Synthesis of (-)-callicarpenal, a potent arthropod repellent

Article abstract of DOI:10.1016/j.tet.2011.02.078

Callicarpenal (1), a natural terpenoid isolated from American beautyberry (Callicarpa americana), has shown significant repellent activities against mosquitoes, ticks, and imported fire ants. Here we report our efficient synthetic approach to this natural product, and preliminary results of the mosquito biting-deterrent effects of callicarpenal as well as its synthetic precursors and related C₈-epimers. The synthetic strategy allows rapid access to various epimers and analogues of the natural product that can be used to explore its structure-activity relationship and optimize its biological properties.

Full text of DOI:10.1016/j.tet.2011.02.078

Tetrahedron 67 (2011) 3023e3029
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of (ꢀ)-callicarpenal, a potent arthropod repellent
,
Taotao Ling a, Jing Xu a, Ryan Smith a, Abbas Ali b, Charles L. Cantrell c, Emmanuel A. Theodorakis a
*
a Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
b National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA
c United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Callicarpenal (1), a natural terpenoid isolated from American beautyberry (Callicarpa americana), has
shown significant repellent activities against mosquitoes, ticks, and imported fire ants. Here we report
our efficient synthetic approach to this natural product, and preliminary results of the mosquito biting-
deterrent effects of callicarpenal as well as its synthetic precursors and related C8-epimers. The synthetic
strategy allows rapid access to various epimers and analogues of the natural product that can be used to
explore its structureeactivity relationship and optimize its biological properties.
Received 15 December 2010
Received in revised form 24 February 2011
Accepted 25 February 2011
Available online 4 March 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Arthropod repellent
Arthropod-borne disease
Natural products
Callicarpenal
Asymmetric synthesis
1. Introduction
new arthropod repellents, led to the evaluation of plants of the
genus Beautyberry (Callicarpa).8 Interestingly, freshly crushed
Blood-feeding arthropods transmit many of the world’s most
dangerous diseases, such as malaria1,2a and dengue fever.2b An es-
timated 243 million malaria cases led to approximately 863,000
deaths in 2008,2a while nearly one third of the world’s population is
at risk of dengue fever.2b Very recently, an outbreak of infection
cases caused by tick biting has been reported resulting in a fatality
rate as high as 30% in some area of central China.3 Research on the
cause of this infection led to the isolation and identification of
a new type of Bunyavirus. As a tick-borne virus, this new species of
Bunyavirus family has been considered as a new emerging deadly
virus.3 Consequently, aside from the urgent need of study for new
antiplasmodium/antivirus agents, the development of new safe and
effective repellents against blood-feeding arthropods is in high
demand.4
American beautyberry leaves have been used in ethnomedicine as
a traditional biting-insect repellent in certain southern states of the
United States.9 Inspired by this information, Cantrell et al. studied
the chemical origins of the bioactivities of the beautyberry extracts
against mosquitoes,9 ticks,10 and imported fire ants.11 These in-
vestigations led to the isolation of three potent mosquito-repellent
The best known insect repellent, due to its potency and simple
synthesis, is N,N-diethyl-3-methylbenzamide (DEET, 4), a com-
pound developed by the US Army in 1946.1 Although it is consid-
ered as safe, extensive use of this compound can lead to skin
irritation.5 Picaridin (KBR3023, 5) is another synthetic repellent
that has been reported to be as effective as DEET but not as toxic in
humans.6 On the other hand, p-menthane-3,8-diol (PMD, 6), the
main component in citrus/eucalyptus solutions, is the best repre-
sentative of natural product-repellents.7 The growing demand for
* Corresponding author. Tel.: þ1 858 822 0456; fax: þ1 858 822 0386; e-mail
address: etheodor@ucsd.edu (E.A. Theodorakis).
Fig. 1. Structures of various insect repellents.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.02.078

3024
T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
compounds: callicarpenal (1) and intermedeol (2), from the
American plant, and spathulenol (3), from the Japanese plant
(Fig. 1).
The low isolation yield of callicarpenal (0.05e0.15% from dry
biomass) together with the tedious bulk isolation and purification
techniques, restrict substantially the availability of this compound
from natural sources. With this in mind, we report here an efficient
stereoselective synthesis of (ꢀ)-callicarpenal. We also report initial
mosquito deterrency studies of this natural product and various
synthetic derivatives.12
electrophilic C-4 carbonyl group of (ꢀ)-7 was selectively protected
with 1.2 equiv of ethylene glycol to form the corresponding C-4
ketal.14 The C-8 enone was then reduced under lithium/ammonia
conditions and the resulting enolate was alkylated in situ with allyl
bromide to afford ketone 8 as a single isomer (70% over two steps).
Conversion of 8 to silyl ether 10 was achieved in three steps: (a)
ozonolysis of the terminal alkene followed by reduction of both
carbonyl groups; (b) selective silylation of the primary alcohol; and
(c) oxidation of the remaining C-8 hydroxyl group. The latter step
was performed using catalytic amount of TPAP (3.5 mol %) and
NMO as the co-oxidant (88% for three steps).15 We also attempted
to replace the ozonolysis reaction with a sequence of alkene dihy-
droxylation followed by oxidative cleavage of the resulting 1,2-diol.
However, treatment of 8 with OsO4/NMO gave rise to hemiacetal 9
complicating subsequent functionalization strategies. The chemical
structure of 9 was unambiguously confirmed via a single crystal
X-ray analysis.16
2. Results and discussion
Our synthesis of (ꢀ)-1 departed from the readily available en-
antiomeric pure diketone (ꢀ)-7 (ee >95%)13 (Scheme 1). The more
We then turned our attention to the installation of the C-8
methyl group. To this end, ketone 10 was converted to alkene 11
under standard Wittig conditions (91% yield). Hydrogenation of 11
under the standard Pd/C-catalyzed conditions proceeded pre-
dominantly from the sterically more accessible bottom face of the
alkene forming the C8-epi-12 as the major product (94%, dr¼10:1.)
