Function-oriented synthesis applied to the anti-botulinum natural product toosendanin

Article abstract of DOI:10.1016/j.bmc.2008.12.042

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 is a traditional Chinese medi

Full text of DOI:10.1016/j.bmc.2008.12.042

Bioorganic & Medicinal Chemistry 17 (2009) 1152–1157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Function-oriented synthesis applied to the anti-botulinum natural
product toosendanin
Yuya Nakai a,b, William H. Tepp c, Tobin J. Dickerson a,d, Eric A. Johnson c, Kim D. Janda a,b,d,
*
a Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b Department of Immunology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
c Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706, USA
d Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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 is a traditional Chinese medicine which has been reported to have anti-botulinum
properties in animal models. To establish what chemical functionalities are necessary for the anti-botu-
linum properties found within toosendanin, a study was initiated with the goal of using function-ori-
Received 30 October 2008
Revised 10 December 2008
Accepted 14 December 2008
Available online 25 December 2008
ented synthesis (FOS) as
a strategy to begin to unravel toosendanin’s powerful anti-botulinum
Keywords:
Botulinum neurotoxins
Toosendanin
Function-oriented synthesis (FOS)
4-Acetoxy CD fragment
properties. From these studies a new synthetic strategy is put forth allowing access to a 4-acetoxy CD
fragment analogue (14) of toosendanin, which was achieved from mesityl oxide and acetylacetone in
14 steps. Animal studies on this fragment are also reported.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and that work by a different mechanism other than inhibiting
the catalytic light chain of the BoNT/A protease.8 Limonoids, 1,
Clostridium botulinum is a rod-shaped, gram-positive, sporulat-
ing anaerobic bacillus that is widely distributed in the environ-
ment.1 The spores, which are resistant to heat, desiccation,
chemicals, radiation, and oxygen, facilitate survival for very long
periods. Under anaerobic conditions, and in the correct nutritional
environment, spore germination and cell division take place. The
global distribution of C. botulinum in the environment varies
widely. The botulinum neurotoxins (BoNTs) comprise a family of
seven immunologically distinct proteins synthesized primarily by
strains of the anaerobic bacteria. These toxins (BoNTs A–G) are
the most lethal poisons known, with BoNT serotype A having a
are tetranortriterpenoids with a 4,4,8-trimethylfuranylsteroid
skeleton (Fig. 1) derived from euphane or tirucallane triterpenoids,
and, in general, have intense bitter taste.9 Meliaceae plants are a
rich source of limonoids. Limonoids from the neem tree Melia aza-
dirachta indica and the bead tree M. azedarach have been well stud-
ied, mainly because of their marked insect anti-feedant
properties.10 Along this same vein of research, Melia toosendan,
which is native to China and known to contain limonoid constitu-
ents, has been found to possess effective antihelmintics.11 A major
limonoid constituent found in M. toosendan is the compound too-
sendanin (2, Fig. 1), which appears to have multiple modes of ac-
tion in insects including damage to midgut tissues and inhibition
of esterases, cytochrome P450-aldrin epoxidase and proteinase
activities.12
LD50 for a 70 kg human of a mere 0.8 l
g by inhalation.2 Historically
associated with food poisoning, these proteinaceous toxins inhibit
the release of acetylcholine at neuromuscular junctions by cleav-
age of SNARE proteins, resulting in progressive flaccid paralysis.3,4
It is as a result of this potent neurotoxic activity that BoNTs are of
substantial concern as potential bioterrorist weapons.5–7
2. Result and discussions
We have embarked on a program to identify new small mole-
cule inhibitors of BoNT/A that are non-proteinaceous in nature
* Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
Figure 1. General structure of a limonoid 1 and toosendanin 2.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.042

Y. Nakai et al. / Bioorg. Med. Chem. 17 (2009) 1152–1157
1153
Toosendanin from a structural diversity standpoint is a fascinat-
ing molecule. The core carbon skeleton of toosendanin comprises a
cyclopentanoperhydrophenanthrene (3, Fig. 2), a molecular frame-
work that consists of four fused, non-planar rings (labeled A–D)
and a densely packed core of 14 stereocenters. Interestingly, this
framework is commonly found in all mammalian steroids and hor-
mones. What has intrigued us besides toosendanin’s anti-parasitic
properties are two reports in the 1980s describing toosendanin as
also having anti-botulinum properties.13 Strikingly, these findings
have remained dormant, as little research has been conducted
upon this molecule from a synthetic standpoint.
To probe toosendanin’s anti-botulinum properties two tactics
were envisioned. The first being a total synthesis of the molecule
that ultimately would allow derivatives to be assessed, and thus
a means to explore toosendanin’s structure in relationship to its
biological activity. A second was what Wender has termed Func-
tion-Oriented Synthesis (FOS).14 Because of the complexity of too-
sendanin and the insurmountable odds of a total synthesis that
would provide large quantities of this natural product or closely re-
lated derivatives, we have set a course on investigating this mole-
cule from a FOS standpoint. The underpinnings of FOS are that the
function of a biologically active lead structure can be emulated,
tuned, or possibly improved by replacement with simpler scaffolds
designed to embrace the key activity-determining structural fea-
tures of the natural product. Because it is unknown which ele-
ments and/or structural features impact toosendanin’s biological
activity, we have decided to break the molecule into two fragments
consisting of the AB and CD rings. In this paper, we report our ini-
tial work exploring the CD ring of toosendanin and its potential
biological function.
