Structure-activity relationship studies on derivatives of eudesmanolides from Inula helenium as toxicants against Aedes aegypti larvae and adults

Article abstract of DOI:10.1002/cbdv.201000031

An Aedes aegypti larval toxicity bioassay was performed on compounds representing many classes of natural compounds including polyacetylenes, phytosterols, flavonoids, sesquiterpenoids, and triterpenoids. Among these compounds, two eudesmanolides, alantol

Full text of DOI:10.1002/cbdv.201000031

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Structure–Activity Relationship Studies on Derivatives of Eudesmanolides
from Inula helenium as Toxicants against Aedes aegypti Larvae and Adults
by Charles L. Cantrell*a), Julia W. Pridgeonb), Frank R. Fronczekc), and James J. Becnelb)
a) USDA-ARS, Natural Products Utilization Research Unit, University, MS 38677, USA
(phone: þ1-662-915-5898; fax: þ1-662-915-1035; e-mail: charles.cantrell@ars.usda.gov)
b) USDA-ARS, Mosquito and Fly Research Unit, Gainesville, FL 32608, USA
c) Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA
An Aedes aegypti larval toxicity bioassay was performed on compounds representing many classes of
natural compounds including polyacetylenes, phytosterols, flavonoids, sesquiterpenoids, and triterpe-
noids. Among these compounds, two eudesmanolides, alantolactone, and isoalantolactone showed
larvicidal activities against Ae. aegypti and, therefore, were chosen for further structure–activity
relationship study. In this study, structural modifications were performed on both alantolactone and
isoalantolactone in an effort to understand the functional groups necessary for maintaining and/or
increasing its activity, and to possibly lead to more effective insect-control agents. All parent compounds
and synthetic modification reaction products were evaluated for their toxic activities against Ae. aegypti
larvae and adults. Structure modifications included epoxidations, reductions, catalytic hydrogenations,
and Michael additions to the a,b-unsaturated lactones. None of the synthetic isomers synthesized and
screened against Ae. aegypti larvae were more active than isoalantolactone itself which had an LC50 value
of 10.0 mg/ml. This was not the case for analogs of alantolactone for which many of the analogs had
larvicidal activities ranging from 12.4 to 69.9 mg/ml. In general, activity trends observed from Ae. aegypti
larval screening were not consistent with observations from adulticidal screening. The propylamine
Michael addition analog of alantolactone was the most active adulticide synthesized with an LC50 value of
1.07 mg/mosquito. In addition, the crystal structures of both alantolactone and isoalantolactone were
determined using CuKa radiation, which allowed their absolute configurations to be determined based on
resonant scattering of the light atoms.
1. Introduction. – The yellow fever mosquito, Aedes aegypti (L.; Diptera:
Culicidae), transmits viral pathogens of humans, including yellow fever [1–4] and
dengue [5–8], both of which can cause severe human morbidity and mortality.
Although there is a safe and effective vaccine for the yellow fever virus, epidemic
transmission still occurs in Africa with sporadic cases in South America [9–13].
Dengue is the most important arboviral disease in the world, causing an undiffer-
entiated fever, dengue fever, dengue hemorrhagic fever, or dengue shock syndrome
[14]. Annually, dengue epidemics cause several million cases and thousands of deaths
worldwide [15].
Mosquito control in many countries relies primarily on insecticides. Following the
introduction of synthetic organic insecticides in the 1940s and 1950s, Ae. aegypti was
eradicated from many areas of the world. The Pan American Health Organization
initiated a campaign to use DDT to eradicate Ae. aegypti in the Western Hemisphere in
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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the late 1940s [16][17]. By 1972, Ae. aegypti had been eradicated from 73% of the land
area and 19 countries [18]. However, insecticide resistance developed [19], and the
campaign ended in 1972 before the eradication goal was achieved. Insecticide
resistance has resulted in significant loss of efficacy to commonly used insecticides
[20–24]. Therefore, there is an urgent need for the development of alternative
insecticides to control these important disease vectors.
One potential source of new pesticides is natural plant chemicals. Not only might
certain natural plant products be a source of new pesticides, but also botanical
derivatives may be more environmentally friendly than synthetic chemicals [25]. This
project began by randomly screening previously isolated and identified natural
products from our personal repositories. The compounds chosen were representative of
particular classes of natural products we had previously reported [26–29]. This
screening approach led to a previously investigated set of eudesmanolides which were
the subject of a structure–activity investigation. In particular, the two eudesmanolides,
alantolactone and isoalantolactone, were the subject of this investigation. The larvicidal
and adulticidal activities of all synthetic isomers, and analogs of alantolactone and
isoalantolactone against Ae. aegypti and the structure–activity relationships are
reported here.
2. Results and Discussion. – Initial Compound Screening. In an effort to identify
novel classes of plant natural products with activity against Ae. aegypti, a high-
throughput larval screening method [30] was performed on compounds representing
many classes of natural compounds (Table 1). Representatives of polyacetylenes,
phytosterols, flavonoids, sesquiterpenoids, and triterpenoids, among others, were
Table 1. Larvicidal Activities of Various Natural Compounds against First Instar Larvae of Ae. aegypti
Mortality [%]a)
125 ppm
62.5 ppm
31.25 ppm
15.625 ppm
8 ppm
(Z,Z)-Matricaria ester
(E)-Cinnamic acid
Locustol
100
0
100
2
100
0
75
0
75
0
0
0
0
0
0
0
0
Cumostrol
0
0
0
Parthenin
0
0
0
0
0
Dihydroparthenolide
Betulin
0
0
0
0
0
0
0
0
0
0
Quercetin
Parthenolide
75
0
75
0
60
0
0
0
0
0
Rutin
0
0
0
0
0
Enhydrin
0
0
0
0
0
Ferulic acid
0
0
0
0
0
(24R)-24,25-Epoxycycloartan-3-one
Alantolactone (1a)
Isoalantolactone (2a)
Ergosterol endoperoxide
0
0
0
0
0
100
0
0
0
20
0
100
100
0
62.5
100
0
5.8
100
0
a) Evaluations also performed at 4, 2, and 1 ppm with 0% mortality for all treatments.

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evaluated from 125 down to 2 ppm in a dose-dependent manner. Percent mortality was
determined for evaluated compounds. Of the 16 compounds evaluated, five of these
compounds gave 75% mortality or higher at 125 ppm. (Z,Z)-Matricaria ester was
active down to 31.25 ppm, locustol down to 62.5 ppm, quercetin down to 31.25 ppm,
alantolactone (1a) down to 31.25 ppm, and isoalantolactone (2a; Fig. 1) down to 8 ppm.
On the basis of the above pre-screen, a structure–activity relationship study was
initiated on the sesquiterpene eudesmanolides alantolactone (1a) and isoalantolactone
(2a) in an effort to both understand the activity of these compounds and quite possibly
produce more active isomers and analogs.
X-Ray Crystal-Structure Determinations. Prior to initiating the structure–activity
Fig. 1. Compounds isolated from Inula helenium. Arbitrary numbering.
relationship study, gram quantities of both alantolactone (1a) and isoalantolactone (2a)
were needed, and purification was performed essentially as described in [27]. As
previously observed, both compounds readily crystallized thus assisting in the
purification of sufficient quantities for this synthetic study. X-Ray crystal-structure
determinations were carried out for both compounds 1a (Fig. 2) and 2a (Fig. 3). The
relatively new Hooft analysis of Bijvoet pairs [31] enabled the conclusive determination
Fig. 2. ORTEP Drawing of alantolactone (1a)

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Fig. 3. ORTEP Drawing of isoalantolactone (2a)
of their absolute configurations by resonant scattering of the light atoms in CuKa
radiation. Details are given in the Exper. Part. Despite the fact that both 1a and 2a have
been reported in the literature for at least half a century, a report on the X-ray crystal-
structure determination was not found and is reported here for the first time.
Peracid Epoxidations. Compounds 11,13-dihydroalantolactone (1b) and 11,13-
dihydroisoalantolactone (2b) were included in this study, as they were natural isomers
which had been previously isolated by the authors [27]. Both compounds demonstrated
LC50 values of >125 mg/ml against larvae of Ae. aegypti indicating the importance of
the a,b-unsaturated lactone moiety (Table 2). Initial synthetic modifications included
the peracid epoxidation of compounds 1a, 2a, 1b, and 2b to their corresponding
epoxides 1c, 2c, 1d, and 2d, respectively (Fig. 4). Details on the synthesis of 1c, 2c, and
1d had been reported in [27]. Similar yields were obtained for the synthesis of 2d as
previously performed and was greater than 100% due to inefficient washing. The
presence of remaining peracid and/or acid was visible in both 1H- and 13C-NMR spectra
of the product. All four epoxy synthetic isomers were also inactive up to the top testing
concentration of 125 mg/ml.
Reductions. Additional modifications were performed on 1b and included a
catalytic hydrogenation to produce the desired product 1e in a 63% yield. LiAlH4
Reduction was also performed on 1b in an attempt to synthesize its corresponding
lactone hydrolysis product. Unfortunately, the diol 1f was instead produced in low yield
(48.7%). Derivatives 1e and 1f were evaluated in the Ae. aegypti larval screens, and
both were inactive up to the top testing concentration of 125 mg/ml.
Michael Additions. The remaining set of modifications during this first batch of
modifications (Table 2) to 1a and 2a targeted the a,b-unsaturated lactone moiety
(Fig. 5). Specifically, Michael addition reactions using the nucleophilic amines

