Alantrypinone and its derivatives: Synthesis and antagonist activity toward insect GABA receptors

Article abstract of DOI:10.1016/j.bmc.2008.11.017

The γ-aminobutyric acid (GABA) receptor bears important sites of action for insecticides. Alantrypinone is an insecticidal alkaloid that acts as a selective antagonist for housefly (vs rat) GABA receptors, and is considered to be a lead compound for the d

Full text of DOI:10.1016/j.bmc.2008.11.017

Bioorganic & Medicinal Chemistry 17 (2009) 94–110
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alantrypinone and its derivatives: Synthesis and antagonist activity toward
insect GABA receptors
c,
Takayuki Watanabe a, Mitsuhiro Arisawa a,b, , Kenji Narusuye , Mohommad Sayed Alam c,à
,
*
Kazumi Yamamoto d, Masaaki Mitomi d, Yoshihisa Ozoe c, Atsushi Nishida a,
*
a Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan
b Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan
c Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan
d Meiji Seika Kaisha, Kohoku-Ku, Yokohama 222-8567, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The c-aminobutyric acid (GABA) receptor bears important sites of action for insecticides. Alantrypinone is an
Received 14 October 2008
Revised 6 November 2008
Accepted 7 November 2008
Available online 17 November 2008
insecticidal alkaloid that acts as a selective antagonist for housefly (vs rat) GABA receptors, and is considered
to be a lead compound for the development of a safer insecticide. In an attempt to obtain compounds with
greater activity, a series of racemic alantrypinone derivatives were systematically synthesized using hetero
Diels–Alderreactions, and atotalof34compounds were examinedfor their abilitytoinhibitthe specific bind-
ing of [3H]40-ethynyl-4-n-propylbicycloorthobenzoate, a high-affinity non-competitive antagonist, to house-
fly-head membranes. The assay results showed that (1) there is no significant difference between the
potencies of natural (+)-alantrypinone and its synthetic racemate; (2) the amide NHs at the 2- and 18-posi-
tions are important for high activity; (3) there is a considerable drop in potency for compounds without an
aromatic ring at the 16-position; and (4) a large substituent at the 3-position is detrimental to high activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keyword:
Alantrypinone
Insect GABA receptors
Hetero Diels–Alder reaction
1. Introduction
be useful for investigating its physiology and pharmacology.2 Insect
GABA receptors are the target of the important insecticide fipronil
Ionotropic
c-aminobutyric acid (GABA) receptors are major neu-
(Fig. 1).3 Since fipronil-resistant insects have recently been re-
ported, structurally different classes of novel insecticide are
needed.
rotransmitter receptors that mediate fast inhibitory synaptic neuro-
transmission in the nervous system of invertebrates as well as
vertebrates. Vertebrate ionotropic GABA receptors are classified
into GABAA-type and GABAC-type receptors.1 However, insect iono-
tropic GABA receptors do not easily fit into the vertebrate GABAA/
GABAC receptor classification. Insect GABA receptors are distin-
guished from GABAA-type vertebrate receptors by their insensitivity
to bicuculline, and differ from GABAC receptors in that insect recep-
tors are subject to allosteric modulation, albeit weak, by benzodi-
azepines and barbiturates. These differences between insect and
vertebrate GABA receptors might provide great opportunities for
the development of safer insecticides. The Drosophila melanogaster
GABA receptor subunit RDL (resistant to dieldrin) can be heterolo-
gously expressed to form functional homo-oligomeric receptors,
the pharmacology of which closely resembles that of most native in-
sect GABA receptors, and thus the RDL receptor has been shown to
Alantrypinone4 ((+)-1, Fig. 1), which is a polycyclic alkaloid that
is biosynthetically derived from anthranilic acid and tryptophan,
was isolated from Penicillium thymicola in 1998. Its biological activ-
ities were not known at the isolation stage. In 2004, one of the
authors (Ozoe) and co-workers reported that (+)-1 and (+)-sera-
ntrypinone5 (Fig. 1), which were isolated through binding assay-
based screening of fungal culture extracts, were insecticidal
alkaloids that were highly selective for insect (vs mammalian)
GABA receptors.6 To the best of our knowledge, there has been no
report on the structure–activity relationships of (+)-1.
To understand the interaction of ( )-1 with GABA receptors,
with the ultimate goal of contributing to the development of a no-
vel insecticide, we designed alantrypinone derivatives (( )-3–( )-
33) and synthesized ( )-1–( )-33 (Fig. 2). In this report, we de-
scribe the design, synthesis, and biological effects of ( )-1 and
these analogues.
* Corresponding authors. Tel.: +81 11 706 3976; fax: +81 11 706 3769 (M.
Arisawa), tel.: +81 43 290 2907; fax: +81 43 290 2909 (A. Nishida).
E-mail addresses: arisawa@pharm.hokudai.ac.jp (M. Arisawa), nishida@p.chiba-
u.ac.jp (A. Nishida).
2. Results and discussion
Present address: Faculty of Pharmaceutical Sciences, Toho University, Funabashi,
Chiba 274-8510, Japan.
à
We designed the analogues shown in Figure 2, based on the
Present address: Department of Chemistry, Jagannath University, Dhaka 1100,
structural features of (+)-1. ( )-3–( )-7 contain halogens and might
Bangladesh.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.017

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
95
H
H
20
19
25
17 N
N
21
18
O
O
Cl
Cl
24
16
22
NC
N
23
N15
N
CF3
Me
NH
7
6
R
N
8
9
3
2
5
NH
4
O
S
1
14
N
N
12
NH2
11
13
CF3
O
O
10
O
O
( )-epi-alantrypinone (2)
fipronil
(+)-alantrypinone (1): R = H
(+)-serantrypinone: R = OH
Figure 1. Structures of quinazolinones and fipronil.
X5
N18
X5
N18
O
N
O
22
22
X3
X3
X2
N
X2
N
N
O
X4
X4
8
9
8
9
2
2
N
N
O
O
X1
X1
O
( )-3 : X1 = 9-Cl, X2 = Me, X3 = H, X4 = H, X5 = H
( )-4 : X1 = 9-F, X2 = Me, X3 = H, X4 = H, X5 = H
( )-5: X1 = 8-Cl, X2 = Me, X3 = H, X4 = H, X5 = H
( )-6: X1 = H, X2 = Me, X3 = Cl, X4 = H, X5 = H
( )-7 : X1 = 9-Cl, X2 = Me, X3 = Cl, X4 = H, X5 = H
( )-8 : X1 = H, X2 = Bn, X3 = H, X4 = H, X5 = H
( )-9 : X1 = H, X2 = iPr, X3 = H, X4 = H, X5 = H
( )-10 : X1 = H, X2 = iBu, X3 = H, X4 = H, X5 = H
( )-11 : X1 = H, X2 = Me, X3 = H, X4 = Me, X5 = H
( )-12: X1 = H, X2 = Me, X3 = H, X4 = H, X5 = Me
( )-13 : X1 = H, X2 = Me, X3 = H, X4 = Me, X5 = Me
( )-14 : X1 = H, X2 = Me, X3 = H, X4 = H, X5 = Bn
( )-15 : X1 = H, X2 = Me, X3 = H, X4 = Me, X5 = Bn
( )-20 : X1 = 9-Cl, X2 = Me, X3 = H, X4 = H, X5 = H
( )-21 : X1 = 9-F, X2 = Me, X3 = H, X4 = H, X5 = H
( )-22: X1 = 8-Cl, X2 = Me, X3 = H, X4 = H, X5 = H
( )-23: X1 = H, X2 = Me, X3 = Cl, X4 = H, X5 = H
( )-24 : X1 = 9-Cl, X2 = Me, X3 = Cl, X4 = H, X5 = H
( )-25 : X1 = H, X2 = Bn, X3 = H, X4 = H, X5 = H
( )-26 : X1 = H, X2 = iPr, X3 = H, X4 = H, X5 = H
( )-27 : X1 = H, X2 = iBu, X3 = H, X4 = H, X5 = H
( )-28 : X1 = H, X2 = Me, X3 = H, X4 = H, X5 = Me
( )-29 : X1 = H, X2 = Me, X3 = H, X4 = Me, X5 = Me
( )-30 : X1 = H, X2 = Me, X3 = H, X4 = H, X5 = Bn
( )-31 : X1 = H, X2 = Me, X3 = H, X4 = Me, X5 = Bn
O
Ph
O
O
R
MeO
N
N
R
Me
NH
OMe
O
Me
Me
NH
N
N
O
N R
N
N
N
O
O
O
O
O
( )-18 : R = H
( )-19 : R = Me
( )-16 : R = Ph
( )-17 : R = H
( )-32 : R = Ph
( )-33 : R = H
Figure 2. Alantrypinone derivatives.
which part of ( )-1 is necessary for a desired biological activity.
Based on a consideration of the efficient and systematic synthesis
of ( )-1 and ( )-3–( )-19 in racemic form, we decided to use a pro-
cedure for the synthesis of ( )-1 developed by Kende and co-work-
ers.4d,e Their key reaction for the synthesis of ( )-1 is a hetero
Diels–Alder reaction between azadiene 34a and dienophile 35a
(Scheme 1). Compounds 34a and 35a are prepared from anthranilic
acid (36a) in 6 steps and isatin (37a) in 2 steps, respectively. This
method is applicable to the synthesis of ( )-3–( )-19. In this syn-
thetic method, we can also obtain the epimers, ( )-2 and ( )-20–
( )-33, and examine their biological activities.
O
P
S
O
O
Figure 3. Structure of IBIPPS.
be easily absorbed into an insect body. ( )-8–( )-10 have a bulky
substituent at the bridgehead of the bicycles and might have a
desired conformational restriction, like the caged antagonist 4-
isobutyl-3-isopropyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-
sulfide (IBIPPS) (Fig. 3).6 ( )-11–( )-15 have an alkyl substituent(s)
on the amide nitrogen at the 2- and/or 18-position, which confers
lower polarity, so these compounds should highlight the necessity
of an amide functional group for sufficient biological activity. Sim-
plified analogues, ( )-16–( )-19, ( )-32, and ( )-33, should clarify
2.1. Synthesis of ( )-1 and ( )-2
Liu and co-workers reported stepwise condensations of anthra-
nilic acid (36a) with two amino acids under microwave conditions
and a one-pot synthesis of quinazolines (Scheme 2).7 The use of

96
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
condensation
of glycine and
alanine
Me
hetero
Diels-Alder reaction
N
O
N
NH2
N
and hydrolysis
OEt
COOH
aromatization
anthranilic acid (36a)
azadiene (34a)
O
O
O
N
N
H
H
Peterson olefination
dienophile (35a)
isatin (37a)
H
N
H
N
O
O
N
Me
NH
Me
NH
N
O
+
N
N
O
O
O
( )-1
( )-2
8 steps overall yield 4.2%
8 steps overall yield 13.5%
Scheme 1. Total synthesis of ( )-alantrypinone (1) by Kende group.
microwave conditions improved the synthesis of 34a so that the
yield of 38a was increased from 31% (4 steps) to 57% (2 steps)
(Scheme 3).4e According to Kende’s method, 38a was converted
to 34a (Scheme 4). Dienophile 35a was prepared by the Peterson
olefination of isatin (37a). The hetero Diels–Alder reaction of 34a
with 35a proceeded regioselectively, and desired ( )-exo-41a and
undesired ( )-endo-41a were obtained in respective yields of 52%
and 18% (Table 1, entry 2). When the amount of 34a in the reaction
was reduced to 1.5 equiv, ( )-endo-41a was obtained in 43% yield
together with 34% of ( )-exo-41a (entry 3). The same reactions un-
der heated conditions (entries 4–6) gave ( )-endo-41a with greater
stereoselectivity. These results indicate that ( )-endo-41a is a ther-
modynamically stable product. Since we needed both isomers, ( )-
exo-41a and ( )-endo-41a, we adopted ‘entry 3’ for our standard
hetero Diels–Alder reaction conditions. ( )-exo-41a and ( )-endo-
41a were converted to ( )-1 and ( )-2, respectively, as reported
by Kende and co-workers. Our synthetic results are summarized
in Scheme 4.
Me
NH
Boc
N
-Boc-L-Ala-OH
P(OPh)3
N
O
Gly methyl ester
36a
O
pyridine (0.2 M)
55 oC, 16 h
Microwave
220 oC, 1.5 min
Me
Me
N
N
O
NHBoc
CO2Me
NH
N
N
O
O
38a
one-pot 57%
Scheme 3. One-pot microwave-promoted synthesis of quinazoline.
R
R
1)
P(OPh)3
HO2C
pyridine
Boc
N
N
NH
H
N
O
36a
CO2Me
2)
O
NH2HCl
N
H
N
H
R = H : glyantrypine (55%)
Microwave
R = Me : fumiquinazoline F (39%)
one-pot reaction
R = i-Pr : fiscalin B (20%)
Scheme 2. Liu’s one-pot synthesis of glyantrypine, fumiquinazoline F, and fiscalin B.

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
97
Me
N
-
Et3O+BF4
Na2CO3
N
DDQ
N
34a
38a
37a
OEt
CH2Cl2
rt, 24 h
60%
benzene
reflux, 30 min
80%
O
39a
(lit. 85%)
(lit. 83%)
HO
TMS
•
BF3 OEt2
TMSCH2MgCl
Et2O
35a
O
N
H
CH2Cl2
-78 ºC, 2 h, 0 ºC, 1 h
quant
-78 ºC to rt, 18 h
77%
40a
(lit. 38%)
(lit. 63%)
EtOAc
EtOAc
1.0
N
HCl
1.0
N
rt
HCl
( )-1
( )-alantrypinone
( )-endo-41a
( )-2
( )-exo-41a
rt
( )-epi-alantrypinone
5 h
3 h
74%
71%
(lit. 85%)
(lit. 89%)
Scheme 4. Synthesis of ( )-1 and ( )-2.
Table 1
Hetero DA reaction
H
N
H
22
X3
N
X3
O
22
X4
X2
15
O
X4
15
azadiene
(34a - g)
X2
N
CHCl3
+
N
N
8
8
9
X1
N
+
N
temp. time
9
N
OEt
dienophile
(35a, b)
X1
OEt
O
O
hetero DA
reaction
(
exo adduct)
(
endo adduct)
( )-41a : X1 = H, X2 = Me, X3 = H, X4 = H
( )-41b : X1 = 9-Cl, X2 = Me, X3 = H, X4 = H
( )-41c : X1 = 9-F, X2 = Me, X3 = H, X4 = H
( )-41d : X1 = 8-Cl, X2 = Me, X3 = H, X4 = H
( )-41e : X1 = H, X2 = Me, X3 = Cl, X4 = H
( )-41f : X1 = 9-Cl, X2 = Me, X3 = Cl, X4 = H
( )-41g : X1 = H, X2 = Bn, X3 = H, X4 = H
( )-41h : X1 = H, X2 = iPr, X3 = H, X4 = H
( )-41i : X1 = H, X2 = iBu, X3 = Cl, X4 = H
Entry
Azadiene
Dienophile (equiv)
Temperature (°C)
Time (h)
Product (yield, %)
Ratio
exo
endo
exo:endo
1a
2
3
4
5
6
7
8
9
10
11
12
13
14
34a
34a
34a
34a
34a
34a
34b
34c
34d
34a
34b
34e
34f
35a (5.0)
35a (5.0)
35a (1.5)
35a (5.0)
35a (1.5)
35a (1.5)
35a (5.0)
35a (1.5)
35a (5.0)
35b (5.0)
35b (1.5)
35a (1.5)
35a (1.5)
35a (1.5)
rt
rt
rt
40
40
Reflux
rt
rt
rt
rt
rt
rt
40
rt
24
24
24
4.5
7
3
24
8
24
24
5
9
24
12
( )-41a
( )-41a
( )-41a
( )-41a
( )-41a
( )-41a
( )-41b
( )-41c
( )-41d
( )-41e
( )-41f
( )-41g
( )-41h
( )-41i
55
52
34
31
30
23
34
40
40
38
31
18
18
43
37
39
46
25
59
36
47
38
3.1:1
2.9:1
1:1.3
1:1.2
1:1.3
1:2
1.4:1
1:1.5
1.1:1
1:1.2
1:1.5
1:1.9b
1:2.5b
1:1.5
67
Quant
34g
26
38
a
Kende’s data.
b
Inseparable mixture was obtained. The ratio was determined by 1H NMR.

