This method was unsuitable for preparation of the amounts of spinochrome E required for biological testing because
of the exceedingly unfavorable substrate–reagent ratio in the 2ꢄ3 conversion step. An approach that was previously described
for sequential conversion of 2,3-dichloro-1,4-naphthoquinones into 2-hydroxy-3-nitro- and then 3-amino-2-hydroxy-1,4-
naphthoquinones [7] and was later adapted to the synthesis of the 2-hydroxy-3-nitro- and 3-amino-2-hydroxynaphthazarines,
respectively, [8] (Scheme 1b) turned out to be more efficient for the preparative synthesis of spinochrome E.
According to Scheme 1b, the starting substrate in the synthesis of spinochrome E (1) was dichlorodiethoxynaphthazarine
4, which was readily transformed in aqueous alcoholic NaNO into hydroxynitro derivative 5 in good yield. Reduction of 5 by
2
Na S O gave aminohydroxynaphthazarine 6. However, attempts at direct conversion of 6 into 1 by conc. HBr or HBr–HOAc
2 2
4
gave complicated product mixtures including those that were not naphthazarines. A much more suitable reagent for this was
a solution of anhydrous AlCl in nitrobenzene. Product 1 obtained as a result of the hydrolysis of 6 by this reagent was in all
3
respects identical to the pigment that was isolated earlier from sea urchins of the genus Echinothrix [9].
EXPERIMENTAL
13
Melting points of products were determined on a Boetius heating stage and are uncorrected. PMR and C NMR
spectra were recorded in CDCl and DMSO-d solutions with TMS internal standard (ꢅ, ppm, J/Hz) at 30°C on Bruker
3
6
Avance-300 (300 and 75 MHz, respectively) and Bruker Avance-700 (700 and 176 MHz, respectively) spectrometers. Mass
spectra (EI) were recorded in an AMD 604S instrument with direct introduction at ionizing electron energy 70 eV.
The course of reactions and purity of products were monitored by TLC on Merck 60F-254 plates impregnated with an
alcohol solution of tartaric acid (0.05 M) [10] using hexane:acetone (1:1, system 1; 2:1, system 2). Pure compounds were
isolated from their mixtures by column chromatography (silica gel, 40–100 ꢆm) and PTLC on plates (20 ꢇ 20 cm) with a loose
layer (silica gel, 5–40 ꢆm). Silica gel acidified by dilute HCl to pH ꢈ 5 was used for chromatographic separation of the
products. R values were determined by chromatography of products on Merck 60F-254 plates using systems 1 and 2. The
f
synthesis of starting dichlorodiethoxynaphthazarine 4 has been reported [11].
2,5,8-Trihydroxy-3-nitro-6,7-diethoxy-1,4-naphthoquinone (5). A solution of 4 (1.04 g, 3 mmol) and NaNO
2
(2.07 g, 30 mmol) in EtOH:H O (2:1, 100 mL) was refluxed for 1 h, cooled, acidified by HCl (5%) to pH ~ 6, and extracted
2
with EtOAc (3 ꢇ 10 mL). The extract was dried over anhydrous Na SO and evaporated at reduced pressure. The crude
2
4
product was purified by column chromatography with elution by hexane:acetone to afford 5 (0.78 g, 77%), mp 118–120°C
(acetone), R 0.60 (system 1). PMR spectrum (300 MHz, CDCl , ꢅ, ppm, J/Hz): 1.44 (3H, t, J = 7.0, CH CH O), 1.45 (3H, t,
f
3
3
2
J = 7.0, CH CH O), 4.34 (2H, q, J = 7.0, CH CH O), 4.47 (2H, q, J = 7.0, CH CH O), 12.39 (1H, s, ꢂ-OH), 13.35 (1H, s,
3
2
3
2
3
2
+
ꢂ-OH). Mass spectrum (m/z, I , %): 339 (53) [M] , 309 (40), 276 (100), 248 (57), 195 (24), 167 (11). High-resolution mass
spectrum: found m/z 339.0599 [M] , C H NO , calcd: MW 339.05903.
rel
+
14 13
9
3-Amino-2,5,8-trihydroxy-6,7-diethoxy-1,4-naphthoquinone (6). A mixture of 5 (680 mg, 2 mmol) and Na S O
2 2
4
(700 mg, 4 mmol) in H O (60 mL) was stirred for 0.5 h (based on the published method [8]). The resulting precipitate was
2
separated by filtration, washed with H O, and dried in air. The product was purified by column chromatography with elution
2
by hexane:acetone to afford 6 (440 mg, 71%), mp 125–127°C (acetone), R 0.52 (system 2). PMR spectrum (700 MHz,
f
CDCl , ꢅ, ppm, J/Hz): 1.42 (3H, t, J = 7.0, CH CH O), 1.42 (3H, t, J = 7.0, CH CH O), 4.28 (2H, q, J = 7.0, CH CH O), 4.32
3
3
2
3
2
3
2
(2H, q, J = 7.0, CH CH O), 4.74 (2H, br.s, NH ), 6.41 (1H, br.s, ꢃ-OH), 12.47 (1H, s, ꢂ-OH), 12.60 (1H, s, ꢂ-OH).
3
2
2
+
Mass spectrum (m/z, I , %): 309 (12) [M] , 280 (8), 255 (100), 191 (16), 159 (37), 127 (42), 95 (11), 63 (42). High-resolution
rel
+
mass spectrum: found m/z 309.0854 [M] , C H NO , calcd: MW 309.08485.
14 15
7
2,3,5,6,7,8-Hexahydroxy-1,4-naphthoquinone (1, spinochrome E). A saturated solution of anhydrous AlCl in
3
anhydrous PhNO (2 mL) was stirred, treated with an equal volume of 6 (30 mg, 0.1 mmol) in the same solvent, held at 70°C
2
for 6 h, and cooled. The product was extracted with HCl (5%, 3 ꢇ 10 mL). The combined extract was washed with hexane
(3 ꢇ 10 mL) to remove traces of nitrobenzene. The resulting HCl solution was refluxed for 10 min to destroy the Al complex
of the product and to hydrolyze the amine. The solution was cooled and extracted with EtOAc (3 ꢇ 10 mL). The extract was
washed with H O (2 ꢇ 10 mL), dried over anhydrous Na SO , and evaporated at reduced pressure. The crude product was
2
2
4
purified by PTLC with elution by hexane:acetone:conc. HCl (10:10:1) to afford spinochrome E (1, 10 mg, 43%), mp >360°C
(acetone); lit. [5] mp 300–320°C (dec), [9] 320°C (MeOH); R 0.46 (system 1). PMR spectrum (700 MHz, DMSO-d ,
f
6
13
ꢅ, ppm): 10.17 (br.s, ꢃ-OH), 12.83 (s, ꢂ-OH).
C NMR spectrum (700 MHz, DMSO-d ): 102.31, 140.72, 166.41.
6
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Borisova
