Design, synthesis, and characterization of BK channel openers based on oximation of abietane diterpene derivatives

Article abstract of DOI:10.1016/j.bmc.2010.09.072

Oxime ether derivatives at the benzylic position of unsubstituted, dichloro, trichloro, and monobromo derivatives of the aromatic C-ring of dehydroabietic acid and podocarpic acid were synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKα channels. Detailed SAR analysis showed that the oximation was particularly effective in the cases of dehydroabietic acid derivatives, and some of these oxime derivatives showed more potent BK channel activities than the standard compound, NS1619. The present studies provide a new structural basis for development of efficient BK channel openers.

Full text of DOI:10.1016/j.bmc.2010.09.072

Bioorganic & Medicinal Chemistry 18 (2010) 8642–8659
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and characterization of BK channel openers based on
oximation of abietane diterpene derivatives
Yong-Mei Cui a,b, Eriko Yasutomi b, Yuko Otani b, Katsutoshi Ido c, Takashi Yoshinaga c, Kohei Sawada c,
Tomohiko Ohwada b,
⇑
a Department of Chemistry, College of Sciences, Shanghai University, 99 Shangda Road, Baoshan District, Shanghai 200444, China
b Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
c Tsukuba Research Laboratories, Eisai Co. Ltd, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxime ether derivatives at the benzylic position of unsubstituted, dichloro, trichloro, and monobromo
derivatives of the aromatic C-ring of dehydroabietic acid and podocarpic acid were synthesized and eval-
uated as BK channel openers in an assay system of CHO-K1 cells expressing hBKa channels. Detailed SAR
analysis showed that the oximation was particularly effective in the cases of dehydroabietic acid deriv-
atives, and some of these oxime derivatives showed more potent BK channel activities than the standard
compound, NS1619. The present studies provide a new structural basis for development of efficient BK
channel openers.
Received 24 August 2010
Accepted 30 September 2010
Available online 8 November 2010
Keywords:
BK channels
Channel openers
Oximation
Ó 2010 Elsevier Ltd. All rights reserved.
Dehydroabietic acid
Podocarpic acid
1. Introduction
with other ion channels (such as Ca2+ ion channels,7,8 chloride
channel,9 TRPC channels10) to serve as a negative feedback path-
Potassium (K+) channels are a structurally diverse family of
transmembrane proteins that are modulated by voltage, cell
metabolism, and calcium or receptor-mediated processes. They
play a key role in the regulation of membrane potential, and in
excitable cells they regulate the frequency and form of the action
potential, the release of neurotransmitters, and contractility.1–3
Calcium-activated potassium (KCa) channels are a subgroup of
these channels that share a dependence on intracellular calcium
ion concentration for activity and are, in addition, regulated by
membrane potential and phosphorylation state.4 On the basis of
single-channel conductance, the KCa channels are further subdi-
vided into three types, BK (maxi-K), IK, and SK channels, referring
to large (100–300 picosiemens, (pS)), intermediate (25–100 pS),
and small conductance (2–25 pS) KCa channels, respectively. These
channels vary in pharmacology, distribution and function, as well
as in sensitivity to voltage and Ca2+ concentration: that is, BK chan-
nels show voltage-dependency, while IK and SK channels are volt-
age-independent. BK channels are of particular interest because of
their large channel conductance and their expression in a range of
excitable cell types, including neurons and smooth muscle cells.5
BK channels are uniquely regulated by changes in both trans-
membrane potential and intracellular Ca2+ level,6 and may couple
way controlling ionic homeostasis, cell excitability, and neuron
activity.11 Structurally, BK channels consist of channel-forming
a-
subunits and accessory b-subunits arranged in tetramers,12 having
a voltage sensor and pore as the membrane-spanning domain and
having a cytosolic domain containing metal-binding sites. Recently
published studies on electron cryomicroscopy (cryo-EM)13 and X-
ray crystallographic structure analysis14 of the BK channel have
provided the first glimpse into the assembly of the quaternary
structure of this massive channel protein, corroborating the close
interactions among these domains during channel gating that were
suggested by the previous functional studies.15 Cloning studies
have revealed the presence of multiple splice variants of
a-sub-
units and multiple subtypes of b-subunits (b1, b2/b3, and b4),16
which may be specific to tissues, organs and functions (e.g., b1:
smooth muscle, b4: brain).17 Because of the large conductance, sig-
nificant dependency on intracellular Ca2+ concentration, and po-
tential organ selectivity due to the variety of b subunits, control
of opening of BK channels is a potentially powerful intervention
in the modulation of muscle contractility or neurotransmitter re-
lease and hormone secretion. Openers of these channels have
emerged as potentially useful agents in the therapy of various dis-
ease states associated with both the central nervous system and
smooth muscle, such as acute stroke, epilepsy, psychoses, erectile
dysfunction, arterial hypertension, asthma, and bladder hyperac-
tivity.18,19 Well-characterized BK channel openers not only are
⇑
Corresponding author. Tel.: +81 3 5841 4730; fax: +81 3 5841 4735.
E-mail address: ohwada@mol.f.u-tokyo.ac.jp (T. Ohwada).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.072

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8643
expected to have therapeutic potential, but also should be of assis-
tance in elucidating the function, structure and role of BK channels.
The range of synthetic and naturally occurring compounds that
exhibit BK channel-opening activity, together with the electro-
physiological and biochemical pharmacological properties of these
compounds, has recently been reviewed.20 Synthetic benzimidazo-
lone derivatives, represented by NS004 and NS1619 (Chart 1), were
the first BK channel openers to be discovered, and have been the
most widely used pharmacological tools in investigations of the
function of BK channels, as well as having been lead compounds
for the design of several novel synthetic BK openers. Biaryl amines,
such as mefenamic and flufenamic acids, biarylureas (NS1608),
biarylthioureas (NS11021), aryloxindoles (BMS-204352), arylpyr-
roles (NS-8), and indole-3-carboxylic acid esters (CGS-7184 and
CGS-7181) have also been described and characterized as BK chan-
nel openers. In addition to these synthetic compounds, a number of
compounds derived from natural products have been evaluated as
BK channel openers, including dehydrosoyasaponin-I (DHS-I), L-
735334, bile acids and diterpenes such as maxikdiol (Chart 1).
However, the range of scaffolds for the molecular design of BK
channel openers is still limited and the BK channel-opening po-
tency of currently available compounds is rather weak.
novel tricyclic compounds has been little explored.26 In a recent
study, we synthesized a series of oxime and oxime ether deriva-
tives of the benzylic ketone of diCl-DHAA and found that the oxime
ether structure significantly increased the BK channel-opening
activity of diCl-DHAA.27
In this paper, we describe detailed SAR studies of introduction
of oximate at the benzylic position of the B ring of dehydroabietic
acid and podocarpic acid derivatives with general structures 3 and
4, and validate the effectiveness of the oximation strategy for
enhancing BK channel-opening activity.
2. Chemistry
2.1. Dehydroabietic acid derivatives
Initially, we synthesized a series of oxime (3a) and oxime ether
derivatives (3b–m) of the benzylic ketone of diCl-DHAA (1b). The
four synthetic routes used to access the target compounds 3a–m
are summarized in Schemes 1–4. The parent oxime 3a was synthe-
sized as shown in Scheme 1. Benzylic oxidation of the methyl ester
derivative 1c with CrO3 in AcOH/Ac2O afforded the 7-keto deriva-
tive 1d. Treatment of the ketone 1d with hydroxylamine hydro-
chloride in EtOH and pyridine under heating at reflux afforded
the corresponding E-oxime 5a (anti) as the sole isolated product.
The E-stereochemistry was confirmed by a single-crystal structure
analysis.27 Finally, the methyl ester of 5a was hydrolyzed with KOH
in methanol in the presence of 18-crown-6 to give the desired car-
boxylic acid 3a. Compound 3b was obtained by condensation of ke-
tone intermediate 1d with methoxylamine hydrochloride,
followed by hydrolysis of the methyl ester with KOH and 18-
crown-6 in methanol.
Compounds 3f, 3g, and 3m were obtained by O-alkylation of the
oxime 5a with various alkyl bromides (allyl bromide, propargyl
bromide and benzyl bromide, respectively) under basic conditions
(KOH) in a mixture of H2O–CH2Cl2 in the presence of TBAB, fol-
lowed by basic hydrolysis of the methyl ester to the carboxylic acid
with KOH in MeOH in the presence of 18-crown-6 by heating at re-
flux, as depicted in Scheme 2. The latter hydrolysis step of the
methyl ester to the acid proceeded very slowly. Compounds 3d,
3e, and 3i–l were synthesized by O-alkylation of the oxime oxygen
atom in a similar manner. According to the literature,28 KOBut in
DMSO can hydrolyze hindered esters. The desired acid compounds
3d, 3e, and 3i–l were obtained by using these hydrolysis conditions
at room temperature for 40 min (Scheme 3).
Our pioneering work on pimarane compounds, which have an
intuitive structural similarity to maxikdiol, has revealed that pim-
aric acid (Chart 1) is a potent BK channel opener.21 Other diterpene
analogues, such as abietic acid, sclareol and methyl pimarate, do
not display significant ability to activate BK channels, despite their
chemical structural similarity. On the other hand, dehydroabietic
acid (DHAA, 1a, Chart 1), which is structurally related to non-aro-
matic abietic acid, showed weak but definite BK channel-opening
activity.22 Chemical modification of DHAA by introduction of
halogen atoms on the benzene ring to afford 12,14-dichlorodehy-
droabietic acid (diCl-DHAA, 1b) markedly increased the BK chan-
nel-opening activity.22 All these BK channel openers are assumed
to interact with the
a
subunit of the BK channels.23 Using DHAA
and diCl-DHAA as starting points, a number of derivatives, wherein
the C ring was modified by introduction of divergent substituents,
have been synthetically prepared and some of them have been
characterized as BK channel openers.24 Also, we found that podo-
carpic acid (2), structurally closely related to dehydroabietic acid,
but bearing the carboxylic acid functionality in a reverse stereo-
chemistry, represents a new scaffold for BK channel openers.25
Pimaric acid, dehydroabietic acid and podocarpic acid are diter-
penes of the abietane chemical class, with common 3-ring phenan-
threne structures. However, modification of the B ring of these
H
F3C
H
N
N
F3C
O
OH
O
N
OH
N
OH
H
H
OH
H
CO2H
F3C
Cl
NS004
NS1619
Pimaric acid
Maxikdiol
R1
OH
O
12
Ph
R1
12
12
C
C
B
1
1
C
C
9
9
14 R2
14
8
B
14
10
10
8
A
B
A
A
A
B
5
5
N
O
N
4
4
6 7
CO2H
H
6 7
H
H
H
CO2H
CO2H
CO2H
O
R2
1a: R1=R2=H
R
2
4
3
1b R1=R2=Cl
Chart 1.

8644
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
Cl
Cl
Cl
Cl
Cl
b
a
Cl
Cl
Cl
Cl
Cl
a
b
N
N
N
H
H
H
H
H
H
CO2H
CO2Me
CO2Me OH
CO2Me OR
CO2H
CO2H
OR
1a
1c
1b
5a
5c R=
3c R=
3h R=
Cl
Cl
Cl
5h R=
Scheme 4. Reagents and conditions: (a) RBr, NaH, DMF, 0 °C to rt, 56% (5c) and 83%
(5h); (b) KOH, 18-crown-6, MeOH, microwave irridiation, 145 °C, 57% (3c) and 63%
(3h).
e
d
Cl
Cl
c
Cl
N
N
O
H
H
H
CO2Me
OR
CO2H
CO2Me
OR
5a R=H
5b R=Me
3a R=H
3b R=Me
1d
Alkylation of the oxime intermediate 5a with 3,3-dimethylallyl
bromide after deprotonation of the oxime hydroxy group of 5a
with NaH afforded the intermediate 5h. However, basic hydrolysis
of the methyl ester with KOH in the presence of crown ether by
heating at reflux failed, probably because of steric hinderance of
the methyl ester. Moreover, alternative hydrolysis conditions
involving KOBut in DMSO led to decomposition to the product
3a. One possibility is that the olefin ether (allyl ether) was first
isomerized to the vinyl ether by the action of KOBut in DMSO
and the latter decomposed under the acidic work-up conditions.29
Finally, the desired compound 3h was obtained in 63% yield by
hydrolysis of 5h with KOH in MeOH in the presence of 18-crown
ether-6 under microwave irradiation at 145 °C for 1 h. Compound
3c was obtained similarly (Scheme 4).
Scheme 1. Reagents and conditions: (a) Cl2, FeCl3/SiO2, DDQ/SiO2, CCl4, 39%; (b)
TMSCHN2, MeOH–toluene, rt, 90%; (c) CrO3, AcOH/Ac2O, 58%; (d) NH2ORꢀHCl, EtOH,
Py, reflux, 87% (R = H) and 100% (R = Me); (e) KOH, 18-crown-6, MeOH, reflux, 88%
(R = H) and 79% (R = Me).
Cl
Cl
Cl
Cl
a
N
N
H
H
CO2Me
CO2Me
OH
OR
To investigate further the effects of substituents in ring C on the
activity, we synthesized oxime and oxime ether derivatives of the
benzylic ketone of DHAA, 12-bromo-DHAA (12-Br-DHAA), and
11,12,14-trichlorodehydroabietic acid (triCl-DHAA). For the syn-
thesis of the DHAA series of compounds, the parent oxime 5n
was synthesized as shown in Scheme 5. Benzylic oxidation of the
methyl ester derivative 1e with CrO3 in AcOH/Ac2O afforded the
7-keto derivative 1f in moderate yield. Similarly, treatment of
the ketone 1f with hydroxylamine hydrochloride in EtOH and pyr-
idine by heating under reflux afforded the corresponding E-oxime
5n (anti) as the sole isolated product. The desired carboxylic acid
3n was obtained by hydrolysis of the methyl ester of 5n with KO-
But in DMSO. O-Alkylation of the oxime 5n with various alkyl bro-
mides and subsequent basic hydrolysis of the methyl ester with
KOH in MeOH in the presence of crown ether by heating under
5a
5f-g, 5m
b
Cl
R
f
Cl
g
m
N
H
CO2H
OR
3f-g, 3m
Scheme 2. Reagents and conditions: (a) RBr, TBAB, 1 N KOH, CH2Cl2, 40 °C, 60–89%;
(b) KOH, 18-crown-6, MeOH, reflux, 43–85%.
Cl
Cl
Cl
Cl
a
a
b
N
N
H
H
O
CO2Me
CO2Me
OH
OR
H
H
H
CO2Me
CO2Me
CO2H
5a
5d-e, 5i-l
1f
1e
1a
R
c
R
b
d
e
o
Cl
CH3
e
p
q
i
j
N
N
Cl
H
H
CO2R1
OR
CO2R
OH
r
N
5o-s R1=CH3
3o-s R1=H
5n R=CH3
3n R=H
H
d
k
l
f
CO2H
OR
s
3d-e, 3i-l
Scheme 5. Reagents and conditions: (a) concd H2SO4, MeOH, 85 °C, 91%; (b) CrO3,
AcOH/Ac2O, 48%; (c) NH2OHꢀHCl, EtOH, Py, reflux, 79%; (d) KOBut, DMSO, rt, 79%; (e)
RBr, NaH, DMF, 0 °C to rt, 70–92%; (f) KOH, 18-crown-6, MeOH, reflux, 57–93%.
Scheme 3. Reagents and conditions: (a) RBr, NaH, DMF, 0 °C to rt, 61–86%; (b)
KOBut, DMSO, rt, 24–63%.

