Synthesis of echinamines A and B, the first aminated hydroxynaphthazarins produced by the sea urchin Scaphechinus mirabilis and its analogues

Article abstract of DOI:10.1021/np0502185

The first total synthesis of two marine animated hydroxynaphthazarins, echinamines A (3-amino-7-ethyl-2,5,6,8-tetrahydroxy-1,4-naphthoquinone) and B (2-amino-7-ethyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), produced by the sea urchin Scaphechinus mirabil

Full text of DOI:10.1021/np0502185

J. Nat. Prod. 2006, 69, 1125-1129
1125
Full Papers
Synthesis of Echinamines A and B, the First Aminated Hydroxynaphthazarins Produced by the
Sea Urchin Scaphechinus mirabilis and Its Analogues†
Nataly D. Pokhilo,‡ Maria I. Shuvalova,§ Maxim V. Lebedko,§ Galina I. Sopelnyak,‡ Alla Ya. Yakubovskaya,‡
Natalia P. Mischenko,‡ Sergey A. Fedoreyev,‡ and Victor Ph. Anufriev*,‡
Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences, 159, Prospect 100 let VladiVostoku, VladiVostok 690022, Russian
Federation, and Institute of Chemistry and Applied Ecology, Far Eastern National UniVersity, 27, Oktyabr’skaya Street,
VladiVostok 690600, Russian Federation
ReceiVed June 16, 2005
The first total synthesis of two marine aminated hydroxynaphthazarins, echinamines A (3-amino-7-ethyl-2,5,6,8-
tetrahydroxy-1,4-naphthoquinone) and B (2-amino-7-ethyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), produced by the
sea urchin Scaphechinus mirabilis is described. This was achieved from 1,2,4-triacetoxybenzene (13) through a sequence
involving double Fries rearrangement of 13, reduction of 3,5-diacetyl-1,2,4-trihydroxybenzene (14), methylation of
3,5-diethyl-1,2,4-trihydroxybenzene (15), simultaneous double acylation of 3,5-diethyl-1,2,4-trimethoxybenzene (16)
with a dichloromaleic anhydride-ethyl radical elimination process, methylation of 6,7-dichloro-3-ethyl-2-hydroxynaph-
thazarin (17), nucleophilic substitution of a chlorine atom by the methoxy group in 6,7-dichloro-3-ethyl-2-
methoxynaphthazarin (18), introduction of an amino group via direct substitution of a chlorine atom in 7-chloro-3-
ethyl-2,6-dimethoxy- (11) and 7-chloro-2-ethyl-3,6-dimethoxynaphthazarins (12) by an azido group, and functional group
deprotection. The synthesis of amino analogues of spinazarin and spinochrome D is also described.
More than 100 years have passed since the first isolation of
quinonoid pigments (spinochromes) from the sea urchin (Echi-
noidea). Some spinochromes and their synthetic analogues are
known today as biologically active compounds that possess high
antimicrobial,1 antialgal,2 antioxidant,3 and cardioprotective activity4
or are actual drugs.5,6 From the sea urchin Scaphechinus mirabilis
(Agassiz) we have recently isolated two novel spinochromes named
echinamines A (1) and B (2).7 Echinamines A and B are the first
polyhydroxylated naphthazarins8 (5,8-dihydroxy-1,4-naphthoquino-
nes) having an amino constituent. In in vitro experiments, com-
pounds 1 and 2 were found to be highly effective antioxidants.7
However, echinamines A and B are not easily accessible on a
preparative scale for extended bioassays due to their very low
natural abundance and separation difficulties.
1,4-naphthoquinones were obtained directly (without the need of a
reduction step of azido derivatives) via an overall intramolecular
oxidation-reduction mechanism.9 However, in our experiments,
mompain dimethyl ether 3, a model substrate, did not add hydrazoic
acid. In the case of naphthopurpurin monomethyl ether 4, the
reaction resulted in an inseparable mixture of products 5 and 6
(Scheme 1).
In our second approach, we introduced an amino group into a
naphthazarin nucleus via direct substitution of a chlorine atom by
an azido group (Scheme 2). The nucleophilic substitution of a
chlorine atom by an azido group in 2-methoxy-3-chloronaphthazarin
(7) prepared from dichloronaphthazarin 8 (see below) by the action
of excess NaN3 in DMSO, followed by treatment with water, gave
the expected 3-amino-2-methoxynaphthazarin (9) in moderate yield.
Product 9 is the result of reduction of the corresponding azido-
1,4-naphthoquinone (derived from 7) with hydrazoic acid that arises
during treatment of the reaction mixture with water (Scheme 2).9
Aminomethoxynaphthazarin 9 was easily converted into the amino
analogue spinazarin (10) by the action of concentrated HBr in acetic
acid.
The second method was employed to obtain echinamines A (1)
and B (2). The necessary chloromethoxynaphthazarins 11 and 12
were obtained from triacetoxybenzene 13 by the route summarized
in Scheme 3, as follows. A double Fries rearrangement of
triacetoxybenzene 13 in rigid conditions (melt AlCl3-NaCl) gave
the green-yellow diacetyl derivative 14 in quantitative yield.
Clemmensen reduction then afforded the corresponding diethyltri-
hydroxybenzene 15, and methylation produced trimethoxy deriva-
tive 16. Intermediate 17 was formed as a result of double acylation
of the trisubstituted hydroquinone derivative 16 with dichloromaleic
anhydride. During the course of this reaction, elimination of ethyl
radical at position 5 takes place. This method of forming the
naphthazarin system constitutes a useful addition to existing
approaches.10 Methylation of hydroxynaphthazarin 17 with trimethyl
Results and Discussion
Two approaches to the synthesis of echinamines A and B have
been investigated. The first is based on the application of a
conjugated addition reaction of hydrazoic acid to 1,4-naphtho-
quinones as the key step. In this case the corresponding 2-amino-
† Dedicated to the memory of Professor George B. Elyakov, who has
been an inspiration to us over the years.
