UV-stability and UV-protective activity of alkaloids from the marine sponge Zyzzya fuliginosa

Article abstract of DOI:10.1007/s10600-006-0040-7

Alkaloids from the marine sponge Zyzzya fuliginosa damirones A (1) and B (2); makaluvamines H (3), C (4), G (5), and L (6); and zyzzyanones A (8) and B (9) were investigated for the ability to protect egg-cell membranes of the sea urchin Strongylocentrotus nudus from UV-radiation. Damirones, zyzzyanones, and tricyclic makaluvamines C (4) and H (3) exhibited the greatest membrane-protective activity. It was shown that makaluvamines G (5) and L (6) were converted by UV-irradiation into damirones A (1), B (2), tricyclic makaluvamines H (3), C (4), and zyzzyanones A (8) and B (9), respectively. 2006 Springer Science+Business Media, Inc.

Full text of DOI:10.1007/s10600-006-0040-7

Chemistry of Natural Compounds, Vol. 42, No. 1, 2006
UV-STABILITY AND UV-PROTECTIVE ACTIVITY OF
ALKALOIDS FROM THE MARINE SPONGE
Zyzzya fuliginosa
A. E. Makarchenko and N. K. Utkina
UDC 547.94+593.4+541.14
Zyzzya fuliginosa
Alkaloids from the marine sponge
G (5), and L (6); and zyzzyanones A (8) and B (9) were investigated for the ability to protect egg-cell
Strongylocentrotus nudus
damirones A ( ) and B ( ); makaluvamines H ( ), C ( ),
1 2 3 4
membranes of the sea urchin
from UV-radiation. Damirones, zyzzyanones, and
tricyclic makaluvamines C (4) and H (3) exhibited the greatest membrane-protective activity. It was shown
that makaluvamines G (5) and L (6) were converted by UV-irradiation into damirones A (1), B (2), tricyclic
makaluvamines H (3), C (4), and zyzzyanones A (8) and B (9), respectively.
Key words: marinemetabolites, alkaloids, makaluvamines, damirones, zyzzyanones,UV-protectors,photolysis, marine
sponge.
sponge
During a search for biologically active metabolites from marine organisms, we isolated from the Australian marine
the known damirones A (1) and B (2); makaluvamines C (4), E (7), G (5), H (3), and L (6); and the
Zyzzya fuliginosa
new zyzzyanones A-D (8-11) [1, 2], which are pyrrolo[4,3,2- ]quinoline alkaloids. Zyzzyanones are the first representatives
de
of a new class of marine alkaloids with the pyrrolo[3,2- ]indol-4,8(1 ,7 )-dione skeleton.
f
H H
O
N
R
R
O
N
R
O
H
N
O
NH
N
N
2
N
+
+
N
OH
R
1
R
1
R
1
1, 2
3, 4
5 - 7
1: R = R = Me
5: R = R = Me
3: R = R = Me
1
1
1
2: R = H, R = Me
6: R = H, R = Me
4: R = H, R = Me
1
1
1
7: R = Me, R = H
1
O
R
H
O
O
R
N
H
N
N
N
O
+
NH
OH
2
OH
N
CHO
Me
8, 9
10, 11
Me
8: R = Me
9: R = H
10: R = Me
11: R = H
Damirones, makaluvamines, and zyzzyanonesarebrightlycolored compounds with strong absorption in the UV range.
Certain pigments are known to act in living organisms as UV filters that protect biologically important molecules from the
harmful action of solar UV-radiation, for example, the aromatic pigment scytonemin in cyanobacteria [3].
Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, 690022, Vladivostok, fax
(4232) 31 40 50, e-mail: utkinan@mail.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 62-64, January-
February, 2006. Original article submitted October 19, 2005.
0009-3130/06/4201-0078 ©2006 Springer Science+Business Media, Inc.
78