98
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
2.2. Synthesis of alantrypinone analogues
ings indicate that the housefly GABA receptor recognizes the ste-
reochemistry of the bicycles. Among the compounds ( )-1–( )-7,
( )-1 and ( )-3 were found to be the most potent, followed by
( )-4 and ( )-7. Based on the assay results of ( )-1 and ( )-8–
Based on the above method for the synthesis of ( )-1 and ( )-
2, other molecules ( )-3–( )-33 were also synthesized. Key
synthons (34b–34g) were efficiently prepared by the one-pot syn-
thesis of quinazolines (Scheme 5) and their conversion to the cor-
responding azadienes (Scheme 6). Dienophile 35b was prepared
from 37b by Peterson olefination (Scheme 7). Hetero Diels–Alder
reactions of these azadienes (34a–34g) with dienophiles (35a,b)
were performed. Although the reactivities and exo–endo selectiv-
ities varied according to the substituent of the azadiene and die-
nophile, hetero Diels–Alder adducts 41b–41i were obtained under
similar conditions (Table 1, entries 7–14). Hydrolysis of exo-ad-
duct and endo-adduct of 41b–41i led to ( )-3–( )-10 and ( )-
20–( )-27 in good to excellent yields, in the same manner as in
the conversion of ( )-exo-41a to ( )-1. Hetero Diels–Alder reac-
tions of azadiene 34a with dienophiles, acrylates 35c and 35d,
are summarized in Table 2. Compound 41j was obtained as an
inseparable mixture of an endo-adduct and an exo-adduct, which
was subjected to hydrolysis to give a less polar product and a
more polar product in respective yields of 10% and 13% (2 steps).
Compound 41k was obtained as separable stereoisomers, which
were also subjected to the same hydrolysis to give the corre-
sponding products in good yields, respectively (Scheme 8). Based
on a comparison of the 1H NMR chemical shifts of aromatic pro-
tons at the 23-position (Fig. 1) of these products (( )-16 and ( )-
32) with the corresponding chemical shifts of ( )-1–( )-10 and
( )-20–( )-27, we assumed that a less polar product from ( )-
41j could be an exo-adduct (( )-16) and a more polar product
could be an endo-adduct (( )-32) (Table 3), as Kende did.4e In
the same manner, by comparing the 1H NMR chemical shifts of
the methoxy protons of ( )-17 and ( )-33 with those of ( )-16
and ( )-32, we assumed that a less polar product from ( )-41k-
LP could be an endo-adduct (( )-33) and a more polar product
could be an exo-adduct (( )-17). The reaction of 34a with N-phen-
ylmaleimide (35e) yielded a less polar and a more polar adducts
in respective yields of 77% and 3% (Scheme 9). Hydrolysis of these
compounds gave the corresponding amides in respective yields of
82% and 65%. The nitrogen at the 2-position of ( )-18 was meth-
ylated to give ( )-19 in 94% yield. 1H NMR analysis of ( )-18 and
( )-19 failed to give any information about the stereochemistry of
these compounds. ( )-11–( )-15 and ( )-28–( )-31 were synthe-
sized from ( )-exo-41a or ( )-endo-41a as shown in Scheme 10.
i
( )-10, high potency is retained with Me, Pr, and Bn substitution
i
at the bridgehead position, while Bu substitution is detrimental
to high potency. We initially hypothesized that bulkier substitu-
ents would lead to higher potency. However, docking studies of
3 using a human b3 GABA receptor homology model, which has
been shown to be a good model of insect GABA receptors,8 pre-
dict that large bridgehead substituents are not tolerated in the
potential binding site within the channel pore (Fig. 6). Figure 6
also shows that 9-Cl of 3 is acceptable within the channel pore,
and that a hydrogen bond between the carbonyl oxygen atom
of the oxindole ring and the hydroxyl group of Thr281 is formed.
( )-11–( )-15 showed low activity, indicating that the amide NHs
at the 2- and 18-positions are important for high activity. Alter-
natively, the Me groups introduced into these positions might
hinder binding to the receptor. Synthetic intermediates and com-
pounds without an aromatic ring at the 16-position showed low
activity.
2.4. Conclusions
In the current study, we systematically synthesized a series of
racemic alantrypinone derivatives, some of which inhibited spe-
cific [3H]EBOB binding to housefly-head membranes. Generally,
exo-adducts had higher activity than endo-adducts in [3H]EBOB
binding assays. However, there was no significant difference be-
tween the potencies of the native enantiomer (+)-1 and its syn-
thetic racemate (( )-1). The inhibitory activities of several
derivatives were found to be comparable to that of (+)-1,
although none showed activity that was dramatically higher
than that of (+)-1. Further modification of ( )-1 based on struc-
ture-activity studies with receptor homology models may help to
improve its potency.
3. Experimental
3.1. Synthesis
All melting points are uncorrected. Infrared absorption spectra
were recorded using a JASCO FT/IR-230 spectrometer. 1H NMR
spectra were recorded in CDCl3 at 25 °C unless otherwise noted,
at 400 MHz, with TMS as an internal standard. 13C NMR spectra
were recorded in CDCl3 at 25 °C unless otherwise noted, at
100 MHz. All moisture-sensitive reactions were performed under
an Ar atmosphere. Flash column chromatography was performed
2.3. Inhibition of GABA-induced currents and [3H]EBOB binding
We first examined whether (+)-1 acts as an antagonist of in-
sect GABA receptors. GABA-induced currents in American cock-
roach (Periplaneta americana L.) neurons were recorded by the
whole-cell patch clamp method. Control inward currents were
with silica gel 60 N (spherical, neutral, 40–50 lm, Kanto Chemical
Co., Inc.). Microwave irradiation experiment was performed in a
CEM Discover system (NC, USA), operating at a frequency of
2.45 GHz with continuous irradiation power from 0 to 150 W.
The reaction was carried out in an open 3 mL glass vial, which
was cooled by air.
induced following the application of 100 lM GABA for 2 s at a
holding potential of ꢀ80 mV, with symmetric chloride concen-
trations (188 mM) between internal and external solutions. As
shown in Figure 4, GABA-induced inward currents were blocked
(by 90.3 1.6%, n = 4) after a 5-min application of 10 lM (+)-1,
indicating that (+)-1 is an antagonist of insect GABA receptors.
We next performed a series of competition assays to deter-
mine the potency of alantrypinone ((+)-1) and its synthetic race-
mate and analogues (( )-1–( )-33) at housefly (Musca domestica
L.) GABA receptors, using the high-affinity non-competitive GABA
receptor antagonist [3H]40-ethynyl-4-n-propylbicycloorthobenzo-
ate ([3H]EBOB). The results summarized in Figure 5 show that
there is no significant difference between the potencies of ala-
ntrypinone ((+)-1) and its racemate (( )-1). The potency of ( )-1
(exo) was found to be higher than that of ( )-2 (endo). Overall,
exo-adducts had a higher potency than endo-adducts. These find-
3.1.1. General procedure for synthesis of azadienes (34)
Step 1: Synthesis of quinazolines (38) using microwave.
To a solution of anthranilic acid (36) and N-Boc-L-Ala-OH
(1.0 equiv) in pyridine (2.0 M) was added P(OPh3)3 (1.2 equiv)
and the mixture was stirred for 16 h at 55 °C. The whole was
cooled to room temperature and to the mixture was added amino
acid methyl ester (1.0 equiv). Microwave irradiation (150 W,
220 °C) was carried out for 1.5 min. After removal of the solvent,
the residue was subjected to column chromatography to give the
corresponding quinazolines 38.

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
99
1) N-Boc-Amino acid (1.0 eq)
P(OPh)3 (1.1 eq)
R
pyridine (0.2 M)
3
N
O
NH2
4
5
8
NH
55 oC, 16 h
N
9
O
CO2H
1
X
X
2) Glymethyl ester (1.0 eq)
Microwave
220 oC, 1.5 min
anthranilic acid
derivatives (36)
quinazoline
derivatives (38)
entry
36 (SM)
product
yield (%)
amino acid
Ala
Ala
Ala
9-Cl (38b)
9-F (38c)
8-Cl (38d)
5-Cl
5-F
1
2
3
48a
56
4-Cl
52
Phe
Val
3-Bn (38e)
3-iPr (38f)
H
H
4
5
51
40
Leu
3-iBu (38g)
H
6
42
a) Overall yield was 22% by 4 steps precedures developed by Kende.
Scheme 5. One-pot microwave-promoted synthesis of various quinazolines.
R
R
R
-
Et3O+BF4
N
3
N
O
N
O
8
NH
N
N
Na2CO3
DDQ
N
N
N
9
O
OEt
OEt
X
CH2Cl2
rt, 24 h
X
benzene
X
O
reflux, 30 min
34b-g
38b-g
39b-g
yield (%)
substrate
X
39a), 1st step
34, 2nd step
entry
R
1
2
38b
38c
38d
Me
Me
83
76
78
9-Cl
9-F
60 (32)
41 (18)
Me
Bn
3
8-Cl
79
60 (40)
48 (25)
67 (33)
4
5
6
H
H
H
68
69
55
38e
38f
i-Pr
i
-Bu
38g
a) The numbers inparentheses indicate the yield of recovered 38.
Scheme 6. Synthesis of various azadienes.
Cl
Cl
Cl
HO
O
TMSCH2MgCl
(2.0 eq)
TMS
BF3 OEt2 (5.0 eq)
CH2Cl2
O
O
O
N
N
N
H
Et2O
-78 oC to rt, 18 h
70%
H
H
-78 oC(2 h), then 0 oC(1 h)
quant
35b
40b
5-Cl-Isatin (37b)
Scheme 7. Synthesis of 35b.
38a9: 57% (2 steps) from 36a, a white solid, 1H NMR (CDCl3,
400 MHz) d: 1.74 (d, J = 6.8 Hz, 3H), 4.68–4.75 (m, 1H), 4.76 (s,
2H), 6.72 (s, 1H), 7.52 (ddd, J = 0.8, 7.6, 7.6 Hz, 1H), 7.68 (dd,
J = 0.6, 8.2 Hz, 1H), 7.79 (ddd, J = 1.4, 7.6, 7.6 Hz, 1H), 8.30 (dd,
J = 1.6, 8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 20.2, 44.6, 51.4,
119.9, 126.8, 127.2, 127.3, 134.8, 147.1, 151.1, 160.6, 166.6; IR
(neat)
m
: 3256, 1668, 1597, 1467, 1380 cmꢀ1; LRMS (EI) m/z 229
[M]+.

100
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
Table 2
4.90 (qui, J = 3.6 Hz, 1H), 6.23 (br s, 1H), 7.10–7.13 (m, 2H), 7.28–
Hetero DA reaction using acrylates
7.34 (m, 3H), 7.54 (ddd, J = 1.2, 7.2, 7.8 Hz, 1H), 7.73–7.75 (m,
1H), 7.83 (ddd, J = 2.0, 7.2, 7.8 Hz, 1H), 8.29 (ddd, J = 0.4, 1.6,
7.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 42.7, 44.2, 57.2, 119.8,
126.8, 127.1, 127.3, 127.9, 129.1 (2C), 129.8 (2C), 134.3, 134.9,
OMe
O
N
R4
Me
N
Me
O
CHCl3
N
R4
N
147.1, 149.9, 160.3, 166.4; IR (KBr)
m: 3062, 1672, 1595, 1473,
+
MeO
1428, 1341 cmꢀ1; LRMS (EI) m/z 305 [M]+; HRMS (FAB) m/z Calcd
for C18H16N3O2 [M+H]+ 306.1243; found: 306.1226.
40 ºC
N
OEt
N
OEt
O
38f: 40% (2 steps) from 36a, a white solid, 1H NMR (CDCl3,
400 MHz) d: 1.00 (d, J = 6.8 Hz, 3H), 1.12 (d, J = 7.2 Hz, 3H), 2.39–
2.50 (m, 1H), 4.38 (d, J = 18.8 Hz, 1H), 4.42 (dd, J = 1.2, 4.8 Hz,
1H), 5.00 (d, J = 18.8 Hz, 1H), 7.50 (ddd, J = 1.2, 7.6, 7.8 Hz, 1H),
7.67 (d, J = 7.6 Hz, 1H), 7.78 (ddd, J = 1.6, 7.6, 7.6 Hz, 1H), 8.28
(dd, J = 1.2, 8.0 Hz, 1H), 8.37 (d, J = 3.2 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 17.0, 19.2, 35.1, 44.6, 61.7, 119.7, 126.6, 127.0,
O
35c: R4 = Ph
35d: R4 = H
( )-41j: R4 = Ph
34a
( )-41k: R4 = H
Entry
Dienophile
Equiv
Time (h)
Yield (%)
LP
21
MP
1
2
35c
35d
1.5
3.0
27
10
30
127.1, 134.8, 147.0, 149.9, 160.7, 166.8; IR (KBr) m: 2963, 1683,
63
1598, 1475, 1438, 1346 cmꢀ1; LRMS (EI) m/z 257 [M]+; HRMS
(FAB) m/z Calcd for C14H16N3O2 [M+H]+ 258.1243; found:
258.1263.
LP, less polar isomer; MP, more polar isomer.
38g: 52% (2 steps) from 36a, a colorless solid, 1H NMR (CDCl3,
400 MHz) d: 1.04 (d, J = 6.0 Hz, 3H), 1.05 (d, J = 6.0 Hz, 3H), 1.67–
1.93 (m, 3H), 4.42 (d, J = 18.4 Hz, 1H), 4.58 (m, 1H), 5.03 (d,
J = 18.4 Hz, 1H), 7.43 (br s, 1H), 7.51 (ddd, J = 0.8, 7.6, 8.0 Hz, 1H),
7.67 (d, J = 8.0 Hz, 1H), 7.78 (ddd, J = 1.6, 7.2, 7.6 Hz, 1H), 8.29
(dd, J = 0.8, 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 21.4, 23.2,
24.6, 44.1, 44.6, 54.8, 120.6, 127.5, 127.90, 127.91, 135.5, 147.9,
38b4e: 48% (2 steps) from 36b, a purple solid, 1H NMR (CDCl3,
400 MHz) d: 1.72 (d, J = 6.8 Hz, 3H), 4.70 (dq, J = 2.4, 6.8 Hz, 1H),
4.73 (s, 1H), 4.76 (s, 1H), 6.59 (br s, 1H), 7.62 (d, J = 8.4 Hz, 1H),
7.71 (dd, J = 2.4, 8.8 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) d: 20.0, 44.7, 51.3, 121.0, 126.1, 129.0,
133.1, 135.2, 145.6, 151.4, 159.6, 166.5; IR (neat)
m: 1679,
1597, 1330, 830, 784 cmꢀ1; LRMS (EI) m/z 263 [M]+; HRMS
(FAB) m/z Calcd for C12H1137ClN3O2 [M+H]+ 266.0514; found:
266.0503.
151.7, 161.6, 167.4; IR (KBr)
m: 3200, 2961, 1700, 1593, 1472,
1324 cmꢀ1; LRMS (FAB) m/z 272 [M+H]+; HRMS (FAB) m/z Calcd
for C15H18N3O2 [M+H]+ 272.1399; found: 272.1380.
38c: 56% (2 steps) from 36c, a purple solid, 1H NMR (CDCl3,
400 MHz) d: 1.73 (d, J = 6.8 Hz, 3H), 4.68 (dq, J = 2.4, 6.8 Hz, 1H),
4.75 (d, J = 9.2 Hz, 2H), 6.52 (br s, 1H), 7.50 (ddd, J = 2.8, 8.0,
8.8 Hz, 1H), 7.70 (dd, J = 4.8, 8.8 Hz, 1H), 7.92 (dd, J = 2.8, 8.0 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d: 20.0, 44.7, 51.2, 111.6 (d,
J = 23.9 Hz), 121.2 (d, J = 9 Hz), 123.4 (d, J = 24 Hz), 129.8 (d,
J = 8.2 Hz), 143.8, 150.5, 159.9 (d, J = 15 Hz), 161.1 (d, J = 236 Hz),
Step 2: Synthesis of 34 fromꢀ38.
A solution of 38, Et3O+ꢁBF4 (1.5 equiv) and Na2CO3 (6.0 equiv)
in CH2Cl2 (20 mM) was stirred for 24 h at room temperature.
The reaction was quenched by the addition of water and the or-
ganic compounds were extracted with CH2Cl2 and the combined
organic layers were washed with brine and dried over Na2SO4.
After removal of the solvent, the residue was subjected to column
chromatography to give the corresponding ethyl enol ether. A
solution of ethyl enol ether and DDQ (1.2 equiv) in benzene
(18 mM) was refluxed for 30 min. The solution was filtered by ba-
sic alumina. After removal of the solvent, the residue was sub-
jected to column chromatography to give the corresponding 34.
34a4e: 48% (2 steps) from 38a, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.13 (t, J = 6.8 Hz, 3H), 2.72 (s, 3H), 3.76 (q,
J = 6.8 Hz, 2H), 7.08 (ddd, J = 1.2, 6.8, 7.6 Hz, 1H), 7.33 (ddd,
J = 1.6, 7.6, 7.6 Hz, 1H), 7.81 (dd, J = 0.8, 8.4 Hz, 1H), 7.88 (s, 1H),
8.54 (dd, J = 0.8, 7.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.6,
21.8, 64.3, 95.0, 116.9, 126.9, 127.0, 128.2, 134.6, 139.0, 147.1,
151.1, 158.0, 161.9; LRMS (EI) m/z 255 [M]+.
166.7; IR (KBr) m
: 3415, 1691, 1610, 1485, 1345, 1141 cmꢀ1; LRMS
(EI) m/z 247 [M]+; HRMS (FAB) m/z Calcd for C12H11FN3O2 [M+H]+
248.0835; found: 248.0827.
38d: 52% (2 steps) from 36d, a purple solid, 1H NMR (CDCl3,
400 MHz) d: 1.72 (d, J = 7.2 Hz, 3H), 4.68 (dq, J = 2.4, 6.6 Hz, 1H),
4.72 (s, 1H), 4.75 (s, 1H), 6.71 (br s, 1H), 7.46 (dd, J = 2.4, 12.6 Hz,
1H), 7.69 (d, J = 2.0 Hz, 1H), 8.21 (d, J = 8.8 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 20.1, 44.6, 51.4, 118.5, 126.9, 128.0, 128.3, 141.1,
148.0, 152.4, 160.0, 166.2; IR (neat) m: 1671, 1599, 1421, 1318,
1100, 786 cmꢀ1; LRMS (EI) m/z 263 [M]+; HRMS (FAB) m/z Calcd
for C12H1137ClN3O2 [M+H]+ 266.0514; found: 266.0526.
38e: 51% (2 steps) from 36a, a white solid, 1H NMR (CDCl3,
400 MHz) d: 3.25 (dd, J = 7.6, 14.0 Hz, 1H), 3.42 (dd, J = 4.0,
14.0 Hz, 1H), 3.51 (d, J = 18.8 Hz, 1H), 4.66 (d, J = 18.8 Hz, 1H),
34b4e: 63% (2 steps) from 38b, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.52 (t, J = 6.8 Hz, 3H), 2.93 (s, 3H), 4.23 (q,
OMe
O
OMe
O
R4
R4
Me
N
AcOEt
Me
1.0
N
HCl
N
N
NH
N
rt
N
O
OEt
O
O
( )-41j : R4 = Ph
( )-41k : R4 = H
( )-16 (R4 = Ph): 10% (2 steps), less polar isomer
( )-32 (R4 = Ph): 13% (2 steps), more polar isomer
( )-17 (R4 = H): 59% (from ( )-41k-MP), more polar isomer
( )-33 (R4 = H): 67% (from ( )-41k-LP), less polar isomer
Scheme 8. Synthesis of simplified alantripinone derivatives (I).

