Structure-activity relationship of α hormones, the mating factors of phytopathogen Phytophthora

Article abstract of DOI:10.1016/j.bmc.2011.12.015

The mating hormones α1 and α2 induce sexual reproduction of the phytopathogenic genus Phytophthora. To demonstrate the structural elements responsible to hormonal activity, 17 derivatives of α1 and α2 were synthesized and their hormonal activity (oospore-

Full text of DOI:10.1016/j.bmc.2011.12.015

Bioorganic & Medicinal Chemistry 20 (2012) 681–686
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of
of phytopathogen Phytophthora
a hormones, the mating factors
Shylaja D. Molli a, Jianhua Qi a,b, Arata Yajima c, Keisuke Shikai c, Tadashi Imaoka c, Tomoo Nukada c,
Goro Yabuta c, Makoto Ojika a,
⇑
a Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
b College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
c Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The mating hormones
a
1 and
a
2 induce sexual reproduction of the phytopathogenic genus Phytophthora.
1 and
were synthesized and their hormonal activity (oospore-inducing activity) was evaluated. The terminal
ester derivatives of 1 (diacetate and dibenzoate) retained the hormonal activity, whereas a dicarbamate
Received 14 October 2011
Revised 8 December 2011
Accepted 9 December 2011
Available online 14 December 2011
To demonstrate the structural elements responsible to hormonal activity, 17 derivatives of
a
a2
a
derivative completely suppressed the activity. Even monocarbamates showed weak activities; among
them the 1-O-carbamate was less active than 16-O-carbamate, suggesting that the 1-OH group is a little
more important than 16-OH. Dihydro, dehydro, and demethyl derivatives exhibited the minimum level of
Keywords:
Phytophthora
Mating hormone
Sexual reproduction
Structure–activity relationship
activity. Surviving activity of 15-epi-
1, not only the terminal diacetate derivative but also monoacetates of
Among the monoacetates, 1-O-acetyl- 2 exhibited little yet relatively better activity than the others. No
activity was observed for mono- and dicarbamoyl derivatives of 2. Dihydro 2 with the saturated dou-
ble bond lost most of the activity. These findings suggest that both the mating hormones 1 and a2
a
1 suggested a less importance of this stereochemistry. Contrary to
a
a2 exhibited no or little activity.
a
a
a
a
require most of the functional (hydroxyl, keto, and olefinic) groups they possess in their natural form
for inducing the sexual reproduction of Phytophthora.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
mating type A2 and
in the A1 mating type.8,9 In 2005, Qi et al. isolated and character-
ized 1 (1) from the A1 mating type strain of Phytophthora
a2 secreted by A2 induces sexual reproduction
The species of the plant-damaging genus Phytophthora are
responsible not only for the enormous loss of agricultural crops
world-wide but also for the environmental damage in natural eco-
systems due to the use of numerous pesticides. One of the
catastrophic moments was the Great Irish Famine in the mid
1840s caused by Phytophthora infestans.1 The sudden oak death,
widespread rots found in tomato, potatoes, tobacco and strawber-
ries are the other foremost diseases caused by Phytophthora.2,3
Generally the heterothallic members of Phytophthora require
both mating types (A1 and A2) for sexual reproduction.4 Ashby,
in 1929, proposed the occurrence of hormone-like compounds
which regulates the sexual reproduction.5 A hormone moves from
one thallus to the other to stimulate the production of oogonia and
antheridia; thus each thallus is bisexual.6 The mating of these two
organs results in the formation of sexual spores known as oosp-
a
nicotianae.10 Yajima et al. described the total synthesis and abso-
lute configuration of this hormone in 2008.11 Recently,
a2 (2)
was also isolated and structurally elucidated.12
Mating hormones a1 (1) and a2 (2) are acyclic oxygenated diter-
penes, with four or three stereogenic centers. The present study
focuses on the relationship between the hormonal activity, that is,
oospore-inducing activity, and chemical structure of the hormones.
Nine derivatives of 1 and eight derivatives of 2 were synthesized
and tested for their ability to induce sexual reproduction in A2
and A1 strains of P. nicotianae. This will help to determine the func-
tional groups responsible for evoking the target biological effect on
the organism, and may help to identify the specific receptors for
these hormones.
ores.7 These hormones were termed as
a1 and a2. The a1 hormone
secreted by A1 mating type strains induces oospores in the counter
HO
1
OH
3
7
11
HO
16
⇑
Corresponding author. Tel./fax: +81 52 789 4284.
E-mail address: ojika@agr.nagoya-u.ac.jp (M. Ojika).
O
α1 (1)
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.015

682
S. D. Molli et al. / Bioorg. Med. Chem. 20 (2012) 681–686
2. K2CO3, MeOH). The 1H NMR spectrum indicates that 9 is a mixture
of three alkenes in the ratio of 1:1:1. The total synthesis of 15-epi-
(10) was performed as summarized in Scheme 1 (Supplementary
data for details). In addition, the total synthesis of 3-demethyl-
(11) was summarized in Scheme 2 (Supplementary data for details).
HO
a
1
OH
HO
α2 (2)
a
1
2. Results and discussion
2.1. Preparation of 1 derivatives
Nine 1 derivatives 3–11 were prepared in the present study
(Fig. 1). Diacetate 3 and dibenzoate 4 were prepared with acylation
reagents under the usual conditions. Two monocarbamates, 5 and
6, and dicarbamate 7 were obtained by the treatment with
p-bromophenylisocyanate followed by chromatographic separa-
tion. Sodium borohydride reduction of the keto group of 1 afforded
2.2. Preparation of a2 derivatives
a
Eight
the important structural elements for the hormonal activity
against the A1 mating type of P. nicotianae. Two monoacetyl- 2s
(12 and 13) and diacetyl- 2 (14) were prepared by partial acetyla-
tion followed by chromatographic separation. 11-O-acetyl- 2 (15)
was prepared by the acetylation of disilyl-
2 (26)13 (Scheme 3).