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8645
reflux afforded compounds 3o–s, respectively, as depicted in
Scheme 5.
generated. Hydrolysis of 6g with KOH in MeOH in the presence of
18-crown-6-ether under microwave irradiation at 145 °C for 1 h or
up to 5 h resulted in practically no reaction; when the reaction was
carried out under microwave irradiation overnight, 6g decomposed
without formation of the desired product. In the similar synthesis
of 4b, this unexpected hydroxylation occurred in the hydrolysis
step of the ester to the acid: O-alkylation of 6f with 1-bromo-3-
phenylpropane gave compound 6j, and then hydrolysis of 6j with
KOBut in DMSO at 60 °C afforded a mixture of the desired 4b as a
minor product and the major by-product 6k. We also studied the
BK channel-opening activities of these hydroxylated carboxylic
acids (6i and 6k). The O-methyl compound 7a was also obtained
by direct basic hydrolysis of the ester 6f (Scheme 10).
Regioselective bromination of 1e with NBS in CH3CN in the
presence of Montmorillonite K-10 afforded a mixture of 12- and
14-monobromo derivatives 1g and 1h in a ratio of 3.1:1. Starting
from the key intermediate 1g, the desired acid derivatives 3t–v
were synthesized according the procedure shown in Scheme 6.
Furthermore, we synthesized the oxime and oximate deriva-
tives of 11,12,14-trichlorodehydroabietic acid (triCl-DHAA). By
controlling the amount of Cl2 and the reaction time, trichlorinated
DHAA (1j) can be obtained in 35% yield. As shown in Scheme 1,
compounds 3w–y were similarly obtained in moderate overall
yields (Scheme 7).
Also, we synthesized the ketone derivatives (1m, 1n and 1p) to
confirm the importance of the oximic group for BK channel-
opening activity. As shown in Scheme 8, direct benzylic oxidation
of the carboxylic acid 1b with CrO3 in Ac2O/AcOH afforded 7-oxo-
12,14-dichlorodehydroabietic acid 1m in 36% yield. Compounds
1n and 1p were obtained by hydrolysis of the corresponding methyl
esters with KOBut in DMSO at rt after benzylic oxidation (1f and 1i).
The synthetic route involving initial alkylation of the oxime hy-
droxyl group of the ester, followed by basic hydrolysis of the ester
to the acid, was accompanied with unexpected hydroxylation. In
order to avoid this hydroxylation reaction, the synthetic route
was modified, beginning with hydrolysis of the ester 6h to the acid
intermediate 4a, followed by selective O-alkylation of the oxime
hydroxyl functionality to afford the desired compounds in moder-
ate conversion yields (Scheme 11).
2.2. Podocarpic acid derivatives
3. Results and discussion
Prior to the benzylic oxidation, the 12-phenol group of com-
pound 6a was firstly protected as an acetate intermediate (6b),
and then oxidized with chromium trioxide to afford the 7-oxo
derivative 6c in 85% yield (Scheme 9). During condensation of 6c
with hydroxylamine hydrochloride in the presence of pyridine by
heating at 100 °C for 3 h, a part of the 12-acetoxy group was re-
moved, and a mixture of two intermediates 6d and 6e was ob-
tained. When a longer heating time of 17 h was applied in the
oximation process, the acetate was completely hydrolyzed to give
the phenol derivative, as in the case of oximation of 6c with O-
methylhydroxyamine hydrochloride, yielding the deprotected
compound 6f, as depicted in Scheme 9.
The activities of all the target compounds as BK channel modu-
lators were evaluated by means of automated patch clamp record-
ing using the 64-hole Population Patch Clamp (PPC) technique.30,31
The BK channel was activated by applying a step pulse to +100 mV
from the holding potential of ꢁ90 mV to CHO-K1 cells expressing
hBKa channels, and the current amplitude in the presence of a test
compound (30
lM) was expressed as percent of the drug-free con-
trol. The values represent an average of data obtained from at least
eight separate measurements. The results for NS1619 are included
for comparison.
Alkylation of 6e with an excess of 1-bromo-3-phenylpropane
gave a mixture of compounds, O,O-dialkylated 6g and O-mono-
alkylated 6h (Scheme 10). Subsequent hydrolysis of isolated 6h
with KOBut in DMSO at 60 °C afforded the desired compound 4a.
On the other hand, hydrolysis of 6g with KOBut in DMSO at 60 °C
afforded no desired carboxylic acid product 4g, instead an unex-
pected new compound, the 6-hydroxy acid derivative 6i, was
3.1. Dehydroabietic acid case
In the present work we mainly focused on the effect of simple
alkyl modifications at the oxime to probe the effect of oximic sub-
stitution on the BK channel-opening activity (Table 1). In our pre-
liminary study, we found that introduction of a phenyl-substituted
carbon chain on the oxime oxygen atom had little influence or
Br
a
Br
+
H
H
H
CO2Me
CO2Me
CO2Me
1g
1h
1e
b
Br
Br
Br
Br
c
e
f
N
N
N
O
H
H
H
H
CO2Me
OR
CO2R
CO2H
OH
OR
CO2Me
5t R=CH3
3t R=H
1i
5u R=CH3
5v R=
3u R=CH3
3v R=
d
Scheme 6. Reagents and conditions: (a) NBS, Mont. K10, CH3CN, rt, 69% (1g/1h = 3.1:1); (b) CrO3, AcOH/Ac2O, 83%; (c) NH2OHꢀHCl, EtOH, Py, reflux, 97%; (d) KOBut, DMSO, rt,
52%; (e) RBr, NaH, DMF, 0 °C to rt, 73% (5u) and 70% (5v); (f) KOH, 18-crown-6, MeOH, reflux, 90% (3u) and 72% (3v).

8646
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
Cl
Cl
Cl
Cl
b
Cl
a
Cl
H
H
H
CO2H
CO2Me
CO2H
1k
1a
1j
Cl
Cl
Cl
Cl
Cl
Cl
e
c
d
Cl
Cl
Cl
N
OH
N
O
x R=CH3
OR
y R=
H
H
H
CO2R1
CO2R
CO2Me
5x-y R1=CH3
5w R=CH3
3w R=H
1l
f
f
3x-y R1=H
Scheme 7. Reagents and conditions: (a) Cl2, FeCl3/SiO2, DDQ/SiO2, CCl4, 35%; (b) TMSCHN2 (2 M, in Et2O), MeOH–toluene, rt, 94%; (c) CrO3, AcOH/Ac2O, 82%; (d) NH2OHꢀHCl,
EtOH, Py, reflux, 84%; (e) RBr, NaH, DMF, 0 °C to rt, 99–100%; (f) KOH, 18-crown-6, MeOH, microwave irridiation, 145 °C, 56–86%.
derivative (3d) showed slightly higher activity than the branched
side chain derivative (3e). A long substituent chain decreased the
activity (e.g., compound 3l showed almost no BK-opening activity).
Introduction of a small unsaturated substituent, such as allyl,
greatly increased the potency (3f vs 3d). However, further append-
ing two terminal methyl groups dramatically decreased the activ-
ity, which became even lower than that of the corresponding
saturated derivative (3h vs 3i). The cyclopropanemethylene deriv-
ative (3j) also showed potent activity. However, introduction of a
large ring dramatically decreased the activity (compound 3k).
Compounds 3b, 3c, 3f, and 3g showed potency comparable to or
higher than that of NS1619.
Cl
Cl
Cl
Cl
a
O
H
H
CO2H
CO2H
1m
1b
R
R
b
For the non-chlorinated DHAA series of compounds (Table 1),
the influence of the substituents on the oxime chain upon the
activity showed a very similar trend to that in the diCl-DHAA ser-
ies, though the range of activity is rather low. The oxime 3n (ion
O
O
H
H
CO2Me
CO2H
1f R = H
1i R = Br
1n R = H
1p R = Br
current = 120.7 9.6% of control at 30 lM) was found to be a mod-
erate BK channel opener, with slightly lower potency than that of
the corresponding diCl-DHAA oxime 3a (137 3.6%). Introduction
of a small saturated substituent such as methyl (3o, 152.8 8.3%)
or small unsaturated substituents (3p, 182.5 8.3%; 3q,
142.1 14.1%) increased the potency. On the other hand, introduc-
tion of a phenyl-substituted carbon chain, such as a benzyl group,
had little influence (3r) as compared with the O-unsubstituted
oxime 3n. It is noteworthy that additional insertion of two methy-
lene spacers (3s) increased the activity. Moreover, the activities of
Scheme 8. Reagents and conditions: (a) CrO3, AcOH/Ac2O, 36%; (b) KOBut, DMSO,
rt, 58% (1n) and 15% (1p).
decreased the BK channel-opening activity as compared with the
O-unsubstituted oxime (3a).
For the diCl-DHAA series of compounds 3a–m, when R changed
from methyl (3b), ethyl (3c), to propyl (3d and 3e), the activity de-
creased. In the case of the propyl substituent, the straight-chain
OAc
OAc
OH
OH
a
b
c
O
COOMe
COOMe
COOH
COOMe
6a
OAc
6b
2
6c
OH
e
OH
d
+
N
N
N
COOMe
COOMe
OH
OH
COOMe
OMe
6e
6f
6d
Scheme 9. Reagents and conditions: (a) TMSCHN2, MeOH, toluene, rt, 100%; (b) CH3COONa, Ac2O, reflux, 90%; (c) CrO3, AcOH/Ac2O, rt, 85%; (d) NH2OHꢀHCl, EtOH, pyridine,
reflux, 3 h; (e) NH2OMeꢀHCl, EtOH, pyridine, reflux, overnight (17 h), 83%.

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8647
O
Ph
O
Ph
OH
a
+
N
N
O
N
COOR
COOMe
OH
R=Me
R=H
COOMe
Ph
OH
6h
4a
6e
6g
b
b
b
O
Ph
O
Ph
N
O
N
HOOC
OH
COOH
4g
OH
6i
Ph
O
Ph
O
Ph
O
Ph
O
Ph
b
a
+
N
N
N
N
COOH
HOOC
COOR
6f R=Me
7a R=H
COOMe
OMe
OH OMe
6k
OMe
OMe
4b
6j
b
Scheme 10. Reagents and conditions: (a) 1-bromo-3-phenylpropane, Cs2CO3, DMF; (b) KOBut, DMSO, 60 °C.
O
Ph
O
Ph
O
Ph
b
a
N
N
N
COOMe
COOH
O
OH
COOMe
O
OR
OR
6h
Ph
Ph
O
Ph
R
c
b
c
d
N
N
OR
N
COOH
4a
COOH
4c-g
COOMe
OH
OH
6h
e
f
g
Scheme 11. Reagents and conditions: (a) RBr, NaH, DMF, 0 °C to rt; (b) KOBut, DMSO, 60 °C; (c) RBr, KOH, NaI (cat.), EtOH, reflux, 61–98% conversion yield.
all the DHAA series were lower than those of the corresponding
diCl-DHAA derivatives, indicating the importance of the chloride
functionality in ring C for the activity.
In the 12-Br-DHAA series (Table 1), the oxime itself 3t (ion cur-
rent = 162.3 11.7% of control at 30 lM) was found to be a potent
BK opener, with much higher potency than that of the correspond-
ing diCl-DHAA oxime 3a (137 3.6%). Introduction of a small satu-
rated substituent such as methyl (3u, 216.5 27.2%) or a small
unsaturated allyl substituent (3v, 237.2 23.3%) greatly increased
the potency.
In the triCl-DHAA series (Table 1), the oxime 3w (ion cur-
rent = 229 15.8% of control at 30 lM) was found to be the most
potent BK opener among the four O-unsubstituted oxime deriva-
tives (3a, 3n, 3t, and 3w). Introduction of a small saturated substi-
tuent such as methyl (3x, 235.3 23.4%) marginally increased the
potency. However, in contrast to the other three series of com-
pounds, introduction of a small unsaturated substituent such as
an allyl group (3y, 183.5 7.3%) decreased the potency compared
with the O-unsubstituted oxime 3w.
As mentioned above, we also synthesized the ketone derivatives
to confirm the importance of the oximic group for BK channel-
opening activity. Interestingly, the 12-Br-DHAA ketone derivative
showed marginally higher activity than the corresponding O-
unsubstituted oxime (1p vs 3t), while the DHAA ketone derivative

8648
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
Table 1
Structure and BKa-opening properties of dehydroabietate derivatives
R2
R1
R3
N
O
H
CO2H
R
Compound
R1
R2
R3
R
Ionic current in the presence of
test compound (30 M) as % of
l
control current (n = 8)
Buffer
NS1619
1m
3a
3b
—
—
—
H
H
—
—
—
Cl
Cl
—
—
—
Cl
Cl
—
—
—
H
100.4 2.3
280.7 15.6
123.5 4.0
137.0 3.6
213.1 9.4
CH3
3c
H
H
Cl
Cl
Cl
Cl
211.8 3.9
3d
191.7 13.8
3e
H
Cl
Cl
183.5 12.2
3f
H
H
Cl
Cl
Cl
Cl
297.4 17.2
260.8 21.6
3g
3h
H
Cl
Cl
125.6 8.4
3i
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
144.8 10.7
180.8 7.9
128.6 7.8
3j
3k
3l
H
H
—
H
H
Cl
Cl
—
H
Cl
Cl
—
H
111.2 7.1
151.1 15.1
100.3 3.7
120.7 9.6
152.8 8.3
3m
1n
3n
3o
CH2Ph
—
H
H
H
CH3
3p
H
H
H
182.5 8.3
3q
3r
H
H
H
H
H
H
142.1 14.1
123.0 7.7
CH2Ph
3s
H
H
H
143.2 6.2
1p
3t
3u
—
H
H
—
Br
Br
—
H
H
—
H
CH3
166.2 11.3
162.3 11.7
216.5 27.2
3v
H
Br
H
237.2 23.3
3w
3x
Cl
Cl
Cl
Cl
Cl
Cl
H
CH3
229.0 15.8
235.3 23.4
3y
Cl
Cl
Cl
183.5 7.3
(1n) showed no activity. The diCl-DHAA ketone derivative (1m)
showed weak activity.
In the podocarpate series of compounds, the oxime itself
4a (ion current = 134.1 13% of control at 30 M) was found to
be moderately active BK opener. Introduction of small
l
a
a
3.2. Podocarpic acid case
saturated substituent such as methyl (4b, 151.1 14%) or small
unsaturated substituents (4c, 159.9 4.4%; 4e, 166.2 9%), as
well as a phenyl-substituted carbon chain (4f, 151.5 14.3%),
increased the potency. However, 3-methyl-2-butenyl (4d) or
phenylpropyl (4g) substitutions had little effect compared with
4a. It is interesting that compound 7a showed almost no
BK channel-opening activity; this suggests the importance of
Our previous study showed that the podocarpic acid structure
represents a new scaffold for BK channel openers.25 A preliminary
SAR study of podocarpic acid derivatives showed that podocarpic
acid itself has BK channel-opening activity, and introduction of a
12-alkoxy substituent, in particular, the phenylpropyl group dra-
matically increased BK channel-opening activity. Thus, we kept
this preponderant functionality at the 12-oxy position and studied
the effect of oximation and the substituent effect at the oxime on
BK channel-opening activity (Table 2).
the O-phenylpropyl group in ring
C for the activity. And
hydroxylation of the 6-position decreased the potency com-
pared with the corresponding carboxylic acids (6i vs 4g; 6k vs
4b).