* To whom correspondence should be addressed. Tel: 7(4232)-346669.
Fax: 7(4232)-314050. E-mail: anufriev@piboc.dvo.ru.
‡ Pacific Institute of Bioorganic Chemistry.
§ Institute of Chemistry and Applied Ecology, Far Eastern National
University.
10.1021/np0502185 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/19/2006

1126 Journal of Natural Products, 2006, Vol. 69, No. 8
Pokhilo et al.
Scheme 1
Scheme 2a
a 1 R1 ) OH, R2 ) Et; 2 R1 ) Et, R2 ) OH; 7 R1 ) R2 ) H; 9, 10 R1 ) R2 ) H; 11 R1 ) OMe, R2)Et; 12 R1 ) Et, R2 ) OMe; 19 R1 ) OMe, R2 ) Et; 20
R1 ) Et, R2 ) OMe; 21 R1 ) OH, R2 ) H; 22 R1 ) H, R2 ) OH; 23 R1 ) OMe, R2 ) H; 24 R1 ) OMe, R2 ) H; 27 R1 ) H, R2 ) OMe. (i) NaN3, DMSO, 50
°C, then H2O; (ii) HBr-HOAc, reflux.
Scheme 3a
a (i) AlCl3-NaCl, 190 °C; (ii) Zn/Hg, HCl, reflux; (iii) Me2SO4, NaOH; (iv) (MeO)3CH, reflux; (v) MeOH/CsF/Al2O3, reflux.
Scheme 4a
orthoformate gave the corresponding methyl ether 18, and nucleo-
philic substitution of chlorine by a methoxy group in that compound
afforded a mixture of 3-chloro-7-ethyl-2,6-dimethoxy- (11) and
3-chloro-6-ethyl-2,7-dimethoxynaphthazarins (12) in high yield.
Isomers 11 and 12 were separable, on a preparative scale, and were
purified by column chromatography. The structures of dimethox-
ynaphthazarins 11 and 12 were assigned on the basis of spectral
analysis and direct comparison with authentic samples.11
Dimethoxynaphthazarins 11 and 12 underwent amination (11 f
19, 12 f 20) via direct substitution of chlorine by an azido group
a (i) MeOH/CsF/Al2O3, reflux; (ii) H2SO4-H3BO3, 200 °C; (iii) CH2N2, Et2O;
and reduction of the corresponding azido-1,4-naphthoquinones
(Scheme 2). Demethylation of dimethyl ethers 19 and 20 gave the
expected aminohydroxynaphthazarins 1 and 2. Synthetic compounds
1 and 2 are identical in all respects with echinamines A and B
isolated from the sea urchin S. mirabilis.7
(iv) Br2, AcOH.
conditions suggests that echinochrome is, at least in part, an artifact
of the isolation procedure.
The same approach was used for the synthesis of amino
analogues of spinochrome D (21 and 22), which were never reported
as a natural product, but are presumed to be present among the
minor pigments of the sea urchin S. mirabilis. The necessary
substrate 23 for the conversion 23 f 24 f 21 (Scheme 2) was
prepared by oxidation of dichloronaphthazarin 8 with sulfuric acid
followed by methylation of dihydroxynaphthazarin 25 with diaz-
omethane according to Scheme 4. Compound 26, in turn, was
obtained by direct bromination of dimethyl ether 3.
In this paper, we describe the first total synthesis of two marine
aminated hydroxynaphthazarins, echinamines A and B, which are
produced by the sea urchin S. mirabilis. The basis for this synthesis
is simultaneous double acylation of 3,5-diethyl-1,2,4-trimethoxy-
benzene with dichloromaleic anhydride followed by an ethyl radical
elimination process and the introduction of an amino group via
direct substitution of a chlorine atom in a naphthazarin moiety by
an azido group. The general utility of this latter transformation has
It should be noted that when echinamines A and B were worked
up using acids (the standard procedure for separation of spinochromes
from sea urchins), the formation of echinochrome (28), a major
pigment of the sea urchin S. mirabilis,7 was observed. The apparent
ease of conversion of compounds 1 and 2 to 28 under acidic

Aminated Hydroxynaphthazarins from Scaphechinus mirabilis
Journal of Natural Products, 2006, Vol. 69, No. 8 1127
In accordance with general procedure 2, 3-amino-5,8-dihydroxy-
2-methoxy-1,4-naphthoquinone (9) was obtained as a brown powder
from 7 (50 mg, 43%): Rf 0.34; mp >260 °C (dec); IR (CHCl3) νmax
3514 m, 3398 m (NH2), 2857 m, 1648 w, 1625 w, 1595 s (CdO),
been demonstrated with amino analogues of spinazarin and
spinochrome D.
