Fungicidal sesquiterpene dialdehyde cinnamates from Pseudowintera axillaris

Article abstract of DOI:10.1021/jf052585s

Bioactivity-directed separation of a foliage extract from the New Zealand shrub Pseudowintera axillaris led to a compound with fungicidal activity against the plant pathogen Phytophthora infestans. This was identified as a new sesquiterpene dialdehyde cin

Full text of DOI:10.1021/jf052585s

468 J. Agric. Food Chem. 2006, 54, 468−473
Fungicidal Sesquiterpene Dialdehyde Cinnamates from
Pseudowintera axillaris
NERIDA J. BRENNAN,† LESLEY LARSEN,† STEPHEN D. LORIMER,†
NIGEL B. PERRY,*,† ELEANOR L. CHAPIN,‡ TODD L. WERK,‡
‡
MATTHEW J. HENRY,‡ AND DONALD R. HAHN
New Zealand Institute for Crop & Food Research Ltd., Department of Chemistry, University of Otago,
P.O. Box 56, Dunedin, New Zealand, and Dow AgroSciences, 9330 Zionsville Road,
Indianapolis, Indiana 46268-1053
Bioactivity-directed separation of a foliage extract from the New Zealand shrub Pseudowintera axillaris
led to a compound with fungicidal activity against the plant pathogen Phytophthora infestans. This
was identified as a new sesquiterpene dialdehyde cinnamate named paxidal. Two 6-hydroxy
derivatives were present at lower levels in the extract. A further nine derivatives were synthesized
from these natural products for a structure-activity study against a range of important food crop
pathogens. The cinnamate group was important for fungicidal effects, and protection of the dialdehyde
as a dimethyl acetal gave more potent, broader spectrum activity.
KEYWORDS: Pseudowintera; sesquiterpene; dialdehyde; cinnamate; antifungal activity; Phytophthora
Table 1. 13C NMR Data for Paxidal (3) and Related Compounds
4
INTRODUCTION
−
14a
Modern agriculture relies on effective control of fungal
diseases to increase crop yield and quality and consequently
increase crop value (1). No single fungicide can be used for all
disease situations, and the widespread use of fungicides can
select for fungicide-resistant pathogens (2). Therefore, there is
a need for safer and more cost-effective fungicides which are
easier to use and provide better performance against resistant
pathogens. During screening of New Zealand plants for biologi-
cally active natural products (3), an extract from foliage of
Pseudowintera axillaris showed in vivo antifungal activity
against the plant pathogen Phytophthora infestans (PHYTIN).
P. axillaris (J. R. et G. Forst.) Dandy (family Winteraceae) is
a shrub found in forests throughout the North Island and the
northwestern South Island of New Zealand (4). It is one of three
species in this endemic genus (5). The more common P. colorata
contains polygodial (1) and 9-deoxymuzigadial (2) (6, 7), which
have antifungal activity, in particular against the human
pathogen Candida albicans (8). However, an extract of P.
axillaris had no activity against C. albicans and did not contain
any detectable polygodial (7). There are no other published
studies of the chemistry of P. axillaris.
atom
3
4
5
6
7
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
9
80.5 81.0 79.8 78.8 87.7 79.3 78.9 79.0 78.1 78.8 78.2 82.1
36.0 35.7 36.4 40.0 34.5 36.8 37.0 36.7 38.7 37.3 38.7 37.2
35.6 35.5 36.0 35.8 35.4 35.5 36.0 35.9 36.3 36.6 36.1 35.9
149.3 146.4 147.0 149.8 150.2 150.4 147.5 147.3 148.0 156.9 147.6 151.1
44.8 49.2 53.9 44.3 44.9 44.5 54.7 54.6 54.1 57.8 50.7 45.5
25.9 64.6 64.7 26.1 26.0 24.6 65.6 65.3 65.3 194.8 73.8 24.4
150.8 148.0 150.4 151.7 150.9 119.9 121.4 121.8 122.8 121.8 118.8 126.8
140.3 140.3 139.1 139.5 140.7 136.8 139.3 138.9 137.5 142.7 138.6 137.2
56.7 56.8 56.2 57.3 57.6 54.6 54.5 53.8 53.8 54.9 53.9 51.2
42.9 42.1 44.1 43.8 44.3 39.0 42.8 42.6 43.7 45.1 43.6 41.0
200.4 199.8 199.7 203.4 200.7 106.9 107.0 106.6 106.5 107.1 106.6 62.7
192.5 193.1 192.3 193.0 192.8 103.6 103.4 103.4 103.3 102.9 103.4 66.9
107.9 110.5 108.4 107.3 107.1 106.3 107.3 107.3 107.1 111.1 107.1 106.4
17.9 17.9 17.6 17.9 18.1 15.0 17.8 17.7 18.0 17.5 18.1 17.8
10.6 13.7 10.9
166.1 166.1 165.9
117.7 117.4 117.3
146.1 146.1 146.3
134.1 134.0 134.0
128.9 128.9 128.9
128.4 128.3 128.3
130.6 130.7 130.7
10
11
12
13
14
15
′
′
′
′
8.2
9.3
7.6
8.7
8.5
7.9
9.5
165.5
118.1
144.9
134.1
129.0
128.0
130.5
7.9
9.0
166.6
118.2
145.4
134.2
128.2
128.9
130.5
1
2
3
4
5
6
7
165.5 165.6 171.7
118.7 118.6 30.8
143.8 144.4 36.0
134.3 134.3 140.4
128.7 129.0 128.5
127.7 128.0 128.2
130.0 130.3 126.2
56.0 56.4 55.2 56.2 56.5 56.2
53.8 54.3 54.4 56.4 54.7 54.9
54.4
′+
′+
′
9
8
′
′
11OMe
12OMe
1/ 6OMe
56.5
We now report the bioactivity-directed isolation of a new
sesquiterpene dialdehyde cinnamate 3 as the fungicidal com-
pound in P. axillaris. Two 6-hydroxy-derivatives 4 and 5 were
present at lower levels in the extract. A further nine derivatives
6-14 were synthesized from these natural products for a
structure-activity study against important food crop pathogens.