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Table 2. Larval LC50 Values [mg/ml] of Various Analogs of Isoalantolactone and Alantolactone against
Ae. aegypti
Batcha)
1st
Compound
LC50 (95% CI)
Slope (SE)
c2
df
1a
2a
2b
1b
1c
2c
2d
1d
1e
1f
36.2 (32.3–40.5)
10.0 (8.63–11.8)
>125
>125
>125
>125
>125
>125
>125
9.04 (1.28)
6.75 (0.92)
13.58
19.12
10
10
>125
4a
3a
4b
3b
14.4 (12.2–17.2)
>125
9.59 (1.54)
25.93
10
55.1 (37.1–92.5)
12.4 (9.75–16.1)
2.50 (0.35)
6.73 (0.86)
5.82
40.09
4
10
2nd
4c
3c
4d
3d
4e
3e
4f
3f
4g
3g
19.3 (15.3–22.5)
21.4 (16.2–27.4)
41.9 (26.5–57.3)
17.3 (13.2–21.3)
64.8 (44.4–95.8)
24.1 (17.4–37.9)
>125
3.82 (0.63)
5.62 (0.72)
2.79 (0.44)
5.87 (0.83)
5.05 (0.62)
5.36 (0.82)
1.66
30.97
6.46
28.82
56.41
24.99
4
9
4
10
10
7
>125
16.6 (14.5–18.9)
42.3 (34.4–51.7)
7.43 (0.95)
6.51 (0.85)
21.42
7.17
13
4
3rd
3h
4h
4i
3i
4j
29.9 (21.8–42.3)
108 (94.1–136)
24.3 (20.4–29.2)
31.5 (22.7–44.4)
55.0 (49.4–61.1)
35.6 (32.3–39.4)
19.7 (17.9–21.9)
20.6 (18.0–23.7)
30.5 (27.3–33.8)
31.0 (22.8–41.8)
35.6 (30.8–41.3)
69.9 (57.7–88.8)
3.36 (0.47)
7.22 (1.33)
6.84 (0.87)
4.81 (0.58)
9.79 (1.57)
6.27 (0.82)
7.44 (1.06)
7.59 (1.03)
8.47 (1.36)
5.07 (.061)
5.95 (0.76)
3.09 (0.45)
7.65
16.71
23.91
46.17
12.93
9.16
4
10
10
10
10
10
9
10
10
10
10
10
3j
4k
3k
4l
3l
4m
3m
6.50
16.56
10.83
42.43
14.91
11.89
a) Data is organized in series by batches tested and synthesized.
piperidine and diethylamine (Et2NH) were performed on both compounds 1a and 2a.
Additions of Et2NH to both 1a and 2a produced the desired products 3a and 4a in yields
of 53.3 and 61.6%, respectively. Additions of piperidine to 1a and 2a provided the
desired products 3b and 4b in yields of 65.7 and 78.4%, respectively. Ae. aegypti
larvicidal activity for the diethylamino analogs were 14.4 and >125 mg/ml for 4a and 3a,
respectively. Compound 4a is only slightly less active than its parent compound 2a.
Piperidine analogs 4b and 3b were both active against Ae. aegypti larvae with LC50

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Fig. 4. Synthetic epoxidation and reduction reaction products
Fig. 5. Michael addition reaction products of alantolactone (1a) and isoalantolactone (2a)
values of 55.1 and 12.4 mg/ml, respectively. On the basis of the above screening for this
first batch of analogs, Michael addition synthetic isomers and analogs will be the target
of the remainder of this SAR study.
The second batch of analogs consists of a variety of nitrogen and sulfur nucleophiles
reacted with both 1a and 2a to produce the desired Michael addition products. Nitrogen
nucleophiles included the primary amine nonylamine, pyrrolidine, benzylamine, and 3-
chloro-N-methylbenzyl amine, and the only sulfur nucleophile in the study was 4-
sulfanylpyridine (Fig. 5). Nonylamine reaction products 3c and 4c were produced in
yields of 70.3 and 77.9%, and larvicidal activities were 21.4 and 19.3 mg/ml, respectively
(Table 2). Pyrrolidine analogs 3d and 4d were produced in yields of 71.2 and 70.0%, and
larvicidal activities were 17.3 and 41.9 mg/ml, respectively. Benzylamine analogs 3e and
4e were produced in yields of 56.7 and 50.8%, and larvicidal activities were 24.1 and
64.8 mg/ml, respectively. These yields were the lowest of any of the Michael addition
products. The reaction of the chlorinated nucleophile, 3-chloro-N-methylbenzyl amine,

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afforded products 3f and 4f in yields of 67.3 and 94.2%, and larvicidal screening
revealed that both compounds were inactive up to 125 mg/ml. The two sulfur-
nucleophile reaction products 3g and 4g were produced in yields of 82.1 and 66.8%, and
larvicidal activities were 42.3 and 16.6 mg/ml, respectively.
The third batch of analogs synthesized contained linear saturated alkylamino
groups from propyl up to dodecyl (Fig. 5). Yields for these reactions ranged from a low
of 42% for the Michael addition reaction of undecylamine with 1a to a high of nearly
100% for the reaction of propylamine with 2a. Larvicidal screening against Ae. aegypti
for these linear alkylamino derivatives revealed that all were active within the range
tested (Table 2). The LC50 values ranged from a low of 19.3 mg/ml for the nonylamine
isoalantolactone adduct, 4c, to a high of 108 mg/ml for the propylamine isoalantolactone
adduct, 4h.
Ae. aegypti Larvicidal Screening. An empirical analysis of the screening results
described above for the reactions of linear amines with both 1a and 2a led us to create a
plot of Ae. aegypti larvicidal LC50 values vs. the number of C-atoms in the linear amines
(Fig. 6). Results of screening propylamino analogs were intentionally omitted due to its
distortion of the trend when plotted; however, all remaining linear alkylamino analogs
from butylamine up to dodecylamine were included. Clearly, there appears to be a
relationship between the Ae. aegypti larvicidal activity and the number of C-atoms in
the linear amine and/or chain length of the linear amine. For the plotting of activity vs.
the number of C-atoms for alantolactone isomers, and subsequent addition of a second-
order polynomial trendline, it becomes clear that seven to eight C-atoms in the amine is
the optimum number needed to maximize activity. Similarly for isoalantolactone
isomers, the trendline and plotting indicates that nine C-atoms would be the optimal
number of C-atoms for the highest activity.
Fig. 6. C4 to C12 aliphatic Michael-addition substituted reaction products vs. larval LC50 activity.
Corresponding second-order polynomial regression lines are overlaid.

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Ae. aegypti Adulticidal Activity. Lastly, all compounds were evaluated for activity
against adult Ae. aegypti mosquitoes (Table 3). The LC50 values for 1a and 2a were 5.16
and 2.28 mg/mosquito, respectively, which follows the trend observed for Ae. aegypti
larvicidal screening that 2a is more active than 1a. The epoxy analogs of 1a and 2a,
namely 1c and 2c, respectively, were both active against adult mosquitoes. Compound
1c was more active than its parent compound alantolactone (1a) with a value of 1.39 mg/
mosquito. The diethylamino analogs of 1a and 2a, namely 3a and 4a, respectively, were
also both active against adult mosquitoes. Again, the analog 3a was more active than its
parent compound 1a with a value of 1.42 mg/mosquito. The propylamine analog of
alantolactone (1a), compound 3h, was the most active adulticide synthesized with an
LC50 value of 1.07 mg/mosquito. The hexylamino analogs of both 1a and 2a were both
highly active against adults with LC50 values of 1.34 and 1.76 mg/mosquito for 3i and 4i,
respectively.
Table 3. Adulticidal Activities of Isoalantolactone and Alantolactone Isomers against Ae. aegypti
Compounda)
LC50 (95% CI) [mg/mosquito]
Slope (SE)
c2 b
)
1a
2a
1c
2c
4a
3a
3h
4i
5.16 (4.66–5.67)
2.28 (1.99–2.56)
1.39 (1.24–1.56)
2.05 (1.82–2.45)
2.29 (1.99–2.99)
1.42 (1.15–1.79)
1.07 (0.95–1.18)
1.76 (1.56–2.07)
1.34 (1.21–1.49)
6.76 (0.95)
5.21 (0.76)
5.52 (0.86)
5.87 (1.13)
5.33 (1.14)
6.98 (1.08)
6.30 (0.97)
4.90 (0.86)
4.80 (0.74)
9.32c)
12.37c)
2.15
0.95
1.66
4.25
1.20
0.42
0.537
3i
a) All isomers in this study were evaluated. All compounds not shown in this Table had LC50 values
higher than 2.34 mg/mosquito. Only compound 1a was evaluated at higher concentrations. b) df¼3.
c) df¼13.
Overall, none of the synthetic isomers screened against Ae. aegypti larvae were
more active than isoalantolactone (2a) itself. This was not the case for analogs of
alantolactone (1a) for which many of the analogs had good larvicidal activity. In
general, activity trends observed from Ae. aegypti larval screening were not consistent
with observations from adulticidal screening. For example, the most active compound
in the adulticidal screen was the propylamino analog of alantolactone (1a), compound
3h, whereas the most active compound in the larvicidal screen was isoalantolactone
(2a). This could be due to the entry-route differences between the two bioassay
systems, i.e., in adult bioassays, compounds enter only through the cuticle, while, in
larval assays, compounds can either be absorbed through the cuticle or enter via the
midgut.
Experimental Part
General. Column chromatography (CC): Biotage, Inc. HorizonTM pump (Charlottesville, Virginia)
equipped with a HorizonTM flash collector and fixed wavelength (254 nm) detector. HPLC Method