Experimental Section
1
1569 m, 1556 s (CdC) cm-1; H NMR (CDCl3, 300 MHz) δ 4.03
General Experimental Procedures. All melting points were
determined with a Boetius apparatus and are uncorrected. The IR
absorption spectra were recorded on a Vector 22 IR-FT spectropho-
tometer. 1H and 13C NMR spectra were recorded on Bruker AVANCE
DPX-300 and DRX-500 NMR spectrometers at 1H and 13C frequencies
of 300 and 75 MHz, or 500 and 125 MHz, respectively. Chemical shifts
in δ are relative to TMS as an internal reference (δ ) 0). Mass spectra
were taken on a LKB-9000S spectrometer (direct sample inlet, ionizing
energy 70 eV, and elevated temperature). Elemental analysis was
performed with a Flash EA1112 CHN/MAS200. The course of reactions
was monitored and the purity of the compounds obtained were checked
by TLC (Merck Kieselgel 60F-254 plates were preliminarily treated
with 0.05 M tartaric acid in MeOH and dried at ∼50 °C for 2-3 h; a
3:1 n-hexane/acetone mixture was used as an eluent). Preparative TLC
and column chromatography were performed on silica gel L (Chemapol,
Czechia), 5/40 and 40/100 µm, respectively, using n-hexane/acetone.
Yields were not optimized.
(3H, s, OCH3), 5.15 (2H, br s, NH2), 7.10, 7.20 (each 1H, d, J ) 9.4
Hz, Harom), 11.97, 12.87 (each 1H, s, R-OH); EIMS m/z 236 [M + 1]+
(46), 235 [M]+ (100), 192 (41), 189 (26), 165 (34), 164 (20); anal. C
56.64%, H 3.72%, N 6.00%, calcd for C11H9NO5, C 56.17%, H 3.86%,
N 5.96%.
General Procedure 3. Hydrolysis of Aminomethoxynaphthaz-
arins 9, 19, 20, 24, and 27. The methyl ether of aminomethoxynaph-
thazarin (0.2 mmol) in concentrated HBr/HOAc, 1:1 (20 mL), was
refluxed for 0.5-1 h. The reaction mixture was diluted with H2O (100
mL) and extracted with EtOAc. The extract was concentrated, and the
product was isolated by preparative TLC (n-hexane/acetone, 2:1).
In accordance with general procedure 3, 3-amino-2,5,8-trihydroxy-
1,4-naphthoquinone (10) was obtained as an orange-brown powder
from 9 (37 mg, 85%); Rf 0.27; mp >300 °C (dec); 1H NMR (DMSO-
d6, 300 MHz) δ 6.36 (2H, br s, NH2), 7.14, 7.20 (each 1H, d, J ) 9.3
Hz, Harom), 8.31 (1H, s, â-OH), 12.01, 12.66 (each 1H, s, R-OH); EIMS
m/z 223 [M + 2]+ (10), 222 [M + 1]+ (65), 221 [M]+ (100), 194 (39),
193 (32); anal. C 54.46%, H 3.07%, N 6.40%, calcd for C10H7NO5, C
54.31%, H 3.19%, N 6.33%.
The following starting compounds were prepared according to
previously described procedures: 5,8-dihydroxy-2,7-dimethoxy-1,4-
naphthoquinone (3),11 5,8-dihydroxy-2-methoxy-1,4-naphthoquinone
(4),12 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (8).10
3,5-Diacetyl-1,2,4-trihydroxybenzene (14). At 140 °C, 1,2,4-
triacetoxybenzene (13) (60 g, 0.24 mol) was added with vigorous
stirring to a melt consisting of anhydrous AlCl3 (234 g, 1.75 mol) and
NaCl (44 g, 0.75 mol). The temperature of the mixture was increased
to 195 °C, and the melt was stirred for an additional 5 min. The reaction
mixture was cooled, hydrolyzed with 5% HCl (2 L), and allowed to
stand for 12 h. From the resulting crude product 14 was separated as
yellow powder and washed with 5% HCl (1 L) and hot H2O (1 L):
6-Amino-5,8-dihydroxy-2-methoxy-1,4-naphthoquinone (5) and
7-Amino-5,8-dihydroxy-2-methoxy-1,4-naphthoquinone (6). To a
stirred solution of naphthopurpurin monomethyl ether 4 (220 mg, 1.0
mmol) in 100 mL of methanol under argon was added a solution of
sodium azide (740 mg, 11.4 mmol) in 10 mL of water, acidified to pH
∼4 (with 1 N HCl). The reaction mixture was stirred at 50 °C for 20
h. Then the mixture was extracted with EtOAc, and the organic layer
was washed with water and brine, dried over Na2SO4, and concentrated.
The mixture of products 5 and 6 in a 1:1.7 ratio was isolated by
preparative TLC (n-hexane/acetone, 3:1) as a brown powder (31 mg,
13%): Rf 0.12; IR (CHCl3) νmax 3515 m, 3395 m (NH2), 1645 w, 1629
1
yield 48.0 g (96%); mp 180-185 °C; H NMR (CDCl3, 500 MHz) δ
2.56, 2.79 (each 1H, s, COCH3), 5.47 (1H, br s, OH), 7.43 (1H, s,
H
arom), 14.39, 14.81 (each 1H, s, OH); 13C NMR (CDCl3, 125 MHz) δ
206.1 (C, C-5-COCH3), 203.0 (C, C-3-COCH3), 161.8 (C, C-4), 158.6
(C, C-2), 137.1 (C, C-1), 119.7 (CH, C-6), 110.3 (C, C-3), 109.5 (C,
C-5), 33.2 (CH3, C-5-COCH3), 26.2 (CH3, C-3-COCH3); EIMS m/z
210 [M]+ (90), 195 (100).
m, 1618 m (CdO), 1593 s, 1564 s (NH2, CdC) cm-1
.