Two monocarbamoyl- 2s (16 and 17) and dicarbamoyl- 2 (18)
were also obtained in a similar manner to that for preparing three
carbamoyl- 1s (5–7). Catalytic hydrogenation of 2 (2) on Rh-
Al2O3 afforded dihydro- 2 (19).
a2 derivatives 12–19 (Fig. 2) were also prepared to obtain
a
a
a
a
a
a
a
dihydro-a1 (8) as a diastereomeric mixture. Dehydrated derivative
9 was obtained from 3 by a series of reactions (1. SOCl2, pyridine;
a
a
a
2.3. Structure–activity relationship
HO
OR2
Thestructure–activity relationshipof the a1 and a2 derivativesis
R1O
3
15
7
11
discussed based on their oospores-inducing competence in A2 and
A1 mating type strains of P. nicotianae, respectively. Natural 1 (1)
or 2 (2), which induced oospores even at a dose of 3 ng per disk,
O
a
3: R1 = R2 = Ac
4: R1 = R2 = p-BrC6H4CO
5: R1 = p-BrC6H4NHCO, R2 = H
6: R1 = H, R2 = p-BrC6H4NHCO
7: R1 = R2 = p-BrC6H4NHCO
a
was used as control in all the experiments. The activity was de-
scribed in the relative values to control (100%). For instance, 10%
activity defines that a 10-fold dosage of the derivative is required
to show the same oospore-inducing activity of
a1 (1) or a2 (2).
The relative hormonal activity of the 1 derivatives 3–11 is
a
HO
shown in Figure 3a. From the results, it is clear that the substitution
of both terminal hydroxyls of 1 with acetyl (3) and benzoyl (4)
groups retained the hormonal (oospore inducing) activity. Com-
pared to all the others assayed, these derivatives exhibited relatively
high activities of approximately 50% and 60% for 3 and 4, respec-
tively. However, no activity was observed when both the terminal
hydroxyls of 1 were replaced with the carbamoyl groups (7). This re-
OH
OH
HO
HO
8
OH
O
9
sult suggests that the ester groups of 3 and 4 were hydrolyzed to a1
(1) or to another active form in the bioassay medium. One possibility
is the presence of non-specific esterase enzymes secreted by the
organism during the bioassay. However, another possibility must
HO
OH
OH
HO
HO
15
10
be discussed because all acetates (12–15) of
as described later (Fig. 3b). This could be explained by the formation
of an active derivative such as 16-O-acetyl(or benzoyl)- 1 possess-
ing a free hydroxyl group at C-1 because, among monocarbamates
(5 and 6), 16-O-carbamoyl- 1 (6) partially retained an activity of
a2 were almost inactive
O
O
HO
a
3
a
11
13% (Fig. 3a). The 1-O-acyl group of 3 and 4 might be more suscepti-
ble to hydrolysis than the 16-O-acyl group due to the participation of
Figure 1. Structures of
a1 derivatives.
OTBS
Br
OTBS
20
(S)-21
TBSO
SO2Ph
OH
OBn
22
23a
OTBS
10
15
TBSO
OBn
24a
Scheme 1. Outline of the total synthesis of 15-epi-
a
1 (10).

S. D. Molli et al. / Bioorg. Med. Chem. 20 (2012) 681–686
683
22
TBSO
SO2Ph
OHC
OPMB
25
OBn
23b
(R)-21
OTBS
11
TBSO
OBn
24b
Scheme 2. Outline of the total synthesis of 3-demethyl-
a1 (11).
R3O
OR2
R1O
12: R1 = Ac, R2 = R3 = H 13: R1 = R3 = H, R2 = Ac
14: R1 = R2 = Ac, R3 = H 15: R1 = R2 = H, R3 = Ac
16: R1 = p-BrC6H4NHCO, R2 = R3 = H
17: R1 = R3 = H, R2 = p-BrC6H4NHCO
18: R1 = R2 = p-BrC6H4NHCO, R3 = H
HO
OH
HO
19
Figure 2. Structures of a2 derivatives.
OH
a, b
OTBS
15
TBSO
26
Scheme 3. Synthesis of 11-O-acetyl-a2 (15). Reagents and conditions: (a) Ac2O,
DMAP, pyridine, rt; (b) TBAF, THF, rt, 7% in two steps.
the neighboring ketone group at C-4 as shown in Scheme 4. A lower
activity of 6 possessing a free 1-OH might be due to its lower solubil-
ity than that of the hypothetical monoesters possessing a free 1-OH.
This mechanism is plausible because (a) the bioassay condition is
Figure 3. Relative hormonal activity of the derivatives of
a
hormones. (a) The
relative activity of the 1 derivatives (3–11). The values are mentioned in
a
percentage of reciprocal of the relative doses required for inducing 100 oospores.
Relative activity = 100 ꢀ [dose of 1 (or 2) required for inducing 100 oospores]/[dose
of a derivative required for inducing 100 oospores]. (b) The relative activity of the
somewhat acidic (pH 6.0), and (b) a1 (1) is actually an equilibrium
a2 derivatives (12–19).
