Toosendanin: Synthesis of the AB-ring and investigations of its anti-botulinum properties (Part II)

Article abstract of DOI:10.1016/j.bmc.2009.12.030

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. The natural product toosendanin, a limonoid, is a traditional

Full text of DOI:10.1016/j.bmc.2009.12.030

Bioorganic & Medicinal Chemistry 18 (2010) 1280–1287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Toosendanin: Synthesis of the AB-ring and investigations
of its anti-botulinum properties (Part II)
a
b
b
b
a,c,*
Yuya Nakai , Sabine Pellett , William H. Tepp , Eric A. Johnson , Kim D. Janda
a
Departments of Chemistry and Immunology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706, USA
Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease character-
ized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. The natural
product toosendanin, a limonoid, is a traditional Chinese medicine that has reported anti-botulinum
properties in animal models. Toosendanin effectively inhibits the biological activity of BoNT/A in neuro-
nal cells at concentrations of 200 nM, and partial inhibition can be observed with concentrations as low
as 8 nM. Mechanistically, toosendanin’s inhibition is due to prevention of transduction of the BoNT LC
through the HC channel. Intriguing questions as to the molecular architecture of toosendanin as related
to its anti-botulinum properties have focused our attention on a synthesis of toosendanin’s unusual AB-
ring, containing a unique bridged hemi-acetal. Within the current work, a synthetic strategy allowing
access to the AB-fragment of toosendanin was achieved from a trans-decalin system. In addition, this
fragment was examined for its modulation of BoNT/A intoxication in a rat spinal cord cellular assay.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 October 2009
Revised 4 December 2009
Accepted 8 December 2009
Available online 22 December 2009
Keywords:
Botulinum neurotoxins
Toosendanin
Semi-synthesis
Rat spinal cord cellular assay
8
1
. Introduction
early 1980s detailing toosendanin’s in vivo activity. Indeed, these
cryptic reports identified toosendanin’s anti-botulinum properties
Botulinum neurotoxins (BoNTs) are the most toxic poisons
known to humans, with a lethal dose (LD50) of approximately
in both monkey and mouse models spanning these serotypes.
Unfortunately, these reports proved to be difficult to substantiate
either due to a lack of pure material (i.e., toosendanin) or inconsis-
tencies with toosendanin in animal assays.
1
1
ng per kg of body weight. There are seven serologically distinct
BoNTs (A–G); serotype A (BoNT/A) is the most potent, possessing a
6
11
toxicity 10 -fold higher than cobra toxin and 10 -fold greater than
cyanide.2 BoNT intoxication is characterized by flaccid paralysis
caused by the proteolytic cleavage of specific SNARE proteins crit-
ical for the release of the neurotransmitter acetylcholine from
To resolve this dilemma, we reported the effects of well charac-
terized toosendanin, using a sensitive and specific spinal cord cell-
based assay, which validated toosendanin’s activity in both BoNT
9
serotypes A and E. Thus, exposure of neurons to BoNT/A in pres-
3
nerve cells.
ence of increasing concentrations of toosendanin resulted in the
gradual preservation of intact, uncleaved synaptosomal associated
protein of 25 kD (SNAP-25), the intracellular BoNT/A and BoNT/E
substrate, becoming practically complete above 200 nM; excit-
ingly, partial inhibition can be observed with concentrations as
low as 8 nM for BoNT/A and 40 nM for BoNT/E, respectively. To
further solidify toosendanin’s BoNT inhibitory activity, we utilized
Limonoids (1 and 2, Fig. 1) are tetranortriterpenoids with a
4
,4,8-trimethylfuranylsteroid skeleton derived from euphane or
4–6
tirucallane triterpenoids.
A major limonoid constituent found
in Melia toosendan is the compound toosendanin (2, Fig. 1) which
appears to have multiple modes of action in insects including dam-
age to midgut tissues, inhibition of esterases, cytochrome P450-al-
drin epoxidase and proteinase activities.7 Interest in the
application of liminoid natural products, and in particular toosend-
anin, in pest management remains high, and while this area of too-
sendanin research remains fertile, we became intrigued by a series
of reports over the past two decades, mainly originating from Chi-
na detailing the special activity of toosendanin as it relates to
BoNT. Most interesting were two reports both published in the
*
Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
Figure 1. General structures of limonoid (1) and toosendanin (2).
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.030

Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
1281
single molecule channel forming experiments that clearly demon-
strated toosendanin’s inhibition mode is due to prevention of trans-
duction of the BoNT light chain (LC) through the heavy chain (HC)
9
,10
channel.
Thus, toosendanin selectively arrests the LC transloca-
tion step of intoxication with subnanomolar potency, and increases
the unoccluded HC channel propensity to open with micromolar
efficacy. Interestingly, these studies also provided strong evidence
that toosendanin has an unprecedented dual mode of action within
the protein-conducting channel acting both as a cargo-dependent
inhibitor of translocation and as cargo-free channel activator. To fur-
ther validate toosendanin’s anti-botulinum properties, we have also
investigated toosendanin’s protective effects in a mouse lethality
model. Positive findings have allowed us to begin to assign which
chemical functionalities are important for the anti-botulinum prop-
erties found within toosendanin.9 As a starting point we initially
turned to semi-synthesis to generate a set of rationallydesigned too-
sendanin analogues (3–6, Fig. 2) that were prepared so as to probe
the most salient functionalities embedded within toosendanin
without perturbing its gross chemical structure. These analogues
were examined in the mouse lethality assay wherein only 4 proved
to have equivalent activity to toosendanin.
Figure 3. AB-ring of toosendanin (7).
on both rings, we found ester (10) to be an ideal candidate for this
study (Scheme 1). This compound is readily available in large scale,
from commercially available starting materials (8 and 9) by a two-
step modification as detailed by Jones and Dodds. Thus, upon
benzylation of the hydroxy group found within 10 under acidic
13
1
4
conditions, the resulting ketone was protected as the ketal (11)
in 51% yield for the two steps. Reduction of 11 with LAH followed
by acid-induced hydrolysis of the dimethyl ketal furnished a hy-
droxy ketone, which could then be utilized for protection of the
primary alcohol with SEMCl affording 12 in 93% overall yield from
11. The preparation of the desired enone (13) was accomplished by
KHMDS-promoted phenyselenylation at À78 °C followed by oxida-
tion and elimination of the resulting selenide using hydrogen
To both potentially improve and ultimately decipher toosenda-
nin’s anti-botulinum properties, the next tactic we envisioned em-
braced function-oriented synthesis (FOS) as advocated by Wender
1
1
15
et al. The underpinnings of FOS are that the function of a biologi-
cally active lead structure can be emulated, tuned, or possibly im-
proved by replacement with simpler scaffolds designed to
encompass the key activity-determining structural features of the
natural product. As stated (vide supra), through our semi-synthetic
efforts, the epoxide and acetoxy moieties were found to be impor-
tant, while the furan ring was not. To further define toosendanin,
we dissected the molecule into two fragments consisting of AB-
and CD-rings. As such, we reported our initial research exploring
the synthesis of the CD-ring of toosendanin and its potential biolog-
peroxide in 83% yield. Subsequent regioselective carboxymethyl-
1
6
ation with Mander’s reagent in the presence of LHMDS at À78 °C
afforded a b-ketomethylester; this was followed by methylation,
thus methyl iodide in the presence of NaH granted the correspond-
1
7
ing
a-methyl-b-ketomethylester. Epoxidation of the enone moi-
ety by TBHP in the presence of benzyltrimethylammonium
1
8
hydroxide (triton B) furnished stereoselectively epoxide (14), in
1
9
65% yield for three steps from 13. The regioselective opening of
2
0
the epoxide moiety found within 14 was established by PhSeNa
(generated in situ from diphenyl diselenide and NaBH ) in 86%
4
1
2
ical function. Our synthetic approach to the 4-acetoxy CD-ring of
toosendanin was achieved starting from mesityl oxide and acetyl-
acetone in 14 steps. This work represented the first semi-synthesis
of the CD-ring of toosendanin correctly displaying all heteroatom
substituents found within the natural product. In continuing with
our general plan, we disclose our successful synthesis of toosenda-
nin’s AB-ring and testing of its biological activity (Fig. 3).
yield followed by silylation of 15 with SEMCl, which led to disilyl
ether (16) in 85% yield from 14 (Scheme 1).
