Synthesis and antiviral activity of scopadulcic acids analogues

Article abstract of DOI:10.1016/S0040-4020(03)01071-8

The synthesis and antiviral properties of several scopadulcic acid analogues functionalized at C-6/C-7 and C-13 is reported. The preparation of advanced intermediates for the synthesis of scopadulcic acid B/scopadulciol analogues is also described. The biological study revealed the importance of polar groups at C-13, while the stereochemistry at C-8 was not critical for activity.

Full text of DOI:10.1016/S0040-4020(03)01071-8

Tetrahedron 59 (2003) 6455–6464
Synthesis and antiviral activity of scopadulcic acids analogues
a
Manuel Arno, Liliana Betancur-Galvis, Juan G. Bueno-Sanchez, Miguel A. Gonzalez
b
b
a
,
*
´
´
a
,
*
´
´
and Ramon J. Zaragoza
a
´
´
Departamento de Quımica Organica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain
´
b
´ ´
Grupo Infeccion y Cancer, Universidad de Antioquia, A.A1226, Medellın, Colombia
Received 11 March 2003; revised 30 May 2003; accepted 7 July 2003
Abstract—The synthesis and antiviral properties of several scopadulcic acid analogues functionalized at C-6/C-7 and C-13 is reported. The
preparation of advanced intermediates for the synthesis of scopadulcic acid B/scopadulciol analogues is also described. The biological study
revealed the importance of polar groups at C-13, while the stereochemistry at C-8 was not critical for activity.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
such as thyrsiflorin B acetate (10, TBA) was isolated from
C. dentata11 together with isovaleroyl esters 11 and 12,
which were also found in C. glabrata (Fig. 1).12
The search for new bioactive compounds from Scoparia
dulcis L. (Scrophulariaceae) species led to the discovery of
three diterpenoids, all possessing a new carbon skeleton (1),
the scopadulcic acids A (2, SDA) and B (3, SDB) which
were isolated from Paraguayan plants1 and scopadulciol (4,
SDC) from plants collected in Taiwan.2 Scopadulciol (4),
originally named dulcinol, was described earlier as a
constituent of S. Dulcis collected in Bangladesh,3 which is
also present in specimens from China and Thailand.4
During the past decade, Hayashi and co-workers
proved that these natural products, and some derivatives,
exhibit an amazing pattern of biological activities,
including antiviral, antitumour, inhibition of gastric acid
secretion and bone resorption as well as potentiation of
known antiviral drugs.5 Recently, SDA (2) has shown in
vitro activity against the malaria parasite Plasmodium
falciparum.6
In view of the broad biological activity shown by the
scopadulcic acids and some derivatives, their production by
leaf culture13 and chemical synthesis14 has attracted the
attention of biochemists and synthetic chemists. Concurrent
to our studies towards the synthesis of TA (5),15 starting
from (þ)-podocarp-(8)14-en-13-one (13) via enone 14,
The systematic study of phytochemicals from plants of the
genus Calceolaria (Scrophulariaceae) led by Professor
Garbarino has resulted in the isolation of 77 new diterpenes,
including thyrsiflorane-type diterpenes having a carbon
framework identical to that of scopadulcic acids.7,8 For
instance, thyrsiflorin A (5, TA), thyrsiflorin B (6, TB),
and thyrsiflorin C (7, TC) were identified as constituents
of C. thyrsiflora, where the acids were isolated as
their corresponding methyl esters.9 More recently, several
new thyrsiflorins were isolated such as 13-hydroxy-
thyrsiflorin 8 and its corresponding acetate 9 from C. recta
and C. paposana.10 Another C7-functionalized member
Keywords: scopadulcic; diterpenes; thyrsiflorin; thyrsifloranes; antivirals.
*
Corresponding authors. Tel.: þ34-963544327; fax: þ34-963544328;
e-mail: miguel.a.gonzalez@uv.es; ramon.j.zaragoza@uv.es
Figure 1.
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01071-8