mixture of the major acyclic form and a trace cyclic hemiacetal as
analyzed by 1H NMR; the latter readily became dominant when trea-
ted with a weakly acidic condition (Supplementary figure). There-
fore, we concluded that the hydroxyl group at C-1 position is more
effectual than 16-OH. The derivatives 8, 9, and 11 exhibited the min-
imal level of activity. It is already known that the stereochemistry of
7R,11R is essential for the hormonal activity,11 whereas the stereo-
O
HO
1
OCOR
4
R
O
O
H+
diester (3 or 4)
isomer 15-epi-
the 15R configuration is not very important. The 3R stereochemistry
is also not important because natural 1 (a mixture of 3R/S isomers)
showed a comparable activity to synthetic 1 with the 3R stereo-
chemistry.11 Hence, we conclude thatthe principle features required
for the oospores-inducing activity of 1 (1) are: (a) two hydroxyl
a1 (10), retained an activity of 85%, suggesting that
a
HO
active form
a
O
R
OH
O
H2O
O
OH
O
a
groups at C-1 and C-11, (b) the ketone group at C-4, and (c) the
methyl group at C-3, as well as the stereochemistry at C-7, C-11
positions.11
Scheme 4. A plausible mechanism of the conversion of diacyl-
a
1 to an active form
during the bioassay. The left terminal structure of the product is the same as that of
natural 1 (1). The equilibration between a hemiacetal and an acyclic structure is
also observed in 1 (1) itself.
a
The relative oospore-inducing activity of the eight
a2 deriva-
a
tives 12–19 is shown in Figure 3b. Contrary to diacetyl-
a1 (3), dia-
cetyl-
were ineffective and induced only a few oospores (3% and 0.2%,
respectively). No activity was observed for 11-O-acetyl- 2 (15),
a
2 (14) showed no activity. Even the monoacetates 12 and 13
(16 and 17) produced any oospores. Very less activity (0.4%) was
observed when the double bond at C-2 position (19) was saturated.
These result indicated that (a) all the three hydroxyl groups of a2
a
indicating 11-OH group is the most important among the three
(2) play a crucial role in the hormonal activity, and (b) the double
hydroxyl groups. Neither dicarbamate 18 nor mono carbamates
bond at C-2 is also essential.

684
S. D. Molli et al. / Bioorg. Med. Chem. 20 (2012) 681–686
with hexane-EtOAc (1:1) to afford 3 (0.05 mg): colorless oil, 1H
3. Conclusion
NMR (CDCl3, 600 MHz): d = 4.05 (2H, m, H-1), 3.96 (1H, dd,
J = 10.8, 6.0 Hz, H-16b), 3.86 (1H, dd, J = 10.8, 7.0 Hz, H-16a), 2.65
(1H, sex, J = 7.0 Hz, H-3), 2.51 (1H, m, H-5b), 2.43 (1H, m, H-5a),
2.06 (3H, s, Ac), 2.05 (1H, m, H-2b), 2.03 (3H, s, Ac), 1.80 (1H, m,
H-15), 1.55–1.65 (2H, m, H-2a, 6b), 1.25–1.45 (12H, m), 1.10–1.18
(2H, m, H-14a, 8a), 1.16 (3H, s, H-18), 1.11 (3H, d, J = 6.6 Hz, H-
20), 0.93 (3H, d, J = 7.0 Hz, H-17), 0.88 (3H, d, J = 6.6 Hz, H-19);
HRMS (ESI): calcd for C24H44O6Na ([M+Na]+) 451.3030, found
451.3018.
Our results about the structure-activity relationship of
mones clearly demonstrate that all the functional groups of
mones other than the 3-C and 15-C stereochemistry of a1 (1) play
a crucial role in the hormonal activity and the modification of the
original structures of these hormones did not show any advantage
over the natural ones. These results suggest the presence of some
specific receptors that recognize the exact chemical structure of
a
a
hor-
hor-
a1 (1) or a2 (2) and are the gate to the signal transduction cascade
leading to the Phytophthora sexual reproduction. In addition the
above findings will provide valuable information for designing effi-
cient molecular probes to identify the receptors.
4.3.2. Bromobenzoyl-
a1 (4)
Hormone 1 (1) (0.13 mg) was converted to 4 (0.24 mg) as
a
described in ref 10. 4: Colorless powder, 1H NMR (CDCl3,
600 MHz): d = 7.90 (2H, d, J = 8.4 Hz, Ar), 7.87 (2H, d, J = 8.4 Hz,
Ar), 7.58 (4H, d, J = 8.4 Hz, Ar), 4.30 (2H, t, J = 6.6 Hz, H-1), 4.21
(1H, dd, J = 10.7, 6.6 Hz, H-16b), 4.11 (1H, dd, J = 10.7, 6.0 Hz, H-
16a), 2.72 (1H, sept, J = 6.6 Hz, H-3), 2.49 (1H, m, H-5b), 2.43 (1H,
m, H-5a), 2.19 (1H, m, H-2b), 1.95 (1H, m, H-15), 1.76 (1H, m, H-
2a), 1.59 (1H, m, H-6b), 1.20–1.60 (13H, m,), 1.16 (3H, d,
J = 6.6 Hz, H-20), 1.15 (3H, s, H-18), 1.10 (1H, m, H-8a), 1.02 (3H,
d, J = 6.6 Hz, H-17), 0.83/0.84 (total 3H, d, J = 6.6 Hz, H-19); MS
(ESI): m/z 731.2/733.2/735.2 (1:2:1) ([M+Na]+); HRMS (ESI): calcd
for C34H4679Br2O6Na ([M+Na]+) 731.1553, found 731.1575.
