In the Laboratory
Synthesis of Diapocynin Mina S. Dasari, Kristy M. Richards, Mikaela L. Alt, Clark F. P. Crawford, Amanda Schleiden, Jai Ingram, Abdel Aziz Amadou Hamidou, Angela Williams, and Patricia A. Chernovitz Chemistry Department, Park University, Parkville, MO 64152 Rensheng Luo Department of Chemistry and Biochemistry, University of Missouri–St. Louis, St. Louis, MO 63121 Grace Y. Sun Biochemistry Department, University of Missouri, Columbia, MO 65212 Ron Luchtefeld Total Diet and Pesticide Research Center, U. S. Food and Drug Administration, 11510 W 80th St., Lenexa, KS 66214 Robert E. Smith* Chemistry Department, Park University, and Total Diet and Pesticide Research Center, U. S. Food and Drug Administration, 11510 W 80th St., Lenexa, KS 66214; *
[email protected] A procedure is described for synthesizing diapocynin, a metabolite of apocynin (Scheme I) that has anti-oxidative and anti-inflammatory properties (1–6). Apocynin (4-hydroxy-3methoxyacetophenone) was identified during activity guided isolation of immunomodulatory constituents from Picrorhiza kurroa, a native plant grown in the mountains of India, Nepal, Tibet, and Pakistan. This compound specifically blocks the activity of NADPH oxidase, a cell membrane enzyme known to protect against reactive oxygen species, or ROS (7). Its effectiveness was also demonstrated against ischemia and hypoxia– reoxygenation-induced injuries (1–6). It has also been suggested that apocynin is metabolized to diapocynin, which may be more active than apocynin. In a dissertation published on the Internet (8), the synthesis of diapocynin was described. It was based on the synthesis of a similar compound, dehydrodivanillin, which was published in 1916 (9), except it started with apocynin and it used the potassium salt of peroxydisulfate and reported a lower yield. Sodium peroxydisulfate can be converted to a sulfate radical, with a standard oxidation–reduction potential of 2.6 V. Iron(II) sulfate is a common initiator. It has been used to oxidize chlorinated and non-chlorinated solvents (10). In the original article (9), vanillin was converted to dehydrodivanillin by oxidation with a mixture of iron(II) sulfate heptahydrate and sodium peroxydisulfate in hot water. The temperature was not specified and the reaction required 30 min. Diapocynin has also been synthesized in smaller quantities using the enzymes horse radish peroxidase and myeloperoxidase (11, 12). Still, diapocynin is not readily available from commercial sources.
O
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2 O OH
CH3
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CH3
CH3 H3C
O
O H
O H
Scheme I. Conversion of apocynin to diapocynin.
O
CH3
Experimental Procedure About 2 g of apocynin (acetovallinone, Sigma-Aldrich, St. Louis, MO) was dissolved in 200 mL of deionized water (in a 250 mL Erlenmeyer flask that was open to the air) by stirring and heating until the solution was boiling gently. About 0.15 g of iron(II) sulfate heptahydrate and 1.6 g of sodium peroxydisulfate were added and stirring was continued. A brown precipitate formed gradually. After 5 min, the mixture was cooled by removing the Erlenmeyer flask from the hot plate and letting it sit on the lab bench and it was filtered using a Büchner funnel and shark skin filter paper. The collected precipitate was dissolved in about 25 mL of 3 M NaOH and then re-precipitated by slowly adding about 15 mL of 6 M HCl with stirring. The precipitate was filtered and washed three times with 100 mL of boiling water. The product was dried overnight in a desiccator. An FTIR spectrum was obtained using a Varian 800 FTIR (Palo Alto, CA) with an attenuated total reflectance (ATR) accessory, from Pike Technologies (Madison, WI). A 1H NMR spectrum of diapocynin in DMSO-d6 was obtained using a Bruker ARX 500 MHz NMR, with a pulse width of 30° and 1 s pulse delay. The chemical shifts were referenced to the DMSO peaks that were set to 2.50 ppm. More experimental details on the LC–MS analysis of the dried product are in the online supplement. Hazards Even though diapocynin may prevent diseases, it is also possible that it is highly toxic, so it should always be handled with care. There are no toxicity data on diapocynin, and data for apocynin are limited. According to the data sheet from Calbiochem (13), apocynin is a cell-permeable, anti-inflammatory phenolic compound that acts as a potent and selective inhibitor of NADPH oxidase; is shown to block peroxynitrite formation in murine macrophages; is reported to increase glutathione synthesis through activation of AP-1 and is reported to prevent phagocytosis of myelin by macrophages. According to the data sheet from Fisher (14) apocynin may cause eye and skin irritation and may cause respiratory and digestive tract irritation. Sodium peroxydisulfate is a strong oxidizing agent and is highly toxic. Similarly, NH3(aq) is a toxic base and produces highly