The stereochemistry of the newly formed C-8 center was confirmed
initially by comparing the NMR data of both epimers to those
reported in the literature17 and ultimately by a single crystal X-ray
analysis16 of the undesired fully deprotected epimer C8-epi-13. In
order to produce the desired C-8 epimer, alkene 11 was initially
desilylated with TBAF and subsequently reduced under Pd/C con-
ditions. This sequence afforded the desired compound 13 as the
major product (97%, dr¼8:1). Importantly, treatment of 11 with
Crabtree’s catalyst18 (3.5 mol %) under H2 atmosphere (70 psi) gave
even higher diastereoselectivity (dr >99:1). Obviously, the better
interaction between the iridium compound and the free alcohol
exclusively delivered the hydrogen from the same face of the
molecule as the alcohol.19 The acetonide was subsequently re-
moved under acidic conditions to form ketone 13 that, after Wittig
olefination, produced alkene 14 (92% for two steps). Isomerization
of the exocyclic double bond of 14 was achieved with RuCl320 under
refluxing ethanolic conditions to form the desired endo-alkene 15
Scheme 1. Reagents and conditions: (a) (CH2OH)2 (1.2 equiv), p-TsOH, PhH, 80 ꢁC, 24 h
(90%); (b) Li/NH3, THF, ꢀ78 to ꢀ30 ꢁC, 2 h, then CH2]CHCH2Br, 2 h (78%); (c) OsO4
(1 mol %), NMO, acetone/H2O (3:1), rt, 12 h (95%); (d) O3, THF, ꢀ78 ꢁC, then PPh3, 2 h,
then 3.0 equiv NaBH4, 0 ꢁC, 2 h; (e) 1.0 equiv TIPSOTf, 2,6-lutidine, CH2Cl2, ꢀ80 ꢁC, 1 h;
(f) TPAP (3.5 mol %), NMO, CH2Cl2, 18 h (88% for three steps); (g) CH3PPh3Br, NaHMDS,
THF, 0 ꢁC to rt, 16 h (91%); (h) Pd/C, H2, rt, 12 h (99%, dr¼10:1); (i) TBAF, THF, 15 h (94%);
(j) 0.1 N HCl, THF, 8 h (97% for 13, 93% for C8-epi-13); (k) H2, Crabtree’s catalyst ([Ir
(COD)(PCy3)(Py)]PF6, 3.5 mol %), CH2Cl2, 70 psi, 15 h (94%, dr >99:1); (l) CH3PPh3Br,
NaHMDS, THF, 0 ꢁC to rt, 18 h (95%); (m) RuCl3$H2O, EtOH, reflux, 18 h (97%); (n) TPAP
(20 mol %), NMO, CH2Cl2, 15 h (76%).
Scheme 2. Reagents and conditions: (a) 0.1 N HCl, THF, 8 h (93%); (b) CH3PPh3Br,
NaHMDS, THF, 0 ꢁC to rt, 18 h (91%); (c) RuCl3$H2O, EtOH, reflux, 18 h (89%); (d) TPAP
(20 mol %), NMO, CH2Cl2, 15 h (90%).

T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
3025
Table 1
the bioactivity. These observations suggest that it is possible to
further optimize the structureeactivity of this natural product and
develop exclusively synthetic analogues of callicarpenal with
a simpler chemical motif, less lengthy synthesis, and optimized
mosquito biting deterrency.
Mosquito biting-deterrent effects of (ꢀ)-callicarpenal (1) and its analogues 13, 14,
and 15 against 7e10 days old adult females of Ae. aegypti
Treatment
(n¼100)
DEET
Amount applied
(nmoles/cm2)
Mean proportion
not biting (SE)
25
25
25
25
25
d
0.91 (0.029)a
0.72 (0.045)b
0.61 (0.049)c
0.61 (0.049)c
0.64 (0.048)c
0.22 (0.041)d
(ꢀ)-Callicarpenal (1)
3. Conclusions
13
14
We have developed an efficient enantioselective synthesis of
(ꢀ)-callicarpenal, a promising arthropod repellent. Key to the
synthesis is the stereoselective reduction of C-8 alkene of 11 that
depending on the conditions can deliver either epimer in excellent
yield and diastereoselectivity. The synthesis of (ꢀ)-callicarpenal
proceeds in 12 steps and 36% overall yield from diketone (ꢀ)-7. The
synthetic strategy utilizes readily available materials and reagents
making the overall approach amenable to scale-up. As such, it al-
lows access to this natural product for further biological in-
vestigation and development. Moreover, mosquito biting-deterrent
studies with callicarpenal and selected analogues suggest that the
chemical structure of the parent molecule can be readily modified
to improve its bioactivity.
15
Ethanol
n¼Number of females tested against each treatment.
SE¼Standard error of the mean.
Means within a column not followed by the same letter are significantly different
(P<0.05, DMRT).
in 97% yield.12e,21 Oxidation of 15 was achieved with TPAP and NMO
and produced callicarpenal (ꢀ)-1 in 76% yield. The analytical and
spectroscopic data (see the Experimental section) of our synthetic
(ꢀ)-1 perfectly matched the reported data of the natural product.22
To evaluate the effect of the C8 stereocenter on the insect-re-
pellent activities of callicarpenal, we synthesized C8-epi-(ꢀ)-1.
Scheme 2 summarizes our approach.
The aldehyde functionality of (ꢀ)-callicarpenal (1) could give rise
to long-term stability issues, thereby limiting its applications. To
overcome such potential limitations we sought to compare its bio-
activity to that of its synthetic precursors. Along these lines, ana-
logues 13, 14, and 15 were chosen due to their availability and
structural similarity with 1. All these compounds were evaluated for
mosquito biting-deterrent effects against 7e10 days old adult fe-
males of Aedes aegypti (Table 1). Analogues were tested side-by-side
against DEETand 1. All of the compounds tested were more effective
than ethanolcontrol at 25nmol/cm2. DEET was moreeffectivethan 1
and all of its analogues 13, 14, and 15. Reduction of the aldehyde
functionality of 1 to the corresponding alcohol decreased consid-
erably the activity of the natural product. On the other hand, isom-
erization of the C4 double bond to the C-3,4 endocyclic position
appears to improve marginally the compound potency. These results
are in agreement with previous observations.23
4. Experimental section
4.1. General experimental procedures
Unless indicated, all commercial reagents were used as received
without further purification. All non-aqueous reactions werecarried
out under argon atmosphere using dry glassware that had been
flame-dried under a stream of argon unless otherwise noted. THF
was dried over sodium/benzophenone, dichloromethane and MeOH
ꢀ
over CaH2. Et3N was dry distilled from flame dried 4 A powdered
molecular sieves. Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230e400 mesh) using hexane/ether
mixtures of increasing polarity. The progress of all the reactions was
monitored by thin-layer chromatography (TLC) using glass plates
precoated with silica gel-60 F254 to a thickness of 0.5 mm (Merck).