a In CDCl3, chemical shift in ppm.
MATERIALS AND METHODS
General Experimental Procedures. All solvents were distilled
before use and were removed by rotary evaporation at temperatures
up to 35 °C. Octadecyl-functionalized silica gel (C18) was used for
reversed-phase (RP) flash chromatography, and Merck silica gel 60,
200-400 mesh, 40-63 µm, was used for silica gel flash chromatog-
raphy. TLC was carried out using Merck DC-plastikfolien Kieselgel
* To whom correspondence should be addressed. Fax: +64 3 4798543.
E-mail: perryn@crop.cri.nz.
† Crop & Food Research.
‡ Dow AgroSciences.
10.1021/jf052585s CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/24/2005

Fungicidal Sesquiterpene Dialdehyde Cinnamates
J. Agric. Food Chem., Vol. 54, No. 2, 2006 469
60 F254, first visualized with a UV lamp, and then by dipping in a
vanillin solution (1% vanillin, 1% H2SO4 in EtOH), and heating. Optical
rotations were measured on a Perkin-Elmer 241 polarimeter. Mass, UV,
and IR spectra were recorded on Kratos MS-80, Shimadzu UV 240,
and Perkin-Elmer 1600 FTIR instruments, respectively. NMR spectra
of CDCl3 solutions at 25 °C were recorded at 500 or 300 MHz for 1H
and at 125 or 75 MHz for 13C on Varian INOVA-500 or -300
spectrometers. Chemical shifts are given in ppm on the δ-scale
referenced to solvent peaks: CHCl3 at 7.25 and CDCl3 at 77.0.
Conformational searching and molecular modeling methods are de-
scribed by Hinkley et al. (9).
Collection and Extraction. P. axillaris twigs and leaves were
collected from the West coast of the South Island, New Zealand, in
January 1997 (collection code 970128-27) and from the Nelson region
in March 1999 (990327-01). Voucher specimens are lodged in the Plant
Extracts Research Unit collection, Crop & Food Research.
Antifungal Assays. Initial biological activity was detected with the
Phytophthora infestans (Bayer code PHYTIN, late tomato blight) in
vitro test (10), which was also used to evaluate chromatographic
fractions. For this assay, cultures of PHYTIN were maintained on rye
seed broth. Assays for growth inhibition were conducted using liquid
minimal media. Conidia were harvested by flooding a culture plate
with assay medium and scraping with a sterile cell lifter. The conidial
suspension was then filtered through two layers of sterile cheesecloth
and sterile glass wool to remove hyphal fragments that may be present.
The conidial suspensions were then adjusted to 3 × 105 conidia/mL
using a hemocytometer. Samples were tested for inhibition of fungal
growth in 96-well polystyrene microtiter plates, after dilution to desired
concentrations in DMSO so that all wells received 2.5 µL of DMSO.
Plates were inoculated by adding 200 µL of the conidial suspension.
The incubation time was 72 h. Plates were evaluated for percent
inhibition of fungal growth using a nephelometer (Nephelostar Galaxy,
BMG Labtechnologies, Offenburg, Germany).
