Structural effects on the bioactivity of dehydroabietic acid derivatives

Article abstract of DOI:10.1055/s-2002-33788

The synthesis and the evaluation of the antimicrobial activity against a filamentous fungus, yeasts and bacteria of 15 hydrophenanthrene compounds derived from dehydroabietic acid, bearing different functional groups and different stereochemistry of the A/B ring junction are disclosed. The results obtained showed how their activity is dependent of the functionality at C-18, which can be increased by deisopropylation or introduction of other groups into the molecule. While the filamentous fungus tested is sensitive to almost all of the compounds under study, the aldehyde function showed to be of major importance to the inhibition of yeast. Alcohols and aldehyde C-18 derivatives also inhibit the growth of a Gram-positive bacteria, whereas Gram-negative are not sensitive.

Full text of DOI:10.1055/s-2002-33788

Bµrbara Gigante1
Ana M. Silva1
Maria Joˆo Marcelo-Curto1
Sónia Savluschinske Feio1
JosØ Roseiro1
Structural Effects on the Bioactivity of Dehydroabietic
Acid Derivatives
Lucinda V. Reis2
Abstract
fungus tested is sensitive to almost all of the compounds under
study, the aldehyde function showed to be of major importance
The synthesis and the evaluation of the antimicrobial activity to the inhibition of yeast. Alcohols and aldehyde C-18 derivatives
against a filamentous fungus, yeasts and bacteria of 15 hydro- also inhibit the growth of a Gram-positive bacteria, whereas
phenanthrene compounds derived from dehydroabietic acid, Gram-negative are not sensitive.
bearing different functional groups and different stereochemis-
try of the A/B ring junction are disclosed. The results obtained Key words
showed how their activity is dependent of the functionality at Bioactivity ´ diterpenes ´ abietic acid ´ dehydroabietic acid ´ anti-
C-18, which can be increased by deisopropylation or introduc- microbial activity
tion of other groups into the molecule. While the filamentous
Introduction
bacteria, we report herein attempts to establish the structural
features that could influence the biological activity of natural
In recent years, many pathogenic microorganisms have as- identical and synthetic diterpenes with a hydrophenanthrene
sumed a serious role, either in human or animal health, due to skeleton.
680
their resistance to the known chemical control agents. This has
occasioned a great effort in the search for novel bioactive In this context, simple and sequential modifications were per-
agents, including available natural products and their deriva- formed in the molecule of dehydroabietic acid 1, compound 1
tives.
and 14 derivatives with or without an isopropyl group at C-13,
different stereoconfiguration in the A/B ring junction (cis or
Naturally occurring diterpenoids with a dehydroabietane ske- trans) and different functional groups, such as acid, ester, alcohol
leton (ketones, alcohols and phenols) are often discovered and or aldehyde at C-18, and also at C-7 or C-12 were tested. All the
isolated from plants and have been reported for their bioactiv- compounds were easily obtained in good yield, by standard che-
ity [1], [2], [3]. While the isolation of natural products from mical procedures. In preliminary bioassays, the microorganisms
plants or animals is a useful way for the discovery of profitable tested were a filamentous fungus, yeasts, and bacteria.
drugs, hemisynthesis or derivatisation of natural products can
be a faster and economical approach to the search for biologi- Some of the synthesized compounds are new and were fully
cally active compounds [4], [5], [6]. Following previous studies characterised.
on the synthesis [5] and bioactivity [6] of C-7 oxidised dealky-
lated resin acid derivatives against fungi and Gram-positive
Affiliation
1 Departamento de Tecnologia de Ind‚strias Quimicas and Laboratório de Microbiologia Industrial, Instituto
Nacional de Engenharia e Tecnologia Industrial, Lisboa, Portugal
2 Departamento de Química, Universidade de Trµs-os-Montes e Alto Douro, Vila Real, Portugal
Correspondence
Bµrbara Gigante, Ph.D. ´ INETI, Departamento de Tecnologia de Ind‚strias Químicas ´
Estrada do PacË o do Lumiar, 22, 1649-038 Lisbon ´ Portugal ´ E-Mail: barbara.gigante@ineti.pt ´
Fax: +351.217.168.100
Received November 16, 2001 ´Accepted March 23, 2002
Bibliography
Planta Med 2002; 68: 680±684 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943

dehydroabietic acid (10a-methyl-8,11,13-podocarpatrien-15-oic
acid (9): Prepared, as a mixture, by dealkylation of 1 with AlCl3
[9]. Repeated recrystallizations from MeOH, allowed the separa-
tion of both isomers as colourless crystals, 5 (25%), m.p. 161 ±
1628C (lit. [9]: 161 ±1638C); [a]D25: + 638 (c 0.51, CHCl3) {lit. [9]:
[a]D25: + 69.48 c 2.7} and 9 (60%), m.p. 172±1738C (lit. [9]: 171 ±
1738C); [a]D25: ±1 .28 (c 0.51, CHCl3) {lit. [9]: [a]D25: + 1 .78 (c 2.96)}.
