Full text of DOI:10.1055/s-0036-1591556

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© Georg Thieme Verlag Stuttgart · New York
2018, 29, A–C
letter
A
en
V. Timokhin et al.
Letter
Synlett
Synthesis of Nepetoidin B
Vitaliy Timokhina
Matthew Regnera
Yukiko Tsujia
John Grabberb
John Ralph*a
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1. Baeyer–Villiger
oxidation
MeO
MeO
OMe
OMe
HO
HO
O
OH
nepetoidin B
17%
2. demethylation
a Department of Energy Great Lakes Bioenergy Research
Center and Department of Biochemistry, the Wisconsin
Energy Institute, University of Wisconsin, Madison, WI,
53726, USA
jralph@wisc.edu
b U.S. Dairy Forage Research Center, USDA-ARS, 1925
Linden Drive West, Madison, WI, 53706, USA
Received: 16.02.2018
Accepted after revision: 26.02.2018
Published online: 28.03.2018
Several studies have demonstrated potential biological
applications for 1: it has shown antibacterial, antifungal,
and free-radical scavenging properties, and it could be used
as a chemotaxonomic marker.8–10,12,15,30–31 Furthermore, 1 is
an inhibitor of xanthine oxidase and thus could help to reg-
ulate hyperuricemia in human gout,12 as well as inhibit the
production of nitric oxide, which is stimulated by bacterial
lipopolysaccharide.27 Surprisingly, in spite of its medicinal
characteristics, there has been no reported synthesis of 1 to
date. The commercial price of nepetoidin B varies from
about $150/mg to about $5,000/g, a price prohibitive to its
use in research studies. In continuation of our work on the
production of bioproducts from biomass, especially with
regard to nepetoidin B and its potential incorporation into
lignin in plant cell walls, we sought to design a synthetic
route for its production.32
DOI: 10.1055/s-0036-1591556; Art ID: st-2018-b0110-l
Abstract The first synthesis of nepetoidin B in an overall yield of 17%
was achieved in two steps through Baeyer–Villiger oxidation of com-
mercially available 1,5-bis(3,4-dimethoxyphenyl)-1,4-pentadien-3-one
with oxone to produce the tetramethylated nepetoidin B, followed by
demethylation using boron tribromide.
Key words Baeyer–Villiger oxidation, demethylation, medicinal chemis-
try, natural products
Nepetoidin B (1) was initially isolated from Plectranthus
caninus in 1975 as the (Z,E)-1 isomer (Figure 1).1 Since then,
(Z,E)-1 and its corresponding stereoisomer, (E,E)-1, have
been isolated from a variety of plant species using solvent
extraction and chromatographic purification.2–31 The amounts
of (Z,E)-1 and (E,E)-1 vary from 9:1 to 1:1 in different
plants,1,10 where the stereochemistry of the two isomers
has historically been elucidated based on the analyses of J-
coupling constants from NMR spectroscopic data.1–9,12,15–20
Nepetoidin’s level in plants is rather low; in a recent iso-
lation from 100 kg of Salvia miltiorrhiza Bunge (Danshen),
extraction and purification yielded just 37 mg.27 Based on
the genes, enzymes, and metabolites that have been associ-
ated with nepetoidin B, several mechanisms have been pro-
posed for its biosynthesis: (1) condensation of the enol
form of dopaldehyde with caffeic acid;1,5,15,19 (2) oxidative
decarboxylation of rosmarinic acid, from which stereo-
specific loss of the pro-R or pro-S methylene proton during
this process would result in the formation of (Z,E)-1 or
(E,E)-1;21,25 and (3) decarboxylation of p-hydroxyphenyl-
pyruvic acid and subsequent esterification with the enol
form of 3,4-dihydroxyphenylacetaldehyde.25
5'
OH
OH
4'
3'
6'
O
9
7
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2
5
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HO
HO
1
(E)
3
4
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7
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OH
Figure 1 The two naturally occurring isomers of nepetoidin B, (Z,E)-1
and (E,E)-1, isolated from plants
Herein, we report the first synthesis of nepetoidin B.