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8649
Table 2
Structure and BK
pure materials. Microwave heating was carried out on a Biotage
Initiator system (300W).
a
-opening properties of podocarpate derivatives
Ph
O
5.1.1. diCl-DHAA series
5.1.1.1. 12,14-Dichlorodehydroabietic acid (1b).
To a sus-
pension of dehydroabietic acid 4a (2.0 g, 6.66 mmol), 2% w/w FeCl3
on SiO2 (1 g), and 1% w/w DDQ on SiO2 (0.1 g) in 25 mL CCl4 was
added a solution of 2.0 M Cl2 in 40 mL CCl4 at 0 °C. The reaction
mixture was vigorously stirred at rt for 1.5 h. The reaction was
quenched with saturated Na2SO3, and the whole was extracted
with CHCl3 three times. The combined organic layer was washed
with brine, and dried over Na2SO4. The residue was recrystallized
from n-hexane to afford 1b (962 mg, yield 39%) as a colorless solid.
1H NMR (CDCl3, 400 MHz): d 7.15 (br s, 1H), 3.91 (br s, 1H), 2.94
(dd of ABX system, J = 18.2, 6.2 Hz, 1H), 2.79–2.70 (m, 1H), 2.24
(d, J = 12.6 Hz, 1H), 2.13 (dd of ABX system, J = 12.6, 1.9 Hz, 1H),
1.86–1.61 (m, 7H), 1.39 (d, J = 7.2 Hz, 6H),1.28 (s, 3H), 1.20 (s, 3H).
N
H
OR
CO2H
Compound
R
Ionic current in the presence of test
compound (30 lM) as % of control current
(n = 8)
Buffer
NS1619
7a
4a
4b
—
—
—
H
100.4 2.3
280.7 15.6
103.0 3.1
134.1 13
151.1 14
CH3
4c
159.9 4.4
4d
137.9 4.7
5.1.1.2. 12,14-Dichlorodehydroabietic acid methyl ester
(1c).
To a solution of 1b (962 mg, 2.605 mmol) in MeOH
4e
4f
166.2
9
(4.4 mL) and PhMe (8.8 mL) was added dropwise 2.0 M TMSCHN2
in Et2O (1.7 mL, 3.4 mmol) at rt over 5 min, and the whole was stir-
red at rt for 30 min. Excess TMSCHN2 was quenched with AcOH,
then the reaction mixture was evaporated in vacuo, and the resi-
due was purified by flash chromatography (n-hexane only to
n-hexane/AcOEt = 10:1) to afford 1c (900 mg, yield 90%) as a color-
less solid. 1H NMR (CDCl3, 400 MHz): d 7.16 (br s, 1H), 3.91 (br s,
1H), 3.67 (s, 3H), 2.92 (dd of ABX system, J = 18.2, 6.2 Hz, 1H),
2.76–2.66 (m, 1H), 2.23 (d, J = 11.4 Hz, 1H), 2.12 (dd of ABX system,
J = 12.8, 2.1 Hz, 1H), 1.83–1.61 (m, 7H), 1.39 (d, J = 7.2 Hz, 6H), 1.26
(s, 3H), 1.19 (s, 3H).
CH2Ph
151.5 14.3
4g
133.6 9.3
6i
6k
—
—
107.0 3.1
124.6 10.6
4. Conclusion
Our results indicate that the oxime structure is a suitable scaf-
fold for designing novel potent BK openers. Detailed SAR analysis
showed that oximation was particularly effective in the case of
dehydroabietic acid derivatives. Unsubstituted, dichloro, trichloro
and monobromo derivatives of the C-ring of dehydroabietic acid
showed similar substituent effects with respect to the oxime O-
substituent, whereas oximation had relatively little effect in the
case of podocarpic acid derivatives. Some of the oxime derivatives
showed more potent BK channel activities than did the standard
compound, NS1619. The present studies provide a new structural
basis for development of efficient BK channel openers with clinical
potential to control pathologically uncontrolled cell excitation.
5.1.1.3. 13-Isopropyl-12,14-dichloro-7-oxopodocarpe-8,11,13-
triene-15-carboxylic acid methyl ester (1d).
of CrO3 (287 mg, 2.867 mmol) in Ac2O (9 mL) and AcOH (4 mL)
was added dropwise suspension of the methyl ester 1c
To a solution
a
(999 mg, 2.606 mmol) in AcOH (15 mL) at 0 °C over 10 min. The
reaction mixture was stirred at 50 °C for 9 h, cooled, and poured
into ice-water (40 mL) and then the whole was extracted with
CHCl3 3 times. The combined organic layer was washed with
water, sat. NaHCO3, and brine, and dried over Na2SO4. The crude
mixture was purified by flash chromatography (n-hexane/
AcOEt = 12:1) to afford 1d (600 mg, yield 58%) as a colorless solid.
1H NMR (CDCl3, 400 MHz): d 7.23 (br s, 1H), 4.09 (br s, 1H), 3.66 (s,
3H), 2.65 (m, 2H), 2.50 (dd of ABX system, J = 16.9, 5.2 Hz, 1H), 2.20
(d, J = 12.4 Hz, 1H), 1.80–1.76 (m, 5H), 1.40 (d, J = 7.2 Hz, 6H), 1.32
(s, 3H), 1.19 (s, 3H).
5. Experimental section
5.1. General procedures
Commercial reagents were used without further purification,
unless otherwise mentioned. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Bruker Avance 400 spec-
trometer in CDCl3, DMSO-d6 or Acetone-d6. Chemical shifts are re-
ported in parts per million (ppm) and coupling constants (J) are
reported in hertz (Hz). Chemical shifts in CDCl3 were reported on
a scale relative to CHCl3 (7.26 ppm) for 1H NMR, and to CDCl3
(77.23 ppm) for 13C NMR, as internal references. The center lines
of the multiplets of DMSO-d6 or acetone-d6 were defined as 2.50
and 2.05 ppm respectively, and used as internal references for 1H
NMR spectra. High-resolution mass spectra (HR-MS) were re-
corded on a Bruker Daltonics MicrOTOF05 in the electro-spray ion-
ization (ESI)-time-of-flight (TOF) detection mode. Elemental
analyses were carried out on a Yanaco CHN CORDER spectrometer
at the Analysis Center of the Graduate School of Pharmaceutical
Sciences, the University of Tokyo. Flash column chromatography
was carried out with silica gel (Kanto Chemical 60 N, particle size
5.1.1.4. 12,14-Dichloro-13-isopropyl-7-hydroxyiminepodocarpe-
8,11,13-triene-15-carboxylic acid methyl ester (5a).
A mix-
ture of the methyl ester 1d (430 mg, 1.08 mmol), pyridine
(0.13 mL), and NH2OHꢀHCl (120 mg, 1.727 mmol in EtOH (4.4 mL)
was stirred at 100 °C for 3 h, then cooled, and evaporated in vacuo.
The residue was purified by flash chromatography (n-hexane/
AcOEt = 5:1) to afford 4e (386 mg, yield 87%) as a colorless solid.
1H NMR (CDCl3, 400 MHz): d 10.02 (br s, 1H), 7.15 (br s, 1H),
4.09 (br s, 1H), 3.66 (s, 3H), 3.08 (dd of ABX system, J = 18.6,
13.1 Hz, 1H), 2.39 (dd of ABX system, J = 18.6, 6.4 Hz, 1H), 2.16
(dd, J = 13.0, 6.4 Hz, 2H), 1.74–1.56 (m, 5H), 1.44 (d, J = 6.0 Hz,
6H),1.36 (s, 3H), 1.04 (s, 3H).
5.1.1.5. 12,14-Dichloro-13-isopropyl-7-methoxyiminepodocarpe-
8,11,13-triene-15-carboxylic acid methyl ester (5b).
ture of the methyl ester 1d (29 mg, 0.073 mmol), pyridine (10
A mix-
L),
l
40–50
lm). Melting points were measured on a Yanaco hot-stage
and NH2OMeꢀHCl (10 mg, 0.120 mmol) in EtOH (1 mL) was stirred
microscope and are uncorrected. Yields represent those of isolated
at 100 °C for 3 h, then cooled, and evaporated in vacuo. The residue

8650
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
was purified by flash chromatography (n-hexane/AcOEt = 8:1) to af-
ford 5b (31 mg, yield 100%) as a colorless solid. 1H NMR (CDCl3,
400 MHz): d 7.13 (br s, 1H), 4.07 (br s, 1H), 4.03 (s, 3H), 3.64 (s,
3H), 2.98 (dd of ABX system, J = 18.5, 13.0 Hz, 1H), 2.29 (dd of ABX
system, J = 18.5, 6.4 Hz, 1H), 2.12 (dd, J = 13.1, 6.4 Hz, 2H), 1.79–
1.52 (m, 5H), 1.41 (d, J = 5.6 Hz, 3H), 1.39 (d, J = 6.4 Hz, 3H), 1.36
(s, 3H), 1.06 (s, 3H).
5.3.1. (E,1R,4aS,10aR)-6,8-Dichloro-1,2,3,4,4a,9,10,10a-octahy-
dro-9-(hydroxyimino)-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylic acid (3a)
Colorless solid, yield 88%. Mp 284–286 °C. 1H NMR (CDCl3,
400 MHz): d 7.14 (br s, 1H), 4.03 (br s, 1H), 3.11 (dd of ABX system,
J = 18.8, 13.0 Hz, 1H), 2.53 (dd of ABX system, J = 18.8, 6.4 Hz, 1H),
2.15 (dd, J = 12.9, 6.5 Hz, 2H), 1.80–1.71 (m, 4H), 1.62–1.53 (m, 1H),
1.40 (d, J = 6.2 Hz, 6H),1.38 (s, 3H), 1.05 (s, 3H). HR-MS: calcd for
C
20H2535Cl2NO3 ([M+H]+), 398.1289; found, 398.1333.
5.2. General procedure for preparation of compounds 5f–g, 5m
Anal. Calcd for C20H25Cl2NO3ꢀ1/2H2O: C, 58.97; H, 6.43; N, 3.44.
Found: C, 58.81; H, 6.19; N, 3.21.
A mixture of the methyl ester 5a (41 mg, 0.1 mmol), the corre-
sponding alkyl or aryl bromide (0.15 mmol), cat. Bun4NþBrꢁ (TBAB)
(3 mg) and 1 N KOH (0.15 mL) in dichloromethane (DCM) (1 mL)
was stirred at 40 °C overnight, then cooled, and diluted with
H2O. The whole was extracted with AcOEt. The combined organic
layer was washed with brine, dried over Na2SO4, filtered, and evap-
orated in vacuo. The residue was purified by flash chromatography
using n-hexane/ethyl acetate as the eluent to afford 5f–g, 5m.
5.3.2. (E,1R,4aS,10aR)-6,8-Dichloro-1,2,3,4,4a,9,10,10a-octahy-
dro-7-isopropyl-9-methoxyimino-1,4a-dimethylphenanthrene-
1-carboxylic acid (3b)
Colorless oil, yield 79%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br s,
1H), 4.09 (br s, 1H),4.04 (s, 3H), 3.00 (dd of ABX system, J = 18.6,
13.1 Hz, 1H), 2.42 (dd of ABX system, J = 18.6, 6.4 Hz, 1H), 2.14
(d, J = 12.2, Hz, 1H), 2.08 (dd of ABX system, J = 13.1, 6.4 Hz, 1H),
1.79–1.72 (m, 4H), 1.69–1.59 (m, 1H), 1.41 (d, J = 6.4 Hz, 1H),
1.36 (s, 3H), 1.07 (s, 3H). HR-MS: calcd for C21H2735Cl2NO3
([M+Na]+), 434.1266; found, 434.1270.
5.2.1. (E,1R,4aS,10aR)-Methyl 9-(allyloxy)imino-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphe-
nanthrene-1-carboxylate (5f)
Anal. Calcd for C21H27Cl2NO3ꢀ2/5H2O: C, 60.12; H, 6.68; N, 3.34.
Found: C, 60.45; H, 6.84; N, 2.93.
Colorless solid, yield 62%. 1H NMR (CDCl3, 400 MHz): d 7.13 (br
s, 1H), 6.13–6.04 (m, 1H), 5.33 (m, 2H), 4.72 (m, 2H), 4.08 (br s, 1H),
3.65 (s, 3H), 3.01 (dd of ABX system, J = 18.6, 13.0 Hz, 1H), 2.32 (dd
of ABX system, J = 18.6, 6.4 Hz, 1H), 2.15 (d, J = 8.4 Hz, 1H), 2.11 (dd
of ABX system, J = 13.0, 6.4 Hz, 1H), 1.77–1.65 (m, 4H), 1.63–1.52
(m, 1H), 1.41 (d, J = 6.7 Hz, 6H), 1.37 (s, 3H), 1.07 (s, 3H).
5.3.3. (E,1R,4aS,10aR)-9-(Allyloxy)imino-6,8-dichloro-1,2,3,4,
4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylic acid (3f)
Colorless oil, yield 85%. 1H NMR (CDCl3, 400 MHz): d 7.15 (br s,
1H), 6.14–6.04 (m, 1H), 5.34 (dd, J = 18.7, 1.6 Hz, 1H), 5.23 (dd,
J = 10.4,1.6 Hz, 1H), 4.74 (m, 2H), 4.09 (br s, 1H), 3.03 (dd of ABX
system, J = 18.6,13.0 Hz, 1H), 2.46 (dd of ABX system, J = 18.6,
6.4 Hz, 1H), 2.15 (d, J = 12.1 Hz, 1H), 2.09 (dd of ABX system,
J = 13.0, 6.4 Hz, 1H), 1.79–1.69 (m, 4H), 1.63–1.53 (m, 1H), 1.40
(d, J = 6.9 Hz, 6H), 1.37 (s, 3H), 1.07 (s, 3H). HR-MS: calcd for
5.2.2. (E,1R,4aS,10aR)-Methyl 9-((prop-2-ynyloxy)imino)-6,8-
dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-di-
methylphenanthrene-1-carboxylate (5g)
Yellow oil, yield 79%. 1H NMR (CDCl3, 400 MHz): d 7.13 (br s,
1H), 4.81 (m, 2H),4.09 (br s, 1H), 3.65 (s, 3H), 3.00 (dd of ABX sys-
tem, J = 18.6, 13.0 Hz, 1H), 2.48 (t, J = 2.4 Hz, 1H), 2.35 (dd of ABX
system, J = 18.6, 6.4 Hz, 1H), 2.16–2.09 (m, 2H), 1.77–1.56 (m,
5H), 1.41 (d, J = 6.8 Hz, 6H), 1.37 (s, 3H), 1.07 (s, 3H).
C
23H2935Cl2NO3 ([M+Na]+), 460.1422; found, 460.1388.
Anal. Calcd for C23H29Cl2NO3ꢀ1/4H2O: C, 62.37; H, 6.71; N, 3.16.
Found: C, 62.44; H, 6.50; N, 2.97.
5.3.4. (E,1R,4aS,10aR)-9-((Prop-2-ynyloxy)imino)-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylic acid (3g)
5.2.3. (E,1R,4aS,10aR)-Methyl 9-(benzyloxy)imino-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylate (5m)
Yellow oil, yield 70%. 1H NMR (CDCl3, 400 MHz): d 7.13 (br s,
1H), 4.82 (m, 2H),4.09 (br s, 1H), 3.03 (dd of ABX system, J = 18.6,
13.0 Hz, 1H), 2.49 (dd of ABX system, J = 18.6, 6.4 Hz, 1H), 2.48 (t,
J = 2.4 Hz, 1H), 2.15 (d, J = 11.9 Hz, 1H), 2.10 (dd of ABX system,
J = 13.0, 6.4 Hz, 1H), 1.80–1.70 (m, 4H), 1.65–1.53 (m, 1H), 1.41
(d, J = 5.8 Hz, 6H), 1.37 (s, 3H), 1.08 (s, 3H). HR-MS: calcd for
Yellow oil, yield 85%. 1H NMR (CDCl3, 400 MHz): d 7.45–7.35
(m, 5H), 7.13 (br s, 1H), 5.29 (d of AB system, J = 12.6 Hz, 1H),
5.25 (d of AB system, J = 12.6 Hz, 1H), 4.06 (br s, 1H), 3.65 (s,
3H), 3.05 (dd of ABX system, J = 18.6, 13.0 Hz, 1H), 2.34 (dd of
ABX system, J = 18.6, 6.4 Hz, 1H), 2.16–2.09 (m, 2H), 1.81–1.63
(m, 4H), 1.62–1.53 (m, 1H), 1.41 (d, J = 6.2 Hz, 6H), 1.37 (s, 3H),
1.05 (s, 3H).
C
23H2735Cl2NO3 ([M+Na]+), 458.1266; found, 458.1242.
Anal. Calcd for C23H27Cl2NO3: C, 63.31; H, 6.24; N, 3.21. Found:
C, 63.26; H, 6.18; N, 2.97.
5.3. General procedure for preparation of compounds 3a–b,
3f–g, 3m (Method A)
5.3.5. (E,1R,4aS,10aR)-9-(Benzyloxy)imino-6,8-dichloro-1,2,3,4,
4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylic acid (3m)
A mixture of the methyl ester 5a–b, 5f–g, 5m (0.1 mmol), KOH
(56 mg, 1 mmol) and 18-crown ether-6 (132 mg, 0.5 mmol) in
MeOH (2 mL) was stirred at 80 °C for 24 h, then cooled, and evap-
orated in vacuo. The residue was diluted with water (10 mL), acid-
ified with 2 N HCl, and extracted with AcOEt. The combined
organic layer was washed with brine, dried over Na2SO4, filtered,
and evaporated in vacuo. The residue was purified by flash chro-
matography using n-hexane/ethyl acetate as the eluent to afford
3a–b, 3f–g, 3m.
Yellow oil, yield 48%. 1H NMR (CDCl3, 400 MHz): d 7.44–7.42
(m, 2H), 7.38–7.30 (m, 3H), 7.11 (br s, 1H), 5.28 (d of AB system,
J = 12.6 Hz, 1H), 5.24 (d of AB system, J = 12.6 Hz, 1H), 4.10 (br s,
1H), 3.06 (dd of ABX system, J = 18.6, 13.0 Hz, 1H), 2.46 (dd of
ABX system, J = 18.6, 6.4 Hz, 1H), 2.14 (d, J = 12.1 Hz, 1H), 2.09
(dd of ABX system, J = 13.0, 6.4 Hz, 1H), 1.79–1.68 (m, 4H), 1.62–
1.52 (m, 1H), 1.39 (d, J = 6.2 Hz, 6H), 1.36 (s, 3H), 1.04 (s, 3H).
HR-MS: calcd for C27H3135Cl2NO3 ([M+Na]+), 510.1579; found,
510.1552.