We envisioned any synthesis emulating the CD rings of too-
sendanin would need to incorporate the epoxide moiety, shared
by rings C and D at the C-14 and C-15 positions. We hypothesized
this functionality to be crucial, as this moiety is not observed with-
in any steroidal skeletons, including mammalian or plant (Fig. 1).
While the epoxide was deemed critical, the furan ring was not,
as we have found through semi-synthesis efforts (Janda et al.
unpublished results) that this ring in a reduced chemical state
did not alter the molecules biological activity. Furthermore, this
functionality was readily susceptible to both oxidation and reduc-
tion conditions, which would clearly hamper any practical synthe-
sis of an abbreviated structure if included within our synthetic
scheme (Janda et al. unpublished data).
Overall, there are few de novo synthetic efforts that describe the
successful syntheses of similar limonoids or key structural frag-
ments. Selected examples are dumsin15 and fragments of epoxyaz-
adiradione.16 Our FOS approach would build on the teachings of
Mateos et al. who have recently described a synthesis of the limo-
noid fragment 4 in ten steps with an overall yield of 44% (Fig. 3).17
We envisioned four crucial cyclization steps in the synthesis of
a FOS toosendanin CD fragment: the Robinson annulation, an aldol
condensation, the Nazarov cyclization, and an epoxidation reac-
tion. Using these series of reactions it was envisioned that at vari-
ous stages appropriate substituents could be embedded within the
CD ring. Importantly, this approach would enable the first synthe-
sis of the CD ring fragment of toosendanin correctly displaying all
heteroatom substituents found within the natural product. Finally,
it is worth noting that a previous paper focusing on the reaction
Figure 3. Limonoid fragment synthesized by Mateos et al. 4.
scope within limonoid construction has considered the possibility
of building such a desired fragment; however, no compound
bearing the
a-acetoxy ketone functionality within ring C was
reported.18
In developing a synthesis, our starting point featured a Robin-
son-type annulation of mesityl oxide 5 and acetylacetone 6 using
BF3 as a catalyst, which bestowed
is appropriate to note that -cyclocitral 7 has been a preparatory
a
-cyclocitral (Scheme 1).16,18 It
a
material for many liminoid derivatives, particularly for SAR stud-
ies of insect anti-feedant development.16–22 The convenience of
starting with 7 is most certainly due to the fact that it can be
readily synthesized on a multi-gram scale with inexpensive start-
ing materials. Selective reduction16 of the conjugated carbonyl of
diketone 7 using NaBH4/CeCl3 at À40 °C furnished the hydroxyk-
etone 8, which could then be utilized for epoxidation with
m-chloroperbenzoic acid affording 9. Aldol condensation of 9 with
benzaldehyde provided dihydroxyketone 10 in 89% overall yield
from 7.
With dihydroxyketone 10, a Nazarov electrocyclization reaction
was envisioned. For comparative purposes we note that the Naza-
rov electrocyclization reaction of a, b, and c (Fig. 4) in the presence
of acid grants the corresponding cyclization compounds.18,22 In
lieu of these reports, we attempted the reaction of d with acid
for the synthesis of a ‘non-functionalized’ moiety at C3, however,
this reaction failed. It is well understood that to accomplish the
required cyclization a keto-group at C4, and a phenyl group at C3
are needed; unfortunately these requirements limit diversity, and
as stated also dictate the stereochemical outcome.
Thus, the Nazarov electrocyclization reaction of 10 in the pres-
ence of acid granted 11a and 11b (inseparable), 11c, and 11d. We
were pleased to observe that the cyclization exclusively formed
the cis-fused product,17,18,21,23 placing the core ring system in
the desired geometry of toosendanin. Reduction of the unsatu-
rated C-ring by catalytic hydrogenation supplied compounds 12a
and 12b (inseparable), 12c, and 12d, respectively (Scheme 2).
We attempted the asymmetric reduction of 11a–d with a plethora
of reagents (e.g., (2S,5S)-(À)-5-benzyl-3-methyl-2-(5-methyl-2-
furyl)-4-imidazolidi-none, binaphthyl derivatives, to name
few) that would have provided the correct C3-stereocenter, but,
a
Scheme 1. Synthesis of compound 10. Reagents and conditions: (a) BF3ÁEt2O, 5 °C;
(b) NaBH4, CeCl3Á7H2O, MeOH, À40 °C; (c) mCPBA, CH2Cl2, rt; 12 h, 98% (d) PhCHO,
NaOH, EtOH, rt, 13 h, 96%.
Figure 2. Toosendanin’s core ring system 3.

1154
Y. Nakai et al. / Bioorg. Med. Chem. 17 (2009) 1152–1157
Figure 4. Nazarov electrocyclization reaction of a–d.
unfortunately the lone reaction that succeeded was palladium–
carbon.
and epoxydation of olefin with m-chloroperbenzoic acid granted
14 in 14% overall yield from 13 (Scheme 3).
The relative stereochemistry of each of these compounds was
determined by 2D-ROESY spectrum (Fig. 5). Thus, for 12a ROESY
correlation was seen between H-4 (geminal with the acetoxy
group) and Me-7 (axial); H-3 (geminal with the phenyl group)
and Me-3a (the angular methyl group); Me-3a and H-7a. For the
case with 12b, we could see a correlation (sv) between H-3 and
Me-3a; Me-3a and H-7a; for 12c, correlation could be seen be-
tween H-3 and Me-3a and H-7a; H-4 and Me-7 (axial); H-5 and
Me-7 (axial); while for 12d, it could be seen between H-3 and
Me-3a; H-7a, Me-3a and H-4 (geminal with the acetoxy group);
H-5 and Me-7 (axial).
