Depsides as non-redox inhibitors of leukotriene B4 biosynthesis and HaCaT cell growth. 1. Novel analogues of barbatic and diffractaic acid

Article abstract of DOI:10.1016/S0223-5234(99)00132-4

A series of barbatic and diffractaic acid analogues has been synthesized and evaluated as inhibitors of leukotriene B₄ (LTB₄) biosynthesis and as antiproliferative agents. The 4-O-demethyl barbatic and diffractaic acid derivatives were among the most active compounds in both assays. In particular, ethyl 4-O-demethylbarbatate was the most potent LTB₄ biosynthesis inhibitor of this series, with an IC₅₀ value in the submicromolar range. Because the compounds did not show appreciable reactivity against a stable free radical, 2,2-diphenyl-1-picrylhydrazyl, and did not produce appreciable amounts of deoxyribose degradation as a measure of their potency to generate hydroxyl radicals, a simple redox effect could not explain their biological activity. Also, there was no nonspecific cytotoxicity as documented by the activity of lactate dehydrogenase released from the cytoplasm of keratinocytes, which was in the control range.

Full text of DOI:10.1016/S0223-5234(99)00132-4

Eur. J. Med. Chem. 34 (1999) 1035−1042
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1035
Original article
Depsides as non-redox inhibitors of leukotriene B4 biosynthesis and HaCaT cell
growth. 1. Novel analogues of barbatic and diffractaic acid
Sunil Kumar KCa, Klaus Müllerb*
aInstitut für Pharmazie, Pharmazeutische Chemie I, Universität Regensburg, D-93040 Regensburg, Germany
bWestfälische Wilhelms-Universität Münster, Institut für Pharmazeutische Chemie, Hittorfstraße 58 – 62, D-48149 Münster, Germany
(Received 25 March 1999; accepted 7 May 1999)
Abstract – A series of barbatic and diffractaic acid analogues has been synthesized and evaluated as inhibitors of leukotriene B4 (LTB4)
biosynthesis and as antiproliferative agents. The 4-O-demethyl barbatic and diffractaic acid derivatives were among the most active
compounds in both assays. In particular, ethyl 4-O-demethylbarbatate was the most potent LTB4 biosynthesis inhibitor of this series, with an
IC50 value in the submicromolar range. Because the compounds did not show appreciable reactivity against a stable free radical,
2,2-diphenyl-1-picrylhydrazyl, and did not produce appreciable amounts of deoxyribose degradation as a measure of their potency to generate
hydroxyl radicals, a simple redox effect could not explain their biological activity. Also, there was no nonspecific cytotoxicity as documented
by the activity of lactate dehydrogenase released from the cytoplasm of keratinocytes, which was in the control range. © 1999 Éditions
scientifiques et médicales Elsevier SAS
barbatic acid / diffractaic acid / antiproliferative activity / keratinocytes / lactate dehydrogenase release / leukotriene biosynthesis
1. Introduction
Depsides are a distinct class of lichen-derived com-
pounds which are formed by condensation of two or more
hydroxybenzoic acids whereby the carboxyl group of one
molecule is esterified with a phenolic hydroxyl group of
a second molecule. One of the most common secondary
metabolites of many lichen species [1] is the didepside
diffractaic acid (1, figure 1). This compound has been
shown by several groups to exhibit antiviral [2], anti-
tumour [3], analgesic and antipyretic [4] properties.
Among several structurally dissimilar lichen-derived me-
tabolites isolated from Parmelia species, we have iden-
tified 1 as a non-redox inhibitor of the biosynthesis of
leukotriene B4 (LTB4) in bovine polymorphonuclear
leukocytes (PMNL) [5]. Leukotrienes are derived from
the biotransformation of arachidonic acid through the
action of 5-lipoxygenase (5-LO) and play an important
role in a variety of pathophysiological states in man,
particularly those involving inflammation [6]. Further-
more, we found that 1 is also a potent antiproliferative
Figure 1. Structures of diffractaic acid (1) and barbatic acid
(2).
agent against the growth of human keratinocytes [7].
