Naphthoquinone derivatives from diospyros maritima

Article abstract of DOI:10.1248/cpb.c17-00178

From the chloroform extract of the fresh fruits of Diospyros maritima BLUME (Ebenaceae), five new naphthoquinone derivatives, 2,7′-dimethyl-2′,3-bijuglone (27), 2,7′-dimethyl-3,3′-bijuglone (28), 2,7′-dimethyl-6,8′-bijuglone (29), 7,7′-dimethyl-3,3′-ethyl

Full text of DOI:10.1248/cpb.c17-00178

Vol. 65, No. 8
Chem. Pharm. Bull. 65, 739–745 (2017)
739
Regular Article
Naphthoquinone Derivatives from Diospyros maritima
Matsutake Higa, Yuji Takashima, Hiroto Yokaryo, Yuta Harie, Toshimasa Suzuka, and
Kazuhito Ogihara*
Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus; 1 Senbaru,
Nishihara-cho, Okinawa 903–0213, Japan.
Received February 28, 2017; accepted May 24, 2017
From the chloroform extract of the fresh fruits of Diospyros maritima BLUME (Ebenaceae), five new naph-
thoquinone derivatives, 2,7′-dimethyl-2′,3-bijuglone (27), 2,7′-dimethyl-3,3′-bijuglone (28), 2,7′-dimethyl-6,8′-
bijuglone (29), 7,7′-dimethyl-3,3′-ethylidenebijuglone (30), and 2′,7-dimethyl-3,6′-ethylidenebijuglone (31),
were isolated, in addition to twenty-one known naphthoquinone derivatives: plumbagin (4), droserone
(5), 2,3-epoxyplumbagin (8), 3,3′-biplumbagin (9), chitranone (10), 3,8′-biplumbagin (11), elliptinone (12),
maritinone (13), isozeylanone (14), methylene-3,3′-biplumbagin (15), ethylidene-3,3′-biplumbagin (16),
ethylidene-3,6′-biplumbagin (17), ethylidene-6,6′-biplumbagin (18), 7-methyl-β-dihydrojuglone (19), 7-meth-
yljuglone (20), 2,3-epoxy-7-methyljuglone (21), neodiospyrin (22), mamegakinone (23), ehretione (24),
isoxylospyrin (25) and β-dihydroplumbagin (26). The structures of the new compounds were established by
spectral analysis. The quinones obtained from the chloroform extract of the fruits were compared with previ-
ously reported quinones obtained from ethanol extracts. The quinones in the fruits were categorized in three
groups: quinones from ethanol extract only, quinones from chloroform extract only, and quinones from both
extracts. The six naphthoquinones, 19–21, 25, 26, and 29, were examined for their ichthyotoxic activity and
germination inhibitory activity. Quinones 19–21, 26, and 29 showed ichthyotoxic activity against Japanese
killifish (Oryzias latipes var.) at 10ppm; quinones 19 to 21 and 26 showed germination inhibitory activity
toward lettuce seeds (Lactuca sativa L. var. Great Lakes) at 100ppm.
Key words Diospyros maritima; Ebenaceae; naphthoquinone; bijuglone; ethylidenebijuglone
Diospyros maritima BLUME (Ebenaceae) is a shrub that and five new quinones (27 to 31) (Chart 2), in addition to qui-
grows in Southeast Asia. We previously reported the isolation nones 4, 5, and 8–18, which were previously isolated from the
of eighteen naphthoquinone derivatives, 1–18, from the ethanol ethanol extract.
extract of the fruits of this plant1,2) (Chart 1). The naphthoqui-
The new quinone 27 was obtained as red prisms with a
none derivatives isolated to date from the genus Diospyros are melting point (mp) of 184–185°C. Its molecular formula was
commonly assumed to be derived from a precursor equivalent determined to be C22H14O6 by high-resolution electrospray
to 4,5-dihydroxy-2-methylnaphthalene or a biogenetic analog.3) ionization (HR-ESI)-MS, showing the pseudomolecular ion
3-Methylplumbagin (3), 3-(2-hydroxyethyl)plumbagin (6), [M−H]− at m/z 373.0711 (calcd for C22H13O6: 373.0712).
6-(1-ethoxyethyl)plumbagin (7), methylene-3,3′-biplumbagin The IR (νmax (KBr) cm−1: 1667, 1643, 1610 sh) and UV (λmax
(15), ethylidene-3,3′-biplumbagin (16), ethylidene-3,6′- (CHCl3) nm (logε): 255 (4.29), 434 (3.90)) spectra showed
biplumbagin (17), and ethylidene-6,6′-biplumbagin (18) isolat- the characteristic bands of juglone (5-hydroxy-1,4-naphtho-
ed from this plant contain an alkyl group that is not found in quinone) derivatives. The MS spectrum showed fragment
the naphthoquinone derivatives isolated from other Diospyros ion peaks at m/z 134 and 106, suggesting the presence of
species. Because it is possible to isolate rare and novel qui- the 7-methyljuglone (20) moiety; the fragment ion peaks at
nones using different extraction solvents, we employed chlo- m/z 120 and 92 exhibited the presence of the plumbagin (4)
roform as the extraction solvent instead of ethanol to examine moiety.4,5) The 1H-NMR spectrum showed the presence of
the naphthoquinone derivatives in the chloroform extract of one aromatic methyl [δ 2.47 (3H, s)], one quinonoid proton [δ
the fresh fruits. Here, we describe the isolation and structure 6.86 (1H, s)], two aromatic protons [δ 7.14 (1H, m), 7.51 (1H,
elucidation of five new naphthoquinone derivatives, 27–31, d, J=1.5Hz)], and one hydrogen-bonded hydroxyl [δ11.84 (1H,
from the chloroform extract; we also discuss the classification s)] attributed to the 7-methyljuglone moiety, one quinonoid
of naphthoquinone derivatives obtained only from the ethanol methyl [δ 2.13 (3H, s)], three aromatic protons [δ 7.28 (1H,
extract of the fresh fruits of this plant, only from the chloro- dd, J=1.0, 8.0Hz), 7.65 (1H, t, J=8.0Hz), 7.71 (1H, dd, J=1.0,
form extract, and from both extracts.