Compound 5: 1H NMR (CDCl3, 300 MHz) δ 3.97 (3H, s, OCH3),
5.33 (2H, br s, NH2), 5.99 (1H, s, Harom), 6.42 (1H, s, Harom), 12.73,
13.81 (each 1H, s, R-OH).13
3,5-Diethyl-1,2,4-trihydroxybenzene (15). In a three-necked 3 L
round-bottom flask equipped with a mechanical stirrer and a reflux
condenser diacetyltrihydroxybenzene 14 (24 g, 0.11 mol), solid zinc
amalgam (500 g), and concentrated HCl (330 mL) were mixed. The
mixture was stirred under reflux for 0.5 h, then a second portion of
substrate 14 (24 g, 0.11 mol) and concentrated HCl (330 mL) was
added, and the mixture was heated to reflux for 3 h. The hot solution
was decanted and allowed to stand for 15 h. The solid was separated,
washed with 35 mL of cold H2O, and successively dried in vacuo.
According to the 1H NMR data, the resulting product, a white
amorphous solid, contained 80% (33 g) of diethyltrihydroxybenzene
15: 1H NMR (CDCl3, 300 MHz) δ 1.18, 1.20 (each 3H, t, J ) 7.8 Hz,
CH3), 2.52, 2.68 (each 2H, q, J ) 7.8 Hz, CH2), 4.35, 4.54 (each 1H,
br s, OH), 5.15 (1H, br s, OH), 6.56 (1H, s, Harom). Crude product 15
was used in the next synthetic step without purification.
Compound 6: 1H NMR (CDCl3, 300 MHz) δ 3.94 (3H, s, OCH3),
5.12 (2H, br s, NH2), 6.02 (1H, s, Harom), 6.55 (1H, s, Harom), 12.49,
13.47 (each 1H, s, R-OH).13
General Procedure 1. Nucleophilic Substitution of Chlorine
Atoms in Chloronaphthazarins 8 and 18. A mixture of the corre-
sponding well-dried substrate (1 mmol), anhydrous CsF (6-7 mmol),
activated neutral alumina (Aldrich, ∼150 mesh, for chromatography)
(1.0-1.5 g), and absolute MeOH (100 mL) was stirred in a closed
flask under reflux for 1 h. After being cooled the absorbent was
separated by filtration and washed successively with 5% HCl (2 mL)
and acetone (5 mL) or hot alcohol. The combined filtrate was
concentrated in vacuo, and the residue was treated with CHCl3. The
organic layer was washed successively with water and brine, dried (Na2-
SO4), filtered, and concentrated. The products were isolated by column
chromatography followed by crystallization from EtOH.
In accordance with general procedure 1, 3-chloro-5,8-dihydroxy-
2-methoxy-1,4-naphthoquinone (7) was obtained as brown-red crystals
from 8 (180 mg, 71%): mp 141-144 °C; 1H NMR (CDCl3, 500 MHz)
δ 4.34 (3H, s, OCH3), 7.25, 7.29 (each 1H, d, J ) 9.6 Hz, Harom),
12.25, 12.44 (each 1H, s, R-OH); 13C NMR (CDCl3, 125 MHz) δ 181.9
(C, C-1), 181.5 (C, C-4), 159.2 (C, C-8), 158.2 (C, C-5), 157.2 (C,
C-2), 130.5 (CH, C-6), 129.6 (CH, C-7), 128.5 (C, C-3), 110.8 (C,
C-8a), 110.1 (C, C-4a), 62.2 (CH3, OCH3); EIMS m/z 255/257 [M +
1]+ (13), 254/256 [M]+ (100), 236/238 (26); anal. C 52.03%, H 2.81%,
calcd for C11H7ClO5, C 51.89%, H 2.77%.
3,5-Diethyl-1,2,4-trimethoxybenzene (16). To a mechanically stirred
dark mixture of 10% aqueous NaOH (290 g) and 3,5-diethyl-1,2,4-
trihydroxybenzene (15) (33 g, 0.18 mol) under argon was added
dropwise Me2SO4 (68.6 g, 0.54 mol), maintaining the temperature of
the mixture below 40 °C. After Me2SO4 was added, the reaction mixture
was heated for 30 min in a boiling water bath to decompose the excess
Me2SO4. After the mixture was cooled, the organic layer was separated,
and the aqueous layer extracted with benzene. The combined organic
layers were washed with 5% aqueous NaOH and H2O, dried over
anhydrous CaCl2, and filtered, and the solvent was removed using a
rotary evaporator. The resulting product, a colorless oil, was purified
1
by vacuum distillation (33.3 g, 82%): bp 133-139 °C, 7 mmHg; H
NMR (CDCl3, 300 MHz) δ 1.19, 1.24 (each 3H, t, J ) 7.8 Hz, CH3),
2.64, 2.66 (each 2H, q, J ) 7.8 Hz, CH2), 3.71, 3.82, 3.83 (each 3H,
s, OCH3), 6.60 (1H, s, Harom).
General Procedure 2. Reaction of Chloronaphthazarins 7, 11,
12, 23, and 26 with NaN3. To a stirred solution of the corresponding
naphthazarin (0.5 mmol) in 40 mL of DMSO was added NaN3 (3.0
mmol). The reaction mixture was stirred at 60-70 °C and monitored
by TLC. Then the reaction mixture was diluted with H2O and extracted
with EtOAc. The organic layer was washed with water and brine, dried
over Na2SO4, and concentrated, and the product was isolated by
preparative TLC (n-hexane/acetone, 3:1).
6,7-Dichloro-3-ethyl-2,5,8-trihydroxy-1,4-naphthoquinone (17).