These combined inhibitory actions against 5-LO and
keratinocyte cell growth suggested a beneficial effect
against inflammatory and hyperproliferative skin diseases
such as psoriasis, since both pathological features were
targeted.
As part of our continuing search for agents suitable for
the treatment of inflammatory and hyperproliferative skin
*Correspondence and reprints

1036
diseases such as psoriasis, we have synthesized several
novel analogues of 1 and its congener barbatic acid (2),
modified at the carboxylic acid function and the
4-methoxy group in the benzoyloxy moieties, to explore
the effect of increased lipophilicity and some redox
properties on the biological activity of the compounds.
The redox properties were evaluated in terms of reactivity
against the stable free radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH), in order to evaluate the antioxi-
dant potential, and deoxyribose degradation was deter-
mined as a measure of hydroxyl radical formation [8].
The ability of the novel compounds to inhibit the growth
of human keratinocytes was evaluated in HaCaT cells [9],
and inhibition of LTB4 biosynthesis was assayed in
bovine polymorphonuclear leukocytes [8].
2. Chemistry
Unambiguous syntheses of the lichen depsides 1 and 2
have been reported [10]. The mononuclear precursors for
the novel depsides were obtained from ethyl 2,4-
dihydroxy-3,6-dimethylbenzoate(3)followingthemethod-
ology of Elix [10] which proved to be particularly suit-
able for the large scale preparation of this starting
material (figure 2). Esters 4a–4e were directly obtained
from 3 by transesterification in the presence of the
corresponding sodium alkoxides and alcohols [10]. Alkyl-
ation of 3 with a one molar proportion of dimethyl sulfate
or the pertinent benzyl chlorides in the presence of
potassium carbonate, selectively yielded the correspond-
ing 4-methoxy derivative 5 or 4-benzyloxy derivatives 9a
and b. Methylation of the second hydroxy group of these
derivatives with dimethyl sulfate gave the 2-methoxy
derivatives 6 and 10a and b. Subsequent hydrolysis of the
esters 5, 6, 9a and b, and 10a and b yielded the requisite
acids 7, 8, 11a and b, and 12a and b, respectively, for the
A-ring of the desired depsides. Depside formation be-
tween these acids and the phenolic esters 3 and 4a–e was
achieved by treatment with trifluoroacetic anhydride in
anhydrous toluene and yielded the barbatic and diffractaic
acid analogues 13a–f, 14a–f, 15a–f and 17a–f, 18a–f,
19a–f, respectively (figure 3). Hydrogenolytic cleavage
of the benzyl ethers 14a–e and 18a–e over palladium/
carbon produced the phenolic analogues 16a–e and
20a–e, respectively, whereas benzyl esters 14f and 18f
were cleaved to the acids 16f and 20f, respectively.
Analogously, benzyl esters 13f and 17f yielded the parent
compounds 2 and 1, respectively.
Figure 2. Reagents: (a) Na, ROH, ∆, 24 h; (b) Me2SO4,
K2CO3, acetone, ∆, 24 h; (c) 4-R≠C6H4CH2Cl, K2CO3, ace-
tone, ∆, 24 h; (d) KOH, H2O, DMSO, 90 °C.
3. Biological results and discussion
Inhibition of LTB4 biosynthesis by the barbatic and
diffractaic acid analogues was determined in bovine
polymorphonuclear leukocytes. As shown in table I, ac-
tivity of barbatic acid (2), with an IC50 value of 7.8 µM,
was similar to that of 1. The biological activity was
reduced when the free carboxylic acid groups were
esterified. The esters 13a–f of 2 were either moderately
active or inactive even at concentrations up to 20 µM.