The D-ring ketone of 12a and 12b was reduced to sec-alcohol by
treatment with 9-BBN (Scheme 3),22 subsequent dehydroxylation
of this sec-alcohol with thionylchloride in pyridine afforded the de-
sired olefin, which upon hydrolysis of the diacetyl functionalities
with potassium carbonate gave dihydroxyolefin 13 in 19% overall
yield from 12. Selective protection of dihydroxyolefin 13 using
TBSOTf at À78 °C provided the 7-TBS-protected compound in 19%
yield. The hydroxyl group at C-6 was oxidized by Dess–Martin’s re-
agent, which on subsequent deprotection of the silyl group with
TBAF, followed by acetylation with acetic anhydride in pyridine,
Figure 5. The relative stereochemistry of compounds 12a–d was determined by
2D-ROESY experiments.
Scheme 2. Synthesis of compounds 12a–d. Reagents and conditions: (a) 10À1 M HClO4/0.5 M Ac2O/AcOEt, rt, 63 h; (b) Pd–alumina, EtOH, rt, 18 h; (c) Pd–carbon, MeOH, rt,
21 h.

Y. Nakai et al. / Bioorg. Med. Chem. 17 (2009) 1152–1157
1155
Scheme 3. Synthesis of compound 14. Reagents and conditions: (a) 9-BBN, THF, rt, 4 h; (b) SOCl2, pyridine, CH2Cl2, rt, 12 h; (c) K2CO3, MeOH, rt, 86 h, 19% for three steps; (d)
TBSOTf, DIPEA, CH2Cl2, À78 °C, 2.5 h; (e) Dess-Martin reagent, CH2Cl2, rt, 13 h; (f) TBAF, THF, rt, 2.5 h; (g) Ac2O, pyridine, cat. DMAP, rt, 24 h; (h) mCPBA, CH2Cl2, rt, 6 h, 14% for
five steps.
Merck Kieselgel 60 F254 silica gel plates (0.25, 0.5, or 1 mm). 1H
NMR spectra were recorded on Bruker DRX-600 (600 MHz), DRX-
500 (500 MHz), or Varian Inova-400 (400 MHz) spectrometers.
13C NMR spectra were recorded on Bruker DRX-600 (150 MHz)
spectrometer. All 2D-ROESY experiments (sm = 200 ms) were also
recorded on Bruker DRX-600 (600 MHz) spectrometer. 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, multiplet). High-resolution mass spectra
were recorded on an Agilent ESI-TOF mass spectrometer.
Figure 6. The relative stereochemistry of compound 14 was determined by 2D-
ROESY experiment.
4.1. 1-(5-Hydroxy-1,3,3-trimethyl-7-oxabicyclo[4.1.0]heptan-2-
yl)ethanone (9)
To determine 14’s relative stereochemistry we again looked to
the 2D-ROESY spectrum (Fig. 6). We could see a ROESY correlation
between H-3 (geminal with the phenyl group) and Me-3a (the
angular methyl group), H-4 (geminal with the acetoxy group)
and H-6 (equatorial).
m-Chloroperbenzoic acid (mCPBA) (77%, 107.5 mg, 0.48 mmol)
was added to a solution of 8 (87.3 mg, 0.48 mmol) in CH2Cl2
(2 mL) at room temperature, and the mixture was stirred at room
temperature for 12 h. Five percentage of Na2SO3 aq was added
and the resulting heterogeneous mixture was vigorously stirred.
The organic layer was separated, and the aqueous phase was ex-
tracted with CH2Cl2. The combined organic extracts were washed
with 5% Na2CO3 aq and brine, and dried over Na2SO4. The solvent
was evaporated under reduced pressure to leave an oil, which
was purified by flash chromatography on SiO2 with Et2O–hexane
(2:1, v/v) to give 9 (93.3 mg, 98%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d: 4.09 (ddd, J = 10.2, 6.3, 2.4 Hz, 1H), 3.17 (d,
J = 2.3 Hz, 1H), 2.49 (s, 1H), 2.29 (s, 3H), 1.78 (dd, J = 13.1,
10.4 Hz, 1H), 1.48–1.41 (m, 1H), 1.47 (s, 3H), 0.94 (s, 3H), 0.92 (s,
3H); 13C NMR (150 MHz, CDCl3) d: 207.2, 66.1, 61.5, 60.5, 60.1,
37.2, 33.7, 32.5, 29.2, 26.9, 24.8; HRMS [MH]+ calcd for C11H19O3,
199.1329; found, 199.1329.
To investigate the potential bioactivity of epoxide 14, we uti-
lized the mouse lethality assay (MLA).8d This assay is considered
the ‘gold standard’ for the field and is used to assess any new pro-
tein or small molecule that may possess anti-botulinum activity.