At 140 °C, a mixture of 1,2,4-trimethoxy-3,5-diethylbenzene (16) (1.75
g, 7.8 mmol) and dichloromaleic anhydride (3 g, 17.9 mmol) was added
with vigorous stirring to a melt consisting of anhydrous AlCl3 (16 g,
120 mmol) and NaCl (3.2 g, 55 mmol). The temperature of the mixture
was increased to 195 °C, and the melt was stirred for an additional 5

1128 Journal of Natural Products, 2006, Vol. 69, No. 8
Pokhilo et al.
min. The reaction mixture was cooled, hydrolyzed with 5% HCl (200
mL), and allowed to stand for 12 h. The resulting crude product was
separated, washed with 100 mL of hot H2O, dried, and purified by
column chromatography to give 17 as wine-red needles (1.39 g, 59%):
mp 156-158 °C (lit.14 mp 156-158 °C); 1H NMR (CDCl3, 300 MHz)
δ 1.18 (3H, t, J ) 7.7 Hz, CH3), 2.66 (2H, q, J ) 7.7 Hz, CH2), 7.42
(1H, br s, â-OH), 12.07, 13.60 (each 1H, s, R-OH); 13C NMR (CDCl3,
75 MHz) δ 187.5 (C, C-4), 181.5 (C, C-1), 154.6 (C, C-5), 154.3 (C,
C-8), 153.5 (C, C-2), 135.3 (C, C-6), 131.3 (C, C-7), 127.7 (C, C-3),
109.3 (C, C-4a), 109.0 (C, C-8a), 16.5 (CH2, CH2CH3), 12.5 (CH3,
CH2CH3); EIMS m/z 302/304/306 [M]+ (71), 286/288/290 (83), 285/
287/289 (65), 267/269 (45), 252/254 (35), 244/246 (31), 230 (100);
anal. C 47.64%, H 2.71%, calcd for C12H8Cl2O5, C 47.55%, H 2.66%.
6,7-Dichloro-3-ethyl-5,8-dihydroxy-2-methoxy-1,4-naphthoquin-
one (18). A mixture of hydroxynaphthazarin 17 (400 mg, 1.32 mmol)
and trimethyl orthoformate (30 mL) was refluxed for 12 h. After being
cooled in the refrigerator to 8 °C, the crystals were separated and dried
in vacuo to give methoxynaphthazarin 18 as wine-red prisms (360 mg).
The filtrate was evaporated and the residue was purified by preparative
TLC using n-hexane/acetone (2:1) to give 40 mg of the same product.
263 (25), 250 (23), 248 (24); anal. C 57.27%, H 5.22%, N 4.66%,
calcd for C14H15O6N, C 57.32%, H 5.16%, N 4.78%.
In accordance with general procedure 3, products 1 and 2 were
obtained as dark brown powders (acetone).
3-Amino-7-ethyl-2,5,6,8-tetrahydroxy-1,4-naphthoquinone (echin-
amine A, 1): obtained from 19 (48 mg, 91%); Rf 0.17; mp 245-246
°C; IR (CHCl3) νmax 3522 m, 3445 w, 3379 m (NH2, â-OH), 1650 m,
1603 m (CdO), 1589 s, 1562 s (NH2, CdC) cm-1; 1H NMR (CDCl3,
300 MHz) δ 1.14 (3H, t, J ) 7.5 Hz, CH3), 2.69 (2H, q, J ) 7.5 Hz,
CH2), 5.36 (2H, br s, NH2), 8.49, 9.20 (each 1H, br s, â-OH), 12.62,
13.03 (each 1H, s, R-OH); 13C NMR (CDCl3, 75 MHz) δ 181.7 (C,
C-1), 177.4 (C, C-4), 161.0 (C, C-8), 154.0 (C, C-5), 152.3 (C, C-6),
137.0 (C, C-2), 132.4 (C, C-3), 126.5 (C, C-7), 108.6 (CH, C-4a), 102.6
(C, C-8a), 16.3 (CH2, CH2CH3), 12.9 (CH3, CH2CH3); EIMS m/z 266
[M + 1]+ (44), 265 [M]+ (100), 264 (15), 223 (12), 222 (40).
2-Amino-7-ethyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone (echin-
amine B, 2): obtained from 20 (37 mg, 69%); Rf 0.19; mp 265-267
°C; IR (CHCl3) νmax 3518 m, 3460 w, 3398 m (NH2, â-OH), 1664 m,
1603 m (CdO), 1580 m, 1560 s (NH2, CdC) cm-1; 1H NMR (CDCl3,
300 MHz) δ 1.13 (3H, t, J ) 7.5 Hz, CH3), 2.67 (2H, q, J ) 7.5 Hz,
CH2), 5.81 (2H, br s, NH2), 8.36, 9.44 (each 1H, br s, â-OH), 13.02
(2H, s, R-OH); 13C NMR (CDCl3, 75 MHz) δ 178.7 (C, C-4), 176.6
(C, C-1), 163.2 (C, C-8), 154.0 (C, C-5), 151.4 (C, C-6), 135.1 (C,
C-3), 134.8 (C, C-2), 124.4 (C, C-7), 107.6 (CH, C-4a), 103.9 (C, C-8a),
16.6 (CH2, CH2CH3), 12.9 (CH3, CH2CH3); EIMS m/z 266 [M + 1]+
(32), 265 [M]+ (77), 250 (25), 237 (17), 222 (100).