Samples were also evaluated for in vivo control of whole plant fungal
infections according to Ricks et al. (11). In these assays, extracts or
compounds were dissolved in acetone, with serial dilutions then made
in acetone to obtain desired concentrations: 6.25, 25, and 100 ppm
for pure compounds (Table 3). Final treatment volumes were obtained
by adding 9 volumes of 0.05% aqueous Tween-20 or 0.01% Triton
X-100, depending upon the pathogen. For each pathogen, host plants
were grown from seed in a soilless peat-based potting mixture (Metro-
Mix) until the seedlings were 10-20-cm tall. These plants were then
sprayed to runoff with the test compound. After 24 h, the test plants
were inoculated by spraying with an aqueous sporangia suspension of
fungus and kept in a dew chamber overnight. The plants were then
transferred to the greenhouse until disease developed in the untreated
control plants. The pathogens and plants were the following: Plasmo-
para Viticola (Bayer code PLASVI, grape downy mildew) on vines
(cultivar Carignane); PHYTIN on tomatoes (cultivar Rutgers); Puccinia
graminis tritici (PUCCRT, wheat stem rust), Septoria tritici (SEPTTR,
wheat leaf spot), and Septoria nodorum (LEPTNO, wheat glume blotch)
on wheat (cultivar Yuma); and Erysiphe graminis tritici (ERYSGT,
wheat powdery mildew) on wheat (cultivar Monon). Disease incidence
was measured relative to untreated control plants at 24-h post
inoculation and, in some tests, at 96 h (Table 3). The C. albicans assay
has been described previously (12).
a
Bioassay-Directed Isolation of Paxidal (3). Foliage (101 g) was
blended with EtOH (1.4 L), then filtered and evaporated to give a green
solid (11.3 g). This crude extract exhibited 100% mean disease control
of PHYTIN in vivo at 1000 ppm. A sample of the crude extract (4.1
g) was dried onto C18 silica gel (8 g) and loaded onto a C18 column
(40 g). Then, 44 fractions were eluted with 21-mL volumes of H2O/
MeCN (1:0, 9:1, 3:1, 1:1, 1:3, 1:9, and 0:1), 1:1 MeCN/CHCl3, and
CHCl3. These were combined on the basis of similarity by TLC.
Fractions eluted with H2O/MeCN (9:1 and 3:1) exhibited 100% killing
of PHYTIN in vitro at 500 ppm.
One active fraction (0.34 g) eluted with H2O/MeCN (9:1) was dried
onto silica gel (0.7 g) and loaded onto a silica gel column (35 g). Then,
40 column fractions were eluted with 30-mL volumes of cyclohexane/
EtOAc (100-25% cyclohexane, in 5% steps). These fractions were
combined on the basis of TLC, and fractions eluted with 70%
a

470 J. Agric. Food Chem., Vol. 54, No. 2, 2006
Brennan et al.
Silica gel chromatography of the crude hydroxy paxidals with 10-
100% EtOAc in CH2Cl2 gave initially pure 6â-hydroxypaxidal (4, 177
mg) as a pale yellow gum: [R]28D -2°, [R]28577nm -10°, [R]28546nm -20°,
[R]28435nm -90°, [R]28405nm -74° (c 0.2, CHCl3); UV (MeOH) λmax (ꢀ)
279 (17 360) nm; IR (film) νmax 3464, 2960, 1708, 1686, 1636, 1173
Table 3. Antifungal Activity of Paxidal (3) and Related Compounds
4−
14 against Crop Diseases in Vivoa
compound dose (ppm) LEPTNO PHYTI N PLASVI PUCCRT SEPTTR
3
3
100
25
17/18
0/0
69/9
57/18
51/0
23/0
0
0
7
0
1
cm-1; H and 13C NMR in Tables 1 and 2; EIMS m/z 394.1769 (M+,
0.1, calcd for C24H26O5, 394.1780), 376.1681 (M+-H2O, 0.4, calcd for
C24H24O4, 376.1675), 131 (100).
4
4
4
100
25
6.25
73
0
0
100/3
82/7
72/0
76/86
28/0
0/0
77/61
8/4
0/0
0
0
0
This was followed by pure 6R-hydroxypaxidal (5, 72 mg) as a pale
yellow gum: [R]28D +58°, [R]28577nm +50°, [R]28546nm +47°, [R]28
435nm
+19°, [R]28
-21° (c 0.2, MeOH); UV (MeOH) λmax (ꢀ) 278
405nm
5
5
5
100
25
6.25
96
73
33
100
91
74
89
19
2
85
15
4
0
0
0
(13 880) nm; IR (film) νmax 3444, 2960, 2870, 1710, 1686, 1636, 1171
1
cm-1; H and 13C NMR in Tables 1 and 2; EIMS m/z 376.1682 (M+-
H2O, 0.3, calcd for C24H24O4, 376.1675), 131 (100).