Methyl 10b-methyl-13-deisopropyldehydroabietate (6): Methy-
lation of 5 with CH2N2 or dealkylation of 2 with HY zeolite [10]
afforded 6 (quantitative yield or 75%, respectively) as colour-
less crystals from methanol, m.p. 107±1098C (lit. [9]: 1 08±
1098C); [a]D25
+ 60.58(c 2.2)}.
:
+ 58.38(c 0.55, CHCl3) {lit. [9]: [a]D25
:
Methyl 10a-methyl-13-deisopropyldehydroabietate (10): Methy-
lation of 9 with CH2N2, afforded 10 (quantitative yield) as colour-
less crystals from methanol, m.p. 92±948C (lit. [9]: 93±948C);
[a]D25: ±4.38(c 0.31, CHCl3) {lit. [9]: [a]D25: ±5.18 (c 2.94)}.
Methyl 10a-methyl-7-oxo-13-deisopropyldehydroabietate (13):
Photoxidation of 10 [5] gave 13 (90%) as a colourless gum recrys-
tallized from MeOH, m.p. 75±778C (lit. [5]: colourless oil); [a]D25
±798 (c 0.2, CHCl3) {lit. [9]: [a]D25: ±78.98 (c 2.2)}.
:
Methyl
12-hydroxy-10a-methyl-13-deisopropyldehydroabietate
Materials and Methods
Chemistry
(16): Obtained from 10 following a procedure early described [11]
as colourless needles from diethyl ether, m.p.123±1258C (lit. [11]:
121 ± 122 8C); Anal. calcd. for C18H24O3 requires C 75.20%, H 8.48%;
Melting points were determined on a Reichert Thermovar melt- found: C 75.22%, H 8.45%; [a]2D5: ±1 28 (c 0.51, CHCl3).
ing point apparatus and are uncorrected. IR spectra were re-
corded on a Perkin-Elmer 457 spectrophotometer. NMR spectra Reductions of 2, 10, 13 and 16: Carried out with LiAlH4 in anhy-
were recorded on a General Electric QE-300 spectrometer at drous ether under N2 atmosphere, by a previously described pro-
300.65 MHz for 1H and at 65.7 MHz for 13C, using a 5-mm dual cedure [8].
681
probe with CDCl3 as solvent. The chemical shifts reported are in
d (ppm, TMS). EIMS were recorded on a Kratos MS 25RF mass Dehydroabietinol (3): (98% from 2), viscous oil (lit. [8], [11]: oil);
spectrometer at 70 eV and HREIMS were determined on Extrell IR (film): nmax = 3320 (OH) cm±1; [a]D25: + 528(c 0.55, CHCl3) {lit.
(Waters) FTMS 2001-DT S.T.I.C.R. instrument. Optical rotations [8]: [a]D25: + 51 .98 (c 0.45, CHCl3)}.
were measured on a Perkin-Elmer 241polarimeter. Elemental
analyses were performed on a Carlo Erba 1106 elemental analy- 10b-Methyl-13-deisopropyldehydroabietinol (7): (95% from 6),
ser. TLC was carried out on Merck precoated silica gel sheets colourless oil (lit. [12]: 85±968C]; [a]D25: + 538(c 0.54, CHCl3); IR
(GF254). Column chromatography was performed using Merck (film) nmax = 3320 (OH) cm±1; EIMS: m/z (rel. int.) = 244 [M]+
60 silica gel (230±400 mesh). UV light was used for the detec- (17), 229 [M±CH3]+ (55), 211 [M±CH ±H2O]+ (75), 131 (100).