Our synthetic route (Scheme 1) began with Baeyer–Villiger
oxidation of commercially available 1,5-bis(3,4-dimethoxy-
phenyl)-1,4-pentadien-3-one (2) with oxone, based on a re-
ported procedure for the transformation of α,β-unsaturated
ketones into their corresponding vinyl acetates.33 We opti-
mized the reaction conditions for the stereospecific trans-
formation of only one of the two symmetrical α,β-unsatu-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–C

B
V. Timokhin et al.
Letter
Synlett
OMe
OMe
O
O
oxone
MeO
MeO
OMe
OMe
MeO
MeO
O
DMF
r.t.
2
3
OH
OH
O
HO
HO
BBr3
O
1
CH2Cl2
–78 °C to r.t.
Scheme 1 Synthesis of nepetoidin B
rated ketones into its corresponding vinyl acetate; our final
conditions afforded tetramethylated nepetoidin B [(E,E)-3
isomer] in an isolated yield of 40% from 2, with the starting
material being recovered in an isolated yield of 37% (i.e., the
overall conversion of starting material to product was about
63%). It is interesting to note that we did not observe iso-
merization of (E,E)-3 to (Z,E)-3 during the Baeyer–Villiger
oxidation of 2. After optimizing the demethylation of 3
using boron tribromide, we obtained 1 in an isolated yield
of 43%, thus completing our two-step synthesis of nepetoi-
din B in an overall yield of 17% (33% when the recovery of
starting compound 2 is taken into account).
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1591556.
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References and Notes
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Synthetic nepetoidin B was obtained as a mixture of the
two isomers in a ratio of 94:6 (E,E)-1/(Z,E)-1 (as estimated
1
by H NMR analysis; Figures S1 and S2 in the Supporting
Information). Considering that the (E,E)-1 isomer was the
predominant product in our synthesis, it appears to be the
thermodynamically favored isomer. The mixture of the iso-
mers of nepetoidin B [(E,E)-1 94% and (Z,E)-1 6%] was sepa-
rated by HPLC.34
In conclusion, we have described the first synthesis of
nepetoidin B. Currently, commercial sources charge about
$5,000/g for the product, and so we expect that this conve-
nient synthesis could allow for nepetoidin B to be manufac-
tured at a significantly reduced cost of about $500/g. Its
potential value as a medicinal compound would seem to
make this a worthwhile venture.
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Funding Information
The project was funded by Stanford University’s Global Climate and
Energy Program (GCEP), with analytical facilities (primarily NMR)
funded in part by the DOE Great Lakes Bioenergy Research Center
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(DOE Office of Science BER DE-FC02-07ER64494).
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oilgcai
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d
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vorin
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atl
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B(
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E
R
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9
4)
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Acknowledgment
The authors are grateful to Justin Mobley for discussion regarding the
Baeyer–Villiger oxidation conditions and Steven D. Karlen for help in
separating the isomers of nepetoidin B.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–C

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V. Timokhin et al.
Letter
Synlett
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course of 1 h, and then to warm to r.t. over the course of 1 h.
After once again cooling the mixture to −78 °C, it was quenched
by addition of saturated aqueous NaHCO3 (18 mL) and then
allowed to warm to r.t. over 20 min. The mixture was parti-
tioned between EtOAc (120 mL) and H2O (150 mL), and the
aqueous layer was extracted with EtOAc (2 x 50 mL). The com-
bined organic layers were washed with brine (50 mL), then
dried over anhydrous Na2SO4, and the solution was concen-
trated in vacuo. The residue was purified by flash column chro-
matography on silica gel (MeOH/CH2Cl2 10:90) to furnish nepe-
toidin B, compound 1, as a yellow solid (490 mg, 43%). Rf = 0.58
(MeOH/CH2Cl2 15:85). The ratio of isomers (E,E)-1/(Z,E)-1 is
94:6, as determined by H NMR spectroscopic analysis (signals
at 5.67 (d, J = 7.4 Hz, H-7) and 6.36 (d, J = 15.8 Hz, H-8) for the
(Z,E)-1 and (E,E)-1 isomers, respectively).