Also, esterification of 1 resulted in less active or inactive
compounds (17a and c–f), except for 17b, where activity
was retained. In general, introduction of a 4-benzyl or
4-methoxybenzyl group into 1 and 2, as in 14a–f, 15a–f,
18a–f, and 19a–f dramatically reduced inhibitory action
against LTB4 biosynthesis. Most of these compounds
were inactive at 20 µM. Exceptions were the

1037
Moreover, the results obtained from the deoxyribose
assay (table I) also suggest that hydroxyl radicals are not
involved in the mechanism of enzyme inhibition by the
novel depsides. The deoxyribose assay is a sensitive test
for the production of hydroxyl radicals [12]. The release
of 2-thiobarbituric acid reactive material is expressed as
malondialdehyde (MDA) and reflects a measure for
hydroxyl-radical generation. However, we did not ob-
serve any deoxyribose degradation from depsides, even
for compounds 16a–f with three phenolic hydroxyl
groups.
In vitro antiproliferative activities were determined in
24-well culture dishes against the growth of HaCaT cells.
This nontransformed human cell line can be used as a
model for highly proliferative epidermis [13]. The parent
compounds 1 and 2 were potent inhibitors of cell growth
with IC50 values of 2.6 and 4.1 µM, respectively (table I).
While the esters 13a–c of 2 showed comparable or
slightly improved activity, the corresponding esters 17a–c
of 1 were inactive. However, butyl ester 17e displayed
potent antiproliferative activity. Furthermore, among the
4-benzylated derivatives of 1 were also some antiprolif-
erative active agents. Similar to the results obtained in the
LTB4 assay, 4-O-demethylation of barbatic and diffrac-
taic acid (16a–f and 20a–f, respectively) generally pro-
duced active compounds, although there were no im-
provements as compared to the parent depsides.
Figure 3. Reagents: (a) (CF3CO)2O, toluene, room tempera-
ture; (b) Pd/C, EtOAc, room temperature. R1, R2, and R3 are
defined in table I.
4-benzyloxydiffractates 14c, e and f of 2 which showed
comparable activity.
Since a major class of leukotriene biosynthesis inhibi-
tors often contains a hydroxylated aromatic ring [11], we
have speculated that demethylation of the 4-methoxy
group of 1 and 2 might improve their activity. As
expected, 4-O-demethyl barbatic and diffractaic acid
derivatives 16a–f and 20a–f, respectively, inhibited LTB4
biosynthesis with IC50 values in the low micromolar
range. With the exception of the free acids 16f and 20f,
these analogues approached the potency of their respec-
tive parent compounds or were even more potent than
these. In particular, the ethyl esters 16b and 20b were the
most potent inhibitors of LTB4 biosynthesis of this series.
Potency of compound 16b, with an IC50 of 0.8 µM, was
comparable to that of the standard inhibitor nordihy-
droguaiaretic acid.
There is little or no correlation between the in vitro
antiproliferative activity of the compounds and their
ability to inhibit LTB4 biosynthesis. With respect to both
features, well-balanced representatives are found among
the esters of diffractaic acids and the 4-O-demethylated
analogues of 1 and 2. Unfortunately, the potent LTB4
biosynthesis inhibitor 20b is inactive at 20 µM. The most
potent inhibitor of keratinocyte growth, ethyl diffractate
(13b), is also an inhibitor of LTB4 biosynthesis. Like-
wise, the most potent inhibitor of LTB4 biosynthesis,
ethyl 4-O-demethylbarbatate (16b), also displays antipro-
liferative activity.
One chemical feature of many inhibitors of LTB4
biosynthesis is their ability to remove free radicals, since
the conversion of arachidonic acid into LTB4 is an
oxidative process. Therefore, we have evaluated the
depsides for their ability to react with the stable free
radical DPPH to give the reduced 2,2-diphenyl-1-
picrylhydrazine. Table I shows that no appreciable
amount of reduced hydrazine was formed by these
compounds, documenting their lack of reactivity against
stable free radicals. This suggests that a simple redox
effect does not explain their activity in the LTB4 assay.