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8651
5.4. General procedure for preparation of compounds 5c–e, 5h–l
(Method B)
5.4.6. (E,1R,4aS,10aR)-Methyl 9-(cyclopropylmethoxy)imino-
6,8-dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylate (5j)
To a solution of compound 5a (0.1 mmol) in 2 mL DMF was
Colorless oil, yield 85%. 1H NMR (CDCl3, 400 MHz): d 7.13 (br s,
1H), 4.07 (m, 3H), 3.65 (s, 3H), 3.02 (dd of ABX system, J = 18.5,
13.0 Hz, 1H), 2.32 (dd of ABX system, J = 18.5, 6.4 Hz, 1H), 2.16–
2.10 (m, 2H), 1.74–1.70 (m, 4H), 1.61 (m, 1H), 1.41 (d, J = 6.2 Hz,
6H), 1.37 (s, 3H), 1.25 (m, 1H), 1.07 (s, 3H), 0.58–0.54 (m, 2H),
0.37–0.34 (m, 1H), 0.31–0.26 (m, 1H). HR-MS: calcd for
added NaH (6 mg, 0.15 mmol, 60% in mineral oil) at about 0 °C,
in a flask fitted with a CaCl2 tube. The mixture was stirred at 0 °C
for 30 min, the corresponding bromide (0.15 mmol) was added,
and the solution was gradually warmed to rt, then stirred over-
night. The reaction was quenched with saturated aqueous NH4Cl,
and the whole was extracted with AcOEt. The combined organic
layer was washed with H2O and brine, dried over Na2SO4, filtered,
and evaporated in vacuo. The residue was purified by flash chro-
matography using n-hexane/ethyl acetate as the eluent to afford
5c–e, 5h–l.
C
24H3135Cl2NO3 ([M+Na]+), 474.1579; found, 474.1576.
5.4.7. (E,1R,4aS,10aR)-Methyl 9-(cyclohexylmethoxy)imino-6,8-
dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylate (5k)
Light yellow oil, yield 73%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.04 (m, 2H), 3.94 (m, 1H), 3.65 (s, 3H), 3.01 (dd of ABX sys-
tem, J = 18.4, 13.0 Hz, 1H), 2.27 (m, 1H), 2.15–2.08 (m, 2H), 1.85–
1.56 (m, 10H), 1.41 (d, J = 6.5 Hz, 6H), 1.37 (s, 3H), 1.27–1.16 (m,
4H), 1.06 (s, 3H), 1.01–0.92 (m, 2H).
5.4.1. (E,1R,4aS,10aR)-Methyl 6,8-dichloro-9-ethoxyimino-1,2,3,
4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylate (5c)
Yellow oil, yield 56%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br s,
1H), 4.25 (m, 2H), 4.09 (br s, 1H), 3.64 (s, 3H), 2.98 (dd of ABX sys-
tem, J = 18.5, 13.0 Hz, 1H), 2.28 (dd of ABX system, J = 18.5, 6.4 Hz,
1H), 2.12 (m, 2H), 1.85–1.58 (m, 5H), 1.41 (d, J = 7.2 Hz, 6H), 1.36
(s, 3H), 1.34 (t, J = 7.2 Hz, 3H), 1.05 (s, 3H).
5.4.8. (E,1R,4aS,10aR)-Methyl 9-(heptyloxy)imino-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylate (5l)
Light yellow oil, yield 86%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.25–4.08 (m, 3H), 3.65 (s, 3H), 2.99 (dd of ABX system,
J = 18.5, 13.0 Hz, 1H), 2.27 (dd of ABX system, J = 18.5, 6.5 Hz,
1H), 2.15–2.08 (m, 2H), 1.76–1.70 (m, 6H), 1.66–1.56 (m, 1H),
1.41 (d, J = 6.5 Hz, 6H),1.36 (s, 3H), 1.38–1.23 (m, 8H), 1.06 (s,
3H), 0.88 (t, J = 6.6 Hz, 3H).
5.4.2. (E,1R,4aS,10aR)-Methyl 6,8-dichloro-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethyl-9-propoxyiminophe-
nanthrene-1-carboxylate (5d)
Light yellow oil, yield 75%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.22–4.07 (m, 3H), 3.65 (s, 3H), 3.00 (dd of ABX system,
J = 18.4, 13.0 Hz, 1H), 2.28 (dd of ABX system, J = 18.4, 6.4 Hz,
1H), 2.15–2.08 (m, 2H), 1.80–1.66 (m, 6H), 1.63–1.56 (m, 1H),
1.41 (d, J = 5.8 Hz, 6H),1.37 (s, 3H), 1.06 (s, 3H), 0.96 (t, J = 7.4 Hz,
3H).
5.5. General procedure for preparation of compounds 3d–e, 3i–l
(Method C)
Compounds 5d–e, 5i–l (0.1 mmol) was treated with KOBut
(1.5 mmol) in DMSO (1.5 mL) at room temperature for about
40 min. Then the reaction mixture was poured into ice-water, acid-
ified with 2 N HCl and extracted with AcOEt. The combined organic
layer was washed with H2O and brine, dried over Na2SO4, filtered,
and evaporated in vacuo. The residue was purified by flash chro-
matography using n-hexane/ethyl acetate as the eluent to afford
3d–e, 3i–l.
5.4.3. (E,1R,4aS,10aR)-Methyl 6,8-dichloro-1,2,3,4,4a,9,10,10a-
octahydro-9-isopropoxyimino-7-isopropyl-1,4a-dimethylphe-
nanthrene-1-carboxylate (5e)
Light yellow oil, yield 61%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.48–4.41 (m, 1H), 4.10 (br s, 1H), 3.65 (s, 3H), 2.97 (dd of
ABX system, J = 18.4, 13.0 Hz, 1H), 2.25 (dd of ABX system,
J = 18.4, 6.4 Hz, 1H), 2.16–2.08 (m, 2H), 1.76–1.65 (m, 4H), 1.63–
1.56 (m, 1H), 1.41 (d, J = 7.6 Hz, 6H), 1.37 (s, 3H), 1.35 (d,
J = 6.4 Hz, 3H), 1.29 (d, J = 6.4 Hz, 3H), 1.06 (s, 3H).
5.5.1. (E,1R,4aS,10aR)-6,8-Dichloro-1,2,3,4,4a,9,10,10a-octahy-
dro-7-isopropyl-1,4a-dimethyl-9-propoxyiminophenanthrene-
1-carboxylic acid (3d)
Light yellow oil, yield 45%. 1H NMR (CDCl3, 400 MHz): d 7.11 (br
s, 1H), 4.21–4.09 (m, 3H), 3.02 (dd of ABX system, J = 18.5, 13.0 Hz,
1H), 2.42 (dd of ABX system, J = 18.5, 6.4 Hz, 1H), 2.14 (d,
J = 12.0 Hz, 1H), 2.09 (dd of ABX system, J = 13.0, 6.4 Hz, 1H),
1.81–1.69 (m, 6H), 1.63–1.60 (m, 1H), 1.39 (d, J = 5.6 Hz, 6H),1.37
(s, 3H), 1.06 (s, 3H), 0.97 (t, J = 7.4 Hz, 3H). HR-MS: calcd for
5.4.4. (E,1R,4aS,10aR)-Methyl 9-((3-methylbut-2-enyloxy)
imino)-6,8-dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-
1,4a-dimethylphenanthrene-1-carboxylate (5h)
Light yellow solid, yield 83%. 1H NMR (CDCl3, 400 MHz): d 7.12
(br s, 1H), 5.50 (m, 1H), 4.76–4.66 (m, 2H), 4.10 (br s, 1H), 3.64 (s,
3H), 2.98 (dd of ABX system, J = 18.5, 13.0 Hz, 1H), 2.30 (dd of ABX
system, J = 18.5, 6.4 Hz, 1H), 2.15–2.08 (m, 2H), 1.78 (s, 3H), 1.73 (s,
3H), 1.69 (m, 4H), 1.61 (m, 1H), 1.41 (d, J = 6.8 Hz, 6H), 1.36 (s, 3H),
1.06 (s, 3H).
C
23H3135Cl2NO3 ([M+H]+), 440.1759; found, 440.1727. Anal. Calcd
for C23H31Cl2NO3: C, 62.73; H, 7.09; N, 3.18. Found: C, 62.71; H,
7.13; N, 3.16.
5.5.2. (E,1R,4aS,10aR)-6,8-Dichloro-1,2,3,4,4a,9,10,10a-octahy-
dro-9-isopropoxyimino-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylic acid (3e)
5.4.5. (E,1R,4aS,10aR)-Methyl 9-(isopentyloxy)imino-6,8-
dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylate (5i)
Light yellow oil, yield 63%. 1H NMR (CDCl3, 400 MHz): d 7.11 (br
s, 1H), 4.47–4.41 (m, 1H), 4.09 (br s, 1H), 2.99 (dd of ABX system,
J = 18.5, 13.0 Hz, 1H), 2.39 (dd of ABX system, J = 18.5, 6.4 Hz,
1H), 2.14 (d, J = 14.9 Hz, 1H), 2.09 (dd of ABX system, J = 13.0,
6.4 Hz, 1H), 1.79–1.69 (m, 4H), 1.62–1.59 (m, 1H), 1.40 (d,
J = 6.9 Hz, 6H), 1.37 (s, 3H), 1.35 (d, J = 6.2 Hz, 3H), 1.29 (d,
J = 6.2 Hz, 3H), 1.06(s, 3H). HR-MS: calcd for C23H3135Cl2NO3
([M+H]+), 440.1759; found, 440.1740. Anal. Calcd for
Light yellow oil, yield 74%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.25 (m, 2H), 4.10 (m, 1H), 3.65 (s, 3H), 2.99 (dd of ABX sys-
tem, J = 18.5, 13.0 Hz, 1H), 2.32–2.23 (m, 1H), 2.15–2.04 (m, 2H),
1.78–1.55 (m, 8H), 1.41 (d, J = 6.0 Hz, 6H), 1.36 (s, 3H), 1.06 (s,
3H), 0.95 (d, J = 6.5 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H). HR-MS: calcd
for C25H3535Cl2NO3 ([M+Na]+), 490.1892; found, 490.1891.

8652
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
C23H31Cl2NO3: C, 62.73; H, 7.09; N, 3.18. Found: C, 62.47; H, 7.14;
N, 3.02.
5.6.1. (E,1R,4aS,10aR)-6,8-Dichloro-9-ethoxyimino-1,2,3,4,4a,
9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3c)
5.5.3. (E,1R,4aS,10aR)-9-(Isopentyloxy)imino-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphe-
nanthrene-1-carboxylic acid (3i)
Yellow oil, yield 57%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br s,
1H), 4.26 (m, 2H), 4.09 (br s, 1H), 3.00 (dd of ABX system,
J = 18.5, 13.1 Hz, 1H), 2.42 (dd of ABX system, J = 18.5, 6.4 Hz,
1H), 2.16–2.07 (m, 2H), 1.79–1.57 (m, 5H), 1.41 (d, J = 6.0 Hz,
6H), 1.37 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.06 (s, 3H). HR-MS: calcd
for C22H2935Cl2NO3 ([M+H]+), 426.1602; found, 426.1602.
Anal. Calcd for C22H29Cl2NO3: C, 61.97; H, 6.86; N, 3.29. Found:
C, 62.28; H, 6.85; N, 2.90.
Light yellow oil, yield 24%. 1H NMR (CDCl3, 400 MHz): d 7.11 (br
s, 1H), 4.25 (m, 2H), 4.09 (m, 1H), 3.01 (dd of ABX system, J = 18.5,
13.0 Hz, 1H), 2.40 (dd of ABX system, J = 18.5, 6.4 Hz, 1H), 2.14 (d,
J = 12.0 Hz, 1H), 2.09 (dd of ABX system, J = 13.0, 6.4 Hz, 1H), 1.79–
1.58 (m, 8H), 1.39 (d, J = 6.0 Hz, 6H), 1.37 (s, 3H), 1.06 (s, 3H), 0.95
(d, J = 6.5 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H).
Anal. Calcd for C25H35Cl2NO3: C, 64.10; H, 7.53; N, 2.99. Found:
C, 63.95; H, 7.61; N, 2.85.
5.6.2. (E,1R,4aS,10aR)-9-((3-Methylbut-2-enyloxy)imino)-6,8-
dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylic acid (3h)
5.5.4. (E,1R,4aS,10aR)-9-(Cyclopropylmethoxy)imino-6,8-dichloro
-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethyl
phenanthrene-1-carboxylic acid (3j)
Yellow oil, yield 57%. 1H NMR (CDCl3, 400 MHz): d 7.11 (br s,
1H), 5.52 (m, 1H), 4.77–4.67 (m, 2H), 4.08 (br s, 1H), 2.99 (dd of
ABX system, J = 18.6, 13.0 Hz, 1H), 2.43 (dd of ABX system,
J = 18.5, 6.4 Hz, 1H), 2.16–2.05 (m, 2H), 1.78 (s, 3H), 1.73 (s, 3H),
1.71 (m, 4H), 1.59 (m, 1H), 1.41 (d, J = 6.8 Hz, 6H), 1.36 (s, 3H),
1.07 (s, 3H). Anal. Calcd for C25H33Cl2NO3: C, 64.37; H, 7.13; N,
3.00. Found: C, 64.47; H, 7.23; N, 2.91.
Colorless oil, yield 43%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br s,
1H), 4.09–3.97 (m, 3H), 3.04 (dd of ABX system, J = 18.5, 13.0 Hz,
1H), 2.45 (dd of ABX system, J = 18.5, 6.4 Hz, 1H), 2.14 (d,
J = 13.4 Hz, 1H), 2.10 (dd of ABX system, J = 13.0, 6.4 Hz, 1H),
1.79–1.72 (m, 4H), 1.62 (m, 1H), 1.40–1.38 (m, 9H), 1.25 (m, 1H),
1.07 (s, 3H), 0.58–0.54 (m, 2H), 0.39–0.32 (m, 1H), 0.31–0.27 (m,
1H).
5.6.3. 13-Isopropyl-12,14-dichloro-7-oxopodocarpe-8,11,13-
triene-15-carboxylic acid (1m)
Anal. Calcd for C24H31Cl2NO3ꢀ1/4H2O: C, 63.09; H, 6.95; N, 3.07.
To a solution of CrO3 (30 mg, 0.298 mmol) in Ac2O (9 mL) and
AcOH (2 mL) was added dropwise a suspension of 1b (100 mg,
0.271 mmol) in AcOH (4 mL) at 0 °C over 10 min. The reaction mix-
ture was stirred at 50 °C for 9 h, then cooled, and poured into ice-
water (20 mL), and the whole was extracted with CHCl3 3 times.
The combined organic layer was washed with water, saturated
aqueous NaHCO3, and brine, and dried over Na2SO4. The crude mix-
ture was purified by flash chromatography (n-hexane/AcOEt = 4:1)
to afford 1m (38 mg, yield 36%) as a colorless oil. 1H NMR (CDCl3,
400 MHz): d 7.22 (br s, 1H), 3.91 (br s, 1H), 2.74–2.64 (m, 2H),
2.6–2.53 (m, 1H), 2.19 (d, J = 12.2 Hz, 1H), 1.82–1.75 (m, 5H),
1.38 (d, J = 7.2 Hz, 6H), 1.33 (s, 3H), 1.15 (s, 3H). HR-MS: calcd for
Found: C, 62.92; H, 6.88; N, 3.09.
5.5.5. (E,1R,4aS,10aR)-9-(Cyclohexylmethoxy)imino-6,8-
dichloro-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylic acid (3k)
Light yellow oil, yield 50%. 1H NMR (CDCl3, 400 MHz): d 7.12 (br
s, 1H), 4.05 (m, 2H), 3.95 (dd of ABX system, J = 10.6, 7.1 Hz, 1H),
3.03 (dd of ABX system, J = 18.5, 13.0 Hz, 1H), 2.40 (dd of ABX sys-
tem, J = 18.5, 6.5 Hz, 1H), 2.14 (d, J = 12.2 Hz, 1H), 2.09 (dd of ABX
system, J = 13.0, 6.5 Hz, 1H), 1.83–1.55 (m, 10H), 1.40 (d, J = 6.5 Hz,
6H), 1.37 (s, 3H), 1.27–1.17 (m, 4H), 1.06 (s, 3H), 0.98 (m, 2H).
Anal. Calcd for C27H37Cl2NO3ꢀ1/4H2O: C, 64.99; H, 7.57; N, 2.81.
Found: C, 65.06; H, 7.45; N, 2.79.
C
20H2435Cl2O3 ([M+Na]+), 405.1001; found, 405.1012.
Anal. Calcd for C20H24Cl2O3ꢀ1/4H2O: C, 61.94; H, 6.37. Found: C,
61.74; H, 6.25.
5.5.6. (E,1R,4aS,10aR)-9-(Heptyloxy)imino-6,8-dichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphe-
nanthrene-1-carboxylic acid (3l)
5.7. DHAA series of compounds
Light yellow oil, yield 42%. 1H NMR (CDCl3, 400 MHz): d 7.11 (br
s, 1H), 4.25–4.07 (m, 3H), 3.02 (dd of ABX system, J = 18.5, 13.0 Hz,
1H), 2.41 (dd of ABX system, J = 18.5, 6.4 Hz, 1H), 2.14 (d,
J = 12.1 Hz, 1H), 2.08 (dd of ABX system, J = 13.0, 6.4 Hz, 1H),
1.79–1.62 (m, 6H), 1.60 (m, 1H), 1.40–1.33 (m, 17H), 1.06 (s, 3H),
0.88 (t, J = 6.7 Hz, 3H). HR-MS: calcd for C27H3935Cl2NO3 ([M+H]+),
496.2385; found, 496.2377.
5.7.1. Dehydroabietic acid methyl ester (1e)
A mixture of dehydroabietic acid 1a (15 g, 0.050 mol) and con-
centrated H2SO4 (24 mL) in MeOH (300 mL) was stirred at 85 °C for
48 h, then cooled, and evaporated in vacuo. The residue was di-
luted with water (300 mL) and extracted with AcOEt 3 times. The
combined organic layer was washed with brine, dried over Na2SO4,
filtered, and evaporated in vacuo. The residue was purified by flash
chromatography (n-hexane/AcOEt = 10:1) to afford 1e (14.3 g,
yield 91%) as a light yellow solid. 1H NMR (CDCl3, 400 MHz): d
7.17 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.89 (s, 1H), 3.67
(s, 3H), 2.89 (m, 2H), 2.83 (m, 1H), 2.30 (d, J = 12.1 Hz, 1H), 2.25
(dd of ABX system, J = 12.4, 2.1 Hz, 1H), 1.89–1.61 (m, 5H), 1.50
(m, 1H), 1.40 (m, 1H), 1.28 (s, 3H), 1.23 (d, J = 6.9 Hz, 6H), 1.22 (s,
3H).
Anal. Calcd for C27H39Cl2NO3: C, 65.31; H, 7.92; N, 2.82. Found:
C, 65.75; H, 7.85; N, 2.85.
5.6. Preparation of compounds 3c and 3h (Method D)
A mixture of the methyl ester 5c/5h (0.048 mmol), KOH (54 mg,
0.96 mmol) and 18-crown ether-6 (63 mg, 0.21 mmol) in MeOH
(1 mL) was stirred at 145 °C for 70 min under microwave irradia-
tion, then cooled, and evaporated in vacuo. The residue was diluted
with water (10 mL), acidified with 2 N HCl, and extracted with
AcOEt. The combined organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated in vacuo. The residue was
purified by flash chromatography using n-hexane/ethyl acetate as
the eluent to afford 3c and 3h.
5.7.2. 13-Isopropyl-7-oxopodocarpe-8,11,13-triene-15-carbox-
ylic acid methyl ester (1f, same procedure as for synthesis of 1d)
Light yellow oil, yield 48%. 1H NMR (CDCl3, 400 MHz): d 7.86 (d,
J = 2.1 Hz, 1H), 7.41 (dd, J = 8.2, 2.1 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H),
3.65(s, 3H), 2.92 (m, 1H), 2.72 (dd, J = 7.3, 3.8 Hz, 2H), 2.38–2.32
(m, 2H), 1.82–1.77 (m, 3H), 1.73–1.64 (m, 2H), 1.34 (s, 3H), 1.26–
1.24 (m, 9H).