4. Experimental procedure
4.1. Phytophthora strains and hormonal activity evaluation
The A1 (ATCC 38607) and A2 (ATCC 38606) mating type strains
of P. nicotianae were purchased from the American Type Culture
Collection (ATCC, YA, USA). The hormonal activity was evaluated
by the method described by Qi et al.10 and Ojika et al.12
4.2. Hormonal activity evaluation
4.3.3. 1-O-Carbamoyl-
dicarbamoyl- 1 (7)
a1 (5), 16-O-carbamoyl-a1 (6), and
To test a1 (1) activity, a loopful of P. nicotianae (ATCC 38606, A2
a
mating type) was inoculated onto a Petri plate (9 cm diameter) con-
taining 20% V-8 juice (Campbell Soup Company, Camden, NJ), 0.3%
CaCO3, and 2% agar and pre-incubated at 25 °C, 60% humidity for
10 days in the dark. A piece (3 ꢀ 3 ꢀ 3 mm) of the colony was then
incubated for 4 days on a Petri plate (9 cm) containing 10% V-8 juice,
0.02% CaCO3, and 2% agar. A solution of a test sample in acetonitrile
To a solution of 1 (0.5 mg) in dry pyridine (0.1 mL) was added
4-bromophenyl isocyanate (6 mg). The mixture was stirred at
room temperature for 14 h, and then quenched by stirring with a
drop of water for 10 min. The mixture was dried and suspended
in 70% MeOH. The soluble part was subjected to HPLC [Develosil
ODS-UG-5 (10 mm id ꢀ 250 mm), 70–100% MeOH, 45 min linear
gradient, 3 mL/min, detected at 244 nm] to give 5 (0.06 mg,
tR = 28.2 min) and 6 (0.07 mg, tR = 30.0 min). 5: UV (MeOH):
(30 lL) was applied to a paper disc (0.7-mm thick, 8 mm diameter;
Advantec, Tokyo, Japan), which was then dried for 30 min in vacuo
and placed on the colony at a distance of 1.5 cm from the colony
center. After incubating for three additional days, the colony around
the paper disc (1.8 cm diameter) was cut out, and the total number
of oospores in the whole area (2.5 cm2) was counted.
kmax = 244 nm (
e
20,000); 1H NMR (CDCl3, 600 MHz): d = 7.41
(2H, d, J = 8.4 Hz, Ar), 7.27 (2H, m, Ar), 6.76 and 6.81 (total 1H, br
s, NH), 4.16 (2H, m, H-1), 3.51 (1H, dd, J = 10.6, 5.7 Hz, H-16b),
3.44 (1H, dd, J = 10.6, 6.6 Hz, H-16a), 2.69 (1H, sex, J = 7.0 Hz, H-
3), 2.30–2.53 (2H, m, H-5), 2.09 (1H, m, H-2b), 1.68 (1H, m, H-
2a), 1.65 (1H, m, H-15), 1.20–1.65 (15H, m), 1.15 (3H, s, H-18),
1.14 (3H, d, J = 7.0 Hz, H-20), 1.13 (2H, m, H-8a, 14a), 0.93 (3H, d,
J = 6.7 Hz, H-17), 0.86/0.87 (total 3H, d, J = 6.2 Hz, H-19); MS
(ESI): m/z 524.2/526.2 (1:1) ([M-OH]+), 564.2/566.2 (1:1)
([M+Na]+); HRMS (ESI): calcd for C27H4479BrNO5Na ([M+Na]+)
564.2295, found 564.2326.
To evaluate a2 (2) activity, after the pre-incubation of P. nicoti-
anae (ATCC 38607, A1 mating type) under the same conditions as
those for the A2 strain, a piece of the colony was incubated for
8 days under the same conditions as those for the A1 strain, except
for adjusting the medium to pH 8. A solution of a test sample in
ethanol (5
l
L) was applied to a mixed cellulose ester membrane
m pore size, White;
filter (No. A020A013A, 13 mm diameter, 0.2
l
Advantec, Tokyo, Japan), which was then dried for 30 min and
placed on the colony at a distance of 1.5 cm from the colony center.
After incubation for 4 days, the colony around the membrane filter
(2.1 cm diameter) was cut out, and the total number of oospores
formed in the entire area was counted under a microscope. Almost
all oospores were formed within the area cut (3.5 cm2).
Three of four duplicate data for each dose (3, 10, 30, 100, and
300 ng/disk) were used for calculating the relative activity shown
inFigure 3. The doses for inducing 100 oospores (as ‘D100’) were ob-
tained from dose-oospore graphs created by linear approximation
method. Relative activity (%) was obtained by the following equa-
tion: 100 ꢀ [D100(1 or 2) sd]/[D100(derivative) sd]. The error
values ( sd) of the relative activity were calculated in the light of
propagation of errors.
6: UV (MeOH): kmax = 244 nm (
e
20,000); 1H NMR (CDCl3,
600 MHz): d = 7.41 (2H, d, J = 8.4 Hz, Ar), 7.29 (2H, br d,
J = 8.4 Hz, Ar), 6.79 (1H, br s, NH), 4.04 (1H, m, H-16b), 3.98 (1H,
m, H-16a), 3.64 (2H, m, H-1), 2.76 (1H, m, H-3), 2.49 (2H, m, H-
5), 1.93/2.01 (total 1H, m, H-2b), 1.84 (1H, m, H-15), 1.20–1.70
(16H, m), 1.20 (1H, m, H-14a), 1.16 (3H, s, H-18), 1.12 (3H, d,
J = 6.6 Hz, H-20), 1.12 (1H, m, H-8a), 0.96 (3H, d, J = 6.6 Hz, H-17),
0.87/0.88 (total 3H, d, J = 6.6 Hz, H-19); MS (ESI): m/z 524.2/526.2
(1:1) ([MꢁOH]+), 564.2/566.2 (1:1) ([M+Na]+); HRMS (ESI): calcd
for C27H4479BrNO5Na ([M+Na]+) 564.2295, found 564.2302.