7.5Hz)], and another hydrogen-bonded hydroxyl (δ 11.75, (1H,
s)) were attributed to the plumbagin moiety. From these re-
sults, the quinone 27 is a dimer that consists of plumbagin (4)
Results and Discussion
Structure Elucidation A chloroform extract of the fresh and 7-methyljuglone (20). The position of the dimeric linkage
fruits of D. maritima afforded, after chromatographic separa- was established by 1H-NMR spectrometry as follows. The
tion, eight known naphthoquinone derivatives: 7-methyl-β- absence of allylic coupling of the quinonoid methyl with the
dihydrojuglone (19), 7-methyljuglone (20), 2,3-epoxy-7-meth- quinonoid proton in 27 indicates that the 3-position of the
yljuglone (21), neodiospyrin (22), mamegakinone (23), plumbagin moiety is occupied by a substituent. Because only
ehretione (24), isoxylospyrin (25), β-dihydroplumbagin (26), one quinonoid proton is observed, the 2- or 3-position of the
*To whom correspondence should be addressed. e-mail: kogihara@sci.u-ryukyu.ac.jp
© 2017 The Pharmaceutical Society of Japan

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Chart 1. Structures of Naphthoquinones Isolated from the EtOH Extracts of Diospyros maritima
Chart 2. Structures and Some Derivatives of Naphthoquinones Isolated from the CHCl3 Extracts of Diospyros maritima
7-methyljuglone moiety is occupied by a substituent. From the spectral analyses. The heteronuclear multiple bond correlation
above results, the dimeric linkages in 27 must be located at heteronuclear multiple bond connectivity (HMBC) spectrum
the 3-position of the plumbagin moiety and the 2- or 3-posi- of 27 (Fig. 1) showed long-range correlations between the
tion of the 7-methyljuglone moiety. Complete assignments of quinonoid proton signal due to the 7-methyljuglone moiety
1
the signals in the H- and 13C-NMR spectra were established and the carbon signals at C-3 and C-10′, which reveals that
by one-dimensional (1D) and two-dimensional (2D)-NMR the 2-position of the 7-methyljuglone moiety is occupied by

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
741
Fig. 1. HMBC Correlations for Compounds 27–31
a substituent. These HMBC data indicate that the location of d, J=8.0Hz)), one aromatic proton (δ 7.26, (1H, s)), and two
the dimeric linkage of 27 is C-3 and C-2′. Based on the above hydrogen-bonded hydroxyls (δ 12.21, 12.43 (each 1H, s)). The
evidence, quinone 27 was characterized as 2,7′-dimethyl-2′,3- presence of the three quinonoid protons and two aromatic
bijuglone.
methyl groups indicates that compound 29 is a dimer com-
The new quinone 28 was obtained as red-brown prisms posed of 4 and 20; it also suggests that linkages exist between
with mp 170°C (dec.). Its molecular formula was determined the aromatic rings, in contrast to 27 and 28. The position of
1
to be C22H14O6 (m/z 373.0695 [M−H]−, calcd for C22H13O6: the dimeric linkage was established by H-NMR spectrometry
373.0712), which is the same as quinone 27, by HR-ESI-MS. as follows. The presence of allylic coupling of the quinonoid
The IR and UV spectra of 28 were almost superimposable methyl group with the quinonoid proton and the presence
with those of 27. The MS spectrum was similar to that of 27. of ortho-coupled aromatic protons show that the position of
1
The H- and 13C-NMR spectra closely coincided with those of the dimeric linkage in the plumbagin moiety must be C-6
27, except for signals due to a quinonoid proton and a carbon or C-8. The presence of two coupled quinonoid protons and
atom. These results suggested that 28 is a dimer consisting of the presence of only one aromatic proton reveal that the po-
compounds 4 and 20, similar to 27, and that the dimeric link- sition of the dimeric linkage in the 7-methyljuglone moiety
ages in 28 must be located at the 3-position of the plumbagin must be C-6′ or C-8′. The HMBC spectrum of 29 (Fig. 1)
moiety and the 2- or 3-position of the 7-methyljuglone moiety. showed long-range correlations between the aromatic proton
The HMBC spectrum of 28 (Fig. 1) showed long-range corre- H-8 and C-1 and between H-7 and C-8′, which indicates that
lations between the quinonoid proton signal due to the 7-meth- the location of the dimeric linkage of the plumbagin moiety
yljuglone moiety and carbon signals at C-3 and 9′, which is C-6. The HMBC spectrum of 29 also showed long-range
reveals that the 3-position of the 7-methyljuglone moiety is correlations between the aromatic proton H-6′ and C-8′ and
occupied by a substituent. These HMBC data indicate that the C-10′. No correlation between the aromatic proton H-6′ and
location of the dimeric linkage of 28 is C-3 and C-3′. Thus, C-1′ was observed. These HMBC data demonstrate that the
quinone 28 was characterized as 2,7′-dimethyl-3,3′-bijuglone.
location of the dimeric linkage of the 7-methyljuglone moiety
Quinones 27 and 28 are mixed dimers of 4 and 20. Al- is C-8′. Thus, the location of the dimeric linkage of 29 was
though many symmetric dimers of quinones 4 and 20 have determined to be C-6 and C-8′; therefore, compound 29 was
been reported from Diospyros species to date, ehretione (24) characterized as 2,7′-dimethyl-6,8′-bijuglone.