3
tion of compounds on TLC. All solvents were purified by stand-
ard methods and anhydrous solvents were dried and freshly 10a-Methyl-13-deisopropyldehydroabietinol (11): (98% from 10),
distilled.
colourless needles from petroleum ether, m.p. 68±698C; [a]D25
:
±12.28 (c 0.35, CHCl3); IR (KBr): nmax = 3320 (OH) cm±1; EIMS:
Dehydroabietic acid (abieta-8,11,13-trien-18-oic acid) (1): Ob- m/z (rel. int.) = 244 [M]+ (17), 229 [M±CH3]+ (55), 211 [M±CH -
tained from dehydrogenated commercial rosin [7]. Recrystalliza- H2O]+ (75), 131 (100); 1H-NMR (CDCl3) d = 0.32 (3H, s, 4-CH3),
3
tion from methanol/water gave colourless prisms, m.p. 166± 1.19 (3H, s, 10-CH ), 1.19±1.24 (1H, m, 3-Ha), 1.32±1.42 (1H, m,
3
1688C (lit. [7]: 162±1658C; lit. [8]: 167±1698C); [a]D25: + 588(c 1-Ha), 1.47±1.57 (3H, m, 2-H , 3-Hb), 1.79 (1H, dd, J = 7.4, 2.0
2
0.50, CHCl3) {lit. [8]: [a]D25 + 57.38(c 1.04, CHCl3)}.
Hz, 5-H), 1.88±1.98 (1H, m, 6-Hb), 2.15±2.28 (1H, m, 6-Ha),
2.42±2.48 (1H, m, 1-Hb), 2.80±3.00 (2H, m, 7-H2), 3.20 (1H, AB
Methyl dehydroabietate (methyl abieta-8,11,13-trien-18-oate) system, J = 11.0 Hz, 4-CH2OH), 3.53 (1H, AB system, J = 11.0 Hz,
(2): Obtained by methylation of 1 with diazomethane. The crude 4-CH2OH), 4.76 (1H, brs, D2O exchange, 4-CH2OH), 7.01±7.16
product affords by recrystallization from methanol colourless (3H, m, Ar-H), 7.27±7.30 (1H, m, Ar-H); 13C-NMR (CDCl3):
prisms, m.p. 63±658C (lit. [8]: 64±658C); [a]D25: + 538(c 0.51, d = 37.5 (C-1), 17.4 (C-2), 36.6 (C-3), 38.8 (C-4), 43.1 (C-5), 18.5
CHCl3) {lit. [8]: [a]D25: + 50.68(c 0.9, CHCl3) EtOH)}.
(C-6), 26.0 (C-7), 137.2 (C-8), 143.7 (C-9), 36.9 (C-10), 123.6 (C-
11), 125.1 (C-12), 125.4 (C-13), 128.7 (C-14), 72.0 (C-4-CH2OH),
10b-Methyl-13-deisopropyldehydroabietic acid (10b-methyl-8,11,13- 18.2 (C-4-CH3), 34.1(C-10- CH3); Anal. calcd. for C17H24O requires
podocarpatrien-15-oic acid) (5) and 10a-methyl-13-deisopropyl- C 83.55, H 9.90%; found: C 83.52, H 9.92%.

7-Hydroxy-10a-methyl-13-deisopropyldehydroabietinol (14): (95% 128.7 (C-14), 206.5 (C-4-CHO), 21.3 (C-4-CH3), 28.3 (C-10-CH3);
from 13) colourless oil; [a]D25: ±558 (c 0.26, CHCl3); HREIMS: m/z
= 260.1745 [M]+ (calcd. for C17H24O2: 260.1770); IR (neat): nmax
3400 (OH) cm±1 1H-NMR (CDCl3): d = 0.28 (3H, s, 4-CH3), 1.21
EIMS: m/z (rel. int.) = 242 [M]+ (5), 227 [M±CH3]+ (17), 209
(23), 143 (42), 131 (100).