Another sample of 1 (0.3 mg) was treated under the same condi-
tions to give 7 (0.24 mg, tR = 40.5 min): UV (MeOH): kmax = 244 nm
(e
40, 000), 1H NMR (CDCl3, 600 MHz): d = 7.40 (4H, d, J = 8.6 Hz, Ar),
7.29 (2H, br d, J = 8.6 Hz, Ar), 7.27 (2H, br d, J = 8.6 Hz, Ar), 6.77 (1H,
br s, NH), 6.70/6.73 (total 1H, br s, NH), 4.16 (2H, m, H-1), 4.02 (1H,
dd, J = 10.4, 6.2 Hz, H-16b), 3.98 (1H, dd, J = 10.4, 6.5 Hz, H-16a),
2.69 (1H, sex, J = 7.0 Hz, H-3), 2.30–2.53 (2H, m, H-5), 2.09 (1H, m,
H-2b), 1.84 (1H, m, H-15), 1.67 (1H, m, H-2a), 1.17–1.70 (15H, m),
1.16 (3H, s, H-18), 1.13 (3H, d, J = 7.0 Hz, H-20), 1.12 (1H, m, H-
8a), 0.96 (3H, d, J = 6.6 Hz, H-17), 0.86/0.87 (3H, d, J = 6.2 Hz,
4.3. Chemical synthesis
4.3.1. Diacetyl-
a1 (3)
Hormone 1 (1) (0.1 mg) was treated with acetic anhydride and
a
pyridine at room temperature for 1.5 h. The reaction mixture was
dried in vacuo, and the residue was chromatographed on silica gel

S. D. Molli et al. / Bioorg. Med. Chem. 20 (2012) 681–686
685
H-19); MS (ESI): m/z 721.2/723.2/725.2 (1:2:1) ([M-OH]+), 761.2/
m
max = 3386 (br), 1705, 1037, 699, 666 cmꢁ1
;
1H NMR (600 MHz,
763.2/765.2 (1:2:1) ([M+Na]+); HRMS (ESI): calcd for C34H4879Br2
N2O6Na ([M+Na]+) 761.1771, found 761.1794.
CD3OD): d = 3.52 (2H, t, J = 6.6 Hz, H-1), 3.41 (1H, dd, J = 10.6,
6.3 Hz, H-16b), 3.32 (1H, dd, J = 10.6, 6.6 Hz, H-16a), 2.54 (2H, t, J
8 6.6 Hz, H-5), 2.42–2.52 (2H, m, H-3), 1.75 (2H, quint, J = 6.6 Hz,
H-2), 1.60 (1H, m, H-6b), 1.58 (1H, m, H-15), 1.28–1.46 (12H, m),
1.13 (1H, m, H-8a), 1.12 (3H, s, H-18), 1.08 (1H, m, H-14a), 0.91
(3H, d, J = 7.0 Hz, H-17), 0.88 (3H, d, J = 6.6 Hz, H-19); 13C NMR
(150 MHz, CD3OD): d = 17.1 (C-17), 19. (C-19), 22.3 (C-9 or 13),
22.4 (C-13 or 9), 26.9 (C-18), 27.7 (C-2), 31.9 (C-6), 33.6 (C-7),
35.1 (C-14), 36.9 (C-15), 38.6 (C-8), 39.8 (C-3), 41.3 (C-5), 43.0
(C-10 and 12), 62.2 (C-1), 68.5 (C-16), 73.4 (C-11), 213.9 (C-4);
HRMS (ESI): calcd for C19H38O4Na ([M+Na]+) 353.2662, found
353.2638.
4.3.4. Dihydro-a1 (8)
To a solution of 1 (0.1 mg) in MeOH (0.1 mL) was added NaBH4
(1 mg), and the mixture was stirred at room temperature for 14 h.
The mixture was diluted with water and extracted with EtOAc. The
combined extracts were washed with water and concentrated. The
residue was chromatographed on silica gel with CHCl3-MeOH
(95:5, 9:1) to give 8 (0.05 mg): 1H NMR (CD3OD, 600 MHz):
d = 3.64 (1H, m, H-1b), 3.56 (1H, m, H-1a), 3.41 (1H, dd, J = 10.8,
.5.4 Hz, H-16b), 3.33 (1H, dd, J = 10.8, 4.2 Hz, H-16a), 3.34/3.42 (to-
tal 1H, m, H-4), 1.69/1.73 (1H, m, H-2b), 1.65 (1H, m, H-3), 1.58
(1H, m, H-15), 1.27–1.55 (15H, m), 1.05–1.17 (2H, m, H-8a, 14a),
1.12 (3H, s, H-18), 0.91 (3H, d, J = 6.6 Hz, H-17), 0.90 (3H, m, H-
19), 0.88/0.91 (total 3H, m, H-20); HRMS (ESI): calcd for
4.3.8. 1-O-Acetyl-
diacetyl- 2 (14)
Hormone 2 (2) (0.55 mg) was treated with acetic anhydride in
a2 (12), 16-O-acetyl-a2 (13), and 1,16-O,O-
a
a
C
20H43O4 ([M+H]+) 347.3156, found 347.3141; calcd for
C20H42O4Na ([M+Na]+) 369.2975, found 369.2968.
pyridine at room temperature for 30 min. TLC analysis [hexane-
EtOAc (1:1)] showed that the most of 2 was converted to diacetate
14 with small amounts of monoacetates. Another 2 (0.55 mg) was
acetylated under the same conditions except for the reaction time
of 5 min, producing additional monoacetates. The crude products
from the two reactions were combined and subjected to prepara-
tive TLC [20 cm ꢀ 10 cm ꢀ 0.25 mm thickness, hexane-EtOAc
(1:1)] to give 12 (0.3 mg, Rf = 0.3), 13 (0.2 mg, Rf = 0.42), and 14
(0.6 mg, Rf = 0.81). 12: 1H NMR (600 MHz, CDCl3): d = 5.33 (1H, t,
J = 7.0 Hz, H-2), 4.59 (2H, d, J = 7.0 Hz, H-1), 3.51 (1H, dd, J = 10.2,
6.0 Hz, H-16b), 3.44 (1H, dd, J = 10.2, 6.6 Hz, H-16a), 2.05 (3H, s,
Ac), 2.00 (2H, m, H-4), 1.69 (3H, s, H-20), 1.65 (1H, m, H-15),
1.20–1.50 (16H, m), 1.07–1.20 (3H, m, H-6a, 8a, and 14a), 1.16
(3H, s, H-18), 0.93 (3H, d, J = 6.6 Hz, H-17), 0.87 (3H, d, J = 6.6 Hz,
H-19)8; HRMS (ESI): calcd for C22H42O4Na ([M+Na]+) 393.2975,
found 393.2997.
