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; *robert.smith@fda.hhs.gov

A procedure is described for synthesizing diapocynin, a Experimental Procedure

metabolite of apocynin (Scheme I) that has anti-oxidative and
anti-inflammatory properties (1–6). Apocynin (4-hydroxy-3-
methoxyacetophenone) 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 effec-
tiveness 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 dehy-
drodivanillin 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 CH3 CH3 O of NADPH oxidase; is shown to block peroxynitrite formation
O CH3
2 phagocytosis of myelin by macrophages. According to the data
CH3
O sheet from Fisher (14) apocynin may cause eye and skin irrita-
OH
H3C O O O O CH3
H H
Scheme I. Conversion of apocynin to diapocynin.
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 peroxydis-
ulfate were added and stirring was continued. A brown precipi-
tate 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 dis-
solved 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 Cal-
biochem (13), apocynin is a cell-permeable, anti-inflammatory
phenolic compound that acts as a potent and selective inhibitor
in murine macrophages; is reported to increase glutathione
synthesis through activation of AP-1 and is reported to prevent
tion 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

© Division of Chemical Education  •  www.JCE.DivCHED.org  •  Vol. 85  No. 3  March 2008  •  Journal of Chemical Education 411
 In the Laboratory

irritating fumes. Also, HCl is a strong acid, highly corrosive, 110

produces highly irritating fumes, and is toxic. Students should
be told that NH3(aq) will leave a soapy feeling on the fingers and

Transmittance (%)
can cause severe eye damage if a contaminated finger is used to 100

touch or scratch one’s eyes. Methanol is flammable and toxic.

90
Results
Only 5 min at 100 ° C was needed for the reaction. Some 80

triapocynin impurity was produced when the reaction proceeded

for 30 min, which was the reaction time used by previous work- 70
ers (8, 9). Small quantities of unreacted apocynin were removed 4000 3000 2000 1000
by thoroughly washing the diapocynin with boiling water three Wavenumber / cm
ź1
times. The most intense peaks in the FTIR spectrum (Figure 1)
were 3318 (OH), 1666 (C=O), 1588 (aryl C=C), 1286, 1204, Figure 1. FTIR spectrum of diapocynin.
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. 10 9 8 7 6 5 4 3 2
Chemical Shift (ppm)
Literature Cited
Figure 2. 1H NMR spectrum of diapocynin in DMSO-d6.
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412 Journal of Chemical Education  •  Vol. 85  No. 3  March 2008  •  www.JCE.DivCHED.org  •  © Division of Chemical Education