Synthesis of Flavone and Initiation with Cytochrome P4501A2
Patrick Curley
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
pbw9@wildcats.unh.edu
Results and Discussion:
Introduction
Flavone is the backbone of an organic group of molecules known as flavonoids.
These flavonoids are found naturally in many plants and have significant biomedical
uses, as they have been shown to be metabolized by cytochrome P450. Flavonoids has
been shown to act as an inhibitor in these reactions.1 Flavone was synthesized as
shown in Scheme 1 and then incubated with cytochrome P4501A2 to observe its
possible inhibition properties.
Flavone was incubated with cytochrome P4501A2 by following a procedure
published in Drug Metabolism & Disposition3. Naphthalene was chosen as a substrate
for the reaction because it is metabolized by cytochrome P4501A2 and its metabolites
are known. Expected results would be to see the metabolites of naphthalene present in
the positive control and see the absence of naphthalene metabolites in the presence of
flavone, as it is excpeted to inhibit the binding site of P4501A2
Table 1: Preparation of Solutions for Inhibition of Flavone
Reagent
Scheme 1: Synthesis of Flavone
Results and Discussion
The first step was the benzoylation of the phenolic –OH group of the starting
material 2-hydroxyacetophenone 1 to yield 2-benzoyloxyacetophenone 2. (84% yield) In
the presence of potassium hydroxide and heat, 2 undergoes the Baker-Venkataraman
rearrangement in order to form 2-hydroxydibenzoylmethane 3.2 (91% yield) The
cyclization of 3 in the presence of sulfuric and acetic acid yields the final product,
flavone 4. (60% yield) Flavone was purified through recrystallization with hexanes and
had melting point range of 96.6 ˚ C-97.9˚C .
Mechanism 1: Baker-Venkataraman Rearrangement
.5 M Phosphate Buffer
Volumes (µL) for .5 mL total
volume
100
Final
Concentrations
100mM
Solution A
25
Solution B
CYP 4501A2 (1pmol/µl)
5
25
1.3 mM NADP+
3.3 mM G6P
3.3 mM MgCl2
.4 U/mL G6PDH
50 pmol/mL
Water
Substrate (78 mM in MeCN)
See Below
See Below
300 µM
Inhibitor (45mM in MeCN)
See Below
Trial Name
1A2 (+ control) 1A2A
Substrate
Concentration
Inhibitor
Concentration
Water
300 µM
(1.9 µL)
0 µM
(0 µL)
343.1 µL
300 µM
(1.9 µL)
300 µM
(3.3 µL)
339.8 µL
150 or 300µM
1A2B
1A2 (control)
300 µM
0 µM
(1.9 µL)
(0 µL)
150 µM
300 µM
(1.6 µL)
(3.3 µL)
341.5 µL
341.7 µL
Future Work
After incubating for 15 minutes in
a 37˚C water bath, the solutions
were centrifuged and the organic
layers were extracted and a gas
chromatography/mass
spectrometry spectrum was
obtained. The chromatogram
shows that the positive control of
naphthalene shows no evidence
of metabolites, which is
unexpected. There are several
unidentified compounds
consistently present in each
spectrum. This data suggests that
there might be a problem with the
procedure or the enzyme itself, as
the peaks are consistent
throughout the results.
Since there is excess product left over from reactions, possible future work should
include editing the incubation procedure and the concentrations of solutions in order to
obtain conclusive results. If this is achieved, additional flavonoids could be synthesized
from remaining product and inhibition experiments could be carried out to gain a
further understanding of the interactions between human cytochrome P450 and
flavonoids.
1H
NMR of Crude 2-hydroxydibenzoylmethane
1H
NMR of Flavone Product
Conclusions:
From the data obtained, it is clear that the incubation procedure must be edited in
order to obtain accurate results. The enzyme could possibly be flawed, as the
inconsistencies in data were present throughout all results.
Acknowledgments :
I would like to thank Holly Guevara, Deepthi Bhogadhi, and Professor Greenberg for guidance
throughout this project, Dr. Stacia Sower for making her laboratory available to us and the Department of
Chemistry for funding.
References:
1.) Sridhar, Jayalakshmi; Ellis, Jamie; Dupart, Patrick; Liu, Jiawang; Stevens, Cheryl L; Foroozesh, Maryam; Drug Metab Lett. 2012; 6(4):
275–284.
2.) M. Hardwood; C.J. Moody; J.M. Percy. Experimental Organic Chemistry; Blackwell Science; Oxford, UK; 1999; Pgs 624-626
3.) Cho,TM; Rose, RL; Hodgson, E; Drug Metab Dispos. 2006 Jan; 34(1):176-83.