4.3.5. Dehydrated
To a solution of diacetyl-
was added thionyl chloride (10
a1 (9)
a
1 (3) (0.6 mg) in dry pyridine (0.3 mL)
L) at 0 °C, and the mixture was
l
stirred for 30 min at 0 °C. The reaction mixture was diluted with
saturated NaHCO3 and extracted with hexane-EtOAc (1:1). The
combined extracts were washed with 1 M HCl and then water.
The organic layer was concentrated to give crude product
(0.4 mg). To a solution of the crude product in MeOH (0.5 mg)
was added K2CO3 (5 mg), and the mixture was stirred at room tem-
perature for 2 h. The reaction was quenched by adding saturated
NH4Cl (1 mL), and the mixture was extracted with EtOAc. The com-
bined extracts were washed with water and concentrated. The
resulting product was purified by HPLC [Develosil ODS-UG-5
(4.6 ꢀ 250 mm), 30–70% (40 min) MeCN, 4 mL/min, detected at
13: 1H NMR (600 MHz, CDCl3): d = 5.41 (1H, t, J = 6.6 Hz, H-2),
4.15 (2H, d, J = 6.6 Hz, H-1), 3.96 (1H, dd, J = 10.5, 5.7 Hz, H-16b),
3.86 (1H, dd, J = 10.5, 7.0 Hz, H-16a), 2.05 (3H, s, Ac), 1.99 (2H,
m, H-4), 1.80 (1H, m, H-15), 1.67 (3H, s, H-20), 1.20–1.50 (16H,
m), 1.16 (3H, s, H-18), 1.05–1.20 (3H, m, H-6a, 8a, and 14a), 0.94
(3H, d, J = 6.6 Hz, H-17), 0.87 (3H, d, J = 6.6 Hz, H-19); HRMS
(ESI): calcd for C22H42O4Na ([M+Na]+) 393.2975, found 393.2995.
14: 1H NMR (600 MHz, CDCl3): d = 5.33 (1H, t, J = 7.0 Hz, H-2),
4.59 (2H, d, J = 7.0 Hz, H-1), 3.96 (1H, dd, J = 10.8, 6.0 Hz, H-16b),
3.86 (1H, dd, J = 10.8, 7.0 Hz, H-16a), 2.05 (6H, s, Ac), 2.01 (2H,
m, H-4), 1.80 (1H, m, H-15), 1.69 (3H, s, H-20), 1.20–1.50 (15H,
m), 1.16 (1H, m, H-14a), 1.16 (3H, s, H-18), 1.10 (2H, m, H-6a
and 8a), 0.94 (3H, d, J = 6.6 Hz, H-17), 0.87 (3H, d, J = 6.0 Hz, H-
19); HRMS (ESI): calcd for C24H44O5Na ([M+Na]+) 435.3081, found
435.3079.
205 nm] to give
9
(tR = 37.1 min, 0.2 mg): 1H NMR (CDCl3,
600 MHz): d = 5.10 (0.66H, t, J = 6.3 Hz, H-10, 12), 4.70 (0.68H, s,
H-18), 3.65 (2H, m, H-1), 3.51 (1H, m, H-16b), 3.44 (1H, m,
H-16a), 2.75 (1H, sex, J = 6.8 Hz, H-3), 2.49 (2H, m, H-5), 1.94
(1H, m, H-2b), 1.92–2.08 (4H, m, H-9b, 10, 12, 13b), 1.63 (1H, m,
H-15), 1.59 (1H, m, H-2a), 1.58 (2H, s, H-18), 1.25–1.65 (9.7H,
m), 1.15 (1H, m, H-8), 1.13 (1H, m, H-14), 1.12 (3H, d, J = 7.0 Hz,
H-20), 0.93 (3H, d, J = 6.6 Hz, H-17), 0.88 (3H, m, H-19); HRMS
(ESI): calcd for C20H37O2 ([MꢁOH]+) 309.2788, found 309.2777;
calcd for C20H38O3Na ([M+Na]+) 349.2713, found 349.2689.
4.3.6. 15-Epi-a1 (10)
Total synthesis of this
a
1 isomer is described in Supplementary
= –12.4 (c 0.6, MeOH); IR (film): max = 3374 (br),
1H NMR
data. 10: ½a 2D3
ꢂ
m
2935, 1705, 1121, 1049, 939, 917, 737, 618 cmꢁ1
;
(400 MHz, CD3OD): d = 3.52 (2H, br. t, J = 6.6 Hz, H-1), 3.41 (1H,
dd, J = 6.6, 10.7 Hz, H-16b), 3.33 (1H, m, H-16a), 2.76 (1H, sex,
J = 6.8 Hz, H-3), 2.53 (2H, t, J = 7.1 Hz, H-5), 1.89 (1H, sex,
J = 6.8 Hz, H-2b), 1.10–1.66 (15H, m), 1.12 (3H, s, H-18), 1.10 (2H,
m, H-8a, 14a), 1.07 (3H, d, J = 6.8 Hz, H-20), 0.91 (3H, d,
J = 6.8 Hz, H-17), 0.89 (3H, d, J = 6.4 Hz, H-19); 13C NMR
(100 MHz, CD3OD): d = 16.9 (C-20), 17.1 (C-17), 19.9 (C-19), 22.3
(C-9 or 13), 22.4 (C-13 or 9), 27.0 (C-18), 31.7 (C-6), 33.6 (C-7),
35.0 (C-14), 36.7 (C-2), 36.9 (C-15), 38.6 (C-8), 40.0 (C-5), 42.9
(C-10 or 12), 43.0 (C-12 or 10), 43.9 (C-3), 60.6 (C-1), 68.4 (C-16),
73.4 (C-11), 217.5 (C-4). These NMR spectra are superimposable
4.3.9. 11-O-Acetyl-
To a stirred solution of bis-TBS-
pyridine (0.5 mL) were added DMAP (15 mg, 0.12 mmol) and acetic
anhydride (10 L, 0.11 mmol), and the mixture was stirred for 84 h
at room temperature. The mixture was poured into water. The
aqueous mixture was extracted with EtOAc, and the combined or-
ganic layers were washed with saturated aqueous CuSO4 solution,
water and brine. The organic layer was dried with Na2SO4, and con-
centrated. The residue was purified by silica gel column chroma-
tography with EtOAc-hexane (1:100) to give crude acetate
(4 mg). This was dissolved in THF (0.5 mL), and TBAF (1.0 M in
a2 (15)
a2 (26) (8 mg, 0.015 mmol) in
l
on those of
a
1 (1). Anal. Calcd for C20H40O4: C, 69.72; H, 11.70.