13C NMR and 1H NMR spectra were recorded on either a 400 MHz
Varian instrument or a 500 MHz JEOL instrument. CDCl3 was treated
The C-8 epimers of callicarpenal and some callicarpenal ana-
logues, synthesized above from the trans-epimer of compound 12,
were also evaluated for mosquito biting-deterrent effects against
7e10 days old adult females of Ae. aegypti (Table 2). Analogues C8-
epi-13, C8-epi-14, C8-epi-15, and C8-epi-(ꢀ)-1 were tested side-by-
side against 1 and ethanol control. Again, all of the compounds
tested were more effective than ethanol control at 25 nM/cm2.
Callicarpenal (1) was more effective than C8-epi-13, C8-epi-14, and
C8-epi-15; while C8-epi-(ꢀ)-1 was as effective as 1.
with flame dried K2CO3, chemical shifts (d) are quoted in parts per
million (ppm) referenced to the appropriate residual solvent peak
(CHCl3), with the abbreviations s, br s, d, t, q, and m denoting singlet,
broad singlet, doublet, triplet, quartet, and multiplet, respectively.
J¼coupling constants given in Hertz (Hz). High resolution Mass
spectra (HRMS) were recorded on a trisector WG AutoSpecQ spec-
trometer. Optical rotation data were collected on a Jasco P-1010
polarimeter using HPLC grade CHCl3 (dried over molecular sieves).
Evaluation of the data shown in Tables 1 and 2 suggests that the
relative stereochemistry at C8 does not play a significant role in
determining the biological activity of the different epimers. More-
over, the functionalities at the C4 center do not appear to influence
4.1.1. (R)-5,8a-Dimethyl-3,4,8,8a-tetrahydronaphthalene-1,6
(2H,7H)-dione ((ꢀ)-7). To
a solution of 2-methyl-1,3-cyclo-
hexadione (0.90 g, 7.17 mmol) in ethyl acetate (50 ml) were added
triethylamine (1.30 ml, 9.32 mmol) and ethyl vinyl ketone (0.78 ml,
7.89 mmol). The reaction mixture was heated at 70 ꢁC for 10 h and
then cooled to 25 ꢁC. The solvent was removed under reduced
pressure and the resulting crude material was chromatographed
(silica, 10% ether in hexanes) to yield the corresponding triketone
(1.45 g, 6.81 mmol, 95%). A solution of the triketone (1.45 g,
Table 2
Mosquito biting-deterrent effects of (ꢀ)-callicarpenal (1) and its analogues C8-epi-
13, C8-epi-14, C8-epi-15, and C8-epi-(ꢀ)-1 against 7e10 days old adult females of Ae.
aegypti
Treatment
(n¼100)
Amount applied
(nmoles/cm2)
Mean proportion
not biting (SE)
6.81 mmol),
L-phenylalanine (1.13 g, 6.81 mmol), and D-cam-
(ꢀ)-Callicarpenal (1)
25
25
25
25
25
d
0.78 (0.048)a
0.56 (0.057)b
0.56 (0.057)b
0.61 (0.056)b
0.76 (0.049)a
0.23 (0.049)c
C8-epi-13
phorsulfonic acid (0.79 g, 3.40 mmol) in DMF (100 ml) was stirred
at rt under argon atmosphere overnight. Then the mixture was
heated at 30 ꢁC for 24 h, and the temperature was raised in 10 ꢁC
intervals every 24 h during 4 days. After the mixture was stirred at
70 ꢁC for 24 h, the oil bath was removed in order to let the reaction
mixture cool down to rt. The resulting solution was then poured
into cold aqueous NaHCO3, extracted with ether (2ꢂ50 ml), then
dried over MgSO4. Evaporation of ether followed by column
C8-epi-14
C8-epi-15
C8-epi-(ꢀ)-1
Ethanol
n¼Number of females tested against each treatment.
SE¼Standard error of the mean.
Means within a column not followed by the same letter are significantly different
(P<0.05, DMRT).

3026
T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
chromatography (silica, 0e20% ether in hexanes) of the residue
afforded the crude enone 7 as a viscous oil, which crystallized on
standing at ꢀ30 ꢁC. Recrystallization from n-hexane in an ice bath
gave (ꢀ)-7 (1.05 g, 5.38 mmol, 79%) as a colorless long needles; mp:
added 4-methylmorpholine-N-oxide (17.5 g, 0.149 mol) followed by
tetrapropylammonium perruthenate (0.50 g, 1.42 mmol). The re-
action mixture was stirred at 25 ꢁC for 18 h, then directly concen-
trated and subjected to flash chromatography (silica, 0e5% ether in
hexanes) to afford ketone 10 (21.5 g, 0.049 mol, 98%) as a colorless
46e48 ꢁC; Rf¼0.46 (35% ether in hexanes); [
a]
þ124.0 (c 1.0,
D25
2952, 1709, 1665, 1610, 1352, 1008; 1H NMR
liquid; Rf¼0.58 (25% ether in hexanes); [
a
]
ꢀ62.3 (c 0.7, CH2Cl2); IR
D25
benzene); IR (film):
n
(400 MHz, CDCl3):
d
2.92e2.81 (m, 1H), 2.73e2.64 (m, 1H),
(film):
n
2939, 2865, 1703, 1461, 1098, 884, 682; 1H NMR (500 MHz,
2.55e2.37 (m, 4H), 2.18e2.02 (m, 4H), 1.81 (d, J¼1.2 Hz, 3H), 1.42 (s,
CDCl3):
d
3.96e3.92 (m, 2H), 3.90e3.84 (m, 2H), 3.64 (m,1H), 3.57 (m,
3H); 13C NMR (100 MHz, CDCl3):
d
212.0, 197.5, 158.1, 130.7, 50.6,
1H), 2.48 (m, 1H), 2.39 (m, 1H), 2.16 (dd, J¼12.0, 3.0 Hz, 1H), 2.07 (m,
37.3, 33.3, 29.5, 27.2, 23.3, 21.5, 11.2; HRMS (ESI): calcd for C12H17O2
1H),1.99 (m,1H),1.71e1.40 (m, 8H, overlapped with water peak),1.16
[MþH]þ 193.1228, found 193.1224.
(s, 3H), 1.04 (m, 21H), 1.02 (s, 3H); 13C NMR (125 MHz, CDCl3):
d (C8
peak is too weak to observe) 112.8, 65.0, 64.8, 59.9, 49.4, 44.6, 42.3,
41.0, 34.7, 30.1, 28.7, 22.6, 21.8, 21.6, 17.9 (6C), 16.3, 11.8 (3C); HRMS
(ESI): calcd for C25H46O4SiNa [MþNa]þ 461.3066, found 461.3068.
