User:ToxicComponents/sandbox
Benzarone
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| Trade names | Benzarone, Fragivix |
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| Formula | C17H14O3 |
| Molar mass | 266.29 g·mol−1 |
| 3D model (JSmol) | |
| Density | 1.2±0.1 g/cm3 |
| Melting point | 126.0 °C (258.8 °F) |
| Boiling point | 493.6 °C (920.5 °F) |
| Solubility in water | Water: 0.02 mg/mL
DMF: 30 mg/mL (20 °C) DMSO: 10 mg/mL Ethanol: 10 mg/mL mg/mL (20 °C) |
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History
[edit]Benzarone is a benzofuran derivative that was initially introduced in the 1950s for the treatment of peripheral venous disorders. Marketed under various brand names, including Vasarex, it was developed as a vasodilator to improve blood circulation in patients suffering from chronic venous insufficiency.
However, by the 1980s, growing concerns over its safety profile emerged due to increasing reports of severe hepatotoxicity, including cases of acute and chronic hepatitis, cirrhosis, and liver enzyme abnormalities. As a result, Benzarone was ultimately withdrawn from the market in multiple countries. Further investigation into its toxicity revealed that Benzarone disrupts mitochondrial function. For instance,a study conducted by Kaufman et al. (2005) in isolated rat hepatocytes revealed that benzarone significantly decreased mitochondrial membrane potential by 54%, indicating mitochondrial toxicity as a possible mechanism of its hepatotoxic effects[1].
Case Reports of Liver Injury
[edit]Several clinical cases have demonstrated benzarone's potential to cause severe liver damage, even in patients without prior liver disease.
Case 1: Acute Hepatitis (Taiwan, National Taiwan University Clinic)
[edit]A 44-year-old woman was hospitalized after experiencing icterus (jaundice) for two months. Upon admission, she exhibited elevated liver function markers, but diagnostic tests—including abdominal sonography, serological examinations, and autoimmune profiles—failed to identify an underlying cause. However, a detailed review of her medication history revealed that she had been taking 200 mg of benzarone (2 tablets per day) for four months without a prescription.
The Roussel Uclaf Causality Assessment Model (RUCAM), which evaluates the likelihood of drug-induced liver injury, yielded a score of 7, suggesting that her liver damage was probably caused by benzarone[2].
Additional Cases
[edit]Three more cases of severe hepatotoxicity linked to benzarone were reported:
- A 35-year-old woman diagnosed with (sub)fulminant hepatitis.
- A 67-year-old woman with macronodular cirrhosis.
- A 68-year-old man with severe chronic active hepatitis and cirrhosis, along with positivity for anti-smooth muscle antibodies (indicating possible autoimmune involvement).
Among these cases, two patients died, further strengthening the evidence for benzarone-induced liver toxicity. [3]
Structure and Reactivity
[edit]Chemical Structure and Biological Activity
[edit]Benzarone is a non-halogenated benzofuran derivative that shares structural similarities with amiodarone, a well-known mitochondrial toxin. Its molecular structure consists of a benzofuran core attached to an aromatic phenyl ring and a hydroxyacetophenone moiety. Additionally, it contains both a hydroxyl (-OH) group and a ketone (C=O) group, which significantly influence its biological activity, solubility, and interactions with biomolecules.
The hydroxyl (-OH) group enhances the water solubility of benzarone, thereby increasing its bioavailability in the body. Meanwhile, the ketone (C=O) group facilitates dipole interactions with proteins and contributes to the drug’s lipophilic properties, enabling it to dissolve in fat tissues. These structural characteristics make benzarone an effective vasodilator and influence its metabolism and clearance.
The hepatotoxicity and metabolism of benzarone is also discussed in later sections.