THF 14 lL, 14 lmol) was added to the resulting solution at 0 °C.
Found: C, 69.92; H, 11.86.
After stirred for 7 h at room temperature, the reaction was
quenched with water. The aqueous mixture was extracted with
EtOAc, and the combined organic layers were washed with brine,
dried with Na2SO4, and concentrated. The residue was purified
by silica gel column chromatography with EtOAc-hexane (3:2)
4.3.7. 3-Demethyl a1 (11)
Total synthesis of this derivative is described in Supplementary
data. 11: Colorless oil; ½a D25
ꢂ
= –2.0 (c 0.10, MeOH), IR (film):

686
S. D. Molli et al. / Bioorg. Med. Chem. 20 (2012) 681–686
and then by silica gel TLC (20 cm ꢀ 10 cm ꢀ 0.25 mm thickness)
with acetone-CHCl3 (1:4) to give 15 (0.4 mg, 7% in two steps): col-
orless oil, 1H NMR (600 MHz, CDCl3): d = 5.41 (1H, t, J = 7.0 Hz, H-
2), 4.15 (2H, d, J = 7.0 Hz, H-1), 3.50 (1H, dd, J = 10.4, 5.8 Hz, H-
16b), 3.42 (1H, dd, J = 10.5, 6.4 Hz, H-16a), 1.99 (2H, m, H-4),
1.97 (3H, s, Ac), 1.83 (1H, dt, J = 4.3, 12.5 Hz, H-12b), 1.81 (1H, dt,
J = 4.1, 12.5 Hz, H-10b), 1.67 (3H, s, H-20), 1.67 (2H, m, H-10a,
12a), 1.63 (1H, m, H-15), 1.41 (1H, m, H-7), 1.39 (3H, s, H-18),
1.17–1.50 (11H, m), 1.05–1.15 (3H, m, H-6a, 8a, 14a), 0.92 (3H, d,
J = 6.6 Hz, H-17), 0.85 (3H, d, J = 6.6 Hz, H-19); HRMS (ESI): calcd
for C22H42O4Na ([M+Na]+) 393.2975, found 393.2965.
(ESI): m/z 490.2/492.2 (1:1) ([M–OCONHC6H4Br]+), 745.2/747.2/
749.2 (1:2:1) ([M+Na]+); HRMS (ESI): calcd for C34H4879Br2N2O5Na
([M+Na]+) 745.1822, found 745.1807.
4.3.11. Dihydro-a2 (19)
To a solution of
a2 (2) (0.1 mg) in EtOAc (0.5 mL) was added 5%
Rh-Al2O3 (1.7 mg), and the mixture was stirred under a hydrogen
atmosphere at room temperature for 1 h. The catalyst was sepa-
rated through a cotton plug and washed with EtOAc. The combined
filtrates were concentrated to give a crude product, which was
purified by TLC (20 cm ꢀ 10 cm ꢀ 0.25 mm thickness) with hex-
ane-EtOAc (1:9) to give pure 19 (0.1 mg, Rf = 0.47): 1H NMR
(600 MHz, CDCl3): d = 3.63–3.76 (2H, m, H-1), 3.51 (1H, dd,
J = 10.4, 6.0 Hz, H-16b), 3.44 (1H, dd, J = 10.4, 6.5 Hz, H-16a),
1.50–1.67 (3H, m), 1.20–1.48 (20H, m), 1.16 (3H, s, H-18), 1.05–
1.20 (3H, m), 0.93 (3H, d, J = 6.6 Hz, H-17), 0.90 (3H, d, J = 6.5 Hz,
H-20), 0.86 (3H, d, J = 6.6 Hz, H-19); HRMS (ESI): calcd for
4.3.10. 1-O-Carbamoyl-
dicarbamoyl- 2 (18)
To a solution of 2 (2) (0.86 mg) in dry pyridine (0.2 mL) was
a2 (16), 16-O-carbamoyl-a2 (17), and
a
a
added p-bromophenyl isocyanate (4 mg), and the mixture was stir-
red at room temperature for 5 min. The reaction was quenched by
adding a drop of water. The mixtures was dryed and suspended in
a mixture of hexane-EtOAc (1:1). The insoluble material was re-
moved by filtration, and the filtrate was concentrtaed and subjected
to silica gel TLC (20 cm ꢀ 10 cm ꢀ 0.25 mm thickness) with hexane-
EtOAc (1:1) to give 16 (0.3 mg, Rf = 0.31), 17 (0.1 mg, Rf = 0.38), and
18 (0.9 mg, Rf = 0.71). 16: 1H NMR (600 MHz, CDCl3): d = 7.40 (2H, d,
J = 8.0 Hz, Ar), 7.29 (2H, d, J = 8.0 Hz, Ar), 6.83 (1H, br s, NH), 5.37
(1H, t, J = 7.0 Hz, H-2), 4.68 (2H, d, J = 7.0 Hz, H-1), 3.51 (1H, dd,
J = 10.4, 6.0 Hz, H-16b), 3.44 (1H, dd, J = 10.4, 6.0 Hz, H-16a), 2.04
(2H, t, J = 7.4 Hz, H-4), 1.72 (3H, s, H-20), 1.64 (1H, m, H-15), 1.41
(1H, m, H-7), 1.36–1.50 (9H, m), 1.20–1.36 (4H, m), 1.16 (3H, s, H-
18), 1.13 (2H, m, H-8a, 14a), 1.07 (1H, m, H-6), 0.93 (3H, d,
J = 6.6 Hz, H-17), 0.86 (3H, d, J = 6.6 Hz, H-19); MS (ESI): m/z
548.3/550.3 (1:1) [M+Na]+; HRMS (ESI): calcd for C27H4479BrNO4Na
([M+Na]+) 548.2346, found 548.2373.