Synthetic Derivatives. Diol (14). NaBH4 (20 mg) was added to a
solution of paxidal (3) (50 mg) in absolute EtOH (2 mL) and left for
1 h. The solution was concentrated, then 10% HCl (2 mL) was added,
and resulting solution was extracted with EtOAc. The organic phase
was washed, dried, and concentrated to give the crude diol. Column
chromatography over silica gel, eluting with 10-100% EtOAc in CH2-
6
6
6
100
25
6.25
60
0
0
28
36
19
24
4
7
0
0
0
0
0
0
7
7
7
100
25
6.25
20
20
20
98
67
3
70
33
9
21
0
0
0
0
0
Cl2 gave the pure diol (14, 24 mg, 48%) as a colorless gum: [R]28
D
-16°, [R]28577nm -1°, [R]28546nm -3°, [R]28435nm -14°, [R]28405nm -17°
(c 0.2, CHCl3); UV (MeOH) λmax (ꢀ) 280 (34 380) nm; IR (film) νmax
3356, 2928, 1838, 1708, 1637, 1174 cm-1; 1H and 13C NMR in Tables
1 and 2; EIMS m/z 364.2032 (M+-H2O, 1, calcd for C24H28O3 364.2038),
362.1873 (M+-H2O-H2, 3, calcd for C24H26O3 362.1882), 131 (100).
Paxidal Dimethyl Acetal (8). A solution of paxidal (3) (150 mg) in
MeOH (5 mL) was run through a column (20 × 20 mm) of DOWEX
50W×8 (100-200 mesh, H+) ion-exchange resin (prewashed with
MeOH) to give the crude dimethyl acetal (172 mg). A sub-sample (50
mg) was chromatographed over silica gel eluted with 5% EtOAc in
CH2Cl2 to give the pure acetal (8, 38 mg, 78%) as a colorless gum:
8
8
8
100
25
6.25
20
0
0
99/14
78/6
47/0
74/57
61/23
7/0
71/19
4/0
4/0
0
0
0
9
9
9
100
25
6.25
0
0
0
0
0
0
0
0
0
7
4
0
78
33
43
10
10
10
100
25
6.25
0
0
0
0
0
0
0
0
0
9
9
42
90
17
0
[R]28D -9°, [R]28577nm -24°, [R]28546nm -38°, [R]28435nm -145°, [R]28
11b
12b
13b
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
11
0
0
405nm
-171° (c 0.2, CHCl3); UV (MeOH) λmax (ꢀ) 280 (13,220) nm; IR (film)
1
νmax 2928, 1711, 1639, 1174 cm-1; H and 13C NMR in Tables 1 and
2; EIMS m/z 392.2001 (M+-CH3OH, 3, calcd for C25H28O4, 392.1983),
197 (100).
14
14
14
100
25
6.25
0
0
0
58
8
-6
70
22
4
0
0
0
0
0
0
1â-Hydroxydial (6). NaOH (0.5 g) in H2O (2 mL) was added to a
solution of paxidal dimethyl acetal (8) (172 mg) in DMSO (4 mL),
and the mixture was kept at 55 °C for 20 h. After cooling, the mixture
was diluted with H2O (50 mL) and extracted with EtOAc (3 × 100
mL). The organic phase was washed with H2O (50 mL), dried, and
concentrated to give the crude 1-â-hydroxydial dimethyl acetal (83 mg).
This was then dissolved in acetone (4 mL) and H2O (1 mL),
toluenesulfonic acid (10 mg) was added, and the mixture was left at
RT for 20 min. Saturated aqueous NaHCO3 (20 mL) was added, and
the solution was extracted with EtOAc (3 × 20 mL). The organic phase
was washed with H2O (20 mL), dried, and concentrated to give the
crude dial. Column chromatography on silica gel eluting with 5-100%
EtOAc in CH2Cl2 gave the pure 1â-hydroxydial (6, 38 mg, 34% from
a Results are % disease inhibition after 24 h, except for some 24 h/96 h results.
LEPTNO
infestans
mildew; PUCCRT
Septoria tritici
against Erysiphe graminis tritici
b No activity at lower levels.
)
)
Septoria nodorum
late tomato blight; PLASVI
)
glume blotch on wheat; PHYTIN
)
)
Phytophthora
grape downy
)
Plasmopara viticola
)
Puccinia graminis tritici ) wheat stem rust; SEPTTR )
)
leaf spot on wheat. None of these compounds showed any activity
) powdery mildew on wheat, at 100 ppm or lower.
cyclohexane showed 100% killing of PHYTIN in vitro at 500 ppm.
These active fractions were subjected to preparative RPLC (Lichrospher
100 RP-18 column, H2O/MeCN 1:9 at 3 mL/min) to give paxidal (3,
20 mg), characterized below.