Synthesis
[edit]Benzarone can be synthesized in a four-step reaction process. First, salicyl aldehyde is converted into 2-acetyl-1-benzofuran. This is achieved by boiling salicyl aldehyde with potassium hydroxide in absolute ethanol. Once the potassium salt is dissolved chloroacetone is added gradually and the mixture is submitted to reflux for two hours. After filtration the residue is distilled to give 2-acetyl-1-benzofurane. 2-ethyl-1-benzofurane is obtained through a reduction reaction of the product after distillation. The next step in the process is a Friedel-Crafts acylation reaction using 2-methoxybenzoyl chloride catalysed by tin tetrachloride, this results in the reaction product 2-ethyl-3-(4-methoxybenzoyl)-1-benzofurane. Lastly, the methoxy side chain is converted into an alcohol side chain. This is done by reacting the product with pyridine hydrochloride. The final product, (2-ethyl-1-benzofuran-3-yl)-(4-hydroxyphenyl)methanone), is recrystallized from acetic acid for purification to obtain the final product, pure benzarone. [4]
The exact experimental can be found in the appendix.
Available forms
[edit]In history, benzarone was administered as an oral tablet for easier ingestion and major availability. [4]
Mechanism of action
[edit]Benzarone has been described to be a mitochondrial toxin as it disrupts the mitochondrial membrane potential required for cellular respiration. This results from uncoupling of oxidative phosphorylation as benzarone causes the leakage of protons across the inner mitochondrial membrane independently of ATP synthase. This inhibits the electron transport chain, causing mitochondrial dysfunction. [1] Inhibition of the electron transport chain by an uncoupler of oxidative phosphorylation stimulates the production of reactive oxygen species (ROS).
Increased ROS levels contribute to oxidative stress and damage to other cellular structures. ROS generation has also been associated with mitochondrial-mediated cell death through the opening of the mitochondrial membrane pore, causing the mitochondria to swell which disrupts the outer membrane. The resultant release of cytochrome c from the mitochondria can activate caspases in the cytosol and signal cell apoptosis. Moreover, due to impaired ATP generation by oxidative phosphorylation of the cell, the apoptotic stimuli could switch towards cellular necrosis. [1]
In addition to causing mitochondrial dysfunction, benzarone is also a potent inhibitor of β-oxidation in hepatocytes. In particular, benzarone has been found to be an inhibitor of the enzymes acyl-CoA dehydrogenase and β-ketothiolase of the β-oxidation pathway. Under conditions where the liver depends mainly on β-oxidation for the production of ATP, the presence of benzarone could therefore further limit cellular ATP content and lead to hepatocyte necrosis. [1]
Currently, benzarone is only being used experimentally for various research purposes, which are discussed under efficacy.
Metabolism
[edit]In humans, it has been found that at low oral concentrations, benzarone is well-absorbed and undergoes extensive phase I metabolism in the liver. Benzarone is hydroxylated in phase I metabolism, forming phenol or alcohol type groups, resulting in more reactive intermediate compounds. [5] These reactive intermediates could also bind to liver proteins, causing hepatocyte damage and trigger immune inflammatory responses.
The hydroxylated metabolites then undergo further glucuronidation by β-glucuronidase in phase II metabolism before excretion in the urine. [5]
Indications
[edit]Currently, benzarone does not have any clinically approved uses. It is only being used for research purposes. [6][7]
Efficacy and side effects
[edit]Benzarone is an EYA (Eyes Absents) inhibitor. EYA’s are multifunctional proteins involved in organogenesis. Benzarone inhibits the tyrosine phosphatase activity of eyes absent homolog 3 (EYA3; IC50 = 17.5 μM), as well as reduces the proliferation and migration of human umbilical vein endothelial cells (HUVECs) when used at a concentration of 7.5 μM. [4] These properties make benzarone a potent anticancer agent in cases of ovarian and breast cancer caused by over expression of EYA’s. [8]
The use of benzarone as an anticancer agent has been researched for Ewing sarcoma. Ewing sarcoma is a rare type of cancer that occurs in bones or in the soft tissue around the bones. [9] In 2021 a study was conducted on the efficacy of EYA3 tyrosine phosphatase inhibition, accomplished by benzarone, in attenuating tumour growth and angiogenesis in an Ewing sarcoma patient-derived tumour xenograft. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumour growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. It appeared that a benzarone treatment was effective, a dose of 25 μg/g reduced tumour growth in an A-673 Ewing sarcoma, mouse xenograft model. [10]