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
101
Table 3
Chemical Shift of ( )-1, ( )-2, ( )-16, ( )-17, ( )-32, and ( )-33 (CDCI3, 400 MHz)
(less polar)
H
N
O
O
MeO
N
H
O
OMe
Me
H23
Me
Me
NH
N
N
NH
NH
N
N
N
O
O
O
O
H
O
O
( )-1
LP: ( )-16
LP: ( )-33
(more polar)
O
H
N
H
O
MeO
O
H23
Me
NH
OMe
Me
N
H
R
NH
N
O
N
N
NH
O
N
N
O
O
O
O
MP: ( )-32
MP: ( )-17
( )-2
Position
Less polar (ppm)
More polar (ppm)
Difference (ppm)
( )-1 (Alantrypinone)
( )-16 or ( )-32
H23
Ph
OMe
OMe
6.81 (exo)
7.19
3.60
5.87 (endo)
6.71
3.79
0.94
0.48
0.19
0.18
( )-17 or ( )-33
3.77
3.59
Ph
O
N
Me
O
O
N
N
CHCl3
N
Me
N
NP h
O
N
+
rt, 30 h.
OEt
N
O
OEt
Hetero DA
reaction
O
35e (5 equiv.)
34a
( )-41l (less polar: 77%)
(more polar: 3%)
Ph
O
Ph
O
N
N
NaH (1.2 eq)
MeI (1.2 eq)
EtOAc
O
1.0
N
HCl
O
Me
Me
N
N
NH
2
N Me
THF
0 oC, 3.5 h
2
rt
N
N
O
3 h
O
O
O
( )-19 (less polar) : 94%
( )-18 (less polar) : 82%
(more polar) : 65%
Scheme 9. Hetero DA reaction of N-phenylmaleimide and synthesis of simplified alantrypinone derivatives (II).
J = 7.2 Hz, 2H), 7.79 (dd, J = 2.4, 4.8 Hz, 1H) 7.87 (d, J = 8.8 Hz, 1H),
8.00 (s, 1H), 8.42 (d, J = 2.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d:
14.6, 21.7, 64.3, 95.1, 117.5, 125.8, 129.9, 132.5, 135.2, 138.8,
J = 24 Hz), 117.8 (d, J = 9.0 Hz), 124.1 (d, J = 26 Hz), 130.9 (d,
J = 8.2 Hz), 138.4, 144.0, 151.4, 157.4 (d, J = 4.0 Hz), 160.8 (d,
J = 244 Hz), 162.0; IR (KBr) m ;
: 1700, 1670, 1636, 1535, 1483 cmꢀ1
145.5, 151.4, 156.9, 161.9; IR (neat)
m
: 1681, 1629, 1462, 1149,
LRMS (EI) m/z 273 [M]+; HRMS (FAB) m/z Calcd for C14H13FN3O2
[M+H]+ 274.0992; found: 274.0993.
1073 cmꢀ1; LRMS (EI) m/z 289 [M]+; HRMS (FAB) m/z Calcd for
C14H1235ClN3O2 [M]+ 289.0618; found: 289.0630.
34d: 32% (2 steps) from 38d, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.52 (t, J = 7.2 Hz, 3H), 2.91 (s, 3H), 4.22 (q,
J = 7.2 Hz, 2H), 7.50 (dd, J = 2.0, 8.8 Hz, 1H), 7.90 (d, J = 1.6 Hz,
1H), 7.95 (br s, 1H), 8.36 (d, J = 8.8 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.6, 21.7, 64.3, 95.2, 115.0, 127.3, 127.6, 128.3,
34c: 47% (2 steps) from 38c, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.52 (t, J = 7.2 Hz, 3H), 2.93 (d, J = 0.8 Hz, 3H), 4.22
(q, J = 7.2 Hz, 2H), 7.62 (ddd, J = 2.8, 8.4, 8.8 Hz, 1H), 7.94 (dd,
J = 5.2, 8.8 Hz, 1H), 7.95 (s, 1H), 8.06 (dd, J = 2.8, 8.4 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.6, 21.7, 64.3, 94.9, 110.9 (d,
139.5, 140.9, 147.9, 151.3, 157.4, 161.8; IR (KBr)
m: 1690,

102
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
Me
Me
N 18
Me
N 18
18
N
O
N
O
N
O
N
EtOAc
NaH
MeI
NaH
MeI
1.0
N
HCl
( )-exo-41a
or
Me
N
Me
Me
NH
N Me
THF
2
rt, 30 min
THF
( )-endo-41a
2
2
N
N
N
0 ºC
0 ºC
OEt
O
O
2.5 h
3 h
O
O
O
( )-exo-42a : quant
( )-endo-42a : quant
( )-12 (exo) : 95%
( )-13 (exo) : quant
( )-29 (endo) : quant
( )-28 (endo) : quant
Bn
Bn
Bn
18
N 18
N 18
N
O
O
O
EtOAc
NaH
NaH
MeI
1.0
N HCl
( )-exo-41a
or
BnBr
Me
N
Me
Me
N
N
N
NH
N Me
THF
2
rt, 3 h
THF
( )-endo-41a
2
2
N
N
N
0 ºC - rt
0 ºC
OEt
O
O
2.5 h
2 h
O
O
O
( )-exo-42b : 83%
( )-endo-42b : 81%
( )-14 (exo) : 86%
( )-30 (endo) : 74%
( )-15 (exo) : 66%
( )-31 (endo) : 83%
Bs
Bs
H
18
N 18
N 18
N
O
O
O
1) EtOAc
1.0
rt
nBuLi
Na anth-
racenide
N HCl
BsCl
Me
Me
N
Me
N
N
N
2
N
( )-exo-41a
N Me
N Me
THF
THF
2) NaH
MeI
THF
0 ºC
2
2
N
N
-78 ºC
-78 ºC
OEt
O
O
5 h
O
O
O
Bs = benzensulfonyl
( )-exo-42c : 69%
( )-43 (exo) : 38% (2 steps)
( )-11 (exo) : 67%
Scheme 10. Synthesis of alantrypinone derivatives (( )-11–( )-15 and ( )-28–( )-31).
134.5, 137.6, 138.3, 147.1, 151.2, 158.0, 162.7; IR (KBr) m: 1685,
1629, 1527, 1454 cmꢀ1; LRMS (EI) m/z 331 [M]+; HRMS (FAB) m/z
Calcd for C20H18N3O2 [M+H]+ 332.1352; found: 332.1376.
34f: 33% (2 steps) from 38f, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.41 (s, 3H), 1.43 (s, 3H), 1.53 (t, J = 7.2 Hz, 3H), 4.26
(q, J = 7.2 Hz, 2H), 4.34 (qui, J = 6.8 Hz, 1H), 7.58 (ddd, J = 1.6, 7.2,
7.6 Hz, 1H), 7.87 (ddd, J = 1.6, 6.8, 7.6 Hz, 1H), 7.91 (ddd, J = 1.6,
7.6, 7.6 Hz, 1H), 7.99 (s, 1H), 8.46 (dd, J = 1.6, 7.6 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.6, 20.9 (2C), 30.6, 64.1, 95.0, 116.9,
126.8, 126.9, 128.3, 134.5, 138.1, 147.2, 151.4, 158.2, 169.0; IR
(KBr) m
: 1687, 1631, 1534, 1458 cmꢀ1; LRMS (EI) m/z 283 [M]+;
HRMS (FAB) m/z Calcd for C16H18N3O2 [M+H]+ 284.1399; found:
284.1395.
34g: 37% (2 steps) from 38g, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.05 (d, J = 6.4 Hz, 6H), 1.52 (t, J = 6.8 Hz, 3H), 2.51
(m, 1H), 3.24 (d, J = 7.2 Hz, 2H), 4.25 (q, J = 6.8 Hz, 2H), 7.58 (ddd,
J = 1.2, 7.2, 7.6 Hz, 1H), 7.87 (ddd, J = 1.6, 6.4, 7.6 Hz, 1H), 7.91
(ddd, J = 0.8, 8.0, 8.8 Hz, 1H), 8.00 (s, 1H), 8.45 (dd, J = 1.6, 8.0 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d: 14.6, 22.7 (2C), 27.4, 42.5,
64.2, 95.1, 116.8, 126.8, 126.9, 128.3, 134.5, 138.9, 147.2, 151.2,
Figure 4. Inhibition of GABA-induced currents by (+)-1 in cockroach neurons.
GABA-evoked currents in cockroach neurons were recorded by the whole-cell
patch–clamp method. (A) Control inward currents induced by 100
l
M GABA. (B)
M (+)-
Inward currents induced by 100 M GABA after the 5-min application of 10
l
l
1. GABA was applied during the period indicated by the black bar. Recordings A and
B were obtained from the same preparations.
1631, 1523, 1449, 1078 cmꢀ1; LRMS (EI) m/z 289 [M]+; HRMS
(FAB) m/z Calcd for C14H1337ClN3O2 [M+H]+ 292.0671; found:
292.0677.
158.2, 164.3; IR (KBr) m
: 1678, 1631, 1531, 1454, 1105 cmꢀ1; LRMS
(EI) m/z 297 [M]+; HRMS (FAB) m/z Calcd for C17H20N3O2 [M+H]+
298.1556; found: 298.1549.
34e: 41% (2 steps) from 38e, a yellow solid, 1H NMR (CDCl3,
400 MHz) d: 1.51 (t, J = 6.8 Hz, 3H), 4.32 (q, J = 7.2 Hz, 2H), 4.68
(s, 2H), 7.18–7.32 (m, 3H), 7.52–7.61 (m, 3H), 7.89 (ddd, J = 1.6,
6.8, 7.6 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.98 (s, 1H), 8.44 (dd,
J = 1.6, 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.6, 40.0, 64.2,
95.6, 116.7, 126.7, 126.8, 126.9, 128.2, 128.3 (2C), 129.7 (2C),
3.1.2. Synthesis of 3-methyleneoxindole
Synthesis of 3-methyleneoxindole (35a): To a solution of isatin
(37a, 1 g, 6.80 mmol) in diethyl ether (70 mL) was added a solution
of TMSCH2MgCl in diethyl ether (1.0 M, 13.6 mL, 13.6 mmol) at
ꢀ78 °C and the mixture was stirred for 15 min at ꢀ78 °C then for

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
103
Figure 5. Potencies of alantrypinone derivatives in inhibiting specific [3H]EBOB binding to housefly-head membranes. Compounds other than (+)-1 were racemates. Data are
means SD of at least three experiments, each performed in triplicate, except the data of exo-41a (n = 1). Data of (+)-1 were taken from Ref. 6.
18 h at room temperature. The reaction was quenched by the addi-
tion of MeOH. After removal of the solvent, the residue was sub-
jected to column chromatography (hexane/AcOEt = 1:1) to give
3-hydroxy-3-trimethylsilylmethyloxindole (1.23 g, 77%) as a yel-
low solid. To a solution of 3-hydroxy-3-trimethylsilylmethyloxin-
dole (1.23 g, 5.23 mmol) in CH2Cl2 (90 mL) was added BF3ꢁOEt2
(3.3 mL, 26.1 mmol) at ꢀ78 °C and the mixture was stirred for
2 h at ꢀ78 °C then for 1 h at 0 °C. The reaction was quenched by
the addition of satd aq NaHCO3 and the organic compounds were
extracted with CH2Cl2 and the combined organic layers were
washed with brine and dried over Na2SO4. After removal of the sol-
vent, 35a10 (750 mg, quant.) was obtained as a yellow solid. 1H
NMR (CDCl3, 400 MHz) d: 6.13 (s, 1H), 6.39 (s, 1H), 6.87 (d,
J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 6.2 Hz, 1H), 7.47
(d, J = 7.6 Hz, 1H), 8.08 (s, 1H).
Synthesis of 5-chloro-3-methyleneoxindole (35b): To a solution of
37b (500 mg, 2.75 mmol) in diethyl ether (30 mL) was added a
solution of TMSCH2MgCl in diethyl ether (1.0 M, 5.5 mL, 5.5 mmol)
at ꢀ78 °C and the mixture was stirred for 15 min at ꢀ78 °C then for
18 h at room temperature. The reaction was quenched by the addi-
tion of MeOH. After removal of the solvent, the residue was sub-
jected to column chromatography (hexane/AcOEt = 1:1) to give
3-hydroxy-3-trimethylsilylmethyloxindole (520 mg, 70%) as a yel-
low solid. To a solution of 3-hydroxy-3-trimethylsilylmethyloxin-
1H), 7.23 (dd, J = 2.0, 8.4 Hz, 1H), 7.44 (s, 1H), 7.75 (br s, 1H); 13C
NMR (CDCl3, 100 MHz) d: 111.0, 120.1, 121.7, 124.0, 127.8, 130.0,
135.0, 139.4, 168.2; IR (KBr) m
: 3410, 1719, 1677 cmꢀ1; LRMS (EI)
m/z 179 [M]+; HRMS (FAB) m/z Calcd for C9H737ClNO [M+H]+
182.0189; found: 182.0188.
3.1.3. General procedure of hetero Diels–Alder reaction
A solution of 34 and 35 in CHCl3 was stirred at the conditions in
Table 1. After removal of the solvent, the residue was subjected to
column chromatography to give the corresponding adducts.
( )-exo-41a4e: 52% from 34a and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.42 (t, J = 7.2 Hz, 3H), 1.56 (s, 3H),
2.25 (dd, J = 2.4, 14 Hz, 1H), 2.57 (dd, J = 3.2, 14 Hz, 1H), 4.32–
4.40 (m, 2H), 6.05 (d, J = 2.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 7.02–
7.09 (m, 2H), 7.23–7.26 (m, 1H), 7.46–7.56 (m, 1H), 7.63 (br s, 1H),
7.72–7.75 (m, 2H), 8.32 (d, J = 7.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.2, 16.8, 37.6, 47.6, 54.7, 63.7, 66.9, 110.1, 120.1,
122.7, 124.1, 126.6, 126.7, 127.8, 128.9, 130.7, 134.2, 141.4,
147.4, 154.2, 159.4, 172.6, 178.6; LRMS (EI) m/z 400 [M]+.
( )-endo-41a4e: 18% from 34a and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.40 (t, J = 7.2Hz, 3H), 1.60 (s, 3H), 2.25
(dd, J = 3.2, 13.8 Hz, 1H), 2.58 (dd, J = 2.0, 13.8 Hz, 1H), 4.13–4.31
(m, 1H), 4.38–4.45 (m, 1H), 5.84 (d, J = 7.6 Hz, 1H), 6.06 (br s,
1H), 6.76–6.74 (m, 1H), 6.85 (d, J = 8.0Hz, 1H), 7.15 (t, J = 8.0, 1H),
7.55 (t, J = 7.2 Hz, 1H), 7.66 (br s, 1H), 7.71 (d, J = 8.4 Hz, 1H),
7.79 (ddd, J = 1.2, 7.2, 7.4 Hz, 1H), 8.38 (d, J = 7.6 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.1, 17.0, 37.1, 48.0, 53.1, 63.8, 67.1,
109.7, 120.1, 122.7, 123.2, 126.9, 127.2, 128.2, 128.8, 130.1,
134.6, 141.1, 147.2, 153.8, 159.1, 171.5, 178.5; LRMS (EI) m/z 400
[M]+.
dole (100 mg, 373
l
mol) in CH2Cl2 (10 mL) was added BF3ꢁOEt2
(240
lL, 1.87 mmol) at ꢀ78 °C and the mixture was stirred for
2 h at ꢀ78 °C then for 1 h at 0 °C. The reaction was quenched by
the addition of satd aq NaHCO3 and the organic compounds were
extracted with CH2Cl2 and the combined organic layers were
washed with brine and dried over Na2SO4. After removal of the sol-
vent, 35b (68 mg, quant.) was obtained as a yellow solid. 1H NMR
(CDCl3, 400 MHz) d: 6.15 (s, 1H), 6.45 (s, 1H), 6.79 (d, J = 8.4 Hz,
( )-exo-41b: 34% from 34b and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.43 (t, J = 7.2 Hz, 3H), 1.60 (s, 3H),

