Cytotoxic activity of intermediates and side products of echinochrome synthesis

Article abstract of DOI:10.1007/s10600-008-9044-9

The cytotoxic activity of the principal intermediates and side products of echinochrome synthesis toward gametes of the sea urchin Strogylocentrotus intermedius was studied. Di-and trichloro naphthazarin derivatives with two vicinal Cl atoms were the most

Full text of DOI:10.1007/s10600-008-9044-9

Chemistry of Natural Compounds, Vol. 44, No. 3, 2008
CYTOTOXIC ACTIVITY OF INTERMEDIATES AND SIDE PRODUCTS
OF ECHINOCHROME SYNTHESIS
N. D. Pokhilo,* M. I. Kiseleva, V. V. Makhan′kov, and V. Ph. Anufriev
UDC 547.655.6+576.315
The cytotoxic activity of the principal intermediates and side products of echinochrome synthesis toward
Strogylocentrotus intermedius
gametes of the sea urchin
was studied. Di- and trichloro naphthazarin
derivatives with two vicinal Cl atoms were the most active.
Key words: naphthazarins, cytotoxic activity.
Echinochrome (2,5,6,7,8-pentahydroxy-3-ethyl-1,4-naphthoquinone) (1), the quinoid pigment of the sea urchin
[1], is the biologicallyactive substance (BAS) ofthe original antioxidant preparation "Gistokhrom" that
Scaphechinus mirabilis
is used to treat acute myocardial infarct, ischemic cardiac disease, and ophthalmologic diseases[2].
One of the most promising synthetic routes to 1 is depicted in Scheme 1. According to it, ethylhydroquinone dimethyl
ether (2) is cycloacylated by dichloromaleic anhydride (3) to form dichloroethylnaphthazarin (4), which is further chlorinated.
A halide atom is replaced by methoxy in the resulting trichloro derivative (5) (yield of the last step 68%) [3]. The final step is
demethylation of the trimethylether (6) of 1 by appropriate reagents [4].
O
Cl
O
OMe
OMe
OH
O
OH
O
Cl
Et
Cl
Cl
Cl
Cl
Cl
Et
O
Cl , H SO
2
2
4
CsF, Al O , MeOH
2 3
3
AlCl −NaCl
Et
3
OH
OH
O
O
OH
OH
O
O
4
5
2
MeO
MeO
OMe
Et
HO
HO
OH
Et
+
H
OH
O
OH
O
6
1
Scheme 1
Our goal was to isolate and establish the chemical structures of the principal side products that are formed during
6
1
preparation of both echinochrome trimethylether ( ) and itself and to determine the cytotoxic activity of the isolated
compounds and intermediates.
Side products were isolated byrepeated chromatographyof the reaction mixture using TLC and HPLC for monitoring.
According toScheme 1 for preparing 6, there was a high probabilityof observing in the reaction mixture products ofincomplete
substitution of Cl by methoxy, i.e., di- and monochloro derivatives of 1. As hypothesized, we isolated and identified
Pacific Institute of Bioorganic Chemistry, Far-East Division, Russian Academy of Sciences, 690022, Vladivostok,
prosp. 100-Letiya Vladivostoka, 159, Russia, fax(4232)31 40 50, e-mail: nat-pokhilo@piboc.dvo.ru. Translated from Khimiya
Prirodnykh Soedinenii, No. 3, pp. 228-231, May-June, 2008. Original article submitted March 5, 2008.
0009-3130/08/4403-0287 ©2008 Springer Science+Business Media, Inc.
287