Before the assay was initiated with 14, we first investigated poten-
tial toxicity issues with this molecule. Gratifyingly, at a concentra-
tion of 2.5 mM this compound showed no propensity to be toxic to
any of the animals it was administered, n = 4. Thus, mice were in-
jected intravenously with 14 followed immediately by ip injection
of approximately 10LD50s of BoNT/A. The mice were observed over
a 48 h period and unfortunately all mice succumbed to BoNT/A
toxicity. In contrast, the natural product toosendanin at this same
concentration extended time to death 7.1 h.
3. Conclusions
4.2. (E)-1-(3,4-Dihydroxy-2,6,6-trimethylcyclohex-1-enyl)-3-
phenylprop-2-en-1-one (10)
The work reported was initiated with a goal of using FOS as a
strategy to begin to unravel toosendanins powerful anti-botulinum
properties. Thus, a new synthetic approach to a 4-acetoxy CD frag-
ment analogue of Toosendanin was achieved from mesityl oxide
and acetylacetone in 14 steps. Unfortunately, this molecule pro-
vided no efficacy in vivo. However, we note that our synthetic
route should allow access to other CD ring analogues, for example,
exchange of benzaldehyde to other aldehydes would allow entry
into other unnatural analogues. In addition, an acetyl group func-
tional change would also provide another series of FOS-molecules.
These compounds as well as the AB ring synthesis will be reported
in due course.
Under argon atmosphere, NaOH (52.6 mg, 1.3 mmol) was
added to a solution of 9 (130.3 mg, 0.66 mmol) and benzaldehyde
(66.8 lL, 0.66 mmol) in EtOH (3 mL) at room temperature, and
the mixture was stirred at room temperature for 13 h. After
evaporation of the solvent, the whole was made acidic by adding
1 N HCl under ice cooling and extracted with Et2O. The extract
was washed with brine, dried over Na2SO4, and evaporated under
reduced pressure to leave an oil, which was purified by flash
chromatography on SiO2 with AcOEt–hexane (5:1, v/v) to give
10 (180.7 mg, 96%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
d: 7.59–7.54 (m, 2H), 7.48 (d, J = 16.2 Hz, 1H), 7.43–7.39 (m,
3H), 6.78 (d, J = 16.2 Hz, 1H), 4.04–3.97 (m, 2H), 1.86–1.76 (m,
1H), 1.77 (s, 3H), 1.63–1.53 (m, 1H), 1.23 (s, 3H), 1.06 (s, 3H);
13C NMR (150 MHz, CDCl3) d: 201.2, 146.6, 144.0, 134.3, 130.9,
129.6, 129.0, 128.6, 127.7, 70.4, 66.7, 40.8, 36.3, 29.7, 29.5,
28.0, 19.1; HRMS [MH]+ calcd for C18H23O3, 287.1642; found,
287.1648.
4. Experimental
In general, reagents and solvents were used as purchased with-
out further purification. All flash column chromatography was
performed using silica gel 60 (230–400 mesh). Analytical and pre-
parative thin-layer chromatography (TLC) was performed using

1156
Y. Nakai et al. / Bioorg. Med. Chem. 17 (2009) 1152–1157
4.3. (3aRS,4RS,5RS,7aSR)-3a,7,7-Trimethyl-1-oxo-3-phenyl-
3a,4,5,6,7,7a-hexahydro-1H-indene-4,5-diyl diacetate (11a),
(3aRS,4SR,5RS,7aSR)-3a,7,7-trimethyl-1-oxo-3-phenyl-3a,4,-
5,6,7,7a-hexahydro-1H-indene-4,5-diyl diacetate (11b),
(3aRS,4RS,5SR,7aSR)-3a,7,7-trimethyl-1-oxo-3-phenyl-
3a,4,5,6,7,7a-hexahydro-1H-indene-4,5-diyl diacetate (11c),
and (3aRS,4SR,5SR,7aSR)-3a,7,7-trimethyl-1-oxo-3-phenyl-
3a,4,5,6,7,7a-hexahydro-1H-indene-4,5-diyl diacetate (11d)
J = 15.3, 3.9 Hz, 1H), 1.68–1.61 (m, 1H), 1.67 (s, 3H), 1.35 (s, 3H),
1.32 (s, 3H), 1.24 (s, 3H); 13C NMR (150 MHz, CDCl3) (mixture of
12a and 12b) d: 214.8, 214.1, 170.5, 170.0, 169.9, 137.5, 136.3,
128.7, 128.2, 128.1, 127.1, 127.0, 71.6, 70.9, 69.2, 68.6, 66.7, 66.5,
50.7, 49.5, 49.0, 47.4, 41.1, 40.9, 40.6, 38.5, 33.7, 32.5, 31.3, 30.3,
29.7, 28.9, 23.2, 23.1, 21.2, 21.0, 20.3; HRMS [MH]+ calcd for
C22H29O5, 373.2009; found, 373.2006. Compound 12b (data ob-
tained from the spectrum of mixture): 1H NMR (500 MHz, CDCl3)
d: 7.32–7.18 (m, 5H), 5.16–5.09 (m, 1H), 5.04 (d, J = 9.5 Hz, 1H),
3.06–2.90 (m, 2H), 2.58–2.51 (m, 1H), 2.05 (d, J = 1.5 Hz, 1H),
1.92 (s, 3H), 1.65–1.61 (m, 2H), 1.59 (s, 3H), 1.40 (s, 3H), 1.25 (s,
3H), 1.19 (s, 3H).
Under argon atmosphere, 10 (484.6 mg, 1.7 mmol) was dis-
solved in 48 mL of 10À1 M HClO4/0.5 M Ac2O/AcOEt reagent.18
The solution was allowed to stand at room temperature for 63 h.