=
;
(3H, s, 10-CH3), 1.21 ±1.28 (1H, m, 3-Ha), 1.45±1.62 (4H, m, 1- 10a-Methyl-7-oxo-13-deisopropyldehydroabietinal (15): (91%
Ha, 2-H2, 3-Hb), 1.66±1.75 (1H, m, 6-Hb), 1.70±1.90 (1H, brd, from 14), colourless needles from CH2Cl2/n-hexane, m.p. 219±
J = 11.0 Hz, D O exchange, 4-CH2OH), 1.88±1.93 (1H, dd,J = 7.8, 2218C; [a]D25: ±608(c 0.26, CHCl3); HREIMS: m/z = 256.1452 [M]+
2
4.5 Hz, 5-H), 2.26±2.31 (1H, m, 1-Hb), 2.40 (1H, brs, D2O ex- (calcd. for C17H20O2: 256.1463); IR (KBr): nmax = 1775 (C = O), 1725
change, 7-OH), 2.62 (1H, td, 6-Ha), 3.30 (1H, AB system, J = 1 1 .0 (C = O) cm±1; 1H-NMR (CDCl3): d = 0.59 (3H, s, 4-CH3), 1.36 (3H,
Hz, 4-CH2OH), 3.47 (1H, AB system, J = 11.0 Hz, 4-CH2OH), 4.91±
s, 10-CH3), 1.20±1.42 (1H, m, 3-Ha), 1.49±1.69 (4H, m, 3-Hb, 2-
4.98 (1H, m, t after D2O exchange, 7-H), 7.20±7.30 (3H, m, Ar-H), H2, 1 -Ha), 2.38±2.53 (3H, m, 6-Ha, 5-H, 1-Hb), 3.07 (1H, dd,
7.56±7.60 (1H, t, Ar-H); 13C-NMR (CDCl3) d = 37.0 (C-1), 18.3 (C- J = 18.9, 6.6 Hz, 6-Hb), 7.31±7.40 (2H, m, Ar-H), 7.54±7.60 (1H,
2), 34.9 (C-3), 38.7 (C-4), 41.8 (C-5), 31.4 (C-6), 66.1 (C-7), 139.8 m, Ar-H), 8.04 (1H, dd, J = 7.8, 1.2 Hz, Ar-H), 9.34 (1H, s, 4-CHO);
(C-8), 143.7 (C-9), 37.5 (C-10), 124.1 (C-11), 124.6 (C-12), 125.6 13C-NMR (CDCl3) d = 36.4 (C-1), 17.5 (C-2), 32.7 (C-3), 50.3 (C-4),
(C-13), 126.9 (C-14), 71.5 (C-4-CH2OH), 19.8 (C-4-CH3), 32.0 (C- 43.6 (C-5), 37.1 (C-6),197.2 (C-7),132.7 (C-8),148.9 (C-9), 36.6 (C-
10-CH3); EIMS: m/z (rel. int.) = 260 [M]+ (26), 242 [M±H2O]+ 10), 124.3 (C-11), 126.4 (C-12), 127.4 (C-13), 134.2 (C-14), 205.1
(5), 224 [M - 2H2O]+ (8), 155 (100), 141 (74), 128 (45), 115 (51).
(C-4-CHO), 16.4 (C-4-CH3), 34.2 (C-10-CH3); FAB-MS: m/z (rel.
int.) = 257 [M + H]+ (46), 239 (19), 227 (21), 211(24), 171(31),
12-Hydroxy-10a-methyl-13-deisopropyldehydroabietinol (17): (95% 165 (40), 137 (40), 115 (73), 89 (100).
from 16) as colourless needles from Et2O/petroleum ether, m.p.