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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development work was performed using an Agilent 1100 system equipped with a quaternary pump,
autosampler, diode-array detector, and vacuum degasser. Semi-prep. HPLC: Waters Delta-Prep system
1
(Milford, MA) equipped with a diode-array detector and a binary pump. H- and 13C-NMR spectra:
Varian ANOVA 400 MHz or Varian Unity INOVA 600 MHz spectrometer (Palo Alto, CA); in CDCl3 or
C6D6; all 13C multiplicities were deduced from 908 and 1358 DEPT experiments. HR-MS: Agilent 1100
HPLC coupled to a JEOL AccuTOF (JMS-T100LC) (Peabody, MA).
High-Resolution LC/MS Analysis. All isolated and synthetic compounds were prepared in MeOH
and injected directly into a 0.3-ml/min stream of either MeOH or 80% MeOH/20% deionized (DI) H2O.
Twenty ml of sample (ca. 0.1 mg/ml) was injected manually at 0.5 min, while mass drift compensation
standards (l-tryptophan (neg. ion), PEG (pos. ion)) were injected at 1.5 min over the course of a 2-min
run isocratic.
GC/MS Analysis. Analogs and reaction intermediates were analyzed by GC/MS on a Varian CP-3800
GC coupled to a Varian Saturn 2000 MS/MS. GC was equipped with a DB-5 column (30 mꢀ0.25 mm
fused silica cap. column, film thickness of 0.25 mm) operated using the following conditions: injector
temp., 2408; column temp., 60–2408 at 38/min then held at 2408 for 5 min; carrier gas, He; injection
volume, 1 ml (splitless). MS Mass range from 40 to 650 m/z, filament delay of 3 min, target TIC of 20,000,
a prescan ionization time of 100 ms, an ion-trap temp. of 1508, manifold temp. of 608, and a transfer line
temp. of 1708.
Alantolactone (¼(3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-Octahydro-5,8a-dimethyl-3-methylidenenaph-
tho[2,3-b]furan-2(3H)-one; 1a), Isoalantolactone (¼(3aR,4aS,8aR,9aR)-Decahydro-8a-methyl-3,5-di-
methylidenenaphtho[2,3-b]furan-2(3H)-one; 2a), 11,13-Dihydroalantolactone (¼(3S,3aR,5S,8aR,9aR)-
3a,5,6,7,8,8a,9,9a-Octahydro-3,5,8a-trimethylnaphtho[2,3-b]furan-2(3H)-one; 1b), and 11,13-Dihydro-
alantolactone (¼(3S,3aR,4aS,8aR,9aR)-Decahydro-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-
2(3H)-one; 2b). Information on the collection, identification, and voucher deposition for Inula helenium
have been reported in [16]. Details on the isolation and identification of compounds 1a, 2a, 1b, and 2b
from I. helenium have been reported in [27].
X-Ray Crystallographic Data for Alantolactone (1a). The crystal structure was determined using X-
ray data collected at 90 K, with CuKa radiation (l¼1.54178 ꢂ) on a Bruker Kappa Apex-II
diffractometer. Crystals are orthorhombic, space group P212121 with Z¼4. All H-atoms were visible in
difference maps, and were placed in calculated positions in the refinement, with a torsional parameter
refined for each Me group, leading to R¼0.049, Rw ¼0.123 for 215 refined parameters and 2272
independent reflections having qmax ¼68.88. The absolute configuration was determined, based on
resonant scattering from light atoms only, to be that shown in Fig. 2, using 904 Bijvoet pairs. The Flack
[32] parameter has a value of x ¼ 0.1(3), and the Hooft [31] parameter has a value of y ¼ 0.11(9),
corresponding to a probability of 1.000 that the reported absolute configuration is correct. The CIF has
been deposited with the Cambridge Crystallographic Data Centre, CCDC-752255.
X-Ray Crystallographic Data for Isoalantolactone (2a). The crystal structure was determined,
including absolute configuration, as for 1a. Crystals are monoclinic, space group P21 with Z¼2, R¼0.023,
Rw ¼0.062 for 215 refined parameters and 2192 independent reflections having qmax ¼68.68. The absolute
configuration determination was based on 956 Bijvoet pairs, Flack parameter x ¼ 0.01(16), Hooft
parameter y ¼ 0.05(5), corresponding to a probability of 1.000 that the reported absolute configuration is
correct. The CIF has been deposited with the Cambridge Crystallographic Data Centre, CCDC-752256.
meta-Chloroperbenzoic Acid (m-CPBA) Oxidation of 1a, 2a, and 1b. Details on the m-CPBA
oxidation of 1a, 2a, and 1b to their corresponding epoxides 1c, 2c, and 1d, resp., have been reported in
[27].
m-CPBA Oxidation of 2b. A soln. of 156.0 mg of 2b in 20 ml of CH2Cl2 was added to 275.6 mg of m-
CPBA in an ice-bath overnight. The mixture was washed two times with 20 ml of 10% aq. NaHCO3 and
twice with 20 ml of deionized H2O. Products were separated by SiO2 CC on a Biotage 40þM column (40–
63 mm, 60 ꢂ, 25ꢀ150 mm) running at 40 ml/min using a hexanes/AcOEt step gradient beginning with
100 :0 to 75 :25 over 1152 ml and finishing with 75 :25 to 50 :50 over 576 ml. 24-ml fractions were
collected and recombined based on TLC similarities into one fraction with one distinct compound.
Drying provided 241.2 mg of pure (3S,3aR,4aR,5R,8aR,9aR)-3,8a-dimethyldecahydro-2H-spiro[naph-
1
tho[2,3-b]furan-5,2’-oxiran]-2-one (2d). H-NMR (600 MHz, CDCl3): 4.41 (br. s, 1 H); 2.71–2.76 (m,

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1 H); 2.66 (d, J ¼ 4.0, 1 H); 2.53 (d, J ¼ 4.0, 1 H); 2.25–2.31 (m, 1 H); 2.11 (d, J¼15.6, 1 H); 1.10 (d, J ¼
7.2, 3 H); 0.90 (s, 3 H). 13C-NMR (150 MHz, CDCl3): 179.6 (s); 77.7 (d); 58.9 (s); 51.0 (t); 44.6 (d); 42.2
(t); 41.8 (d); 41.5 (t); 40.4 (d); 35.4 (t); 34.9 (s); 20.5 (t); 18.8 (q); 17.0 (t); 9.4 (q). EI-MS (70 eV): 250 (3,
Mþ ), 236 (15), 235 (100), 147 (11), 91 (9). HR-ESI-MS: 273.1423 ([MþNa]þ , C15H22NaOþ3 ; calc.
273.1467), 523.3046 ([2MþNa]þ , C30H44NaOþ6 ; calc. 523.3036).
Catalytic Hydrogenation of 1b. Compound 1b (51.9 mg) was dissolved in 10 ml of MeOH in a 250-ml
round-bottom flask. Two spatula tips full of 5% Pd/C was added to the mixture and charged with H2 with
stirring for 3 d. The mixture was filtered through a bed of Celite 545 and washed with CH2Cl2. The
products were purified using normal-phase chromatography Biotage 40þM column (40–63 mm, 60 ꢂ,
40ꢀ150 mm) running at 40 ml/min hexanes/AcOEt step gradient beginning with 100 :0 to 75 :25 over
1152 ml and finishing with 75 :25 to 50 :50 over 576 ml. 24-ml fractions were collected and recombined
based on TLC similarities into two distinct compounds. Drying provided 33.6 mg of pure
(3S,3aR,4aS,5S,8aR,9aR)-3,5,8a-trimethyldecahydronaphtho[2,3-b]furan-2(3H)-one (1e). 1H-NMR
(600 MHz, CDCl3): 4.36 (br. s, 1 H); 2.65–2.72 (m, 1 H); 2.30–2.33 (m, 1 H); 1.87 (d, J¼15.0, 1 H);
1.12 (d, J¼6.6, 3 H); 0.89 (s, 3 H); 0.80 (d, J¼7.8, 3 H). 13C-NMR (150 MHz, CDCl3): 179.7 (s); 78.3 (d);
45.3 (t); 44.1 (d); 42.2 (t); 41.6 (d); 41.2 (d); 33.6 (t); 33.2 (d); 33.0 (s); 24.5 (t); 21.1 (q); 16.8 (t); 14.7 (q);
9.3 (q). EI-MS (70 eV): 237 (64, Mþ ), 219 (25), 192 (19), 177 (100), 163 (86), 147 (38), 135 (26), 109
(50). HR-ESI-MS: 237.1899 ([MþH]þ , C15H25O2þ ; calc. 237.1854, 259.1688 ([MþNa]þ , C15H24NaOþ2 ;
calc. 259.1674, 495.3470 ([2MþNa]þ , C30H48NaO4; calc. 495.3450).
LiAlH4 Reduction of 1b. Compound 1b (90.2 mg) was dissolved in 5 ml of anh. THF and transferred
to a dry 100-ml round-bottom flask with a magnetic stirrer. To this soln., 215.5 mg of LiAlH4 were slowly
added in an ice-bath, followed by 8 ml of THF. The mixture was refluxed overnight with the addition of
205.6 mg of LiAlH4 dissolved in 2 ml of THF. The mixture was stirred under reflux overnight. The
reaction was complete in 2 d at which time ca. 25 ml of deionized H2O was added dropwise to quench the
reaction. This mixture was extracted three times with 25 ml of Et2O. After removal of organics in vacuo,
the residue (79.6 mg) was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ
150 mm) running at 40 ml/min using a hexane/AcOEt step gradient beginning with 100 :0 to 50 :50 over
1728 ml, followed by 50 :50 to 0 :100 over 576 ml. 24-ml fractions were collected and recombined based
on TLC similarities into three distinct fractions (A–C). Fr. B provided 45.9 mg of pure (2R,3R,5S,8aR)-
1,2,3,5,6,7,8,8a-octahydro-3-[(2S)-1-hydroxypropan-2-yl]-5,8a-dimethylnaphthalen-2-ol (1f). 1H-NMR
(600 MHz, CDCl3): 5.20 (br. s, 1 H); 4.06 (br. s, 1 H); 3.55–3.65 (m, 1 H); 3.47 (dd, J¼3.6, 9.0, 1 H);
2.41–2.48 (m, 1 H); 2.25–2.27 (m, 1 H); 1.29 (s, 3 H); 1.12 (d, J¼7.2, 3 H); 0.99 (d, J¼7.2, 3 H).
13C-NMR (150 MHz, CDCl3): 148.4 (s); 118.0 (d); 68.3 (d); 65.2 (t); 48.2 (t); 45.1 (d); 43.1 (t); 39.4 (d);
37.2 (d); 34.5 (s); 33.8 (t); 28.6 (q); 22.5 (q); 17.4 (t); 17.3 (q). EI-MS (70 eV): 239 (100, Mþ ), 221 (78),
203 (49), 189 (53), 161 (31), 139 (21), 105 (27). HR-ESI-MS: 477.3973 ([2MþH]þ , C30H53O4þ ; calc.
477.3943).
Michael Addition of Et2NH and 2a. A soln. of 51.5 mg of 2a in 4 ml of 100% anh. EtOH was added to
100 ml of Et2NH, and the soln. was stirred for 1 h at r.t. 400 ml of Et2NH was added, and the mixture was
refluxed overnight. The reaction was quenched with 3 ml of deionized H2O, and the mixture was
extracted three times with 20 ml of AcOEt. Products were dried (MgSO4) and decanted into a 250-ml
round-bottom flask. After removal of organics in vacuo, the residue was separated by SiO2 CC on a
Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/min using a hexane/Et2O step
gradient beginning with 100 :0 to 0 :100 over 1728 ml, followed by 100% Et2O over 1728 ml. 24-ml
fractions were collected and recombined based on TLC similarities into one distinct compound. Drying
provided 41.7 mg of pure (3aR,4aS,8aR,9aR)-3-[(diethylamino)methyl]decahydro-8a-methyl-5-methyl-
idenenaphtho[2,3-b]furan-2(3H)-one (4a). 1H-NMR (600 MHz, CDCl3): 4.70 (s, 1 H); 4.43 (br. s, 1 H);
4.39 (s, 1 H); 2.93–2.96 (m, 1 H); 1.02 (t, J¼7.2, 6 H), 0.736 (s, 3 H). 13C-NMR (150 MHz, CDCl3): 177.9
(s); 149.4 (s); 106.4 (t); 78.4 (d); 47.9 (t); 47.1 (t); 46.5 (d); 45.6 (d); 42.3 (t); 41.6 (t); 39.2 (d); 36.8 (t); 34.8
(s); 22.7 (t); 21.4 (t); 17.9 (q); 11.2 (q). EI-MS (70 eV): 306 (12, Mþ ), 290 (19), 276 (11), 86 (100), 72 (17),
58 (8). HR-ESI-MS: 306.2420 ([MþH]þ , C19H32NO2þ, calc. 306.2433).
Michael Addition of Et2NH and 1a. A soln. of 53.4 mg of 1a in 4 ml of 100% abs. EtOH was added to
600 ml of Et2NH, and the mixture was stirred at r.t. over 3 d. 400 ml of Et2NH was added, and the mixture
was stirred for 4 h. The reaction was quenched with 3 ml of deionized H2O, and the mixture was extracted