Saturated NaHCO3 aq was added to quench the reaction. The or-
ganic layer was separated, and the aqueous phase was extracted
with AcOEt. The extract was washed with 5% Na2CO3 aq and brine,
dried over Na2SO4, and evaporated under reduced pressure to leave
a solid. The residue was recrystallized from AcOEt–hexane to give a
mixture of 11a and 11b (311.3 mg, 50%) as a colorless powder. The
mother liquor was purified by p-TLC on SiO2 with AcOEt–hexane
(1:4, v/v) to give 11c (109.0 mg, 17%) as a yellow oil and 11d
(77.8 mg, 12%) as a colorless oil in the order of elution. Compound
11a: 1H NMR (400 MHz, CDCl3) d: 7.52–7.43 (m, 5H), 6.05 (s, 1H),
5.73 (d, J = 3.3 Hz, 1H), 4.91 (ddd, J = 11.1, 4.7, 3.4 Hz, 1H), 2.25
(s, 1H), 1.99 (s, 3H), 1.95–1.87 (m, 1H), 1.93 (s, 3H), 1.61–1.53
(m, 1H), 1.51 (s, 3H), 1.28 (s, 3H), 1.01 (s, 3H); 13C NMR
(150 MHz, CDCl3) (mixture of 11a and 11b) d: 208.5, 206.4,
180.7, 179.3, 170.7, 170.2, 170.0, 169.8, 136.3, 134.0, 133.2,
130.2, 130.0, 128.9, 128.2, 127.6, 127.3, 74.6, 71.9, 70.1, 68.4,
64.3, 63.7, 50.9, 50.4, 43.4, 39.2, 34.3, 33.9, 32.7, 32.3, 29.0, 25.9,
25.1, 22.2, 21.1, 20.9, 20.6, 20.0; HRMS [MH]+ calcd for C22H27O5,
371.1853; found, 371.1854. Compound 11b (data obtained from
the spectrum of mixture): 1H NMR (400 MHz, CDCl3) d: 7.39–
7.34 (m, 3H), 7.32–7.28 (m, 2H), 6.08 (s, 1H), 5.50 (d, J = 8.0 Hz,
1H), 5.14 (dt, J = 8.0, 2.1 Hz, 1H), 2.27 (s, 1H), 2.16–2.04 (m, 1H),
1.94 (s, 3H), 1.67 (s, 3H), 1.61–1.53 (m, 1H), 1.34 (s, 3H), 1.29 (s,
3H), 1.17 (s, 3H). Compound 11c: 1H NMR (400 MHz, CDCl3) d:
7.44–7.37 (m, 5H), 6.27 (s, 1H), 5.65 (d, J = 2.1 Hz, 1H), 5.30 (ddd,
J = 12.1, 5.5, 2.3 Hz, 1H), 2.12 (s, 1H), 2.00 (s, 3H), 1.93–1.83 (m,
1H), 1.75 (s, 3H), 1.60 (s, 3H), 1.58–1.51 (m, 1H), 1.40 (s, 3H),
1.27 (s, 3H); 13C NMR (150 MHz, CDCl3) d: 205.5, 174.0, 170.7,
169.5, 134.0, 131.2, 129.8, 128.8, 127.7, 71.7, 68.6, 62.4, 51.6,
36.2, 33.5, 32.7, 28.7, 25.6, 21.0, 20.5; HRMS [MH]+ calcd for
C22H27O5, 371.1853; found, 371.1861. Compound 11d: 1H NMR
(400 MHz, CDCl3) d: 7.44–7.33 (m, 5H), 6.26 (s, 1H), 5.32 (d,
J = 3.6 Hz, 1H), 5.05 (ddd, J = 9.3, 7.2, 3.6 Hz, 1H), 2.25 (s, 1H),
2.09–2.02 (m, 1H), 2.07 (s, 3H), 1.71 (s, 3H), 1.55–1.50 (m, 1H),
1.54 (s, 3H), 1.34 (s, 3H), 1.14 (s, 3H); 13C NMR (150 MHz, CDCl3)
d: 207.0, 177.5, 169.7, 169.3, 135.3, 132.2, 129.6, 128.6, 127.6,
75.9, 72.1, 63.4, 50.6, 41.6, 33.6, 33.0, 29.7, 28.4, 23.7, 21.2, 20.6;
HRMS [MH]+ calcd for C22H27O5, 371.1853; found, 371.1856.
