Establishment of benzodioxazine core structure for sarcodonin class of natural products by X-ray analysis

Article abstract of DOI:10.1021/ol400595k

Sarcodonin ε (2), isolated from Sarcodon scabrosus, was treated with trimethylsilyldiazomethane to yield a crystalline methylated derivative 6. The structure of 6 was determined by X-ray analysis, which confirmed the presence of an unprecedented N(1b)-OMe group, the configuration at N(1b) and the 1,3,4-substituted phenyl ring structure of 2. More importantly, the structures of 6 and 2 have answered the intriguing problem of solving the core structure of the sarcodonin class of natural products, establishing that sarcodonins have a benzodioxazine core structure, rather than the recently proposed benzodioxane aminal core structure.

Full text of DOI:10.1021/ol400595k

ORGANIC
LETTERS
2013
Vol. 15, No. 9
2076–2079
Establishment of Benzodioxazine Core
Structure for Sarcodonin Class of Natural
Products by X‑ray Analysis
Hironori Masubuti,† Yasuhisa Endo,‡ Hiroshi Araya,‡ Hidehiro Uekusa,† and
Yoshinori Fujimoto*,†
Department of Chemistry and Materials Science, Tokyo Institute of Technology,
Meguro, Tokyo 152-8551, Japan, and School of Agriculture, Meiji University,
Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
fujimoto.y.aa@m.titech.ac.jp
Received March 5, 2013
ABSTRACT
Sarcodonin ε (2), isolated from Sarcodon scabrosus, was treated with trimethylsilyldiazomethane to yield a crystalline methylated derivative 6.
The structure of 6 was determined by X-ray analysis, which confirmed the presence of an unprecedented N(1b)-OMe group, the configuration at
N(1b) and the 1,3,4-substituted phenyl ring structure of 2. More importantly, the structures of 6 and 2 have answered the intriguing problem of
solving the core structure of the sarcodonin class of natural products, establishing that sarcodonins have a benzodioxazine core structure, rather
than the recently proposed benzodioxane aminal core structure.
Since the first isolation of the nitrogenous p-terphenyl
natural product sarcodonin (1a) in 2000,1 more than
10 sarcodonin classes of natural products have been
reported.2ꢀ5 These compounds have been isolated from
mushrooms, Sarcodon leucopus, S. scabrosus, Hydnellum
suaveolens, H. geogerirum and Phellodon niger and have
been reported to show anti-HIV, antioxidant, and anti-
cancer activity.2ꢀ5 All of these isolation studies have de-
scribed a characteristic benzodioxazine core structure of
sarcodonins as well as an N,N-dioxide ring junction, which
were deduced from 2D-NMR studies and chemical degra-
dation studies leading to a diketopyperazine composed of
two isoleucines. In contrast, Baran and co-workers recently
reported the possibility of an alternative benzodioxane
aminal core structure through synthetic studies of this
class of compounds (1b for sarcodonin) (Figure 1).6
We recently investigated the secondary metabolites of
S. scabrosus7 and isolated a sizable amount of a new com-
pound named sarcodonin ε (2) that yielded NMR data
similar to those of phellodonin5 (3) and a minor substance
that was identified as sarcodonin δ3 (reported structure 4,
revised structure in the present study 5 in Figure 1),
obtained from the fruiting bodies of S. scabrosus, from
which sarcodonin δ was previously isolated. Compounds 2
and 5 yielded an identical acetate (FABMS, m/z 863
[M þ H]þ) upon typical acetylation.
The availability of 2 and 5 prompted us to investigate
the intriguing structure problem of the sarcodonin class
of natural products. In this article we describe evidence
in favor of the benzodioxazine core structure and the
N,N-dioxide ring junction.
† Tokyo Institute of Technology.
‡ Meiji University.
(1) Geraci, C.; Neri, P.; Paterno, C.; Rocco, C.; Tringali, C. J. Nat.
Prod. 2000, 63, 347.
(2) Cali, V.; Spatafora, C.; Tringali, C. Eur. J. Org. Chem. 2004, 592.
(3) Ma, B.-J.; Liu, J.-K. Z. Naturforsch., B: J. Chem. Sci. 2005, 60b,
565.
The structure of sarcodonin ε (molecular formula
C39H42N2O15 was deduced from HRFABMS data) was
ꢀ
(4) Hashimoto, T.; Quang, D. N.; Kuratsune, M.; Asakawa, Y.
Chem. Pharm. Bull. 2006, 54, 912.
(5) Fang, S.-T.; Zhang, L.; Li, Z.-H.; Li, B.; Liu, J.-K. Chem. Pharm.
(6) Lin, D. W.; Masuda, T.; Biskup, M. B.; Nelson, J. D.; Baran, P. S.
J. Org. Chem. 2011, 76, 1013.
(7) Endo, Y.; Minowa, A.; Kanamori, R.; Araya, H. Biochem. Syst.
Bull. 2010, 58, 1176.
Ecol. 2012, 44, 286.
r
10.1021/ol400595k
Published on Web 04/15/2013
2013 American Chemical Society