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1691
three times with 20 ml of AcOEt. Products were dried (MgSO4) and decanted into a 250-ml round-
bottom flask. After removal of organics in vacuo, the residue was separated by SiO2 CC on a Biotage 40þ
M column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/min using a hexane/AcOEt step gradient
beginning with 50 :50 to 0 :100 over 1152 ml, followed by 100% AcOEt over 576 ml. 24-ml fractions were
collected and recombined based on TLC similarities into one distinct compound. Drying provided
36.1 mg of pure (3aR,5S,8aR,9aR)-3-[(diethylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethyl-
1
naphtho[2,3-b]furan-2(3H)-one (3a). H-NMR (600 MHz, CDCl3): 5.37 (s, 1 H); 4.66–4.69 (m, 1 H);
3.05–3.08 (m, 1 H); 2.95–2.99 (m, 1 H); 1.18 (s, 3 H); 1.08 (d, J¼7.2, 3 H); 0.98 (t, J ¼ 7.2, 7.2, 3 H).
13C-NMR (150 MHz, CDCl3): 177.9 (s); 150.7 (s); 116.1 (d); 77.5 (d); 49.3 (t); 46.8 (t); 44.1 (d); 43.1 (t);
42.5 (t); 38.8 (d); 38.1 (d); 33.2 (s); 33.1 (t); 28.8 (q); 23.2 (q); 17.1 (d); 11.5 (q). EI-MS (70 eV): 306 (7,
Mþ ), 290 (13), 276 (4), 86 (100), 72 (6), 58 (8). HR-ESI-MS: 306.2428 ([MþH]þ , C19H32NOþ2 ; calc.
306.2433.
Michael Addition of Piperidine and 2a. A soln. of 51.2 mg of 2a in 4 ml of 100% abs. EtOH was added
to 200 ml of piperidine, and the mixture was stirred overnight at 08. The reaction was quenched with 3 ml
of deionized H2O, and the mixture was extracted three times with 20 ml of AcOEt. Products were dried
(MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo, the residue
was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/
min using a hexane/AcOEt step gradient beginning with 50 :50 to 0 :100 over 1152 ml, followed by 100%
AcOEt over 576 ml. 24-ml fractions were collected and recombined based on TLC similarities into one
distinct compound. Drying provided 54.9 mg of pure (3aR,4aS,8aR,9aR)-decahydro-8a-methyl-5-
methylidene-3-(piperidin-1-ylmethyl)naphtho[2,3-b]furan-2(3H)-one (4b). 1H-NMR (600 MHz, CDCl3):
4.71 (s, 1 H); 4.39–4.42 (m, 1 H); 4.41 (s, 1 H); 2.87–2.92 (m, 1 H); 2.65 (dd, J ¼ 3.6, 13.2, 1 H), 2.55 (t,
J ¼ 10.8, 1 H); 0.73 (s, 3 H). 13C-NMR (150 MHz, CDCl3): 178.1 (s); 149.5 (s); 106.5 (t); 78.3 (d); 54.8 (t);
53.9 (t); 46.6 (d); 45.6 (d); 42.3 (t); 41.7 (t); 39.5 (d); 36.8 (t); 34.9 (s); 26.0 (t); 24.3 (t); 22.8 (t); 21.1 (t);
17.9 (q). EI-MS (70 eV): 318 (32, Mþ ), 98 (100). HR-ESI-MS: 318.2452 ([MþH]þ , C20H32NOþ2 ; calc.
318.2433).
Michael Addition of Piperidine and 1a. A soln. of 51.4 mg of 1a in 4 ml of 100% abs. EtOH was added
to 600 ml of piperidine, and the mixture was stirred overnight at 08. The reaction was quenched with 3 ml
of deionized H2O and extracted three times with 20 ml of AcOEt. Products were dried (MgSO4) and
decanted into a 250-ml round-bottom flask. After removal of organics in vacuo, the residue was separated
by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/min using a
hexane/AcOEt step gradient beginning with 50 :50 to 0 :100 over 1152 ml, followed by 100% AcOEt
over 576 ml. 24-ml fractions were collected and recombined based on TLC similarities into one distinct
compound. Drying provided 46.2 mg of pure (3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-
dimethyl-3-(piperidin-1-ylmethyl)naphtho[2,3-b]furan-2(3H)-one (3b). 1H-NMR (600 MHz, CDCl3):
5.28 (s, 1 H); 4.65 (br. s, 1 H); 3.05–3.08 (m, 1 H); 2.98–3.01 (m, 1 H); 1.16 (s, 3 H); 1.06 (d, J¼7.8, 3 H).
13C-NMR (150 MHz, CDCl3): 177.9 (s); 150.7 (s); 116.0 (d); 77.4 (d); 55.0 (t); 43.8 (d); 43.0 (t); 42.4 (t);
38.7 (d); 38.2 (d); 33.2 (t); 33.0 (s); 28.7 (q); 26.1 (t); 24.4 (t); 23.1 (q); 17.1 (t). EI-MS (70 eV): 318 (27,
Mþ ), 98 (100). HR-ESI-MS: 318.2439 ([MþH]þ , C20H32NOþ2 ; calc. 318.2433.
Michael Addition of Pyrrolidine and 2a. A soln. of 52.3 mg of 2a in 4 ml of 100% abs. EtOH was
added to 600 ml of pyrrolidine, and the mixture was stirred overnight at 08. The reaction was quenched
with 3 ml of deionized H2O and extracted three times with 20 ml of AcOEt. Products were dried
(MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo, the residue
was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/
min using an isocratic method of 100% acetone over 1152 ml. 24-ml fractions were collected and
recombined based on TLC similarities into two distinct fractions (A and B). Fr. A provided 47.8 mg of
pure (3aR,4aS,8aR,9aR)-decahydro-8a-methyl-5-methylidene-3-(pyrrolidin-1-ylmethyl)naphtho[2,3-
b]furan-2(3H)-one (4d). 1H-NMR (600 MHz, CDCl3): 4.72 (br. s, 1 H); 4.43 (s, 1 H); 4.40–4.45 (m,
1 H); 2.85–2.90 (m, 1 H); 2.81 (t, J¼12.6, 1 H); 2.72 (dd, J¼4.2, 12.6, 1 H); 0.75 (s, 3 H). 13C-NMR
(150 MHz, CDCl3): 177.7 (s); 149.4 (s); 106.4 (t); 78.3 (d); 54.4 (t); 50.9 (t); 47.1 (d); 46.6 (d); 42.3 (t); 41.6
(t); 39.3 (d); 36.8 (t); 34.9 (s); 23.6 (t); 22.8 (t); 21.1 (t); 17.9 (q). EI-MS (70 eV): 304 (15, Mþ ), 84 (100),
70 (5), 42 (6). HR-ESI-MS: 629.4299 ([2MþNa]þ , C38H58N2NaO4þ ; calc. 629.4294).