1
Total yield of 18 was 94%; mp 138-141 °C; H NMR (CDCl3, 500
MHz) δ 1.14 (3H, t, J ) 7.1 Hz, CH3), 2.65 (2H, q, J ) 7.1 Hz, CH2),
4.17 (3H, s, OCH3), 12.86, 13.32 (each 1H, s, R-OH); 13C NMR (CDCl3,
125 MHz) δ 181.9 (C, C-4), 177.4 (C, C-1), 159.5 (C, C-8), 158.8 (C,
C-5), 157.2 (C, C-2), 138.3 (C, C-3), 135.5 (C, C-7),15 134.1 (C, C-6),15
110.0 (C, C-8a), 108.9 (C, C-4a), 61.8 (CH3, OCH3), 17.0 (CH2, CH2-
CH3), 13.2 (CH3, CH2CH3); EIMS m/z 316/318/320 [M]+ (100), 301/
303/305 (73), 283/285/287 (10), 273/275/277 (15); anal. C 49.54%, H
3.23%, calcd for C13H10Cl2O5, C 49.23%, H 3.18%.
3-Bromo-5,8-dihydroxy-2,7-dimethoxy-1,4-naphthoquinone (26).
To a stirred solution of 5,8-dihydroxy-2,7-dimethoxy-1,4-naphtho-
quinone (3) (250 mg, 1.0 mmol) in 60 mL of AcOH was added dry
bromine (325 µL). The reaction mixture was kept at room temperature
for 48 h and monitored by TLC (n-hexane/acetone, 2:1). Then the
reaction mixture was diluted with H2O and extracted with EtOAc. The
organic layer was washed with water and brine, dried over Na2SO4,
and concentrated, and the product was isolated by preparative TLC
(n-hexane/acetone, 2:1) as a red-brown solid (118 mg, 36%): Rf 0.42;
mp >230 °C (dec); IR (CCl4) νmax 2856 s, 1614 s, 1602 s (CdO),
In accordance with general procedure 1, products 11 and 12 were
obtained as red solids.
3-Chloro-7-ethyl-5,8-dihydroxy-2,6-dimethoxy-1,4-naphthoquin-
one (11): 156 mg (50%); Rf 0.62; mp 137-139 °C (acetone); 1H NMR
(CDCl3, 500 MHz) δ 1.15 (3H, t, J ) 7.5 Hz, CH3), 2.69 (2H, q, J )
7.5 Hz, CH2), 4.12, 4.25 (each 3H, s, OCH3), 13.09, 13.14 (each 1H s,
R-OH); 13C NMR (CDCl3, 125 MHz) δ 172.5 (C, C-4), 170.6 (C, C-1),
168.4 (C, C-5), 166.2 (C, C-8), 156.6 (C, C-2), 156.2 (C, C-6), 137.0
(C, C-3), 126.0 (C, C-7), 108.3 (C, C-8a), 108.5 (C, C-4a), 61.9 (CH3,
C-6-OCH3), 61.6 (CH3, C-2-OCH3), 17.0 (CH2, CH2CH3), 13.4 (CH3,
CH2CH3); EIMS m/z 312/314 [M]+ (100), 311/313 (92), 297/299 (12),
296/298 (25), 294 (10), 293 (11), 256 (9); anal. C 53.79%, H 4.30%,
calcd for C14H13ClO6, C 53.84%, H 4.20%.
6-Chloro-3-ethyl-5,8-dihydroxy-2,7-dimethoxy-1,4-naphthoquin-
one (12): 81 mg (26%); Rf 0.59; mp 129-131 °C (acetone); 1H NMR
(CDCl3, 500 MHz) δ 1.17 (3H, t, J ) 7.5 Hz, CH3), 2.71 (2H, q, J )
7.5 Hz, CH2), 4.11, 4.22 (each 3H, s, OCH3), 12.83, 13.34 (each 1H,
s, R-OH); 13C NMR (CDCl3, 125 MHz) δ 171.8 (C, C-4), 169.3 (C,
C-5), 169.0 (C, C-1), 167.8 (C, C-8), 156.1 (C, C-2), 155.3 (C, C-7),
138.2 (C, C-3), 127.6 (C, C-6), 110.3 (C, C-8a), 106.6 (C, C-4a), 61.8
(CH3, C-7-OCH3), 61.6 (CH3, C-2-OCH3), 17.3 (CH2, CH2CH3), 13.5
(CH3, CH2CH3); EIMS m/z 312/314 [M]+ (100), 311/313 [M - 1]+-
(70), 297/299 (27), 296/298 (30), 278 (12), 223 (16); anal. C 53.77%,
H 4.28%, calcd for C14H13ClO6, C 53.84%, H 4.20%.
1
1570 m, 1554 m, 1550 m (CdC) cm-1; H NMR (CDCl3, 500 MHz)
δ 3.96, 4.18 (each 3H, s, OCH3), 6.34 (1H, s, Harom), 12.67, 13.37 (each
1H, s, R-OH); 13C NMR (CDCl3, 125 MHz) δ 177.7 (C, C-4), 171.4
(C, C-1), 164.4 (C, C-5), 163.4 (C, C-8), 159.7 (C, C-2), 156.0 (C,
C-7), 120.2 (C, C-3), 111.3 (C, C-8a), 108.9 (CH, C-6), 105.5 (C, C-4a),
61.5 (CH3, C-7-OCH3), 56.9 (CH3, C-2-OCH3); EIMS m/z 328/330
[M]+ (92), 330 (33), 237 (62), 236 (100).