Another cancer that is linked to EYA’s is medulloblastoma. It is one of the most common malignant brain tumours of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signalling pathway. [11] EYA1, a halo acid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). EYA1 is not inhibited by benzarone, but benzarone was used as a point of departure for development of possible functional inhibitors for EYA1. A panel of 35 derivatives were developed and tested in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signalling. [12]
Benzarone was also proven to be effective as a drug for chloroform edema on mice. On the back skin of a mouse a wheal was produced by applying chloroform containing a fluid rich in protein. It was investigated whether a series of edema-protective drugs could inhibit the development of wheal edema. Only benzarone proved a statistically significant inhibition. [13]
The effect of benzarone on serum lipids and arterial wall of cholesterol fed rats was researched as well. Rats of BD X strain and SHR/NIH Montreal Ingelheim strain received a diet containing 3.9% cholesterol or 3.7% cholesterol plus 0.6% benzarone, respectively, and libitum for 5 or 9 months. The cholesterol-benzarone diet caused a body weight reduction of 10%, a relative increase of serum HDL and a corresponding decrease of serum LDL and VLDL, as compared with the effects of the cholesterol diet. The conclusion of the study was that benzarone application to cholesterol fed rats effects a statistically significant decrease of liver cholesterol and triglycerides. [14]
Benzarone is also used for the treatment of venous vascular disorders. [15] The influence of benzarone on the energy metabolism of the wall of the rabbit common carotid artery and the rat portal vein was investigated by measuring the oxygen consumption of in vitro incubated vessel segments. [16] The research indicated that benzarone can increase the oxygen consumption of the carotid artery in a concentration-dependent manner, without altering the basal tone of the artery. This suggests that benzarone may directly affect the metabolism of smooth muscle cells. Additionally, benzarone has been observed to decrease the oxygen consumption of the portal vein, while also blocking spontaneous phasic activity and lowering basal tone. These effects imply that benzarone may have an uncoupling effect on oxidative phosphorylation, which could be consistent with its anti-inflammatory properties. [16]
Finally, benzarone is an active metabolite of the urate anion transporter 1 (URAT1) inhibitor benzbromarone. This inhibits URAT1 in Xenopus oocytes expressing the human enzyme, which helps lower serum uric acid levels and thus can be used to treat kidney diseases. [4]
The drug was initially ‘successfully’ tested on animals. But this is a stark example of a drug where animal research findings were not translatable to humans and posed great risks to human health. [17]
Toxicity
[edit]Several case studies of benzarone-associated hepatotoxicity have been reported. Liver biopsies and assessments conducted revealed liver lesions of chronic active hepatitis or acute hepatitis in most cases. [2][3][18] Consistent with the mechanism of action of benzarone, examinations also revealed various patterns of liver necrosis and fibrosis, as well as hepatocyte apoptosis. [2][18] This could be explained by the blocking of mitochondrial electron transport chains by benzarone, causing an accumulation of ROS in the liver. [2]
The combination of toxic metabolite formation, oxidative stress, and prolonged hepatic accumulation makes benzarone a hepatotoxic compound. These adverse effects contributed to the withdrawal of four pharmaceutical products containing benzarone in 1992 by the Federal Health Office from the market in various countries due to safety concerns.[19]
Effects on animals
[edit]In a study into the hepatotoxicity of benzarone isolated rat liver cells were subjected to several tests. In hepatocytes 20 micromol/L benzarone caused a decrease in mitochondrial membrane potential by 54%. In isolated mitochondria benzarone decreased state 3 oxidation and respiratory control ratios for L-glutamate (a 50% decrease of respiratory control ratio at 10.8 micromol/L). Benzarone also uncoupled oxidative phosphorylation. In the presence of 100 micromol/L benzarone mitochondrial β-oxidation was decreased by 87%, whereas ketogenesis was not affected. Furthermore, reactive oxygen species production was increased, mitochondrial leakage of cytochrome c was induced in HepG2 cells, and permeability transition was induced. At the same concentration, benzarone induced apoptosis and necrosis of isolated rat hepatocytes. [1]