4.5. (3RS,3aSR,4RS,5SR,7aSR)-3a,7,7-Trimethyl-1-oxo-3-
phenyloctahydro-1H-indene-4,5-diyl diacetate (12c)
A solution of 11c (102.7.0 mg, 0.28 mmol) in MeOH (6 mL) was
hydrogenated in the presence of Pd–carbon (10 wt%, 50.5 mg) at
room temperature, and 1 atom for 21 h. Pd–carbon was filtered
off, and the filtrate was evaporated under reduced pressure to
leave an oil, which was purified by p-TLC on SiO2 with AcOEt–hex-
ane (1:3, v/v) to give 12c (89.9 mg, 87%) as a colorless oil. 1H NMR
(600 MHz, CDCl3) d: 7.33–7.29 (m, 2H), 7.28–7.24 (m, 1H), 7.14–
7.10 (m, 2H), 5.13 (ddd, J = 12.7, 4.4, 2.2 Hz, 1H), 4.71 (d,
J = 1.9 Hz, 1H), 3.23–3.18 (m, 1H), 2.82 (dd, J = 18.8, 12.0 Hz, 1H),
2.56 (ddd, J = 18.7, 10.4, 1.0 Hz, 1H), 2.14 (s, 3H), 1.97–1.89 (m,
2H), 1.86 (s, 3H), 1.55 (s, 3H), 1.53 (s, 3H), 1.33 (dd, J = 12.9,
4.4 Hz, 1H), 1.21 (s, 3H); 13C NMR (150 MHz, CDCl3) d: 213.6,
170.5, 169.0, 135.4, 129.0, 128.3, 127.6, 72.8, 68.9, 62.8, 50.7,
49.1, 42.3, 36.0, 33.7, 31.2, 29.4, 26.2, 21.7, 20.9; HRMS [MNa]+
calcd for C22H28O5Na, 395.1829; found, 395.1829.
4.6. (3RS,3aSR,4SR,5SR,7aSR)-3a,7,7-Trimethyl-1-oxo-3-
phenyloctahydro-1H-indene-4,5-diyl diacetate (12d)
A solution of 11d (135.7 mg, 0.37 mmol) in MeOH (6 mL) was
hydrogenated in the presence of Pd–carbon (10 wt%, 60.5 mg) at
room temperature, and 1 atom for 21 h. Pd–carbon was filtered
off, and the filtrate was evaporated under reduced pressure to
leave an oil, which was purified by p-TLC on SiO2 with AcOEt–hex-
ane (1:3, v/v) to give 12d (82.5 mg, 61%) as a colorless oil. 1H NMR
(500 MHz, CDCl3) d: 7.35–7.30 (m, 2H), 7.29–7.20 (m, 3H), 4.87–
4.80 (m, 1H), 4.24 (s, 1H), 3.23 (dd, J = 13.9, 8.2 Hz, 1H), 3.04 (dd,
J = 17.6, 13.9 Hz, 1H), 2.51 (ddd, J = 17.6, 8.2, 1.6 Hz, 1H), 2.15 (s,
1H), 2.07 (s, 3H), 2.02–1.95 (m, 1H), 1.99 (s, 3H), 1.58–1.48 (m,
1H), 1.56 (s, 3H), 1.28 (s, 3H), 1.19 (s, 3H); 13C NMR (150 MHz,
CDCl3) d: 216.7, 169.2, 168.2, 136.3, 128.4, 127.4, 75.5, 72.0, 62.8,
51.3, 46.7, 43.9, 39.3, 34.6, 32.1, 29.7, 28.4, 24.8, 21.7, 21.1; HRMS
[MNa]+ calcd for C22H28O5Na, 395.1829; found, 395.1834.
4.7. (1RS,6RS,7RS,7aSR)-4,4,7a-Trimethyl-1-phenyl-2,4,5,6,7,7a-
hexahydro-1H-indene-6,7-diol (13)
4.4. (3RS,3aSR,4RS,5RS,7aSR)-3a,7,7-Trimethyl-1-oxo-3-
phenyloctahydro-1H-indene-4,5-diyl diacetate (12a) and
(3RS,3aSR,4SR,5RS,7aSR)-3a,7,7-trimethyl-1-oxo-3-
phenyloctahydro-1H-indene-4,5-diyl diacetate (12b)
To a solution of 12a and 12b (935.0 mg, 2.5 mmol) in THF
(20 mL) was slowly added 9-BBN (0.5 M, 10 mL, 5.0 mmol). The
reaction mixture was stirred under argon atmosphere at room
temperature for 4 h, and then MeOH (10 mL) was slowly added un-
der stirring for 3 h. Removal of the solvent afforded a crude prod-
uct. To a solution of crude product in CH2Cl2 (10 mL) at 0 °C under
argon atmosphere were added pyridine (0.8 mL, 10 mmol) and
SOCl2 (0.4 mL, 5.0 mmol). The reaction mixture was stirred at room
temperature for 12 h, and then poured into ice-water. The organic
layer was separated, and the aqueous phase was extracted with
CH2Cl2. The whole was washed with 5% Na2CO3 and brine, and
dried over Na2SO4. Removal of the solvent afforded a crude prod-
uct, which was purified by flash chromatography on SiO2 with
A solution of 11a and 11b (34.0 mg, 0.09 mmol) in EtOH (2 mL)
was hydrogenated in the presence of Pd–alumina (5 wt%, 34.0 mg)