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Michael Addition of Pyrrolidine and 1a. A soln. of 54.2 mg of 1a in 4 ml of 100% abs. EtOH was
added to 600 ml of pyrrolidine, and the mixture was stirred overnight at 08. The reaction was quenched
with 3 ml of deionized H2O and extracted three times with 20 ml of AcOEt. Products were dried
(MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo, the residue
was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/
min using an isocratic method of 100% acetone over 1152 ml. 24-ml fractions were collected and
recombined based on TLC similarities into two distinct fractions (A and B). Fr. B provided 50.4 mg of
pure (3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethyl-3-(pyrrolidin-1-ylmethyl)naph-
tho[2,3-b]furan-2(3H)-one (3d). 1H-NMR (600 MHz, CDCl3): 5.30 (d, J¼3.0, 1 H); 4.66–4.70 (m,
1 H); 3.11–3.15 (m, 1 H); 2.95–3.00 (m, 1 H); 2.84 (t, J¼12.6, 1 H); 2.65 (dd, J¼4.2, 12.6, 1 H); 1.19 (s,
3 H); 1.09 (d, J¼7.8, 3 H). 13C-NMR (150 MHz, CDCl3): 177.4 (s); 150.8 (s); 115.8 (d); 77.4 (d); 54.2 (t);
52.0 (t); 45.4 (d); 43.0 (t); 42.4 (t); 38.7 (d); 38.1 (d); 33.2 (d); 33.0 (t); 28.7 (q); 23.7 (t); 23.1 (q); 17.0 (t).
EI-MS (70 eV): 304 (14, Mþ ), 288 (9), 84 (100), 42 (6). HR-ESI-MS: 629.4304 ([2MþNa]þ
,
C38H58N2NaOþ4 , 629.4294).
Michael Addition of PhCH2NH2 and 2a. A soln. of 49.4 mg of 2a in 4 ml of 100% abs. EtOH was
added to 600 ml of PhCH2NH2, and the mixture was stirred overnight at 08. The reaction was quenched
with 3 ml of deionized H2O, and the mixture was extracted three times with 20 ml of AcOEt. Products
were dried (MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo,
the residue was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm)
running at 40 ml/min using a hexane/AcOEt step gradient beginning with 50 :50 to 0 :100 over 1152 ml,
followed by 100% AcOEt over 576 ml. 24-ml fractions were collected and recombined based on TLC
similarities into one distinct compound. Drying provided 36.7 mg of pure (3aR,4aS,8aR,9aR)-3-
[(benzylamino)methyl]decahydro-8a-methyl-5-methylidenenaphtho[2,3-b]furan-2(3H)-one
(4e).
1H-NMR (600 MHz, CDCl3): 7.30–7.34 (m, 4 H); 7.21–7.26 (m, 1 H); 4.74 (s, 1 H); 4.44–4.46 (m,
1 H); 4.36 (s, 1 H); 3.79 (d, J¼13.8, 1 H); 3.70 (d, J¼13.2, 1 H); 0.69 (s, 3 H). 13C-NMR (150 MHz,
CDCl3): 178.1 (s); 149.2 (s); 128.6 (d); 128.3 (d); 127.2 (d); 106.6 (t); 78.4 (d); 54.1 (t); 47.4 (d); 46.5 (d);
44.7 (t); 42.3 (t); 41.5 (t); 39.1 (d); 36.8 (t); 34.8 (s); 22.7 (t); 21.1 (t); 17.9 (q). EI-MS (70 eV): 340 (33, Mþ
), 120 (14), 106 (100), 91 (29). HR-ESI-MS: 340.2297 ([MþH]þ , C22H30NO2þ ; calc. 340.2276), 679.4456
([2MþH]þ , C44H59N2O4þ ; calc. 679.4474). 701.4275 ([2MþNa]þ , C44H59N2NaOþ4 ; calc. 701.4294).
Michael Addition of PhCH2NH2 and 1a. A soln. of 52.5 mg of 1a in 4 ml of 100% abs. EtOH was
added to 600 ml of PhCH2NH2 and the mixture was stirred overnight at 08. The reaction was quenched
with 3 ml of deionized H2O, and the mixture was extracted three times with 20 ml of AcOEt. Products
were dried (MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo
the residue was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm)
running at 40 ml/min using a hexane/AcOEt step gradient beginning with 50 :50 to 0 :100 over 1152 ml,
followed by 100% AcOEt over 576 ml. 24-ml fractions were collected and recombined based on TLC
similarities into one distinct compound. Drying provided 43.5 mg of pure (3aR,5S,8aR,9aR)-3-
[(benzylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethylnaphtho[2,3-b]furan-2(3H)-one (3e).
1H-NMR (600 MHz, CDCl3): 7.29–7.36 (m, 4 H); 7.21–7.26 (m, 1 H); 5.04–5.06 (m, 1 H); 4.69–4.71 (m,
1 H); 3.86 (d, J¼13.2, 1 H); 3.75 (d, J ¼ 13.2, 1 H); 1.18 (s, 3 H); 1.03 (d, 3 H, J¼7.8 ) . 13C-NMR
(150 MHz, CDCl3): 177.9 (s); 150.9 (s); 128.5 (d); 128.3 (d); 127.1 (d); 115.2 (d); 77.5 (d); 54.1 (t); 45.9
(d); 45.8 (t); 42.8 (t); 42.9 (t); 38.4 (d); 37.7 (d); 33.0 (q); 32.9 (s); 28.7 (t); 23.0 (t); 16.9 (q). EI-MS
(70 eV): 340 (22, Mþ ), 322 (16), 120 (26), 106 (100), 91 (29), 65 (13). HR-ESI-MS: 340.2302 ([MþH]þ
C22H30NOþ2 ; calc. 340.2276), 679.4467 ([2MþH]þ , C44H59N2Oþ4 ; calc. 679.4474), 701.4291 ([2MþNa]þ
C44H59N2NaOþ4 ; calc. 701.4294).
,
,
Michael Addition of 1-(3-Chlorophenyl)-N-methylmethanamine and 2a. A soln. of 56.5 mg of 2a in
4 ml of 100% abs. EtOH was added to 600 ml of 3-chloro-N-methylbenzylamine and stirred overnight at
08. The reaction was quenched with 3 ml of deionized H2O and extracted three times with 20 ml of
AcOEt. Products were dried (MgSO4) and decanted into a 250-ml round-bottom flask. After removal of
organics in vacuo, the residue was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ,
40ꢀ150 mm) running at 40 ml/min using a hexane/AcOEt step gradient beginning with 100 :0 to 0 :100
over 1152 ml, followed by 100% AcOEt over 576 ml. 24-ml fractions were collected and recombined
based on TLC similarities into one distinct compound. Drying provided 88.9 mg of pure (3aR,4a-