The relative stereochemistry of 15 was ascertained through
analysis of its 2D-ROESY NMR. Thus, for 15, a ROESY correlation
was observed between Me-1 (equatorial) and H-8a (axial); H-7
(axial) and H-8a; no long range interactions were observed be-
tween H-4 (equatorial) and H-8a protons (Fig. 4).
2
.1. Synthesis of 7-epi AB-ring of toosendanin (TSDN)
2
. Results and discussion
To construct the diol directly, we attempted the stereoselective
In the search for a suitable trans-decalin system as a starting
reduction of the ketone and methyl ester moiety of 16 with LAH or
DIBAL at À78 °C, but unfortunately the undesired stereochemistry
at C-2 position was obtained as a major isomer; the lone reaction
material with an angular substituent and oxygen functionalities
4 2 2
that succeeded was NaBH in CH Cl /MeOH (15:1). Again, how-
ever, the desired compound 17 was obtained as a minor isomer
yet, the total yield was quantitative, hence, we invoked a recycling
method via oxidation of 2b-form (epi-17) with Dess–Martin re-
agent that granted 16 in 96% yield. Reduction of ester (17) to the
primary alcohol using LAH proceeded in an excellent yield. The
resulting diol was selectively oxidized with 2,2,6,6-tetramethyl-
1
-piperidinyloxy free radical (TEMPO) under biphasic conditions
to furnish an aldehyde followed by acetylation of the free second-
ary alcohol granted 18 in 85% yield for three steps from 17.21
Hydrogenation of 18 with palladium hydroxide on carbon provided
1
9 as a hemi-acetal moiety in 94% yield; as expected 20 was not
observed. While, removal of SEM group with TFA furnished 7-epi
AB-ring of toosendanin (epi-7) in 80% yield (Scheme 2).
With the first hurdle in the synthesis in place, that is, construc-
tion of the AB-ring, we were now faced with the challenge of
inverting the stereochemistry at the C-7 position. Our initial at-
tempt engaged the Mitsunobu reaction. Thus, 16’s benzyl ether
Figure 2. Structures of toosendanin’s analogues (3–6).

1
282
Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
Scheme 1. Synthesis of 16. Reagents and conditions: (a) NaOEt, EtOH; (b) H
cat. H SO , MeOH, 51% for two steps; (e) LAH, THF, reflux; (f) 5 N HCl, THF; (g) SEMCl, DIPEA, CH
PhSeCl, CH Cl , THF, 83% for three steps; (k) LHMDS, THF, À78 °C, then NCCO Me, À78 °C; (l) NaH, MeI, THF; (m) TBHP, triton B, THF, 65% for three steps;
, À78 °C; (j) 30% H
n) (PhSe) , NaBH , EtOH, 86%; (o) SEMCl, DIPEA, CH Cl , 99%.
2
, Pd/C, ether; (c) benzyl 2,2,2-trichloroacetimidate, cat. TfOH, cyclohexane/CH
2 2
Cl (2:1, v/v); (d)
2
4
2
Cl
2
93% for three steps; (h) KHMDS, THF, À78 °C, then TESCl, À78 °C; (i)
2
2
2
O
2
2
(
2
4
2
2
2
.2. Synthesis of AB-ring of toosendanin (TSDN)
Since the necessary inversion of stereochemistry at C-7 was
found untenable using a Mitsunobu sequence we were forced to
rethink our strategy. We sought an advanced intermediate, and
1
9 appeared well-suited to meet this challenge. Thus, 19’s hemi-
acetal was protected by treatment with CbzCl, however, to im-
prove yields due to over reaction, (21), was recycled providing
the desired alcohol (22). Oxidation of 22 using Dess–Martin re-
agent afforded ketone (23) in 96% yield. Stereoselective reduction
was attempted using a plethora of reagents, however, none were
Figure 4. The relative stereochemistry of compound (15) as determined by 2D-
ROESY experiment.
was removed by hydrogenation with palladium on carbon, and
subsequent Mitsunobu reaction with 4-nitrobenzoic acid afforded
satisfactory. Hence, we simply relied on reduction using NaBH
4
resulting in a mixture of 22 and the desired compound 24. These
compounds were readily separated and again recycling was used
on 22 to obtain the correct stereochemistry at C-7. The correspond-
ing AB-ring (7), as embedded within toosendanin was finally se-
cured by hydrogenation of 24, followed by SEM removal with
TFA (Scheme 3).
4
-nitrobenzylester with the desired stereochemistry at C-7. Unfor-
tunately, all attempts to hydrolyze this ester failed; we surmise
that either the ester was resistant to hydrolysis or when hydrolysis
was seen it coincided with elimination of SEM group resulting in
the a,b-unsaturated ketone.
Scheme 2. Synthesis of 7-epi AB-ring of toosendanin (epi-7). Reagents and conditions: (a) NaBH
periodinane, CH Cl , 96%; (c) LAH, ether; (d) NaOCl, TEMPO, KBr, CH Cl /satd NaHCO aq (2:1, v/v); (e) Ac
CH Cl /MeOH (1:1, v/v), 94%; (g) TFA, CH Cl , 0 °C, 80%.
4
, CH
2
Cl
2
/MeOH (15:1, v/v), 99% (ratio of epi-17/17 = 2/1); (b) Dess–Martin
2
2
2
2
3
2
O, pyridine, cat. DMAP, 85% for three steps; (f) H , Pd(OH) /C,
2
2
2
2
2
2

Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
1283
Scheme 3. Synthesis of AB-ring of toosendanin (7). Reagents and conditions: (a) CbzCl, TMEDA, CH
2
Cl
2
, À78 °C, 49% of 22; (b) H
2
, Pd/C, EtOH, 49% for two steps; (c) Dess–
Martin periodinane, CH Cl , 96%; (d) NaBH , CH Cl /MeOH (15:1, v/v), 95% (ratio of 22/24 = 2.6/1); (e) H
2
2
4
2
2
2
, Pd/C, EtOH; (f) TFA, CH Cl , 0 °C, 82% for two steps.
2 2
2
.3. Testing toosendanin analogues 7 and epi-7 in an RSC assay
tection from BoNT/A intoxication within the RSC assay. However,
noteworthy is that the merging of these studies with both our
semi-synthetic and CD-ring analogues provides a unified picture
emphasizing the importance of toosendanin’s ABCD nucleus in
the blocking of BoNT/A intoxication. Taken together a logical point
for future studies, we will focus on the furan moiety found within
toosendanin as its requirements appear more adaptable for ana-
logues preparation and biological testing.
To examine the biological potency of toosendanin analogues 7
and epi-7, a primary rat spinal cord cellular assay (RSC assay)
2
2
was engaged. The value of this assay is it avoids the use of ani-
mals and lethality as an end-point. Here western blot analysis
can be employed where visualization of intact SNAP-25 is a mea-
sure of a compounds efficacy against botulinum neurotoxin A. In
these regards and based on our previous findings in terms of data
accrued from our semi-synthetic and CD-ring analogues, we antic-
ipated little or no biological potency. Yet, we felt it incumbent to
examine these structures in this cellular assay both to contrast,
and validate our previous results/hypotheses as well as provide a
solid grounding for future FOS studies focused upon utilizing the
AB-ring nucleus. Thus, primary rat spinal cord cells were exposed
to 5.6 pM BoNT/A1 combined with 200
1
tain toxin only, or no toxin. Cell lysates were analyzed by Western
blot using a monoclonal anti-SNAP-25 antibody (Synaptic Sys-
tems). As expected, addition of toosendanin at 200 lM resulted
4
4
. Experimental
.1. Chemistry
2 2 2
In general, CH Cl and MeOH were distilled from CaH . Tetrahy-
drofuran (THF) was distilled from Sodium. Reagents were pur-
chased from commercial sources and used without further
purification. Synthetic reactions were monitored by analytical thin
layer chromatography (TLC) carried out on 0.25 mm silica gel
plates (60F-254). All flash column chromatography was performed
using Silica Gel 60 (230–400 mesh). Preparative TLC was also per-
lM toosendanin (2),
mM AB-ring (7), or 1 mM 7-epi AB-ring (epi-7). The controls con-
in complete inhibition of BoNT/A1 activity, as evidenced by the
appearance of uncleaved SNAP-25 only (Fig. 5). Addition of 7 or
epi-7 at 1 mM concentrations consistently resulted in the same ex-
tent of SNAP-25 cleavage as the positive (toxin only) control. Thus,
as expected, 7 and epi-7 did not result in any detectable inhibition
of BoNT/A1 activity in primary neuronal cells.