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8653
5.7.3. 13-Isopropyl-7-hydroxyiminepodocarpe-8,11,13-triene-
15-carboxylic acid methyl ester (5n, same procedure as for
synthesis of 5a)
(m, 1H), 1.23 (s, 3H), 1.18 (d, J = 6.9 Hz, 6H), 1.03 (s, 3H). HR-MS:
calcd for C20H27NO3 ([M+Na]+), 352.1889; found, 352.1885.
Anal. Calcd for C20H27NO3ꢀ1/4H2O: C, 71.93; H, 8.30; N, 4.19.
Found: C, 72.08; H, 8.31; N, 4.09.
Colorless solid, yield 79%. 1H NMR (CDCl3, 400 MHz): d 7.69 (s,
1Y, 7.21 m, 2H), 3.66 (s, 3H), 2.91(m, 1H), 2.68–2.65 (m, 2H),
2.36–2.28 (m, 2H), 1.77–1.61 (m, 5H), 1.38 (s, 3H), 1.25 (d,
J = 6.9 Hz, 6H), 1.12 (s, 3H).
5.9. Synthesis of compounds 3o–s (Method A)
5.9.1. (E,1R,4aS,10aR)-1,2,3,4,4a,9,10,10a-Octahydro-7-
isopropyl-9-methoxyimino-1,4a-dimethylphenanthrene-1-
carboxylic acid (3o)
5.8. Synthesis of compounds 5o–s
Starting from compound 5n, Method B was employed to obtain
compounds 5o–s.
Colorless solid, yield 93%. Mp 108–110 °C. 1H NMR (CDCl3,
400 MHz): d 7.75 (s, 1H), 7.19 (br s, 2H), 4.02 (s, 3H), 2.90 (m,
1H), 2.65 (m, 2H), 2.26 (m, 2H), 1.76 (m, 4H), 1.64 (m, 1H),1.36
(s, 3H), 1.25 (d, J = 6.9 Hz, 3H), 1.24 (d, J = 6.9 Hz, 3H), 1.10 (s, 3H).
Anal. Calcd for C21H29NO3: C, 73.44; H, 8.51; N, 4.08. Found: C,
73.04; H, 8.55; N, 3.99.
5.8.1. (E,1R,4aS,10aR)-Methyl 1,2,3,4,4a,9,10,10a-octahydro-7-
isopropyl-9-methoxyimino-1,4a-dimethylphenanthrene-1-
carboxylate (5o)
Colorless oil, yield 91%. 1H NMR (CDCl3, 400 MHz): d 7.76 (s,
1H), 7.19 (d, J = 1.2 Hz, 2H), 4.00 (s, 3H), 3.64 (s, 3H), 2.91 (m,
1H), 2.56 (m, 2H), 2.32–2.26 (m, 2H), 1.76–1.60 (m, 5H), 1.36 (s,
3H), 1.26 (d, J = 6.9 Hz, 3H), 1.25 (d, J = 6.9 Hz, 3H), 1.10 (s, 3H).
5.9.2. (E,1R,4aS,10aR)-9-(Allyloxy)imino-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-carbox-
ylic acid (3p)
Light yellow oil, yield 84%. 1H NMR (CDCl3, 400 MHz): d 7.73 (s,
1H), 7.18 (m, 2H), 6.14–6.05 (m, 1H), 5.36 (dd, J = 17.3, 1.2 Hz, 1H),
5.23 (dd, J = 10.4, 1.2 Hz, 1H), 4.72 (d, J = 5.6 Hz, 2H), 2.90 (m, 1H),
2.68 (m,2H), 2.31–2.27 (m, 2H), 1.77 (m, 4H), 1.64–1.61 (m, 1H),
1.36 (s, 3H), 1.24 (d, J = 6.9 Hz, 3H), 1.23 (d, J = 6.9 Hz, 3H), 1.11
(s, 3H). HR-MS: calcd for C23H31NO3 ([M+Na]+), 392.2202; found,
392.2155.
5.8.2. (E,1R,4aS,10aR)-Methyl 9-(allyloxy)imino-1,2,3,4,4a,
9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylate (5p)
Light yellow oil, yield 92%. 1H NMR (CDCl3, 400 MHz): d 7.75 (s,
1H), 7.20 (m, 2H), 6.13–6.04 (m, 1H), 5.33 (dd, J = 17.2, 1.6 Hz, 1H),
5.23 (dd, J = 10.4, 1.6 Hz, 1H), 4.72 (d, J = 5.6 Hz, 2H), 3.64 (s, 3H),
2.92 (m, 1H), 2.63 (m,2H), 2.34–2.27 (m, 2H), 1.76–1.60 (m, 5H),
1.37 (s, 3H), 1.25 (d, J = 7.0 Hz, 6H), 1.11 (s, 3H).
Anal. Calcd for C23H31NO3: C, 74.76; H, 8.46; N, 3.79. Found: C,
74.51; H, 8.48; N, 3.69.
5.8.3. (E,1R,4aS,10aR)-Methyl 9-((prop-2-ynyloxy)imino)-1,2,3,
4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylate (5q)
5.9.3. (E,1R,4aS,10aR)-9-((Prop-2-ynyloxy)imino)-1,2,3,4,4a,9,10,
10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3q)
Light yellow oil, yield 70%. 1H NMR (CDCl3, 400 MHz): d 7.77 (s,
1H), 7.20 (m, 2H), 4.80 (d, J = 2.4 Hz, 2H), 3.65 (s, 3H), 2.91 (m, 1H),
2.63–2.59 (m, 2H), 2.49 (t, J = 2.4 Hz, 1H), 2.33–2.26 (m, 2H), 1.76–
1.59 (m, 5H), 1.36 (s, 3H), 1.25 (d, J = 6.9 Hz, 3H), 1.24 (d, J = 6.9 Hz,
3H), 1.11 (s, 3H).
Red brown oil, yield 57%. 1H NMR (CDCl3, 400 MHz): d 7.75 (s,
1H), 7.19 (m, 2H), 4.81 (d, J = 2.4 Hz, 2H), 2.90 (m, 1H), 2.69 (m,
2H), 2.50 (t, J = 2.4 Hz, 1H), 2.31–2.26 (m, 2H), 1.77 (br s, 4H),
1.63 (m, 1H), 1.36 (s, 3H), 1.25 (d, J = 6.9 Hz, 3H), 1.24 (d,
J = 6.9 Hz, 3H), 1.11 (s, 3H).
Anal. Calcd for C23H29NO3ꢀ1/4H2O: C, 74.26; H, 7.99; N, 3.77.
5.8.4. (E,1R,4aS,10aR)-Methyl 9-(benzyloxy)imino-1,2,3,4,4a,
9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylate (5r)
Found: C, 74.33; H, 8.01; N, 3.65.
5.9.4. (E,1R,4aS,10aR)-9-(Benzyloxy)imino-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3r)
Light yellow oil, yield 77%. 1H NMR (CDCl3, 400 MHz): d 7.79 (s,
1H), 7.40 (m, 5H), 7.22 (m, 2H), 5.28 (s, 2H), 3.64 (s, 3H), 2.92 (m,
1H), 2.65 (m, 2H), 2.36–2.29 (m, 2H), 1.76–1.62 (m, 5H), 1.37 (s,
3H), 1.27 (d, J = 6.9 Hz, 6H), 1.13 (s, 3H).
Light yellow oil, yield 91%. 1H NMR (CDCl3, 400 MHz): d 7.75 (s,
1H), 7.42 (m, 2H), 7.36 (m, 2H), 7.29 (m, 1H), 7.19 (br s, 2H), 5.25 (s,
2H), 2.90 (m, 1H), 2.77 (dd of ABX system, J = 18.8, 5.2 Hz, 1H), 2.66
(dd of ABX system, J = 18.8, 13.2 Hz, 1H), 2.33–2.27 (m, 2H), 1.77
(br s, 4H), 1.62 (m, 1H), 1.36 (s, 3H), 1.25 (d, J = 6.9 Hz, 6H), 1.11
(s, 3H).
5.8.5. (E,1R,4aS,10aR)-Methyl 9-((3-phenylpropoxy)imino)-1,2,3,
4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanth-
rene-1-carboxylate (5s)
Colorless oil, 60% Yield. 1H NMR (CDCl3, 400 MHz): d 7.78 (s,
1H), 7.33–7.13 (m, 7H), 4.24 (t, J = 6.5 Hz, 2H), 3.65 (s, 3H), 2.92
(m, 1H), 2.76 (m, 2H), 2.65 (m, 2H), 2.31 (m, 2H), 2.07 (m,
2H),1.77 (m, 5H), 1.39 (s, 3H), 1.27 (d, J = 7.0 Hz, 6H), 1.13 (s, 3H).
Anal. Calcd for C27H33NO3: C, 77.29; H, 7.93; N, 3.34. Found: C,
77.09; H, 7.95; N, 3.32.
5.9.5. (E,1R,4aS,10aR)-9-((3-Phenylpropoxy)imino)-1,2,3,4,4a,9,
10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3s)
5.8.6. (E,1R,4aS,10aR)-1,2,3,4,4a,9,10,10a-Octahydro-9-
(hydroxyimino)-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3n)
Compound 3n was obtained from 5n according to Method C.
Colorless solid, yield 79%. Mp 270–271 °C. 1H NMR (CDCl3,
400 MHz): d 12.32 (s, 1H), 11.16 (s, 1H), 7.68 (d, J = 1.6 Hz, 1H),
7.24 (d, J = 8.2 Hz, 1H), 7.20 (dd, J = 8.2, 1.6 Hz, 1H), 2.85 (m, 1H),
2.56 (dd of ABX system, J = 18.6, 4.8 Hz, 1H), 2.41 (dd of ABX sys-
tem, J = 18.6, 13.6 Hz, 1H), 2.30 (d, J = 13.0 Hz, 1H), 2.07 (dd of
ABX system, J = 13.6,4.8 Hz, 1H), 1.68–1.63 (m, 4H), 1.48–1.42
Colorless oil, 77% Yield. 1H NMR (CDCl3, 400 MHz): d 7.78 (s,
1H), 7.31–7.17 (m, 7H), 4.25 (t, J = 6.5 Hz, 2H), 2.93 (m, 1H), 2.75
(m, 3H), 2.64 (dd of ABX system, J = 18.8, 13.2 Hz, 1H), 2.30 (d,
J = 5.2 Hz, 2H), 2.08 (m, 2H),1.76 (br s, 4H), 1.64 (m, 1H), 1.38 (s,
3H), 1.28 (d, J = 6.9 Hz, 3H), 1.27 (d, J = 6.9 Hz, 3H), 1.14 (s, 3H).
HR-MS: calcd for C29H37NO3 ([M+H]+), 448.2851; found, 448.2832.
Anal. Calcd for C29H37NO3: C, 77.82; H, 8.33; N, 3.13. Found: C,
77.56; H, 8.45; N, 3.12.

8654
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
5.9.6. 13-Isopropyl-7-oxopodocarpe-8,11,13-triene-15-carboxylic
acid (1n, Method C)
5.11. Synthesis of compounds 5u–v (Method B)
Colorless solid, yield 79%. Mp 90–91 °C. 1H NMR (CDCl3,
400 MHz): d 7.87 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 8.2, 2.1 Hz, 1H),
7.29 (d, J = 8.2 Hz, 1H), 2.91 (m, 1H), 2.71 (m, 2H), 2.49 (d,
J = 15.0 Hz, 1H), 2.36 (d, J = 12.5 Hz, 1H), 1.86–1.77 (m, 4H), 1.64
(m, 1H), 1.35 (s, 3H), 1.26 (s, 3H), 1.24 (d, J = 6.9 Hz, 3H), 1.23 (d,
J = 6.9 Hz, 3H). HR-MS: calcd for C20H26O3 ([M+Na]+), 337.1780;
found, 337.1781.
5.11.1. (E,1R,4aS,10aR)-Methyl 6-bromo-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-9-methoxyimino-1,4a-dimethyl
phenanthrene-1-carboxylate (5u)
Colorless oil, yield 73%. 1H NMR (CDCl3, 400 MHz): d 7.80 (s,
1H), 7.40 (s, 1H), 3.99 (s, 3H), 3.62 (s, 3H), 3.29 (m, 1H), 2.54 (m,
2H), 2.27–2.20 (m, 2H), 1.78–1.69 (m, 4H), 1.62 (m, 1H), 1.34 (s,
3H), 1.27 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 6.8 Hz, 3H), 1.09 (s, 3H).
Anal. Calcd for C20H26O3ꢀ1/8H2O: C, 75.86; H, 8.36. Found: C,
75.71; H, 8.09.
5.11.2. (E,1R,4aS,10aR)-Methyl 9-(allyloxy)imino-6-bromo-1,2,3,
4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethyl
phenanthrene-1-carboxylate (5v)
5.10. 12-Bromo-DHAA series
Light yellow oil, yield 70%. 1H NMR (CDCl3, 400 MHz): d 7.80 (s,
1H), 7.40 (s, 1H), 6.12–6.02 (m, 1H), 5.33 (m, 1H), 5.23 (m, 1H),
4.71 (dt, J = 5.8, 1.3 Hz, 2H), 3.64 (s, 3H), 3.29 (m, 1H), 2.58 (d,
J = 9.4 Hz, 2H), 2.29–2.21 (m, 2H), 1.75–1.69 (m, 4H), 1.65–1.58
(m, 1H), 1.35 (s, 3H), 1.26 (d, J = 6.9 Hz, 3H), 1.23 (d, J = 6.9 Hz,
3H), 1.10 (s, 3H).
5.10.1. (1R,4aS,10aR)-Methyl 6-bromo-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylate (1g)
To a stirred solution of 1e (13.7 g, 43.8 mmol) in dry CH3CN
(350 mL) was added N-bromosuccinimide (NBS, 8.57 g, 48.2 mmol)
and Montmorillonite K10 (42.48 g, 0.97 g/mmol) at rt under pro-
tection from light. After 24 h, the catalyst was removed by filtra-
tion and the solid was washed with AcOEt at least three times.
The combined filtrates were washed with saturated aqueous
Na2SO3, brine and dried over Na2SO4. The crude mixture was puri-
fied by flash chromatography (n-hexane/AcOEt = 10:1) to afford a
mixture of 1g and 1h (11.9 g, conversion rate 69%, 1g/1h = 3.1:1)
as a yellow solid. Compound 1g: 1H NMR (CDCl3, 400 MHz): d
7.36 (s, 1H), 6.91 (s, 1H), 3.66 (s, 3H), 3.29–3.22 (m, 1H), 2.85–
2.81 (m, 2H), 2.24 (d, J = 12.3 Hz, 1H), 2.18 (dd of ABX system,
J = 12.5, 2.1 Hz, 1H), 1.86–1.65 (m, 5H), 1.56–1.34 (m, 2H), 1.27
(s, 3H), 1.21 (d, J = 7.1 Hz, 3H), 1.20 (s, 3H), 1.20 (d, J = 7.1 Hz, 3H).
5.12. Synthesis of compounds 3u–v (Method A)
5.12.1. (E,1R,4aS,10aR)-6-Bromo-1,2,3,4,4a,9,10,10a-octahydro-
7-isopropyl-9-methoxyimino-1,4a-dimethylphenanthrene-1-
carboxylic acid (3u)
Colorless solid, yield 90%. Mp 128–131.5 °C. 1H NMR (CDCl3,
400 MHz): d 7.80 (s, 1H), 7.39 (s, 1H), 4.01 (s, 3H), 3.29 (m, 1H),
2.69–2.54 (m, 2H), 2.24 (m, 2H), 1.76 (m, 4H), 1.61 (m, 1H), 1.34
(s, 3H), 1.27 (d, J = 6.9 Hz, 3H), 1.24 (d, J = 6.9 Hz, 3H), 1.10 (s,
3H). HR-MS: calcd for C21H2879BrNO3 ([M+Na]+), 444.1151; found,
444.1152.
Anal. Calcd for C21H28BrNO3ꢀ1/4H2O: C, 59.09; H, 6.73; N, 3.28.
5.10.2. (1R,4aS,10aR)-Methyl 6-bromo-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethyl-9-oxophenanthrene-1-
carboxylate (1i, same procedure as 1d)
Found: C, 58.91; H, 6.60; N, 3.16.
Colorless oil, yield 83%. 1H NMR (CDCl3, 400 MHz): d 7.87 (s,
1H), 7.51 (s, 1H), 3.62 (s, 3H), 3.32–3.22 (m, 1H), 2.68–2.65 (m,
2H), 2.36–2.31 (m, 2H), 1.78–1.67 (m, 4H), 1.64–1.58 (m, 1H),
1.30 (s, 3H), 1.24 (s, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d,
J = 7.0 Hz, 3H).
5.12.2. (E,1R,4aS,10aR)-9-(Allyloxy)imino-6-bromo-1,2,3,4,4a,
9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3v)
Light yellow oil, yield 72%. 1H NMR (CDCl3, 400 MHz): d 7.79 (s,
1H), 7.39 (s, 1H), 6.13–6.03 (m, 1H), 5.33 (m, 1H), 5.23 (m, 1H),
4.72 (dt, J = 5.8, 1.3 Hz, 2H), 3.29 (m, 1H), 2.74–2.56 (m, 2H),
2.27–2.21 (m, 2H), 1.76 (m, 4H), 1.61(m, 1H), 1.34 (s, 3H), 1.26
(d, J = 6.8 Hz, 3H), 1.23 (d, J = 6.8 Hz, 3H), 1.10 (s, 3H).
Anal. Calcd for C23H30BrNO3: C, 61.61; H, 6.74; N, 3.12. Found: C,
61.39; H, 6.71; N, 2.75.
5.10.3. (E,1R,4aS,10aR)-Methyl 6-bromo-1,2,3,4,4a,9,10,10a-
octahydro-9-(hydroxyimino)-7-isopropyl-1,4a-dimethyl
phenanthrene-1-carboxylate (5t, same procedure as 5a)
Light yellow solid, yield 97%. 1H NMR (CDCl3, 400 MHz): d 7.75
(s, 1H), 7.52 (s, 1H), 3.65 (s, 3H), 3.30 (m, 1H), 2.64–2.62 (m, 2H),
2.29 (dd of ABX system, J = 10.4, 8.1 Hz, 1H), 2.24 (d, J = 12.4 Hz,
1H), 1.77–1.62 (m, 5H), 1.36 (s, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.23
(d, J = 6.8 Hz, 3H), 1.12 (s,3H).
5.12.3. (1R,4aS,10aR)-6-Bromo-1,2,3,4,4a,9,10,10a-octahydro-7-
isopropyl-1,4a-dimethyl-9-oxophenanthrene-1-carboxylic acid
(1p)
Compound 1i (84 mg, 0.206 mmol) was treated with KOBut
(3.1 mmol) in DMSO (3.1 mL) at rt for about 40 min. The reaction
mixture was poured into ice-water, acidified with 2 N HCl and ex-
tracted with AcOEt. The combined organic layer was washed with
H2O and brine, dried over Na2SO4, filtered, and evaporated in va-
cuo. The crude mixture was purified by flash chromatography (n-
hexane/AcOEt = 2:1) to afford 1p (12 mg, 15% yield) as a yellow
oil. 1H NMR (CDCl3, 400 MHz): d 7.90 (s, 1H), 7.53 (s, 1H), 3.31
(m, 1H), 2.69 (m, 2H), 2.48 (d, J = 15.2 Hz, 1H), 2.31 (d,
J = 12.6 Hz, 1H), 1.81 (m, 4H), 1.64 (m, 1H), 1.34 (s, 3H), 1.27 (s,
3H), 1.25 (d, J = 6.9 Hz, 3H), 1.22 (d, J = 6.9 Hz, 3H). HR-MS: calcd
for C20H2579BrO3 ([M+H]+), 393.1065; found, 393.1034. Anal. Calcd
for C20H25BrO3: C, 61.07; H, 6.41. Found: C, 61.30; H, 6.49.
5.10.4. (E,1R,4aS,10aR)-6-Bromo-1,2,3,4,4a,9,10,10a-octahydro-
9-(hydroxyimino)-7-isopropyl-1,4a-dimethylphenanthrene-1-
carboxylic acid (3t, Method C)
Colorless solid, yield 52%. Mp 268–271 °C. 1H NMR (CDCl3,
400 MHz): d 7.56 (s, 1H), 7.42 (s, 1H), 3.24 (m, 1H), 2.84 (dd of
ABX system, J = 19.2, 5.0 Hz, 1H), 2.69 (dd of ABX system, J = 19.2,
13.4 Hz, 1H), 2.32 (dd of ABX system, J = 13.4, 5.0 Hz, 1H), 2.24
(d, J = 12.7 Hz,1H), 1.78 (m, 4H), 1.63–1.57 (m, 1H), 1.39 (s, 3H),
1.13 (d, J = 7.2 Hz, 6H), 1.12 (s,3H).
HR-MS: calcd for C20H2679BrNO3 ([M+Na]+), 408.1174; found,
408.1165.