6)
isolated from Diospyros ehretioides WALL is only one ex-
ample of a mixed dimer of quinones 4 and 20.
Compound 30 was obtained as an orange powder with
mp 223−225°C. Its molecular formula was determined to
Compound 29 was obtained as orange leaflets with be C24H18O6 (m/z 401.1043 [M−H]−, calcd. for C24H17O6:
mp 176–178°C. Its molecular formula was determined to 401.1025) by HR-ESI-MS. The IR (νmax (KBr) cm−1: 1668,
be C22H14O6 (m/z 373.0705 [M−H]−, calcd for C22H13O6: 1636, 1614) and UV (λmax (CHCl3) nm (logε): 256 (4.21), 432
373.0712) by HR-ESI-MS. The IR (νmax (KBr) cm−1: 1665, (3.75)) spectra showed the characteristic peaks of juglone de-
1636, 1614) and UV (λmax (CHCl3) nm (logε): 256 (4.31), rivatives. The MS spectrum showed the molecular ion at m/z
435 (3.83)) spectra showed the characteristics of juglone de- 402; fragment ions at m/z 134 and 106 demonstrated the pres-
rivatives. The H-NMR spectrum showed the presence of one ence of the 7-methyljuglone moiety.4,5) The H-NMR spectrum
quinonoid methyl (δ 2.23 (3H, d, J=1.5Hz)), one aromatic showed the presence of two aromatic methyls (δ 2.43 (6H, s)),
methyl (δ 2.10 (3H, s)), one quinonoid proton (δ 6.84 (1H, two quinonoid protons (δ 6.72 (2H, d, J=1.5Hz)), four aro-
q, J=1.5Hz)), two coupled quinonoid protons (δ 6.72 (1H, matic protons (δ 7.07, 7.43 (2H, d, J=1.5Hz)), two hydrogen-
d, J=10.0Hz), 6.91 (1H, d, J=10.0Hz)), one pair of ortho- bonded hydroxyls (δ 11.89 (2H, s)), and one ethylidene group
coupled aromatic protons (δ 7.29 (1H, d, J=8.0Hz), 7.74 (1H, (δ 4.68 (1H, brq, J=7.0Hz, >CHCH3), 1.45 (3H, d, J=7.0Hz,
1
1

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Vol. 65, No. 8 (2017)
>CHCH3)) in 30. From the above results, compound 30 is a the rotation values were significantly different between both
symmetric dimer of 20 linked by an ethylidene bridge. Be- reports (−7° and +24°).3,8) Therefore, the cis-isomer (32) was
cause only two quinonoid protons were observed in addition treated with benzoyl chloride in pyridine to afford its dibenzo-
to all four aromatic protons, the position of the linkage must ate (34) as a pale yellow semi-solid, [α]D +71.5° (CHCl3). The
1
be C-2,2′ or C-3,3′. Thus, compound 30 is ethylidene-2,2′-di-7- signals in the H-NMR spectrum and the circular dichroism
methyljuglone or ethylidene-3,3′-di-7-methyljuglone. The (CD) curve of the dibenzoate (34) were almost superimposable
HMBC spectrum (Fig. 1) showed long-range correlations be- with those in previous reports.3,8) Thus, cis-tetralone (32) must
tween the methyl protons of the ethylidene group and C-3,3′. be isoshinanolone, and the absolute configuration at C-2 of 26
These HMBC correlations indicate that the ethylidene group was established to be R.
is located at C-3 and C-3′. Based on the above evidence, the
Categorization of Isolates by Extraction Solvent By
location of the dimeric linkage of 30 was determined to be changing the extraction solvent, the quinones in the fruit of D.
C-3 and C-3′; thus, 30 was characterized as 7,7′-dimethyl-3,3′- maritima were categorized in three groups: quinones isolated
ethylidenebijuglone.
Compound 31 was obtained as orange needles with mp chloroform extract, and quinones isolated from both extracts.