formed using Merck Kieselgel 60F-254 silica gel plates (0.5 or
1
1
(
mm). H NMR spectra were recorded on Bruker DRX-600
13
600 MHz) or DRX-500 (500 MHz) spectrometers, and C NMR
spectra was recorded on Bruker DRX-600 (150 MHz) or DRX-500
125 MHz) spectrometers. Chemical shifts were reported in parts
(
per million (ppm) on the d scale from an internal standard (NMR
descriptions: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet). Electrospray ionization (ESI) time-of-flight reflectron experi-
ments were performed at The Scripps Research Institute on an
Agilent ESI-TOF mass spectrometer. Samples were electrosprayed
into the TOF reflectron analyzer at a ESI voltage of 4000 V and a
3
. Conclusion
We have achieved the synthesis of the AB-ring (7) of toosenda-
nin (5.8% overall yield in 23 steps from 10) and the 7-epi AB-ring
epi-7) of toosendanin (10% overall yield in 19 steps from 10). In
(
flow rate of 200 lL/min. Toosendanin was purchased from Ava-
Chem Scientific. Subsequently, the purity of the toosendanin was
accord with our previous findings neither structure afforded pro-
confirmed by both TLC and HPLC. Toosendanin by TLC with
EtOAc–hexane (3:1, v/v) runs as two spots with R values of 0.38
f
and 0.56 representing a mixture of endo and exo isomers. Finally,
we note that reported NMR data displays compound 7, epi-7,
and 19 as a mixture of both endo and exo isomers.
4
.1.1. (4aRS,7RS,8aSR)-Ethyl 7-(benzyloxy)-2,2-dimethoxydeca-
hydronaphthalene-4a-carboxylate (11)
Catalytic amount of trifluoromethanesulfonic acid was added to
a solution of (2RS,4aRS,8aSR)-ethyl 2-hydroxy-7-oxodecahydro-
1
3
Figure 5. Western blot of RSC assay using toosendanin (2) and analogues 7/epi-7.
naphthalene-4a-carboxylate (10) (1.37 g, 5.7 mmol) and benzyl

1
284
Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
2
(
,2,2-trichloroacetimidate (1.2 mL, 6.3 mmol) in cyclohexane
20 mL) and CH Cl (10 mL) at room temperature, and the mixture
was stirred at room temperature for 9 h. The precipitate was fil-
tered off, and the filtrate was washed with satd NaHCO aq and
brine, dried over Na SO . The solvent was evaporated under re-
NaHCO
brine, dried over Na
duced pressure to leave an oil. The crude was dissolved in dry
CH Cl
(75 mL), cooled to À78 °C, and addition of a solution of
PhSeCl (98%, 2.1 g, 10.5 mmol) in dry CH Cl (15 mL). The mixture
was stirred at À78 °C for 30 min. After 30 min, the mixture was
washed with satd NaHCO aq and brine, dried over Na SO . The
3
aq, the whole was extracted with ether, washed with
2
2
2
SO . The solvent was evaporated under re-
4
3
2
2
2
4
2
2
duced pressure to leave an oil. The crude was dissolved in dry
MeOH (40 mL), then addition of catalytic amount of concentrated
3
2
4
H
2
SO
perature for 14 h (over night). The reaction mixture was poured
into satd K CO aq under ice-cooling. The mixture was extracted
with CHCl , washed with brine, dried over Na SO . The solvent
was evaporated under reduced pressure to leave an oil, which
was purified by flash chromatography on SiO with EtOAc–hexane
1:4, v/v) to give 11 (1.10 g, 51%, for two steps from 10) as a yellow
4
at room temperature. The mixture was stirred at room tem-
solvent was evaporated under reduced pressure to leave an oil.
The crude was dissolved in THF (75 mL), cooled at 0 °C, then
2
3
2
H O
2
(30 wt % in water, 2.7 mL, 26.3 mmol) was added. The mix-
ture was stirred at room temperature for 1 h. After addition of
EtOAc, the mixture was washed with 5% Na CO aq and water,
the aqueous layer was extracted with EtOAc. The combined organic
layer was washed with brine, dried over Na SO . The solvent was
evaporated under reduced pressure to leave an oil, which was puri-
fied by flash chromatography on SiO with EtOAc–hexane (1:5, v/v)
to give 13 (3.02 g, 83%, for three steps from 12) as a pale yellow oil.
3
2
4
2
3
2
(
2
4
1
oil: H NMR (CDCl
H), 4.54 (q, J = 12.4 Hz, 2H), 4.17–4.12 (m, 2H), 3.39 (sept,
J = 5.1 Hz, 1H), 3.18 (s, 3H), 3.12 (s, 3H), 2.17–2.14 (m, 1H), 2.06
t, J = 13.2 Hz, 1H), 1.98–1.79 (m, 5H), 1.75–1.71 (m, 1H), 1.48–
3
, 600 MHz) d 7.33–7.30 (m, 4H), 7.27–7.23 (m,
1
2
1
(
3
H NMR (CDCl , 600 MHz) d 7.34–7.33 (m, 4H), 7.29–7.24 (m, 1H),
1
.43 (m, 1H), 1.30–1.12 (m, 7H, including 1.25 (t, J = 7.2 Hz, 3H));
C NMR (CDCl , 150 MHz) d 174.6, 138.9, 128.3, 127.5, 127.4,
3
6.83 (d, J = 10.0 Hz, 1H), 5.97 (d, J = 10.0 Hz, 1H), 4.60–4.54 (m, 4H),
3.81 (d, J = 9.4 Hz, 1H), 3.77 (d, J = 9.4 Hz, 1H), 3.56–3.53 (m, 2H),
3.45 (sept, J = 5.1 Hz, 1H), 2.54 (dd, J = 17.7, 14.6 Hz, 1H), 2.24
(dd, J = 17.7, 4.3 Hz, 1H), 2.11–1.97 (m, 3H), 1.92–1.89 (m, 1H),
1.67–1.61 (m, 1H), 1.51 (q, J = 12.3 Hz, 1H), 1.30–1.24 (m, 1H),
1
3
1
3
C
00.1, 77.1, 69.8, 60.0, 47.6, 47.5, 47.3, 38.9, 36.2, 35.5, 34.8,
3.6, 29.6, 29.4, 14.3; ESI-TOF MS (m/z): [MNa] calcd for
+
22
H
32
O
5
Na 399.2142, found 399.2144.
1
3
0
3
.97–0.90 (m, 2H), 0.01 (s, 9H); C NMR (CDCl , 150 MHz) d
4
.1.2. (4aRS,7RS,8aSR)-7-(Benzyloxy)-4a-(((2-(trimethylsilyl)-
199.2, 157.7, 138.5, 128.8, 128.3, 127.5, 127.4, 94.8, 76.5, 70.1,
ethoxy)methoxy)methyl)octahydronaphthalen-2(1H)-one (12)
LiAlH (2.0 M in THF, 2.1 mL, 4.1 mmol) was added to a solu-
67.9, 65.1, 40.7, 40.1, 39.2, 33.4, 31.2, 27.7, 17.9, À1.5; ESI-TOF
+
4
MS (m/z): [MNa] calcd for
C
24
H
36
O
4
SiNa 439.2275, found
tion of 11 (779 mg, 2.1 mmol) in dry THF (8 mL) at 0 °C, and
the mixture was stirred at 100 °C (reflux condition) for 4 h. After
addition of EtOAc, 1 N HCl, and Rochelle salt, the precipitate was
filtered off, then the whole was washed with 1 N HCl, satd NaH-
439.2285.