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8655
5.13. 11,12,14-Trichloro-DHAA series
5.14. General procedure for preparation of compounds 5x–y
5.13.1. 11,12,14-Trichlorodehydroabietic acid (1j)
To a solution of compound 5w (0.1 mmol) in 2 mL DMF was
added NaH (6 mg, 0.15 mmol, 60% in mineral oil) at about 0 °C,
in a flask fitted with a CaCl2 tube. The mixture was stirred at 0 °C
for 30 min, then the corresponding bromide (0.15 mmol) was
added. The solution was gradually warmed to rt and stirred over-
night. The reaction was quenched with saturated aqueous NH4Cl,
and then the whole was extracted with AcOEt. The combined or-
ganic layer was washed with H2O and brine, dried over Na2SO4, fil-
tered, and evaporated in vacuo. The residue was purified by flash
chromatography using n-hexane/ethyl acetate as the eluent to af-
ford 5x–y.
To a solution of 1a (2.008 g, 6.689 mmol) in CCl4 (7 mL) was
added FeCl3 (503 mg, 3.10 mmol) and 1% DDQ on SiO2
(119.1 mg). The reaction mixture was stirred at rt, and a solution
of Cl2 in CCl4 (2.4 M, 40 mL) was added in one portion at 0 °C in
the dark. Stirring was continued at rt for 17 h, then the solution
was poured into
a saturated aqueous solution of Na2SO3
(100 mL), and extracted with CHCl3 (70 ꢂ 3 mL). The organic layer
was washed with water (20 mL), dried over Na2SO4 and evaporated
in vacuo to give a residue, which was purified by flash chromatog-
raphy (n-hexane/AcOEt = 7:1) to afford 1j (0.973 g, 2.34 mmol,
yield 35%) as a colorless powder. Mp: 252.8–253.0 °C (recrystal-
lized from AcOEt/n-hexane). Colorless needles.
5.14.1. (E,1R,4aS,10aR)-Methyl 5,6,8-trichloro-1,2,3,4,4a,9,10,
10a-octahydro-7-isopropyl-9-methoxyimino-1,4a-dimethyl-
phenanthrene-1-carboxylate (5x)
1H NMR (CDCl3, 400 MHz): d 4.01 (m, 1H), 3.45 (d, J = 1.24 Hz,
1H), 2.90–2.81 (m, 2H), 2.06 (d, J = 11.2 Hz, 1H), 1.90–1.60 (m,
5H), 1.59–1.51 (m, 1H), 1,48 (s, 3H), 1.38 (d, J = 7.20 Hz, 6H), 1.29
(s, 3H), 1.25–1.10 (m, 1H). 13C NMR (CDCl3, 100 MHz): 184.9,
146.1, 141.3, 134.6, 48.2, 46.7, 40.9, 36.1, 34.9, 32.3, 21.5, 19.2,
Yellow oil, yield 99%. 1H NMR (CDCl3, 400 MHz): d 4.07 (br s,
1H), 3.97 (s, 3H), 3.67 (s, 3H), 3.40 (m, 1H), 2.92 (dd of ABX system,
J = 18.5, 13.7 Hz, 1H), 2.28 (dd of ABX system, J = 18.5, 5.6 Hz, 1H),
2.18 (dd of ABX system, J = 13.7, 5.6 Hz, 1H), 1.69–1.39 (m, 5H),
1.42 (m, 6H), 1.35 (s, 3H), 1.26 (s, 3H).
19.0, 18.6, 16.9. HR-MS: calcd for
C
20H2435Cl3O2 [M-H]ꢁ
401.0842; found, 401.0844. Anal. Calcd for C20H25Cl3O2: C; 59.49,
H; 6.24. Found: C; 59.24, H; 6.24.
5.14.2.(E,1R,4aS,10aR)-Methyl9-(allyloxy)imino-5,6,8-trichloro-
1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethyl
phenanthrene-1-carboxylate(5y)
5.13.2. 11,12,14-Trichlorodehydroabietic acid methyl ester (1k)
To a solution of 1j (300 mg, 0.74 mmol) in MeOH (1 mL) and
PhMe (2 mL) was added dropwise 2.0 M TMSCHN2 in Et2O
(0.5 mL, 1 mmol) at rt over 5 min, and the whole was stirred at rt
for 30 min. Excess TMSCHN2 was quenched with AcOH, then the
reaction mixture was evaporated in vacuo, and the residue was
purified by flash chromatography (n-hexane only to n-hexane/
AcOEt = 10:1) to afford 1k (292 mg, yield 94%) as a colorless solid.
1H NMR (CDCl3, 400 MHz): d 4.02 (m, 1H), 3.69 (s, 3H), 3.47 (m,
1H), 2.89–2.74 (m, 2H), 2.07 (d, J = 11.6 Hz, 1H), 1.76–1.58 (m,
6H), 1.48 (s, 3H), 1.41 (s, 3H), 1.40 (s, 3H), 1.29 (s, 3H), 1.15 (m, 1H).
Colorless prisms, yield 100%. 1H NMR (CDCl3, 400 MHz): d 6.09–
6.00 (m, 1H), 5.26 (m, 2H), 4.67 (m, 2H), 4.09 (br s, 1H), 3.67 (s, 3H),
3.41 (d, J = 12.4 Hz, 1H), 2.96 (dd of ABX system, J = 18.5, 13.7 Hz,
1H), 2.31 (dd of ABX system, J = 18.5, 5.7 Hz, 1H), 2.20 (dd of ABX
system, J = 13.7, 5.7 Hz, 1H), 1.69–1.62 (m, 5H), 1.42 (d, J = 6.5 Hz,
6H), 1.36 (s, 3H), 1.27 (s, 3H).
5.15. General procedure for preparation of compounds 3w–y
A mixture of the methyl ester 5w–y (0.048 mmol), KOH (54 mg,
0.96 mmol) and 18-crown ether-6 (63 mg, 0.21 mmol) in MeOH
(1 mL) was stirred at 145 °C for 70 min under microwave irradia-
tion, then cooled, and evaporated in vacuo. The residue was diluted
with water (10 mL), and the mixture was acidified with 2 N HCl,
then extracted with AcOEt. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and evaporated in
vacuo. The residue was purified by flash chromatography using
n-hexane/ethyl acetate (2/1) as the eluent to afford 3w–y.
5.13.3. (1R,4aS,10aR)-Methyl 5,6,8-trichloro-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-1,4a-dimethyl-9-oxophenanthrene-1-
carboxylate (1l)
To a solution of CrO3 (80 mg, 0.8 mmol) in Ac2O (2 mL) and
AcOH (1 mL) was added dropwise a suspension of the methyl ester
1k (292 mg, 0.7 mmol) in AcOH (2 mL) at 0 °C over 10 min. The
reaction mixture was stirred at 50 °C for 9 h, then cooled, poured
into ice-water (40 mL) and extracted with CHCl3 3 times. The com-
bined organic layer was washed with water, saturated NaHCO3,
and brine, and dried over Na2SO4. The crude mixture was purified
by flash chromatography (n-hexane/AcOEt = 12:1) to afford 1l
(247 mg, yield 82%) as a yellow solid. 1H NMR (CDCl3, 400 MHz):
d 4.03 (br s, 1H), 3.67 (s, 3H), 3.47 (m, 1H), 2.73–2.63 (m, 2H),
1.76–1.65 (m, 5H), 1.42 (s, 3H), 1.40 (s, 6H), 1.32 (s, 3H).
5.15.1. (E,1R,4aS,10aR)-5,6,8-Trichloro-1,2,3,4,4a,9,10,10a-
octahydro-9-(hydroxyimino)-7-isopropyl-1,4a-
dimethylphenanthrene-1-carboxylic acid (3w)
light yellow solid, yield 86%. 1H NMR (DMSO-d6, 400 MHz): d
4.05 (br s, 1H), 3.40 (m, 1H), 2.85 (dd of ABX system, J = 18.6,
14.0 Hz, 1H), 2.23 (dd of ABX system, J = 18.6, 5.3 Hz, 1H), 2.07
(dd of ABX system, J = 14.0, 5.3 Hz, 1H), 1.62–1.57 (m, 5H), 1.37
(d, J = 6.9 Hz, 6H), 1.23 (s, 3H), 1.19 (s, 3H).
HR-MS: calcd for C20H2435Cl3NO3 ([M+H]+), 432.0900; found,
432.0886.
Anal. Calcd for C20H24Cl3NO3: C, 55.51; H, 5.59; N, 3.24. Found:
C, 55.41; H, 5.66; N, 3.11.
5.13.4. (E,1R,4aS,10aR)-Methyl 5,6,8-trichloro-1,2,3,4,4a,9,10,
10a-octahydro-9-(hydroxyimino)-7-isopropyl-1,4a-dimethyl-
phenanthrene-1-carboxylate (5w)
A mixture of the methyl ester 1l (179 mg, 0.415 mmol), pyridine
(0.05 mL), and NH2OHꢀHCl (50 mg, 0.72 mmol) in EtOH (1 mL) was
stirred at 100 °C for 3 h, then cooled, and evaporated in vacuo. The
residue was purified by flash chromatography (n-hexane/
AcOEt = 5:1) to afford 5w (156 mg, yield 84%) as a colorless solid.
1H NMR (CDCl3, 400 MHz): d 4.09 (br s, 1H), 3.68 (s, 3H), 3.42
(m, 1H), 3.00 (dd of ABX system, J = 18.7, 13.8 Hz, 1H), 2.36 (dd
of ABX system, J = 18.7, 5.6 Hz, 1H), 2.23 (dd of ABX system,
J = 13.8, 5.6 Hz, 1H), 1.69–1.63 (m, 5H), 1.44 (d, J = 6.5 Hz,
6H),1.36 (s, 3H), 1.25 (s, 3H).
5.15.2. (E,1R,4aS,10aR)-5,6,8-Trichloro-1,2,3,4,4a,9,10,10a-
octahydro-7-isopropyl-9-methoxyimino-1,4a-
dimethylphenanthrene-1-carboxylic acid (3x)
Yellow oil, yield 56%. 1H NMR (CDCl3, 400 MHz): d 4.06 (br s,
1H), 3.99 (s, 3H), 3.42 (d, J = 13.2 Hz, 1H), 2.95 (dd of ABX system,
J = 18.6, 13.8 Hz, 1H), 2.43 (dd of ABX system, J = 18.6, 5.6 Hz, 1H),
2.18 (dd of ABX system, J = 13.8, 5.6 Hz, 1H), 1.70 (m, 5H), 1.42 (br
s, 6H), 1.36 (s, 3H), 1.27 (s, 3H).