Table 1. 1H and 13C NMR Spectroscopic Data of 2 and 6
(500/125 MHz, in CDCl3)
2
6
no.
δ
H (mult, J in Hz)
δC
δ
H (mult, J in Hz)
δC
1
2
3
4
5
6
10
ꢀ
128.0
ꢀ
127.5
6.95 (d, 1.8) 118.5
7.02 (d, 1.8) 118.6
ꢀ
ꢀ
140.8
141.6
ꢀ
ꢀ
139.7
139.1
7.13 (d, 8.3) 117.0
7.07 (d, 8.4) 116.5
7.02 (dd, 8.3, 1.8) 125.1
6.97 (dd, 8.4, 1.8) 124.6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
128.8
139.1
139.4
130.9
123.1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
128.9
139.3
139.1
130.7
123.3
20, 60
300, 50
400
1
200,600
300,500
400
7.16 (d, 8.6) 130.9
6.84 (d, 8.6) 115.3
7.23 (d, 8.5) 130.7
6.92 (d, 8.5) 113.7
ꢀ
ꢀ
156.0
159.1
166.4
33.3
26.8
11.9
15.5
92.9
159.3
42.5
24.6
12.7
13.3
20.1
168.2b)
20.1
167.7b)
67.0
ꢀ
ꢀ
ꢀ
159.5
141.8
158.9
33.5
24.1
12.4
14.1
92.2
105.1
40.6
23.9
13.0
13.9
20.1d)
167.3e)
20.0d)
167.6e)
64.6
54.3
55.2
ꢀ
2a
3a
ꢀ
ꢀ
4a
3.11 (m)
3.40 (m)
5a
1.42 (m), 1.59 (m)
0.87 (t, 7.5)
1.07 (d, 7.1)
ꢀ
1.61 (m), 1.96 (m)
0.84 (t, 7.5)
1.24 (d, 7.0)
ꢀ
6a
7a
2b
3b
ꢀ
ꢀ
4b
2.41 (m)
2.36 (m)
5b
1.61 (m), 1.79 (m)
0.99 (t, 7.5)
1.21 (d, 6.9)
1.99a) (s)
1.32 (m), 1.96 (m)
0.90 (t, 7.5)
1.10 (d, 6.9)
1.98c) (s)
6b
7b
20/60- OAc
30/50- OAc
1.96a) (s)
1.94c) (s)
(1b)-OMe
3b-OMe
400-OMe
1a-OH
3.95 (s)
ꢀ
3.71 (s)
3.76 (s)
3.84 (s)
ꢀ
ꢀ
ꢀ
7.92 (bs)
6.02 (bs)
ꢀ
400-OH
ꢀ
ꢀ
ꢀ
a)ꢀd) Interchangeable within the column.
and C-3a (δ 164.2, shifted upfield compared to that
(δ 166.4) of 2)], with both chemical shifts being in agreement
with an acylation shift.8 The MeO signal on N(1b) of the
acetate also showed an NOE to H-4b. Finally, the methy-
lated compound 7 (vide infra) exhibited NOE correlations
between N(1b)-OMe and C(3a)-OMe and between N(1b)-
OMe and H-4b. It is of note that the presumed structure of 2
has an unprecedented N(1b)-OMe group instead of an
N(1a)-OMe group, as found in phellodonin (3).
Generally, the sarcodonin class of compounds is non-
crystalline, which has hampered definitive structure deter-
mination. Our attempt to obtain a crystalline derivative
such as p-nitrobenzoyl and camphorsulfonyl esters of 2
was not successful, because the reactions did not proceed
cleanly. Fortunately, we have found that treatment of 2
with TMSCHN2 (hexane solution) in MeOHꢀEt2O at
room temperature overnight under N2 yielded a crystalline
Figure 1. Structures of sarcodonins 1ꢀ5.
found to be distinct from phellodonin, as revealed by
careful NMR spectral comparison (Table 1), and was
thought to be 2 (a most likely tautomeric/resonance struc-
ture is depicted) on the basis of the followings. The OMe
group was determined to be attached to the nitrogen atom
rather than the carbon atom because of the lack of HMBC
correlation to any carbon atom. The strongly chelated
hydrogen of OH (δ 7.92) showed a clear HMBC correla-
tion to C-2a, but not to C-3a. The MeO signal showed an
NOE to H-4b. Compound 2 was not susceptible to epimeri-
zation at N(1b),2 suggesting the presence of N(1b)-OMe
group. Compound 2 formed a C(3a)-OAc,C-400-OAc deri-
vative in good yield, which exhibited 13C data [C-2a
(δ 165.6, shifted downfield compared to that (δ 159.1) of 2)
(8) Terui, Y.; Tori, K.; Tsuji, N. Tetrahedron Lett. 1976, 621. Jones,
R. A.; Stokes, M. J. Tetrahedron 1984, 40, 1051.
Org. Lett., Vol. 15, No. 9, 2013
2077