4.1.4. (1aRS,3RS,3aRS,5RS,7aRS,7bRS)-Methyl 5-(Benzyloxy)-3-
methyl-2-oxo-7a-(((2-(trimethylsilyl)ethoxy)methoxy)meth-
yl)decahydronaphtho[2,1-b]oxirene-3-carboxylate (14)
3 2 4
CO aq, and brine, dried over Na SO . The solvent was evaporated
under reduced pressure to leave an oil. The crude was dissolved
in THF (16 mL), then addition of 5 N HCl (4 mL) at room temper-
ature. The mixture was stirred at room temperature for 1 h. After
LiHMDS (1.0 M in THF, 10.5 mL, 10.5 mmol) was added in a sin-
gle portion to a solution of 13 (2.18 g, 5.2 mmol) in dry THF
(80 mL) at À78 °C. After 40 min of stirring at À78 °C, methyl cyano-
formate (0.63 mL, 7.7 mmol) was added, and the mixture was stir-
red at À78 °C for 4 h. After addition of water and EtOAc, the whole
addition of solid NaHCO
the sediment dissolved (pH 9). The whole was extracted with
CH Cl , washed with brine, dried over Na SO . The solvent was
evaporated under reduced pressure to leave an oil. The crude
was dissolved in dry CH Cl (8 mL), then DIPEA (0.75 mL,
.6 mmol) and SEMCl (0.73 mL, 4.1 mmol) were added at 0 °C.
3
, water was added to the mixture until
2
2
2
4
2 4
was extracted with EtOAc, washed with brine, dried over Na SO .
The solvent was evaporated under reduced pressure to leave an
oil, which was purified by flash chromatography on SiO with
2
2
2
4
EtOAc–hexane (1:4, v/v) to give b-ketomethylester (2.33 g, 94%)
as a pale yellow oil. The b-ketomethylester (2.33 g, 4.9 mmol)
was dissolved in dry THF (80 mL), cooled at 0 °C, and addition of
NaH (60%, 0.24 g, 5.9 mmol). After 5 min of stirring at 0 °C, methyl
iodide (1.5 mL, 24.6 mmol) was added, and the mixture was stirred
at room temperature for 14 h (over night). After addition of satd
The mixture was stirred at room temperature for 15 h (over
night). After addition of EtOAc, the whole was washed with 1 N
HCl, satd NaHCO
vent was evaporated under reduced pressure to leave an oil,
which was purified by flash chromatography on SiO with
EtOAc–hexane (1:5, v/v) to give 12 (809 mg, 93%, for three steps
from 11) as a pale yellow oil. 1H NMR (CDCl
, 600 MHz) d 7.34–
.32 (m, 4H), 7.29–7.26 (m, 1H), 4.69 (s, 2H), 4.56 (s, 2H), 3.80
3 2 4
aq, and brine, then dried over Na SO . The sol-
2
NH
4
Cl aq, the whole was extracted with EtOAc, washed with brine,
SO . The solvent was evaporated under reduced
3
dried over Na
2
4
7
pressure to leave an oil. The crude was dissolved in THF (80 mL),
cooled at 0 °C, and addition of triton B (40 wt % in water, 3.9 mL,
9.8 mmol) and TBHP (70 wt % in water, 1.4 mL, 9.8 mmol). The
mixture was stirred at room temperature for 3 h. After addition
(
(
s, 2H), 3.62–3.59 (m, 2H), 3.41 (sept, J = 5.3 Hz, 1H), 2.52–2.46
m, 1H), 2.34–2.29 (m, 2H), 2.22–2.14 (m, 2H), 2.06–1.99 (m,
2
1
0
1
3
C
H), 1.80–1.78 (m, 1H), 1.73–1.62 (m, 1H), 1.61–1.56 (m, 1H),
.40 (q, J = 12.2 Hz, 1H), 1.27–1.21 (m, 1H), 0.98–0.91 (m, 3H),
of EtOAc, the whole was washed with 10% Na
aqueous layer was extracted with EtOAc. The combined organic
layer was washed with brine, dried over Na SO . The solvent was
evaporated under reduced pressure to leave an oil, which was puri-
fied by flash chromatography on SiO with EtOAc–hexane (1:5, v/v)
to give 14 (1.71 g, 65%, for three steps from 13) as a very pale yel-
2 2 3
S O aq, then the
1
3
3
.02 (s, 9H); C NMR (CDCl , 150 MHz) d 210.8, 138.7, 128.4,
27.5, 95.1, 76.9, 70.1, 65.2, 63.6, 44.2, 42.4, 37.9, 36.1, 34.6,
2
4
+
4.5, 32.7, 27.6, 18.1, À1.5; ESI-TOF MS (m/z): [MNa] calcd for
24
H
38
O
4
SiNa 441.2431, found 441.2448.
2
1
4
.1.3. (4aSR,7RS,8aSR)-7-(Benzyloxy)-4a-(((2-(trimethylsilyl)-
3
low oil. H NMR (CDCl , 600 MHz) d 7.34–7.24 (m, 5H), 4.63 (s, 2H),
ethoxy)methoxy)methyl)-4a,5,6,7,8,8a-hexahydronaphthalen-2-
4.56–4.51 (m, 2H), 3.85 (d, J = 10.4 Hz, 1H), 3.66–3.58 (m, 6H,
including 3.64 (s, 3H)), 3.55–3.51 (m, 1H), 3.42 (d, J = 4.4 Hz, 1H),
3.34 (sept, J = 5.2 Hz, 1H), 2.32–2.29 (m, 1H), 2.06–2.03 (m, 2H),
1.84–1.82 (m, 1H), 1.56–1.49 (m, 1H), 1.41–1.36 (m, 4H, including
(
1H)-one (13)
KHMDS (0.5 M in toluene, 26.4 mL, 13.2 mmol) was added
dropwise over the course of 10 min) to a solution of 12 (3.67 g,
(
8
.8 mmol) in dry THF (75 mL) at À78 °C. After 40 min of stirring
1.40 (s, 3H)), 1.24–1.18 (m, 1H), 0.95–0.89 (m, 2H), 0.01 (s, 9H); 13
C
at À78 °C, TESCl (2.2 mL, 13.2 mmol) was added, and the mixture
was stirred at À78 °C for 40 additional min. After addition of satd
3
NMR (CDCl , 150 MHz) d 205.3, 170.9, 138.4, 128.4, 127.6, 127.5,
95.0, 77.0, 70.3, 66.9, 65.2, 64.2, 57.4, 55.9, 52.2, 42.6, 38.8, 29.4,

Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
1285
+
orless oil in the order of elution. Compound 17: 1H NMR (CDCl
600 MHz) d 7.38–7.34 (m, 4H), 7.29–7.26 (m, 1H), 4.75–4.74 (m,
H), 4.62–4.53 (m, 5H), 4.10 (br s, 1H), 4.07 (br s, 1H), 3.94 (br s,
2
C
8.9, 27.1, 23.8, 18.1, À1.4; ESI-TOF MS (m/z): [MNa] calcd for
3
,
27
H
40
O
7
SiNa 527.2435, found 527.2441.