A clinical report on benzarone, written between 1967 and 1997 and published by Kindai Igakusha and Yubunsha [20], showcases the results on several toxicity tests executed on rodents. Acute toxicity data obtained by LD50 testing showed that 12 mg/kg administered subcutaneously it causes dermatitis in skin and appendages in both species. Rats were also exposed intraperitoneally to a dosage of 3400 mg/kg which gave rise to behavioural effects, namely somnolence and general depressed activity. [21]
The toxicological effects of multiple doses were also analysed with TDLo, Lowest published toxic dose testing. A dosage of 52500 mg/kg of body weight was administered to rats orally over a period of 5 weeks. Which resulted in several toxic effects. Gastrointestinal hypermotility and diarrhoea was observed as well as changes in tubules (including acute renal failure, acute tubular necrosis) in the kidney, urethra and bladder. The experiment was repeated over a period of 30 weeks which resulted in nutritional and gross metabolic changes. Weight loss or decreased weight gain and changes in calcium were observed. In both studies several fatal cases were reported as well. [21]
The effect of benzarone on reproductivity was also tested with TDLo, Lowest published toxic dose, tests. 1020 mg/kg was administered orally to female rats 9-14 day(s) after conception, which led to maternal effects in the ovaries and fallopian tubes and to specific developmental abnormalities in the musculoskeletal system. A second test was done with the same conditions, but the dose was changed to 6 gm/kg. This resulted in effects on embryo or fetus, fetotoxicity was observed (except death, e.g., stunted fetus). A test conducted on female mice, where 600 mg/kg was administered orally 7-12 day(s) after conception, resulted in specific developmental abnormalities in the musculoskeletal system as well as physical effects on newborn (e.g. reduced weight gain). [21]
Appendix
[edit]Experimental process on how to prepare Benzarone:
[edit]The process of preparation of benzarone, (2-ethyl-1-benzofuran-3-yl)-(4-hydroxyphenyl)methanone, includes the following steps: First, one mole of salicylic aldehyde is added to one mole of potassium hydroxide in absolute ethanol. The mixture is brought to boiling in a water bath until the potassium salt formed is dissolved. One mole of chloroacetone is gradually added and the solution is boiled in a reflux condenser for 2 hours. The mixture is then cooled, and the formed potassium chloride precipitate is separated off by filtration. The residue is distilled to give 2-acetyl-1-benzofurane (BP: 135°C/15 mm Hg). The 2-acetyl-1-benzofurane product is then reduced by hydrazine hydrate in an alkaline medium (by process of Hyuang-Minlon, J.A.C.S., 1946, 68, 2487) to give 2-ethyl-1-benzofurane (BP: 211°-212°C). This (2-Ethyl-1-benzofurane) is condensed with 2-methoxybenzoyl chloride in the presence of tin tetrachloride (according to the process described by Bisagni, J.C.S., 1955, 3694). Thus 2-ethyl-3-(4-methoxybenzoyl)-1-benzofurane is obtained (BP: 226°C/15 mm Hg).
One part of 2-ethyl-3-(4-methoxybenzoyl)-1-benzofurane is mixed with two parts of pyridine hydrochloride and heated with an oil bath at 210°C under nitrogen flow for 1 hour. After cooling 10 parts of 0.5 N HCl are added. A water layer is mixed with 20 parts of 1% NaOH. The alkaline layer is separated and acidified with dilute HCl. The dropped precipitate (2-ethyl-1-benzofuran-3-yl)-(4-hydroxyphenyl)methanone) is recrystallized from acetic acid (MP: 124.3°C) for purification.
Use of AI
[edit]Copilot was used in the section of “efficacy and side effects” to expedite the search of sources discussing the use of benzarone and its efficacy. Finding proper sources on this subject was rather difficult because most sources on benzarone focus on its hepatotoxic character and on the reasons why it has been withdrawn.
ChatGPT was utilized primarily for grammar and spelling corrections to ensure clarity and readability. Like Copilot, it was also employed to expedite the search for sources discussing the use of Benzarone, particularly its available forms, as obtaining comprehensive information online proved to be challenging. The AI-assisted search helped streamline the research process by identifying relevant scientific literature and references more efficiently.