4.1.2. (4a0R,50R,8a’R)-50-Allyl-50,8a0-dimethylhexahydro-20H-spiro
[[1,3]dioxolane-2,10-naphthalen]-60(70H)-one (8). A solution of enone
(ꢀ)-7 (1.05 g, 5.38 mmol) and p-toluenesulfonic acid (0.20 g,
1.08 mmol) in benzene (100 ml) was treated with ethylene glycol
(0.36 ml, 6.46 mmol), then heated at 80 ꢁC for 24 h under an atmo-
sphere ofargon. Aftercooling inanice bath, the resultingsolutionwas
poured into aqueous NaHCO3 and the mixture was extracted with
ether (2ꢂ50 ml). The combined extracts were washed with water
(20 ml) and brine (20 ml) and dried over MgSO4. Evaporation of the
solvent followed by flash column chromatography (silica, 0e10%
ether in hexanes) yielded the corresponding ketal (1.1 g, 4.84 mmol,
90%) as a colorless liquid. To a solution of lithium (0.15 g, 21.15 mmol)
in liquid ammonia (100 ml) at ꢀ78 ꢁC was added dropwise a solution
of the ketal obtained above (0.98 g, 4.23 mmol) in THF (2 ml). After
4.1.4. (2-((4a0R,50R,8a0R)-50,8a0-Dimethyl-60-methyleneoctahydro-
20H-spiro[[1,3]dioxolane-2,10-naphthalen]-50-yl)ethoxy)
triisopro-
pylsilane (11). A solution of methyltriphenylphosphonium bromide
(37.0 g, 0.068 mol) in THF (400 ml) was treated dropwise with so-
dium bis(trimethylsilyl)amide (90.0 ml of a 1.0 M solution in THF,
0.09 mol) at 0 ꢁC and stirred at this temperature for 30 min. The
resulting orange solution was treated dropwise with a solution of
ketone 10 (30.0 g, 0.068 mmol) in THF (50 ml) at 0 ꢁC. After stirring
at 65 ꢁC for 15 h, the mixture was quenched with saturated aqueous
NaHCO3 (500 ml), diluted with ether (500 ml), washed with brine
(3ꢂ300 ml). The organic solution was dried over MgSO4, concen-
trated, then subjected to flash chromatography (silica, 0e5% ether
in hexanes) to afford alkene 11 (27.0 g, 0.062 mol, 91%) as a color-
reflux for 1 h at ꢀ30 ꢁC, water (76.2
ml, 4.23 mmol) was added drop-
wise. The solution was refluxed for another hour, then allyl bromide
(1.83 ml, 21.15 mmol) was added as rapidly as possible, and the re-
action mixture was heated at reflux for 2 h. The ammonia was evap-
orated and the reaction was quenched with saturated NH4Cl solution
(20 ml). The resulting mixture was then extracted with ether
(3ꢂ30 ml) and the organic solutions were combined, dried over
MgSO4, concentrated, and subjected to column chromatography
(silica, 0e10% ether in hexanes) to afford ketone 8 (0.91 g, 3.3 mmol,
less liquid; Rf¼0.74 (25% ether in hexanes); [
a
]
D25
þ75.1 (c 1.0,
CH2Cl2); IR (film):
n
d
2939, 2865, 1461, 1085, 1072, 884; 1H NMR
(500 MHz, CDCl3):
3.82 (m, 1H), 3.72 (m, 1H), 3.58 (m, 1H), 2.33 (m, 1H), 2.14 (m, 1H),
4.74 (s, 1H), 4.69 (s, 1H), 3.92e3.88 (m, 3H),
1.83e1.71 (m, 2H), 1.67e1.31 (m, 11H), 1.24 (s, 1H), 1.11 (s, 3H), 1.04
(m, 18H), 0.96 (s, 3H); 13C NMR (125 MHz, CDCl3):
d 154.4, 113.3,
106.3, 65.1, 64.7, 59.5, 44.9, 43.3, 41.9, 41.5, 31.4, 30.2, 29.4, 23.7,
22.8, 21.4, 18.0 (6C), 17.7, 11.9 (3C); HRMS (ESI): calcd for
C26H48O3SiCs [MþCs]þ 569.2429, found 569.2432.
78%)as a colorless liquid; Rf¼0.63(50% ether in hexanes); [
a
]
ꢀ64.4
D25
(c 1.0, CH2Cl2); IR (film):
n
d
2946, 2872,1703, 1441, 1186, 1045, 910; 1H
NMR (500 MHz, CDCl3):
5.66 (m,1H), 5.03e4.96 (m, 2H), 3.96e3.83
(m, 4H), 2.57e2.50 (m, 2H), 2.33 (m, 1H), 2.13 (m, 1H), 2.01e1.90 (m,
2H), 1.70e1.31 (m, 7H), 1.20 (s, 3H), 1.03 (s, 3H); 13C NMR (125 MHz,
CDCl3): 215.8,135.2,117.3,112.8, 65.3, 64.8, 50.9, 44.1, 42.6, 42.5, 35.0,
30.3, 28.8, 22.6, 21.7, 21.3, 16.3; HRMS (ESI): calcd for C17H26O3Na
[MþNa]þ 301.3814, found 301.3827.
4.1.5. 2-((4a0R,50S,60R,8a0R)-50,60,8a0-Trimethyloctahydro-20H-spiro
[[1,3]dioxolane-2,10-naphthalen]-50-yl)ethanol (12). Olefin 11 (1.1 g,
2.52 mmol) in THF (20 ml) and was treated with TBAF (1.0 M in THF,
7.56 ml, 7.56 mmol) at 25 ꢁC for 15 h. Then the reaction was
quenched with water (50 ml) and extracted with ether (3ꢂ50 ml).
The organic layers were dried over MgSO4, concentrated and
chromatographed (silica, 10% ether in hexanes) to afford the cor-
responding alcohol (0.664 g, 2.37 mmol, 94%). This alcohol (0.30 g,
1.07 mmol) and the Crabtree’s catalyst ([Ir(COD)(PCy3)(Py)]PF6,
30 mg, 0.037 mmol) in 30 ml of CH2Cl2 was hydrogenated in a Parr
apparatus (70 psi) for 15 h at 70 psi. The catalyst was then removed
by a short silica pad to afford the reduced product 12 (286 mg,
1.01 mmol) as a single diastereomer at the C8 center (dr >99:1,
4.1.3. (4a0R,50R,8a0R)-50,8a0-Dimethyl-50-(2-((triisopropylsilyl) oxy)
ethyl)hexahydro-20H spiro[[1,3]dioxolane-2,10-naphthalen]-60(70H)-
one (10). Asolutionofketone8(12.0 g, 0.043 mol)inTHF(50 ml)was
exposed to ozone at ꢀ78 ꢁC until the starting material was consumed.