8) as a colorless oil: [R]28D 25°, [R]28577nm 11°, [R]28546nm 9°, [R]28
435nm
-26°, [R]28405nm -4° (c 0.2, CHCl3); UV (MeOH) λmax (ꢀ) 227 (13 980)
Improved Isolation of Paxidal (3) plus 6-Hydroxypaxidals (4)
and (5). Dried P. axillaris foliage (50 g) was crushed and shaken with
CHCl3 (200 mL) at room temperature overnight, then filtered, and the
solvent evaporated to give a green gum (4.65 g). Flash vacuum
chromatography on silica gel 60H started with CH2Cl2, then elution
with 5 and 10% EtOAc in CH2Cl2 gave crude paxidal (1.5 g), and
20-100% EtOAc in CH2Cl2 gave crude mixed hydroxy paxidals (400
mg). To remove the chlorophylls and carotenoids from these fractions,
MeCN solutions were passed through short columns of RP C-18, to
give the crude products as pale yellow oils. Column chromatography
over silica gel on the crude paxidal fraction eluting with 5% EtOAc in
CH2Cl2 gave paxidal mixed 10:1 with an unidentified dialdehyde as a
pale yellow oil (0.96 g). Recrystallization from methanol gave pure
paxidal (3) as an off-white crystalline solid: mp 120 °C (MeOH);
analysis found C 76.33, H 7.05, C24H26O4 requires C 76.19, H 6.88;
1
nm; IR (film) νmax 3460, 2872, 1714, 1682, 1644 cm-1; H and 13C
NMR in Tables 1 and 2; EIMS m/z 248.1410 (M+, 1, calcd for
C15H20O3, 248.1412), 91 (100).
1â-Methoxydial (7). A solution of 1â-hydroxydial (6) (35 mg) in
MeI (5 mL) containing silver oxide (10 mg) and 4-Å molecular sieves
(10 mg) under N2 was sonicated for 15 s and then refluxed for 2 h.
Filtration and evaporation gave a mixture of an acetal and the required
methyl ether, which was separated by column chromatography on silica
gel eluting with 0-20% EtOAc in CH2Cl2 to give 1â-methoxydial (7,
5 mg, 14%) as a colorless oil: [R]28 64°, [R]28
57°, [R]28
D
577nm
546nm
56°, [R]28435nm 53°, [R]28405nm 98° (c 0.2, CHCl3); UV (MeOH) λmax (ꢀ)
225 (14 400), 280 (3900) nm; IR (film) νmax 2934, 1715, 1682, 1096
1
cm-1; H and 13C NMR in Tables 1 and 2; EIMS m/z 262.1576 (M+,
5, calcd for C16H22O3 262.1569), 91 (100). This was followed by
recovered compound 6 (15 mg).
6R-Hydroxypaxidal Dimethyl Acetal (9). A solution of 6R-hydroxy-
paxidal (5) (48 mg) in MeOH (5 mL) was run through a column (20
× 20 mm) of DOWEX 50W×8 (100-200 mesh, H+) ion-exchange
resin (pre-washed with MeOH) to give the crude dimethyl acetal.
[R]28 +11°, [R]28
+0.3°, [R]28
-10°, [R]28
-81°,
D
577nm
546nm
435nm
[R]28
-74° (c 0.2, MeOH); UV (MeOH) λmax (ꢀ) 280 (18 270)
405nm
nm; IR (film) νmax 1705, 1680, 1640, 1170 cm-1; 1H and 13C NMR in
Tables 1 and 2; EIMS m/z 378 (M+, 2), 350 (22), 247 (22), 230 (42),
202 (55), 187 (19), 173 (24), 131 (100), 103 (76), 91 (30), 77 (45).

Fungicidal Sesquiterpene Dialdehyde Cinnamates
J. Agric. Food Chem., Vol. 54, No. 2, 2006 471
Purification by column chromatography over silica gel eluting with
increasing proportions of EtOAc in CH2Cl2 gave the pure 6R-
hydroxypaxidal dimethyl acetal (9, 41 mg, 80%) as a colorless oil:
[R]28D 31°, [R]28577nm 22°, [R]28546nm 14°, [R]28435nm -57°, [R]28405nm -69°
(c 0.2, CHCl3); UV (MeOH) λmax (ꢀ) 280 (18 040) nm; IR (film) νmax
3450, 2923, 1705, 1640, 1177 cm-1; 1H and 13C NMR in Tables 1 and
2; EIMS m/z 408.1939 (M+-CH3OH, 3, calcd for C25H28O5 408.1937),
131 (100).
Acetal (11). To a solution of the acetal (9) (32 mg) in DMSO (0.6
mL), aqueous NaOH (25%, 0.3 mL) was added, and the mixture was
left at 55 °C for 20 h. After cooling, the reaction mixture was diluted
with H2O (30 mL) and extracted with EtOAc (3 × 50 mL). The organic
phase was washed with H2O (30 mL), dried, and concentrated to give
the acetal (11, 19 mg, 60%) as a colorless oil: [R]28 112°, [R]28
D
577nm
107°, [R]28
110°, [R]28
107°, [R]28
133° (c 0.2, CHCl3);
405nm
UV (MeOH) λmax (ꢀ) 270 (2010) nm; IR (film) νmax 3460, 2896, 971
546nm
435nm
1
cm-1; H and 13C NMR in Tables 1 and 2; EIMS m/z 279.1603 (M+-
Figure 1. Predicted most stable conformation of paxidal (3) from molecular
modeling, showing key NOE interactions (Me14, Me15, and phenyl protons
omitted for clarity)
OCH3, 33, calcd for C16H23O4, 279.1596), 200 (100).