215–216°C (dec.) and [α]D (26)+0.42° (c=4.0, CHCl3). Its mo-
The naphthoquinone derivatives isolated only from the
only from the ethanol extract, quinones isolated only from the
lecular formula was determined to be C24H18O6 (m/z 401.1030 chloroform extract of the fresh fruits are listed as 19–26, in
[M−H]−, calcd for C24H17O6: 401.1025) by HR-ESI-MS. The addition to five new quinones, 27–31. The chloroform extract
IR (νmax (KBr) cm−1: 1667, 1639, 1614) and UV (λmax (CHCl3) newly afforded seven naphthoquinone derivatives, 7-methyl-β-
nm (logε): 260 (4.33), 430 (3.88)) spectra showed the charac- dihydrojuglone (19), 7-methyljuglone (20), 7-methyl-2,3-
1
teristic peaks of juglone derivatives. The H-NMR spectrum epoxyjuglone (21), neodiospyrin (22), mamegakinone (23),
showed the presence of the plumbagin moiety (one quinonoid ehretione (24), isoxylospyrin (25), and β-dihydroplumbagin
methyl [δ 2.19 (3H, d, J=1.5Hz)], a quinonoid proton [δ 6.80 (26), in comparison with naphthoquinones isolated from the
(1H, q, J=1.5Hz)], two ortho-coupled aromatic protons [δ ethanol extract. 7-Methyljuglone (20) has been shown to be
7.54, δ 7.62 (each 1H, d, J=7.5Hz)], one hydrogen-bonded readily converted into 2- and 3-methoxy-7-methyljuglone at
hydroxyl [δ 12.49 (1H, s)]), one 7-methyljuglone moiety (one room temperature in methanol.9) Therefore, quinones 20–25,
aromatic methyl [δ 2.42 (3H, s)], another quinonoid protons which contain 7-methyljuglone moieties, are expected to be
[δ 6.66 (1H, d, J=1.5Hz)], two meta-coupled aromatic protons unstable or labile in ethanol. 7-methyl-β-dihydrojuglone (19) is
[δ 7.04 (1H, m), 7.41 (1H, d, J=1.5Hz)], another hydrogen- quite rare; its isolation has been reported only from Diospyros
bonded hydroxyl [δ 11.93 (1H, s)]), and one ethylidene group hebecarpa by Cooke and Dowd in 1952.10) When quinones 19
(δ 4.83 (1H, br q, J=7.0Hz, >CHCH3), 1.55 (3H, d, J=7.0Hz, and 26 were refluxed in chloroform, no new products were
>CHCH3)) in 31. These results indicate that 31 is a mixed obtained. However, these quinones were converted completely
dimer of 4 and 20 linked by an ethylidene bridge. Because one into the corresponding naphthoquinones, 7-methyljuglone (20)
quinonoid proton coupled with a quinonoid methyl and two and plumbagin (4), respectively, by refluxing in 50% aque-
ortho-coupled aromatic protons were observed, the position ous ethanol for 12h. Quinones 19 and 26 may be isolated
of the dimeric linkage in the plumbagin moiety must be C-6′ from other Diospyros species by extraction of the fresh plant
or C-8′. Because only one quinonoid proton and two meta- with chloroform. Quinone 26 is believed to be the biogenetic
coupled aromatic protons were observed, the position of the precursor of zeylanone and isozeylanone (14), which have
dimeric linkage in the 7-methyljuglone moiety must be C-2 been isolated from Plumbago zeylanica.11) However, quinone
or C-3. The HMBC spectrum (Fig. 1) showed long-range cor- 26 has not been isolated from any species belonging to the
relations between the methyl protons of the ethylidene group genus Plumbago. β-Dihydrojuglone (35) has been shown to be
and C-3 and C-6′. These HMBC correlations indicate that involved in the biosynthesis of juglone (36) in the genus Jug-
the ethylidene group is located at C-3 and C-6′. Based on the lans (Juglandaceae).12) Therefore, quinones 19 and 26 may be
above evidence, the location of the dimeric linkage of 31 was intermediates in the biosynthesis of 7-methyljuglone (20) and
determined to be C-3 and C-6′; thus, 31 was characterized as plumbagin (4), respectively, in the genus Diospyros.
2′,7-dimethyl-3,6′-ethylidenebijuglone.
The naphthoquinone derivatives isolated only from the
β-Dihydroplumbagin (26) was obtained as pale yellow leaf- ethanol extract of the fresh fruits were listed as 1, 2, 3, 6, and
lets (hexane), mp 80–82°C, [α]D (23) −88.1° (c=0.78, CHCl3). 7, and those from both extracts were as 15–18, in addition to
Compound 26 was previously isolated from Juglans nigra and quinones 4, 5, and 8–14. Meanwhile, quinones 1, 2, 3, 6, and
J. regia by Binder et al.7); however, they did not describe its 7 were isolated only from the ethanol extract. 3-Chloroplum-
optical properties. Therefore, the absolute configuration at C-2 bagin (2) has been also isolated from Plumbago zeylanica13)
of 26 was determined as follows. Quinone 26 was reduced and Drosera intermadia.14) Lillie et al. suggested that 2 is an
with zinc in 80% acetic acid to afford the cis-tetralone (32) as artifact formed by the reaction of plumbagin-2,3-epoxide (8)
a pale yellow oil, [α]D +14.3° (CHCl3), and the trans-tetralone with chloroform.4) Quinone 2 was obtained as a byproduct
(33) as colorless needles with mp 102°C, [α]D 0° (CHCl3). of the conversion of 4 with NaBO3 and HCl into 8.15) The
1
The H-NMR spectrum of the cis-tetralone (32) was almost reaction mechanism can be described as follows: compound
superimposable on that of isoshinanolone (4R,8-dihydroxy-3R- 8 underwent SN2 substitution with HCl followed by dehydra-