104
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
1H), 8.38 (s, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.2, 16.8, 37.7,
47.7, 54.6, 63.8, 66.9, 110.0, 111.6 (d, J = 24 Hz), 121.4 (d,
J = 8 Hz), 122.7 (d, J = 24 Hz), 122.9, 124.3, 129.0, 130.3 (d,
J = 8 Hz), 130.6, 141.3, 144.2, 153.6, 158.7 (d, J = 3 Hz), 160.9 (d,
J = 247 Hz), 172.5, 178.2; IR (KBr)
m: 3214, 1722, 1685, 1655,
1632, 1487 cmꢀ1; LRMS (EI) m/z 418 [M]+; HRMS (FAB) m/z Calcd
for C23H20FN4O3 [M+H]+ 419.1519; found: 419.1488.
( )-endo-41c: 59% from 34c and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.41 (t, J = 7.2 Hz, 3H), 1.58 (s, 3H),
2.24 (dd, J = 3.2, 13.6 Hz, 1H), 2.58 (dd, J = 2.0, 13.6 Hz, 1H), 4.23–
4.31 (m, 1H), 4.39–4.47 (m, 1H), 5.80 (d, J = 7.6 Hz, 1H), 6.04 (dd,
J = 2.0, 3.2 Hz, 1H), 6.73 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H),
7.17 (t, J = 7.6 Hz, 1H), 7.51 (ddd, J = 2.8, 8.0, 8.4 Hz, 1H), 7.56 (s,
1H), 7.72 (dd, J = 4.8, 8.4 Hz, 1H), 8.00 (dd, J = 2.8, 8.4 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.1, 16.9, 37.0, 48.2, 53.2, 63.9, 67.1,
109.9, 111.8 (d, J = 24 Hz), 121.3 (d, J = 9 Hz), 122.6, 123.0, 123.2
(d, J = 24 Hz), 128.9, 130.1, 130.5 (d, J = 8 Hz), 141.3, 143.8, 153.2,
158.4 (d, J = 3 Hz), 161.1 (d, J = 248 Hz), 171.3, 178.9; IR (KBr) m:
3239, 1646, 1475, 1382, 1343 cmꢀ1; LRMS (EI) m/z 418 [M]+; HRMS
(FAB) m/z Calcd for C23H20FN4O3 [M+H]+ 419.1519; found:
419.1484.
( )-exo-41d: 40% from 34d and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.43 (t, J = 7.2 Hz, 3H), 1.52 (s, 3H),
2.25 (dd, J = 2.0, 14 Hz, 1H), 2.55 (dd, J = 3.2, 14 Hz, 1H), 4.36 (dq,
J = 4.0, 7.2 Hz, 2H), 6.02 (t, J = 2.8 Hz, 1H), 6.77 (d, J = 7.6 Hz, 1H),
7.00–7.08 (m, 2H), 7.24–7.26 (m, 1H), 7.43 (dd, J = 2.0, 8.4 Hz,
1H), 7.74 (d, J = 1.2 Hz, 1H), 8.03 (br s, 1H), 8.24 (d, J = 8.4 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d: 14.2, 16.8, 37.7, 47.7, 54.7,
63.8, 67.1, 110.0, 118.8, 123.0, 124.3, 125.6, 127.2, 127.6, 128.3,
129.1, 130.6, 141.3, 148.5, 155.6, 158.8, 172.6, 178.2; IR (KBr) m:
1687, 1600, 1471, 1422, 1330 cmꢀ1; LRMS (EI) m/z 434 [M]+; HRMS
(FAB) m/z Calcd for C23H2035ClN4O3 [M+H]+ 435.1224; found:
435.1232.
( )-endo-41d: 36% from 34d and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.41 (t, J = 7.2 Hz, 3H), 1.53 (s, 3H),
2.24 (dd, J = 3.2, 13.6 Hz, 1H), 2.57 (dd, J = 2.4, 13.6 Hz, 1H), 4.24–
4.31 (m, 1H), 4.39–4.45 (m, 1H), 5.83 (d, J = 7.6 Hz, 1H), 6.04 (t,
J = 2.8 Hz, 1H), 6.75 (t, J = 7.6 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 7.17
(t, J = 7.6 Hz, 1H), 7.50 (dd, J = 2.0, 8.4 Hz, 1H), 7.72 (d, J = 2.0 Hz,
1H), 8.30 (d, J = 8.4 Hz, 1H), 8.42 (s, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.1, 16.9, 37.1, 48.2, 53.1, 64.0, 67.2, 109.7, 118.6,
122.8, 123.1, 127.7, 127.8, 128.3, 129.0, 129.9, 140.9, 141.1,
Figure 6. A homopentameric b3 GABA receptor channel model with 3 docked into
the putative binding site. A 3 isomer having the same stereochemistry as that of (+)-
1 was docked into the model. Only the pore-facing region surrounded by five
Ala277s, five Thr281s, and five Leu284s from five subunits, which is thought to be
the antagonist-binding site,8 is shown. (A) Side view. (B) Top view of the channel.
2.25 (dd, J = 2.4, 14 Hz, 1H), 2.55 (dd, J = 3.2, 14 Hz, 1H), 4.32–4.40
(m, 2H), 6.02 (s, 1H), 6.75–6.81 (m, 1H), 7.04–7.13 (m, 3H), 7.67 (s,
2H), 7.90 (s, 1H), 8.28 (s, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.2,
16.8, 37.7, 47.8, 54.7, 63.9, 66.9, 110.1, 121.3, 123.0, 124.2, 126.1,
129.1, 129.6, 130.6, 132.4, 134.6, 141.3, 146.0, 154.6, 158.4,
148.1, 155.2, 158.5, 171.4, 178.2; IR (KBr) m: 1722, 1643, 1471,
1343, 1179 cmꢀ1; LRMS (EI) m/z 434 [M]+; HRMS (FAB) m/z Calcd
for C23H2037ClN4O3 [M+H]+ 437.1205; found: 437.1218.
172.6, 178.5; IR (neat)
m
: 1700, 1654, 1472, 1027, 991 cmꢀ1; LRMS
( )-exo-41e: 38% from 34a and 35b, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.44 (t, J = 7.2 Hz, 3H), 1.57 (s, 3H),
2.23 (dd, J = 2.0, 14 Hz, 1H), 2.57 (dd, J = 3.2, 14 Hz, 1H), 4.33–
4.41 (m, 2H), 6.05 (dd, J = 2.0, 3.2 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H),
7.06 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 2.0, 8.4 Hz, 1H), 7.49 (m, 1H),
7.74 (d, J = 4.0 Hz, 3H), 8.32 (d, J = 7.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.2, 16.9, 37.7, 47.5, 55.0, 63.9, 66.9, 111.0, 120.2,
124.8, 126.8, 127.9, 128.1, 128.9, 132.0, 132.4, 134.3, 139.9,
(EI) m/z 434 [M]+; HRMS (FAB) m/z Calcd for C23H2035ClN4O3
[M+H]+ 435.1224; found: 435.1220.
( )-endo-41b: 25% from 34b and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.41 (t, J = 7.2 Hz, 3H), 1.58 (s, 3H),
2.24 (dd, J = 3.2, 13.8 Hz, 1H), 2.58 (dd, J = 2.0, 13.6 Hz, 1H), 4.23–
4.31 (m, 1H), 4.39–4.47 (m, 1H), 5.80 (d, J = 7.2 Hz, 1H), 6.04 (d,
J = 5.6 Hz, 1H), 6.73 (t, J = 6.8 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 7.17
(t, J = 6.8 Hz, 1H), 7.65–7.77 (m, 3H), 8.34 (d, J = 2.4 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 14.1, 16.9, 37.1, 48.3, 53.2, 63.9, 67.2,
109.8, 121.2, 122.7, 123.1, 126.3, 129.0, 129.8, 130.0, 133.1,
147.5, 153.8, 159.3, 172.8, 178.1; IR (KBr) m: 3261, 1632, 1477,
1385, 1300, 1192, 1155 cmꢀ1; LRMS (EI) m/z 434 [M]+; HRMS
(FAB) m/z Calcd for C23H2035ClN4O3 [M+H]+ 435.1224; found:
435.1191.
135.1, 141.2, 145.7, 154.1, 158.2, 171.4, 178.5; IR (neat) m: 1655,
1560, 1473, 1025, 993 cmꢀ1; LRMS (EI) m/z 434 [M]+; HRMS
(FAB) m/z Calcd for C23H2035ClN4O3 [M+H]+ 435.1224; found:
435.1192.
( )-endo-41e: 48% from 34a and 35b, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.42 (t, J = 7.6 Hz, 3H), 1.60 (s, 3H),
2.24 (dd, J = 3.6, 14 Hz, 1H), 2.57 (dd, J = 2.2, 14 Hz, 1H), 4.24–
4.32 (m, 1H), 4.38–4.46 (m, 1H), 5.76 (d, J = 2.0 Hz, 1H), 6.08 (t,
J = 2.8 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 2.0, 8.0 Hz,
1H), 7.57 (ddd, J = 0.8, 7.5, 8.0 Hz, 1H), 7.74 (dd, J = 0.8, 8.0 Hz,
1H), 7.81 (ddd, J = 1.2, 7.2, 7.7 Hz, 1H), 8.38 (dd, J = 1.6, 8.0 Hz,
1H), 9.70 (s, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.2, 17.1, 37.1,
48.0, 53.7, 64.0, 67.2, 110.9, 120.1, 124.0, 126.9, 127.5, 128.0,
( )-exo-41c: 40% from 34c and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.42 (t, J = 7.2 Hz, 3H), 1.51 (s, 3H),
2.24 (dd, J = 2.0, 14.0 Hz, 1H), 2.55 (dd, J = 3.2, 14.0 Hz, 1H), 4.29–
4.41 (m, 2H), 6.03 (br s, 1H), 6.67 (d, J = 8.0 Hz, 1H), 7.01–7.07
(m, 2H), 7.21 (ddd, J = 1.6, 7.6, 7.6 Hz, 1H), 7.44 (ddd, J = 2.8, 8.4,
8.6 Hz, 1H), 7.72 (dd, J = 4.8, 8.6 Hz, 1H), 7.94 (dd, J = 2.8, 8.4 Hz,