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2. Results and discussion
Enone 14, ketal 17 and 7,13-dienyl acetate 18, which were
used as precursors for the synthesis of the C6- and C7-
functionalized scopadulanes, were prepared from podo-
carpenone (þ)-13 following our reported synthetic strategy
(Schemes 1 and 2).18
Scheme 2. Synthesis of intermediates 17 and 18.
The synthesis of several scopadulcic acids analogues
functionalized at C-7 and C-13 starting from 17 and 18 is
outlined in Scheme 3. First, hydroboration of 17 followed by
oxidation with H2O2/NaOH gave the 7b-alcohol 19 in 71%
yield together with 7a-alcohol 20 (22%). Benzoylation of
19 with freshly distilled BzCl and 4-pyrrolidinopyridine
(4-PP) as catalyst in Et3N followed by hydrolysis with
aqueous HCl gave benzoate 21 in 65% overall yield.
Reduction of 21 using NaBH4 in MeOH gave 13a-alcohol
22 as the major product in 70% yield. Similarly, the 8a-
scopadulane alcohol 20 gave 7a-benzoate 23 (70% over two
steps). Reduction of 23 with NaBH4/MeOH gave 13b-
alcohol 24 in 62% yield accompanied by a 22% of the 13a-
epimer 25. The stereochemistry at C-13 was determined by
NOE experiments. In particular, irradiation of the proton
signal H-7 in 25 gave a clear NOE enhancement of the
signal assigned to proton H-13. On the other hand,
stereoselective epoxidation of dienyl acetate 18 with
m-chloroperoxybenzoic acid (MCPBA) (see Fig. 2)
followed by treatment with Na2S2O3/NaHCO3 gave the
7a-hydroxy enone 26 in 67% yield. Assignment of the
a-orientation of the 7-OH group in 26 may be inferred from
its spectroscopic data, for example the 13C NMR signal due
to C-5 which is shifted upfield with respect to the similar
signal in enone 14 (41.2 and 47.2, respectively) due to the
g-effect exerted by the OH group on C-7. Also the splitting
pattern showed for H-7 (dd, J¼3.4, 2.9 Hz) is in agreement
with an equatorial orientation. Enone 26 was next
benzoylated to afford benzoate 27 in 70% yield.
Scheme 1. Conversion of Abietic acid into scopadulenones 14 and 16, via
podocarpenones 13 and 15.
Tagat and co-workers described the chiral preparation of the
enone 16 using a different strategy from podocarpenone
15,16 which was prepared from abietic acid using a
modification of the protocol developed in our laboratory
(Scheme 1).17 A few years later, we reported the
versatility of enone 14 for the synthesis of C7-functiona-
lized thyrsiflorins such as TBA (10) and TC (7).18 A lack
of reports on the biological activity of the
thyrsiflorins prompted us to carry out a biological
evaluation of several compounds prepared during the
course of our synthetic studies.19 As a result, four of
these compounds showed moderate in vitro antiviral
activity against Herpes simplex virus type II (HSV-2),
likely due to their cytotoxicity. The cytotoxic
activity against two human tumour cell lines (HeLa and
HEp-2), however, was too weak to prompt further
investigation.
Following the preparation of scopadulcic acids analogues
22–27, we turned our attention towards the functionaliza-
tion of compounds 17 and 18 at position C-6 to prepare
additional analogues and to explore the introduction of a
6b-benzoyl moiety via allylic oxidation (Scheme 4). We
attempted several methods for the allylic oxidation at C-6 in
alkene 17 to obtain enone 28. Reduction of the enone system
in 28 would give an equivalent intermediate used by Ziegler
and Wallace to synthesize SDB and SDC.14c Treatment of
17 with N-bromosuccinamide (NBS) and CaCO3 in aqueous
dioxane,20 chromium trioxide/3,5-dimethylpyrazole (CrO3
3,5-DMP) complex,21 hexacarbonyl chromium (Cr(CO)6)/
In this paper, we report the synthesis of several
scopadulcic acids analogues (21–25, 27, and 34)
functionalized at C-7 and C-13, and the chemical
studies towards functionalization at the C-6 position of
enone 14 en route to prepare SDB and SDC. The in vitro
anti-HSV-2 activity of these analogues and several C6-
functionalized scopadulanes (35–38, and 41) is also
described.

