in diabetic patients. Nevertheless, the inhibitory activity of sulfuretin against AGE formation was 10 times lower than that of the drug aminoguanidine. Animal experiments demonstrated that sulfuretin protects pancreatic β-cells from damage induced by streptozotocin, which was used in this study to induce experimental diabetes. This protective effect is achieved through the suppression of nuclear factor kappa B (NF-κB) activity.

Aurone derivatives conjugated with Fatty acids (aurone fatty acid esters) are capable of significantly reducing the proliferation of adipocytes (fat cells). Concurrently, a decrease in glucose uptake by these cells is observed, which potentially could be utilized in the Prevention of obesity, although The Mechanism of this action has not yet been thoroughly investigated.

1.10.6. Defense Against Protozoan Parasites

The ability of aurones to protect against the causative agents of leishmaniasis and malaria was discovered in the late 1990s to early 2000s. The most toxic compounds against leishmaniasis pathogens were hydrophobic aurones possessing a small number of hydroxyl groups. For instance, 4’,6’-dihydroxyaurone and 6-methoxyaurone were the most active in disrupting the mitochondrial respiratory Functions of Leishmania parasites (Fig. 29). The Introduction of additional hydroxyl groups into the molecule resulted

in a marked decrease in the activity of these substances. The antiparasitic activity of aurones can be attributed to the inhibition of the mitochondrial enzyme fumarate reductase, which is essential for Anaerobic Metabolism, wherein energy is derived via the reduction of fumarate to succinate. Aurones are capable of suppressing the activity of this enzyme by more than 90%, whereas chalcones reduced activity by only 46.6%.

Fig. 29. Synthetic aurones exhibiting the highest antiparasitic activity.

Aurones also disrupted the erythrocytic stage of the malaria parasite's life cycle. The most active aurones contained multiple methoxy and acetoxy substituents. For example, 4,6,4’-triacetoxy-3’,5’-dimethoxyaurone (Fig. 29) exhibited high activity against both chloroquine-sensitive and chloroquine-resistant strains of the parasite. The replacement of the endocyclic oxygen atom with a nitrogen atom significantly enhanced the potency of these agents. In a study of 35 different aurone derivatives, it was demonstrated that activity increased in the presence of a 4,6-dimethoxy group as well as hydrophobic substituents at the 4’ position.

1.10.7. Antibacterial Activity

Like many flavonoids, aurones are capable of exhibiting antibacterial activity. For example, 6,7-dihydroxyaurones target chorismate synthase, an enzyme involved in the shikimate pathway for the synthesis of essential aromatic acids (phenylalanine, Tyrosine, Tryptophan) in plants, Fungi, and Bacteria, but absent in animals. This makes components of this pathway convenient targets for the development of human-safe Antibiotics. The presence of a hydroxyl group at the 2’ position and an ether linkage at the 4’ position (Fig. 30) yields agents with an IC₅₀ < 1 μM.

It has been shown that replacing the B-ring in aurones with imidazole or furan groups (Fig. 30) yields potent growth inhibitors against Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis, although their mechanism of action remains to be elucidated. The presence of the benzofuran ring, characteristic of aurones, was found to be the most critical structural element determining the activity of these compounds. In addition, the 2-configuration of the aurone molecule is preferred.

Fig. 30. Aurone derivatives with antibacterial activity. The top row shows chorismate synthase inhibitors of Streptococcus pneumoniae. The bottom row shows growth inhibitors of Staphylococcus epidermidis. The MIC value represents the concentration of the substance required to inhibit Microbial growth after overnight incubation.

1.10.8. Antiviral activity

Aurones are currently considered the most promising natural scaffolds for developing synthetic agents targeting Influenza virus neuraminidase—one of the key surface envelope Proteins responsible for viral entry into the respiratory tract, as well as the release of mature virions from infected cells, which facilitates the spread of infection. Consequently, neuraminidase remains the most extensively studied target for antiviral drug discovery. The aurones sulfuretin (IC₅₀ = 30–50 µM) and hispidol (IC₅₀ = 22 µM) exhibit significantly higher activity against influenza A and B Viruses than flavonoids from other classes, such as flavones, flavanones, flavonols, or isoflavones. High activity requires the presence of specific functional groups in the molecule: 4’-OH, 7-OH, and 4-O.

Aurones also demonstrate inhibitory activity against the RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus. This enzyme plays a crucial role in viral RNA Replication and is regarded as a primary target for Antiviral Therapy. Hydrophobic aurone molecules can inhibit NS5B at micromolar concentrations (Fig. 31). Mutagenesis and molecular docking studies have revealed that aurones bind not to the active site of the enzyme, but to an allosteric pocket (Thumb Site I). These naturally occurring, non-toxic compounds offer a distinct advantage over previously developed fully synthetic and potentially toxic pharmaceutical agents.

Fig. 31. Natural and synthetic aurones as Inhibitors of the hepatitis C virus NS5B enzyme.