Rather, activity appears to be due to specific enzyme
interaction.
Keratinocytes were also tested for their susceptibility
to the action of the most potent depsides on plasma
membrane integrity. As a measure of cytotoxicity, release
of lactate dehydrogenase into the culture medium was
determined [14]. In these experiments, all potent inhibi-
tors of keratinocyte growth showed values in the control
range, documenting that their activity was due to cyto-
static rather than cytotoxic effects. This may be advanta-
geous as compared with the topical antipsoriatic agent
anthralin, which is known to induce inflammation of the
healthy skin surrounding a psoriatic lesion. As a result of
the strong hydroxyl radical generating activity of this

1038
Table I. Redox properties, inhibition of LTB4 biosynthesis, antiproliferative activity and cytotoxicity against HaCaT cells of barbatic and
diffractaic acid derivatives.
a
c
kDPPH
DD ( OH)b
LTB4
AAd
LDHe
(mU)
Compound
R1
R2
R3
(M–1 s–1)
IC50 (µM)
IC50 (µM)
1
2
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Prop
CHMe2
Bu
CH2Ph
Me
0.63 ± 0.01
0.14 ± 0.07
0.93 ± 0.01
0.31 ± 0.05
0.29 ± 0.01
ND
ND
0.45 ± 0.13
ND
0.12 ± 0.07
0.09 ± 0.01
0.16 ± 0.04
0.18 ± 0.06
0.21 ± 0.03
ND
ND
0.08 ± 0.01
ND
7.6
7.8
2.6
4.1
4.8
1.9
136
148
148
143
143
ND
ND
ND
150
142
ND
ND
150
140
ND
ND
ND
ND
ND
ND
143
149
149
146
140
140
ND
ND
ND
170f
167f
ND
ND
ND
ND
ND
ND
ND
13a
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
14f
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
18.6
11.3
10.5
> 20
> 20
19.2
> 20
> 20
9.0
16.2
9.0
7.9
> 20
> 20
> 20
15.0
> 20
> 20
2.1
3.2
> 20
> 20
> 20
19.0
16.7
> 20
> 20
9.0
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
Et
ND
ND
Prop
CHMe2
Bu
CH2Ph
Me
0.55 ± 0.04
0.78 ± 0.01
0.51 ± 0.01
0.96 ± 0.01
ND
ND
ND
0.72 ± 0.01
ND
ND
0.34 ± 0.01
0.52 ± 0.07
0.91 ± 0.60
0.43 ± 0.14
0.67 ± 0.09
0.81 ± 0.03
0.85 ± 0.03
0.57 ± 0.01
0.93 ± 0.01
ND
ND
ND
ND
0.97 ± 0.02
ND
0.19 ± 0.03
0.23 ± 0.01
0.11 ± 0.03
0.26 ± 0.01
ND
ND
ND
0.19 ± 0.01
ND
ND
0.21 ± 0.02
0.02 ± 0.01
0.12 ± 0.01
0.16 ± 0.01
0.72 ± 0.01f
0.01 ± 0.01
0.19 ± 0.01
0.21 ± 0.01
0.06 ± 0.02
ND
8.4
> 20
> 20
> 20
> 20
> 20
> 20
8.2
8.4
3.6
8.0
3.8
Et
Prop
CHMe2
Bu
CH2Ph
Me
Et
0.8
5.7
2.6
5.0
14.0
13.2
5.3
Prop
CHMe2
Bu
H
Me
H
8.2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
> 20
> 20
> 20
14.0
4.1
> 20
> 20
> 20
ND
Et
Prop
CHMe2
Bu
CH2Ph
Me
19
> 20
> 20
> 20
> 20
18.3
> 20
> 20
> 20
> 20
ND
ND
ND
Et
0.44 ± 0.02f
ND
Prop
CHMe2
Bu
ND
ND
ND
ND
ND
ND
ND
ND
ND
CH2Ph
a
Reducing activity against 2,2-diphenyl-1-picrylhydrazyl with equimolar amounts of test compound. bDeoxyribose degradation as a measure
of hydroxyl-radical formation. Indicated values are µmol of malondialdehyde/mmol of deoxyribose released by 75 µM of test compound
c
(controls < 0.1). Inhibition of LTB4 biosynthesis in bovine PMNL. Inhibition was significantly different with respect to that of the control;
n = 3 or more, P < 0.01. Nordihydroguaiaretic acid was used as the standard inhibitor (IC50 = 0.4 µM) [8]. dAntiproliferative activity against
HaCaT cells. Inhibition of cell growth was significantly different with respect to that of the control, n = 3, P < 0.01. eActivity of LDH (mU)
release in HaCaT cells after treatment with 2 µM test compound (n = 3, SD < 10%). fValues are significantly different with respect to vehicle
g
control (P < 0.05). ND = not determined. Positive control [8].