155±1578C; [a]2D5: ±1 83 (c 0.22, CHCl3); IR (KBr): nmax = 3470 Biological evaluation
(OH), 3190 (OH) cm±1; EIMS: m/z (rel. int.) = 260 [M]+ (59), 245 In vitro antimicrobial activity: The microorganisms assayed were
[M±CH3]+ (36), 227 [M±CH3 ±H2O]+ (100), 211(24), 171(31), 165
Trichophyton mentagrophytes CCMI 226, Staphylococcus aureus
(40), 137 (40), 115 (73), 89 (100);1H-NMR (CDCl3) d = 0.36 (3H, s, CCM I335, Serratia marcescens CCMI 638, Pseudomonas
4-CH3), 1.17 (3H, s, 10-CH), 1.18±1.24 (1H, m, 3-Ha), 1.32±1.40 aeruginosa CCMI 331, Candida parapsilosis CL 2, Candida kruzei CL
3
(1H, m, 1-Ha), 1.48±1.56 (3H, m, 2-H , 3-Hb), 1.76 (1H, dd,J = 7.2, 6 and two strains of Candida albicans, CMI 209 and CMI 110. The
2
1.8 Hz, 5-H), 1.86±1.95 (1H, m, 6-Hb), 2.11 ±2.24 (1H, m, 6-Ha), bioassays with a filamentous fungus were performed according
2.35 (1H, d, J = 12.9 Hz, 1-Hb), 2.58 (1H, brs, D2O exchange, 4- to Henricks [14], using compounds dissolved in acetone and tested
CH2OH), 2.78±2.84 (2H, m, 7-H2), 3.21(1H, AB system, J = 1 1 .1 at 0.1% (w/w, compound/culture medium). Blank plates containing
Hz, , after D2O exchange, 4-CH2OH), 3.53 (1H, AB system, J = 1 1 .1 acetone were equally prepared. Amphotericin B was used as posi-
Hz, after D2O exchange, 4-CH2OH), 6.58 (1H, dd, J = 8.1 , 2.4 Hz,1 3- tive control. The results were expressed as Relative Inhibition (RI).
H), 6.79 (1H, dd, J = 2.4 Hz, 11-H), 6.88 (1H, dd, J = 8.1Hz, 14-H),
8.01(1H, s, D 2O exchange, 12-OH); 13C-NMR (CDCl3) d = 37.6 (C- Yeast and bacteria bioassays were performed on multiwell plates
1), 17.5 (C-2), 36.7 (C-3), 38.9 (C-4), 42.9 (C-5), 18.6 (C-6), 25.1 (C- according to a previously described procedure [6]. The test com-
7), 128.0 (C-8), 145.0 (C-9), 36.9 (C-10), 110.7 (C-11), 154.6 (C-12), pounds, dissolved in acetone at a concentration of 50 mg/ml,
112.3 (C-13), 129.3 (C-14), 71.5 (C-4C-H2OH), 18.2 (C-4-CH3), 34.2 were applied to the multiwell plates containing the micro-
(C-10-CH3); Anal. calcd. for C17H24O2 requires C 78.42, H 9.29%; organism incorporated in the culture medium. 5-Fluorocytosine
682
found: C 78.44, H 9.28%.
and rifampicin were used as positive controls, respectively, for
yeasts and bacteria.
Oxidation of 3, 11 and 14: Done with PCC in dichloromethane in
accordance with a procedure previously described [8]. Pure alde-
hydes were obtained after column chromatography (silica gel, Results and Discussion
petroleum ether with gradual addition of dichloromethane) and
recrystallization from methanol/petroleum ether:
Synthesis of the compounds
The dehydroabietic acid 1, isolated from dehydrogenated rosin
Desidroabietinal (4): (94% from 3), colourless needles from me- (65±70% yield [7]), was the starting material from which all the
thanol/petroleum ether, m.p. 85±878C (lit. [8]: 86±888C); others compounds, natural identical (2±4) [15], [16], [17], [18],
[a]D25: + 568(c 0.51, CHCl3) {lit. [12]: [a]D25 + 55.88(c 0.95, CHCl3)}.
[19] or synthetic (5±7 and 9±17) diterpenes were successively
obtained.
10a-Methyl-13-deisopropyldehydroabietinal (12): (94% from 11),
thick gum from petroleum ether (lit. [14]: oil, bp 858C/0.7 Torr); The dealkylated isomers, 10b-methyl-13-deisopropyldehydro-
[a]D25: ±1 .28(c 0.24, CHCl3) {lit. [14]: [a]D25: ±1 .88(c 1.6, EtOH)}; abietic acid 5 (25%) and 10a-methyl-13-deisopropyldehydro-
HREIMS: m/z = 242.1659 [M]+ (calcd. for C17H22O: 242.1670); IR abietic acid (9) (60%) were obtained from 1 by a Friedel-Crafts
±1
(KBr): nmax = 1715 (C = O) cm ; 1H-NMR (CDCl3) d = 0.88 (3H, reaction with AlCl3 in toluene [11]. Methylation of the acids1, 5
s, 4-CH3), 1.23 (3H, s, 10-CH ), 1.15±1.25 (1H, m, H-3a), 1.47±1.70 and 9 with diazomethane gave the corresponding esters 2, 6 and
3
(4H, m, 6-Hb, 2-Hb, 1 -Ha, 5-H), 1.77±1.92 (2H, m, 1-Hb, 2-Ha), 10 in quantitative yield. Methyl 10b-methyl-13-deisopropylde-
1.97±2.08 (2H, m, 3-Hb, 6-Ha), 2.85±2.89 (2H, m, 7-H2), 7.03± hydroabietate (6) can also be obtained selectively (75%) by deal-
7.17 (3H, m, Ar-H), 7.25±7.28 (1H, m, Ar-H), 9.55 (1H, d, J = 1 .2 kylation of 2 using an acidic HY zeolite as catalyst [10].