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1693
S,8aR,9aR)-3-{[(3-chlorobenzyl)(methyl)amino]methyl}decahydro-8a-methyl-5-methylidenenaph-
tho[2,3-b]furan-2(3H)-one (4f). 1H-NMR (600 MHz, CDCl3): 7.24 (br. s, 1 H); 7.11–7.19 (m, 3 H); 4.67
(s, 1 H); 4.39 (s, 1 H); 4.20 (s, 1 H); 3.57 (d, J¼13.2, 1 H); 3.27 (d, J ¼ 12.6, 1 H); 2.18 (s, 3 H); 0.70 (s,
3 H). 13C-NMR (150 MHz, CDCl3): 177.5 (s); 148.9 (s); 129.6 (d); 129.0 (d); 127.3 (d); 127.1 (d); 106.5 (t);
78.2 (t); 62.2 (t); 52.2 (t); 46.4 (d); 45.7 (d); 42.4 (q); 42.2 (t); 41.5 (t); 38.9 (d); 36.7 (t); 34.7 (s); 22.7 (t);
20.7 (t); 17.8 (q). EI-MS (70 eV): 388 (28, Mþ ), 262 (10), 168 (100), 154 (21), 125 (24). HR-ESI-MS:
388.2040 ([MþH]þ , C23H31ClNO2þ ; calc. 388.2043), 410.1867 ([MþNa]þ , C23H30ClNNaOþ2 ; calc.
410.1862), 797.3837 ([2MþNa]þ , C46H60Cl2N2NaO4þ ; calc. 797.3827).
Michael Addition of 1-(3-Chlorophenyl)-N-methylmethanamine and 1a. A soln. of 46.7 mg of 1a in
4 ml of 100% abs. EtOH was added to 600 ml of 3-chloro-N-methylbenzylamine, and stirred overnight at
08. The reaction was quenched with 3 ml of deionized H2O and extracted three times with 20 ml of
AcOEt. Products were dried (MgSO4) and decanted into a 250-ml round-bottom flask. After removal of
organics in vacuo, the residue was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ,
40ꢀ150 mm) running at 40 ml/min using a hexane/AcOEt step gradient beginning with 100 :0 to 0 :100
over 1152 ml, followed by 100% AcOEt over 576 ml. 24-ml fractions were collected and recombined
based on TLC similarities into one distinct compound. Drying provided 52.5 mg of pure (3aR,5-
S,8aR,9aR)-3-{[(3-chlorobenzyl)(methyl)amino]methyl}-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethyl-
naphtho[2,3-b]furan-2(3H)-one (3f). 1H-NMR (600 MHz, CDCl3): 7.30 (br. s, 1 H); 7.15–7.22 (m, 3 H);
5.10 (s, 1 H); 4.68 (s, 1 H); 3.63 (d, J¼13.2, 1 H); 3.26 (d, J ¼ 12.6, 1 H); 2.24 (s, 3 H); 1.14 (s, 3 H); 0.92
(d, J ¼ 7.8, 3 H). 13C-NMR (150 MHz, CDCl3): 177.3 (s); 150.9 (s); 129.6 (d); 129.2 (d); 127.5 (d); 127.4
(d); 115.5 (d); 77.4 (d); 62.4 (t); 53.4 (t); 44.0 (d); 42.9 (t); 42.4 (q); 42.3 (t); 38.4 (d); 37.8 (d); 33.0 (t); 32.9
(s); 28.7 (q); 23.1 (q); 16.9 (t). EI-MS (70 eV): 388 (16, Mþ ), 262 (4), 170 (33), 168 (100), 154 (11), 125
(15). HR-ESI-MS: 388.2025 ([MþH]þ
,
C23H31ClNOþ2 ; 388.2043), 410.1870 ([MþNa]þ
,
C23H30ClNNaOþ2 ; calc. 410.1862), 797.3826 ([2MþNa]þ , C46H60Cl2N2NaOþ4 ; calc. 797.3827).
Michael Addition of 4-Sulfanylpyridine and 2a. A soln. of 55.2 mg of 2a in 4 ml of 100% abs. EtOH
was added to 600 ml of a 25.0 mg/ml soln. of 4-sulfanylpyridine, and the mixture was stirred at 08
overnight. 2.4 ml of 25.0 mg/ml soln. of 4-sulfanylpyridine was added, and the mixture was stirred for 3 d.
With no disappearance of starting material (i.e., 2a), 105.3 mg of 4-sulfanylpyridine powder was added,
and the mixture was stirred overnight under reflux. The reaction was quenched with 3 ml of deionized
H2O and extracted three times with 20 ml of AcOEt. Products were dried (MgSO4) and decanted into a
250-ml round-bottom flask. After removal of organics in vacuo, the residue was separated by SiO2 CC on
a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/min using a hexane/AcOEt
step gradient beginning with 100 :0 to 0 :100 over 1152 ml, followed by 100% AcOEt over 576 ml. 24-ml
fractions were collected and recombined based on TLC similarities into two distinct fractions (A and B).
Fr. B provided 54.5 mg of pure (3aR,4aS,8aR,9aR)-decahydro-8a-methyl-5-methylidene-3-[(pyridin-4-
ylsulfanyl)methyl]naphtho[2,3-b]furan-2(3H)-one (4g). 1H-NMR (600 MHz, CDCl3): 8.33 (s, 2 H); 7.06
(s, 2 H); 4.68 (s, 1 H); 4.40 (br. s, 1 H); 4.36 (s, 1 H); 3.49 (d, J¼9.0, 1 H); 0.70 (s, 3 H). 13C-NMR
(150 MHz, CDCl3): 176.2 (s); 149.4 (d); 148.9 (s); 147.6 (s); 120.8 (d); 106.6 (t); 78.2 (d); 46.2 (d); 46.1
(d); 42.0 (t); 41.3 (t); 38.5 (d); 36.6 (t); 34.7 (s); 25.7 (t); 22.6 (t); 20.5 (t); 17.7 (q). EI-MS (70 eV): 344
(100, Mþ ), 182 (34), 147 (18), 105 (26), 79 (13), 41 (9). HR-ESI-MS: 344.1654 ([MþH]þ , C20H26NO2Sþ
calc. 344.1684), 366.1498 ([MþNa]þ , C20H25NNaO2Sþ ; calc. 366.1503), 687.3634 [2MþH]þ
C40H51N2O4S2þ ; calc. 687.3290), 709.3169 [2MþNa]þ , C40H50N2NaO4Sþ2 ; calc. 709.3109).
;
,
Michael Addition of 4-Sulfanylpyridine and 1a. A soln. of 48.5 mg of 1a in 4 ml of 100% abs. EtOH
was added to 202.4 mg of 4-sulfanylpyridine, and the mixture was stirred at r.t. overnight. 50.0 mg of 4-
sulfanylpyridine was added, and the mixture was stirred overnight under reflux. The reaction was
quenched with 3 ml of deionized H2O and extracted three times with 20 ml of AcOEt. Products were
dried (MgSO4) and decanted into a 250-ml round-bottom flask. After removal of organics in vacuo, the
residue was separated by SiO2 CC on a Biotage 40þM column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running
at 40 ml/min using a hexane/AcOEt step gradient beginning with 50 :50 to 0 :100 over 1152 ml, followed
by 100% AcOEt over 576 ml. 24 ml fractions were collected and recombined based on TLC similarities
into one distinct compound. Drying provided 58.9 mg of pure (3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-
octahydro-5,8a-dimethyl-3-[(pyridin-4-ylsulfanyl)methyl]naphtho[2,3-b]furan-2(3H)-one
(3g).
1H-NMR (600 MHz, CDCl3): 8.35 (s, 2 H); 7.10 (s, 2 H); 5.15 (s, 1 H); 4.67–4.69 (m, 1 H); 3.50 (dd,

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
J¼3.6, 13.2, 1 H); 1.15 (s, 3 H); 1.04 (d, J ¼ 7.8, 3 H). 13C-NMR (150 MHz, CDCl3): 175.8 (s); 152.7 (s);
149.1 (s); 149.0 (d); 120.9 (d); 113.6 (d); 77.4 (d); 44.7 (d); 42.6 (t); 42.2 (t); 38.5 (d); 37.2 (d); 33.1 (t); 32.7
(s); 28.6 (q); 27.0 (t); 23.0 (q); 16.7 (t). EI-MS (70 eV): 344 (100, Mþ ), 162 (39), 105 (26), 91 (19), 51
(10). HR-ESI-MS: 344.1667 ([MþH]þ
,
C20H26NO2Sþ
;
344.1684), 366.1501 ([MþNa]þ
,
C20H25NNaO2Sþ ; calc. 366.1503), 687.3324 ([2MþH]þ , C40H51N2O4S2; calc. 687.3290), 709.3169
([2MþNa]þ , C40H50N2NaO4Sþ2 ; calc. 709.3147).
Michael Addition of 1a or 2a and Linear Amines to Form Amine Adducts. A soln. of the appropriate
eudesmanolide (40–50 mg) in 4 ml of 100% abs. EtOH was added to 600 ml of amine, and the mixture
was stirred overnight at 08. The reaction was quenched with 3 ml of deionized H2O and extracted three
times with 20 ml of AcOEt. Products were dried (MgSO4) and decanted into a 250-ml round-bottom
flask. After removal of organics in vacuo, the residue was separated by SiO2 CC on a Biotage 40þM
column (40–63 mm, 60 ꢂ, 40ꢀ150 mm) running at 40 ml/min using an isocratic method of 100% acetone
over 1152 ml. 24-ml fractions were collected and recombined based on TLC similarities into one distinct
compound. Drying provided pure compounds: 3h (44.6 mg), 4h (64.3 mg), 4i (49.5 mg), 3i (58.2 mg), 4j
(42.3 mg), 3j (49.4 mg), 4k (64.3 mg), 3k (65.2 mg), 4l (72.0 mg), 3l (38.9 mg), 4m (71.7 mg), and 3m
(74.1 mg), 4c (64.7 mg), and 3c (58.0 mg).
(3aR,4aS,8aR,9aR)-Decahydro-8a-methyl-5-methylidene-3-[(nonylamino)methyl]naphtho[2,3-
b]furan-2(3H)-one (4c). 1H-NMR (600 MHz, CDCl3): 4.69 (br. s, 1 H); 4.39–4.44 (m, 1 H); 43.9 (br. s,
1 H); 2.92–2.96 (m, 1 H); 2.82–2.86 (m, 1 H); 2.67–2.72 (m, 1 H); 0.80 (t, 3 H, J ¼ 7.2); 0.73 (s, 3 H).
13C-NMR (150 MHz, CDCl3): 178.2 (s); 149.3 (s); 106.5 (t); 51.0 (t); 78.3 (d); 50.2 (t); 47.5 (d); 46.5 (d);
45.4 (t); 42.3 (t); 41.5 (t); 39.2 (d); 36.8 (t); 34.8 (t); 31.9 (t); 30.0 (t); 29.6 (t); 29.3 (t); 27.3 (t); 22.76 (t);
22.73 (t); 21.1 (t); 17.8 (q); 14.1 (q). EI-MS (70 eV): 376 (27, Mþ ), 262 (100), 156 (6), 142 (14), 91 (6), 44
(26). HR-ESI-MS: 376.3239 ([MþH]þ , C24H42NO2þ ; calc. 376.3215).
(3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-Octahydro-5,8a-dimethyl-3-[(nonylamino)methyl]naphtho[2,3-
b]furan-2(3H)-one (3c). 1H-NMR (600 MHz, CDCl3): 5.11 (d, J¼2.4, 1 H); 4.67–4.71 (m, 1 H); 3.05–
3.10 (m, 1 H); 1.18 (s, 3 H); 1.07 (d, J¼7.2, 3 H); 0.82 (t, J¼7.2, 3 H). 13C-NMR (150 MHz, CDCl3): 178.1
(s); 150.9 (s); 115.2 (d); 77.4 (d); 50.2 (t); 46.7 (t); 45.8 (d); 42.8 (t); 42.3 (t); 38.6 (d); 37.8 (d); 33.1 (t);
32.9 (t); 31.9 (t); 30.0 (t); 29.6 (t); 29.3 (q); 28.7 (t); 27.4 (t); 23.0 (q); 22.7 (t); 16.9 (t); 14.1 (q). EI-MS
(70 eV): 376 (27, Mþ ), 262 (100), 156 (15), 142 (12), 105 (11), 91 (10), 44 (29). HR-ESI-MS: 376.3189
([MþH]þ , C24H42NO2þ ; calc. 376.3215), 751.6324 ([2MþH]þ , C48H83N2Oþ4 ; calc. 751.6352.
(3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-Octahydro-5,8a-dimethyl-3-[(propylamino)methyl]-
1
naphtho[2,3-b]furan-2(3H)-one (3h). H-NMR (400 MHz, CDCl3): 5.07 (s, 1 H); 4.67 (s, 1 H); 3.05 (s,
1 H); 2.82–2.92 (m, 2 H); 2.65–2.73 (m, 1 H); 2.50–2.59 (m, 2 H); 2.40 (s, 1 H); 2.02 (d, J¼14.4, 1 H);
1.69–1.85 (m, 2 H); 1.05 (s, 3 H); 1.03 (d, J¼7.2, 3 H); 0.83 (t, J ¼ 7.6, 3 H). 13C-NMR (100 MHz, CDCl3):
178.0; 150.7; 115.1; 76.7; 51.9; 46.5; 45.6; 42.6; 42.1; 38.4; 37.7; 32.9; 32.8; 28.6; 23.0; 22.9; 16.8; 11.7. EI-MS
(70 eV): 262 (100, Mþ ), 72 (38), 292 (25), 105 (24), 91 (23), 44 (21). HR-ESI-MS: 292.2275 ([MþH]þ
C18H30NOþ2 ; calc. 292.2276), 583.4438 ([2MþH]þ , C36H59N2Oþ4 ; calc. 583.4474), 605.4254 ([2MþNa]þ
C36H58N2NaOþ4 ; calc. 605.4294).
,
,
(3aR,4aS,8aR,9aR)-Decahydro-8a-methyl-5-methylidene-3-[(propylamino)methyl]naphtho[2,3-
b]furan-2(3H)-one (4h). 1H-NMR (400 MHz, CDCl3): 4.69 (s, 1 H); 4.41 (br. s, 1 H); 4.38 (s, 1 H); 3.32
(s, 1 H); 2.93 (dd, J¼7.2, 11.6, 1 H); 2.83 (dd, J ¼ 6.4, 13.6, 1 H); 2.67 (dd, J¼7.2, 11.2, 1 H); 2.48–2.56
(m, 2 H); 2.38–2.48 (m, 1 H); 2.07 (d, J¼15.2, 2 H); 1.85–1.94 (m, 1 H); 1.70 (d, J¼12, 1 H); 0.85 (t, J ¼
7.6, 3 H); 0.72 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 178.4; 149.3; 106.5; 78.4; 52.1; 47.4; 46.5; 45.3; 42.2;
41.4; 39.2; 36.8; 34.8; 23.0; 22.7; 21.2; 17.9; 11.8. EI-MS (70 eV): 292 (100, Mþ ), 262 (88), 44 (25), 293
(18), 72 (13), 58 (9). HR-ESI-MS: 292.2270 ([MþH]þ , C18H30NO2þ ; calc. 292.2276), 583.4436 ([2Mþ
H]þ , C36H59N2O4þ ; calc. 583.4474), 605.4253 ([2MþNa]þ , C36H58N2NaOþ4 ; calc. 605.4294).
(3aR,4aS,8aR,9aR)-3-[(Hexylamino)methyl]decahydro-8a-methyl-5-methylidenenaphtho[2,3-
1
b]furan-2(3H)-one (4i). H-NMR (400 MHz, CDCl3): 4.71 (s, 1 H); 4.41–4.45 (m, 1 H); 4.40 (s, 1 H);
2.96 (dd, J¼6.8, 11.6, 1 H); 2.85 (dd, J¼7.2, 13.6, 1 H); 2.70 (dd, J¼6.8, 11.6, 1 H); 2.52–2.62 (m, 1 H);
2.40–2.48 (m, 2 H); 2.22–2.30 (m, 1 H); 2.08 (d, J ¼ 14.0, 1 H); 1.88–1.98 (m, 1 H); 1.73 (d, J¼12.4,
1 H); 0.82 (t, J¼7.2, 3 H); 0.74 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 178.4; 149.4; 106.6; 78.4; 50.4; 47.8;
46.6; 45.5; 42.3; 41.5; 39.2; 36.8; 34.9; 31.9; 30.0; 27.1; 22.8; 22.7; 21.2; 17.9; 14.1. EI-MS (70 eV): 262 (100,