at room temperature, and 1 atom for 18 h. Pd–alumina was filtered
off, and the filtrate was evaporated under reduced pressure to
leave an oil, which was purified by p-TLC on SiO2 with Et2O–hex-
ane (1:1, v/v) to give a mixture of 12a and 12b (34.2 mg, 100%)
as a colorless oil. Compound 12a: 1H NMR (500 MHz, CDCl3) d:
7.32–7.19 (m, 5H), 5.37 (q, J = 4.0 Hz, 1H), 4.88 (d, J = 4.4 Hz, 1H),
2.99 (dd, J = 13.1, 8.1 Hz, 1H), 2.86 (dd, J = 18.8, 13.2 Hz, 1H), 2.56
(dd, J = 18.8, 8.2 Hz, 1H), 2.16 (s, 1H), 1.98 (s, 3H), 1.78 (dd,

Y. Nakai et al. / Bioorg. Med. Chem. 17 (2009) 1152–1157
1157
AcOEt–hexane (1:7, v/v) to give diacetoxyolefin. A 1 N aqueous
solution of K2CO3 (3 mL) was added to a solution of diacetoxyolefin
in MeOH (10 mL) and CH2Cl2 (2 mL) at room temperature, and the
mixture was stirred at room temperature for 86 h. After evapora-
tion of the solvent, the whole was made acidic by adding 2 N HCl
under ice cooling and was extracted with AcOEt. The extract was
washed with brine, dried over Na2SO4, and evaporated under re-
duced pressure to leave an oil, which was purified by flash chroma-
tography on SiO2 with AcOEt–hexane (1:4, v/v) to give 13
(128.6 mg, 19%, three steps from mixture of 12a and 12b) as a col-
orless amorphous substance. 1H NMR (600 MHz, CDCl3) d: 7.28–
7.16 (m, 5H), 5.65–5.59 (m, 1H), 3.88 (q, J = 3.6 Hz, 1H), 3.14 (dd,
J = 7.9, 1.7 Hz, 1H), 3.08 (d, J = 3.6 Hz, 1H), 2.92 (ddd, J = 16.8, 7.9,
1.7 Hz, 1H), 2.43–2.38 (m, 1H), 1.80 (dd, J = 14.6, 3.3 Hz, 1H), 1.51
(s, 3H), 1.33 (s, 3H), 1.27 (dd, J = 14.7, 3.6 Hz, 1H), 1.16 (s, 3H);
13C NMR (150 MHz, CDCl3) d: 154.9, 144.9, 128.4, 128.0, 126.5,
122.2, 72.2, 72.0, 57.0, 55.8, 43.3, 37.3, 33.0, 32.1, 30.9, 23.3; HRMS
[MNa]+ calcd for C18H24O2Na, 295.1668; found, 295.1665.
and the aqueous phase was extracted with CH2Cl2. The combined
organic extracts were washed with 5% Na2CO3 aq and brine, and
dried over Na2SO4. The solvent was evaporated under reduced
pressure to leave an oil, which was purified by p-TLC on SiO2 with
AcOEt–hexane (1:5, v/v) to give 14 (18.7 mg, 14%, five steps from
13) as a colorless amorphous substance: 1H NMR (600 MHz,
CDCl3) d: 7.30–7.25 (m, 2H), 7.25–7.19 (m, 1H), 7.17–7.12 (m,
2H), 5.07–5.06 (m, 1H), 3.80 (s, 1H), 2.85 (dd, J = 10.5, 8.3 Hz,
1H), 2.59 (d, J = 13.2 Hz, 1H), 2.42 (d, J = 13.2 Hz, 1H), 2.37–2.27
(m, 2H), 1.36 (s, 3H), 1.23 (s, 3H), 1.16 (s, 3H), 1.00 (s, 3H); 13C
NMR (150 MHz, CDCl3) d: 202.4, 169.6, 138.0, 128.6, 128.3,
127.0, 76.2, 70.9, 61.2, 51.3, 50.5, 50.0, 36.5, 30.1, 27.4, 24.3,
19.6, 17.3; HRMS [MH]+ calcd for C20H25O4, 329.1747; found,
329.1750.
Acknowledgments
This work was supported by National Institute of Health (NIH)
Grant (AI 072358) and The Skaggs Institute for Chemical Biology.
4.8. (1SR,3RS,3aRS,4RS,7aRS)-4-Acetoxy-1,7a-epoxy-3-phenyl-
3a,7,7-trimethyl-1,2,3a,4,7,7a-hexahydro-3H-inden-5-one (14)
References and notes
1. Johnson, E. A.; Bradshaw, M. Toxicon 2001, 39, 1703.
2. Schantz, E. J.; Johnson, E. A. Microbiol. Rev. 1992, 56, 80.
3. Simpson, L. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 167.
4. Dickerson, T. J.; Janda, K. D. ACS Chem. Biol. 2006, 1, 359.
5. Madsen, J. M. Clin. Lab. Med. 2001, 21, 593.
6. Christopher, G. W.; Cieslak, T. J.; Pavlin, J. A.; Eitzen, E. M., Jr. JAMA 1997, 278,
412.