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6457
Figure 2. Steric interactions during the MCPBA epoxidation of the C7–C8
double bond of dienolacetate 18. It can be seen a major steric hindrance in
the attack from the b-face.
cobalt salts for allylic oxidations with TBHP encouraged us
to carry out further experimentation in this line. Thus, we
attempted the oxidation of 17 with TBHP in the presence of
either ruthenium trichloride (RuCl3) in acetonitrile and
dichloroethane,24 copper iodide (CuI) in acetonitrile,25 or
cobalt acetate (Co(OAc)2·4H2O) in acetonitrile.26 In spite of
observing some reaction using Co(OAc)2, the results did not
merit further optimization. We also considered the
possibility of carrying out a Karasch–Sosnovsky benzoyl-
oxylation27 on 17 with the hope that this metal-mediated
reaction would favour, at least in some extent, the allylic
hydrogen abstraction at C-6. If successful, we could then
obtain directly the desired 6b-benzoate group (30). Not
surprisingly, in all attempts the substrate 17 failed to react
under the conditions used.28
Scheme 3. Synthesis of scopadulcic acids analogues functionalised at C-7,
C-8, and C-13. Reagents and conditions: (a) (i) BH3/THF, (ii) H2O2/NaOH;
(b) 4-PP, BzCl, Et3N; (c) 9:1 acetone/12 M HCl; (d) BzCl, pyridine;
(e) NaBH4/MeOH; (f) (i) m-CPBA, (ii) NaS2O3/NaHCO3.
tert-butylhydroperoxide (TBHP),22 and pyridium dichro-
mate (PDC)/TBHP in the presence of celite,23 led to
complex mixtures of products. Among the reaction products
of 17 with PDC/TBHP we observed some compounds
without the ketal moiety. Therefore, we repeated this
oxidation in the presence of pyridine, and under these
basic conditions the reaction was cleaner giving as major
product the ketal enone 29 in 50% yield, accompanied by
other products, including a compound whose 1H NMR
spectrum was assigned to the desired structure 28. However,
28 was obtained in less than 5% yield. The structure
assignment of compound 29 was confirmed after deketali-
zation giving rise to g-diketone 34 (80%), which was also
obtained from enol acetate 18 as we describe later.
After our fruitless efforts to oxidize alkene 17 to the
unsaturated ketone 28, or ester 30, we subjected the alkene
17 to typical allylic hydroxylation conditions, which would
lead to the desired enone 28 after oxidation of the alcohol
31. The starting alkene 17 was recovered after treatment
with trimethylamine N-oxide and selenium dioxide (SeO2)
in dioxane,29 however, the use of catalytic SeO2 and TBHP
in DCM30 gave a single allylic alcohol in excellent yield
(95%). Unfortunately, a comparison between the NMR data
of the starting material and this alcohol revealed that the
Recent reports on the use of catalytic ruthenium, copper and