1039
Table I. (continued).
DD ( OH)b
LTB4
AAd
LDHe
a
c
kDPPH
Compound
R1
R2
R3
(M–1 s–1)
IC50 (µM)
IC50 (µM)
(mU)
19a
19b
19c
19d
19e
19f
20a
20b
20c
20d
20e
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
4-MeOPhCH2
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
ND
ND
ND
ND
ND
ND
0.93 ± 0.01
0.64 ± 0.01
0.89 ± 0.05
0.97 ± 0.01
0.51 ± 0.01
0.24 ± 0.03
24.2 ± 4.2f
ND
ND
ND
ND
ND
ND
0.18 ± 0.01
0.10 ± 0.02
0.01 ± 0.01
0.09 ± 0.01
0.16 ± 0.01
0.15 ± 0.01
2.89 ± 0.14f
> 20
> 20
> 20
> 20
> 20
> 20
7.8
1.4
8.5
5.8
7.8
> 20
> 20
ND
ND
ND
ND
9.0
> 20
> 20
7.2
ND
ND
ND
ND
ND
ND
138
ND
ND
168
ND
114
294f
Prop
CHMe2
Bu
CH2Ph
Me
Et
Prop
CHMe2
Bu
> 20
9.8
0.7
20f
H
11.0
37.0
anthraling
agent [15], LDH release by anthralin significantly ex-
ceeded that of the vehicle control.
1 630 cm–1; 1H-NMR (250 MHz, CDCl3) δ 12.14 (s, 1H),
6.20 (s, 1H), 4.97 (s, 1H), 4.30 (t, J = 6.5 Hz, 2H), 2.48
(s, 3H), 2.10 (s, 3H), 1.80 (m, 2H), 1.03 (t, J = 7.4 Hz,
3H); Anal. (C12H16O4) C, H.
Analogously, compounds 4a and c–e were prepared
from 3 (table II).
In conclusion, barbatic acid analogues were consis-
tently more active against the biosynthesis of LTB4 and
the growth of HaCaT keratinocytes than the correspond-
ing diffractaic acid analogues. Though this may be related
to their additional phenolic hydroxyl group, determina-
tion of the antioxidant and pro-oxidant potential of the
compounds did not reveal any appreciable redox activity.
Barbatic acid analogue 16b has been identified as a potent
non-redox inhibitor of LTB4 biosynthesis which also
displays antiproliferative activity against keratinocyte
growth.