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1695
Mþ ), 44 (33), 334 (16), 263 (15), 100 (14), 91 (10). HR-ESI-MS: 334.2744 ([MþH]þ , C21H36NOþ2 ; calc.
334.2746), 667.5442 ([2MþH]þ , C42H71N2Oþ4 ; calc. 667.5413).
(3aR,5S,8aR,9aR)-3-[(Hexylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethylnaphtho[2,3-
b]furan-2(3H)-one (3i). 1H-NMR (400 MHz, CDCl3): 5.04 (br. s, 1 H); 4.67 (br. s, 1 H); 3.96 (t, J¼7.6,
1 H); 3.02–3.10 (m, 2 H); 2.95 (dd, J¼7.6, 15.2, 1 H); 2.83 (t, J¼7.6, 1 H); 2.70 (dd, J ¼ 6.8, 11.6, 1 H);
2.52–2.59 (m, 1 H); 2.32–2.44 (m, 1 H); 1.99 (dd, J ¼ 2.8, 14.4, 1 H); 1.95 (s, 1 H); 1.87 (s, 1 H); 1.14 (s,
3 H); 1.03 (d, J¼7.6, 3 H); 0.84 (t, J¼7.6, 3 H). 13C-NMR (100 MHz, CDCl3): 178.3; 151.1; 115.1; 77.6;
50.1; 45.5; 42.7; 42.2; 38.5; 37.8; 33.1; 32.8; 31.8; 29.7; 29.7*; 28.7; 26.9; 22.9; 22.6; 16.9; 14.1. EI-MS
(70 eV): 262 (100, Mþ ), 44 (36), 334 (25), 114 (20), 263 (19), 105 (18). HR-ESI-MS: 334.2688 ([MþH]þ
,
C21H36NOþ2 ; calc. 334.2746), 667.5447 ([2MþH]þ , C42H71N2Oþ4 ; calc. 667.5413).
(3aR,4aS,8aR,9aR)-3-[(Butylamino)methyl]decahydro-8a-methyl-5-methylidenenaphtho[2,3-b]-
furan-2(3H)-one (4j). 1H-NMR (600 MHz, CDCl3): 4.69 (s, 1 H); 4.40 (br. s, 1 H); 4.38 (s, 1 H); 2.93 (dd,
J¼6.8, 11.6, 1 H); 2.82 (dd, J¼7.2, 14.0, 1 H); 2.65 (dd, J ¼ 7.2, 11.6, 1 H); 2.50–2.60 (m, 2 H); 2.38–2.44
(m, 1 H); 2.24 (br. d, J¼12.0, 1 H); 2.08 (dd, J¼1.6, 15.6, 1 H); 1.85–1.95 (m, 1 H); 1.70 (d, J ¼ 12.0,
1 H); 0.83 (t, J¼7.6, 3 H); 0.72 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 178.3; 149.3; 106.5; 78.3; 49.9; 47.5;
46.5; 45.4; 42.2; 41.4; 39.1; 36.8; 34.8; 32.1; 22.7; 21.1; 20.5; 17.9; 14.1. EI-MS (70 eV): 306 (100, Mþ ), 262
(31), 307 (21), 44 (12), 263 (5). HR-ESI-MS: 306.2415 ([MþH]þ , C19H32NO2þ ; calc. 306.2433), 611.4831
([2MþH]þ , C38H63N2Oþ4 ; calc. 611.4787).
(3aR,5S,8aR,9aR)-3-[(Butylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethylnaphtho[2,3-
b]furan-2(3H)-one (3j). 1H-NMR (400 MHz, CDCl3): 5.07 (br. s, 1 H); 4.67 (br. s, 1 H); 3.02–3.08 (m,
1 H); 2.81–2.86 (m, 2 H); 2.69 (dd, J¼7.2, 11.6, 1 H); 2.50–2.62 (m, 1 H); 2.34–2.44 (m, 1 H); 2.00 (dd,
J¼2.8, 14.8, 1 H); 1.68–1.80 (m, 1 H); 1.15 (s, 3 H); 1.04 (d, J¼7.2, 3 H); 0.84 (t, J ¼ 7.2, 3 H). 13C-NMR
(100 MHz, CDCl3): 178.0; 150.7; 115.1; 77.4; 49.7; 46.5; 45.6; 42.6; 42.1; 38.4; 37.6; 32.9; 32.7; 31.9; 28.6;
22.8; 20.3; 16.7; 13.9. EI-MS (70 eV): 262 (100, Mþ ), 306 (47), 44 (35), 105 (19), 86 (16). HR-ESI-MS:
306.2442 ([MþH]þ , C19H32NOþ2 ; calc. 306.2433).
(3aR,4aS,8aR,9aR)-3-[(Decylamino)methyl]decahydro-8a-methyl-5-methylidenenaphtho[2,3-b]-
furan-2(3H)-one (4k). 1H-NMR (400 MHz, CDCl3): 4.68 (s, 1 H); 4.41 (br. s, 1 H); 4.37 (s, 1 H); 2.93 (dd,
J¼7.2, 11.6, 1 H); 2.83 (dd, J¼6.4, 13.2, 1 H); 2.67 (dd, J ¼ 6.8, 11.6, 1 H); 2.50–2.60 (m, 2 H); 2.24 (br. d,
J¼12.4, 2 H); 2.06 (br. d, J ¼ 16.4, 1 H); 1.85–1.95 (m, 1 H); 1.70 (d, J¼12.0, 1 H); 0.78 (t, J ¼ 6.8, 3 H);
0.71 (s, 3 H). 13C-NMR (100 MHz, CDCl3): 178.4; 149.3; 106.5; 78.3; 50.2; 47.4; 46.5; 45.4; 42.2; 41.4; 39.1;
36.8; 34.8; 31.9; 29.9; 29.6 (br., 4 C); 29.5; 29.4; 27.3; 22.7; 21.2; 17.8; 14.2. EI-MS (70 eV): 262 (100, Mþ ),
390 (80), 44 (27), 391 (21), 263 (18). HR-ESI-MS: 390.3333 ([MþH]þ , C25H44NO2þ ; calc. 390.3372).
(3aR,5S,8aR,9aR)-3-[(Decylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethylnaphtho[2,3-
b]furan-2(3H)-one (3k). 1H-NMR (400 MHz, CDCl3): 4.99 (br. s, 1 H); 4.54–4.58 (m, 1 H); 2.92–2.98
(m, 1 H); 2.72–2.82 (m, 1 H); 2.57 (dd, J¼6.8, 10.8, 1 H); 2.40–2.50 (m, 2 H); 2.25–2.35 (m, 1 H); 1.91
(dd, J¼2.4, 14.8, 1 H); 1.60–1.68 (m, 1 H); 1.05 (s, 3 H); 0.95 (d, J ¼ 7.2, 3 H); 0.70 (t, J ¼ 6.8, 3 H).
13C-NMR (100 MHz, CDCl3): 177.9; 150.6; 115.4; 77.7; 50.1; 46.7; 45.7; 42.8; 42.2; 38.5; 37.7; 32.9; 32.8;
31.9; 30.0; 29.6 (br., 4 C); 29.4; 28.7; 27.4; 22.9; 22.7; 16.9; 14.1. EI-MS (70 eV): 262 (100, Mþ ), 390 (66),
44 (30), 158 (24), 391 (17). HR-ESI-MS: 390.3351 ([MþH]þ , C25H44NO2þ ; calc. 390.3345).
(3aR,4aS,8aR,9aR)-Decahydro-8a-methyl-5-methylidene-3-[(undecylamino)methyl]naphtho[2,3-
b]furan-2(3H)-one (4l). 1H-NMR (400 MHz, CDCl3): 4.71 (s, 1 H); 4.44 (br. s, 1 H); 4.40 (s, 1 H); 2.89–
3.00 (m, 2 H); 2.70–2.80 (m, 1 H); 2.55–2.63 (m, 1 H); 2.42–2.50 (m, 1 H); 2.26 (d, J¼12.8, 1 H); 2.10
(d, J ¼ 15.6, 1 H); 1.90–1.99 (m, 1 H); 1.72 (d, J ¼ 12.0, 1 H); 0.82 (t, J ¼ 7.2, 3 H); 0.74 (s, 3 H).
13C-NMR (150 MHz, CDCl3): 178.5; 149.4; 106.7; 78.5; 50.3; 47.4; 46.7; 45.5; 42.4; 41.6; 39.3; 36.9; 34.9;
32.1; 29.9 (br., 6 C); 29.5; 27.5; 22.9; 21.3; 18.0; 14.4. EI-MS (70 eV): 262 (100, Mþ ), 44 (37), 41 (22), 170
(19), 172 (17). HR-ESI-MS: 404.3529 ([MþH]þ , C26H46NO2þ ; calc. 404.3528).
(3aR,5S,8aR,9aR)-3a,5,6,7,8,8a,9,9a-Octahydro-5,8a-dimethyl-3-[(undecylamino)methyl]naphtho-
[2,3-b]furan-2(3H)-one (3l). 1H-NMR (400 MHz, CDCl3): 5.09 (s, 1 H); 4.68 (br. s, 1 H); 3.10–3.02 (m,
1 H); 2.86–2.98 (m, 2 H); 2.70 (dd, J¼6.4, 10.8, 1 H); 2.50–2.57 (m, 2 H); 2.38–2.48 (m, 1 H); 2.03 (d,
J¼15.2, 1 H); 1.17 (s, 3 H); 1.06 (d, J¼8.0, 3 H); 0.81 (t, J ¼ 6.8, 3 H). 13C-NMR (150 MHz, CDCl3):
178.3; 151.2; 115.2; 77.5; 50.3; 46.7; 45.8; 42.9; 42.4; 38.7; 37.9; 33.2; 33.1; 32.1; 29.9; 29.8 (br., 4 C); 29.6;
28.9; 27.5; 23.1; 22.9; 17.1; 14.4. EI-MS (70 eV): 262 (100, Mþ ), 207 (81), 171 (72), 217 (67), 91 (54). HR-
ESI-MS: 404.3542 ([MþH]þ , C26H46NO2þ ; calc. 404.3528).