The resulting mixture was flushed with argon, treated with PPh3
(22.5 g, 0.086 mol) and allowed to warm up to 25 ꢁC. After stirring for
2 h, NaBH4 (4.88 g, 0.129 mol) was added to the above reaction mix-
ture at 0 ꢁC, followed by MeOH (5 ml), then the reaction mixture was
allowed to slowly warm upto 25 ꢁC and stirred at this temperature for
2 h, then quenched with saturated brine (100 ml). The organic layer
was extracted with ether (3ꢂ100 ml), combined and dried over
MgSO4, concentrated, and subjected to flash chromatography (silica,
50% ether in hexanes) to afford the corresponding diol (11.2 g,
0.039 mol, 92%). A solution of the above diol in CH2Cl2 (150 ml) was
treated with 2,6-lutidine (6.81 ml, 0.058 mol) and triisopropylsilyl
trifluoromethanesulfonate (12.6 ml, 0.047 mmol) at ꢀ78 ꢁC. The re-
action mixture was stirred for 1 h and then diluted with saturated
aqueous NaHCO3 (100 ml) and extracted with ether (3ꢂ100 ml). The
combined organic extracts were dried over MgSO4, concentrated, and
subjected to flash chromatography (silica, 0e20% ether in hexanes) to
afford the corresponding secondary alcohol (16.9 g, 0.038 mol, 98%).
A solution of above alcohol (22 g, 0.0499 mol) in CH2Cl2 (200 ml) was
D25
94%); [
a
]
þ52.1 (c 1.0, CH2Cl2); IR (film):
n
3382, 2939, 2872,1710,
3.93e3.88 (m,
1448, 1387, 1139, 1051; 1H NMR (500 MHz, CDCl3)
d
3H), 3.83e3.79 (m, 1H), 3.69e3.58 (m, 2H), 1.68e1.29 (m, 15H), 1.02
(s, 3H), 0.83 (d, J¼6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz,
CDCl3)
d 113.4, 65.1, 64.6, 58.1, 44.4, 43.4, 40.5, 38.4, 37.1, 30.2, 30.0,
26.7, 22.7, 20.4, 17.6, 16.9, 16.0; HRMS (ESI): calcd for C17H31O3
[MþH]þ 283.2275, found 283.2273.
4.1.6. (4aR,5S,6R,8aR)-5-(2-Hydroxyethyl)-5,6,8a-trimethylocta hy-
dronaphthalen-1(2H)-one (13). To a solution of ketal 12 (10.0 g,
0.035 mol) in THF (100 ml) was added hydrochloric acid (10.0 ml,
0.1 N). The reaction was stirred at rt for 8 h, then neutralized with
saturated aqueous NaHCO3 (150 ml). The two layers were separated
and the aqueous layer was extracted with ether (3ꢂ200 ml). The
organic solutions were combined, dried over MgSO4, concentrated,

T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
3027
and subjected to flash chromatography (silica, 10e50% ether in
hexanes) to afford hydroxy ketone 13 (8.10 g, 0.034 mmol, 97%) as
treated with TBAF (1.0 M in THF, 0.75 ml, 0.75 mmol) at 25 ꢁC for
12 h. Then the reaction was quenched with water (5 ml) and
extracted with ether (3ꢂ10 ml). The organic layers were dried over
MgSO4, concentrated, and chromatographed (silica, 10% ether in
hexanes) to afford the corresponding alcohol (67 mg, 0.24 mmol,
a white solid; Rf¼0.25 (50% ether in hexanes); [
benzene); IR (film): 3441, 2938, 1702, 1455, 1381, 1257, 1135, 1027,
952; 1H NMR (500 MHz, CDCl3):
3.40e3.70 (m, 2H), 2.01e2.60 (m,
3H), 1.05e1.95 (m, 12H), 1.12 (s, 3H), 0.85 (d, 3H, J¼4.80 Hz), 0.81 (s,
3H); 13C NMR (125 MHz, CDCl3):
215.8, 57.9, 49.3, 48.9, 40.6, 39.6,
a
]
þ14.5 (c 1.0,
D25
n
d
94%). Colorless liquid; Rf¼0.33 (silica, 70% ether in hexanes); [
þ16.8 (c 2.2, CH2Cl2); IR (film) 3355, 2952, 2878, 1454, 1381, 1186,
1031, 937; 1H NMR (500 MHz, CDCl3)
3.94 (m, 3H), 3.81 (m, 1H),
3.68 (m, 2H), 1.89e1.22 (m, 15H), 1.07 (s, 3H), 0.98 (d, J¼7.0 Hz, 3H),
0.93 (s, 3H); 13C NMR (125 MHz, CDCl3)
113.8, 65.5, 65.0, 59.4,
a]
D25
d
n
37.4, 37.1, 32.9, 26.5, 26.0, 20.8, 19.1, 18.1, 16.1; HRMS (ESI): calcd for
d
C15H27O2 [MþH]þ 239.2011, found 239.2030.
d
4.1.7. 2-((1S,2R,4aR,8aR)-1,2,4a-Trimethyl-5-methylenedeca hydro-
43.9, 43.1, 42.7, 37.5, 36.1, 30.5, 25.0, 23.7, 23.2, 21.3, 20.1, 17.7, 15.0;
naphthalen-1-yl)ethanol (14). To
a
suspension of methyl-
HRMS (ESI): calcd for C17H31O3 [MþH]þ 283.2275, found 283.2271.