4.1.3. Ethyl 2-hydroxy-4-(4-methoxybenzyloxy)-3,6-
dimethylbenzoate 9b
A suspension of 3 (6 g, 28.57 mmol), anhydrous po-
tassium carbonate (11.25 g, 81.39 mmol) and
4-methoxybenzylchloride (4.47 g, 28.57 mmol) in dry
acetone (75 mL) was refluxed for 24 h, then cooled,
acidified with cold 10% HCl and extracted with ether (3
× 200 mL). The combined organic phase was washed
with water, dried over MgSO4 and evaporated. The crude
product was purified by column chromatography (SiO2)
using hexane/ethyl acetate (9:1) to afford colourless
crystals; FTIR 3 438, 1 720, 1 636 cm–1; 1H-NMR
(CDCl3) δ 11.91 (s, 1H), 7.35 (d, J = 9.5 Hz, 2H), 6.93 (d,
J = 9.5 Hz, 2H), 6.34 (s, 1H), 5.03 (s, 2H), 4.38 (q, J =
7.1 Hz, 2H), 3.82 (s, 3H), 2.52 (s, 3H), 2.11 (s, 3H), 1.41
(t, J = 7.1 Hz, 3H). Anal. (C19H22O5) C, H.
4. Experimental protocols
4.1. Chemistry
4.1.1. General
For analytical instruments and methods see refer-
ence [16].
Compounds 1–3 and 5–8 were prepared as de-
scribed [10].
4.1.4. Ethyl 2-methoxy-4-(4-methoxybenzyloxy)-3,6-
dimethylbenzoate 10b
4.1.2. Propyl 2,4-dihydroxy-3,6-dimethylbenzoate 4b
Sodium (0.5 g, 21.73 mmol) was dissolved in absolute
propanol (50 mL) and stirred at room temperature. Then
3 (3 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; FTIR 3 396, 2 980, 2 957,
A suspension of 9b (2.20 g, 6.67 mmol), anhydrous
potassium carbonate (2.76 g, 20 mmol) and dimethyl
sulfate (0.6 mL, 6.67 mmol) in dry acetone (50 mL) was
refluxed for 24 h, then cooled, acidified with cold 10%
HCl and extracted with ether (3 × 200 mL). The com-
bined organic phase was washed with water, dried over
MgSO4 and evaporated. The crude product was purified
by column chromatography (SiO2) to afford a colourless

1040
Table II. Chemical data of starting materials, barbatic and diffractaic acid derivatives.
Compounda
Formulab
M.p. (°C)
Yield (%)
Solventc,d (vol%)
Anal.e
4a
4b
4c
4d
4e
9a
9b
C10H12O4
C12H16O4
C12H16O4
C13H18O4
C16H16O4
C18H20O4
C19H22O5
C19H22O4
C20H24O5
C16H16O5
C17H18O5
C17H18O4
C18H20O5
C20H22O7
C21H24O7
C22H26O7
C22H26O7
C23H28O7
C26H26O7
C26H26O7
C27H28O7
C28H30O7
C28H30O7
C29H32O7
C32H30O7
C27H28O8
C28H30O8
C29H32O8
C29H32O8
C30H30O8
C33H32O8
C19H20O7
C20H22O7
C21H24O7
C21H24O7
C22H26O7
C18H18O7
C21H24O7
C22H26O7
C23H28O7
C23H28O7
C24H30O7
C27H28O7
C27H28O7
C28H30O7
C29H32O7
C29H32O7
C30H34O7
C33H32O7
C28H30O8
C29H32O8
C30H34O8
C30H34O8
C31H32O8
C34H34O8
144; ref. [18] 145
135; ref. [19] 139
88; ref. [19, 20] 92
120; ref. [19, 20] 123
118; ref. [10] 113
67; ref. [10] 67–68
80
oil; ref. [17] oil
oil
167; ref. [10] 165–167
198
120; ref. [17] 122
136
166; ref. [17] 170
186; ref. [21] 189
137; ref. [20] 138–139