2,6-dimethoxy-7-chloro-3-ethylnaphthazarin (7) [5], 2,7-dimethoxy-6-chloro-3-ethylnaphthazarin (8) [5], 7-hydroxy-2,6-
dichloro-3-ethylnaphthazarin (9), 6-hydroxy-2,7-dichloro-3-ethylnaphthazarin (10), 2,6,7-trimethoxy-3-(1′-
methoxyethyl)naphthazarin (11), and 2,7-dimethoxy-6-ethylnaphthazarin (12) [6]. The last compound is the dimethylether of
ethylmompain (2,7-dihydroxy-3-ethylnaphthazarin). Ethylmompain (13) itself, which was first isolated from spines of the sea
urchin Echinus diadema [7], was observed among the impurities of natural BAS echinochrome [8].
OH
O
OH
O
OH
O
R
1
R
1
R
R
R
1
R
3
R
1
Et
R
2
Et
OH
O
OH
O
OH
O
R
2
7 - 10, 14, 17 - 19
11, 15, 16
12, 13
7: R1 = Cl, R2 = R3 = OMe; 8: R1 = R3 = OMe, R2 = Cl; 9: R1 = OH, R2 = R3 = Cl; 10: R1 = R3 = Cl, R2 = OH
11: R1 = R2 = OMe; 12: R = OMe; 13: R = OH; 14: R1 = R2 = Cl, R3 = OH; 15: R1 = R2 = Cl
16: R1 = OMe; R2 = OH; 17: R1 = R2 = OMe, R3 = OH; 18: R1 = R3 = OH, R2 = OMe; 19: R1 = OMe, R2 = R3 = OH
Naphthazarins 9-11 are new compounds, the structures of which were established using IRand NMRspectroscopyand
mass spectrometry. However, the mutual locations of the Cl atom and the hydroxyl on C-6 and C-7 could not be definitively
13
assigned for the two isomeric hydroxychloronaphthazarins 9 and 10. The proposed assignments in the PMR and C NMR
spectra of the compounds are ambiguous. The third possible hydroxychloronaphthazarin isomer 2-hydroxy-6,7-dichloro-3-
ethylaphthazarin (14) was synthesized previously[9] and is a keyintermediate in the synthesis ofechinochrome bythe literature
method [10] and was not observed among the studied side products.
2,6,7-Trimethoxy-3-(1′-methoxyethyl)naphthazarin (11) was apparently formed via substitution of the Cl atoms by
methoxyls in 2,6,7-trichloro-3-(1′-chloroethyl)naphthazarin (15). According to the scheme [3], it can be assumed that 15 was
formed as a side product via chlorination of 2,6,7-trichloro-3-ethylnaphthazarin (5). Compound 11 is the first synthesized
methylether of lomazarin [2,6,7-trimethoxy-3-(1′-hydroxyethyl)naphthazarin, 16], which was isolated earlier from roots of
Lomandra hastilis (Liliaceae) [11].
Side products from hydrolysis ofechinochrome trimethylether (6) [4], as expected, were dimethoxyand monomethoxy
echinochromederivatives2-hydroxy-6,7-dimethoxy-3-ethylnaphthazarin (17), 2,7-dihydroxy-6-methoxy-3-ethylnaphthazarin
(18), and 2,6-dihydroxy-7-methoxy-3-ethylnaphthazarin (19). Monomethylethers 18 and 19 are natural compounds that were
isolated earlier from Diadema antillarum [12].
Gametes of sea urchin, which are widely used for biological testing because of the timing of their isolation [13], were
used to study the cytotoxic activity of the synthetic naphthazarins. Natural echinochrome is known not to be cytotoxic toward
Strogyloctytrotus intermedius sea urchin nymphs [14]. We investigated the cytotoxic activity of the isolated side products 7-12
and 17-19 and samples of intermediates in the synthesis of echinochrome 4-6 [3, 4] and 14 [4, 10]. Table 1 gives the results.
With the exception of 6, all tested naphthazarins, depending on the concentration, inhibited partially or completely
embryo development. In contrast with known cytostatics, which block division of ova and turnover of sea urchin embryos
immediatelyafter adding them [15], the inhibiting action of the tested naphthazarins on developing embryos was observed both
immediatelyin the earlystages and graduallyduring the whole incubation period. Lysis ofblastomers was observed after adding
high doses of the compounds; anomalous development of embryos, lower doses. Nevertheless, all compounds could be divided
into two groups. These were 7-12 and 17-19 that were weakly cytotoxic and 4, 5, and 14 with two vicinal Cl atoms that were
1-2 orders of magnitude more active than the others. In this instance, the mutual locations of the Cl atoms in the naphthazarins
had a decisive effect on their biological properties. Therefore, the presence of even traces of 4, 5, and 14 in the final product,
i.e., echinochrome, can affect its quality.
Thus, structures of the principal side products formed during synthesis ofechinochrome were established. Compounds
with the highest cytotoxic activity were identified.
288