1
4
.1.5. (1R,4S,4aR,7R,8aR)-methyl 7-(benzyloxy)-4-hydroxy-1-
1H), 3.76–3.71 (m, 1H), 3.67–3.54 (m, 5H, including 3.65 (s, 3H)),
3.53–3.48 (m, 2H), 3.36–3.32 (m, 1H), 3.08 (d, J = 10.2 Hz, 1H),
2.25–2.18 (m, 2H), 2.09–2.06 (m, 1H), 2.03–1.84 (m, 3H), 1.79–
1.77 (m, 1H), 1.53–1.44 (m, 2H), 1.29 (s, 3H), 0.96–0.90 (m, 4H),
0.03–0.01 (m, 18H); C NMR (CDCl , 150 MHz) d 177.2, 138.9,
3
128.4, 127.6, 127.5, 95.2, 94.8, 78.5, 77.7, 72.0, 70.1, 66.0, 65.0,
methyl-2-oxo-4a-(((2-(trimethylsilyl)ethoxy)methoxy)-
methyl)decahydronaphthalene-1-carboxylate (15)
Acetic acid (7.1
tion of PhSeNa which prepared by the reduction of (PhSe)
19 mg, 0.37 mmol) with NaBH (28 mg, 0.75 mmol) in EtOH
2 mL), and the mixture was stirred at room temperature for few
lL, 0.12 mmol) was added to an ethanolic solu-
1
3
2
(98%,
1
(
4
64.0, 51.5, 48.2, 42.5, 38.5, 29.3, 29.1, 27.4, 26.9, 23.6, 18.1, 18.0,
+
minutes until the bright yellow solution turned colorless. The
resulting solution was added to a solution of 14 (125 mg,
À1.4; ESI-TOF MS (m/z): [MNa] calcd for
33 58 8 2
C H O Si Na
1
3
661.3562, found 661.3562. Compound epi-17: H NMR (CDCl ,
0
3
.25 mmol) in EtOH (3 mL), and stirred at room temperature for
0 min. After addition of EtOAc, the whole was washed with satd
Cl aq and brine, dried over Na SO . The solvent was evaporated
4 2 4
600 MHz) d 7.37–7.33 (m, 4H), 7.29–7.26 (m, 1H), 4.73 (d,
J = 7.0 Hz, 1H), 4.62–4.54 (m, 5H), 3.89 (br s, 1H), 3.68 (s, 3H),
3.65–3.60 (m, 3H), 3.57–3.50 (m, 3H), 3.29 (br sept, J = 5.0 Hz,
1H), 3.22 (d, J = 9.9 Hz, 1H), 2.23–2.15 (m, 2H), 2.07–1.95 (m,
NH
under reduced pressure to leave an oil, which was purified by flash
chromatography on SiO with EtOAc–hexane (1:3, v/v) to give 15
2
2H), 1.78–1.64 (m, 4H), 1.54–1.37 (m, 6H, including 1.40 (s, 3H)),
1
13
(
108 mg, 86%) as a yellow oil. H NMR (CDCl
3
, 600 MHz) d 7.37–
0.94–0.92 (m, 4H), 0.03–0.01 (m, 18H);
3
C NMR (CDCl ,
7
.33 (m, 4H), 7.29–7.26 (m, 1H), 4.63 (s, 2H), 4.59 (s, 2H), 4.26
150 MHz) d 178.2, 138.8, 128.4, 127.6, 127.5, 94.8, 94.2, 78.2,
(
br s, 1H), 3.85 (d, J = 10.4 Hz, 1H), 3.68 (s, 3H), 3.62 (d,
75.5, 73.1, 70.1, 65.4, 64.9, 63.9, 51.6, 49.2, 44.6, 42.2, 32.7, 29.7,
+
J = 10.4 Hz, 1H), 3.58–3.55 (m, 2H), 3.34–3.31 (m, 1H), 3.27 (dd,
J = 15.1, 2.9 Hz, 1H), 2.55 (dd, J = 15.1, 3.3 Hz, 1H), 2.22–2.20 (m,
27.1, 26.7, 23.2, 18.1, 18.0, À1.4; ESI-TOF MS (m/z): [MNa] calcd
for
33 58 8 2
C H O Si Na 661.3562, found 661.3576. The epi-17
1
1
H), 2.05–2.02 (m, 2H), 1.89–1.80 (m, 2H), 1.71 (br s, 1H), 1.59–
2 2
(826 mg, 1.3 mmol) was dissolved in dry CH Cl (10 mL), then
Dess–Martin periodinane (97%, 679 mg, 1.6 mmol) was added at
0 °C. The mixture was stirred at room temperature for 1 h. After
1
3
.54 (m, 2H), 1.36 (s, 3H), 0.98–0.89 (m, 2H), 0.02 (s, 9H);
, 150 MHz) d 206.6, 173.5, 138.6, 128.4, 127.6, 127.5,
4.9, 77.8, 71.7, 70.2, 65.2, 64.7, 57.3, 52.3, 45.2, 44.4, 42.0, 29.4,
C
NMR (CDCl
3
9
2
C
addition of sat. NaHCO
washed with brine, dried over Na
under reduced pressure to leave an oil, which was purified by flash
chromatography on SiO with EtOAc–hexane (1:5, v/v) to give 16
3
aq, the whole was extracted with CH
2 2
Cl ,
+
7.2, 26.7, 20.8, 18.1, À1.5; ESI-TOF MS (m/z): [MNa] calcd for
2
SO . The solvent was evaporated
4
27
42 7
H O
SiNa 529.2592, found 529.2596.
2
4
.1.6. (1RS,4SR,4aRS,7RS,8aRS)-Methyl 7-(benzyloxy)-1-methyl-
(790 mg, 96%) as a colorless oil.
2-oxo-4-((2-(trimethylsilyl)ethoxy)methoxy)-4a-(((2-(trimeth-
ylsilyl)ethoxy)methoxy)methyl)decahydronaphthalene-1-car-
boxylate (16)
4.1.8. (1RS,2RS,4SR,4aRS,7RS,8aRS)-7-(Benzyloxy)-1-formyl-1-
methyl-4-((2-(trimethylsilyl)ethoxy)methoxy)-4a-(((2-(trime-
thylsilyl)ethoxy)methoxy)methyl)decahydronaphthalen-2-yl
acetate (18)
2 2
The 15 (108 mg, 0.21 mmol) was dissolved in dry CH Cl (3 mL),
then DIPEA (0.19 mL, 1.2 mmol) and SEMCl (0.19 mL, 1.1 mmol)
were added at 0 °C. The mixture was stirred at room temperature
for 39 h. After addition of EtOAc, the whole was washed with 1 N
LiAlH
4
(1.0 M in Et
2
O, 3.6 mL, 3.6 mmol) was added to a solution
O (12 mL) at 0 °C, and the mix-
of 17 (1.16 g, 1.8 mmol) in dry Et
2
HCl, satd NaHCO
vent was evaporated under reduced pressure to leave an oil, which
was purified by flash chromatography on SiO with EtOAc–hexane
3
aq, and brine, then dried over Na
2
SO
4
. The sol-
ture was stirred at room temperature for 1.5 h. After careful addi-
tion of 1 mL of water, 3 mL of 15% NaOH aq, and 1 mL of water, the
white precipitate was filtrated off. The aqueous layer was extracted
2
1
(
(
(
2
(
1:5, v/v) to give 16 (134 mg, 99%) as a colorless oil. H NMR
CDCl , 600 MHz) d 7.37–7.33 (m, 4H), 7.29–7.27 (m, 1H), 4.70
d, J = 7.2 Hz, 1H), 4.62 (s, 2H), 4.60 (d, J = 7.2 Hz, 1H), 4.59 (s,
H), 4.09 (br t, J = 2.9 Hz, 1H), 3.86 (d, J = 10.4 Hz, 1H), 3.69–3.58
m, 5H, including 3.67 (s, 3H)), 3.57–3.53 (m, 3H), 3.35–3.29 (m,
with ether, washed with brine, dried over Na
evaporated under reduced pressure to leave an oil, which was puri-
fied by flash chromatography on SiO with EtOAc–hexane (1:2, v/v)
to give diol (1.02 g, 91%) as a colorless oil. H NMR (CDCl
2 4
SO . The solvent was
3
2
1
3
,
600 MHz) d 7.35–7.33 (m, 4H), 7.29–7.26 (m, 1H), 4.75 (d,
J = 7.0 Hz, 1H), 4.62–4.54 (m, 5H), 3.95 (br s, 1H), 3.83 (br s, 1H),
3.76–3.71 (m, 2H), 3.64–3.56 (m, 3H), 3.50 (d, J = 11.0 Hz, 1H),
3.46–3.43 (m, 2H), 3.41–3.37 (m, 1H), 2.06–1.97 (m, 4H), 1.89–
1.82 (m, 2H), 1.51–1.42 (m, 2H), 1.34 (br q, J = 12.3 Hz, 1H), 1.25
1
2
1
9
1
5
H), 3.11 (dd, J = 15.3, 2.8 Hz, 1H), 2.72 (dd, J = 15.3, 3.3 Hz, 1H),
.20–2.18 (m, 1H), 2.01–1.96 (m, 2H), 1.85–1.75 (m, 2H), 1.60–
.54 (m, 2H), 1.35 (s, 3H), 0.98–0.89 (m, 4H), 0.03 (s, 9H), 0.03 (s,
H); C NMR (CDCl , 150 MHz) d 205.9, 173.5, 138.7, 128.3,
3
27.5, 127.5, 94.9, 94.2, 77.7, 77.4, 70.1, 65.4, 65.2, 64.3, 57.1,
1
3
(s, 1H), 1.12 (s, 3H), 0.96–0.92 (m, 4H), 0.03–0.02 (m, 18H); 13
C
2.2, 45.1, 42.2, 42.0, 29.5, 27.2, 26.6, 20.9, 18.1, 18.0, À1.5; ESI-
3
NMR (CDCl , 150 MHz) d 138.8, 128.4, 127.6, 127.5, 95.0, 95.0,
+
TOF MS (m/z): [MNa] calcd for C33
H
56
O
8
Si
2
Na 659.3406, found
78.4, 77.4, 71.1, 70.1, 66.0, 65.9, 65.8, 65.2, 43.1, 42.5, 39.7, 28.4,
+
6
59.3406.