C
20H42O3Na ([M+Na]+) 353.3026, found 353.3042.
Acknowledgments
The authors are thankful to Paul W. L. Lai, Institute of Liberal
Arts and Sciences, Nagoya University, for English correction of this
manuscript. This work was supported by JSPS KAKENHI (22248012
and 21710239) and S.D.M is a recipient of a Japanese Government
Foreign Student Scholarship (MEXT, No. 080028).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.015.
17: 1H NMR (600 MHz, CDCl3): d = 7.41 (2H, d, J = 8.6 Hz, Ar), 7.29
(2H, d, J = 8.6 Hz, Ar), 6.74 (1H, br s, NH), 5.41 (1H, t, J = 7.0 Hz, H-2),
4.16 (2H, d, J = 7.0 Hz, H-1), 4.04 (1H, dd, J = 10.4, 5.6 Hz, H-16b),
3.98 (1H, dd, J = 10.4, 6.6 Hz, H-16a), 1.99 (1H, m, H-4), 1.85 (1H,
m, H-15), 1.66 (3H, s, H-20), 1.16 (3H, s, H-18), 1.15–1.50 (15H,
m), 1.05–1.15 (2H, m, H-6a, 8a), 0.97 (3H, d, J = 6.6 Hz, H-17), 0.86
(3H, d, J = 6.6 Hz, H-19); MS (ESI): m/z 490.2/492.2 (1:1) ([M–
H2O–OH]+), 548.2/550.2 (1:1) ([M+Na]+); HRMS (ESI): calcd for
References and notes
1. Fry, W. E.; Goodwin, S. B. Bioscience 1997, 47, 363.
2. Erwin, D. C.; Ribeiro, O. K. Phytophthora Diseases Worldwide; American
Phytopathological Society: St. Paul, MN, 1996.
3. Rizzo, D. M.; Garbelotto, M.; Davidson, J. M.; Slaughter, G. M.; Koike, S. T. Plant
Dis. 2002, 86, 205.
4. Savage, E. J.; Clayton, C. W.; Hunter, J. H.; Brenneman, J. A.; Laviola, C.; Gallegly,
M. E. Phytopathology 1968, 58, 1004.
5. Ashby, S. F. Trans. Br. Mycol. Soc. 1929, 14, 18.
C
27H4479BrNO4Na ([M+Na]+) 548.2346, found 548.2361.
6. Judelson, H. S. Fungal Genet. Biol. 1997, 21, 188.
7. Judelson, H. S.; Blanco, F. A. Nat. Rev. Microbiol. 2005, 3, 47.
8. Ko, W. H. J. Gen. Microbiol. 1978, 107, 15.
18: 1H NMR (600 MHz, CDCl3): d = 7.40 (d, J = 8.6 Hz, 2H, Ar), 7.39
(2H, d, J = 8.6 Hz, Ar), 7.29 (4H, d, J = 8.0 Hz, Ar), 6.74 (1H, br s, NH),
6.83 (1H, br s, NH), 5.37 (1H, t, J = 7.0 Hz, H-2), 4.68 (2H, d, J = 7.0 Hz,
H-1), 4.03 (1H, dd, J = 10.4, 6.2 Hz, H-16b), 3.97 (1H, dd, J = 10.4,
6.6 Hz, H-16a), 2.02 (2H, t, J = 7.4 Hz, H-4), 1.72 (3H, s, H-20), 1.83
(1H, m, H-15), 1.41 (1H, m, H-7), 1.20–1.50 (13H, m), 1.19 (1H, m,
H-14a), 1.17 (3H, s, H-18), 1.12 (1H, m, H-8a), 1.07 (1H, m, H-6a),
0.96 (3H, d, J = 6.8 Hz, H-17), 0.86 (3H, d, J = 6.6 Hz, H-19); MS
9. Ko, W. H. Bot. Stud. 2007, 48, 365.
10. Qi, J.; Asano, T.; Jinno, M.; Matsui, K.; Atsumi, K.; Sakagami, Y.; Ojika, M. Science
2005, 309, 1828.
11. Yajima, A.; Qin, Y.; Zhou, X.; Kawanishi, N.; Xiao, X.; Wang, J.; Zhang, D.; Wu, Y.;
Nukada, T.; Yabuta, G.; Qi, J.; Asano, T. Nat. Chem. Biol. 2008, 4, 235.
12. Ojika, M.; Molli, S. D.; Kanazawa, H.; Yajima, A.; Toda, K.; Nukada, T.; Mao, H.;
Murata, R.; Asano, T.; Qi, J.; Sakagami, Y. Nat. Chem. Biol. 2011, 7, 591.
13. Yajima, A.; Toda, K.; Molli, S. D.; Ojika, M.; Nukada, T. Tetrahedron 2011, 67,
8887.