8656
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
HR-MS: calcd for C21H2635Cl3NO3 ([M+Na]+), 468.0876; found,
5.17. Synthesis of compounds 6d and 6e
468.0874. Anal. Calcd for C21H26Cl3NO3ꢀ1/4H2O: C, 55.89; H, 5.92;
N, 3.10. Found: C, 55.90; H, 6.02; N, 2.92.
A mixture of the methyl ester 6c (39 mg, 0.113 mmol), pyridine
(25
l
L), and NH2OHꢀHCl (13 mg, 0.187 mmol) in EtOH (1 mL) was
stirred at 100 °C for 3 h, then cooled, and evaporated in vacuo.
The residue was purified by flash chromatography (n-hexane/
AcOEt = 2:1–1:1) to afford 6d (7 mg, 17% yield) as a colorless oil
and 6e (30 mg, 83% yield) as a colorless solid.
5.15.3. (E,1R,4aS,10aR)-9-(Allyloxy)imino-5,6,8-trichloro-1,2,3,4,
4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethyl
phenanthrene-1-carboxylic acid (3y)
Light yellow oil, yield 56%. 1H NMR (CDCl3, 400 MHz): d 6.10–
6.01 (m, 1H), 5.34–5.21 (m, 2H), 4.68 (m, 2H), 4.06 (br s, 1H),
3.42 (d, J = 13.2 Hz, 1H), 2.98 (dd of ABX system, J = 18.6, 13.8 Hz,
1H), 2.46 (dd of ABX system, J = 18.6, 5.6 Hz, 1H), 2.19 (dd of ABX
system, J = 13.8, 5.6 Hz, 1H), 1.70 (m, 5H), 1.42 (d, J = 6.4 Hz, 6H),
1.36 (s, 3H), 1.28 (s, 3H). Anal. Calcd for C23H28Cl3NO3: C, 58.42;
H, 5.97; N, 2.96. Found: C, 58.71; H, 6.08; N, 2.70.
5.17.1. (E,1S,4aS)-Methyl 6-acetoxy-1,2,3,4,4a,9,10,10a-
octahydro-9-(hydroxyimino)-1,4a-dimethylphenanthrene-1-
carboxylate (6d)
1H NMR (CDCl3, 400 MHz): d 7.96 (br s, 1H), 7.94 (d, J = 8.6 Hz,
1H), 7.04 (d, J = 2.3 Hz, 1H), 6.93 (dd, J = 8.6, 2.3 Hz, 1H), 3.71 (s,
3H), 3.42 (dd of ABX system, J = 18.8, 4.4 Hz, 1H), 3.07 (dd of ABX
system, J = 18.8, 13.9 Hz, 1H), 2.30 (s,3H), 2.25 (m, 2H), 2.00 (m,
1H), 1.75–1.66 (m, 2H), 1.53 (td, J = 13.4, 4.1 Hz, 1H), 1.32 (s, 3H),
1.11 (td, J = 13.6, 3.8 Hz, 1H), 1.01 (s, 3H).
5.16. Podocarpate series
5.16.1. Podocarpic acid methyl ester (6a)
To a solution of podocarpic acid 2 (500 mg, 1.82 mmol) in
MeOH (3 mL) and PhMe (6 mL) was added dropwise 2.0 M
TMSCHN2 (2.4 mmol) in Et2O (1.2 mL) at rt over 5 min, and the
whole was stirred at rt for 1 h. Excess TMSCHN2 was quenched
with AcOH, then the reaction mixture was evaporated in vacuo,
and the residue (6a) was used for the following procedures without
further purification. Mp 213.3–214.5 °C. 1H NMR (CDCl3,
5.17.2. (E,1S,4aS)-Methyl 1,2,3,4,4a,9,10,10a-octahydro-6-
hydroxy-9-(hydroxyimino)-1,4a-dimethylphenanthrene-1-
carboxylate (6e)
1H NMR (CDCl3, 400 MHz): d 7.80 (d, J = 8.6 Hz, 1H), 6.80 (d,
J = 2.5 Hz, 1H), 6.67 (dd, J = 8.6, 2.5 Hz, 1H), 3.71 (s, 3H), 3.42 (dd
of ABX system, J = 18.7, 4.4 Hz, 1H), 3.04 (dd of ABX system,
J = 18.7, 13.8 Hz, 1H), 2.25 (m, 2H), 2.00 (m, 1H), 1.73–1.64 (m,
2H), 1.49 (td, J = 13.2, 4.0 Hz, 1H), 1.31 (s, 3H), 1.09 (td, J = 13.4,
3.8 Hz, 1H), 0.98 (s, 3H).
400 MHz):
d 8.94 (s, 1H), 6.78 (d, J = 8.2 Hz, 1H), 6.63 (d,
J = 2.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.2 Hz, 1H), 3.58 (s, 3H), 2.72 (dd
of ABX system, J = 15.9, 4.3 Hz, 1H), 2.61 (dt, J = 12.5, 5.7 Hz, 1H),
2.14–2.04 (m, 3H), 1.92–1.75 (m, 2H), 1.54 (m, 1H), 1.49 (d,
J = 11.8 Hz, 1H), 1.26 (dt, J = 13.2, 3.8 Hz, 1H), 1.21 (s, 3H), 1.08
(dt, J = 13.2, 4.0 Hz, 1H), 0.91 (s, 3H). HR-MS: calcd for C18H24O3
([M+Na]+), 311.1623; found, 311.1615.
5.18. Synthesis of compounds 6g and 6h
A solution of 6e (30 mg, 0.095 mmol) and Cs2CO3 (62 mg,
0.189 mmol) in 1 mL DMF was stirred at r.t for 30 min, then 1-bro-
mo-3-phenylpropane (25 lL, 0.164 mmol) was added. The mixture
5.16.2. 12-Acetoxypodocarpic acid methyl ester (6b)
was stirred overnight at rt, then poured into ice-water, and ex-
tracted with AcOEt 3 times. The combined organic layer was
washed with water, and brine, and dried over Na2SO4. The crude
mixture was purified by flash chromatography (n-hexane/
AcOEt = 4:1) to afford 6g (24 mg, 46% yield) as a colorless oil and
6h (30 mg, 46% yield) as a yellow solid.
A
solution of 6a (4.03 g, 0.014 mol), CH3COONa (0.976 g,
11.9 mmol) in 80 mL Ac2O was refluxed for 3 h. After removal of
the excess Ac2O under reduced pressure, the residue was dissolved
in ether. The organic layer was washed with 5% NaHCO3, dried over
Na2SO4 and evaporated to dryness. Recrystallization from MeOH-
H2O afforded 6b (4.16 g, 90% yield) as a colorless solid. 1H NMR
(CDCl3, 400 MHz): d 7.03 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 2.4 Hz,
1H), 6.81 (dd, J = 8.3, 2.4 Hz, 1H), 3.66 (s, 3H), 2.92–2.87 (m, 1H),
2.81–2.72 (m, 1H), 2.29 (m, 1H), 2.26 (s,3H), 2.20–2.16 (m, 2H),
2.04–1.91 (m, 2H), 1.62 (m, 1H), 1.53 (dd of ABX system, J = 12.3,
1.6 Hz, 1H), 1.40 (td, J = 13.3, 4.2 Hz, 1H), 1.27 (s, 3H), 1.09 (td,
J = 13.5, 4.2 Hz, 1H), 1.03 (s, 3H).
5.18.1. (E,1S,4aS,10aR)-Methyl 9-((3-phenylpropoxy)imino)-6-
(3-phenylpropoxy)-1,2,3,4,4a,9,10,10a-octahydro-1,4a-
dimethylphenanthrene-1-carboxylate (6g)
1H NMR (CDCl3, 400 MHz): d 7.97 (d, J = 8.7 Hz, 1H), 7.35–7.28
(m, 4H), 7.25–7.23 (m, 6H), 6.86 (d, J = 2.4 Hz, 1H), 6.76 (dd,
J = 8.7, 2.4 Hz, 1H), 4.25 (t, J = 6.4 Hz, 2H), 4.01 (t, J = 6.2 Hz, 2H),
3.74 (s, 3H), 3.45–3.39 (m, 3H), 3.04 (dd of ABX system, J = 18.6,
13.8 Hz, 1H), 2.87–2.78 (m, 4H), 2.31 (m, 2H), 2.24–1.99 (m, 5H),
1.76–1.70 (m, 2H), 1.54 (m, 1H), 1.34 (s, 3H), 1.14 (m, 1H), 1.04
(s, 3H).
5.16.3. 12-Acetoxy-7-oxo-podocarpic acid methyl ester (6c)
To a solution of CrO3 (1.468 g, 14.7 mmol) in Ac2O (40 mL) and
AcOH (30 mL) was added dropwise a suspension of the methyl
ester 6b (4.043 g, 12.2 mmol) in AcOH (15 mL) at 0 °C over
10 min. The reaction mixture was stirred at rt overnight, and
poured into ice-water. The whole was extracted with AcOEt 3
times. The combined organic layer was washed with water, satu-
rated NaHCO3, and brine, dried over Na2SO4. The crude mixture
was purified by flash chromatography (n-hexane/AcOEt = 4:1) to
afford 6c (3.597 g, yield 85%) as a colorless solid. 1H NMR (CDCl3,
400 MHz): d 8.06 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 7.03
(dd, J = 8.6, 2.2 Hz, 1H), 3.69 (s, 3H), 3.21 (dd of ABX system,
J = 18.0, 14.5 Hz, 1H), 2.98 (dd of ABX system, J = 18.0, 3.2 Hz,
1H), 2.32–2.26 (m, 2H), 2.31 (s,3H), 2.04 (m, 2H), 1.70 (m, 1H),
1.53 (td, J = 13.4, 4.2 Hz, 1H), 1.25 (s, 3H), 1.13 (td, J = 13.6,
4.0 Hz, 1H), 1.11 (s, 3H).
5.18.2. (E,1S,4aS,10aR)-Methyl 6-(3-phenylpropoxy)-1,2,3,4,4a,
9,10,10a-octahydro-9-(hydroxyimino)-1,4a-dimethylphe-
nanthrene-1-carboxylate (6h)
1H NMR (CDCl3, 400 MHz): d 7.85 (d, J = 8.8 Hz, 1H), 7.32–
7.28 (m, 2H), 7.23–7.19 (m, 3H), 6.86 (d, J = 2.5 Hz, 1H), 6.75
(dd, J = 8.8, 2.5 Hz, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.72 (s, 3H),
3.45 (dd of ABX system, J = 18.7, 4.4 Hz, 1H), 3.07 (dd of ABX
system, J = 18.7, 13.9 Hz, 1H), 2.82 (t, J = 7.8 Hz, 2H), 2.30 (m,
2H), 2.15–2.08 (m, 2H), 2.00 (m, 1H), 1.76–1.67 (m, 2H), 1.53
(td, J = 13.3, 4.0 Hz, 1H), 1.33 (s, 3H), 1.12 (td, J = 13.5, 3.8 Hz,
1H), 1.01 (s, 3H).

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8657
5.18.3. (E,1S,4aS,10aR)-6-(3-Phenylpropoxy)-1,2,3,4,4a,9,10,10a-
octahydro-9-(hydroxyimino)-1,4a-dimethylphenanthrene-1-
carboxylic acid (4a)
The crude mixture was purified by flash chromatography (n-hex-
ane/AcOEt = 8:1) to afford 6j (30 mg, colorless oil) in 92% yield.
1H NMR (CDCl3, 400 MHz): d 7.92 (d, J = 8.7 Hz, 1H), 7.31–7.27
(m, 2H), 7.21 (m, 3H), 6.82 (d, J = 2.5 Hz, 1H), 6.72 (dd, J = 8.7,
2.5 Hz, 1H), 3.99 (s, 3H), 3.97 (t, J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.31
(dd of ABX system, J = 18.7, 4.3 Hz, 1H), 2.97 (dd of ABX system,
J = 18.7, 13.8 Hz, 1H), 2.81 (t, J = 7.3 Hz, 2H), 2.26 (m, 1H), 2.09
(m, 2H), 1.97 (m,1H), 1.71–1.64 (m, 2H), 1.53 (m, 1H), 1.29 (s,
3H), 1.09 (td, J = 13.6, 3.8 Hz, 1H), 0.98 (s, 3H).
Compound 6h (197 mg, 0.452 mmol) was treated with KOBut
(6.78 mmol) in DMSO (6 mL) at 60 °C for about 2 h. Then the reac-
tion mixture was poured into ice-water, acidified with 2 N HCl, and
extracted with AcOEt. The combined organic layer was washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated
in vacuo. The crude mixture was purified by flash chromatography
(n-hexane/AcOEt = 2:1) to afford 4a (170 mg, 89% yield) as a light
yellow solid. 1H NMR (CDCl3, 400 MHz): d 7.76 (d, J = 8.7 Hz, 1H),
7.31–7.27 (m, 2H), 7.20–7.18 (m, 3H), 6.86 (d, J = 2.5 Hz, 1H),
6.76 (dd, J = 8.7, 2.5 Hz, 1H), 3.98 (t, J = 6.3 Hz, 2H), 3.40 (dd of
ABX system, J = 19.1, 4.8 Hz, 1H), 3.27 (dd of ABX system, J = 19.1,
13.5 Hz, 1H), 2.81 (t, J = 7.3 Hz, 2H), 2.30 (m, 2H), 2.14–2.00 (m,
3H), 1.77–1.68 (m, 2H), 1.53 (td, J = 13.3, 4.0 Hz, 1H), 1.38 (s, 3H),
1.12 (td, J = 13.4, 3.4 Hz, 1H), 1.10 (s, 3H). HR-MS: calcd for
5.19. Synthesis of compounds 4b and 6k
Compound 6j (183 mg, 0.407 mmol) was treated with KOBut
(684 mg, 6.1 mmol) in DMSO (6.00 mL) at 60 °C for about 2 h.
The reaction mixture was poured into ice-water, acidified with
2 N HCl and extracted with AcOEt. The combined organic layer
was washed with H2O and brine, dried over Na2SO4, filtered, and
evaporated in vacuo. The crude mixture was purified by flash chro-
matography (n-hexane/AcOEt = 4:1) to afford 4b (38 mg, 21%
yield) and 6k (65 mg, 35% yield) as yellow oil.
C
26H31NO4 ([M+H]+), 422.2331; found, 422.2351.
Anal. Calcd for C26H31NO4: C, 74.08; H, 7.41; N, 3.32. Found: C,
73.91; H, 7.55; N, 3.31.
5.18.4. (E,1S,4aS)-Methyl 1,2,3,4,4a,9,10,10a-octahydro-6-
hydroxy-9-methoxyimino-1,4a-dimethylphenanthrene-1-
carboxylate (6f)
5.19.1. (E,1S,4aS)-6-(3-Phenylpropoxy)-1,2,3,4,4a,9,10,10a-
octahydro-9-methoxyimino-1,4a-dimethylphenanthrene-1-
carboxylic acid (4b)
A mixture of the methyl ester 6c (98 mg, 0.285 mmol), pyridine
1H NMR (CDCl3, 400 MHz): d 7.93 (d, J = 8.7 Hz, 1H), 7.31–7.27
(m, 2H), 7.21 (m, 3H), 6.82 (d, J = 2.5 Hz, 1H), 6.73 (dd, J = 8.7,
2.5 Hz, 1H), 3.99 (s, 3H), 3.97 (t, J = 6.4 Hz, 2H), 3.31 (dd of ABX sys-
tem, J = 18.8, 4.1 Hz, 1H), 3.05 (dd of ABX system, J = 18.8, 13.9 Hz,
1H), 2.81 (t, J = 7.3 Hz, 2H), 2.29–2.25 (m, 2H), 2.12 (m, 2H), 2.08
(m,1H), 1.74–1.67 (m, 2H), 1.52 (td, J = 13.3, 3.7 Hz, 1H), 1.36 (s,
3H), 1.14 (td, J = 13.6, 3.7 Hz, 1H), 1.08 (s, 3H). HR-MS: calcd for
(63
l
L), and NH2OMeꢀHCl (38 mg, 0.456 mmol) in EtOH (1 mL) was
stirred at 100 °C overnight, then cooled, and evaporated in vacuo.
The residue was purified by flash chromatography (n-hexane/
AcOEt = 4:1) to afford 6f (78 mg, 83% yield) as a light yellow solid.
1H NMR (CDCl3, 400 MHz): d 7.87 (d, J = 8.6 Hz, 1H), 6.75 (d,
J = 2.5 Hz, 1H), 6.64 (dd, J = 8.6, 2.5 Hz, 1H), 3.98 (s, 3H), 3.70 (s,
3H), 3.31 (dd of ABX system, J = 18.8, 4.4 Hz, 1H), 2.98 (dd of ABX
system, J = 18.8, 13.8 Hz, 1H), 2.27–2.18 (m, 2H), 1.96 (m, 1H),
1.69–1.62 (m, 2H), 1.46 (td, J = 13.3, 4.2 Hz, 1H), 1.28 (s, 3H), 1.08
(td, J = 13.5, 3.8 Hz, 1H), 0.95 (s, 3H).
C
27H33NO4 ([M+H]+), 436.2488; found, 436.2444.
Anal. Calcd for C27H33NO4ꢀ1/4H2O: C, 73.69; H, 7.67; N, 3.18.
Found: C, 73.79; H, 7.50; N, 3.17.
5.19.2. (E,1S,4aS)-10-Hydroxy-6-(3-Phenylpropoxy)-1,2,3,4,4a,
9,10,10a-octahydro-9-methoxyimino-1,4a-dimethylphenanth-
rene-1-carboxylic acid (6k)
5.18.5. (E,1S,4aS)-1,2,3,4,4a,9,10,10a-Octahydro-6-hydroxy-9-
methoxyimino-1,4a-dimethylphenanthrene-1-carboxylic acid
(7a)
1H NMR (CDCl3, 400 MHz): d 7.70 (d, J = 8.6 Hz, 1H), 7.29 (m,
2H), 7.21 (m, 3H), 6.83 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 8.6, 2.4 Hz,
1H), 6.05 (d, J = 9.6 Hz, 1H), 4.07 (s, 3H), 3.98 (t, J = 6.2 Hz, 2H),
2.81 (t, J = 7.3 Hz, 1H), 2.28 (d, J = 13.2 Hz, 1H), 2.19 (d,
J = 13.3 Hz, 1H), 2.11 (m, 2H), 1.97 (m,1H), 1.74–1.64 (m, 3H),
1.55 (s, 3H), 1.18 (td, J = 13.5, 3.2 Hz, 1H), 0.99 (s, 3H). HR-MS:
calcd for C27H33NO5 ([M+H]+), 452.2430; found, 452.2230.
Anal. Calcd for C27H33NO5: C, 71.82; H, 7.37; N, 3.10. Found: C,
71.56; H, 7.36; N, 3.05.
Compound 6f (25 mg, 0.075 mmol) was treated with KOBut
(1.13 mmol) in DMSO (1.13 mL) at 60 °C for about 2 h, then the
reaction mixture was poured into ice-water, the whole was acidi-
fied with 2 N HCl, and extracted with AcOEt. The combined organic
layer was washed with H2O and brine, dried over Na2SO4, filtered,
and evaporated in vacuo. The crude mixture was purified by flash
chromatography (n-hexane/AcOEt = 2:1) to afford 7a (22 mg, 92%
yield) as a colorless oil. 1H NMR (Acetone-d6, 400 MHz): d 8.47
(br s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 6.67
(dd, J = 8.6, 2.5 Hz, 1H), 3.90 (s, 3H), 3.29 (dd of ABX system,
J = 18.9, 4.5 Hz, 1H), 3.06 (dd of ABX system, J = 18.9, 13.8 Hz,
1H), 2.30–2.21 (m, 2H), 2.11–1.96 (m, 1H), 1.69–1.60 (m, 2H),
1.49 (td, J = 13.3, 4.1 Hz, 1H), 1.31 (s, 3H), 1.14 (td, J = 13.4,
3.8 Hz, 1H), 1.05 (s, 3H). HR-MS: calcd for C18H23NO4 ([M+H]+),
318.1705; found, 318.1694.
5.19.3. (E,1S,4aS,10aR)/-10-Hydroxy-9-((3-phenylpropoxy)imino) -
6-(3-phenylpropoxy)-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimeth
ylphenanthrene-1-carboxylate (6i, same procedure as 4a)
Colorless oil. 1H NMR (CDCl3, 400 MHz): d 7.69 (d, J = 8.6 Hz,
1H), 7.31–7.27 (m, 4H), 7.21 (m, 6H), 6.82 (d, J = 2.4 Hz, 1H), 6.76
(dd, J = 8.6, 2.4 Hz, 1H), 6.09 (d, J = 9.6 Hz, 1H), 4.27 (t, J = 6.4 Hz,
2H), 3.98 (t, J = 6.2 Hz, 2H), 2.81 (t, J = 7.3 Hz, 2H), 2.75 (t,
J = 7.7 Hz, 2H), 2.25 (d, J = 13.0 Hz, 1H), 2.18–2.05 (m, 5H), 1.93
(m, 1H), 1.69 (m, 3H), 1.53 (s, 3H), 1.17 (m, 1H), 0.98 (s, 3H). HR-
MS: calcd for C35H41NO5 ([M+H]+), 578.2883; found, 578.2923.
Anal. Calcd for C18H23NO4ꢀ3/4H2O: C, 65.34; H, 7.46; N, 4.23.
Found: C, 65.56; H, 7.50; N, 4.15.
5.18.6. (E,1S,4aS)-Methyl 6-(3-phenylpropoxy)-1,2,3,4,4a,9,10,
10a-octahydro-9-methoxyimino-1,4a-dimethylphenanthrene-
1-carboxylate (6j)
5.20. General procedure for synthesis of compounds 4c–g
A solution of 6f (24 mg, 0.072 mmol) and Cs2CO3 (35 mg,
0.108 mmol) in 1 mL DMF was stirred at r.t for 30 min, then 1-bro-
mo-3-phenylpropane (14 lL, 0.094 mmol) was added. The mixture
was stirred overnight at r.t, and poured into ice-water, then the
whole was extracted with AcOEt 3 times. The combined organic
layer was washed with water, and brine, and dried over Na2SO4.
A mixture of 4a (1 mmol), KOH (10 mmol), cat. NaI and an excess
of the the corresponding alkyl bromide (5 mmol) in EtOH (2 mL) was
refluxed overnight. The whole was cooled, and evaporated. The res-
idue was diluted with water (10 mL), acidified with 2 N HCl, and ex-
tracted with AcOEt. The combined organic layer was washed with