triphenylphosphonium bromide (16.60 g, 46.6 mmol) in THF
(300 ml) was added dropwise sodium bis-trimethylsilyl amide (1.0 M
in THF, 38.80 ml, 38.80 mmol) and the suspension was stirred at 0 ꢁC
for 30 min. Hydroxy ketone 13 (3.70 g, 15.50 mmol) in THF (50.0 ml)
was then added to the above reaction and the mixture and stirred at
25 ꢁC for 18 h. The reaction was quenched with aqueous saturated
NaHCO3 (400 ml) and the mixture was extracted with ethyl ether
(3ꢂ400 ml). The organic layers were collected, dried over MgSO4,
concentrated, and chromatographed (silica,10e20% ether in hexane)
to afford olefin 14 (3.50 g, 14.80 mmol, 95%) as a white solid; Rf¼0.30
4.1.11. (4aR,5S,6S,8aR)-5-(2-Hydroxyethyl)-5,6,8a-trimethylocta hy-
dronaphthalen-1(2H)-one (C8-epi-13). See Section 4.1.6; white
solid, 93% yield; mp: 95e98 ꢁC; Rf¼0.49 (silica, 80% ethyl ether in
D25
hexanes); [
a
]
þ44.7 (c 1.0, benzene); IR (film):
n 3445, 2935, 1701,
1455, 1381, 1255, 1136, 1026, 950; 1H NMR (400 MHz, CDCl3):
3.62e3.76 (m, 2H), 2.52e2.61 (m, 1H), 2.15e2.22 (m, 1H),
d
2.02e2.10 (m, 1H), 1.84e1.89 (m, 2H), 1.22e1.68 (m, 10H), 1.17 (s,
3H), 1.03 (s, 3H), 0.93 (d, 3H, J¼6.8 Hz); 13C NMR (100 MHz, CDCl3):
d
216.0, 58.4, 49.2, 47.0, 42.4, 38.3, 37.3, 35.5, 26.1, 26.1, 24.5, 21.5,
D25
(50% ether in hexane); [
2931, 2859, 1633, 1447, 1379, 1026, 889; 1H NMR (500 MHz, CDCl3):
4.49 (d, 2H, J¼1.60 Hz), 3.40e3.70 (m, 2H), 1.84e2.40 (m, 3H),
1.05e1.80 (m, 13H), 1.03 (s, 3H), 0.86 (d, 3H, J¼6.40 Hz), 0.74 (s, 3H);
13C NMR (125 MHz, CDCl3)
160.2, 102.5, 58.7, 49.8, 40.9, 40.2, 39.4,
a
]
þ56.2 (c 1.0, benzene); IR (film):
n
3321,
20.2, 19.6, 14.6; HRMS (ESI): calcd for C15H27O2 [MþH]þ 239.2011,
found 239.2030.
d
4.1.12. 2-((1S,2S,4aR,8aR)-1,2,4a-Trimethyl-5-methylenedeca hydro-
naphthalen-1-yl)ethanol (C8-epi-14). See Section 4.1.7; white solid,
91% yield; mp: 53e57 ꢁC; Rf¼0.62 (silica, 80% ethyl ether in hex-
d
37.8, 37.3, 33.1, 28.7, 27.6, 22.1, 21.0, 18.0, 16.4; HRMS (ESI): calcd for
C16H29O [MþH]þ 237.2218, found 237.2221.
ane); [
a
]
þ78.0 (c 0.33, benzene); IR (film):
n
3323, 2932, 2856,
4.53 (br s,
1H), 4.49 (br s, 1H), 3.62e3.75 (m, 2H), 1.02e2.36 (m, 15H), 1.08 (s,
3H), 0.99 (s, 3H), 0.97 (s, 3H); 13C NMR (100 MHz, CDCl3):
160.4,
D25
1632, 1445, 1372, 1026, 890; 1H NMR (400 MHz, CDCl3):
d
4.1.8. 2-((1S,2R,4aR,8aR)-1,2,4a,5-Tetramethyl-1,2,3,4,4a,7,8,8a-octa-
hydronaphthalen-1-yl)ethanol (15). To a solution of olefin 14 (0.72 g,
3.05 mmol) in ethanol (30 ml) was added Rhodium (III) chloride hy-
drate (0.14 g, 0.67 mmol) and the mixture was refluxed for 18 h. The
reaction mixture was cooled to 25 ꢁC, concentrated, and chromato-
graphed (10e20% ether in hexanes) to afford alcohol 15 (0.70 g,
d
102.1, 59.5, 47.9, 42.8, 40.3, 38.1, 36.2, 33.0, 30.1, 28.8, 25.8, 21.7, 21.6,
21.5, 15.3; HRMS (ESI): calcd for C16H29O [MþH]þ 237.2218, found
237.2221.
2.96 mmol, 97%) as a clear oil; Rf¼0.30 (50% ether in hexanes); [
ꢀ39.7 (c 1.0, benzene); IR (film): 3329, 2939, 1743, 1266, 1174, 1134,
1028; 1H NMR (500 MHz, CDCl3):
5.17 (s, 1H), 3.60 (m, 2H),
a
]
4.1.13. 2-((1S,2S,4aR,8aR)-1,2,4a,5-Tetramethyl-1,2,3,4,4a,7,8,8a-oc-
tahydronaphthalen-1-yl)ethanol (C8-epi-15). See Section 4.1.8; clear
D25
n
d
oil, 92% yield; Rf¼0.60 (silica, 80% ethyl ether in hexane); [
ꢀ28.8 (c 0.50, benzene); IR (film): 3320, 2942, 1741, 1269, 1179,
1131, 1028; 1H NMR (400 MHz, CDCl3):
5.17 (br s, 1H), 3.68e3.79
(m, 2H), 1.25e2.09 (m, 16H), 1.03 (s, 3H), 0.97 (d, 3H, J¼6.8 Hz), 0.96
(s, 3H); 13C NMR (100 MHz, CDCl3):
144.2, 120.2, 59.5, 45.1, 42.8,
a]
D25
1.85e2.20 (m, 2H), 1.02e1.90 (m, 14H), 0.99 (s, 3H), 0.86 (d, 3H,
n
J¼6.0 Hz), 0.73(s, 3H); 13C NMR (125 MHz,CDCl3):
d
143.9,120.3, 58.2,
d
47.4, 40.8, 38.7, 38.2, 37.3, 36.7, 27.5, 26.8, 19.9, 18.6, 18.1, 17.9, 16.2;
HRMS (ESI): calcd for C16H29O [MþH]þ 237.2218, found 237.2229.
d
38.4, 37.6, 36.2, 30.2, 27.1, 25.9, 21.4, 20.9, 18.3, 18.2, 15.3; HRMS
4.1.9. (ꢀ)-Callicarpenal ((ꢀ)-1). A solution of alcohol 15 (40.0 mg,
0.169 mmol) in CH2Cl2 (5.0 ml) was added 4-methylmorpholine-N-
oxide (39.6 mg, 0.338 mmol) followed by tetrapropylammonium
perruthenate (11.90 mg, 0.034 mmol). The reaction mixture was
stirred at 25 ꢁC for 15 h, then directly concentrated and subjected to
flash chromatography (silica, 0e5% ether in hexanes) to afford al-
dehyde (ꢀ)-1 ((ꢀ)-callicarpenal) (30.0 mg, 0.128 mmol, 76%) as
(ESI): calcd for C16H29O [MþH]þ 237.2218, found 237.2229.