28.0, 27.7, 27.4, 22.2, 18.2, 18.1, À1.4; ESI-TOF MS (m/z): [MH]
59 7 2
calcd for C32H O Si 611.3794, found 611.3795. The diol (1.02 g,
4
.1.7. (1RS,2RS,4SR,4aRS,7RS,8aRS)-Methyl 7-(Benzyloxy)-2-
1.7 mmol) was dissolved in CH
2
Cl
2
(40 mL), then TEMPO
hydroxy-1-methyl-4-((2-(trimethylsilyl)ethoxy)methoxy)-4a-
((2-(trimethylsilyl)ethoxy)methoxy)methyl)decahydronaph-
thalene-1-carboxylate (17)
NaBH (226 mg, 5.8 mmol) was added to a solution of 16
929 mg, 1.5 mmol) in CH Cl (15 mL) and MeOH (1 mL) at 0 °C,
the mixture was stirred at room temperature for 24 h. After addi-
tion of CH Cl , the whole was washed with satd NH Cl aq and
brine, then dried over Na SO . The solvent was evaporated under
reduced pressure to leave an oil, which was purified by flash chro-
matography on SiO with EtOAc–hexane (1:5, v/v) to give 17
286 mg, 31%) as a colorless oil and epi-17 (644 mg, 68%) as a col-
(21.0 mg, 0.13 mmol), KBr (31.7 mg, 0.27 mmol), and satd NaHCO
3
(
aq (20 mL) ware added at room temperature. The resulting bipha-
sic mixture was cooled at 0 °C, and then treated with NaOCl (4% in
water, 3.0 mL, 1.8 mmol), the mixture was stirred at room temper-
4
(
2
2
ature for 20 min. After addition of brine and satd NaHCO
whole was extracted with CH Cl , washed with brine, dried over
Na SO . The solvent was evaporated under reduced pressure to
leave an oil. The crude was dissolved in pyridine (6 mL), and then
Ac O (3 mL) and catalytic amount of DMAP were added at room
3
aq the
2
2
2
2
4
2
4
2
4
2
2
temperature. The mixture was stirred at room temperature for
17 h (over night). The solvent was removed under reduced
(

1
286
Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
pressure to leave an oil, which was purified by flash chromatogra-
phy on SiO with EtOAc–hexane (1:4, v/v) to give 18 (1.01 g, 85%
for three steps from 17) as a colorless oil. H NMR (CDCl
rated under reduced pressure to leave an oil, which was purified
by flash chromatography on SiO with EtOAc–hexane (1:1, v/v) to
give 21 (165 mg, including di-Cbz and/or undesired mono-Cbz
products) as a colorless oil, 22 (115 mg, 49%) as a colorless oil,
2
2
1
3
,
6
5
1
00 MHz) d 9.65 (s, 1H), 7.37–7.32 (m, 4H), 7.31–7.28 (m, 1H),
.36 (br s, 1H), 4.65–4.63 (m, 2H), 4.59–4.52 (m, 4H), 3.82 (br s,
H), 3.68 (d, J = 9.8 Hz, 1H), 3.62–3.48 (m, 4H), 3.44 (br sept,
and incompletely purified recovery of 19 (14.3 mg) as a colorless
1
oil in the order of elution. Compound 22: H NMR (CDCl
3
,
J = 5.1 Hz, 1H), 3.18 (d, J = 9.8 Hz, 1H), 2.23–2.16 (m, 3H), 2.08–
.02 (m, 4H, including 2.07 (s, 3H)), 1.95–1.91 (m, 1H), 1.76–1.66
m, 3H), 1.54–1.47 (m, 1H), 1.04 (s, 3H), 0.94–0.88 (m, 4H), 0.02
600 MHz) d 7.38–7.33 (m, 5H), 5.71 (s, 1H), 5.19–5.15 (m, 3H),
4.71 (d, J = 7.0 Hz, 1H), 4.54 (d, J = 7.0 Hz, 1H), 4.22 (d, J = 12.4 Hz,
1H), 3.68–3.54 (m, 3H), 3.49 (d, J = 3.6 Hz, 1H), 3.39 (d,
J = 12.4 Hz, 1H), 2.54–2.43 (m, 2H), 2.10–1.83 (m, 7H, including
2.04 (s, 3H)), 1.60–1.54 (m, 1H), 1.39–1.32 (m, 1H), 1.10–1.08 (m,
2
(
(
1
3
s, 9H), 0.01 (s, 9H); C NMR (CDCl
3
, 150 MHz) d 201.7, 170.4,
38.6, 128.4, 127.6, 127.6, 94.7, 93.6, 77.9, 73.1, 71.2, 70.2, 65.3,
5.2, 64.8, 51.0, 42.0, 39.3, 27.2, 27.1, 27.1, 26.2, 21.3, 19.3, 18.1,
1
6
1
6
1H), 0.96–0.84 (m, 5H, including 0.86 (s, 3H)), 0.01 (s, 9H); 13
C
+
8.0, À1.4, À1.4; ESI-TOF MS (m/z): [MNa] calcd for C34
H
58
O
8
Si
2
Na
NMR (CDCl
3
, 150 MHz) d 170.3, 154.2, 134.7, 128.6, 128.6, 128.4,
73.3562, found 673.3562.
97.9, 93.2, 77.7, 72.8, 70.8, 70.1, 67.4, 65.5, 38.9, 36.4, 34.1, 31.2,
+
3
0.9, 30.3, 26.8, 21.3, 18.2, 18.1, À1.4; ESI-TOF MS (m/z): [MNa]
4
.1.9. 4-((2-Trimethylsilyl)ethoxy)methoxy-7-epi AB-ring of
calcd for C29 SiNa 587.2647, found 587.2648. A solution of
44 9
H O
TSDN (19)
A solution of 18 (135 mg, 0.21 mmol) in dry CH
dry MeOH (2 mL) was hydrogenated in the presence of Pd(OH)
carbon (20 wt %, 60 mg) at room temperature, and 1 atom for 1 h.
Pd(OH) on carbon was filtered off, and the filtrate was evaporated
under reduced pressure to leave an oil, which was purified by flash
chromatography on SiO with EtOAc–hexane (2:1, v/v) to give 19
84.0 mg, 94%) as a colorless oil. Compound 19 is an inseparable
21 (165 mg) in dry EtOH (5 mL) was hydrogenated in the presence
of Pd on carbon (10 wt %, 80 mg) at room temperature, and 1 atom
for 1.5 h. Pd on carbon was filtered off, and the filtrate was com-
bined with incompletely purified recovery of 19 (14.3 mg), then
evaporated under reduced pressure to leave an oil, which was puri-
fied by flash chromatography on SiO with EtOAc–hexane (2:1, v/v)
2
to give 19 (86.1 mg, 49% for two steps from 19 via 21) as a colorless
oil.