External links
[edit]- Benzarone (no date) ChemicalBook. Available at: https://www.chemicalbook.com/ChemicalProductProperty_EN_CB6893840.htm (Accessed: March 6, 2025).
- Benzarone (no date) Chemspider.com. Available at: https://www.chemspider.com/Chemical-Structure.224468.html (Accessed: March 6, 2025).
- Benzarone (no date) Drugcentral.org. Available at: https://drugcentral.org/drugcard/317 (Accessed: March 6, 2025).
- Hcs, A. to O. (no date) Safety Data Sheet, Caymanchem.com. Available at: https://cdn.caymanchem.com/cdn/msds/34903m.pdf (Accessed: March 6, 2025).
- PubChem (no date) Benzarone, Nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/255968 (Accessed: March 6, 2025).
- TCI (no date) Benzarone Tcichemicals.com. Available at: https://www.tcichemicals.com/OP/en/p/E1289 (Accessed: March 6, 2025).
References
[edit]- ^ a b c d e Kaufmann, Priska; Török, Michael; Hänni, Anya; Roberts, Paul; Gasser, Rodolfo; Krähenbühl, Stephan (2005-04). "Mechanisms of benzarone and benzbromarone-induced hepatic toxicity". Hepatology (Baltimore, Md.). 41 (4): 925–935. doi:10.1002/hep.20634. ISSN 0270-9139. PMID 15799034.
{{cite journal}}: Check date values in:|date=(help) - ^ a b c d Tseng, Hsu-Hua; Tseng, Liang-Wei; Yang, Hung-Chih (2020-09). "Benzarone-induced liver injury: First case report in Asia". Journal of the Formosan Medical Association. 119 (9): 1445–1447. doi:10.1016/j.jfma.2020.02.002.
{{cite journal}}: Check date values in:|date=(help) - ^ a b Babany, Gérard; Larrey, Dominique; Pessayre, Dominique; Degott, Claude; Rueff, Bernard; Benhamou, Jean-Pierre (1987-01). "Chronic active hepatitis caused by benzarone". Journal of Hepatology. 5 (3): 332–335. doi:10.1016/S0168-8278(87)80039-9.
{{cite journal}}: Check date values in:|date=(help) - ^ a b "Benzarone | 1477-19-6". ChemicalBook. Retrieved 2025-03-14.
- ^ a b c Wood, S. G.; John, B. A.; Chasseaud, L. F.; Bonn, R.; Grote, H.; Sandrock, K.; Darragh, A.; Lambe, R. F. (1987-01). "Metabolic fate of the thrombolytic agent benzarone in man: comparison with the rat and dog". Xenobiotica. 17 (7): 881–896. doi:10.3109/00498258709043998. ISSN 0049-8254.
{{cite journal}}: Check date values in:|date=(help) - ^ "Benzarone (Fragivix) | hURAT1 Inhibitor | MedChemExpress". MedchemExpress.com. Retrieved 2025-03-14.
- ^ Wempe, Michael F.; Jutabha, Promsuk; Quade, Bettina; Iwen, Timothy J.; Frick, Morin M.; Ross, Ian R.; Rice, Peter J.; Anzai, Naohiko; Endou, Hitoshi (2011-04-28). "Developing Potent Human Uric Acid Transporter 1 (hURAT1) Inhibitors". Journal of Medicinal Chemistry. 54 (8): 2701–2713. doi:10.1021/jm1015022. ISSN 0022-2623. PMC 3124071. PMID 21449597.
{{cite journal}}: CS1 maint: PMC format (link) - ^ Vartuli, Rebecca L.; Zhou, Hengbo; Zhang, Lingdi; Powers, Rani K.; Klarquist, Jared; Rudra, Pratyaydipta; Vincent, Melanie Y.; Ghosh, Debashis; Costello, James C.; Kedl, Ross M.; Slansky, Jill E.; Zhao, Rui; Ford, Heide L. (2018-06-01). "Eya3 promotes breast tumor–associated immune suppression via threonine phosphatase–mediated PD-L1 upregulation". Journal of Clinical Investigation. 128 (6): 2535–2550. doi:10.1172/JCI96784. ISSN 0021-9738.