methyl-3,4-dihydro-1(2H)-naphthalenone).3,8) The cis- and tion to afford 2. It is known that chloroform is decomposed
trans-isomers can be distinguished by comparing the cou- into COCl2 and HCl by daylight. Therefore, 8 was refluxed
pling constants of the carbinyl protons of the two isomers in chloroform containing a small amount of ethanol16) under
(cis: J=2.5Hz, trans: J=7.5 Hz).3) The optical rotations for daylight for 48h; however, no quinone 2 was obtained ini-
cis-isomer (32) were reported by two research groups, but tially. If this treatment is continued for a long time (more

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
743
than 48h), 2 may be detected. However, this is longer than UV on a Hitachi 100–50 spectrophotometer and a JASCO
the time required for chromatographic separation. Compound V-630 spectrophotometer; EI-MS on a Hitachi RMU-6L mass
2 would first be isolated from the chloroform extract if it was spectrometer and a Hitachi M-2500 apparatus (70eV, direct
formed by this reaction. Thus, it is shown that the chlorine inlet system); HR-ESI-MS on a JEOL JMS-T100LP mass
1
atom of 2 did not originate from the chloroform used for spectrometer; H, 13C, and 2D-NMR on a Hitachi R-24 NMR
partition between solvents during the isolation procedure. spectrometer (1H: 60MHz), a JEOL α500 NMR spectrometer
3-Bromoplumbagin (1) is easily obtained by treatment of 4 (1H: 500MHz, 13C: 125MHz), and a Bruker Avance III 500
with bromine.1,15) Therefore, it is possible that 1 is an artifact NMR spectrometer (1H: 500MHz, 13C: 125MHz). Chemical
formed during ethanolic extraction, although the origins of shifts are given on a δ (ppm) scale with tetramethylsilane as
the bromine are not clear. If quinones 1 and 2 are artifacts, an internal standard. The symbols s, d, t, q, dd, dq, m, and
it is suggested that the quinonoid moiety of 4 can be read- br denote singlet, doublet, triplet, quartet, doublet of doublets,
ily substituted with chlorine and bromine and converted into double quartet, multiplet, and broad, respectively. Column
quinones 1 and 2 by reaction in ethanol. However, quinones 1 chromatography (CC) and flash-column chromatography (FC)
and 2 may not be extracted from the fruits using chloroform, were carried out with Wakogel C-300 (Wako Pure Chemical
as the solubilities of the precursors, which are expected to Industries, Ltd.) and Kieselgel 60N 40−50µm (Merck), re-
be equivalent to 4,5-dihydroxy-2-methylnaphthalene or the spectively. Preparative TLC (PTLC) was performed on a pre-
biogenetic analogs of 1 and 2 in chloroform, are lower than coated Kieselgel 60F254 plate (Merck). HPLC was performed
their solubilities in ethanol. 3-Methylplumbagin (3) has also with a Shimadzu LC-8A.
been isolated from the acetone extracts of Juglans nigra and
Extraction and Isolation The fresh fruits (82.0kg) of
J. regia (Juglandaceae)7); however, the possibility that 3 is an D. maritima, collected at Chinen, Okinawa in April 1988,
artifact has not been suggested. 3-(2-Hydroxyethyl)plumbagin were extracted with CHCl3 (25L), after crushed by using a
(6) and 6-(1-ethoxyethyl)plumbagin (7) contain ethanolic moi- Waring blender in the same solvent, in the day. The extract
eties, as does the extraction solvent. However, it is believed to was evaporated to dryness, and the residue was partitioned
be unlikely that 6 and 7 are artifacts formed during ethanolic between CHCl3 and H2O. The CHCl3 layer was evaporated,
extraction, as experimental evidence for this has not been and the residue (963.0g) was divided into C6H6-soluble and
demonstrated.
C6H6-insoluble portions (247g). The C6H6-soluble portion was
The naphthoquinone derivatives isolated from both subjected to CC on silica gel with a gradient of C6H6−EtOAc
the chloroform and ethanol extracts of the fresh fruits to afford nine fractions (I to IX). The procedure was finished
were listed as 15–18, in addition to 4, 5, and 8–14. in a day and the fractions I to IX were stored at −5 or −20°C
Methylene-3,3′-biplumbagin (15), ethylidene-3,3′-biplumbagin until chromatographic separations and purifications.
(16), ethylidene-3,6′-biplumbagin (17), and ethylidene-6,6′-
Fraction I (30.11g) contained only non-quinonoid material.
biplumbagin (18) were isolated from both extracts. This fact Fraction II (408.26g) was purified by FC on silica gel (hex-
indicates that quinones 15–18 are not artifacts formed during ane−C6H6 gradient) to yield 4 (321.1g) and 8 (726mg). Frac-
ethanol extraction. In other words, the results clearly excluded tion III (76.35g) was purified by FC on silica gel (hexane−
the possibility that quinones 15–18 were formed during the C6H6 gradient) and PTLC on silica gel (5:5 hexane−C6H6)
isolation procedure. Gunaherath et al. suggested that the bio- to yield 26 (9.78g), 19 (26.50g), 15 (45mg), 20 (5.27g), 21
synthesis of quinone 15 in the plant may involve the participa- (87mg), 22 (143mg), 23 (780mg), 9 (105mg), and 24 (378mg).
tion of formaldehyde.17) The origin of the ethylidene group of Fraction IV (22.08g) was purified by FC on silica gel (hex-
quinones 16–18 is not yet clear.