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In the Laboratory
Results Only 5 min at 100 ° C was needed for the reaction. Some triapocynin impurity was produced when the reaction proceeded for 30 min, which was the reaction time used by previous workers (8, 9). Small quantities of unreacted apocynin were removed by thoroughly washing the diapocynin with boiling water three times. The most intense peaks in the FTIR spectrum (Figure 1) were 3318 (OH), 1666 (C=O), 1588 (aryl C=C), 1286, 1204, 1127, 1083, 910 cm‒1. The 1H NMR spectrum of diapocynin (Figure 2) contained peaks with the following chemical shifts, in ppm, and assignments in parentheses: 2.494 (CH3), 3.895 (OCH3), 7.451 and 7.463 (aromatic CH) and 9.465 (OH). (The peak at ~3.2 ppm is due to HOD, moisture in the sample that exchanged one of its hydrogens with DMSO-d6.) This work should not be taken as having an impact on FDA policy or regulations. Literature Cited 1. Wang, Q.; Tompkins, K. D.; Simonyi, A.; Korthuis, R. J.; Sun, G. Y. Brain Res. 2006, 1090, 182–189. 2. Yang, Z.; Asico, L. D.; Yu, P.; Wang, Z.; Quinn, M. T.; Sibley, D. R.; Romero, G. G.; Felder, R. A.; Jose, P. A. Am. J. Physiol. Regul. Comp. Physiol. 2006, 290, 96–104. 3. Hashimoto, Y.; Niikura, T.; Ito, Y.; Terracita, K.; Nishimoto, I. J. Pharmacol. Exp. Therap. 2002, 300, 736–745. 4. Dodd-o, J. M.; Welsh, L. E.; Salazar, J. D.; Walinsky, P. L.; Zweier, J. L.; Baumgartner, W. A.; Pearse, D. B. AJP-Heart. 2004, 287, 927–936. 5. Lafeber, F. P. J. G.; Beukelman, C. J.; van den Worm, E.; van Roy, J. L. A. M.; Vianene, M. E.; van Roon, J. A. G.; van Dijk, H.; Bijlsma, J. W. J. Rheumetol. 1999, 38, 1088–1093. 6. Klees, R. F.; DeMarco, P. C.; Salazsnyk, R. M.; Ahuja, D.; Hogg, M.; Antoniotti, S.; Kamath, L.; Dordick, J. S.; Plopper, G. E. J. Biomed. Biotechnol. 2006, 2006, 1–10. 7. Stolk, J.; Hilterman, T. J.; Dijkman, J. H. Am. J. Respir. Cell. Mol. Biol. 1994, 11, 95–102. 8. van den Worm, E.; van den Berg, A. J. J.; Kemeling, G. M.; Beukelman, C. J.; Halkes, S. B. A.; Labadie, R. P.; van Dijk, H. Isolation, Characterization and Activity of Diapocynin, An Apocynin Metabolite. Chapter 5. http://igitur-archive.library.uu.nl/ dissertations/1957866/c5.pdf (accessed Nov 2007). 9. Elbs, K.; Lerch, H. J. Prakt. Chem. 1916, 93, 1–6. 10. Block, P. A.; Brown, R. A.; Robinson, D. Novel Activation Technologies for Sodium Persulfate In Situ Chemical Oxidation. Proceedings of the Fourth International Conference on Remediation of Chlorinated and Recalcitrant Compounds, Monterey, CA, Battelle Me-
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irritating fumes. Also, HCl is a strong acid, highly corrosive, produces highly irritating fumes, and is toxic. Students should be told that NH3(aq) will leave a soapy feeling on the fingers and can cause severe eye damage if a contaminated finger is used to touch or scratch one’s eyes. Methanol is flammable and toxic.
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Wavenumber / cm
ź1
Figure 1. FTIR spectrum of diapocynin.
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Chemical Shift (ppm)
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Figure 2. 1H NMR spectrum of diapocynin in DMSO-d6.
11. 12. 13. 14.
morial Institute, May, 2004. http://www.geo-log.de/uploads/media/ novel_persulfate_activation_technologies.pdf (accessed Nov 2007). van den Worm, E.; Beukelman, C. J.; van den Berg, A. J. J.; Kroes, B. H.; Labadie, R. P.; van Dijk, H. Eur. J. Pharmacol. 2001, 433, 225–230. Antoniotti, S.; Santhanam, L.; Ahuja, D.; Hogg, M. G.; Dordick, J. S. Org. Lett. 2004, 6, 1975–1978. Calbiochem. Apocynin Material Safety Data Sheet. http://www. emdbiosciences.com/Products/ProductDisplay asp?catno=178385 (accessed Nov 2007). Fisher Scientific. Apocynin Material Safety Data Sheet.https:// fscimage.fishersci.com/msds/99894.htm (accessed Nov 2007).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Mar/abs411.html Abstract and keywords Full text (PDF) Links to cited URLs and JCE articles Supplement Additional experimental details MS and UV spectra of diapocynin Structures of diapocynin and apocynin generated by Spartan
Journal of Chemical Education • Vol. 85 No. 3 March 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