To a solution of 13 (115.9 mg, 0.43 mmol) in CH2Cl2 (5 mL) at
À78 °C under argon atmosphere were added diisopropylethyl-
amine (DIPEA) (0.1 mL, 0.86 mmol) and t-butyldimethylsilyl tri-
fluoromethanesulfonate (TBSOTf) (98%, 0.2 mL, 0.86 mmol). The
reaction mixture was stirred at À78 °C for 2.5 h. After addition
of H2O, the whole was extracted with CH2Cl2. The extract was
washed with brine, dried over Na2SO4, and evaporated under re-
duced pressure to leave an oil, which was purified by p-TLC on
SiO2 with AcOEt–hexane (1:9, v/v) to give 7-TBS-protected prod-
uct, which was desired (30.6 mg, 19%) as a colorless oil, 6-TBS-
protected product (69.0 mg, 42%) as a colorless oil, and unreacted
13 (25.8 mg, 22%) as a colorless oil in the order of elution. To a
solution of 7-TBS-protected product (30.6 mg, 0.08 mmol) in
CH2Cl2 (3 mL) at room temperature under argon atmosphere
was added Dess–Martin reagent (97%, 41.6 mg, 0.1 mmol). The
reaction mixture was stirred at room temperature for 13 h. After
addition of saturated NaHCO3 aq, the whole was extracted with
CH2Cl2. The extract was washed with brine, dried over Na2SO4,
and evaporated under reduced pressure to leave an oil. To a solu-
tion of crude oil in THF (2 mL) at room temperature under argon
atmosphere was added TBAF (1.0 M, 0.1 mL, 0.12 mmol). The
reaction mixture was stirred at room temperature for 2.5 h. Re-
moval of the solvent afforded a crude product, which was purified
by p-TLC on SiO2 with AcOEt–hexane (1:9, v/v) to give 7-hydrox-
yketone (16.0 mg, 75%, two steps from 7-TBS protected product)
as a colorless oil. To a solution of 7-hydroxyketone (16.0 mg,
0.06 mmol) in pyridine (0.5 mL) at room temperature were added
Ac2O (0.25 mL) and catalytic amount of 4-dimethylaminopyridine
(DMAP). The reaction mixture was stirred at room temperature
for 24 h. Removal of the solvent afforded a crude product, which
was purified by p-TLC on SiO2 with AcOEt–hexane (1:7, v/v) to
give 7-acetoxyketone (17.8 mg, 96%) as a colorless oil. mCPBA
(77%, 15.3 mg, 0.07 mmol) was added to a solution of 7-acetoxyk-
etone (17.8 mg, 0.06 mmol) in CH2Cl2 (1.0 mL) at room tempera-
ture, and the mixture was stirred at room temperature for 6 h.
Na2SO3 aq (5%) was added and the resulting heterogeneous
mixture was vigorously stirred. The organic layer was separated,
7. Zilinskas, R. A. JAMA 1997, 278, 418.
ˇ
8. (a) Capková, K.; Yoneda, Y.; Dickerson, T. J.; Janda, K. D. Bioorg. Med. Chem. Lett.
2007, 17, 6463; (b) Boldt, G. E.; Eubanks, L. M.; Janda, K. D. Chem. Commun.
2006, 3063; (c) Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Org. Lett. 2006, 8, 1729;
(d) Eubanks, L. M.; Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.; 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; (e) Silvaggi, N. R.; Boldt, G. E.; Hixon, M. S.; Kennedy, J. P.; Tzipori, S.;
Janda, K. D.; Allen, K. N. ACS Chem. Biol. 2007, 14, 533.
9. Nakatani, M. Heterocycles 1999, 50, 595.
10. Carpinella, C.; Ferrayoli, C.; Valladares, G.; Defago, M.; Palacios, S. Biosci.
Biotechnol. Biochem. 2002, 66, 1731.
11. Xie, Y. S.; Fields, P. G.; Isman, M. B.; Chen, W. K.; Zhang, X. J. Stored. Prod. Res.
1995, 31, 259.
12. (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;
(c) Zhou, J. B.; Minami, Y.; Yagi, F.; Tadera, K.; Nakatani, M. Heterocycles 1997,
45, 1781.
13. (a) Li, P.; Zou, J.; Miao, W.; Ding, F.; Meng, J.; Jai, G.; Li, J.; Ye, H.; He, X.
Zhongcaoyao 1982, 13, 28; (b) Zou, J.; Miao, W.; Ding, F.; Meng, J.; Ye, H.; Jia, G.;
He, X.; Sun, G.; Li, P. J. Trad. Chin. Med. 1985, 5, 29.
14. Wender, P. A.; Verma, V. A.; Paxton, T. J.; Pillow, T. H. Acc. Chem. Res. 2008, 41,
40.
15. Paquette, L. A.; Hong, F. T. J. Org. Chem. 2003, 68, 6905.
16. Fernandez-Mateos, A.; Martin de la Nava, E. M.; Pascual Coca, G.; Rubio
Gonzalez, R.; Ramos Silvo, A. I.; Simmonds, M. S. J.; Blaney, W. M. J. Org. Chem.
1998, 63, 9440.
17. Fernandez-Mateos, A.; Martin de la Nava, E. M.; Rabanedo Clemente, R.; Rubio
Gonzalez, R.; Simmonds, M. S. J. Tetrahedron 2005, 61, 12264.
18. Fernandez-Mateos, A.; Martin de la Nava, E. M.; Rubio Gonzalez, R. Tetrahedron
2001, 57, 1049.
19. Fernandez-Mateos, A.; Pascual Coca, G.; Rubio Gonzalez, R.; Tapia Hernandez,
C. J. Org. Chem. 1996, 61, 9097.
20. Fernandez-Mateos, A.; Lopez Barba, A. M. J. Org. Chem. 1995, 60, 3580.
21. Fernandez-Mateos, A.; Lopez Barba, A.; Pascual Coca, G.; Rubio Gonzalez, R.;
Tapia Hernandez, C. Synthesis 1997, 1381.
22. Fernandez-Mateos, A.; Mateos Buron, L.; Martin de la Nava, E. M.; Rubio
Gonzalez, R. J. Org. Chem. 2003, 68, 3585.
23. Fernandez-Mateos, A.; Lopez Barba, A. M.; Martin De La Nava, E.;
Pascual Coca, G.; Perez Alonso, J. J.; Ramos Silvo, A. I. Tetrahedron
1997, 53, 14131.