2
Cl
2
(2 mL) and
on
2
2
2
(
1
mixture of diastereomers (dr = 5:2). H NMR (CDCl
.22 (d, J = 3.7 Hz, 1H), 4.95 (s, 1H), 4.74 (s, 1H), 4.72 (d,
J = 3.7 Hz, 1H), 4.70 (d, J = 7.0 Hz, 1H), 4.69 (d, J = 7.0 Hz, 1H),
3
, 500 MHz) d
5
4.1.12. Hemi-acetal-protected 4-((2-trimethylsilyl)ethoxy)me-
thoxy-7-keto AB-ring of TSDN (23)
4
1
3
2
2
0
1
7
3
.53 (d, J = 7.0 Hz, 1H), 4.53 (d, J = 7.0 Hz, 1H), 4.28 (d, J = 12.0 Hz,
H), 4.12 (d, J = 12.0 Hz, 1H), 3.85 (br s, 1H), 3.66–3.44 (m, 8H),
.41 (br s, 1H), 3.30 (d, J = 12.0 Hz, 1H), 3.07 (d, J = 12.0 Hz, 1H),
.56–2.51 (m, 2H), 2.36–1.24 (m, 20H, including 2.04 (s, 3H) and
.03 (s, 3H)), 1.06–1.03 (m, 2H), 0.95–0.83 (m, 10H, including
2 2
The 22 (134 mg, 0.24 mmol) was dissolved in dry CH Cl (5 mL),
then Dess–Martin periodinane (97%, 311 mg, 0.71 mmol) was
added at 0 °C. The mixture was stirred at room temperature for
17 h (over night). After addition of 10% Na
was extracted with CH Cl , washed with sat. NaHCO
dried over Na SO . The solvent was evaporated under reduced
pressure to leave an oil, which was purified by flash chromatogra-
phy on SiO with EtOAc–hexane (1:2, v/v) to give 23 (127 mg, 96%)
as a colorless oil. H NMR (CDCl
S
2 2
O
3
aq, the whole
2
2
3
aq and brine,
.91 (s, 3H) and 0.86 (s, 3H)), 0.00 (s, 18H); 13C NMR (CDCl
3
,
2
4
25 MHz) d 170.7, 96.3, 95.8, 93.3, 93.2, 78.1, 78.0, 76.0, 72.7,
1.1, 70.9, 66.7, 65.4, 61.8, 40.1, 39.6, 36.5, 36.5, 33.9, 32.4, 31.8,
2
1
1.7, 31.5, 31.1, 30.8, 30.1, 27.8, 27.0, 21.3, 21.3, 18.9, 18.2, 18.0,
3
, 600 MHz) d 7.39–7.33 (m, 5H),
+
À1.5; ESI-TOF MS (m/z): [MNa] calcd for
C
H
21 38
O
7
SiNa
5.70 (s, 1H), 5.20–5.16 (m, 3H), 4.70 (d, J = 7.1 Hz, 1H), 4.54 (d,
J = 7.1 Hz, 1H), 4.27 (d, J = 12.6 Hz, 1H), 3.64–3.59 (m, 3H), 3.55–
4
53.2279, found 453.2283.
3
.50 (m, 1H), 2.90 (dd, J = 14.2, 4.7 Hz, 1H), 2.55–2.51 (m, 2H),
4
.1.10. 7-Epi AB-ring of TSDN (epi-7)
2.45–2.33 (m, 3H), 2.25–2.20 (m, 1H), 2.16–2.04 (m, 1H), 2.02 (s,
3H), 1.37–1.33 (m, 1H), 0.95–0.83 (m, 5H, including 0.84 (s, 3H)),
0.00 (s, 9H); C NMR (CDCl , 150 MHz) d 210.3, 170.1, 154.1,
3
TFA (2 mL) was added to a solution of 19 (45.4 mg, 0.11 mmol)
in CH Cl (2 mL) at 0 °C, the mixture was stirred at 0 °C for 1.5 h.
The solvent was removed under reduced pressure to leave an oil,
which was purified by flash chromatography on SiO with
1
3
2
2
134.5, 128.7, 128.6, 128.4, 97.6, 92.9, 77.7, 72.3, 70.2, 67.4, 65.7,
2
39.0, 37.4, 36.5, 36.4, 35.5, 31.0, 28.3, 21.2, 18.1, 18.0, À1.5; ESI-
+
EtOAc–MeOH (95:5, v/v) to give epi-7 (25.2 mg, 80%) as a colorless
TOF MS (m/z): [MNa] calcd for C29
42
H O
9
SiNa 585.2490, found
oil. Compound epi-7 is an inseparable mixture of diastereomers
585.2501.
1
(
dr = 7:1). H NMR (CD
3
OD, 600 MHz) d 5.17 (d, J = 3.6 Hz, 1H),
4
.87 (s, 1H), 4.67 (d, J = 3.6 Hz, 1H), 4.62 (s, 1H), 4.27 (d,
4.1.13. Hemi-acetal-protected 4-((2-trimethylsilyl)ethoxy)me-
thoxy AB-ring of TSDN (24)
J = 12.0 Hz, 1H), 4.11 (d, J = 12.0 Hz, 1H), 3.63–3.62 (m, 1H), 3.49–
3
2
1
1
.43 (m, 3H), 3.24 (d, J = 12.0 Hz, 1H), 2.98 (d, J = 12.0 Hz, 1H),
.69–2.63 (m, 2H), 2.33 (dd, J = 13.5, 3.8 Hz, 1H), 2.23–1.72 (m,
5H, including 2.03 (s, 3H) and 2.02 (s, 3H)), 1.52–1.46 (m, 1H),
.37–1.24 (m, 3H), 1.05–1.01 (m, 2H), 0.87 (s, 3H), 0.81 (s, 3H);
NaBH
(127 mg, 0.23 mmol) in CH
0 °C, the mixture was stirred at room temperature for 16 h (over
night). After addition of CH Cl , the whole was washed with satd
NH Cl aq and brine, then dried over Na SO . The solvent was evap-
orated under reduced pressure to leave an oil, which was purified
by flash chromatography on SiO with CH Cl –MeOH (93:7, v/v) to
4
(17.3 mg, 0.45 mmol) was added to a solution of 23
2
2
Cl (5.6 mL) and MeOH (0.4 mL) at
2
2
1
3
3
C NMR (CD OD, 150 MHz) d 172.8, 172.7, 97.4, 96.8, 78.2, 74.8,
4
2
4
7
3
4.2, 74.2, 72.2, 72.0, 67.8, 62.7, 41.7, 41.2, 38.0, 37.9, 36.9, 36.7,
4.7, 33.4, 32.5, 32.0, 31.7, 31.2, 29.2, 28.4, 21.4, 21.3, 19.6, 19.0;
2
2
2
+
ESI-TOF MS (m/z): [MNa] calcd for C15
H
24
O
6
Na 323.1465, found
give mixture of 22 and 24 (122 mg, 95%, 22/24 = 2.6/1 by analytical
3
23.1452.
HPLC) as a colorless oil. The mixture was completely separated by
1
p-TLC and HPLC technique. Compound 24: H NMR (CDCl
3
,
4
.1.11. Hemi-acetal-protected 4-((2-trimethylsilyl)ethoxy)me-
600 MHz) d 7.38–7.33 (m, 5H), 5.68 (s, 1H), 5.19–5.15 (m, 3H),
4.74 (d, J = 7.1 Hz, 1H), 4.58 (d, J = 7.1 Hz, 1H), 4.21–4.19 (m, 2H),
3.67–3.63 (m, 1H), 3.60–3.55 (m, 1H), 3.44 (d, J = 3.7 Hz, 1H),
3.40 (d, J = 12.2 Hz, 1H), 2.80–2.77 (m, 1H), 2.54–2.50 (m, 1H),
2.34–2.28 (m, 1H), 2.11–2.01 (m, 5H, including 2.04 (s, 3H)),
1.80–1.64 (m, 3H), 0.95–0.79 (m, 6H, including 0.83 (s, 3H)), 0.01
thoxy-7-epi AB-ring of TSDN (22)
CbzCl (95%, 0.10 mL, 0.70 mmol) was added to a solution of 19
(
177 mg, 0.41 mmol) in dry CH
2
Cl
.37 mmol) at À78 °C, and the mixture was stirred at À78 °C for
h. After addition of CH Cl , The whole was washed with water,
N HCl aq, and brine, dried over Na SO . The solvent was evapo-
2
(5 mL) and TMEDA (55.5 lL,
0
2
1
2
2
1
3
2
4
3
(s, 9H); C NMR (CDCl , 150 MHz) d 170.5, 154.3, 134.7, 128.6,

Y. Nakai et al. / Bioorg. Med. Chem. 18 (2010) 1280–1287
1287
1
3
28.6, 128.3, 98.1, 93.2, 78.1, 73.1, 70.0, 67.1, 66.0, 65.3, 38.5, 36.8,
1.2, 29.0, 27.9, 27.2, 21.9, 21.4, 18.1, À1.4; ESI-TOF MS (m/z):
Acknowledgment
+
[
MNa] calcd for C29
H
44
O
9
SiNa 587.2647, found 587.2646.