4.1.14. C8-epi-(ꢀ)-Callicarpenal (C8-epi-(ꢀ)-1). See Section 4.1.9;
clear oil, 71% yield; Rf¼0.85 (silica, 25% ether in hexanes); [
a]
D25
ꢀ36.4 (c 1.0, benzene); IR (film):
n 2933, 1710, 1460, 1381, 1060,
1000; 1H NMR (400 MHz, CDCl3):
d
9.91 (d, 1H, J¼3.6 Hz), 5.16 (br s,
1H), 2.18e2.40 (m, 2H), 1.80e2.10 (m, 3H), 1.20e1.58 (m, 10H), 1.18
a colorless liquid; Rf¼0.81 (25% ether in hexanes); [
a
]
ꢀ75.0 (c
(s, 3H), 1.03 (s, 3H), 1.01 (d, 3H, J¼6.8 Hz); 13C NMR (100 MHz,
D25
0.50, benzene); IR (film):
n
2938, 1710, 1450, 1385, 1055, 1010; 1H
CDCl3): d 204.4, 144.2, 120.5, 54.0, 44.5, 39.8, 38.7, 36.7, 30.2, 26.7,
NMR (400 MHz, CDCl3):
d
9.83 (d, 1H, J¼3.6 Hz), 5.17 (br s, 1H),
26.0, 22.6, 20.6, 18.6, 18.1, 15.8; HRMS (ESI): calcd for C16H27O
2.30e2.48 (m, 2H), 1.90e2.08 (m, 2H), 1.15e1.76 (m, 11H), 1.00 (s,
[MþH]þ 235.2056, found 235.2049.
3H), 0.94 (d, 3H, J¼6.8 Hz), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3):
d
203.8, 143.6, 120.5, 51.7, 49.4, 41.7, 39.0, 38.4, 36.4, 27.3, 26.6, 19.9,
4.2. Biological experimental procedures
18.9, 17.9, 17.2, 16.3; HRMS (ESI): calcd for C16H27O [MþH]þ
235.2056, found 235.2059.
4.2.1. Insects. Ae. aegypti (L.) used in these studies were from a labo-
ratory colony maintained at Mosquito and Fly Research Unit at Center
for Medical, Agricultural and Veterinary Entomology, USDA-ARS,
Gainesville, Florida. Thiscolonyhasbeenmaintained since1952 using
standard procedures.24 We received the eggs and stored these in our
laboratory to use as needed. Mosquitoes were reared to the adult
stage by feeding the larvae on a diet of 2:1 alfalfa pellets (US Nutrition
Inc. Bohemia, NY) and hog chow (Ware Milling 150 ALF Drive,
4.1.10. 2-((4a0R,50S,60S,8a0R)-50,60,8a0-Trimethyloctahydro-20H-spiro
[[1,3]dioxolane-2,10-naphthalen]-50-yl)ethanol (C8-epi-12). Olefin 11
(110 mg, 0.25 mmol) and 10% Pd/C (11 mg) in MeOH was hydro-
genated in a Parr apparatus (70 psi) for 12 h. The catalyst was then
removed by a short silica pad, the corresponding reduced product
was then concentrated and re-dissolved in THF (2 ml) and was

3028
T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
Houston, MS 38851). The diet contents were ground in a grinder and
passed through sieve no. 40, 425 m (USA Standard Sieve, Humboldt
instrumentation grants CHE-9709183 and CHE-0741968. We also
acknowledge the assistance of Dr. Anthony Mrse (UCSD NMR Fa-
cility), Dr. Yongxuan Su (UCSD MS Facility), Dr. Arnold L. Rheingold,
and Dr. Curtis E. Moore (UCSD X-Ray facility). We thank Dr. James J.
Becnel, Mosquito and Fly Research Unit, Center for Medical, Agri-
cultural and Veterinary Entomology, USDA-ARS, Gainesville, for
supplying Ae. aegypti eggs. This study was supported, in part, by
a Deployed War-Fighter Protection Research Program Grant funded
by the U.S. Department of Defense through the Armed Forces Pest
Management Board.
m
MFG. Co. Norridge, IL 60706). Eggs were hatched under vacuum
(z1 h) by placing a piece of a paper towel with eggs in a cup filled
with50 ml de-ionized watercontaining a small quantityoflarvaldiet.
Larvae were removed from vacuum and held overnight in the cup.
These larvae were then transferred into 500 ml cups (about 50 larvae
per cup) filled with de-ionized water. Larval diet was added every day
until pupation and the insects were kept in an environment con-
trolled room. Both the larvae and adults were maintained at a tem-
perature of 27 ꢁCꢃ2 ꢁC and 70ꢃ5% RH in a photoperiod regimen of
12:12 (L:D) h. The adults were fed on 10% sucrose solution. Cotton
pads moistened with the sucrose solution were placed on the top of
screens of 4 L cages. 5e9 days old mated females were used in these
bioassays. Females were deprived ofany food for24 h prior tothe test.
Timings of these tests were centered around 1200 h.
Supplementary data
1H NMR and 13C NMR spectra for compounds 8, 10, 11e15, (ꢀ)-1,
C8-epi-12, C8-epi-13, C8-epi-14, C8-epi-15, C8-epi-(ꢀ)-1 as well as X-
ray data of compounds 9 and C8-epi-13. Supplementary data as-
sociated with this article can be found in online version at
doi:10.1016/j.tet.2011.02.078. These data include MOL files and
InChIKeys of the most important compounds described in this
article.
4.2.2. Chemicals. CPDA-125 was prepared by dissolving 3.33 g so-
dium citrate, 0.376 g citric acid, 4.02 g dextrose, 0.28 g monobasic
sodium phosphate (Fisher Scientific Chemical Co. Fairlawn, NJ), and
0.346 g of adenine (SigmaeAldrich, St. Louis, MO) in 1026 ml of de-
ionized water. ATP was added to CPDA-1 to yield 10ꢀ3 M ATP. CPDA-
1 and ATP preparations were freshly mixed on the day of the test.
CPDA-1þATP is reported to be as attractive to mosquito females for
feeding as blood.26 DEET (N,N-diethyl-m-toluamide 97%) was
obtained from SigmaeAldrich (St. Louis, MO). Molecular biology
grade ethanol was obtained from Fisher Scientific Chemical Co.