125; ref. [20] 128–129
134; ref. [20] 133
132; ref. [10] 136–138
137; ref. [22] 133–134
148
108
124
84
93
62
88
55
65
85
91
86
84
92
71
76
77
71
89
64
90
69
63
73
73
65
61
78
69
73
65
68
63
71
87
95
90
90
89
91
69
73
82
66
88
83
74
67
63
74
63
69
82
73
62
80
77
65
MCc
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
MCc
MCc
MCc
MCc
H/EAc (9 + 1); Md
H/EAc (9 + 1); Md
H/EAc (9 + 1)
10a
10b
11a
11b
12a
12b
13a
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
14f
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
19a
19b
19c
19d
19e
19f
H/EAc (9 + 1)
H/EAc (1 + 1)
H/EAc (1 + 1)
H/EAc (1 + 1)
H/EAc (1 + 1)
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAd
114
128; ref. [10] 131–132
98
107
88
97
119
103
108; ref. [23] 108–112
142
146
123
H/EAd
H/EAd
H/EAd
153
H/EAd
172; ref. [10] 136–138
133; ref. [24] 127–128
144; ref. [25] 141–144
126; ref. [19] 127
124; ref. [19] 127
115; ref. [19] 115
123; ref. [10] 119
124
124
141
113
142
118; ref. [17] 118–120
130
98
122
117
111
89
H/EAd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd
H/EAc (9 + 1); M/Cd

1041
Table II. (continued).
Compounda
Formulab
M.p. (°C)
Yield (%)
Solventc,d (vol%)
Anal.e
20a
20b
20c
20d
20e
20f
C20H22O7
C21H24O7
C22H26O7
C22H26O7
C23H28O7
C19H20O7
161
170
143
124
98
93
97
94
91
95
H/EAd
H/EAd
H/EAd
H/EAd
H/EAd
H/EAd
C, H
C, H
C, H
C, H
C, H
C, H
112
203; ref. [17] 207–209
b
1
aAll compounds were obtained as colourless crystals except where stated otherwise. All new compounds displayed H-NMR and FTIR
c
d
consistent with the assigned structure. Eluant used for column chromatography. Solvent for recrystallization; C = chloroform; EA = ethyl
acetate; H = hexane; M = methanol; MC = methylene chloride. Elemental analyses were within ± 0.4% of calculated values except where
e
stated otherwise.
1
oil; FTIR 3 423, 1 700, 1 638 cm–1; 1H-NMR (CDCl3) δ
7.32 (d, J = 9.5 Hz, 2H), 6.93 (d, J = 9.5 Hz, 2H), 6.53
(s, 1H), 4.98 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 3.82 (s,
3H), 3.76 (s, 3H), 2.29 (s, 3H), 2.13 (s, 3H), 1.38 (t, J =
7.1 Hz, 3H). Anal. (C20H24O5) C, H.
2 965, 2 900, 1 665, 1 618 cm–1; H-NMR (CDCl3) δ
11.99 (s, 1H), 11.53 (s, 1H), 7.32–7.47 (m, 5H), 6.51 (s,
1H), 6.44 (s, 1H), 5.17 (s, 2H), 4.44 (q, J = 7.1 Hz, 2H),
2.66 (s, 3H), 2.55 (s, 3H), 2.17 (s, 3H), 2.08 (s, 3H), 1.43
(t, J = 7.13 Hz, 3H); Anal. (C27H28O7) C, H.
Analogously, 13a–f were prepared from 7 [10] and 3
and 4a–c; 14a and c–f were prepared from 11a [10] and
4a–c; 15a–f were prepared from 11b and 3 and 4a–c;
17a–f were prepared from 8 [10] and 3 and 4a–c; 18a–f
were prepared from 12a [17] and 3 and 4a–c; 19a–f were
prepared from 12b and 3 and 4a–c (table II).