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
(3aR,4aS,8aR,9aR)-3-[(Dodecylamino)methyl]decahydro-8a-methyl-5-methylidenenaphtho[2,3-
b]furan-2(3H)-one (4m). 1H-NMR (400 MHz, CDCl3): 4.70 (s, 1 H); 4.41 (br. s, 1 H); 4.39 (s, 1 H); 2.90–
2.96 (m, 1 H); 2.82–2.89 (m, 1 H); 2.65–2.72 (m, 1 H); 2.53–2.62 (m, 2 H); 2.41–2.45 (m, 1 H); 2.25 (d,
J ¼ 12.4, 1 H); 1.70 (d, J ¼ 12.0, 1 H); 0.79 (t, J ¼ 6.8, 3 H); 0.73 (s, 3 H). 13C-NMR (150 MHz, CDCl3):
178.4; 149.3; 106.5; 78.3; 50.3; 47.4; 46.5; 45.4; 42.3; 41.5; 39.2; 36.8; 34.8; 31.9; 29.9; 29.7; 29.6 (br., 5 C);
29.4; 27.4; 22.8; 21.2; 17.9; 14.2. EI-MS (70 eV): 262 (100, Mþ ), 207 (62), 44 (43), 91 (63), 281 (32). HR-
ESI-MS: 418.3720 ([MþH]þ , C27H48NO2þ ; calc. 418.3685).
(3aR,5S,8aR,9aR)-3-[(Dodecylamino)methyl]-3a,5,6,7,8,8a,9,9a-octahydro-5,8a-dimethylnaph-
tho[2,3-b]furan-2(3H)-one (3m). 1H-NMR (400 MHz, CDCl3): 5.06 (br. s, 1 H); 4.67 (br. s, 1 H); 3.01–
3.10 (m, 1 H); 2.88–2.90 (m, 2 H); 2.69 (dd, J¼7.2, 12.0, 1 H); 2.56–2.59 (m, 2 H); 2.36–2.42 (m, 1 H);
2.22–2.30 (m, 2 H); 2.02 (dd, J ¼ 2.8, 14.8, 1 H); 1.16 (s, 3 H); 1.05 (d, J ¼ 7.6, 3 H); 0.79 (t, J ¼ 7.0, 3 H).
13C-NMR (150 MHz, CDCl3): 178.3; 150.9; 115.2; 77.5; 50.2; 46.7; 45.7; 42.8; 42.3; 38.6; 37.8; 33.1; 32.9;
31.9; 29.9; 29.7 (br., 5 C); 29.4; 28.7; 27.4; 23.0; 22.8; 16.9; 14.2. EI-MS (70 eV): 262 (100, Mþ ), 207 (95),
281 (49), 209 (43), 217 (40). HR-ESI-MS: 418.3734 ([MþH]þ , C27H48NO2þ ; calc. 418.3685).
Aedes aegypti. The Orlando strain of Ae. aegypti was reared in the insectary of the Mosquito and Fly
Research Unit at Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), USDA-
ARS. The Orlando strain of Ae. aegypti has been established in CMAVE since 1952. Female adults were
used for all experiments, since only females take blood meals and are the main concern of the general
public. Eggs were hatched by placing a square of a paper towel with eggs in a flask filled with 1000 ml of
dist. H2O containing 40 mg of larval diet (3 :2 brewerꢃs yeast/liver powder (MP Biomedicals, Irvine,
CA)). The hatched larvae were held overnight in the flask, and 200 larvae were transferred to a 4-l plastic
tray containing 2 l of dist. H2O. Larval diet was added to each tray according to the following schedule:
day 1, 80 mg; day 3, 40 mg; day 4, 80 mg; day 5, 120 mg; and day 6, 150 mg. Mosquitoes were reared in an
environmental chamber set with a temp. profile representing a simulated summer day regime (ranging
from 22 to 308) and 80% relative humidity (RH). Incandescent lighting was set to a crepuscular profile
with a photoperiod of 14 :10 light/dark (L/D) h, including 2 h of simulated dawn and 2 h of simulated
dusk. Adults were held in a screened cage and provided 10% sucrose ad libitum. Bovine blood in 1%
heparin that had been placed in a pig intestine and warmed to 378 was provided to adults twice a week.
Eggs were collected on paper towels (Vasco Brands, Elmira, NY) that lined the rim of H2O containers.
These egg-laden papers were air-dried at 278 and 80% RH for 24 h, and stored in containers with 100%
humidity for 3–30 d. When needed, eggs were hatched under vacuum and larvae were reared in
containers as described above.
Larvae Bioassays. Larval bioassays were performed as described in [30]. Briefly, five first instar
larvae of Ae. aegypti were added to each well of 24-well plates. Deionized H2O (950 ml) and larval diet
(40 ml) were then added to each well. All chemicals to be evaluated were diluted in acetone. Decreased
concentrations were used to further group the chemicals as highly active, moderately active, slightly
active, or highly inactive. Dil. chemicals (10 ml) were then added to each well containing a total volume of
1 ml of larvae, food, and H2O. As control treatments, 10 ml of acetone alone was added to each well.
Larval mortality was recorded after 24 h of exposure. The larval assays were repeated several times on
different days with six concentrations providing a range of 0–100% mortality.
Adult Bioassays. To determine the toxicity of each chemical against female Ae. aegypti, chemicals
were serially diluted in acetone and topically applied to individual mosquitoes. Prior to topical
application, 5–7-day-old females were briefly anaesthetized for 30 s with CO2 and placed on a 48 chill
table (BioQuip Products, Rancho Dominguez, CA). A droplet of 0.5 ml of chemical soln. was applied to
the dorsal thorax using a 700 series syringe and a PB 600 repeating dispenser (Hamilton, Reno, NV). Six
concentrations providing a range of 0–100% mortality were used on 25–30 females per concentration.
Tests were replicated three times. Control treatments with 0.5 ml of acetone alone gave control mortality
with less than 10%. After treatment, mosquitoes were kept in plastic cups and supplied with 10% sucrose
soln. for 24 h before mortality was recorded. Temp. and humidity were maintained at 268 and 80% RH,
resp. Every bioassay was conducted at 278 and 80% RH and replicated three times. Bioassay data were
analyzed using PoloPlus probit and logit analysis software (LeOra Software, Petaluma, CA). Chi-
squared goodness of fit test was performed, and LD50/LD95 values were calculated using PoloPlus
program.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1697
The authors thank Amber Reichley, Solomon Green III, and Willian Reid for technical assistance.
This study was supported, in part, by Deployed War-Fighter Protection Research Program Grant funded
by the U.S. Department of Defense through the Armed Forces Pest Management Board.
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Received February 1, 2010