ane−C6H6 gradient) to yield 17 (150mg). Fraction V (24.43g)
Bioassay of Quinones Two bioassays, ichthyotoxicity and was purified by FC on silica gel (hexane−C6H6 gradient) to
seed germination tests, were performed with six compounds yield 27 (322mg), 16 (99mg), 10 (399mg), 14 (210mg), 28
(19–21, 25, 26, and 29). Compounds 19–21, 26, and 29 dis- (482mg), 11 (99mg), and 18 (161mg). Fraction VI (38.65g)
played ichthyotoxic activity against Japanese killifish (Oryzias was purified by FC on silica gel (hexane−C6H6 gradient) to
latipes var.) when tested at 10ppm. Compound 25 did not yield 30 (58mg), 31 (294mg), and 12 (1.43g). Fraction VII
display any ichthyotoxic activity. Compounds 19–21 and 26 (10.20g) was purified by FC on silica gel (hexane−C6H6 gra-
showed germination inhibitory activity against lettuce seeds dient) and PTLC (C6H6) to yield 13 (268mg), 29 (420mg),
(Lactuca sativa L. var. Great Lakes) when tested at 100ppm. and 25 (484mg). Fraction VIII (1.59g) was purified by FC on
Compounds 25 and 29 did not show any germination inhibi- silica gel (hexane−C6H6 gradient) to yield 5 (531mg). Fraction
tory activity. The ichthyotoxic and germination inhibitory ac- IX (18.33g) contained only non-quinonoid material.
tivities of 22–24, 27, 28, 30, and 31 could not be determined
Compounds 4, 5, and 8–18 were identified by direct com-
due to the low quantities of the samples or low solubility in parison with authentic samples.
the solvent.
7-Methyl-β-dihydrojuglone (19)
Yellow-green leaflets (C6H6), mp 113–114°C (lit.9)
112–113°C). IR νmax (KBr) cm−1: 1680, 1635, 1605. UV λmax
Experimental
1
General Procedures Melting points were measured on (CHCl3) nm (logε): 241 (4.19), 277 (3.84), 350 (3.77). H-NMR
a Yanagimoto MP-S3 micro melting point apparatus and (CDCl3) δ: 2.41 (3H, s, CH3-7), 3.01 (4H, s, –CH2CH2–), 7.02
are uncorrected. Specific rotations were recorded using a (1H, m, H-6), 7.31 (1H, m, H-8), 12.09 (1H, s, OH-5). MS m/z
JASCO DIP-1000 digital polarimeter. Spectral data were (rel. int. %): 190 [M]+ (100), 175 (9), 162 (13), 147 (7), 134
obtained using the following instruments: CD on a JASCO (54), 106 (13). Compound 19 was identified by direct compari-
MOE-1 spectropolarimeter; IR on JASCO A-302, Shimadzu son with synthetic sample.11)
FTIR-8200A, and JASCO FT/IR-4100 spectrophotometers;

744
Chem. Pharm. Bull.
Vol. 65, No. 8 (2017)
Table 1. 1H-NMR Spectral Data for Compounds 27–31 (500MHz, CDCl3, δ)
27
28
29
30
31
H-2
6.72 (d, 1.5)
6.66 (d, 1.5)
H-3
6.84 (q, 1.5)
H-6
H-7
H-8
H-2′
H-3′
H-6′
7.28 (dd, 1.0, 8.0)
7.65 (t, 8.0)
7.71 (dd, 1.0, 7.5)
7.29 (dd, 1.0, 8.5)
7.66 (t, 8.0)
7.71 (dd, 1.0, 7.5)
6.84 (s)
7.07 (m)
7.04 (m)
7.29 (d, 8.0)
7.74 (d, 8.0)
6.72 (d, 10.0)
6.91 (d, 10.0)
7.26 (s)
7.43 (d, 1.5)
6.72 (d, 1.5)
7.41 (d, 1.5)
6.80 (q, 1.5)
6.86 (s)
7.14 (m)
7.12 (m)
7.07 (m)
H-7′
H-8′
7.54 (d, 7.5)
7.62 (d, 7.5)
7.51 (d, 1.5)
2.13 (s)
7.51 (d, 1.5)
2.15 (s)
7.43 (d, 1.5)
CH3-2
CH3-2′
CH3-7
CH3-7′
OH-5
OH-5′
>CHCH3
>CHCH3
2.23 (d, 1.5)
2.19 (d, 1.5)
2.42 (s)
2.43 (s)
2.43 (s)
11.89 (s)
11.89 (s)
4.68 (brq, 7.0)
1.45 (d, 7.0)
2.47 (s)
11.75 (s)
11.84 (s)
2.47 (s)
11.75 (s)
11.72 (s)
2.10 (s)
12.21 (s)
12.43 (s)
11.93 (s)
12.49 (s)
4.83 (brq, 7.0)
1.55 (d, 7.0)
7-Methyljuglone (20)
Table 2. 13C-NMR Spectral Data for Compounds 27−31 (125MHz,
CDCl3, δ)
Orange needles (C6H6), mp 108°C (lit.3) 120–121°C). The
1H-NMR spectral data were identical to those in ref. 3.
2,3-Epoxy-7-methyljuglone (21)
27
28
29
30
31
Orange-red plates (C6H6), mp 95–96°C (lit.16) 94–95°C). The
1H-NMR spectral data were identical to those in ref. 16.