Hz, 4-CHO); 13C-NMR (CDCl3): d = 36.4 (C-1), 18.9 (C-2), 30.0
(C-3), 49.5 (C-4), 45.2 (C-5), 19.9 (C-6), 29.3 (C-7), 135.9 (C-8), In order to improve the activity of these compounds additional
145.6 (C-9), 38.2 (C-10), 125.4 (C-11), 125.9 (C-12), 126.1 (C-13), functions were introduced. Methyl 10a-methyl-7-oxo-13-deiso-

propyldehydroabietate (13) and the phenol, methyl 12-hydroxy- are expressed as relative inhibition RI (%). For yeasts and bacter-
10a-methyl-13-deisopropyldehydroabietate (16) were obtained ia a multiwell plates assay was performed according to a proce-
from 10, respectively, by a selective photo-oxygenation proce- dure previously described [6] and the results expressed in
dure [5] or by a sequential acetylation, Bayer-Villiger oxidation terms of minimum inhibitory concentration MIC (mmol/ml).
and hydrolysis methodology [11]. Due to the differences in the The amounts of compounds tested by these techniques are
experimental procedure used and in the melting point of the high and are related with the density of the microbial popula-
compound obtained with that previously reported in the litera- tion used in the test, 108 CFU/ml for fungi and 105 CFU/ml for
ture, the characterisation of 16 was also done. Esters 2, 6, 10, 13 bacteria [1], [6].
and 16 on reduction with lithium aluminium hydride (LiAlH4)
furnished the respective alcohols, 3, 7, 11, 14 and 17, which by As summarized in Table 1, all compounds showed activity
oxidation with pyridinium chlorochromate (PCC) gave the re- against T. mentagrophytes. Antifungal activity is enhanced by
spective aldehydes 4, 8, 12 and 15. 1 0b-Methyl-13-deisopropyl- the alcohols without an isopropyl group at C-13, 7 and 11 show-
dehydroabietinal (8) could not be characterised and tested due ing RI of 91% and 90%, which improved to 100% when another
to its quick degradation during the purification step.
hydroxy group is present in the molecule (14 and 17). No signifi-
cant differences in activity against T. mentagrophytes were in-
The new compounds (11, 14, 15 and 17) and the aldehyde 12 were duced by the difference on the A/B ring junction (5±7 vs. 9±11).
characterised by different physical and spectrometric methods In acetone the microorganisms grew normally, while amphoter-
(m.p., IR, NMR, MS and/or elemental analysis), while the struc- icin B inhibited the fungal growth (RI = 100%).
tures of the other compounds, already known and described,
were established by comparison with data found in the litera- The assays against several Candida strains show that only the
ture.
compounds containing an aldehyde group at C-18 (4, 12 and 15)
inhibited their growth. The dehydroabietinal 4 shows the same
MIC (> 70 mmol/ml) for all the strains, decreasing significantly
Antimicrobial activity assays
The antimicrobial activity of deisopropylated resin acid deriva- for the deisopropylated analogue with a cis A/B ring junction,
tives 5±7 and 9±12 was tested and compared with that of C-13 12, with MIC between 26.9 and 53.8 mmol/ml. That value is two-
isopropylated analogous 1±4. The results were also compared fold reduced for C. albicans 110,C. albicans 407, C. kruzei, or even
with those obtained with the bifunctional derivatives 13 ± 15 lower (6.3 mmol/ml) for C. parapsilosis when the aldehydic com-
and 17 (Table 1).
pound possesses a ketone group at C-7 (15). In spite of this in-
creasing effect on the activity, those compounds are less active
With the filamentous fungus the bioassays were performed in than the control (5-fluorocytosine) which showed a lower MIC
acetone solution using the solvent as control [1] and the results (0.8 mmol/ml).