derivative 6 with 52% yield, together with a minor oily
product 7 (m/z 807 [M þ H]þ) in 23% (Scheme 1).
The 1H NMR and FABMSdata (m/z807 [M þ H]þ) of 6
indicated that two methyl groups were newly introduced
onto 2. 2D NMR analysis of 6 (Table 1) indicated that a
methyl group was located at the C-400 position and the
other methyl group was assigned as C(3b)-OMe because
both the OMe group (δ 3.76) and H-4b (δ 2.36) had
HMBC correlation to C-3b (δ 105.1). It is notable that
C-3b (δ 105.1) was significantly shifted upfield compared
to that (δ 159.3) of 2. The 1H and 13C shifts of the MeO
group at N(1b) were shifted only slightly compared to
those of 2 (see Table 1). We were not able to obtain
definitive evidence of the existence of the presumed N(1b)-
OMe linkage in 6.
Thus, compound 6 was recrystallized from hexaneꢀ
EtOAc, and a resulting needle (mp 208 °C) was analyzed
by X-ray crystallography. The ORTEP drawing is illu-
strated in Figure 2, which permitted us to determine the
structure of 6 including the benzodioxazine core structure.
Absolute stereochemistry of the molecule was deduced
based on the S chirality of the isoleucine-derived moiety.1
The configuration at N(1b) and the structure of the 1,3,4-
substituted phenyl ring, previously reported for sarco-
donins,1ꢀ5 were substantiated unequivocally for the first
time. Furthermore, the uniquestructure bearing the N(1b)-
OMe group of 2 was ascertained.
Figure 2. Single-crystal X-ray structure of 6.
C(3b)ꢀN(1a)ꢀC(2a) and N(1a)ꢀC(2a)ꢀC(3a) were
122.8(6), 117.3(6) and 118.9(6)o, respectively. These data
suggested that the iminium N-oxide system bears C(3b)d
N(1a)þꢀOꢀ and C(3b)ꢀꢀN(1a)þdO resonance forms. It
appears that the latter form contributes the above-
mentioned upfield shift of C-3b (δ 105.1) of 6.9
To shed light on the fate of the N(1b)-OMe group in 2
during the methylation reaction, 2 was treated with
TMSCHN2 in CD3OD (known to act as a “CD3” source10)
instead of MeOH. The 1H NMR analysis of the resulting
product 6a indicated that the OMe group on N(1b) was
essentially unlabeled (δH 3.71), but the OMe group at
C(3b) was exclusively deuterium labeled (residual CD2H
at δH 3.73), as in the OMe group at C-400. This finding
implied that the OMe group at N(1b) in 2 remained at the
original position and eliminated the possibility of an
unexpected migration during the TMSCHN2 treatment.
The minor product 7a had deuterium-labeled OMe groups
at C-400 and C-3a, whereas N(1b)-OMe was unlabeled.
Here, we would like to comment on the structure of