Neodiospyrin (22)
C-1
183.51
147.36
139.92
187.73a)
161.51
124.51
136.58
119.59
131.84
114.66
182.30
144.47
138.34
188.38a)
161.83
124.51
148.89
121.40
131.30
113.07
14.78
183.45
147.55
139.55
187.72
161.53
124.52a)
136.60
119.62
131.60
114.61
183.51
138.80
143.77
187.16
162.17
124.54a)
149.11
120.77
131.81
112.75
14.80
184.65
149.92
135.35a)
190.60
158.61
136.00
135.42a)
119.67
131.22
115.03
184.86
140.23
137.98
190.37
161.89
125.32
148.38
129.48
129.07
113.93
16.57
184.31
134.96
152.96
188.16
162.09
124.23
148.78
120.42
131.63
113.05
184.31
134.96
152.96
188.16
162.09
124.23
148.78
120.42
131.63
113.05
184.76
135.06
153.82
188.79
161.98
124.10
148.43
120.14
131.72
113.14
184.53
149.81
135.40
190.50
158.67
138.70
133.62
119.12
130.72
114.92
C-2
C-3
Orange-red needles (C6H6), mp 221–223°C (dec.) (lit.18)
C-4
1
C-5
205–210°C (dec.)). The H-NMR spectral data were identical
to those in ref. 18.
C-6
C-7
Mamegakinone (23)
C-8
Orange-red needles (C6H6), mp 250–255°C (dec.) (lit.19)
C-9
1
256°C (dec.)). The H-NMR spectral data were identical to
C-10
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-7′
C-8′
those in ref. 19.
Ehretione (24)
Orange needles (CHCl3), mp 242–243°C (lit.6) 232°C (dec.)).
1
The H-NMR spectral data were identical to those in ref. 6.
Isoxylospyrin (25)
A yellow powder (C6H6), mp >300°C (lit.20) >350°C). The
1H-NMR spectral data were identical to those in ref. 21.
(−)-2R-β-Dihydroplumbagin (26)
Pale yellow leaflets (hexane), mp 80–82°C. [α]D (23) −88.1°
(c=0.78, CHCl3). IR νmax (KBr) cm−1: 1685, 1640, 1605. UV
λmax (CHCl3) nm (logε): 242 (4.08), 266 (3.74), 349 (3.74). MS
m/z (rel. int. %): 190 [M]+ (76), 175 (100), 162 (17), 147 (11),
C-9′
C-10′
CH3-2
CH3-2′
CH3-7
CH3-7′
>CHCH3
>CHCH3
21.26
16.48
22.16
1
120 (37), 92 (22). H-NMR (CDCl3) δ: 1.31 (3H, d, J=6.0Hz,
22.20
22.20
30.53
17.86
22.28
22.28
Me-2), 2.1–3.5 (3H, m, H-2,3,3), 7.0–7.8 (3H, m, H-6,7,8),
12.01 (1H, s, OH-5). Compound 26 was identified by direct
comparison with synthetic sample.11)
31.65
18.21
2,7′-Dimethyl-2′,3-bijuglone (27)
a) Signals with the same superscript may be interchanged.
Red plates (C6H6), mp 184–185°C. HR-ESI-MS m/z:
373.0711 ([M−H]−, calcd for C22H13O6: 373.0712). IR νmax 254 (4.27), 434 (3.87). MS m/z (rel. int. %): 374 [M]+ (100),
(KBr) cm−1: 1667, 1643, 1610 sh. UV λmax (CHCl3) nm (logε): 359 (10), 357 (17), 346 (5), 187 (4), 134 (8), 120 (7), 106 (13),
255 (4.29), 434 (3.90). MS m/z (rel. int. %): 374 [M]+ (100), 92 (14). H-NMR: Table 1. 13C-NMR: Table 2.
1
359 (27), 357 (24), 346 (7), 187 (6), 134 (3), 120 (8), 106 (5),
2,7′-Dimethyl-6,8′-bijuglone (29)
1
92 (14). H-NMR: Table 1. 13C-NMR: Table 2.
Orange leaflets (hexane), mp 176–178°C. HR-ESI-MS m/z:
2,7′-Dimethyl-3,3′-bijuglone (28)
373.0705 ([M−H]−, calcd for C22H13O6: 373.0712). IR νmax
Orange-red plates (C6H6), mp 170°C (dec.). HR-ESI-MS (KBr) cm−1: 1665, 1636, 1614. UV λmax (CHCl3) nm (logε): 256
m/z: 373.0695 ([M−H]−, calcd for C22H13O6: 373.0712). IR νmax (4.31), 435 (3.83). MS m/z (rel. int. %): 374 [M]+ (100), 357
(KBr) cm−1: 1667, 1633, 1610 sh. UV λmax (CHCl3) nm (logε): (19), 346 (9), 329 (21), 319 (3), 303 (6), 278 (5), 250 (6), 166

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
745
1
(4), 140 (4). H-NMR: Table 1. 13C-NMR: Table 2.
bonded OH).
cis-Tetralone Dibenzoate (34)
cis-Tetralone (32, 18mg) in pyridine (1mL) was treated with
7,7′-Dimethyl-3,3′-ethylidenebijuglone (30)
Orange powder (C6H6), mp 223–225°C. HR-ESI-MS m/z:
m/z 401.1043 ([M−H]−, calcd for C24H17O6: 401.1025). IR νmax benzoyl chloride (0.5mL). The reaction product was separated
(KBr) cm−1: 1668, 1636, 1614. UV λmax (CHCl3) nm (logε): by PTLC (C6H6) as a pale yellow semi-solid (21mg), [α]D (22)
256 (4.21), 432 (3.75). MS m/z (rel. int. %): 402 [M]+ (100), +71.5° (c=1.01, CHCl3). IR νmax (neat) cm−1: 1740, 1720, 1690.