683
Table 1 Bioactivity of dehydroabietic acid derivatives against a filamentous fungus (F.F.), yeasts and bacteriaa,b
F.F.c
Yeastsd,e
Bacteriad),f)
RI (%)
(mmol/ml)
(mmol/ml)
Compound
T. mentagrophytes C. albicans 110
C. albicans 407
C. kruzei
C. parapsilosis
S. aureus
1
6.8
7.0
na
na
na
na
> 70
na
na
na
na
na
na
na
> 70
na
na
na
na
na
na
na
> 70
na
na
na
na
na
2
na
na
3
56
na
5.6
5.6
na
4
44
77
78
91
77
4
> 70
na
5
6
na
na
7
na
6.5
na
9
na
10
9
na
na
na
na
na
11
90
na
na
na
na
6.5
6.6
12
nt
26.9
na
26.9
na
53.8
na
26.9
na
13
88
100
39
22.7
3.5
14
na
na
na
na
15
12.7
na
12.7
na
25.46.3
na
3.9
na
17
100
100
nt
1.5
nt
Amphotericin B
5-Fluorocytosine
Rifampicin
nt
nt
nt
nt
0.8
nt
0.8
nt
0.8
nt
0.8
nt
nt
nt
0.001
a No inhibition of growth was observed at 40 mmol/ml for any of the tested compounds against the Gram-negative bacteria, P. aeroginosa and S. marcencens, also tested.
b nt ± not tested.
c Acetone as control.
d Values of MIC (mmol/ml.
e na ± not active below 80 mmol/ml.
f na ± not active below 40 mmol/ml.

In the case of bacteria, none of the tested compounds inhibited References
the growth of Gram-negative bacteria P. aeruginosa and S.
1
Franich RA, Gadgil PD, Shain L. Fungistatic effects of Pinus radiata nee-
dle epicuticular fatty and resin acids on Dothistroma pini. Physiol.
Plant Pathol. 1983; 23: 183±95
marcescens. The presence of alcohols or aldehydes at C-4 (3, 4, 7,
11 and 12) seems to be important for the inhibition of Gram-po-
sitive bacteria S. aureus. MIC has quite similar values (ca. 6 mmol/
ml) for compounds either with (3, 4) or without the isopropyl
group at C-13 (7, 11, 12), and is independent of the stereoconfi-
guration (3, 4, 7 vs. 11, 12). The MIC is reduced two-fold when an-
other hydroxy or ketone group is present in the molecule (14, 15)
at C-7 or C-12. For S. aureus the activity of rifampicin, used as a
control, showed a lower MIC (0.001 mmol/ml) than the com-
pounds under study.
2 Ulubelen A, Oksuz S, Kolac U, Bozok-Johansson C, Þelik C, Voelter W.
Antibacterial diterpenes from the roots of Salvia viridis. Planta Medica
2000; 66: 458±62
3 Mensah AY, Houghton PJ, Bloomfield S, Vlietinck A, Berghe DV. Known
and novel terpenes from Buddleja globosa displaying selective anti-
fungal activity against dermatophytes. J. Nat. Prod. 2000; 63: 1210±3
4 Feliciano AS, Gordaliza M, Salinero MA, Corral JMM. Abietane acids:
Sources, biological activities, and therapeutic uses. Planta Medica
1993; 59: 485±98, and references cited therein
5 Gigante B, Lobo AM, Prabhakar S, Marcelo-Curto MJ. A new selective
synthesis of oxidized resin acid derivatives. Synthetic Commun.
1991; 21: 1959±66
The lack of activity of dehydroabietic acid derivatives towards
Gram-negative bacteria has already been observed [1], [6]. In a
previous work [6], only combined methyl 10a-methyl-7-oxo-13-
deisopropyldehydroabietate (13) and its 10b-methyl isomer inhib-
ited the growth of Escherichia coli and Klebsiella pneumoniae.
