409
malondialdehyde (MDA) and reflects a measure for
hydroxyl-radical generation. However, we did not ob-
serve any deoxyribose degradation from the novel dep-
sides (table I) that would approximately reach the amount
of hydroxyl radicals generated by the standard agent
anthralin [20].
4.1.1.1. Propyl 2,4-dihydroxy-6-methylbenzoate 11c
Sodium (0.5 g, 21.73 mmol) was dissolved in absolute
propanol (50 mL) and stirred at room temperature. Then
11b [15] (2.8 g, 14.28 mmol) was added to the solution
and refluxed under nitrogen for 24 h. The solution was
cooled, acidified with cold 10% HCl and extracted with
ether (3 × 100 mL). The combined organic phase was
dried over MgSO4 and evaporated. The crude product
was purified by flash chromatography (SiO2, CH2Cl2) to
give colourless crystals; 92% yield; m.p. 123 °C; FTIR
3 428, 2 968, 1 615 cm–1; 1H-NMR (250 MHz, CDCl3) δ
11.86 (s, 1H), 6.28 (d, J = 2.6 Hz, 1H), 6.21 (d, J = 2.6
Hz, 1H), 5.37 (s, 1H), 3.92 (s, 3H), 4.38 (t, 3H), 2.50 (s,
3H), 1.79 (m, 2H), 1.04 (t, 3H). Anal. (C11H14O4) C, H.
Analogously, compounds 11a and 11c–f [14, 15, 26]
were prepared from 11b.
Mounting evidence suggests that leukotrienes have a
profound influence on the development and progression
of human cancers, and some pharmacological agents that
inhibit the LO-mediated signalling pathways are avail-
able to treat inflammatory and hyperproliferative diseases
such as psoriasis [21]. Furthermore, lichen depsides are
inhibitors of keratinocyte growth [22]. Therefore, se-
lected compounds were evaluated in vitro for their ability
to inhibit the growth of HaCaT cells, a non-transformed
human cell line that can be used as a model for highly
proliferative epidermis [23]. Unfortunately, the most po-
tent inhibitors of LTB4 biosynthesis, depsides 13c and
15c, were inactive at 20 µM concentrations. There was no
correlation found between the potency of the depsides to
inhibit LTB4 biosynthesis and their in vitro antiprolifera-
tive activity. 2-O-Methylobtusatic acid 17 and its butyl
ester 16e were the most potent inhibitors of cell growth,
and also the 4-O-demethylated compounds 15f and 20a
displayed antiproliferative activity.
To determine cytotoxic effects of the potent antiprolif-
erative agents, release of lactate dehydrogenase into the
culture medium of HaCaT cells was studied [24]. This is
commonly used as an indicator of plasma membrane
damage. In this assay, LDH release by the standard
anthralin significantly exceeded that of the vehicle con-
trol. On the other hand, LDH release of the test com-
pounds (table I) was unchanged as compared to controls
at 2 µM concentration, documenting that their antiprolif-
erative activity was due to cytostatic rather than cytotoxic
effects.
4.1.2. General procedure for the condensation of
benzoic acids with phenolic esters
4.1.2.1. Ethyl 2-hydroxy-4-(2-hydroxy-4-methoxy-3,6-di-
methylbenzoyloxy)-6-methylbenzoate 12b
A solution of 5 (100 mg, 0.6 mmol) and 11b [14]
(126 mg, 0.6 mmol) in anhydrous toluene (2 mL) and
trifluoroacetic anhydride (0.5 mL) was stirred at room
temperature for 2.5 h (TLC control). The solvent was
removed under reduced pressure and the residue was
purified by column chromatography (SiO2) using hexane/
ethyl acetate (9 + 1). The product was recrystallized from
MeOH/CHCl3 to give colourless crystals; FTIR 3 380,
1
2 944, 1 750, 1 661 cm–1; H-NMR (CDCl3) δ 11.66 (s,
1H), 11.39 (s, 1H), 6.71 (d, 1H), 6.59 (d, 1H), 6.35 (s,
1H), 4.46 (q, 2H), 3.89 (s, 3H), 2.65 (s, 3H) 2.58 (s, 3H),
2.09 (s, 3H), 1.44 (s, 3H); Anal. (C20H22O7) C, H.
Analogously, 12a and 12c–f were prepared from 5 and
11a, 11c–f; 13a–f were prepared from 6 and 11a–f; 14a–f
were prepared from 7 and 11a–f; 16a–f were prepared
from 8 and 11a–f; 18a–f were prepared from 9 and 11a–f;
19a–f were prepared from 10 and 11a–f (table II).
In conclusion, we have identified 13c and 15c as potent
inhibitors of LTB4 biosynthesis which, in contrast to
many known compounds, do not function primarily as
redox-based inhibitors. As a consequence, toxicity which
may be attributed to non-specific redox enzyme inhibition
can be ruled out.
4.1.3. General procedure for hydrogenolysis
4.1.3.1. 2-Hydroxy-4-(2,4-dimethoxy-3,6-dimethyl-
benzoyloxy)-6-methylbenzoic acid 17
4. Experimental protocols
A suspension of 16f (100 mg, 0.22 mmol) and 10%
palladium/carbon (25 mg) in dry ethyl acetate (2 mL) was
stirred in H2 for 2 h (TLC control). The suspension was
then filtered through celite, and the filtrate was evapo-
rated. The residue was purified by column chromatogra-
phy (SiO2) using hexane/ethyl acetate (9 + 1) to give
colourless crystals; FTIR 3 446, 2 953, 1 675, cm–1;
1H-NMR (CDCl3) δ 11.42 (s, 1H), 6.67 (d, 1H), 6.61 (d,
4.1. Chemistry
4.1.1. General
For analytical instruments and methods see refer-
ence [25].
Starting compounds 4, 5 [14], 6, 7 [10], 8 [14], 9 and
10 [10] were prepared as described.
Kumar Kc, Sunil