(Fairlawn, NJ). (ꢀ)-Callicarpenal used as the positive control in
bioassays was isolated in bulk as previously described.23
References and notes
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3. Stone, R. Science 2010, 330, 20e21.
4. Insect Repellents: Principles, Methods, and Uses; Debboun, M., Frances, S. P.,
Strickman, D., Eds.; CRC Press: Boca Raton, FL, 2007; ISBN 978-0-8493-7196-7.
5. Petherick, A. How DEET jams insects’ smell sensors, Nature News; http://www.
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6. Scheinfeld, N. J. Drugs Dermatol. 2004, JaneFeb.
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8. For some investigations of beautyberry’s other bioactivities see: (a) Tellez, M.
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2000, 48, 3008e3012; (b) Kobaisy, M.; Tellez, M. R.; Dayan, F. E.; Duke, S. O.
Phytochemistry 2002, 61, 37e40.
9. (a) Cantrell, C. L.; Klun, J. A.; Bryson, C. T.; Kobaisy, M.; Duke, S. O. J. Agric. Food
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in Invertebrate and Vertebrate Repellents; Paluch, G., Coats, J., Eds.; American
Chemical Society: Washington, DC, 2011.
10. Carroll, J. F.; Cantrell, C. L.; Klun, J. A.; Kramer, M. Exp Appl. Acarol. 2007, 41,
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12. For other synthetic work of callicarpenal and its isomers see: (a) Tokoroyama,
T.; Tsukamoto, M.; Asada, T.; Iio, H. Tetrahedron Lett. 1987, 28, 6645e6648; (b)
Tokoroyama, T.; Fujimori, K.; Shimizu, T.; Yamagiwa, Y.; Monden, M.; Iio, H.
Tetrahedron 1988, 44, 6607e6622; (c) Hagiwara, H.; Inome, K.; Uda, H. Tetra-
hedron Lett. 1994, 35, 8189e8192; (d) Hagiwara, H.; Inome, K.; Uda, H. A. J.
Chem. Soc., Perkin Trans. 1 1995, 7, 757e764; (e) Ling, T. T.; Poupon, E.; Rueden,
E. J.; Kim, S. H.; Theodorakis, E. A. J. Am. Chem. Soc. 2002, 124, 12261e12267; (f)
Watanabe, H.; Oguro, D. Jpn. Kokai Tokkyo Koho, JP 2010053039, 2010.
13. Hagiwara, H.; Uda, H. J. Org. Chem. 1988, 53, 2308e2311.
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CCDC798139 contains the supplementary crystallographic data for compound
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4.2.3. Mosquito biting bioassays. Experiments were conducted by
using a six-celled in vitro Klun and Debboun (K and D) module bio-
assay system developed by Klun et al.26 for quantitative evaluation of
bite deterrentpropertiesofcandidatecompounds forhumanuse. This
bioassay method determines specifically measured biting (feeding)
deterrent properties of the chemicals. Briefly the assay system con-
sists of a six well blood reservoir with each of the 3 cmꢂ4 cm wells
containing 6 ml of blood. As reported earlier,26 female mosquitoes
feed as well on the CPDA-1þATP as they do on blood. Therefore, we
used the CPDA-1þATP instead of blood. The temperature of the so-
lution in the reservoir was maintained at 37 ꢁC by continuously
passingthewarmwaterthroughthereservoirusingacirculatorybath.
The reservoirs were covered with a layer of collagen membrane
(Devro, SandyRun, SC). This blood membraneunitsimulateda human
host for mosquito feeding. The test compounds were randomly ap-
plied to six 4 cmꢂ5 cm areas of organdy cloth (G Street Fabrics,
Rockville, MD) and positioned over the membrane-covered CPDA-
1þATPsolutionwitha separatorplaced betweenthe treatedclothand
the six-celled module. A six-celled K and D module containing five
females per cell was positioned over cloth treatments covering the six
blood membrane wells, and trap doors were opened to expose the
treatments to these females. After a 3 min exposure, the number of
mosquitoes biting through cloth treatments in each cell was recorded
and mosquitoes were prodded back into the cells. These mosquitoes
were then squashed to determine the number, which has actually
engorged the solution. A replicate consisted of six treatments: four
test compounds, DEET (a standard bite deterrent compound) or cal-
licarpenaland95%ethanol-treatedclothasthecontrol.The25 nmoles
DEET/cm2 cloth dose was used as a standard, because it suppresses
mosquitobiting by 80% ascompared tocontrols.27 A setof replications
were conducted on different days using new lots of mosquitoes.28
18. Crabtree, R. H. Acc. Chem. Res. 1979, 12, 331e337.
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Am. Chem. Soc. 2001, 123, 11586e11593; (c) Nakatani, M.; Nakamura, M.; Su-
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21. For some other examples of exoeendo alkene isomerisation in natural product
Acknowledgements
€
synthesis see: (a) Giannis, A.; Heretsch, P.; Sarli, V.; Stoûel, A. Angew. Chem., Int.
Ed. 2009, 48, 7911e7914; (b) Piers, E.; Roberge, J. Y. Tetrahedron Lett. 1992, 33,
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Financial support by the NIH (5R01GM081484) is gratefully ac-
knowledged. We thank the National Science Foundation for

T. Ling et al. / Tetrahedron 67 (2011) 3023e3029
3029
22. There is a small difference of optical rotations between the synthetic material
([
benzene), probably due to the difference between measuring concentrations.
23. Cantrell, C. L.; Klum, J. A.; Pridgeon, J.; Becnel, J.; Green, I. I. I. S.; Fronczek, F. R.
Chem. Biodiv. 2009, 6, 447e458.
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25. AABB [American Association of Blood Banks] Technical Manual, 15th ed.;
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26. Klun, J. A.; Kramer, M.; Zhang, A.; Wang, S.; Debboun, M. J. Am. Mosq. Control
a
]
D25
ꢀ75.0, c 0.5, benzene) and the natural product ([
a
]
D25
ꢀ45.3, c 0.0053,
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27. Klun, J. A.; Kramer, M.; Debboun, M. J. Am. Mosq. Control Assoc. 2005, 21, 64e70.
28. Since we are dealing with an assay on a whole organism we use statistics as
a tool to explain the variation in data. These bioassays have been replicated 20
times, which is sufficient for conclusive data. For instance, increase in means
from 0.6 to 0.7 is quite substantial in terms of anti-biting effects.