HPLC Separation of the Isomers of Nepetoidin B [(E,E)-1 and
(Z,E)-1]
1
(28) Odonbayar, B.; Murata, T.; Matsumoto, N.; Batkhuu, J.; Sasaki, K.
Mongolian Journal of Chemistry 2016, 17, 14.
The mixture of the isomers of nepetoidin B [(E,E)-1 94% and
(Z,E)-1 6%] obtained from the demethylation of (E,E)-3 was sep-
arated by HPLC. To collect a sufficient quantity for 1H and 13C
NMR analysis, 1 (12 mg) was dissolved in MeOH (350 μL), and
the method described above was run five times using 50 μL
injections of the solution. The appropriate fractions were com-
bined and concentrated in vacuo to afford each purified isomer
for further analysis. (E,E)-1 (7 mg): 1H NMR (500 MHz, acetone-
d6): δH = 6.36 (d, J = 15.8 Hz, H-8), 6.38 (d, J = 12.8 Hz, H-7'),
6.77−6.78 (m, H-6' and H-5), 6.89 (d, J = 8.2 Hz, H-5'), 6.93 (d, J =
1.8 Hz, H-2), 7.11 (dd, J = 8.2, 2.1 Hz, H-6), 7.22 (d, J = 2.1 Hz,
H-2'), 7.68 (d, J = 15.9 Hz, H-7), 7.81 (d, J = 12.8 Hz, H-8'), 8.21
(br. s, 4 x OH). The obtained NMR spectroscopic data for (E,E)-1
matched literature values.10,15,19 (Z,E)-1 (0.3 mg): 1H NMR (500
MHz, acetone-d6): δH = 5.67 (d, J = 7.4 Hz, H-7'), 6.51 (d, J = 15.9
Hz, H-8), 6.81 (d, J = 8.2 Hz, H-5'), 6.90 (d, J = 8.2 Hz, H-5), 7.00
(dd, J = 8.2, 2.1 Hz, H-6'), 7.13 (dd, J = 8.2, 2.1 Hz, H-6), 7.26 (d,
J = 2.0 Hz, H-2), 7.26 (d, J = 7.3 Hz, H-8'), 7.37 (d, J = 2.1 Hz, H-2'),
7.73 (d, J = 15.9 Hz, H-7), 8.05 (br. s, 4 x OH).1,10,15,19,30
(29) Venuprasad, M. P.; Kandikattu, H. K.; Razack, S.; Amruta, N.;
Khanum, F. Biomed. Pharmacother. 2017, 91, 1.
(30) Beladjila, K. A.; Cotugno, R.; Berrehal, D.; Kabouche, Z.; De Tom-
masi, N.; Braca, A.; De Leo, M. Nat. Prod. Res. 2017, 20, 1.
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tother. Res. 2018, 32, 185.
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Bruijnincx, P. C. A.; Weckhuysen, B. M. Angew. Chem. Int. Ed.
2016, 55, 8164.
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Llavona, R.; Concellon, C.; del Amo, V. Org. Lett. 2013, 15, 2810.
(34) Synthesis of 2-(3,4-Dihydroxyphenyl)ethenyl-3-(3,4-dihy-
droxyphenyl)-2-propenoate (1)
To a stirred solution of (E,E)-3 (1.341 g, 3.620 mmol, 1.0 equiv)
in dry CH2Cl2 (20 mL) under argon was added neat BBr3 (2.09
mL, 21.722 mmol, 6.0 equiv) dropwise at −78 °C. The solution
was stirred at the same temperature for another 30 min, at
which point the mixture was allowed to warm to 0 °C over the
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–C