- ^ "Ewing Sarcoma", SpringerReference, Berlin/Heidelberg: Springer-Verlag, retrieved 2025-03-14
- ^ Wang, Yuhua; Pandey, Ram Naresh; Roychoudhury, Kaushik; Milewski, David; Kalin, Tanya V.; Szabo, Sara; Pressey, Joseph G.; Hegde, Rashmi S. (2021-05-01). "Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis". Molecular Cancer Therapeutics. 20 (5): 803–815. doi:10.1158/1535-7163.MCT-20-0749. ISSN 1535-7163.
- ^ "Medulloblastoma: Diagnosis and Treatment - NCI". www.cancer.gov. 2018-09-17. Retrieved 2025-03-14.
- ^ Hwang, Grace H.; Pazyra-Murphy, Maria F.; Seo, Hyuk-Soo; Dhe-Paganon, Sirano; Stopka, Sylwia A.; DiPiazza, Marina; Sutter, Nizhoni; Gero, Thomas W.; Volkert, Alison; Ombelets, Lincoln; Dittemore, Georgia; Rees, Matthew G.; Ronan, Melissa M.; Roth, Jennifer A.; Agar, Nathalie Y.R. (2024-03-15). "A Benzarone Derivative Inhibits EYA to Suppress Tumor Growth in SHH Medulloblastoma". Cancer Research. 84 (6): 872–886. doi:10.1158/0008-5472.CAN-22-3784. ISSN 0008-5472.
- ^ "Die Wirkungen von Iscador auf den praecancerösen Zustand und auf das Carzinom". Der Merkurstab. 1958. doi:10.14271/dms-11468-de. ISSN 0935-798X.
- ^ Rechholtz, Charlotte Hanna Maren. Der Einfluss von vertikaler Ganzkörpervibration, Alendronat und 8-Prenylnaringenin auf die Muskulatur der ovariektomierten Ratte (Thesis). University Goettingen Repository.
- ^ "Benzarone (Fragivix) | hURAT1 Inhibitor | MedChemExpress". MedchemExpress.com. Retrieved 2025-03-14.
- ^ Beer, A.-M.; Lukanov, J.; Sagorchev, P. (2000-09). "Die Wirkung von Caulophyllum D4 auf die spontane kontraktile Aktivität der glatten Muskulatur - The Effects of Caulophyllum D4 on the Spontaneous Contractile Activity of Smooth Muscle -". Geburtshilfe und Frauenheilkunde. 60 (9): 456–458. doi:10.1055/s-2000-8022. ISSN 0016-5751.
{{cite journal}}: Check date values in:|date=(help) - ^ "Dangerous Drugs". Animal-Free Science Advocacy. Retrieved 2025-03-14.
- ^ a b Hautekeete, Marc Leopold; Henrion, Jean; Naegels, Serge; DeNeve, André; Adler, Michael; Deprez, Carlne; Devis, Ghislain; Klöppel, Günter (1995-02). "Severe hepatotoxicity related to benzarone: a report of three cases with two fatalities". Liver. 15 (1): 25–29. doi:10.1111/j.1600-0676.1995.tb00102.x. ISSN 0106-9543.
{{cite journal}}: Check date values in:|date=(help) - ^ "Evaluation of certain veterinary drug residues in food. Thirtyeight report of the Joint FAO/WHO Expert Committee on Food Addition (WHO Technical Report Series 815). 64 Seiten, 16 Tab. World Health Organisation, Geneva 1991. Preis: 9,– Sw. fr. (developing countries: 6,30 Sw. fr.)". Food / Nahrung. 36 (4): 419–420. 1992-01. doi:10.1002/food.19920360427. ISSN 0027-769X.
{{cite journal}}: Check date values in:|date=(help) - ^ "Kiso to rinsho. The clinical report. - NLM Catalog - NCBI". www.ncbi.nlm.nih.gov. Retrieved 2025-03-14.
- ^ a b c "Benzarone". www.chemsrc.com. Retrieved 2025-03-14.