387 (47), 359 (5), 216 (5), 201 (3), 188 (5), 135 (6), 134 (4), 106 UV λmax (CHCl3) nm (logε): 243 (4.03), 282 (3.21), 292 sh
(7), 77 (7), 57 (3), 43 (4). H-NMR: Table 1. 13C-NMR: Table (3.12). MS m/z (%): 400 [M]+ (5), 295 (1), 278 (3), 174 (3), 105
1
1
2.
(100), 77 (18). H-NMR (CDCl3) δ: 1.17 (3H, brd, J=6.5Hz,
2′,7-Dimethyl-3,6′-ethylidenebijuglone (31)
–CH(CH3)–), 2.75 (3H, m, –CH2CH(CH3)–), 6.40 (1H, brs,
Orange needles (C6H6), mp 215–216°C, [α]D (26)+0.42° –CH(OBz)–), 7.1–8.4 (13H, m, Ar H). CD λext (MeOH) nm
(c=4.0, CHCl3). HR-ESI-MS m/z: 401.1030 ([M−H]−, calcd (∆ε): 290 (+1.3), 239 (+10.4), 230 (0.0), 222 (−6.2).
for C24H17O6: 401.1025). IR νmax (KBr) cm−1: 1667, 1639, 1614. Ichthyotoxicity Tests According to a previous paper,1)
UV λmax (CHCl3) nm (logε): 260 (4.33), 430 (3.88). MS m/z ichthyotoxicity tests were performed on 150mL of aqueous so-
(rel. int. %): 402 [M]+ (100), 384 (27), 374 (4), 356 (6), 227 (8), lution containing the test sample using male Japanese killifish
1
201 (5), 135 (3), 106 (2), 40 (14). H-NMR: Table 1. 13C-NMR: (Oryzias latipes var.).
Table 2.
Seed Germination Tests According to the previous
Reflux of 2,3-Epoxyplumbagin (8) in CHCl3 A solu- paper,1) seed germination tests were conducted in 10mL of
tion of 8 (50mg) in CHCl3 containing 7% EtOH (50mL) was aqueous solution containing the test sample against Lettuce
refluxed for 48h in daylight. The reaction mixture was ex- seeds (Lactuca sativa L. var. Great Lakes).
amined by HPLC (Shim-pack SIL (H), n-hexane–C6H6 (7:3)
containing 0.2% AcOH, UV 430nm). Compound 2 was not
detected in the reaction mixture.
Conflict of Interest The authors declare no conflict of
interest.
Reflux
of
7-Methyl-β-dihydrojuglone
(19)
and
β-Dihydroplumbagin (26) in CHCl3 and in 50% EtOH
Compounds 19 (20mg) and 26 (20mg) were refluxed in chlo-
roform (10mL) and in 50% aqueous EtOH (10mL) for 12h.
The resulting mixtures were examined by HPLC (Shim-pack
SIL (H), C6H6 containing 0.2% AcOH, UV 340, 430nm). No
change was observed in the reaction mixtures of 19 and 26 in
CHCl3. In the reaction mixtures of 19 and 26 in 50% aque-
ous EtOH, both 19 and 26 disappeared and 20 and 4 were
detected, respectively.
Reduction of (−)-β-Dihydroplumbagin (26) To 26
(100mg) in 80% acetic acid (20mL) was added zinc (5g);
the mixture was refluxed for 2h. The zinc was removed by
filtration. After the addition of water (100mL), the mixture
was extracted with EtOAc. The extract was washed with 1%
NaHCO3 and water and dried over Na2SO4. After evaporation
of the solvent, the residue was subjected to preparative TLC
(CHCl3−C6H6 (2:1)). The yellow fluorescent band afforded a
mixture of cis- and trans-tetralones. The mixture was sepa-
rated into cis-tetralone (32) (18mg, 18%) and trans-tetralone
(33) (35mg, 34%) by HPLC (Shim-pack PREP SIL (H), CHCl3
containing 0.2% AcOH, UV 340nm).
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cis-Tetralone (32)
Pale yellow oil, [α]D (23) +14.3° (c=0.105, CHCl3). IR νmax
(neat) cm−1: 3450, 1635. UV λmax (CHCl3) nm (logε): 259
(3.93), 332 (3.59). MS m/z (%): 192 (M+, 100), 177 (13), 174
1
(11), 163 (9), 159 (5), 150 (51), 122 (40), 121 (83). H-NMR
(CDCl3) δ: 1.13 (3H, d, J=6.5Hz, –CHCH3–), 2.2–3.2 (3H,
m, –CH2CH(CH3)–), 4.69 (1H, d, J=2.5Hz, –CHOH–), 6.8–7.6
(3H, m, Ar H), 12.36 (1H, s, bonded OH).
trans-Tetralone (33)
Colorless needles (C6H6), mp 102°C, [α]D (30) 0° (c=0.075,
CHCl3). IR νmax (KBr) cm−1: 3200, 1630. UV λmax (CHCl3)
nm (logε): 260 (3.97), 334 (3.60). MS m/z (rel. int. %): 192
[M]+ (100), 177 (15), 174 (10), 163 (9), 159 (7), 150 (40), 122
(29), 121 (57). 1H-NMR (CDCl3) δ: 1.18 (3H, d, J=6.0Hz,
–CH(CH3)–), 2.1–3.2 (3H, m, –CH2CH(CH3)–), 4.47 (1H, brd,
J=7.5Hz, –CHOH–), 6.7–7.7 (3H, m, Ar H), 12.32 (1H, s,