TABLE 1. Cytotoxic Activity of 4-12, 14, and 17-19
MED, μg/mL*
1-8 blastomers
Compound
MW
LD50, μg/mL**
1 blastomer
4
5
6
7
8
9
10
11
12
14
17
18
19
287.1
321.54
308.29
312.71
312.71
303.10
303.10
338.310
278.26
303.10
294.26
280.23
280.23
2.40 0.02 (L)
3.3 0.3 (L)
>1925
80.0 7.0 (L)
110.0 7.0 (L)
42.0 1.5
115.0 9.5 (L)
163.0 5.0 (L)
400 100 (L)
3.0 0.13 (L)
<500
0.60 0.01 (1-4 bl)
0.38 0.02 (1-2, L)
>1925
0.30 0.01
0.048 0.002
>1925
58.5 5.5 (1-4 bl, an)
82.5 3.5 (1-4 bl, an)
24.0 1.0 (1-4 bl, an)
60 2.8 (1-8 bl, an)
95.5 4.5 (1-2 bl, an)
150 25 (1-2 bl, an)
1.55 0.06 (1-4 bl, an)
450 50 (1-8 bl, an)
80.0 6.5 (1-2 bl, an)
160.0 5.0 (1-2 bl, an, L)
50.0 3.0
25.0 6.0
10.0 0.3
38.0 0.2
27.0 1.5
58.0 7.5
0.23 0.11
250 50
130.0 8.0
195.0 5.0 (L)
60.0 2.5
97.5 0.5
______
*Minimal effective dose stopping division and killing fertilized ova in 1 and 1-8 blastomers.
**Concentration lethal to 50% of embryos within a day. >1925 means that the compound at a concentration of 1925 μg/mL
did not exhibit cytotoxic activity. The activity of the compounds at concentrations >1925 μg/mL was not investigated. bl,
blastomers; L, lysis ofblastomers; an, anomalous development ofblastomers. Data from 5-6 experiments are given. Confidence
range p ≤ 0.05.
EXPERIMENTAL
13
Melting points were determined on a Boetius heating stage and are uncorrected. PMR and C NMR spectra in CDCl
3
(internal standard Me Si) were recorded on a Bruker DRX-500 spectrometer (500.13 MHz and 125 MHz). Mass spectra (EI)
4
were obtained in an LKB-9000S instrument with direct sample introduction at ionizing-electron energy 70 eV. The purity of
the compounds was monitored by TLC on Merck 60F-254 plates using hexane:acetone (3:1) and HPLC. Pure compounds were
+
isolated by preparative TLC on plates (20 × 20 cm) with a fixed layer of silica gel (H -form, 5-40 μm) [16] or column
+
chromatography over a column of silica gel (L 40/100 μm, H -form). Elemental analyses were performed on a Flash EA1112
C,H,N-analyzer and agree with those calculated for all compounds.
HPLC was carried out on a LaChrom (Merck Hitachi) liquid chromatograph equipped with an L-7100 pump,
L-7400 UV detector, L-7300 thermostat, D-7500 integrator, and Agilent Technologies Zorbax Eclipse XDB-C18 column
(3.5 μm, 75 cm × 4.6 mm) with a Hypersil ODS guard column (4.0 cm × 4.0 mm). The column was thermostatted at 30°C.
Impurities were separated by a mixture of solvents A (water + glacial acetic acid, 1%) and B (CH CN + glacial acetic acid,
3
1%) using the program isocratic, 90% A, 10% B, 0-5 min; gradient 90% A, 10% B to 10% A, 90% B, 5-35 min. The analysis
time was 35 min.
Compounds 4-8, 12, 14, and 17-19 wereidentifiedbydirect comparison with authenticsamplesandcomparison oftheir
physical chemical properties with the literature values.
7-Hydroxy-2,6-dichloro-3-ethylnaphthazarin(9). Retention time20.20min, mp278-280°C(dec.)(hexane:acetone).
IR spectrum (CHCl , ν, cm−1): 3398 (OH), 1607 (C=O), 1457, 1426. PMR spectrum (500 MHz, CDCl , δ, ppm, J/Hz): 1.22
3
3
13
(3H, t, J = 7.6, CH ), 2.97 (2H, q, J = 7.6, CH ), 12.09 (1H, s, α-OH), 13.06 (1H, s, α-OH). C NMR spectrum (125 MHz,
3
2
CDCl ): 181.17, 178.9, 157.7, 155.7, 153.5, 146.9, 132.2, 119.3, 107.97, 107.76, 21.5, 11.9. Mass spectrum (EI, 70 eV, m/z,
3
+
I , %): 306/304/302 (100) [M] .
rel
6-Hydroxy-2,7-dichloro-3-ethylnaphthazarin (10). Retention time 19.30 min, mp 170-174°C (hexane:acetone).
PMR spectrum (500 MHz, CDCl , δ, ppm, J/Hz): 1.22 (3H, t, J = 7.6, CH ), 2.98 (2H, q, J = 7.6, CH ), 12.14 (1H, s, α-OH),
3
3
2
13
13.09 (1H, s, α-OH). C NMR spectrum (125 MHz, CDCl ): 181.4, 179.1, 157.6, 155.6, 153.6, 147.9, 132.6, 132.1, 108.0,
107.8, 21.6, 12.1. Mass spectrum (EI, 70 eV, m/z, I , %): 306/304/302 (100) [M] .
3
+
rel
289