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
105
128.2, 128.9, 131.9, 134.9, 139.9, 147.1, 153.3, 159.2, 171.6, 178.7;
2.12–2.19 (m, 1H), 2.35 (td, J = 2.8, 13.6 Hz, 1H), 2.84 (dd, J = 4.0,
13.6 Hz, 1H), 3.77 (s, 3H), 4.12–4.30 (m, 2H), 5.89 (s, 1H), 7.47–
7.50 (m, 1H), 7.75–7.78 (m, 2H), 8.27 (d, J = 7.6 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) d: 13.9, 20.8, 30.0, 45.0, 47.5, 52.2, 63.4, 63.6,
120.1, 126.7, 126.9, 127.8, 134.4, 147.5, 155.7, 159.0, 170.6,
IR (KBr) m
: 3218, 1719, 1642, 1609, 1473, 1176 cmꢀ1; LRMS (EI) m/z
434 [M]+; HRMS (FAB) m/z Calcd for C23H2037ClN4O3 [M+H]+
437.1205; found: 437.1199.
( )-exo-41f: 31% from 34b and 35b, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.45 (t, J = 7.2 Hz, 3H), 1.54 (s, 3H),
2.23 (dd, J = 2.4, 14 Hz, 1H), 2.55 (dd, J = 3.2, 14 Hz, 1H), 4.35–
4.39 (m, 2H), 6.03 (dd, J = 2.4, 3.2 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H),
7.05 (d, J = 1.6 Hz, 1H), 7.23 (dd, J = 2.0, 8.0 Hz, 1H), 7.66-7.67 (m,
2H), 8.07 (s, 1H), 8.28 (dd, J = 1.6, 1.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.2, 16.8, 37.7, 47.6, 54.9, 64.0, 66.8, 110.9, 121.3,
125.0, 126.2, 128.3, 129.0, 129.6, 132.2, 132.6, 134.7, 139.6,
171.9; IR (KBr) m
: 1674, 1641, 1473, 1431, 1373 cmꢀ1; LRMS (EI)
m/z 341 [M]+; HRMS (FAB) m/z Calcd for C18H20N3O4 [M+H]+
342.1454; found: 342.1451.
( )-41k-MP: 63% from 34a and 35d, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.31 (t, J = 7.2 Hz, 3H), 1.89 (s, 3H),
2.19–2.22 (m, 2H), 2.86 (dd, J = 7.2, 9.2 Hz, 1H), 3.59 (s, 3H),
4.11–4.22 (m, 2H), 5.87 (t, J = 6.8 Hz, 1H), 7.45–7.49 (m, 1H), 7.73
(d, J = 3.6 Hz, 2H), 8.29 (d, J = 8.0 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 13.9, 20.8, 29.8, 46.7, 47.5, 52.3, 63.4, 63.8, 120.2,
126.6, 126.7, 127.9, 134.2, 147.6, 154.4, 159.2, 171.5, 172.2; IR
146.0, 154.1, 158.3, 172.7, 177.5; IR (KBr) m: 1720, 1628, 1475,
1383, 1192 cmꢀ1; LRMS (FAB) m/z 469 [M+H]+; HRMS (FAB) m/z
Calcd for C23H1935Cl2N4O3 [M+H]+ 469.0834; found: 469.0846.
( )-endo-41f: 48% from 34b and 35b, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.40 (t, J = 7.2 Hz, 3H), 1.57 (s, 3H), 2.20
(dd, J = 3.2, 14 Hz, 1H), 2.57 (dd, J = 2.0, 14 Hz, 1H), 4.23–4.31 (m,
1H), 4.38–4.46 (m, 1H), 5.73 (d, J = 2.0 Hz, 1H), 6.04 (dd, J = 2.0,
3.2 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.0, 8.4 Hz, 1H),
7.54 (br s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.4, 8.8 Hz,
1H), 8.34 (dd, J = 0.4, 2.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d:
14.0, 16.9, 36.9, 48.0, 53.5, 64.0, 67.1, 110.8, 121.1, 123.8, 126.2,
128.0, 128.9, 129.7, 131.6, 133.3, 135.3, 139.7, 145.5, 153.6,
(KBr) m
: 1720, 1655, 1637, 1508, 1475 cmꢀ1; LRMS (EI) m/z 341
[M]+; HRMS (FAB) m/z Calcd for C18H20N3O4 [M+H]+ 342.1454;
found: 342.1469.
( )-41l-LP: 77% from 34a and 35d, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.29 (t, J = 7.2 Hz, 3H), 2.26 (s, 3H),
3.17 (d, J = 8.0 Hz, 1H), 3.57 (dd, J = 4.0, 8.0 Hz, 1H), 4.12–4.26
(m, 2H), 6.34 (d, J = 4.0 Hz, 1H), 7.21–7.24 (m, 2H), 7.41–7.45
(m, 1H), 7.49 (ddd, J = 1.6, 6.2, 6.7 Hz, 2H), 7.54 (ddd, J = 2.4,
5.5, 5.8 Hz, 1H), 7.78–7.83 (m, 2H), 8.32 (dd, J = 1.2, 8.2 Hz, 1H);
13C NMR (CDCl3, 100 MHz) d: 13.9, 19.3, 45.3, 46.2, 48.1, 64.0,
64.1, 120.0, 126.0 (2C), 126.9, 127.5, 128.0, 129.1, 129.3 (2C),
158.1, 171.4, 178.3; IR (KBr)
m: 1687, 1473, 1387, 1281,
1174 cmꢀ1; LRMS (FAB) m/z 469 [M+H]+; HRMS (FAB) m/z Calcd
for C23H1935Cl2N4O3 [M+H]+ 469.0834; found: 469.0846.
( )-exo-41g and ( )-endo-41g were obtained as an inseparable
mixture (67%) from 34e and 35a.
131.2, 134.9, 147.2, 154.3, 158.8, 168.7, 171.5, 172.2; IR (KBr) m:
1721, 1686, 1624, 1384 cmꢀ1; LRMS (EI) m/z 428 [M]+; HRMS
(FAB) m/z Calcd for C24H21N4O4 [M+H]+ 429.1563; found:
429.1595.
( )-exo-41h and ( )-endo-41h were obtained as an inseparable
mixture (quant.) from 34f and 35a.
( )-41l-MP: 3% from 34a and 35d, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.36 (t, J = 3.2 Hz, 3H), 2.21 (s, 3H),
3.32 (d, J = 8.8 Hz, 1H), 3.60 (dd, J = 3.2, 8.8 Hz, 1H), 4.20–4.32 (m,
2H), 6.37 (d, J = 2.8 Hz, 1H), 6.62–6.66 (m, 2H), 7.17–7.23 (m,
3H), 7.49 (ddd, J = 2.4, 3.0, 10.9 Hz, 1H), 7.74-7.79 (m, 2H), 8.26
(dd, J = 1.2, 7.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 13.9, 20.4,
44.9, 47.8, 49.1, 64.2, 64.3, 119.7, 126.0 (2C), 127.1, 127.5, 128.1,
129.0, 129.1 (2C), 130.7, 134.8, 147.0, 152.9, 158.5, 170.5, 171.5,
( )-exo-41i: 26% from 34g and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 0.89 (d, J = 6.8 Hz, 3H), 1.01 (d,
J = 6.8 Hz, 3H), 1.13 (dd, J = 6.8, 13.2 Hz, 1H), 1.42 (t, J = 7.2 Hz,
3H), 2.00 (dq, J = 2.8, 6.8 Hz, 1H), 2.21 (dd, J = 2.0, 14.0 Hz, 1H),
2.32 (dd, J = 2.8, 13.6 Hz, 1H), 2.48 (dd, J = 2.8, 13.6 Hz, 1H), 4.37
(dq, J = 0.8, 6.8 Hz, 2H), 6.03 (dd, J = 2.8, 2.8 Hz, 1H), 6.76 (d,
J = 8.0 Hz, 1H), 7.00–7.09 (m, 2H), 7.23 (ddd, J = 1.6, 7.2, 7.2 Hz,
1H), 7.48 (ddd, J = 3.2, 5.6, 7.6 Hz, 1H), 7.71–7.76 (m, 2H), 7.88
(br s, 1H), 8.32 (d, J = 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d:
14.2, 24.4, 24.8, 25.6, 36.1, 38.6, 47.0, 55.2, 63.4, 70.4, 109.7,
120.2, 122.7, 124.4, 126.5, 126.7, 128.2, 128.7, 131.3, 134.1,
171.9; IR (KBr) m
: 1718, 1685, 1638, 1381 cmꢀ1; LRMS (EI) m/z
428 [M]+; HRMS (FAB) m/z Calcd for C24H21N4O4 [M+H]+
429.1563; found: 429.1530.
141.3, 147.6, 153.4, 159.6, 171.9, 178.1; IR (KBr)
m
: 3226, 1715,
3.1.4. General procedure: hydrolysis of 41 to ( )-1–( )-10, ( )-
16–( )-18, ( )-20–( )-27, ( )-32, and ( )-33
1645, 1472, 1338, 1193 cmꢀ1; LRMS (EI) m/z 442 [M]+; HRMS
(FAB) m/z Calcd for C26H27N4O3 [M+H]+ 443.2083; found:
443.2089.
A solution of 41 in AcOEt (15 lM) and 1.0 N HCl (1.5 lM) was
stirred at room temperature. After removal of the solvent, the res-
idue was washed with MeOH to give the corresponding hydrolyzed
product.
( )-endo-41i: 38% from 34g and 35a, a yellow amorphous, 1H
NMR (CDCl3, 400 MHz) d: 0.97 (d, J = 6.8 Hz, 3H), 1.05 (d,
J = 6.8 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.54 (dd, J = 6.8, 13.6 Hz,
1H), 1.97 (dd, J = 4.0, 14.0 Hz, 1H), 2.09 (dq, J = 2.4, 6.8 Hz, 1H),
2.14 (dd, J = 3.2, 13.6 Hz, 1H), 2.53 (dd, J = 2.0, 13.6 Hz, 1H), 4.15–
4.45 (m, 2H), 5.77 (d, J = 6.8 Hz, 1H), 6.04 (dd, J = 2.4, 3.2 Hz, 1H),
6.70 (ddd, J = 0.8, 7.6, 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 7.15
(ddd, J = 0.8, 7.6, 7.6 Hz, 1H), 7.55 (ddd, J = 1.2, 7.6, 7.6 Hz, 1H),
7.58 (br s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.78 (ddd, J = 1.2, 7.6,
7.6 Hz, 1H), 8.37 (dd, J = 1.2, 7.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 14.1, 24.6, 24.8, 25.4, 36.6, 38.1, 47.4, 53.8, 63.6,
70.3, 109.4, 120.1, 122.6, 123.5, 126.8, 127.0, 128.3, 128.7, 130.2,
( )-14e: 74% from ( )-exo-41a, a white solid, 1H NMR (DMSO-d6,
400 MHz) d: 1.16 (s, 3H), 2.39 (s, 2H), 5.56 (d, J = 2.0Hz, 1H), 6.91
(d, J = 7.6Hz, 1H), 7.10 (t, J = 7.2Hz, 1H), 7.17 (d, J = 6.8Hz, 1H),
7.31 (t, J = 6.8Hz, 1H), 7.59 (t, J = 7.2, 1H), 7.69 (d, J = 8.0Hz, 1H),
7.86 (ddd, J = 1.2, 6.8, 7.2Hz, 1H), 8.20 (d, J = 6.8Hz, 1H), 9.56 (s,
1H), 10.65 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d: 13.4, 35.9,
51.9, 54.7, 61.8, 109.8, 120.0, 122.3, 123.7, 126.3, 127.2, 127.7,
129.1, 129.9, 134.7, 142.5, 146.8, 152.9, 158.3, 169.6, 176.6; IR
(KBr) m
: 3176, 1680, 1627, 1469 cmꢀ1; LRMS (EI) m/z 372 [M]+.
( )-24e: 71% from ( )-endo-41a, a white solid, 1H NMR (DMSO-
d6, 400 MHz) d: 1.22 (s, 3H), 2.31 (dd, J = 4.0, 14.4Hz, 1H), 2.54
(d, J = 14.0, 1H), 5.56 (s, 1H), 5.86 (d, J = 7.2Hz, 1H), 6.65 (t,
J = 7.6Hz, 1H), 6.86 (d, J = 7.6Hz, 1H), 7.14 (t, J = 7.6Hz, 1H), 7.61–
7.69 (m, 2H), 7.87 (t, J = 8.4Hz, 1H), 8.25 (d, J = 8.0Hz, 1H), 9.28
(s, 1H), 10.73 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d: 13.3, 35.2,
52.2, 52.6, 61.9, 109.5, 120.1, 121.6, 123.3, 126.6, 127.7, 127.8,
128.9, 129.2, 135.0, 142.3, 146.3, 152.4, 158.2, 168.9, 177.1; IR
134.5, 140.8, 147.1, 153.2, 159.2, 170.5, 178.3; IR (KBr) m: 3193,
1702, 1605, 1467, 1335 cmꢀ1; LRMS (EI) m/z 442 [M]+; HRMS
(FAB) m/z Calcd for C26H27N4O3 [M+H]+ 443.2083; found:
443.2089.
( )-exo-41j and ( )-endo-41j were obtained as an inseparable
mixture (30%) from 34a and 35c.
( )-41k-LP: 21% from 34a and 35d, a colorless amorphous, 1H
NMR (CDCl3, 400 MHz) d: 1.34 (t, J = 7.2 Hz, 3H), 1.96 (s, 3H),
(KBr) m
: 3192, 1691, 1620, 1469 cmꢀ1; LRMS (EI) m/z 372 [M]+.

106
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
( )-3: 62% from ( )-exo-41b, a white solid, mp >300 °C (MeOH);
m/z 448 [M]+; HRMS (FAB) m/z Calcd for C27H21N4O3 [M+H]+
449.1614; found: 449.1570.
1H NMR (DMSO-d6, 400 MHz) d: 1.16 (s, 3H), 2.40 (s, 2H), 5.55 (s,
1H), 6.91 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.17 (d,
J = 6.8 Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.89
(d, J = 8.8 Hz, 1H), 8.15 (s, 1H), 9.60 (s, 1H), 10.66 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz) d: 13.3, 35.9, 52.2, 54.7, 61.8, 109.9,
121.4, 122.4, 123.8, 125.3, 129.2, 129.8, 129.9, 131.5, 134.8,
( )-9: 27% (2 steps) from 34f and 35a, a white solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.14 (d, J = 6.8 Hz, 3H),
1.22 (d, J = 6.4 Hz, 3H), 2.01 (qui, J = 6.8 Hz, 1H), 2.24 (d,
J = 14.0 Hz, 1H), 2.43 (dd, J = 4.0, 14.0 Hz, 1H), 5.56 (br s, 1H),
6.92 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 7.6 Hz,
1H), 7.33 (d, J = 6.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.69 (d,
J = 8.0 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 9.52
(br s, 1H), 10.65 (br s, 1H); 13C NMR (DMSO-d6, 100 MHz) d:
16.4, 18.3, 30.7, 48.5, 51.2, 54.1, 66.9, 110.1, 119.9, 122.2, 123.9,
126.1, 127.1, 127.8, 129.0, 130.2, 134.5, 141.7, 146.5, 150.9,
142.6, 145.6, 153.5, 157.4, 169.4, 176.6; IR (KBr) m: 3217, 1726,
1619, 1470 cmꢀ1; LRMS (EI) m/z 406 [M]+; HRMS (FAB) m/z Calcd
for C21H1635ClN4O3 [M+H]+ 407.0911; found: 407.0920.
( )-4: 90% from ( )-exo-41c, a white solid, mp >300 °C (MeOH);
1H NMR (DMSO-d6, 400 MHz) d: 1.18 (s, 3H), 2.41 (s, 2H), 5.57 (d,
J = 2.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.19
(d, J = 6.8 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.76–7.80 (m, 2H), 7.90
(dd, J = 2.4, 8.8 Hz, 1H), 9.58 (s, 1H), 10.65 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 13.3, 35.8, 52.1, 54.6, 61.7, 109.8, 111.0
(d, J = 23 Hz), 121.3 (d, J = 8.0 Hz), 122.3, 123.0, 123.7, 129.1,
129.8, 130.4 (d, J = 9 Hz), 142.5, 143.6, 152.4, 157.6 (d, J = 3 Hz),
158.5, 169.7, 177.8; IR (KBr)
m: 3183, 1709, 1617, 1471, 1370,
1331 cmꢀ1; LRMS (EI) m/z 400 [M]+; HRMS (FAB) m/z Calcd for
C23H21N4O3 [M+H]+ 401.1614; found: 401.1637.
( )-10: quant. from ( )-exo-41i, a colorless solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 0.81 (d, J = 6.4 Hz, 3H),
0.88 (d, J = 6.4 Hz, 3H), 1.02 (dd, J = 5.2, 14.0 Hz, 1H), 2.07–2.18
(m, 2H), 2.34 (s, 2H), 5.59 (s, 1H), 6.91 (d, J = 7.6 Hz, 1H), 7.10 (t,
J = 7.6 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.61
(t, J = 7.6 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H),
8.22 (d, J = 7.6 Hz, 1H), 9.17 (s, 1H), 10.61 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 23.1, 23.9 (2C), 33.6, 37.0, 51.5, 55.9,
65.3, 109.6, 119.9, 122.3, 124.6, 126.2, 127.1, 127.7, 129.1, 129.9,
160.2 (d, J = 244 Hz), 169.4, 176.6; IR (KBr) m: 3252, 1705, 1629,
1486, 1334 cmꢀ1; LRMS (EI) m/z 390 [M]+; HRMS (FAB) m/z Calcd
for C21H16FN4O3 [M+H]+ 391.1206; found: 391.1202.
( )-5: 60% from ( )-exo-41d, a white solid, mp >300 °C (MeOH);
1H NMR (DMSO-d6, 400 MHz) d: 1.15 (s, 3H), 2.40 (d, J = 2.4 Hz,
2H), 5.55 (d, J = 2.4 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 7.10 (ddd,
J = 1.2, 7.5, 7.6 Hz, 1H), 7.17 (d, J = 6.8 Hz, 1H), 7.31 (ddd, J = 1.2,
7.6, 7.8 Hz, 1H), 7.63 (dd, J = 2.0, 8.4 Hz, 1H), 7.77 (d, J = 2.0 Hz,
1H), 8.20 (d, J = 8.8 Hz, 1H), 9.59 (d, J = 1.6 Hz, 1H), 10.67 (s, 1H);
13C NMR (DMSO-d6, 100 MHz) d: 13.3, 35.8, 52.0, 54.7, 61.8,
109.9, 118.9, 121.4, 122.4, 123.8, 126.9, 127.5, 128.4, 129.2,
134.6, 142.5, 146.5, 151.4, 158.3, 170.0, 176.4; IR (KBr) m: 3318,
1700, 1665, 1619, 1467 cmꢀ1; LRMS (EI) m/z 414 [M]+; HRMS
(FAB) m/z Calcd for C24H23N4O3 [M+H]+ 415.1770; found:
415.1751.
( )-16: 10% (2 steps) from from 34a and 35c, a colorless solid,
mp 275-277 °C (MeOH);1H NMR (CDCl3, 400 MHz) d: 1.72 (s, 3H),
2.94–3.02 (m, 2H), 3.60 (s, 3H), 5.84 (d, J = 2.4 Hz, 1H), 6.45 (s,
1H), 7.19 (d, J = 6.8 Hz, 2H), 7.30–7.39 (m, 3H), 7.52 (ddd, J = 1.2,
7.4, 7.4 Hz, 1H), 7.78 (ddd, J = 1.2, 7.4, 7.6 Hz, 1H), 7.82 (t,
J = 7.6 Hz, 1H), 8.30 (d, J = 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz)
d: 16.3, 37.8, 52.1, 52.7, 58.5, 62.6, 120.3, 126.9, 127.4, 128.1
(2C), 128.2, 128.3, 128.5 (2C), 134.6, 137.0, 147.4, 153.2, 159.1,
129.8, 139.3, 142.6, 147.9, 154.4, 169.4, 176.6; IR (KBr) m: 3199,
1712, 1662, 1599 cmꢀ1; LRMS (EI) m/z 406 [M]+; HRMS (FAB) m/z
Calcd for C21H1537ClN4O3 [M+H]+ 409.0890; found: 409.0902.
( )-6: 72% from ( )-exo-41e, a white solid, 1H NMR (DMSO-d6,
400 MHz) d: 1.21 (s, 3H), 2.41 (dd, J = 3.6, 14.4 Hz, 1H), 2.50 (d,
J = 14.4 Hz, 1H), 5.58 (s, 1H), 6.95 (d, J = 8.0 Hz, 1H), 7.17 (d,
J = 2.0 Hz, 1H), 7.39 (dd, J = 2.0, 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz,
1H), 7.70 (d, J = 8.0 Hz, 1H), 7.88 (ddd, J = 1.2, 7.8, 8.4 Hz, 1H),
8.21 (dd, J = 1.2, 8.0 Hz, 1H), 9.69 (d, J = 1.2 Hz, 1H), 10.81 (s, 1H);
13C NMR (DMSO-d6, 100 MHz) d: 13.3, 35.6, 51.7, 55.0, 61.7,
111.2, 119.9, 123.9, 126.0, 126.3, 127.2, 127.6, 129.0, 131.7,
169.7, 172.7; IR (KBr)
m: 1739, 1626, 1458, 1389, 1331,
1242 cmꢀ1; LRMS (EI) m/z 389 [M]+; HRMS (FAB) m/z Calcd for
C22H20N3O4 [M+H]+ 390.1454; found: 390.1436.
( )-17: 59% from ( )-41j-MP, a colorless amorphus solid, 1H
NMR (CDCl3, 400 MHz) d: 1.34 (t, J = 7.2 Hz, 3H), 1.96 (s, 3H),
2.12–2.19 (m, 1H), 2.35 (td, J = 2.8, 13.6 Hz, 1H), 2.84 (dd, J = 4.0,
13.6 Hz, 1H), 3.77 (s, 3H), 4.12–4.30 (m, 2H), 5.89 (s, 1H), 7.47–
7.50 (m, 1H), 7.75–7.78 (m, 2H), 8.27 (d, J = 7.6 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) d: 13.9, 20.8, 30.0, 45.0, 47.5, 52.2, 63.4, 63.6,
120.1, 126.7, 126.9, 127.8, 134.4, 147.5, 155.7, 159.0, 170.6,
134.6, 141.5, 146.7, 152.5, 158.2, 169.5, 176.2; IR (KBr) m: 1719,
1686, 1624, 1475 cmꢀ1; LRMS (FAB) m/z 407 [M+H]+; HRMS
(FAB) m/z Calcd for C21H1635ClN4O3 [M+H]+ 407.0911; found:
407.0887.
( )-7: 90% from ( )-exo-41f, a white solid, mp >300 °C (MeOH);
1H NMR (DMSO-d6, 400 MHz) d: 1.20 (s, 3H), 2.41 (dd, J = 3.2,
14.8 Hz, 1H), 2.49 (d, J = 14.8 Hz, 1H), 5.57 (s, 1H), 6.95 (d,
J = 8.4 Hz, 1H), 7.17 (s, 1H), 7.40 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H),
7.73 (dd, J = 0.8, 8.4 Hz, 1H), 7.90 (ddd, J = 1.6, 2.4, 8.4 Hz, 1H),
8.15 (dd, J = 1.2, 2.4 Hz, 1H), 9.71 (s, 1H), 10.82 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 13.2, 35.5, 51.9, 55.0, 61.6, 111.3, 121.3,
123.9, 125.3, 126.1, 129.0, 129.8, 131.5, 131.6, 134.8, 141.5,
171.9; IR (KBr) m
: 1674, 1641, 1473, 1431, 1373 cmꢀ1; LRMS (EI)
m/z 341 [M]+; HRMS (FAB) m/z Calcd for C18H20N3O4 [M+H]+
342.1454; found: 342.1451.
( )-18: 82% from ( )-exo-41l-LP, a colorless solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.94 (s, 3H), 3.42 (d,
J = 8.4 Hz, 1H), 4.02 (dd, J = 4.0, 8.0 Hz, 1H), 5.62 (dd, J = 2.0,
4.0 Hz, 1H), 7.21 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.56 (t,
J = 7.6 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.90
(ddd, J = 1.2, 7.2, 7.6 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 9.64 (d,
J = 1.2 Hz, 1H); 13C NMR (DMSO-d6, 150 MHz) d: 16.4, 44.6, 48.3,
53.1, 59.1, 121.0, 127.0, 127.4 (2C), 128.2, 128.3, 129.5, 129.7
(2C), 132.4, 135.4, 147.3, 154.1, 158.9, 166.8, 173.3, 173.8; IR
145.4, 153.0, 157.3, 169.3, 176.2; IR (KBr) m: 1672, 1626, 1464,
1319 cmꢀ1; LRMS (EI) m/z 440 [M]+; HRMS (FAB) m/z Calcd for
C21H1535Cl2N4O3 [M+H]+ 441.0521; found: 441.0509.
( )-8: 17% (2 steps) from from 34e and 35a, a colorless solid, mp
>300 °C (MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 2.37–2.47 (m,
2H), 2.71 (d, J = 14.8 Hz, 1H), 3.45 (d, J = 14.8 Hz, 1H), 5.60 (br s,
1H), 6.87 (d, J = 7.6 Hz, 1H), 7.12–7.15 (m, 4H), 7.27 (d, J = 7.6 Hz,
1H), 7.28 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.35 (t,
J = 7.6 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.89
(t, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 9.11 (s, 1H), 10.51 (s,
1H); 13C NMR (DMSO-d6, 100 MHz) d: 32.4, 37.5, 51.8, 55.3, 65.2,
110.0, 120.0, 122.4, 124.3, 126.4, 126.6, 127.4, 127.5 (2C), 127.7,
129.3, 129.7, 131.3 (2C), 134.0, 134.8, 142.8, 146.5, 151.5, 158.4,
(KBr) m
: 1717, 1623, 1468, 1389, 1182 cmꢀ1; LRMS (FAB) m/z 401
[M+H]+; HRMS (FAB) m/z Calcd for C22H17N4O4 [M+H]+ 401.1250;
found: 401.1221.
( )-20: 60% from ( )-endo-41b, a white solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.22, (s, 3H), 2.31 (dd,
J = 3.6, 14. Hz, 1H), 2.55 (d, J = 14 Hz, 1H), 5.55 (s, 1H), 5.92 (d,
J = 7.6 Hz, 1H), 6.68 (t, J = 7.6 Hz, 1H), 6.86 (t, J = 7.6 Hz, 1H), 7.16
(t, J = 7.6 Hz, 1H), 7.72 (t, J = 8.8 Hz, 1H), 7.90 (dd, J = 2.8, 8.8 Hz,
169.7, 170.4; IR (KBr) m
: 3264, 1701, 1628, 1473 cmꢀ1; LRMS (EI)