100
90
8
1
80
3
70
60
50
40
5
C ontrol
0
10
20
30
40
Irradiation tim e, m in
Fig. 1. Effect of UV-irradiation on survival rate of egg
cells of the sea urchin S. nudus covered by filters of
1 3 5
8
(0.05 M) and solvent
solutions of
(control).
,
,
, and
The goal ofthe work was toinvestigate the UV-protector activityof certain aromatic alkaloids isolated from the marine
sponge Z. fuliginosa and to study their stability during UV-irradiation.
We used egg cells of the sea urchin Strongylocentrotus nudus as a model for investigating the ability of alkaloids to
protect cell membranes from the action of UV-radiation. A suspension ofegg cells in marine water was irradiated bya UV lamp
through 1-mm quartz cuvettes containing alcohol solutions of the tested compounds. Irradiation for 45 min was sufficient to
reach 50% death of egg cells in the control (Fig. 1). The amount of dead egg cells was calculated after dyeing with trypan blue
solution (0.1%) because this procedure is a direct indicator of cell-membrane destruction [4].
1 3 5
8
Figure 1 shows the survival rate of egg cells protected by filters of , , , and as functions of time of UV irradiation.
2 4 6
9
The survival rate of egg cells protected by filters of , , , and (not shown in Fig. 1) were practically the same as that of egg
1 3 5
8
cells protected by filters of , , , and , respectively. This is due to the same chromophore in each pair of compounds. The
8
9
1
2
survival rate of egg cells protected by filters of solutions of zyzzyanones A ( ) and B ( ) and damirones A ( ) and B ( ) and
3
4
tricyclic makaluvamines H ( ) and C ( ) increased approximately 1.5-1.7 times compared with the control. Despite the fact
5
6
that tetracyclic makaluvamines G ( ) and L ( ) absorb strongly in the UV range, they were much less protective than tricyclic
4
3
1
2
8
9
makaluvamines C ( ) and H ( ), damirones A ( ) and B ( ), and zyzzyanones A ( ) and B ( ) (Fig. 1). This may be due to the
5
6
photosensitivity of tetracyclic makaluvamines G ( ) and L ( ). Recently a pyrrolo[4,3,2-de]quinoline alkaloid, discorhabdine
B, was shown to be photosensitive and capable of forming dimeric discorhabdine W upon UV irradiation [5].
1 9
We determined the photostability of alcohol solutions of - by studying their absorption spectra before and after UV
5
6
irradiation for 2 h. In fact, the absorption peak at 273-278 nm in the UV spectra of tetracyclic makaluvamines G ( ), L ( ), and
7
E ( ) disappeared whereas the peak at 343-357 nm increased in strength. The strength of the UV absorption decreased only
1
2
4
3
8
insignficantly after irradiation for 2 h of damirones A ( ) and B ( ), makaluvamines C ( ) and H ( ), and zyzzyanones A ( )
9
and B ( ).
Because the tetracyclic makaluvamines - changed significantly, we investigated the products formed by UV
5 7
5
6
irradiation of alcohol solutions of makaluvamines G ( ) and L ( ), the amounts of which were adequate to perform the
experiment. The compounds were irradiated until the green color typical of solutions of makaluvamines G and L completely
1
3
8
disappeared. Parts of the initial solutions were not irradiated and served as controls. Compounds (15%), (5.2%), (3.5%),
10
5
and (10%) were isolated from the reaction mixtureafter irradiation of . The reaction products were identified bycomparison
1 3 8 10
of spectra data with authentic samples of , , , and the ethyl ester of p-hydroxybenzoic acid ( ).
10
6
2
4
9
Irradiation of makaluvamine L ( ) formed (12.5%), (4.5%), (2.7%), and
(10.7%), which were identified by
2 4 9 10
comparison of spectral data with authentic samples of , , , and
.
5
6
Thus, we found that and are photosensitive compounds. One of the photodegradation pathways is homolytic
3
4
cleavage of the N-(p-hydroxystyryl) bond, as a result of which tricyclic makaluvamines H( ) and C ( ), respectively, are formed.
3
4
1
2
Makaluvamines H ( ) and C ( ), in turn, are readily converted into the corresponding damirones A ( ) and B ( ), the principal
photolysis products, probably through formation of o-iminoquinones [6]. The ethyl ester of p-hydroxybenzoic acid is formed
79