sarcodonin δ (5). Compound 5 should have the same core
structure as 2 because both compounds 2 and 5 exhibited
essentially identical NMR data for the benzodioxazine
core moiety and yielded the same hexa-acetate (acetylated
20, 30, 50, 60, 400, C-3a). Furthermore, TMSCHN2 treatment
of 5 proceeded in the same manner as that of 2 to yield an
oily compound 8 (m/z 751 [M þ H]þ) as a major product
(Scheme 2), which gave essentially identical NMR spectra
(N(1b)-OMe δH 3.71, δC 64.7; C(3b)-OMe δH 3.80, δC
54.5) for the core moiety as did 6, thus confirming the
presence of the N(1b)-OMe and C(3b)-OMe groups in
sarcodonin δ. Hence, the structure of sarcodonin δ was
revised as 5, shown in Figure 1.
Scheme 1. Methylated Products 6 and 7 Obtained from 2 upon
TMSCHN2 Treatmenta
a Compounds 6a and 7a were obtained using CD3OD solvent instead
of MeOH.
(9) Bohle, D. S.; McQuade, L. E.; Perepichka, I.; Zhang, L. J. Org.
Chem. 2007, 72, 3625.
(10) Kuhnel, E.; Laffan, D. D. P.; Lloyd-Jones, G. C.; del Campo,
T. M.; Shepperson, I. R.; Slaughter, J. L. Angew. Chem., Int. Ed. 2007,
46, 7075.
(11) Baran and coworkers have recently completed the synthsis of
sarcodonin ε and phellodonin. See the following paper in this issue:
Usui, I.; Lin, D. W.; Masuda, T.; Baran, P. S. Org. Lett. 2013, DOI:
10.1021/ol400709f.
The observed bond lengths for the core part of 6 in the
X-ray structure are as follows: N(1a)ꢀC(2a) 1.519(9),
C(2a)ꢀC(3a) 1.281(8), C(3a)ꢀN(1b) 1.519(8), N(1b)ꢀ
C(2b) 1.532(8), C(2b)ꢀC(3b) 1.417(7), C(3b)ꢀN(1a)
1.357(9), N(1a)-O 1.253(7), C(3a)ꢀO 1.286(6), and C(3b)ꢀO
˚
1.423(7) A. The bond angles for C(2b)ꢀC(3b)ꢀN(1a),
2078
Org. Lett., Vol. 15, No. 9, 2013

evidence for the core structure of the sarcodonin class
compounds. It is concluded that sarcodonins have a
benzodioxazine core structure (such as 1a) as originally
reported, but not a benzodioxane aminal core structure.11
Scheme 2. Methylated Product 8 Obtained from 5 upon
TMSCHN2 Treatment
Acknowledgment. We thank Ms. Satsuki Itoh of Tokyo
InstituteofTechnologyfor FABMSmeasurements. A part
of this work was supported by the Foundation of Meiji
University.
Supporting Information Available. Detailed experimen-
tal procedures, NMR spectra of compounds 2, 5, per-
acetate of 2, 6, 6a, 7, 7a and 8, and CIF file of 6. This
material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, X-ray analysis of compound 6, derived
from sarcodonin ε (2), established the structures of 6
as well as 2. The present study has provided conclusive
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 9, 2013
2079