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
107
1H), 8.20 (d, J = 2.4 Hz, 1H), 9.34 (s, 1H), 10.95 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 12.3, 35.1, 52.4, 52.6, 62.0, 109.5, 121.7,
122.2, 123.5, 125.6, 128.9, 129.1, 130.0, 131.9, 135.0, 142.4,
( )-26: 68% (2 steps) from 34f and 35a, a colorless solid, mp
>300 °C (MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 0.98 (d,
J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 2.12 (dd, J = 3.2, 14.0 Hz,
1H), 2.44 (qui, J = 6.8 Hz, 1H), 2.53 (dd, J = 2.4, 14.0 Hz, 1H), 5.52
(br s, 1H), 5.97 (d, J = 7.2 Hz, 1H), 6.70 (t, J = 7.6 Hz, 1H), 6.86 (d,
J = 7.6 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.75
(d, J = 8.0 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H),
9.20 (br s, 1H), 10.77 (br s, 1H); 13C NMR (DMSO-d6, 100 MHz) d:
17.1, 18.4, 28.9, 51.6, 51.9 (2C), 67.9, 109.7, 120.1, 121.7, 123.5,
126.5, 127.8, 128.0, 128.8, 130.2, 135.1, 141.7, 146.2, 151.5,
145.1, 152.9, 157.3, 168.7, 177.1; IR (KBr) m: 3202, 1739, 1622,
1469 cmꢀ1; LRMS (EI) m/z 406 [M]+; HRMS (FAB) m/z Calcd for
C21H1637ClN4O3 [M+H]+ 409.0890; found: 409.0889.
( )-21: 67% from ( )-endo-41c, a white solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.23 (s, 3H), 2.32 (dd,
J = 3.2, 14 Hz, 1H), 2.56 (d, J = 14 Hz, 1H), 5.56 (s, 1H), 5.90 (d,
J = 7.6 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 7.16
(t, J = 7.6 Hz, 1H), 7.77 (d, J = 6.0 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H),
9.31 (s, 1H), 10.75 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d: 13.3,
158.2, 169.3, 178.3; IR (KBr)
m: 3206, 1709, 1616, 1468, 1373,
1333 cmꢀ1; LRMS (EI) m/z 400 [M]+; HRMS (FAB) m/z Calcd for
C23H21N4O3 [M+H]+ 401.1614; found: 401.1617.
35.1, 52.3, 52.6, 61.9, 109.5, 111.3 (d,
J =29 Hz), 121.5 (d,
J = 8 Hz), 121.6, 123.3 (d, J = 24 Hz), 123.4, 128.9, 129.2, 130.7 (d,
J = 9 Hz), 142.4, 143.2, 151.9, 157.6 (d, J = 3 Hz), 160.5 (d,
( )-27: 91% from ( )-endo-41i, a colorless solid, mp >300 °C
(MeOH); 1H NMR (CDCl3, 400 MHz) d: 0.99 (d, J = 6.8 Hz, 6H),
1.47 (dd, J = 6.8, 14.8 Hz, 1H), 2.03 (dd, J = 4.8, 14.8 Hz, 1H), 2.16–
2.22 (m, 1H), 2.29 (dd, J = 4.0, 14.4 Hz, 1H), 2.77 (dd, J = 1.6,
14.4 Hz, 1H), 5.85 (d, J = 7.2 Hz, 1H), 5.95 (dd, J = 1.6, 1.6 Hz, 1H),
6.60 (s, 1H), 6.75 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 7.20
(t, J = 7.6 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H),
7.83 (ddd, J = 1.2, 7.6, 7.6 Hz, 1H), 7.93 (s, 1H), 8.41 (dd, J = 1.2,
8.0 Hz, 1H); 13C NMR (DMSO-d6, 100 MHz) d: 23.1, 23.6, 23.8,
33.6, 36.2, 54.0, 57.8, 65.3, 109.5, 120.0, 121.5, 123.5, 126.4,
127.6, 127.8, 128.8, 129.1, 134.9, 147.2, 145.9, 151.0, 158.1,
J = 245 Hz), 168.7, 177.1; IR (KBr)
m: 3188, 1740, 1709, 1626,
1484 cmꢀ1; LRMS (EI) m/z 390 [M]+; HRMS (FAB) m/z Calcd for
C21H16FN4O3 [M+H]+ 391.1206; found: 391.1220.
( )-22: 86% from ( )-endo-41d, a white solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.20 (s, 3H), 2.31 (dd,
J = 3.6, 14.4 Hz, 1H), 2.53 (dd, J = 1.6, 14.4 Hz, 1H), 5.53–5.54 (m,
1H), 5.93 (d, J = 7.2 Hz, 1H), 6.69 (ddd, J = 0.8, 7.6, 7.7 Hz, 1H),
6.85 (d, J = 7.6 Hz, 1H), 7.14 (ddd, J = 0.8, 7.8, 8.0 Hz, 1H), 7.66
(dd, J = 2.0, 8.8 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 8.8 Hz,
1H), 9.32 (d, J = 1.6 Hz, 1H), 10.77 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) d: 13.3, 35.0, 52.2, 52.5, 62.0, 109.5, 119.1, 121.7,
123.4, 127.0, 128.0, 128.6, 128.9, 129.0, 139.5, 142.3, 147.5,
169.3, 177.2; IR (KBr) m
: 2920, 1700, 1619, 1560, 1467 cmꢀ1; LRMS
(EI) m/z 414 [M]+; HRMS (FAB) m/z Calcd for C24H23N4O3 [M+H]+
415.1770; found: 415.1786.
153.8, 157.6, 168.7, 177.0; IR (KBr)
m
:
3445, 1735, 1707,
( )-32: 13% (2 steps) from 34a and 35c, a colorless solid, mp
257–259 °C (MeOH);1H NMR (CDCl3, 400 MHz) d: 1.87 (s, 3H),
2.98 (dd, J = 2.4, 15.6 Hz, 1H), 3.04 (dd, J = 3.2, 15.6 Hz, 1H), 3.79
(s, 3H), 5.88 (d, J = 2.4 Hz, 1H), 6.51 (s, 1H), 6.71 (d, J = 7.6 Hz,
2H), 7.11 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.19 (t,
J = 7.2 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.72
(ddd, J = 1.2, 7.6, 7.6 Hz, 1H), 8.35 (dd, J = 1.2, 8.0 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 15.4, 37.6, 51.7, 52.8, 59.0, 63.6, 120.3,
126.9 (2C), 127.0, 127.5, 128.0, 128.2, 128.4 (2C), 134.6, 137.4,
1603 cmꢀ1; LRMS (EI) m/z 406 [M]+; HRMS (FAB) m/z Calcd for
C21H1637ClN4O3 [M+H]+ 409.0890; found: 409.0873.
( )-23: 83% from ( )-endo-41e, a white solid, 1H NMR (DMSO-
d6, 400 MHz) d: 1.24 (s, 3H), 2.43 (dd, J = 4.0, 10.0 Hz, 1H), 2.52
(d, J = 10.0 Hz, 1H), 5.55 (s, 1H), 5.93 (d, J = 1.6 Hz, 1H), 6.88 (d,
J = 8.4 Hz, 1H), 7.21 (dd, J = 2.0, 8.4 Hz, 1H), 7.66 (t, J = 7.6 Hz,
1H), 7.72 (d, J = 8.0 Hz, 1H), 7.90 (ddd, J = 1.2, 7.2, 7.6 Hz, 1H),
8.27 (d, J = 8.0 Hz, 1H), 9.35 (s, 1H), 10.92 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 13.9, 34.7, 52.6, 53.6, 55.5, 62.6, 111.4,
120.8, 124.7, 125.9, 127.1, 128.3, 129.3, 131.6, 135.5, 141.9,
146.9, 152.2, 159.0, 169.0, 173.0; IR (KBr) m: 1697, 1604, 1560,
1450, 1369 cmꢀ1; LRMS (EI) m/z 389 [M]+; HRMS (FAB) m/z Calcd
for C22H20N3O4 [M+H]+ 390.1454; found: 390.1417.
146.8, 152.7, 158.7, 169.4, 177.3; IR (KBr) m: 1734, 1690, 1626,
1468, 1375, 1311 cmꢀ1; LRMS (FAB) m/z 407 [M+H]+; HRMS
(FAB) m/z Calcd for C21H1635ClN4O3 [M+H]+ 407.0911; found:
407.0887.
( )-33: 67% from ( )-41j-LP, a colorless solid, mp 214–218 °C
(MeOH); 1H NMR (CDCl3, 400 MHz) d: 1.25 (s, 3H), 1.84 (s, 3H),
2.38–2.52 (m, 2H), 2.88 (dd, J = 5.2, 9.6 Hz, 1H), 5.77–5.79 (m,
1H), 6.38 (br s, 1H), 7.53 (ddd, J = 1.2, 7.4, 7.4 Hz, 1H), 7.72 (d,
J = 7.2 Hz, 1H), 7.78 (ddd, J = 1.2, 7.6, 9.5 Hz, 1H), 8.30 (dd, J = 1.6,
8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 17.2, 29.3, 45.7, 52.0,
52.6, 59.1, 120.7, 127.0, 127.6, 127.9, 134.7, 147.1, 153.4, 158.9,
( )-24: 92% from ( )-endo-41f, a white solid, mp >300 °C
(MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 1.23 (s, 3H), 2.45 (dd,
J = 3.6, 14.4 Hz, 1H), 2.53 (d, J = 14.4 Hz, 1H), 5.54 (s, 1H), 6.03 (d,
J = 1.6 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 1.6, 8.4 Hz,
1H), 7.75 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 2.4, 8.8 Hz, 1H), 8.17 (d,
J = 2.4 Hz, 1H), 9.36 (s, 1H), 10.92 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) d: 13.3, 34.5, 52.3, 53.1, 62.1, 110.9, 121.7, 124.2,
125.5, 125.6, 128.8, 129.9, 130.9, 131.9, 135.0, 141.4, 145.1,
169.0, 171.3; IR (KBr) m
: 1709, 1626, 1367, 1213, 1176 cmꢀ1; LRMS
(EI) m/z 313 [M]+; HRMS (FAB) m/z Calcd for C16H16N3O4 [M+H]+
314.1141; found: 314.1139.
152.6, 157.3, 168.7, 176.8; IR (KBr)
m
:
1693, 1626, 1475,
3.1.5. Synthesis of 2-methyl-alantrypinone (( )-11)
1373 cmꢀ1; LRMS (EI) m/z 440 [M]+; HRMS (FAB) m/z Calcd for
To a solution of ( )-exo-41e (80 mg, 200
was added a solution of n-BuLi (210
mol) at ꢀ78 °C and the mix-
ture was stirred for 30 min at ꢀ78 °C. To the mixture was added
benzenesulfonyl chloride (130 L, 210
mol) for 5 h at ꢀ78 °C.
lmol) in THF (1 mL)
C21H1535Cl2N4O3 [M+H]+ 441.0521; found: 441.0509.
l
( )-25: 32% (2 steps) from 34e and 35a, a colorless solid, mp
>300 °C (MeOH); 1H NMR (DMSO-d6, 400 MHz) d: 2.31 (dd,
J = 3.6, 14.0 Hz, 1H), 2.58 (d, J = 14.0 Hz, 1H), 2.97 (d, J = 14.8 Hz,
1H), 3.18 (d, J = 14.8 Hz, 1H), 5.59 (br s, 1H), 5.89 (d, J = 7.6 Hz,
1H), 6.70 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 7.13 (t,
J = 7.6 Hz, 2H), 7.19 (t, J = 7.6 Hz, 2H), 7.40 (d, J = 7.2 Hz, 2H), 7.65
(t, J = 7.6 Hz, 2H), 7.88 (t, J = 7.6 Hz, 1H), 8.25 (d, J = 7.2 Hz, 1H),
8.71 (br s, 1H), 10.82 (br s, 1H); 13C NMR (DMSO-d6, 100 MHz) d:
32.0, 36.5, 51.9, 53.7, 65.1, 109.7, 120.1, 121.6, 123.7, 126.3,
126.5, 127.5 (2C), 127.6, 127.8, 128.9, 129.1, 131.2 (2C), 134.3,
l
l
The reaction was quenched by the addition of satd aq NH4Cl and
the organic compounds were extracted with AcOEt and the com-
bined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was subjected
to column chromatography (hexane/AcOEt = 2:1) to give ( )-exo-
42c (75 mg, 69%) as a colorless amorphous solid. 1H NMR (CDCl3,
400 MHz) d: 1.15 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H), 2.16 (dd, J = 2.0,
14 Hz, 1H), 2.47 (dd, J = 3.2, 14 Hz, 1H), 4.25–4.27 (m, 2H), 5.98
(t, J = 2.8 Hz, 1H), 7.05 (dd, J = 0.4, 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz,
1H), 7.41 (ddd, J = 1.2, 8.0, 8.2 Hz, 1H), 7.49 (ddd, J = 1.2, 6.8,
7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.68 (t, J = 7.6 Hz, 2H), 7.76
135.0, 142.4, 145.9, 150.5, 158.1, 169.0, 179.1; IR (KBr) m: 3229,
1686, 1625, 1471 cmꢀ1; LRMS (EI) m/z 448 [M]+; HRMS (FAB) m/z
Calcd for C27H21N4O3 [M+H]+ 449.1614; found: 449.1580.