via cleavage of the phenol of makaluvamines G (5) and L (6) with its subsequent oxidation and esterification by ethyl alcohol,
which was used as the solvent. Another photolysis pathwayis radical intramolecular cyclization with accompanying hydrolysis
of the imine bond in the p-iminoquinoid fragment of the molecule, which forms zyzzyanones A (8) and B(9), respectively. This
pathway is probably not the main one judging from the low yield of 8 and 9. Damirones A and B, makaluvamines C and H,
and zyzzyanones A and B did not form in the control alcohol solutions of 5 and 6 that were not irradiated, even after prolonged
storage for three months.
Thus, it was confirmed that the low activity of 5 and 6 for protecting sea urchin egg-cell membranes from the action
of UV radiation is due to the photosensitivity of these compounds. Despite the fact that damirones, tricyclic makaluvamines,
and zyzzyanones act as UV protectors, their concentrations among the formed photoproducts are toolowfor protecting egg cells
from UV radiation.
EXPERIMENTAL
Sephadex LH-20 (Pharmacia Fine Chemicals) was used for column chromatography. Absorption was measured on
a UV-mini 1240 spectrophotometer (Shimadzu). UV radiation was from an OKH-11-M mercury—quartz irradiator. NMR
1
spectra were recorded on a Bruker AVANCE DRX-500 spectrometer at working frequency 500 MHz for H and 125 MHz for
13
1
13
C. Chemical shifts for H are given relative to TMS internal standard (δ
= 0); for C, relative to the residual signal of
TMS
CD OD (δ = 49.0 ppm). Low-resolution mass spectra were measured in an LKB-9000 S instrument with direct sample
3
introduction into the ion source at ionizing potential 70 eV; high-resolution mass spectra, in an AMD-604 S instrument
+
(Intectra, Germany) with Cs atom bombardment at 8 kV.
Determination of the Photosensitivity of the Compounds. The photosensitivity of the compounds was determined
bystudying absorption of compounds before and after irradiation. Quartz cuvettes with EtOH solutions of the compounds were
irradiated in UV light for 2 h. The lamp was 75 cm from the cuvette.
Photolysis of Makaluvamine G (5). A solution of 5 (23 mg) in EtOH (200 mL) was placed in a quartz flask and
irradiated for 8 h until the green color disappeared completely. The reaction mixture was concentrated in vacuo and separated
over a column ofSephadexLH-20usingCHCl :EtOH:TFA(4:1:0.05)toproducedamironeA (1, 3.45 mg, 15%), makaluvamine
3
H (3, 1.2 mg, 5.2%), zyzzyanone A (8, 0.8 mg, 3.5%), and 10 (2.3 mg, 10%). The spectral properties of the isolated compounds
agreed with those published [1, 6-8].
Damirone A (1). UV spectrum (MeOH, λ , nm, log ε): 240 (4.30), 347 (3.86), 516 (3.80). PMR spectrum (CDCl ,
3
max
δ, ppm, J/Hz): 2.85 (2H, t, J = 7.1, CH ), 3.06 (3H, s, CH ), 3.57 (2H, t, J = 7.1, CH ), 3.93 (3H, s, CH ), 5.29 (1H, s, H), 6.62
2
3
2
3
+
(1H, s, H). Mass spectrum (m/z): 216 [M] [6, 8].
Makaluvamine H (3). UV spectrum (MeOH, λ , nm, log ε): 240 (4.30), 345 (4.10), 522 (3.00). PMR spectrum
max
(DMSO-d , δ, ppm, J/Hz): 2.86 (2H, t, J = 7.5, CH ), 3.27 (3H, s, CH ), 3.81 (2H, t, J = 7.5, CH ), 3.83 (3H, s, CH ), 5.65 (1H,
6
2
3
2
3
+
s), 7.26 (1H, s), 8.60, 9.40 (2H, NH ). Mass spectrum (m/z): 216 [M + H] [7].
2
Zyzzyanone A (8). UV spectrum (MeOH, λ , nm, log ε): 242 (4.26), 286 (3.90), 342 (3.59), 484 (3.44). PMR
max
spectrum (CD OD, δ, ppm, J/Hz): 2.67 (3H, s, CH ), 3.01 (2H, t, J = 7.1, CH ), 3.19 (2H, t, J = 7.1, CH ), 3.89 (3H, s, CH ),
3
3
2
2
3
+
6.77 (2H, d, J = 8.7, H ), 6.80 (1H, s), 7.01 (1H, s), 7.54 (2H, d, J = 8.7, H ). FABMS (m/z): 350.1483 [M + H] , calc. for
ar
ar
C H N O , 350.1504 [1].
20 20
3 3
Ethyl Ester of p-Hydroxybenzoic Acid (10). UV spectrum (MeOH, λ , nm, log ε): 208 (3.46), 257 (3.56). PMR
max
spectrum (DMSO-d , δ, ppm, J/Hz): 1.35 (3H, t, J = 7.1, CH ), 4.30 (2H, q, J = 7.1, CH O), 6.81 (2H, d, J = 8.8, H ), 7.87 (2H,
6
3
2
ar
13
d, J = 8.8, H ). C NMR spectrum (CD OD): 15.3 (q), 62.3 (t), 116.7 (d), 123.1 (s), 133.3 (d), 164 (s), 168.9 (s). Mass
spectrum (m/z): 166 [M] .
ar
3
+
Photolysis of Makaluvamine L (6). A solution of 6 (22.4 mg) in EtOH (200 mL) was placed in a quartz flask and
irradiated for 6 h until the green color disappeared completely. The reaction mixture was concentrated in vacuo and separated
over a column of Sephadex LH-20 using CHCl :EtOH:TFA (4:1:0.05) to produce damirone B (2, 2.8 mg, 12.5%),
3
makaluvamine C (4, 1.0 mg, 4.5%), zyzzyanone B (9, 0.6 mg, 2.7%), and 10 (2.4 mg, 10.7%). The spectral properties of the
isolated compounds agreed with those published [2, 6, 9].
80