2,6,7-Trimethoxy-3-(1′-methoxyethyl)naphthaarin (11). Retention time 21.94 min, mp 53-63°C (hexane:acetone).
PMR spectrum (500 MHz, CDCl , δ, ppm, J/Hz): 1.60 (3H, d, J = 6.9, CH ), 4.97 (1H, q, J = 6.9, CH), 3.31 (3H, s, OCH ), 4.07
3
3
3
13
(3H, s, OCH ), 4.09 (3H, s, OCH ), 4.14 (3H, s, OCH ), 12.91 (1H, s, α-OH), 13.28 (1H, s, α-OH).
C NMR spectrum
3
3
3
(125 MHz, CDCl ): 181.0, 179.7, 162.6, 156.8, 156.7, 148.7, 147.2, 133.0, 110.1, 106.4, 71.2, 61.644, 61.638, 61.605, 57.1,
3
+
19.4. Mass spectrum (EI, 70 eV, m/z, I , %): 338 (100) [M] , 323 (52), 306 (52), 292 (93), 263 (25).
rel
Determination of Cytotoxic Activity. Cytotoxic activity of synthetic naphthazarins was determined using fertilized
ova of S. intermedius sea urchin collected in Peter the Great Bay (Sea of Japan). Male and female gametes and further
fertilization were performed as before [17]. Compounds in various concentrations were added to an incubated mixture 3-5 min
after fertilization of ova in the zygote stage. The incubation mixture contained an ethanolic solution (0.01 mL) of the studied
compound or ethanol (0.01 mL, control) and a suspension (0.99 mL, 2500/mL ofseawater) offertilized ova. Fertilized ova were
incubated with the tested compounds in open containers with filtered seawater for 24 h until late gastrula under optimal (20°C)
temperature conditions. The activities of the compounds were followed using a microscope. The condition of fertilized ova and
embryos was determined visually by comparison with the control (embryos in seawater and embryos in seawater with 1-2%
alcohol). Cytotoxic activityofthenaphthazarinswas estimated from their abilitytoinduce blockage, blastomer lysis, anomalies,
or reduced division of ova and to delay embryo development 3, 12, and 24 h after fertilization [18].
ACKNOWLEDGMENT
The work was financed partially by a grant of the RAS Presidium (Molecular and Cellular Biology Program), an
interdisciplinary integrated project of the Far-East and Siberian Division of the RAS (No. 07-11-CO-05-020), a grant of the
RFBR No. 06-04-96974, and government contract No. 02.522.11.2013.
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