108
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
(ddd, J = 1.2, 7.6, 8.0 Hz, 1H), 7.99 (t, J = 7.6 Hz, 3H), 8.30 (dd, J = 1.2,
8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.1, 16.6, 38.6, 47.2,
54.6, 63.8, 67.5, 113.6, 120.2, 124.3, 125.3, 126.7, 126.8, 127.9
(2C), 128.1, 129.0, 129.2 (2C), 129.7, 134.3, 134.7, 137.7, 139.6,
(s, 3H), 2.48 (dd, J = 2.0, 14.4 Hz, 1H), 2.69 (dd, J = 3.6, 14.4 Hz, 1H),
3.17 (s, 3H), 5.95 (br s, 1H), 6.39 (br s, 1H), 6.91 (d, J = 7.6 Hz, 1H),
7.14 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz,
1H), 7.52 (ddd, J = 2.4, 6.8, 7.6 Hz, 1H), 7.73–7.79 (m, 2H), 8.34
(d, J = 7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.2, 26.7, 30.4,
36.6, 54.9, 62.7, 108.7, 120.7, 123.6, 127.1, 127.2, 127.3, 128.1,
129.1, 129.7, 134.4, 144.2, 147.1, 151.7, 159.1, 170.2, 175.0; IR
147.5, 152.8, 159.2, 172.6, 174.6; IR (KBr) m: 1751, 1687, 1628,
1460, 1389 cmꢀ1; LRMS (EI) m/z 540 [M]+; HRMS (FAB) m/z Calcd
for C29H25N4O5S [M+H]+ 541.1546; found: 541.1502.
A solution of ( )-exo-42c (75 mg, 139
lmol) in AcOEt (5 mL)
(KBr) m
: 3186, 1706, 1656, 1610, 1468, 1350 cmꢀ1; LRMS (EI) m/z
and 1.0 N HCl (0.5 mL) was stirred at room temperature for 3 h.
After removal of the solvent, the residue was washed with MeOH
to give 18-benzenesulfonyl-alantrypinone (70 mg, 98%) as a color-
less solid. To a solution of 18-benzenesulfonyl-alantrypinone
386 [M]+; HRMS (FAB) m/z Calcd for C22H19N4O3 [M+H]+
387.1457; found: 387.1440.
To a solution of ( )-12 (6 mg, 15.5
l
mol) and NaH (0.7 mg,
L, 18.6 mol) at
18.6 mol) in THF (1.0 mL) was added MeI (1.2
l
l
l
(70 mg, 137
was added MeI (10
l
mol) and NaH (4 mg, 165
l
mol) in THF (1.0 mL)
0 °C and the mixture was stirred for 2 hours at 0 °C. The reaction
was quenched by the addition of satd aq NH4Cl and the organic
compounds were extracted with AcOEt and the combined organic
layers were washed with brine and dried over Na2SO4. After re-
moval of the solvent, the residue was subjected to column chroma-
tography (hexane/AcOEt = 1:1) to give ( )-13 (6.2 mg, quant.) as a
colorless solid. Mp 294–296 °C (AcOEt);1H NMR (CDCl3, 400 MHz)
d: 1.47 (s, 3H), 2.43 (dd, J = 2.4, 14.0 Hz, 1H), 2.64 (dd, J = 3.6,
14.0 Hz, 1H), 2.98 (s, 3H), 3.17 (s, 3H), 6.01 (dd, J = 2.8, 2.8 Hz,
1H), 6.91 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.15 (t,
J = 7.6 Hz, 1H), 7.40 (ddd, J = 0.8, 7.6, 8.0 Hz, 1H), 7.51 (ddd,
J = 2.4, 7.6, 8.0 Hz, 1H), 7.70–7.78 (m, 2H), 8.34 (dd, J = 0.8,
8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 12.7, 26.6, 28.7, 35.7,
51.2, 55.5, 65.8, 108.7, 120.8, 123.5, 123.6, 127.0, 127.2, 128.0,
129.3, 129.7, 134.3, 144.2, 147.1, 151.6, 159.0, 169.2, 175.1; IR
l
L, 165 mol) at 0 °C and the mixture was stir-
l
red for 2 h at 0 °C. The reaction was quenched by the addition of
satd aq NH4Cl and the organic compounds were extracted with
AcOEt and the combined organic layers were washed with brine
and dried over Na2SO4. After removal of the solvent, the residue
was subjected to column chromatography (hexane/AcOEt = 1:1)
to give 43 (28 mg, 39%) as a colorless solid. 1H NMR (CDCl3,
400 MHz) d: 1.25 (s, 3H), 2.34 (dd, J = 2.4, 14.4 Hz, 1H), 2.53 (dd,
J = 3.2, 14.4 Hz, 1H), 2.88 (s, 3H), 5.94 (dd, J = 2.4, 3.2 Hz, 1H),
7.05 (dd, J = 1.2, 7.6 Hz, 1H), 7.26–7.30 (m, 1H), 7.48 (ddd, J = 1.2,
7.6, 8.0 Hz, 1H), 7.52–7.56 (m, 3H), 7.67–7.72 (m, 2H), 7.79 (ddd,
J = 1.6, 7.2, 7.6 Hz, 1H), 8.00–8.04 (m, 3H), 8.32 (dd, J = 1.6,
7.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 12.7, 28.6, 36.4, 50.9,
55.8, 66.5, 114.1, 120.8, 123.9, 126.0, 127.0, 127.4, 127.9 (2C),
128.0, 128.2, 129.3 (2C), 130.5, 134.5, 134.9, 137.4, 139.7, 147.1,
(KBr) m
: 1697, 1610, 1467, 1378 cmꢀ1; LRMS (EI) m/z 400 [M]+;
150.4, 158.9, 168.8, 173.6; IR (KBr)
m
: 1687, 1624, 1459, 1374,
HRMS (FAB) m/z Calcd for C23H21N4O3 [M+H]+ 401.1614; found:
401.1584.
1232, 1185, 1154, 1084, 957, 755 cmꢀ1; LRMS (EI) m/z 526 [M]+;
HRMS (FAB) m/z Calcd for C28H22N4O5S [M+H]+ 527.1389; found:
527.1385.
3.1.7. Synthesis of ( )-14 and ( )-15
To a solution of 43 (25 mg, 47
0.4 M solution of Na-anthracenide in THF (0.7 mL, 280
l
mol) in THF (1.0 mL) was added
To a solution of ( )-exo-41a (20 mg, 50
l
mol) and NaH (2.4 mg,
mol) in THF (1.0 mL) was added benzyl bromide (12 L,
mol) at 0 °C and the mixture was stirred for 3.5 h at 0 °C.
lmol) at
100
100
l
l
l
ꢀ78 °C and the mixture was stirred for 30 min at ꢀ78 °C. The reac-
tion was quenched by the addition of satd aq NH4Cl and the organ-
ic compounds were extracted with AcOEt and the combined
organic layers were washed with brine and dried over Na2SO4.
After removal of the solvent, the residue was subjected to column
chromatography (hexane/AcOEt = 1:1) to give 11 (12 mg, 67%) as a
colorless solid. 1H NMR (CDCl3, 400 MHz) d: 1.54 (s, 3H), 2.43 (dd,
J = 2.0, 14.0 Hz, 1H), 2.65 (dd, J = 3.2, 14 Hz, 1H), 2.97 (s, 3H), 6.00
(dd, J = 2.4, 2.8 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 7.2 Hz,
1H), 7.10 (t, J = 7.2 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.52 (t,
J = 7.2 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 8.24
(br s, 1H), 8.34 (d, J = 7.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d:
12.7, 28.7, 35.7, 51.2, 55.9, 65.9, 110.3, 120.9, 123.6, 123.9, 127.1,
127.2, 127.9, 129.6, 129.7, 134.3, 141.3, 147.2, 151.6, 159.0,
The reaction was quenched by the addition of satd aq NH4Cl and
the organic compounds were extracted with AcOEt and the com-
bined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was subjected
to column chromatography (hexane/AcOEt = 2:3) to give ( )-exo-
42b (20.4 mg, 83%) as a colorless amorphous solid. A solution of
( )-exo-42b (20 mg, 41 lmol) in AcOEt (5 mL) and 1.0 N HCl
(0.5 mL) was stirred at room temperature for 3.5 h. After removal
of the solvent, the residue was washed with MeOH to give ( )-14
(16.4 mg, 86%) as a colorless solid. Mp >300 °C (MeOH); 1H NMR
(CDCl3, 400 MHz) d: 1.38 (s, 3H), 2.51 (dd, J = 2.0, 14 Hz, 1H),
2.75 (dd, J = 3.6, 14 Hz, 1H), 4.78 (d, J = 15.6 Hz, 1H), 4.88 (d,
J = 15.6 Hz, 1H), 5.97 (ddd, J = 1.6, 1.6, 2.8 Hz, 1H), 6.43 (br s, 1H),
6.83 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.25–7.35 (m, 7H),
7.52 (ddd, J = 3.2, 5.2, 8.0 Hz, 1H), 7.75–7.80 (m, 2H), 8.35 (dd,
J = 0.4, 8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 14.2, 36.9, 44.3,
52.1, 54.8, 62.8, 109.7, 120.7, 123.6, 123.7, 127.1, 127.3, 127.5
(2C), 127.9, 128.1, 128.9 (2C), 129.2, 129.6, 134.5, 135.3, 143.3,
169.1, 177.0; IR (KBr) m
: 1687, 1610, 1469, 1385, 1329 cmꢀ1; LRMS
(EI) m/z 386 [M]+; HRMS (FAB) m/z Calcd for C22H19N4O3 [M+H]+
387.1457; found: 387.1443.
3.1.6. Synthesis of ( )-12 and ( )-13
To a solution of ( )-exo-41a (10 mg, 25
l
mol) and NaH (0.7 mg,
147.2, 151.7, 159.2, 170.7, 175.2; IR (KBr) m: 3434, 1710, 1629,
30
lmol) in THF (1.0 mL) was added MeI (1.9
l
L, 30 mol) at 0 °C
l
1466, 1362 cmꢀ1; LRMS (EI) m/z 462 [M]+; HRMS (FAB) m/z Calcd
and the mixture was stirred for 1.5 h at 0 °C. The reaction was
quenched by the addition of satd aq NH4Cl and the organic com-
pounds were extracted with AcOEt and the combined organic lay-
ers were washed with brine and dried over Na2SO4. After removal
of the solvent, the residue was subjected to column chromatogra-
phy (hexane/AcOEt = 2:3) to give ( )-exo-42a (10.4 mg, quant.) as a
colorless amorphous solid. A solution of ( )-exo-42a (10 mg,
for C28H23N4O3 [M+H]+ 463.1770; found: 463.1783.
To a solution of ( )-14 (15 mg, 32
lmol) and NaH (1.0 mg,
39
lmol) in THF (1.0 mL) was added MeI (2.4
l
L, 39 mol) at
l
0 °C and the mixture was stirred for 2 h at room temperature.
The reaction was quenched by the addition of satd aq NH4Cl and
the organic compounds were extracted with AcOEt and the com-
bined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was subjected
to column chromatography (hexane/AcOEt = 1:1) to give ( )-15
(10.5 mg, 69%) as a colorless solid. Mp 290–291 °C (AcOEt); 1H
NMR (CDCl3, 400 MHz) d: 1.51 (s, 3H), 2.46 (dd, J = 2.0, 14 Hz,
24 lmol) in AcOEt (1 mL) and 1.0 N HCl (0.5 mL) was stirred at
room temperature for 30 min. After removal of the solvent, the res-
idue was washed with MeOH to give ( )-12 (8.8 mg, 95%) as a col-
orless solid. Mp >300 °C (MeOH); 1H NMR (CDCl3, 400 MHz) d: 1.35

