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D. J. Hawranik et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2383–2385
Scheme 1. Proposed biosynthesis of usnic acid (1) based upon Shibata.3 Methylphloracetophenone (2) is produced by a polyketide synthase and then oxidized first to 7,
followed by elimination of H2O to produce 1. S-adenosyl methionine (SAM) is the source of the methyl group bonded directly to the aromatic ring.
ing our yield was to dissolve 6 in anhydrous acetone and then
remove the solvent by rotary evaporation. We found it necessary
to repeat this process at least twice to optimize the yield. On a
1 mmol scale our conditions gave a 17% yield of 2, with the side
products being the result of oxygen methylation. However, when
we increased our reaction to a 10 mmol scale, the yield improved
to a more preparatively useful 45% (Scheme 4).7 Again the major
Scheme 2. Acylation of trihydroxytouene (3) forming 4. Reagents and conditions:
(i) BF3–OEt2, HOAc, Ac2O, 100 °C, 3 h, anhydrous.
side products were the result of methylation at the oxygen atoms.
Reducing the number of equivalents of iodomethane resulted in an
overall erosion of the isolated yield of 2, without any significant
change in the ratio of this desired product to the side products.
It was also possible to recover more than 20% of the starting
material under the reaction conditions described in Scheme 4
implying that recycling of starting material could further increase
our yield. Furthermore, although we have not yet demonstrated
this, it should be possible to improve the overall yield of (2) from
(6) by removing the O-methyl groups from the side products. We
have developed a straightforward methodology for the synthesis
of the key usnic acid (1) intermediate, 2, in one-step from commer-
cially available precursors.
Our next efforts focused on attempting to convert our synthetic
methylphloracetophenone (2) to usnic acid (1) by using horserad-
ish peroxidase (HRP) as a model system for the oxidative enzyme.
Using HRP with H2O2 as oxidant and 5 mg of 2 we were able to de-
tect the production of usnic acid (1) (Scheme 5) using a combina-
tion of LC–MS and 1H NMR. We chose to use HRP as a model
system as this had been successfully used in a previous synthesis
of usnic acid (1)8 and is commercially available. Key to our success
in this effort was the realization that it was critical to add the H2O2
stepwise over an extended period of time (2.5 h). After a total incu-
bation time of 5.5 h at 37 °C a 40% yield, of hydrated usnic acid (7)
was obtained from the enzyme assay mixture as determined by 1H
NMR.9 This identity of this molecule was confirmed by LC–MS and
by comparison of the 1H NMR with the spectral data for a commer-
cial standard sample of usnic acid (1). A modified enzyme assay
work-up10 using acetic anhydride followed by the addition of sul-
furic acid11 resulted in the detection of 1 in our enzyme assay in
addition to a significant amount of 7. A significant amount of unre-
acted starting material was recovered and we could not detect any
other oxidation products in our HRP enzyme assay mixture. In
In an effort to deactivate the aromatic ring towards acylation,
we decided to attempt to protect the hydroxyl groups by methyl-
ation. We treated trihydroxytoluene (3) with an excess of iodo-
methane, anticipating trimethoxytoluene as the sole product.
However, somewhat unexpectedly, the only product isolated from
this reaction was compound 5 (Scheme 3) with no trace of trimeth-
oxytoluene detected. Compound 5 is the result of extensive carbon
methylation of the aromatic ring of 3 by iodomethane. Trihydroxy-
toluene (3) is an ambident nucleophile whose alcohol oxygen
atoms are stronger nucleophiles than the carbon atoms in the ring.
Methylation with iodomethane occurs at the carbon atom rather
than the oxygen atom since iodide is a weak base and a good leav-
ing group and thus considered a soft anion.
We used the result in Scheme 3 as a suggestion that it might be
possible to use a similar reaction to alkylate the aromatic ring of
phloracetophenone (6) (Scheme 4). The direct alkylation of 6 with
iodomethane would provide an efficient one-step synthesis of 2
from an atom economy view. In addition, 6 is commercially avail-
able and considerably less expensive than 3. This would improve
the overall attractiveness of this approach. Initial attempts at this
reaction, at room temperature, were successful in producing a
small amount of 2 (<10%). The major side products of this reaction
are the result of methylation of the oxygen atoms producing mono-
, di- and tri methoxy versions of 2. By reducing the temperature to
0 °C, with slow addition (5 min) of the excess iodomethane
(4 equiv), we could improve our yield of 2 while minimizing the
production of the oxygen methylated products. Trihydroxyaceto-
phenone (6) is sold as the monohydrate and a key step in improv-
Scheme 3. Methylation of trihydroxytoluene. Reagents and conditions: (i) CH3I,
K2CO3, DMF, 100 °C.
Scheme 4. Methylation of phloracetophenone (6) with excess ioodomethane.
Reagents and conditions: (i) CH3I, K2CO3, acetone, 0 °C, 9 h (45%) see Ref. 7.
Scheme 5. Oxidation of methylphloracetophenone (2) by horseradish peroxidase
(HRP). Reagents and conditions: (i) See Ref. 9; (ii) See Ref. 10.