8658
Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
brine, dried over Na2SO4, filtered, and evaporated in vacuo. The res-
idue was purified by flash chromatography using n-hexane/ethyl
acetate (4/1–2/1) as eluent to afford the desired compounds 4c–g.
6.82 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.8, 2.4 Hz, 1H), 4.21 (t,
J = 6.5 Hz, 2H), 3.97 (t, J = 6.3 Hz, 2H), 3.37 (dd of ABX system,
J = 18.7, 4.3 Hz, 1H), 3.06 (dd of ABX system, J = 18.7, 13.9 Hz,
1H), 2.81 (t, J = 7.4 Hz, 2H), 2.75 (t, J = 7.4 Hz, 2H), 2.27 (m, 2H),
2.11 (m, 6H), 2.06 (m, 1H), 1.71 (m, 2H), 1.51 (m, 1H), 1.36 (s,
3H), 1.11 (m, 1H), 1.08 (s, 3H). HR-MS: calcd for C35H41NO4
([M+H]+), 540.3114; found, 540.3071.
5.20.1. (E,1S,4aS)-9-(Allyloxy)imino-6-(3-phenylpropoxy)-
1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethylphenanthrene-1-
carboxylic acid (4c)
Yellow oil, 98% conversion yield. 1H NMR (CDCl3, 400 MHz): d
7.93 (d, J = 8.8 Hz, 1H), 7.29 (m, 2H), 7.21 (m, 3H), 6.82 (d,
J = 2.5 Hz, 1H), 6.72 (dd, J = 8.8, 2.5 Hz, 1H), 6.15–6.05 (m, 1H),
5.35 (m, 1H), 5.23 (m, 1H), 4.70 (m, 2H), 3.97 (t, J = 6.2 Hz, 2H),
3.37 (dd of ABX system, J = 18.8, 4.3 Hz, 1H), 3.07 (dd of ABX sys-
tem, J = 18.7, 13.8 Hz, 1H), 2.81 (t, J = 7.3 Hz, 2H), 2.28 (m, 2H),
2.10 (m, 2H), 2.05 (m,1H), 1.70 (m, 2H), 1.51 (m, 1H), 1.36 (s,
3H), 1.11 (m, 1H), 1.08 (s, 3H). HR-MS: calcd for C29H35NO4
([M+H]+), 462.2644; found, 462.2678. Anal. Calcd for C29H35NO4:
C, 75.46; H, 7.64; N, 3.03. Found: C, 75.16; H, 7.64; N, 3.12.
5.21. Biological assay
Effects of compounds on hBK currents were evaluated in CHO
cells stably expressing hBK
a channel by population patch clamp
methods with automated patch clamp system, Ion Works™ Quatro
(Molecular devices, Inc., Sunnyvale, CA). Composition of internal
solution was (in mM) 140 KCl, 1.0 MgCl2/6H2O, 20 HEPES (pH
7.25–7.3 with KOH). And extracellular solution used was (in mM)
137 NaCl, 4.0 KCl, 1.0 MgCl2/6H2O, 2.0 CaCl2, 10 glucose, 20 HEPES
(pH 7.4 with NaOH). Cells were suspended in extracellular solution
5.20.2. (E,1S,4aS)-9-((3-Methylbut-2-enyloxy)imino)-6-(3-
phenylpropoxy)-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl
phenanthrene-1-carboxylic acid (4d)
containing 20 lM BAPTA-AM just prior loading to the cell boat in
IonWorks. The internal solution was substituted to that containing
amphotericin B (25 mg/mL) to establish the perforated patch
clamp mode. Cells were voltage clamped at a holding potential of
ꢁ90 mV and the currents were elicited by a step pulse to
+100 mV pulse (350 msec) followed by a ramp pulse from
ꢁ90 mV to 100 mV (1650 msec). The external solution contained
Light yellow oil, 73% conversion yield. 1H NMR (CDCl3,
400 MHz): d 7.95 (d, J = 8.7 Hz, 1H), 7.29 (m, 2H), 7.20 (m, 3H),
6.82 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 8.7, 2.4 Hz, 1H), 5.53 (t,
J = 6.9 Hz, 1H), 4.70 (d, J = 6.9 Hz, 2H), 3.97 (t, J = 6.3 Hz, 2H), 3.36
(dd of ABX system, J = 18.7, 4.3 Hz, 1H), 3.04 (dd of ABX system,
J = 18.7, 13.9 Hz, 1H), 2.81 (t, J = 7.3 Hz, 2H), 2.28 (m, 2H), 2.10
(m, 2H), 2.05 (m,1H), 1.79 (s, 3H), 1.76 (s, 3H), 1.69 (m, 2H), 1.51
(m, 1H), 1.36 (s, 3H), 1.10 (m, 1H), 1.08 (s, 3H). HR-MS: calcd for
1
l
M ionomycin to activate hBK current. The currents elicited by
a step pulse to 100 mV before and after the application of test com-
pounds (30 M) were used to evaluate the current activation by
l
compounds. The effects of compounds were shown by the % of
pre-value. The data of each compound represent the average of 8
wells.
C
31H39NO4 ([M+H]+), 490.2957; found, 490.2939.
5.20.3. (E,1S,4aS)-9-((Prop-2-ynyloxy)imino)-6-(3-phenylprop-
oxy)-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethylphenanth-
rene-1-carboxylic acid (4e)
Acknowledgments
Light yellow oil, 69% conversion yield. 1H NMR (CDCl3,
400 MHz): d 7.97 (d, J = 8.8 Hz, 1H), 7.29 (m, 2H), 7.20 (m, 3H),
6.82 (d, J = 2.5 Hz, 1H), 6.73 (dd, J = 8.8, 2.5 Hz, 1H), 4.79 (d,
J = 2.4 Hz, 2H), 3.98 (t, J = 6.4 Hz, 2H), 3.37 (dd of ABX system,
J = 18.8, 4.3 Hz, 1H), 3.08 (dd of ABX system, J = 18.8, 13.9 Hz,
1H), 2.81 (t, J = 7.3 Hz, 2H), 2.48 (t, J = 2.4 Hz, 1H), 2.28 (m, 2H),
2.11 (m,2H), 2.00 (m, 1H), 1.70 (m, 2H), 1.51 (m, 1H), 1.37 (s,
3H), 1.11 (m, 1H), 1.09 (s, 3H). HR-MS: calcd for C29H33NO4
([M+H]+), 460.2488; found, 460.2483.
Y.-M.C. is grateful for a postdoctoral fellowship for a foreign re-
searcher from the Japan Society for the Promotion of Science and
for the support of the Innovation Foundation of Shanghai
University, the Shanghai Pujiang Program (No. 10PJ1403700) and
the Project of Shanghai Municipal Education Commission (No.
J50102). Thanks are also due to Professor Masakatsu Shibasaki,
Graduate School of Pharmaceutical Sciences, The University of To-
kyo, for valuable discussions and encouragement during the later
stage of this work. Y.O. also thanks Shorai Foundation for Science
and Technology for a financial support in part. We thank Dr. Yoi-
chiro Ezaki, Arakawa Chemical Industry, for a gift of podocarpic
acid. We also thank Mr. Yoji Kabasawa of our group for help in
the synthesis of triCl-DHAA.
Anal. Calcd for C29H33NO4: C, 75.79; H, 7.24; N, 3.05. Found: C,
75.49; H, 7.23; N, 2.98.
5.20.4. (E,1S,4aS)-9-(Benzyloxy)imino-6-(3-phenylpropoxy)-
1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethylphenanthrene-1-
carboxylic acid (4f)
References and notes
Yellow oil, 92% conversion yield. 1H NMR (CDCl3, 400 MHz): d
7.93 (d, J = 8.8 Hz, 1H), 7.45 (m, 2H), 7.37 (m, 2H), 7.29 (m, 3H),
7.20 (m, 3H), 6.82 (d, J = 2.5 Hz, 1H), 6.72 (dd, J = 8.8, 2.5 Hz, 1H),
5.24 (s, 2H), 3.97 (t, J = 6.3 Hz, 2H), 3.41 (dd of ABX system,
J = 18.7, 4.3 Hz, 1H), 3.09 (dd of ABX system, J = 18.7, 13.8 Hz,
1H), 2.81 (t, J = 7.3 Hz, 2H), 2.28 (t, J = 13.4 Hz, 2H), 2.10 (m, 2H),
2.04 (m, 1H), 1.70 (m, 2H), 1.51 (m, 1H), 1.36 (s, 3H), 1.11 (m,
1H), 1.08 (s, 3H). HR-MS: calcd for
512.2801; found, 512.2771.
Anal. Calcd for C33H37NO4: C, 77.47; H, 7.29; N, 2.74. Found: C,
77.19; H, 7.24; N, 2.64.
1. Coghlan, M. J.; Carroll, W. A.; Gopalakrishnan, M. J. Med. Chem. 2001, 44, 1627.
2. Atwal, K. S. Med. Res. Rev. 1992, 12, 569.
3. Wulff, H.; Zhorov, B. S. Chem. Rev. 2008, 108, 1744.
4. Ledoux, J.; Werner, M. E.; Brayden, J. E.; Nelson, M. T. Physiology 2006, 21, 69.
5. Kaczorowski, G. J.; Knaus, H. G.; Leonard, R. J.; McManus, O. B.; Garcia, M. L. J.
Bioenerg. Biomembr. 1996, 28, 255.
6. Toro, L.; Wallner, M.; Meera, P.; Tanaka, Y. News Physiol. Sci. 1998, 13, 112.
7. Berkefeld, H.; Sailer, C. A.; Bildl, W.; Rohde, V.; Thumfart, J. O.; Eble, S.;
Klugbauer, N.; Reisinger, E.; Bischofberger, J.; Oliver, D.; Knaus, H. G.; Schulte,
U.; Fakler, B. Science 2006, 314, 615.
8. Loane, D. J.; Lima, P. A.; Marrion, N. V. J. Cell Sci. 2007, 120, 985.
9. Greenwood, I. A.; Miller, L. J.; Ohya, S.; Horowitz, B. J. Biol. Chem. 2002, 277,
22119.
10. Kim, E. Y.; Alvarez-Baron, C. P.; Dryer, S. E. Mol. Pharmacol. 2009, 75, 466.
11. Brayden, J. E.; Nelson, M. T. Science 1992, 256, 532.
12. Vergara, C.; Latorre, R.; Marrion, N. V.; Adelman, J. P. Curr. Opin. Neurobiol. 1998,
8, 321.
13. Wang, L.; Sigworth, F. J. Nature 2009, 461, 292.
14. Yuan, P.; Leonetti, M. D.; Pico, A. R.; Hsiung, Y.; MacKinnon, R. Science 2010,
329, 182.
C
33H37NO4 ([M+H]+),
5.20.5. (E,1S,4aS)-9-((3-Phenylpropoxy)imino)-6-(3-phenyl
propoxy)-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl
phenanthrene-1-carboxylic acid (4g)
Light yellow oil, 61% conversion yield. 1H NMR (CDCl3,
400 MHz): d 7.93 (d, J = 8.8 Hz, 1H), 7.29 (m, 4H), 7.22 (m, 6H),
15. Lee, U. S.; Cui, J. Trends Neurosci. 2010. doi:10.1016/j.tins.2010.06.004.

Y.-M. Cui et al. / Bioorg. Med. Chem. 18 (2010) 8642–8659
8659
16. Salkoff, L.; Butler, A.; Ferreira, G.; Santi, C.; Wei, A. Nat. Rev. Neurosci. 2006, 7,
921.
23. Sakamoto, K.; Nonomura, T.; Ohya, S.; Muraki, K.; Ohwada, T.; Imaizumi, Y. J.
Pharmacol. Exp. Ther. 2006, 316, 144.
17. Orio, P.; Rojas, P.; Ferreira, G.; Latorre, R. News Physiol. Sci. 2002, 17,
156.
24. Cui, Y.-M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.; Sawada, K.; Ohwada, T.
Bioorg. Med. Chem. Lett. 2008, 18, 5201.
18. Ghatta, S.; Nimmagadda, D.; Xu, X.; O’Rourke, S. T. Pharmacol. Ther. 2006, 110,
103.
25. Cui, Y.-M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.; Sawada, K.; Ohwada, T.
Bioorg. Med. Chem. Lett. 2008, 18, 5197.
19. Gribkoff, V. K.; Starrett, J. E., Jr,; Dworetzky, S. I.; Hewawasam, P.; Boissard, C. G.
J. R.; Huston, K.; Johnson, G.; Krishman, B. S.; Kinney, G. G.; Lombardo, L. A.;
Meanwell, N. A.; Molinoff, P. B.; Myers, R. A.; Moon, S. L.; Ortiz, A.; Pajor, L.;
Pieschl, R. L.; Post-Munson, D. J.; Signor, L. J.; Srinivas, N.; Taber, M. T.; Thalody,
G.; Trojnacki, J. T.; Wiener, H.; Yeleswarm, K.; Yeola, S. W. Nat. Med. 2001, 7,
471.
20. Nardi, A.; Olesen, S.-P. Curr. Med. Chem. 2008, 15, 1126.
21. Imaizumi, Y.; Sakamoto, K.; Yamada, A.; Hotta, A.; Ohya, S.; Muraki, K.;
Uchiyama, M.; Ohwada, T. Mol. Pharmacol. 2002, 62, 836.
22. Ohwada, T.; Nonomura, T.; Maki, K.; Sakamoto, K.; Ohya, S.; Muraki, K.;
Imaizumi, Y. Bioorg. Med. Chem. Lett. 2003, 13, 3971.
26. Tashima, T.; Toriumi, Y.; Mochizuki, Y.; Nonomura, T.; Nagaoka, S.; Furukawa,
K.; Tsuru, H.; Adachi-Akahane, S.; Ohwada, T. Bioorg. Med. Chem. 2006, 14,
8014.
27. Cui, Y.-M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.; Sawada, K.; Ohwada, T.
Bioorg. Med. Chem. Lett. 2008, 18, 6386.
28. Chang, F. C.; Wood, N. F. Tetrahedron Lett. 1964, 40, 2969.
29. Gigg, J.; Gigg, R. J. Chem. Soc. C 1996, 82.
30. Finkel, A.; Wittel, A.; Yang, N.; Handran, S.; Hughes, J.; Costantin, J. J. Biomol.
Screen. 2006, 11, 488.
31. John, V. H.; Dale, T. J.; Hollands, E. C.; Chen, M. X.; Partington, L.; Downie, D. L.;
Meadows, H. J.; Trezise, D. J. J. Biomol. Screen. 2007, 12, 50.