T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
109
1H), 2.70 (dd, J = 3.6, 14 Hz, 1H), 2.99 (s, 3H), 4.77 (d, J = 15.6 Hz,
1H), 4.89 (d, J = 15.6 Hz, 1H), 6.03 (dd, J = 2.0, 3.6 Hz, 1H), 6.83 (d,
J = 8.0, Hz, 1H), 7.08–7.13 (m, 2H), 7.25–7.35 (m, 6H), 7.52 (ddd,
J = 2.8, 6.0, 6.8 Hz, 1H), 7.74–7.79 (m, 2H), 8.35 (dd, J = 2.8,
8.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d: 13.0, 28.7, 36.1, 44.3,
51.2, 55.4, 65.9, 109.8, 120.9, 123.65, 123.69, 127.1, 127.3, 127.5
(2C), 128.0, 128.1, 128.9 (2C), 129.4, 129.6, 134.4, 135.3, 143.4,
orless solid. Mp 285–288 °C (AcOEt); 1H NMR (CDCl3, 400 MHz) d:
1.47 (s, 3H), 2.33 (dd, J = 4.0, 14.4 Hz, 1H), 2.74 (dd, J = 1.6, 14.4 Hz,
1H), 3.07 (s, 3H), 3.27 (s, 3H), 5.87 (d, J = 7.6 Hz, 1H), 6.00 (dd,
J = 4.0, 7.6 Hz, 1H), 6.75 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H),
7.25 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8.0 Hz,
1H), 7.80 (ddd, J = 1.6, 8.0, 8.0 Hz, 1H), 8.40 (dd, J = 1.6, 8.0 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d: 12.8, 26.6, 29.0, 35.6, 51.5,
53.1, 65.9, 108.3, 120.7, 123.1, 123.2, 127.2, 127.7, 128.1, 128.9,
147.2, 151.6, 159.0, 169.2, 175.3; IR (KBr) m: 1687, 1604, 1469,
1375cmꢀ1; LRMS (EI) m/z 476 [M]+; HRMS (FAB) m/z Calcd for
129.3, 134.7, 143.8, 146.8, 151.7, 158.8, 168.2, 176.2; IR (KBr) m:
C29H25N4O3 [M+H]+ 477.1927; found: 477.1927.
2926, 1685, 1610, 1468, 1376 cmꢀ1; LRMS (EI) m/z 400 [M]+; HRMS
(FAB) m/z Calcd for C23H21N4O3 [M+H]+ 401.1614; found:
401.1595.
3.1.8. Synthesis of 12,13-Dimethyl-2-phenyl-3a,4,12,
12a-tetrahydro-12,4-iminomethano-5-azaphthalimido[2,1,
b]-quinazoline-6,14-dione (19)
3.1.10. Synthesis of ( )-30 and ( )-31
To a solution of 18 (20 mg, 50.0
l
mol) and NaH (1.5 mg,
mol) in diethyl ether (1.0 mL) was added MeI (3 L,
mol) at 0 °C and the mixture was stirred for 3.5 h at 0 °C.
To a solution of ( )-endo-41a (30 mg, 75
(4.0 mg, 150 mol) in THF (1.0 mL) was added benzyl bromide
(18 L, 150 mol) at 0 °C and the mixture was stirred for 3.5 h at
lmol) and NaH
60.0
60.0
l
l
l
l
l
l
The reaction was quenched by the addition of satd aq NH4Cl and
the organic compounds were extracted with AcOEt and the com-
bined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was washed with
hexane to give 19 (19 mg, 94%) as a colorless solid. Mp 287–289 °C
(Hexane); 1H NMR (CDCl3, 400 MHz) d: 2.29 (s, 3H), 3.00 (s, 3H),
3.24 (d, J = 8.4 Hz, 1H), 3.65 (dd, J = 4.0, 8.4 Hz, 1H), 6.26 (d,
J = 4.0 Hz, 1H), 7.20 (d, J = 7.6 Hz, 2H), 7.44 (t, J = 7.6 Hz, 1H), 7.50
(t, J = 7.6 Hz, 2H), 7.57 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H),
7.82 (t, J = 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 15.5, 27.8, 44.3, 48.2, 52.3, 62.5, 86.5, 120.7, 126.5
(2C), 127.3, 128.1, 129.4, 129.5 (2C), 131.0, 135.0, 146.9, 152.0,
0 °C. The reaction was quenched by the addition of satd aq NH4Cl
and the organic compounds were extracted with AcOEt and the
combined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was subjected
to column chromatography (hexane/AcOEt = 2:3) to give ( )-
endo-42b (31 mg, 84%) as a colorless amorphous solid. A solution
of ( )-endo-42b (44 mg, 90 lmol) in AcOEt (3 mL) and 1.0 N HCl
(0.5 mL) was stirred at room temperature for 3.5 h. After removal
of the solvent, the residue was washed with MeOH to give ( )-30
(29 mg, 74%) as a colorless solid. Mp >300 °C (MeOH); 1H NMR
(CDCl3, 400 MHz) d: 1.38 (s, 3H), 2.40 (dd, J = 3.6, 13.6 Hz, 1H),
2.83 (d, J = 13.6 Hz, 1H), 4.83 (d, J = 15.6 Hz, 1H), 5.07 (d,
J = 15.6 Hz, 1H), 5.91 (d, J = 7.6 Hz, 1H), 5.96 (br s, 1H), 6.28 (s,
1H), 6.74 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 7.16 (t,
J = 8.0 Hz, 1H), 7.30-7.35 (m, 5H), 7.59 (t, J = 8.0 Hz, 1H), 7.67 (d,
J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d: 13.6, 35.5, 43.3, 52.2, 52.4, 62.2, 109.3,
120.2, 122.4, 123.4, 126.7, 127.6 (2C), 127.7, 127.8, 127.9, 128.5,
128.8 (2C), 129.1, 135.1, 136.3, 143.0, 146.4, 152.3, 158.2, 168.9,
158.4, 165.1, 170.8, 172.3; IR (KBr)
m: 1730, 1619, 1499, 1370,
1164 cmꢀ1; LRMS (EI) m/z 414 [M]+; HRMS (FAB) m/z Calcd for
C23H19N4O4 [M+H]+ 415.1406; found: 415.1416.
3.1.9. Synthesis of ( )-28 and ( )-29
To a solution of ( )-endo-41a (20 mg, 250
lmol) and NaH
(1.4 mg, 60 mol) in THF (1.0 mL) was added MeI (3.7
l
l
L, 60 mol)
l
at 0 °C and the mixture was stirred for 1.5 h at 0 °C. The reaction
was quenched by the addition of satd aq NH4Cl and the organic
compounds were extracted with AcOEt and the combined organic
layers were washed with brine and dried over Na2SO4. After re-
moval of the solvent, the residue was subjected to column chroma-
tography (hexane/AcOEt = 2:3) to give ( )-endo-42a (10.4 mg,
quant.) as a colorless amorphous solid. A solution of ( )-endo-42a
175.7; IR (KBr) m
: 3229, 1716, 1677, 1618, 1468, 1371 cmꢀ1; LRMS
(EI) m/z 462 [M]+; HRMS (FAB) m/z Calcd for C28H23N4O3 [M+H]+
463.1770; found: 463.1747.
To a solution of ( )-30 (20 mg, 43
lmol) and NaH (1.2 mg,
52
lmol) in THF (1.0 mL) was added MeI (3.9
l
L, 52 mol) at
l
0 °C and the mixture was stirred for 2 h at room temperature.
The reaction was quenched by the addition of satd aq NH4Cl and
the organic compounds were extracted with AcOEt and the com-
bined organic layers were washed with brine and dried over
Na2SO4. After removal of the solvent, the residue was subjected
to column chromatography (hexane/AcOEt = 1:1) to give ( )-31
(17 mg, 83%) as a colorless solid. Mp 138–140 °C (AcOEt); 1H
NMR (CDCl3, 400 MHz) d: 1.47 (s, 3H), 2.36 (dd, J = 4.0, 14 Hz,
1H), 2.78 (dd, J = 2.0, 14 Hz, 1H), 3.07 (s, 3H), 4.81 (d, J = 15.2 Hz,
1H), 5.07 (d, J = 15.2 Hz, 1H), 5.86 (d, J = 7.6 Hz, 1H), 6.02 (dd,
J = 2.0, 4.0 Hz, 1H), 6.72 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H),
7.15 (ddd, J = 0.8, 7.6, 7.6 Hz, 1H), 7.29–7.38 (m, 5H), 7.58 (ddd,
J = 0.8, 7.6, 7.6 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.80 (ddd, J = 1.6,
7.6, 7.6 Hz, 1H), 8.39 (dd, J = 0.8, 8.0 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d: 12.9, 29.0, 35.9, 44.3, 51.5, 53.0, 66.1, 109.3, 120.7,
123.1, 123.2, 127.2, 127.5 (2C), 127.7, 128.0, 128.1, 128.87,
128.90 (2C), 129.2, 134.7, 135.4, 143.0, 146.7, 151.7, 158.8, 168.1,
(20 mg, 48 lmol) in AcOEt (1.5 mL) and 1.0 N HCl (0.5 mL) was
stirred at room temperature for 30 min. After removal of the sol-
vent, the residue was washed with MeOH to give ( )-28
(18.6 mg, quant.) as a colorless solid. Mp 295–298 °C (MeOH); 1H
NMR (CDCl3, 400 MHz) d: 1.40 (s, 3H), 2.37 (dd, J = 3.2, 14.4 Hz,
1H), 2.77 (d, J = 14.4 Hz, 1H), 3.29 (s, 3H), 5.93 (d, J = 7.6 Hz, 1H),
5.94 (s, 1H), 6.71 (br s, 1H), 6.77 (t, J = 7.6 Hz, 1H), 6.86 (d,
J = 7.6 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.70
(d, J = 7.6 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 8.41 (d, J = 7.6 Hz, 1H);
13C NMR (CDCl3, 100 MHz) d: 13.9, 26.7, 36.4, 52.4, 53.0, 62.7,
108.4, 123.1, 123.3, 127.2, 127.7, 128.2, 128.6, 129.4, 134.8,
140.0, 143.8, 146.8, 151.7, 158.9, 169.4, 176.1; IR (KBr) m: 3232,
1718, 1675, 1619, 1468, 1375 cmꢀ1; LRMS (EI) m/z 386 [M]+; HRMS
(FAB) m/z Calcd for C22H19N4O3 [M+H]+ 387.1457; found:
387.1440.
To a solution of ( )-28 (20 mg, 52
lmol) and NaH (1.5 mg,
176.4; IR (KBr) m
: 1718, 1610, 1473, 1375 cmꢀ1; LRMS (EI) m/z
62
l
mol) in THF (1.0 mL) was added MeI (3.9
l
L, 62 mol) at
l
476 [M]+; HRMS (FAB) m/z Calcd for C29H25N4O3 [M+H]+
0 °C and the mixture was stirred for 2 h at 0 °C. The reaction was
quenched by the addition of satd aq NH4Cl and the organic com-
pounds were extracted with AcOEt and the combined organic lay-
ers were washed with brine and dried over Na2SO4. After removal
of the solvent, the residue was subjected to column chromatogra-
phy (hexane/AcOEt = 1:1) to give ( )-29 (20.8 mg, quant.) as a col-
477.1927; found: 477.1946.
3.2. Patch–clamp recording procedures
The thoracic and the sixth abdominal ganglia were excised from
male adult American cockroaches (P. americana L.) and incubated

110
T. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 94–110
for 60 min at room temperature in a cockroach saline solution con-
taining collagenase (0.5 mg/mL; Sigma–Aldrich) and trypsin
(0.5 mg/mL; Sigma–Aldrich) without Ca2+ and Mg2+. The ganglia
were then rinsed twice in a normal saline solution (in mM): 167
NaCl, 3.1 KCl, 5 CaCl2, 4 MgCl2, 10 Hepes, 10 glucose, and 10 ppm
phenol red (pH adjusted to 7.4 with NaOH), and mechanically dis-
sociated by gentle repeated trituration through a fire-polished Pas-
teur pipette.
Membrane currents were recorded with the whole-cell record-
ing configuration11 using a patch–clamp amplifier (EPC-8; HEKA,
Lambrecht/Pfalz, Germany) linked to a computer. Recording proce-
dures were controlled by pCLAMP software (Axon Instruments,
Sunnyvale, CA). Data were low-pass filtered with a cutoff fre-
quency of 1 kHz and then digitized at 2 kHz by an analog-to-digital
interface. Fast and slow capacitative transients were canceled by
the compensation circuitry of EPC-8. Cells were voltage-clamped
at a membrane potential of ꢀ80 mV. Patch pipettes were pulled
from borosilicate glass capillaries (Warner Instruments, Hamden,
CT) on a puller (P-97; Sutter Instrument, Novato, CA) and had a
the nicotinic acetylcholine receptor of Torpedo marmorata ob-
tained at 4 Å resolution (PDB entry 1OED).13 The multiple se-
quence alignment of both receptor subunits was performed
with the MOE 2006.08 program (Chemical Computing Group,
Montreal) using the PAM250 substitution matrix, and the long
loop between TM3 and TM4 was deleted because of the lack of
structure data in the template. About 20 intermediary models
were generated by MOE, and the raw model with the best pack-
ing quality was further refined until the root mean square gradi-
ent became 0.01, according to the molecular mechanics
minimization protocol using the Merck molecular field force
MMFF94x14 in MOE. The validation tests of stereochemical qual-
ity were conducted using MOE. Only three residues from the con-
structed model fell outside of the acceptable regions of the
Ramachandran plot (data not shown). All bond angles, bond
lengths, and C chiralities were also found to be adequate. The
a
structure of 3 having the same stereochemisty as that of (+)-1
was built using the molecular modeling software SYBYL ver. 7.1
(Tripos, St. Louis, MO) and was fully optimized by the semi-
empirical molecular orbital method AM1. Compound 3 was auto-
matically docked into the potential binding site8 of the b3 recep-
tor model using the dock program in MOE. Compound 3 was
subjected to conformational analysis, placement, and pharmaco-
phore filtering, and was scored in terms of hydrophobic, ionic,
and hydrogen bond contacts, which were generally favorable
interactions. A final representation of the potential binding mode
of (+)-3 was chosen based on selection of the compound that pos-
sessed the best docking score within the most populated cluster
with the lowest docking energy.
resistance of 5 MX when filled with a pipette solution, which con-
tained (in mM): 15 NaCl, 170 KCl, 0.5 CaCl2, 1 MgCl2, 10 EGTA, 10
Hepes, 5 ATP–Tris, and 20 sodium pyruvate (pH adjusted to 7.4
with KOH). GABA was dissolved in the cockroach saline solution
and ejected through the hole of the polyethylene tubing that was
located close to the cell (ꢂ100
lm). (+)-1 dissolved in dimethyl
sulfoxide (DMSO) was diluted with the saline solution at final
DMSO concentrations of 0.1% (v/v) or less. Solutions of control
and (+)-1 were applied by perfusion using a VC-6M valve control
system (Warner Instruments) at a flow rate of 1 mL/min.
3.3. [3H]EBOB binding assays using housefly-head membranes
Acknowledgment
The heads of adult houseflies (Musca domestica L.) were homog-
enized in 10 mM Tris–HCl buffer, pH 7.5, containing 0.25 M sucrose
(buffer A) with a glass-Teflon homogenizer; then, the homogenate
This research was partially supported by a Grant-in-Aid from
the Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan. We thank Professors T. Ishikawa and T. Kumamoto
for their help with microwave irradiation experiments.
was filtered through two layers of 64-
centrifuged at 500g for 5 min. The supernatant was filtered
through two layers of 64- m mesh nylon screen and centrifuged
lm mesh nylon screen and
l
References and notes
at 25,000g for 30 min. The pellets were suspended in buffer A
and were kept on an ice bath for 30 min. The suspension was cen-
trifuged at 25,000g for 30 min. The pellets were suspended in
10 mM sodium phosphate buffer, pH 7.5, containing 0.3 M NaCl
(buffer B) and were used immediately for the binding assays.
The homogenates containing housefly-head membranes
1. Bormann, J. B. Trends Pharmacol. Sci. 2000, 21, 16.
2. Buckingham, S. D.; Biggin, P. C.; Sattelle, B. M.; Brown, L. A.; Sattelle, D. B. Mol.
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4. Isolation: (a) Larse, T. O.; Frydenvang, K.; Frisvad, J. C.; Christophersen, C. J. Nat.
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Tetrahedron Lett. 1999, 40, 5429; (c) Hart, D. J.; Magomedov, N. A. J. Am. Chem.
Soc. 2001, 123, 5892; Synthesis of ( )-1 (d) Kende, A. S.; Fan, J.; Chen, Z. Org.
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Christophersen, C.; Barrero, A. F. J. Nat. Prod. 2001, 64, 1590; Synthesis of
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(200 lg as protein) were incubated with various concentrations
of test compounds and 0.5 nM [3H]EBOB in 1 mL of buffer B at
22 °C for 70 min. Test compounds were added as DMSO solutions
(4 lL) in buffer B. The protein content was measured by the meth-
od of Bradford.12 After the incubation, the mixture was filtered
through Whatman GF/B filters and rapidly rinsed twice with
5 mL of cold buffer B using a Brandel M-24 cell harvester. The
radioactivity of [3H]EBOB on the filters was measured with a liquid
scintillation counter. Nonspecific binding was determined in the
8. Alam, M. S.; Huang, J.; Ozoe, F.; Matsumura, F.; Ozoe, Y. Bioorg. Med. Chem.
2007, 15, 5090.
9. Hernandez, F.; Buenadicha, F. L.; Avendano, C.; Sollhuber, M. Tetrahedron:
Asymmetry 2001, 12, 3387.
presence of 1
lM a-endosulfan. Experiments were repeated at
least three times.
10. Rossiter, S. Tetrahedron Lett. 2002, 43, 4671.
11. Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Pflugers Arch.
3.4. Homology modeling and docking
1981, 391, 85.
12. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
13. Miyazawa, A.; Fujiyoshi, Y.; Unwin, N. Nature 2003, 423, 949.
14. Halgren, T. A. J. Comput. Chem. 1996, 17, 490.
The transmembrane segments of the human b3 GABA receptor
were modeled using the cryo-electron microscopy structure of