3-Chloro-2,5,6,8-tetrahydroxy-1,4-naphthoquinone (25). A mix-
ture of dichloronaphthazarin 8 (2.0 g, 7.9 mmol), H3BO3 (1.0 g, 16.1
mmol), and 20 mL of concentrated H2SO4 was heated at 210-220 °C
for 20 min. After being cooled the reaction mixture was diluted with
H2O. The solid was separated, dried in vacuo, and diluted with EtOAc
(1 L). Then this solution was filtered through the column with silica
gel and dried over Na2SO4, and the product was isolated by column
chromatography (n-hexane/acetone, 2:1) as a dark wine-red powder
(1.4 g, 69%): mp 227-230 °C (dec); 1H NMR (acetone-d6, 300 MHz)
δ 6.62 (1H, s, Harom), 10.07, 10.52 (each 1H, br s, â-OH), 12.29, 13.00
(each 1H, s, R-OH); 13C NMR (CDCl3, 125 MHz) δ 180.1 (C, C-4),
174.8 (C, C-1), 166.0 (C, C-6), 159.3 (C, C-8), 157.6 (C, C-2), 154.1
(C, C-5), 116.8 (C, C-3), 110.4 (C, C-4a), 109.1 (CH, C-7), 105.1 (C,
C-8a); EIMS m/z 256/258 [M]+ (100), 228/230 (42), 200 (5), 193 (12),
188 (6), 186 (17), 158 (7); anal. C 47.02%, H 2.02%, calcd for C10H5-
ClO6, C 46.81%, H 1.96%.
7-Chloro-5,8-dihydroxy-2,6-dimethoxy-1,4-naphthoquinone (23):
obtained by reaction of corresponding naphthazarin 25 (0.5 mmol) in
Et2O with a solution of CH2N2 in Et2O monitored by TLC. The reaction
mixture was concentrated in vacuo to give a residue, which was purified
by preparative TLC (n-hexane/acetone, 3:1). Compound 23: dark red
needles; yield 110 mg (78%); Rf 0.43; mp 190-194 °C; 1H NMR
(CDCl3, 500 MHz) δ 3.97, 4.26 (each 3H, s, OCH3), 6.35 (1H, s, Harom),
13.03, 13.09 (each 1H, s, R-OH); 13C NMR (CDCl3, 125 MHz) δ 177.4
(C, C-4), 168.6 (C, C-1),15 167.2 (C, C-8),15 163.6 (C, C-5), 160.2 (C,
C-2), 156.3 (C, C-6), 124.9 (C, C-7), 108.6 (C, C-8a), 108.3 (CH, C-3),
107.7 (C, C-4a), 61.9 (CH3, C-6-OCH3), 56.9 (CH3, C-2-OCH3); EIMS
m/z 284/286 [M]+ (100), 283/285 [M - 1]+ (92), 266/268 (25), 255
(19), 249 (12), 236 (9); anal. C 50.50%, H 3.45%, calcd for C12H9-
ClO6, C 50.63%, H 3.19%.
In accordance with general procedure 2, products 19 and 20 were
obtained as yellow-brown needles.
3-Amino-7-ethyl-5,8-dihydroxy-2,6-dimethoxy-1,4-naphthoquin-
one (19): obtained from 11 (63 mg, 43%); Rf 0.32; mp 300 °C (dec);
IR (CHCl3) νmax 3514 m, 3398 m (NH2), 1684 w, 1641 m, 1616 m
(CdO), 1593 s, 1556 s (NH2, CdC) cm-1; 1H NMR (CDCl3, 500 MHz)
δ 1.17 (3H, t, J ) 7.6 Hz, CH3), 2.74 (2H, q, J ) 7.6 Hz, CH2), 4.00,
4.01 (each 3H, s, OCH3), 5.06 (2H, br s, NH2), 12.52, 13.48 (each 1H,
s, R-OH); EIMS m/z 293 [M]+ (100), 292 (30), 278 (76), 263 (22),
250 (31), 248 (37), 235 (27), 221 (26); anal. C 57.25%, H 5.25%, N
4.90% calcd for C14H15O6N, C 57.32%, H 5.16%, N 4.78%.
3-Amino-6-ethyl-5,8-dihydroxy-2,7-dimethoxy-1,4-naphthoquin-
one (20): obtained from 12 (66 mg, 45%); Rf 0.36; mp 118-120 °C;
IR (CHCl3) νmax 3514 m, 3396 m (NH2), 1639 m, 1618 m (CdO),
1
1590 s, 1555 (NH2, CdC) cm-1; H NMR (CDCl3, 500 MHz) δ 1.15
(3H, t, J ) 7.5 Hz, CH3), 2.69 (2H, q, J ) 7.5 Hz, CH2), 3.99, 4.06
(each 3H, s, OCH3), 5.17 (2H, br s, NH2), 12.67, 13.56 (each 1H, s,
R-OH); 13C NMR (CDCl3, 125 MHz) δ 181.1 (C, C-1), 181.2 (C, C-4),
159.6 (C, C-5), 156.3 (C, C-7), 153.4 (C, C-8), 140.5 (C, C-3), 136.5
(C, C-2), 133.5 (C, C-6), 108.7 (C, C-8a), 106.4 (C, C-4a), 61.4 (CH3,
C-7-OCH3), 60.4 (CH3, C-2-OCH3), 17.0 (CH2, CH2CH3), 13.6 (CH3,
CH2CH3); EIMS m/z 293 [M]+ (100), 292 (86), 279 (22), 278 (86),
In accordance with general procedure 2, products 24 and 27 were
obtained as yellow-brown powders.

Aminated Hydroxynaphthazarins from Scaphechinus mirabilis
Journal of Natural Products, 2006, Vol. 69, No. 8 1129
3-Amino-5,8-dihydroxy-2,6-dimethoxy-1,4-naphthoquinone
(24): from 23 (15% yield); Rf 0.29; mp 190-194 °C; IR (CHCl3) νmax
3514 m, 3399 s (NH2), 2854 m, 1647 w, 1620 sh m, 1597 s (CdO),
1580 sh m, 1557 m (CdC), cm-1; 1H NMR (CDCl3, 300 MHz) δ 3.97,
4.26 (each 3H, s, OCH3), 6.35 (1H, s, Harom), 13.03, 13.10 (each 1H,
s, R-OH); EIMS m/z 266 [M + 1]+ (46), 265 [M]+ (100); anal. C
54.25%, H 4.40%, N 5.20%, calcd for C12H11NO6, C 54.34%, H 4.18%,
N 5.28%.
the Russian Federation of Support of Leading Scientific Schools, the
Integration Project of Far-Eastern and Siberian Branches of RAS (No.