6 Savluchinske-Feio S, Roseiro JC, Gigante B, Marcelo-Curto MJ. Method
on multiwell plates for the evaluation of the antimicrobial activity of
resin acid derivatives. J. Microbiol. Methods 1997; 28:201 ±6 and
1999; 35: 201 ±6
7 Halbrook NJ, Lawrence RV. The isolation of dehydroabietic acid from
disproportionated rosin. J. Org. Chem. 1966; 31: 4246±7
8 Shyong Li W, McChesney JD. Preparation of potential anti-inflamma-
tory agents from dehydroabietic Acid. J. Pharm Sci. 1992; 81: 646±51
9 Ohta M, Ohmori L. Studies on abietic acid derivatives. deisopropyla-
tion of dehydroabietic acid. Chem. Pharm. Bull. 1957; 5: 91 ±95 and
96±100
Comparison and correlation with other studies [1], [2], [3],
where the bioactivity of diterpenes with a dehydroabietane ske-
leton was evaluated, is not straightforward, mainly due to the
difference on the biological methodologies used.
10
Pereira C, Alvarez F, Marcelo-Curto MJ, Gigante B, Ribeiro FR, Guisnet
M. Stereoselectivity of the deisopropylation of methyl dehydroabie-
tate. Stud. Surf. Sci. Cat. 1993; 78: 581 ±6
From this study we can conclude that simple derivatisation of re-
sin acid derivatives can increase their bioactivity. The alcohol or
aldehyde C-18 derivatives of dehydroabietic acid are more active
against T. mentagrophytes, a filamentous fungus, several Candida
strains (C. albicans 110,C. albicans 407, C. kruzei, C. parapsilosis)
and S. aureus, than the acid or its methyl ester themselves. Their
activity can be increased by deisopropylation or by the introduc-
tion of an alcohol or ketone function at C-7 or C-12, depending on
the micro-organism under study. The stereochemistry of the A/B
ring junction seems not to display a significant role.
11
Tahara A, Akita H. Diterpenoids. XXX. Reaction of methyl dehydro-
abietate derivatives with aluminum chloride under effect of electron-
donating group. Chem. Pharm. Bull. 1975; 23:1976±83 and Ohta M.,
Yakugaku Zasshi 1957; 77: 924; Chem Abstr. 1958, 1110
Spencer TA, Weaver TD, Villarica RM, Friary RJ, Posler J, Schwartz MA.
Syntheses of methyl deisopropyldehydroabietate. Diterpenoid syn-
thesis by the AB-ABC approach. J. Org. Chem. 1968; 33: 712±9
Wenkert E, Beak P, Carney RWJ, Chamberlain JW, Johnston DBR, Roth
CD, Tahara A. Derivatives of dehydroabietic and podocarpic acids. Can.
J. Chem. 1963; 41: 1924±36
Henriks ML, Ekman R, von Weissenberg K. Bioassay of some resin and
fatty acids with Fomes annosus. Acta Acad. Aboensis 1979; 39B: 1 ±7
Harris GC. Resin acids. V. The composition of the gum oleoresin acids
of Pinus palustris. J. Am. Chem. Soc.1948; 70: 3671 ±4
Buratti L, Allais JP, Barbier M. A resin acid from Pinus sylvestris needles.
Phytochemistry 1990; 29: 2708±9
12
13
684
14
15
16
17
18
Nevertheless, the low activity found for the compounds under
study, the structural effects observed in this work may be an im-
portant basis for further investigations on resin acid derivatives
towards bioactive compounds.
Norin T, Winell B. Extractives from the bark of common spruce, Picea
abies L. Karst. Acta Chem. Scand. 1972; 26: 2280±96
Hafizoglu H, Reunanen M. Composition of oleoresins from bark and
cones of Abies nordmanniana and Picea orientalis. Holzforschung,
1994; 48: 7±11
Acknowledgements
19
Fraga BM, Mestres T, Diaz CE, Arteaga JM. Dehydroabietane diterpenes
from Nepeta teydea. Phytochemistry 1994; 35: 1509±12
The authors are grateful to C. Santos for the optical rotation
measurements and the financial support of FundacË ˆo para a
Ci†ncia e Tecnologia (Proj. PRAXIS/PCNA/C/BIA/178/96).