4.1.5. 2-Hydroxy-4-(4-methoxybenzyloxy)-3,6-dimethyl-
benzoic acid 11b
A solution of aqueous potassium hydroxide (2.86 g in
7 mL H2O, 51.0 mmol) was added to a solution of 9b
(3.00 g, 8.82 mmol) in DMSO (40 mL) and heated on a
water bath for 2.5 h (TLC control). The solution was
cooled to room temperature, diluted with excess water
(100 mL), acidified with cold 10% HCl, and extracted
with ether (3 × 100 mL). The combined organic phase
was washed with water (3 × 200 mL), dried over MgSO4
and evaporated. The residue was purified by column
chromatography (SiO2) using hexane/ethyl acetate (1:1)
to afford colourless crystals; FTIR 3 053, 1 700, cm–1;
1H-NMR (CDCl3, DMSO-d6) δ 12.49 (s, 1H), 7.30
(d, J = 9.5 Hz, 2H), 6.99 (d, J = 9.5 Hz, 2H), 6.32 (s, 1H),
5.09 (s, 2H), 3.82 (s, 3H), 2.55 (s, 3H), 2.13 (s, 3H), Anal.
(C17H18O5) C, H.
4.1.7. General procedure for hydrogenolysis
4.1.7.1. Ethyl 4-(2,4-dihydroxy-3,6-dimethylbenzoyloxy)-
2-hydroxy-3,6-dimethylbenzoate 16b
A suspension of 14b (112 mg, 0.25 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 3 456, 2 943, 1 665, cm–1;
1H-NMR (CDCl3) δ 12.00 (s, 1H), 11.71 (s, 1H), 6.50 (s,
1H), 6.30 (s, 1H), 5.29 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H),
2.61 (s, 3H), 2.54 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H), 1.43
(t, J = 7.1 Hz, 3H); Anal. (C20H22O7) C, H.
Analogously, 12b was prepared from 10b (table II).
4.1.6. General procedure for the condensation of benzoic
acids with phenolic esters
Analogously, 16a and c–f were prepared from 14a and
c–f; 20a–f were prepared from 18a–f; 1 and 2 were
prepared from 17f and 13f, respectively (table II).
4.1.6.1. Ethyl 4-(4-benzyloxy-2-hydroxy-3,6-dimethyl-
benzoyloxy)-2-hydroxy-3,6-dimethylbenzoate 14b
A solution of 11a (136 mg, 0.5 mmol) and 3 [10]
(107 mg, 0.5 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 3 430,
4.2. Biological assay methods
The procedures for the biological assays presented in
table I were described previously in full detail: determin-
ation of the reducing activity against 2,2-diphenyl-1-
picrylhydrazyl [8], deoxyribose degradation [8], inhibi-

1042
[9] Müller K., Leukel P., Ziereis K., Gawlik I., J. Med. Chem. 37 (1994)
1660–1669.
tion of LTB4 biosynthesis [8], inhibition of HaCaT cell
proliferation [9], and release of LDH into culture me-
dium [14].
[10] Elix J.A., Norfolk S., Aust. J. Chem. 28 (1975) 1113–1124.
[11] Fitzsimmons B.J., Rokach J., in: Rokach J. (Ed.), Leukotrienes and
Lipoxygenases, Elsevier, Amsterdam, 1989, pp. 427–502.
[12] Halliwell B., Grootveld M., Gutteridge J.M.C., Methods Biochem.
Anal. 33 (1988) 59–90.
Acknowledgements
[13] Boukamp P., Petrussevska R.T., Breitkreutz D., Hornung J.,
Markham A., Fusenig N.E., J. Cell Biol. 761 (1988) 761–771.
We thank Mr K. Ziereis for his excellent technical
assistance. S.K. KC thanks the German Academic Ex-
change Service for a scholarship.
[14] Müller K., Huang H.S., Wiegrebe W., J. Med. Chem. 39 (1996)
3132–3138.
[15] Müller K., Biochem. Pharmacol. 53 (1997) 1215–1221.
[16] Müller K., Reindl H., Gawlik I., Eur. J. Med. Chem. 33 (1998)
969–973.
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