This work was supported by National Institutes of Health Grant
AI 072358) and The Skaggs Institute for Chemical Biology.
(
4
.1.14. AB-ring of TSDN (7)
A solution of 24 (12.0 mg) in dry EtOH (2 mL) was hydrogenated
References and notes
in the presence of Pd on carbon (10 wt %, 5 mg) at room tempera-
ture, and 1 atom for 1 h. Pd on carbon was filtered off, then evap-
orated under reduced pressure to leave an oil. The crude was
1
.
(a) Johnson, E. A.; Bradshaw, M. Toxicon 2001, 39, 1703; (b) Schantz, E. J.;
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Singh, B. R. Nat. Struct. Biol. 2000, 7, 617.
2.
dissolved in CH
2
Cl
2
(2 mL), cooled at 0 °C, then TFA (2 mL) was
3. (a) Willis, B.; Eubanks, L. M.; Dickerson, T. J.; Janda, K. D. Angew. Chem., Int. Ed.
008, 47, 8360; (b) Dickerson, T. J.; Janda, K. D. ACS Chem. Biol. 2006, 1, 359; (c)
2
added. The mixture was stirred at 0 °C for 1.5 h. The solvent was
removed under reduced pressure to leave an oil, which was puri-
Simpson, L. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 167.
. Nakatani, M. Heterocycles 1999, 50, 595.
4
fied by flash chromatography on SiO
2
with EtOAc–MeOH (95:5,
5. Carpinella, C.; Ferrayoli, C.; Valladares, G.; Defago, M.; Palacios, S. Biosci.
Biotechnol. Biochem. 2002, 66, 1731.
v/v) to give 7 (5.2 mg, 82% for two steps from 24) as a colorless
6.
Xie, Y. S.; Fields, P. G.; Isman, M. B.; Chen, W. K.; Zhang, X. J. Stored Prod. Res.
995, 31, 259.
oil. Compound 7 is an inseparable mixture of diastereomers
1
1
(
dr = 2:1). H NMR (CDCl
3
, 600 MHz) d 5.44 (d, J = 3.3 Hz, 1H),
7
.
(a) Akhtar, Y.; Isman, M. B. J. Chem. Ecol. 2003, 29, 1853; (b) Carpinella, M. C.;
Defago, M. T.; Valladares, G.; Palacios, S. M. J. Agric. Food Chem. 2003, 51, 369;
4
1
.96 (d, J = 2.5 Hz, 1H), 4.94 (d, J = 3.3 Hz, 1H), 4.79 (d, J = 2.5 Hz,
H), 4.29 (d, J = 12.1 Hz, 1H), 4.21 (br s, 1H), 4.17 (br s, 1H), 4.12
(
4
c) Zhou, J.-B.; Minami, Y.; Yagi, F.; Tadera, K.; Nakatani, M. Heterocycles 1997,
5, 1781.
(
d, J = 12.1 Hz, 1H), 3.67 (br s, 2H), 3.47 (br s, 2H), 3.32 (d,
J = 12.1 Hz, 1H), 3.09 (d, J = 12.1 Hz, 1H), 2.85–2.79 (m, 2H), 2.69
d, J = 3.5 Hz, 1H), 2.59 (dd, J = 13.3, 4.2 Hz, 1H), 2.47 (dd, J = 13.3,
8
.
.
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Tradit. Chin. Med. 1985, 5, 29; (b) Li, P. Z.; Zhou, J.; Miao, W. Y.; Ding, F. H.;
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2009, 106, 1330.
(
9
4
1
.2 Hz, 1H), 2.41–2.33 (m, 3H), 2.12 (s, 3H), 2.11 (s, 3H), 1.93–
.62 (m, 10H), 0.92–0.84 (m, 8H, including 0.91 (s, 3H) and 0.87
1
3
ˇ
10. (a) Capcová, K.; Yoneda, Y.; Dickerson, T. J.; Janda, K. D. Bioorg. Med. Chem. Lett.
(
s, 3H)); C NMR (CDCl
3
, 150 MHz) d 169.9, 169.7, 96.5, 95.9,
4.6, 74.5, 74.1, 66.3, 66.1, 66.1, 61.4, 60.4, 40.3, 39.6, 37.5, 35.7,
5.6, 29.7, 29.1, 28.2, 28.1, 28.1, 26.9, 25.8, 22.9, 22.2, 21.5, 21.4,
2007, 17, 6463; (b) Eubanks, L. M.; Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.;
7
3
1
3
Tepp, W. H.; Baldwin, M. R.; Malizio, C. J.; Goodnough, M. C.; Barbieri, J. T.;
Johnson, E. A.; Boger, D. L.; Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A.
+
2007, 104, 2602; (c) Silvaggi, N. R.; Boldt, G. E.; Hixon, M. S.; Kennedy, J. P.;
24 6
8.6, 18.0; ESI-TOF MS (m/z): [MNa] calcd for C15H O Na
Tzipori, S.; Janda, K. D.; Allen, K. N. ACS Chem. Biol. 2007, 14, 533; (d) Boldt, G.
E.; Eubanks, L. M.; Janda, K. D. Chem. Commun. 2006, 3063; (e) Boldt, G. E.;
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23.1465, found 323.1463.
4
4
.2. Biological evaluation
11. Wender, P. A.; Verma, V. A.; Paxton, T. J.; Pillow, T. H. Acc. Chem. Res. 2008, 41, 40.
12. Nakai, Y.; Tepp, W. H.; Dickerson, T. J.; Johnson, E. A.; Janda, K. D. Bioorg. Med.
Chem. 2009, 17, 1152.
.2.1. Details of assay procedures
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2002, 41, 2103.
23
ium botulinum strains Hall A hyper as described. The specific
activity was determined via the mouse bioassay24 to be 7.8 pg/
mouse LD50. The toxins were stored in 40% glycerol, 15 mM sodium
phosphate, 90 mM NaCl at À20 °C.
15. (a) Ghosh, S.; Rivas, F.; Fischer, D.; González, M. A.; Theodorakis, E. A. Org. Lett.
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1
1
1
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4
.2.2. RSC assay
1
2
9. Nicolaus, K. C.; Sasmal, P. K.; Roecker, A. J.; Sun, X.-W.; Mandal, S.; Converso, A.
Angew. Chem., Int. Ed. 2005, 44, 3443.
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Primary rat spinal cord cells were prepared as described previ-
2
2
ously, and seeded into collagen coated 96-well plates (BD Biosci-
ences) at a density of 75,000 cells/well. The RSC assay was
essentially performed as described,9,22 after the cells had matured
for at least three weeks. In short, the cells were exposed to a mix-
ture of the indicated concentrations of toosendanin or analogues
and 5.6 pM BoNT/A1 (42 pg or about 5.5 mouse LD50 Units) in
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5
0
l
L of culture medium containing 1% DMSO. The positive control
contained toxin and 1% DMSO, and the negative control contained
% DMSO only in culture medium. All samples were tested in rep-
licates of three. After 24 h of incubation at 37 °C, 5% CO , the cells
22. Pellett, S.; Tepp, W. H.; Clancy, C. M.; Borodic, G. E.; Johnson, E. A. FEBS Lett.
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1
23. Malizio, C. J.; Goodnough, M. C.; Johnson, E. A. Methods Mol. Biol. 2000, 145, 27.
2
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2
were harvested by lysis in 1 Â LDS sample buffer (Invitrogen), and
analyzed by Western blot as described.9
,22