06-II-CO-05-020), and the Program Grants of the Presidium of RAS
“Molecular and Cell Biology” (No. 06-I-P10-019). The authors are
grateful to Dr. V. A. Denisenko (Pacific Institute of Bioorganic
Chemistry) for measurements of NMR spectra.
References and Notes
3-Amino-5,8-dihydroxy-2,7-dimethoxy-1,4-naphthoquinone (27):
from 26 (29% yield); Rf 0.29; mp 100-110 °C; IR (CCl4) νmax 3520
m, 3400 m (NH2), 2856 m, 1650 m, 1619 m, 1591 s (CdO), 1580 sh
s, 1545 m (CdC) cm-1; 1H NMR (acetone-d6, 300 MHz) δ 3.89, 3.98
(each 3H, s, OCH3), 6.48 (2H, br s, NH2), 6.58 (1H, s, Harom), 12.67,
13.83 (each 1H, s, R-OH); EIMS m/z 266 [M + 1]+ (17), 265 [M]+
(100), 250 (20), 247 (14); anal. C 54.12%, H 4.52%, N 5.07%, calcd
for C12H11NO6, C 54.34%, H 4.18%, N 5.28%.
(1) Service, M.; Wardlaw, A. C. Comp. Biochem. Physiol. 1984, 79,
161-163.
(2) GBR Patent 2159056, 1985; Chem. Abstr. 1986, 104, 83795.
(3) Boguslavskaya, L. V.; Khrapova, N. G.; Maximov, O. B. IzV. Akad.
Nauk USSR. Ser. Khim. 1985, 7, 1471-1476 (in Russian).
(4) Anufriev, V. Ph.; Novikov, V. L.; Maximov, O. B.; Elyakov, G. B.;
Levitsky, D. O.; Lebedev, A. V.; Sadretdinov, S. M.; Shvilkin, A.
V.; Afonskaya, N. I.; Ruda, M. Ya.; Cherpachenko, N. M. Bioorg.
Med. Chem. Lett. 1998, 8, 587-592.
In accordance with general procedure 3, products 21 and 22 were
obtained as dark brown powders.
(5) U.S. Patent 6384084, 2002.
(6) U.S. Patent 6410601, 2002.
(7) Mischenko, N. P.; Fedoreyev, S. A.; Pokhilo, N. D.; Anufriev, V.
Ph.; Denisenko, V. A.; Glazunov, V. P. J. Nat. Prod. 2005, 68,
1390-1393.
(8) For convenience naphthazarins are written in one tautomeric form
only.
(9) Couladouros, E. A.; Plyta, Z. F.; Haroutounian, S. A., Papageorgiou,
V. P. J. Org. Chem. 1997, 62, 6-10.
(10) Huot, R.; Brassard, P. Can. J. Chem. 1974, 54, 838-842.
(11) Glazunov, V. P.; Tchizhova, A. Ya.; Shestak, O. P.; Sopelnyak, G.
I.; Anufriev, V. Ph. Russ. Chem. Bull., Int. Ed. 2001, 50, 95-100.
(12) Donaldson, N. The Chemistry and Technology of Naphthalene
Compounds; Arnold: London, 1960; p 461.
(13) The NMR spectral assignment for 5 and 6 is exchangeable.
(14) Tchizhova, A. Ya.; Kochergina, T. Yu.; Anufriev, V. Ph.; Denisenko,
V. A.; Glazunov, V. P. Russ. Chem. Bull., Int. Ed. 1999, 48, 938-
943.
3-Amino-2,5,7,8-tetrahydroxy-1,4-naphthoquinone (21): from 24
1
(66% yield); Rf 0.05; mp >300 °C (dec); H NMR (acetone-d6, 300
MHz) δ 5.90 (2H, br s, NH2), 6.53 (1H, s, Harom), 8.32, 9.84 (1H each,
both br s, â-OH), 12.54, 12.58 (1H each, both s, R-OH); EIMS m/z
(%) 238 (12) [M + 1]+, 237 (8) [M]+, 236 (50), 235(100), 223 (7),
218 (5), 205 (5); anal. C 50.40%, H 3.10%, N 5.70%, calcd for C10H7-
NO6, C 50.64%, H 2.97%, N 5.91%.
3-Amino-2,5,6,8-tetrahydroxy-1,4-naphthoquinone (22): from 27
1
(48% yield); Rf 0.06; mp >300 °C (dec); H NMR (acetone-d6, 300
MHz) δ 5.90 (2H, br s, NH2), 6.46 (1H, s, Harom), 8.32, 9.84 (1H each,
both br s, â-OH), 12.50, 13.03 (1H each, both s, R-OH); EIMS m/z
(%) 240 (13), 239 (39), 238 (92) [M + 1]+, 237 (100) [M]+, 236 (19),
211 (12), 210 (25), 209 (26); anal. C 50.75%, H 3.00%, N 6.10%,
calcd for C10H7NO6, C 50.64%, H 2.97%, N 5.91%.
(15) Signals may be interchanged with each other.
Acknowledgment. This research was supported by Grant No. 06-
04-48068 from the RFBR, Grant SSh. 1237.2003.3 of the President of
NP0502185