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

Eur. J. Med. Chem. 34 (1999) 1035−1042

1035

Original article

Depsides as non-redox inhibitors of leukotriene B₄biosynthesis and HaCaT cell

growth. 1. Novel analogues of barbatic and diffractaic acid

Sunil Kumar KC^a, Klaus Müller^b*

^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 LTB₄biosynthesis inhibitor of this series, with an

IC₅₀value 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

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 LTB₄biosynthesis 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 LTB₄biosynthesis 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 IC₅₀value 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 LTB₄biosynthesis. 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 IC₅₀values 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

LTB₄assay, 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 R³are

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 IC₅₀values 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 LTB₄biosynthesis of this series.

Potency of compound 16b, with an IC₅₀of 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 LTB₄biosynthesis. 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 LTB₄biosynthesis. Like-

wise, the most potent inhibitor of LTB₄biosynthesis,

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 LTB₄is 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 LTB₄assay.

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 LTB₄biosynthesis, antiproliferative activity and cytotoxicity against HaCaT cells of barbatic and

diffractaic acid derivatives.

a

c

k_DPPH

DD ( OH)^b

LTB₄

AA^d

LDH^e

(mU)

Compound

R¹

R²

R³

(M^–1s^–1)

IC₅₀(µM)

1

2

Me

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

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

0.08 ± 0.01

ND

7.6

7.8

2.6

4.1

4.8

1.9

136

148

143

ND

150

142

ND

150

140

ND

143

149

146

140

ND

170^f

167^f

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

19.2

> 20

9.0

16.2

9.0

7.9

> 20

15.0

> 20

2.1

3.2

> 20

19.0

16.7

> 20

9.0

PhCH₂

4-MeOPhCH₂

H

Me

PhCH₂

Et

ND

Prop

CHMe₂

Bu

CH₂Ph

Me

0.55 ± 0.04

0.78 ± 0.01

0.51 ± 0.01

0.96 ± 0.01

ND

0.72 ± 0.01

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

0.97 ± 0.02

ND

0.19 ± 0.03

0.23 ± 0.01

0.11 ± 0.03

0.26 ± 0.01

ND

0.19 ± 0.01

ND

0.21 ± 0.02

0.02 ± 0.01

0.12 ± 0.01

0.16 ± 0.01

0.72 ± 0.01^f

0.01 ± 0.01

0.19 ± 0.01

0.21 ± 0.01

0.06 ± 0.02

ND

8.4

> 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

> 20

14.0

4.1

> 20

ND

Et

Prop

CHMe₂

Bu

CH₂Ph

Me

19

> 20

18.3

> 20

ND

Et

0.44 ± 0.02^f

ND

Prop

CHMe₂

Bu

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 LTB₄biosynthesis 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

AA^d

LDH^e

a

c

k_DPPH

Compound

R¹

R²

R³

(M^–1s^–1)

IC₅₀(µM)

(mU)

19a

19b

19c

19d

19e

19f

20a

20b

20c

20d

20e

4-MeOPhCH₂

H

Me

Et

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 ± 4.2^f

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 ± 0.14^f

> 20

7.8

1.4

8.5

5.8

7.8

> 20

ND

9.0

> 20

7.2

ND

138

ND

168

ND

114

294^f

Prop

CHMe₂

Bu

CH₂Ph

Me

Et

Prop

CHMe₂

Bu

> 20

9.8

0.7

20f

H

11.0

37.0

anthralin^g

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 LTB₄and

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 LTB₄biosynthesis 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 MgSO₄and 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 MgSO₄and 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

MgSO₄and 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.

Compound^a

Formula^b

M.p. (°C)

Yield (%)

Solvent^c,d(vol%)

Anal.^e

4a

4b

4c

4d

4e

9a

9b

C₁₀H₁₂O₄

C₁₂H₁₆O₄

C₁₃H₁₈O₄

C₁₆H₁₆O₄

C₁₈H₂₀O₄

C₁₉H₂₂O₅

C₁₉H₂₂O₄

C₂₀H₂₄O₅

C₁₆H₁₆O₅

C₁₇H₁₈O₅

C₁₇H₁₈O₄

C₁₈H₂₀O₅

C₂₀H₂₂O₇

C₂₁H₂₄O₇

C₂₂H₂₆O₇

C₂₃H₂₈O₇

C₂₆H₂₆O₇

C₂₇H₂₈O₇

C₂₈H₃₀O₇

C₂₉H₃₂O₇

C₃₂H₃₀O₇

C₂₇H₂₈O₈

C₂₈H₃₀O₈

C₂₉H₃₂O₈

C₃₀H₃₀O₈

C₃₃H₃₂O₈

C₁₉H₂₀O₇

C₂₀H₂₂O₇

C₂₁H₂₄O₇

C₂₂H₂₆O₇

C₁₈H₁₈O₇

C₂₁H₂₄O₇

C₂₂H₂₆O₇

C₂₃H₂₈O₇

C₂₄H₃₀O₇

C₂₇H₂₈O₇

C₂₈H₃₀O₇

C₂₉H₃₂O₇

C₃₀H₃₄O₇

C₃₃H₃₂O₇

C₂₈H₃₀O₈

C₂₉H₃₂O₈

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

65

61

78

69

73

65

68

63

71

87

95

90

89

91

69

73

82

66

88

83

74

67

63

74

63

69

82

73

62

80

77

65

MC^c

C, H

MC^c

H/EA^c(9 + 1); M^d

H/EA^c(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/EA^c(9 + 1)

H/EA^c(1 + 1)

H/EA^c(9 + 1); M/C^d

H/EA^d

114

128; ref. [10] 131–132

98

107

88

97

119

103

108; ref. [23] 108–112

142

146

123

H/EA^d

153

H/EA^d

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

141

113

142

118; ref. [17] 118–120

130

98

122

117

111

89

H/EA^d

H/EA^c(9 + 1); M/C^d

1041

Table II. (continued).

Compound^a

Formula^b

M.p. (°C)

Yield (%)

Solvent^c,d(vol%)

Anal.^e

20a

20b

20c

20d

20e

20f

C₂₀H₂₂O₇

C₂₁H₂₄O₇

C₂₂H₂₆O₇

C₂₃H₂₈O₇

C₁₉H₂₀O₇

161

170

143

124

98

93

97

94

91

95

H/EA^d

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 H₂for 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/CHCl₃to 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 LTB₄biosynthesis [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.

References

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Article Doi

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

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