Damirone B (2). UV spectrum (MeOH, λ , nm, log ε): 242 (4.47), 346 (4.30), 492 (3.80). PMR spectrum
max
(DMSO-d , δ, ppm, J/Hz): 2.79 (2H, t, J = 7.0, CH ), 3.03 (3H, s, CH ), 3.59 (2H, t, J = 7.0, CH ), 5.12 (1H, s), 7.06 (1H, d,
6
2
3
2
+
J = 2.5), 12.40 (1H, br.s, NH). Mass spectrum (m/z): 202 [M] [2, 6].
Makaluvamine C (4). UV spectrum (MeOH, λ , nm, log ε): 241 (4.40), 357 (4.20), 520 (3.60). PMR spectrum
max
(DMSO-d , δ, ppm, J/Hz): 2.92 (2H, t, J = 7.5, CH ), 3.31 (3H, s, CH ), 3.90 (2H, t, J = 7.5, CH ), 5.73 (1H, s), 7.28 (1H, d,
6
2
3
2
+
J = 2.5), 8.65, 9.53 (2H, NH ), 13.10 (1H, br.s, NH). Mass spectrum (m/z): 202 [M + H] [9].
2
Zyzzyanone B (9). UV spectrum (MeOH, λ , nm, log ε): 240 (3.99), 281 (3.64), 342 (3.33), 328 (3.08). PMR
max
spectrum (CD OD, δ, ppm, J/Hz): 2.69 (3H, s, CH ), 3.09 (2H, t, J = 6.4, CH ), 3.26 (2H, t, J = 6.4, CH ), 6.77 (2H, d, J = 8.6,
3
3
2
2
+
H ), 6.95 (1H, s), 7.04 (1H, s), 7.54 (2H, d, J = 8.6, H ). FABMS (m/z): 336.1339 [M + H] , calc. for C H N O , 336.1343
ar
ar
19 18 3 3
[2].
Determination of UV-Protective Activity. Sea urchin S.nudus egg cells were obtained by injection of KCl solution
(3 mL, 0.5 M) into the perivisceral cavity. The obtained egg cells were purified of mechanical impurities by filtration, washed,
and suspended in marine water to a concentration of 2000 egg/mL.
The suspension of egg cells (0.1 mL aliquots) was placed on a planchet, covered with a quartz cuvette (1-mm) with
the tested compound in EtOH (0.05 M), and irradiated by a UV lamp for 45 min. The lamp was 75 cm from the planchet. After
each 5 min, a trypan blue solution (0.1%, 0.02 mL) was added to the suspension (0.1 mL) and held for 2 min. The number of
colored egg cells was counted using a microscope and Goryaev chamber. The survival rate of the egg cells was estimated by
comparison with the control that was irradiated through a cuvette containing EtOH. The percent survival rate was calculated
using the formula survival rate (%) = 100 - (n
× 100/n ), where n
is the number of colored cells in the sample
colored
colored
total
and n
is the total number of cells. The average of three determinations was used to contruct the curves.
total
ACKNOWLEDGMENT
Theworkwassupportedbygrantsofthe RFBR, RAS Presidium Program "Molecular andCellular Biology,"grant NSh-
725.2003.4 (RF President), and FED RAS grant 06-III-V-05-132.
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