Acetal (13). A solution of acetal (11) (19 mg) in MeI (5 mL)
containing silver oxide (10 mg) and 4-Å molecular sieves (10 mg) under
N2 was sonicated for 15 s and then refluxed for 2 h. Filtration and
evaporation gave the monomethyl ether, which was purified by column
chromatography on silica gel eluting with 0-20% EtOAc in CH2Cl2
istic double bond 1H signals at 6.42 and 7.69 (both d, J 16 Hz),
and a 13C signal at 166.1 for the ester carbonyl (Tables 1 and
2). Other NMR signals (Tables 1 and 2) were very similar to
those reported for 9-deoxymuzigadial (2) (13), especially the
dialdehyde and exocyclic methylene signals. 2-D NMR studies
(Supporting Information), especially an HMBC correlation
between H1 and the ester carbonyl, showed that the cinnamate
was linked to the sesquiterpene at C1. The relative stereochem-
istry was proven by NOE interactions between axial protons
H1, H3, H5, and H9 (Figure 1). The absolute stereochemistry
was assumed to be the same as that for polygodial (1) and
9-deoxymuzigadial (2) in P. colorata (7, 14), giving the
proposed structure 3, a new compound for which we propose
the name paxidal.
to give acetal (13, 15 mg, 70%) as a colorless oil: [R]28D 187°, [R]28
577nm
186°, [R]28
199°, [R]28
265°, [R]28
327° (c 0.2, CHCl3);
405nm
546nm
435nm
UV (MeOH) λmax (ꢀ) 280 (972) nm; IR (film) νmax 3370, 1640 cm-1
;
1H and 13C NMR in Tables 1 and 2; EIMS m/z 293.1753 (M+-OCH3,
32, calcd for C17H25O4 293.1757), 45 (100).
6-Ketopaxidal (12). PCC (3 × 10 mg) was added to a solution of
the 6R-hydroxypaxidal acetal (28 mg) in dry CH2Cl2 (3 mL) over 30
min. The solution was put down a short column of silica, and 10%
EtOAc in CH2Cl2 eluted the pure ketone (12, 17 mg, 61%) as a colorless
oil: [R]28D 97°, [R]28577nm 89°, [R]28546nm 86°, [R]28435nm 17°, [R]28
405nm
-92° (c 0.2, CHCl3); UV (MeOH) λmax (ꢀ) 280 (25 186) nm; IR (film)
1
νmax 3370, 1705, 1673, 1640 cm-1; H and 13C NMR in Tables 1 and
2; EIMS m/z 438.2034 (M+, 1, calcd for C26H30O6 438.2034), 131 (100).
Dihydrocinnamate (10). A solution of the acetal (9) (17 mg) in
MeOH (4 mL) was stirred with Pd on C (5%, 5 mg) under an
atmosphere of H2 for 2 h. The suspension was filtered through Celite
and evaporated to give the pure dihydrocinnamate (10, 16 mg, 97%)
The two other compounds isolated from P. axillaris had NMR
spectra very similar (Tables 1 and 2) to that of paxidal (3), and
MS showed them both to have one more oxygen than 3. Both
new compounds showed oxygenated methine signals (4.81/4.76
ppm) coupled to H5 and H7 (Table 2 and Supporting Informa-
tion), indicating a 6-CHOH group. The minor compound showed
a diaxial coupling of 9.5 Hz between H6 and H5 which, together
with NOE interactions between the H6 proton and Me15,
showed this compound to be the 6R-hydroxypaxidal (5). The
other compound showed only a weak H6-H5 coupling, and
an NOE interaction between the H6 and one H13, so this is
6â-hydroxypaxidal (4).
Compounds 3-5 are new examples of the rare muzigadial
skeleton and the first with oxygenation at C1 or C6 (8). The
only examples of drimane dialdehydes oxygenated at C1 are
1â-p-coumaroyloxypolygodial (15) and a few simple derivatives,
isolated from three different Drimys species (also in the family
Winteraceae) (15-18). The NMR signals for H1 and C1 of 15
(18) were similar to those for paxidal (3, Tables 1 and 2).
Drimane dialdehydes oxygenated at C6 are more common, but
none oxygenated at both C1 and C6 have been reported
previously (8).
as a colorless oil: [R]28 82°, [R]28
75°, [R]28
73°, [R]28
435nm
1
D
577nm
546nm
50°, [R]28
72° (c 0.2, CHCl3); UV (MeOH) end adsorption; H
405nm
and 13C NMR in Tables 1 and 2; EIMS m/z 440.2203 (M+-H2, 1, calcd
for C26H32O6 440.2199), 91 (100).
RESULTS AND DISCUSSION
New Natural Products. An ethanol extract of P. axillaris
foliage showed in vitro activity against Phytophthora infestans
(late blight of tomatoes and potatoes, PHYTIN) in high
throughput screening. This activity was confirmed as interesting
when the extract exhibited 100% control of PHYTIN in vivo
at 1000 ppm. The in vitro PHYTIN assay was used to direct
the isolation of the fungicidal compounds. Reversed-phase (RP)
C18 column chromatography followed by silica gel column
chromatography gave an active fraction, which was subjected
to preparative RPLC to give the main active compound 3. This
compound was of medium-to-low polarity, and it was found
that a chloroform extract of foliage gave a better yield of 3
(1.9 wt % of dry foliage). This also gave two related compounds,
4 (0.35%) and 5 (0.14%), showing even greater activity against
PHYTIN than 3 (see below).
The structure of 3 was proven by a combination of NMR
experiments. The 13C NMR spectrum (Table 1) showed 22
carbon signals, whereas the mass spectrum and elemental
composition suggested the molecular formula C24H26O4. This
deficit was accounted for by the presence of two pairs of
equivalent carbons in a trans cinnamate group, shown by the
aromatic signals of a monosubstituted benzene ring, character-
We have found that dialdehydes 1 and 2 are quite unstable
on storage, as have others (19), but compounds 3-5 were stable
under the same conditions. This may be due to steric congestion
around the C11 aldehyde, shown by molecular modeling (Figure
1), which restricts access to this reactive center. There is a
change in the average conformation about the C9 to C11 bond,
since the H9 to H11 couplings in compounds 1 and 2 are about
4.5 Hz (7, 13), whereas the corresponding couplings in 3-5
are around 2 Hz (Table 2). The stability of these compounds

472 J. Agric. Food Chem., Vol. 54, No. 2, 2006
Brennan et al.
features, especially H1 appearing at higher field than in the
cinnamate esters. Methylation of the highly hindered alcohol
(6) was achieved, albeit in a low yield, to give the methoxy
ether (7). Unfortunately all attempts to reduce the conjugated
7-8 double bond of paxidal (3) proved unsuccessful. Catalytic
hydrogenation gave complex mixtures with the aldehyde groups
and the side-chain double bond reduced, but with the 7-8
double bond intact. Dissolving metal reductions, and other 1,4-
reducing agents, also gave complex mixtures.
The next series of derivatives were formed from the less
abundant but more active (see below) 6R-hydroxypaxidal (5).
First the dimethyl acetal 9 was formed using the same
ion-exchange resin conditions as before. Hydrolysis of the
cinnamate group by the same method as before gave the diol
11. Methylation led to a single monomethyl product, which was
shown to be the 6-methoxy product (13) by an HMBC
correlation between C6 and one methoxy signal, plus the
downfield shift of the C6 signal (Table 1). This regioselectivity
was explained by the much less hindered environment of C6.
Other attempts to differentiate between C1 and C6 led to the
formation of the 6-keto product (12), but this proved unstable
to the strongly basic conditions needed to hydrolyze the
cinnamate ester to take the route further. Variation in the
cinnamate group was achieved by catalytic hydrogenation of
dimethyl acetal 9 to give the dihydrocinnamate (10), with no
sign of reduction of the 7-8 double bond.
Fungicidal Activities. The natural products 3-5 and syn-
thetic derivatives 6-14 were tested in vivo against some
important food crop pathogens (Table 3). In addition to the
activity of paxidal (3) against PHYTIN (late blight of tomatoes
and potatoes), which was used to direct its isolation, compound
3 was also active against Plasmopara Viticola (PLASVI, grape
downy mildew). However, inhibition of both of these diseases
fell off after 4 days (Table 3).
Figure 2.
3-5 and their different fungicidal activities (see below) encour-
aged us to synthesize derivatives of the natural products for
testing.
Synthesis of Derivatives. The first set of derivatives was
based on the most abundant paxidal (3), probing the importance
of the obvious functional groups, that is, the dialdehyde and
cinnamate moieties. Reduction of the dialdehyde 3 to the diol
14 was readily achieved with sodium borohydride. Compound
14 showed NMR signals (Tables 1 and 2) around the diol
moiety similar to the corresponding signals in the naturally
occurring diol derivative of polygodial (20).
This initial result prompted us to prepare and test synthetic
derivatives of paxidal (3), plus the co-occurring natural products.
The natural 6-hydroxy compounds 4 and 5 were more active
against PHYTIN and showed a broader spectrum of activity,
inhibiting Septoria nodorum (LEPTNO, glume blotch on wheat)
and Puccinia graminis tritici (PUCCRT, wheat stem rust, Table
3). The synthetic paxidal derivatives 6 and 14 also showed
interesting antifungal activities (Table 3), so we synthesized
derivatives of 6R-hydroxypaxidal (5), the most active natural
product.
The dialdehyde functionality of paxidal (3) was converted
to a dimethyl acetal using ion-exchange resin. Only one major
diastereoisomer 8 was formed, although four are possible. The
same treatment of polygodial (1) has been found to give two
dimethyl acetals, with the major product having both methoxy
groups R, and the minor product having 11R and 12â (19). The
paxidal dimethyl acetal 8, and the other dimethyl acetals 9-13,
all showed NOE interactions between H11 and Me15, suggest-
ing 11R methoxy groups. No NOE interaction was observed
between H11 and H12, but molecular modeling suggested that
these protons would be separated by over 3.5 Å for either
geometry at C12. We propose 12R methoxy groups in dimethyl
Surprisingly, even though paxidal dimethyl acetal (8) had
shown a potency and spectrum of activity similar to that of 6R-
hydroxypaxidal (5), 6R-hydroxypaxidal dimethyl acetal (9)
showed a different spectrum of activity (Table 3). Compound
9, and its dihydrocinnamate analogue 10, were inactive against
PHYTIN, LEPTNO, and PUCCRT, but were active against
Septoria tritici (SEPTTR, leaf spot on wheat). This change in
selectivity may be related to the greater stability of 6R-
hydroxypaxidal dimethyl acetal (9) to mild acid hydrolysis,
compared to paxidal dimethyl acetal (8). This was shown by
the greater stability of 6R-hydroxypaxidal dimethyl acetal (9)
in deuteriochloroform, compared to paxidal dimethyl acetal (8).
1
acetals 8-13, on the basis of the similarity of our H NMR
data (Table 2) to that reported for the corresponding polygodial
11R,12R-dimethyl acetal (19). We found that even the small
amount of acid present in some samples of deuteriochloroform
was enough to cause hydrolysis of the acetal 8 back to the free
dialdehyde.
Considering the potencies of the antifungal activities overall
(Table 3), some structure-activity relationships can be sug-
gested. Substitution and orientation at C6 is important, with
antifungal potencies in the following order: 6R-hydroxy 5 >
6â-hydroxy 4 > 6-deoxy 3; and 6R-hydroxy 9 > 6-keto 12. A
bulky and/or low polarity substituent at C1 is also important:
methoxy 7 > cinnamate 3 > hydroxy 6; and cinnamate 9 ≈
dihydrocinnamate 10 > hydroxy 11. The free dialdehyde is not
Protection of the dialdehyde in dimethyl acetal 8 allowed
removal of the highly hindered cinnamate ester by hydrolysis
under the relatively drastic conditions of heating a dimethyl-
sulfoxide solution with 25% potassium hydroxide to 55 °C
overnight. This gave the 1â-hydroxy acetal, which was depro-
tected by mild acid hydrolysis to give the 1â-hydroxydial (6).
The NMR spectra of 6 (Tables 1 and 2) showed the expected

Fungicidal Sesquiterpene Dialdehyde Cinnamates
J. Agric. Food Chem., Vol. 54, No. 2, 2006 473
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essential for antifungal activity: dimethyl acetal 8 > dialdehyde
3 > diol 14.
This last finding was surprising, since the general biological
activity of polygodial (1) is ascribed to the unsaturated dialde-
hyde functionality and its reactivity toward biological nucleo-
philes (8). Given the sensitivity of the acetal 8 to acid-catalyzed
hydrolysis (see above), the activity of 8 may be due to a sort of
controlled release of 3, that is, 8 is a pro-drug, itself not very
active but slowly releasing the potent 3.
Sesquiterpene dialdehydes such as 1 are especially active
against the yeasts C. albicans and Saccharomyces cereVisiae.
However, cinnamate-substituted sesquiterpene dialdehydes 3-5
showed no activity against C. albicans in a disk diffusion assay
in which compounds 1 and 2 showed strong activity (all at 60
µg/ disk). On the other hand, compounds 1 and 2 are not active
against PHYTIN, since an extract of P. colorata rich in these
compounds (6) was not active against this plant pathogen. There
do not seem to be any other reports of sesquiterpene dialdehydes
being active against the important food crop pathogens studied
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ACKNOWLEDGMENT
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and insecticidal activity of compounds from Pseudowintera
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We thank A. Evans for plant collections and identifications;
Timberlands West Coast Ltd. and P. Butler for permission to
collect; W. Redmond and M. Thomas for NMR assistance; B.
Clark for MS; G. Ellis for C. albicans assays.
Supporting Information Available: Tables of 2D NMR
correlation data for compounds 3-5. This material is available
free of charge via the Internet at http://pubs.acs.org.
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