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M. Arno et al. / Tetrahedron 59 (2003) 6455–6464
6458
reported the synthesis of SDB from a similar dienone
system.14d Compounds with C6-hydroxy functionalization
can be obtained by selective epoxidation of the C6–C7
double bond in 35 and regioselective opening of the epoxide
moiety (Scheme 5). Initial treatment of dienyl acetate 18
using N-bromoacetamide in acetone followed by hydrolysis
with CaCO3 in DMF32 failed to give the dienone 35 leading
to a mixture of products. Eventually, the treatment of enone
14 with dichlorodicyanoquinone (DDQ) in refluxing
benzene gave the desired dienone 35 in high yield (89%).
However, epoxidation of the C6–C7 double bond in 35 was
low yielding, though highly stereoselective, in agreement
with the observations (a-epoxide in 40% yield) by Tagat
and co-workers for the epoxidation of the 18-carbomethoxy
analogue of dienone 35 with MCPBA/NaHCO3.16b They
pointed out that this result could arise from partial
deprotonation of H-5 leading to by-products of oxidation
in ring C. Initial attempts to epoxidise 35 were conducted
using p-nitroperoxybenzoic acid in chloroform,33 however,
Scheme 4. Allyic oxidation of intermediates 17, 18 and 14. Reagents and
conditions: (a) PDC, TBHP, celite, pyridine, ,5% of 28, 50% of 29; (b) cat.
SeO2, TBHP, 95%; (c) Ac2O, 4-PP, Et3N; (d) DDQ, PhH, 89%.
structure of the latter was actually 32. The two H-14 signals
of 17 disappeared in the 1H NMR spectrum of the product,
proton H-14 in 32 was shown as a singlet at 3.86 ppm, and
the chemical shifts (13C) for carbons C-5, C-6, and C-7
remained unchanged. The stereochemistry of C-14 was
determined by NOE difference studies of the corresponding
acetate (33).31
Parallel experiments of allylic oxidation of 7,13-dienyl
acetate 18 using the conditions applied to compound 17
were also attempted, but most often gave rise to complex
mixtures of products. For example, when compound 18 was
treated with PDC/TBHP/celite/pyridine the g-diketone 34
was obtained in 63% yield. This transformation probably
occurs through the enone 14 since treatment of enone 14,
under the same conditions, gave the diketone 34 as major
product.
The failure of our own attempts of allylic oxidation to
incorporate an oxygenated function at C-6 forced us to
abandon this approach and focus our attention on the
preparation of dienone 35, since Overman and co-workers
Scheme 5. Functionalization of scopadulenone 14 at C-6. Reagents and
conditions: (a) m-CPBA, pH¼8, DCM, reflux, 50%; (b) NaTeH, EtoH,
60%; (c) Li, NH3, THF, 2358C; (d) PCC, alumina, 70%; (e) MOMCI,
iPr2NEt, 75%.

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6459
the desired epoxide 36 was not obtained. Little improve-
ment was observed when the reaction mixture was buffered
at pH¼8, so we then changed the oxidizing agent to
MCPBA. After some optimization we were able to obtain
50% yield of the epoxide 36. For this result the MCPBA was
added to a mixture of dienone 35 in DCM and pH¼8 buffer
in several portions (2.4 equiv.) over a period of 48 h heating
at 408C. The stereochemistry of the epoxide 36 was assured
by NOE difference experiments, in particular, irradiation of
the C-20 methyl signal gave NOE enhancement of the
signals due to protons H-6, H-7 and H-19, and therefore
confirming the a-orientation of the oxirane ring.
Table 1. Cytotoxicity and anti-HSV-2 activity of scopadulane-type
diterpenes on vero cells determined the end-point titration technique
(EPTT)
Compound
CC100
(mg/mL)a
Viral reduction factorb
Antiviral activity
(mg/mL)c
8d
32
40
.50
25
.50
50
50
28
50
35
.50
50
50
25
50
.600
102.0
102.0
100.5
100.5
NA
7.5
20
12.5
12.5
NA
25
25
14
25
17.5
25
25
25
12.5
25
6.0
14d
21
22
23
24
101.5
101.0
101.0
101.5
101.0
101.0
100.5
101.5
100.5
100.5
104
25
26d
27
With the epoxide 36 in hand, we studied the regioselective
opening of the oxirane ring at the allylic carbon (Scheme 5).
Acid-catalysed opening of the epoxide 36 was attempted
with PTSA leading to a mixture of products.34 Following the
precedent of Overman and co-workers,14d epoxide 36 was
treated with sodium hydrogen telluride (NaTeH),35 a known
reducing agent for the opening of a,b-epoxy-ketones and
esters at the a-position. To our surprise, we obtained alcohol
3736 as a single stereoisomer in modest yield (60%) instead
of the expected alcohol 38. This anomalous ‘Luche-type’
reduction could be controlled by the significant steric
hindrance of the b-face, preventing the attack of the hydride
species to either the oxirane ring or even the enone
system.37 Regioselective epoxide opening of similar
epoxy-enone systems such as 6a,7a-epoxy–steroid deriva-
tives using lithium–ammonia is a known method for
introducing a hydroxy group at C-7 in the testosterone
skeleton.38 The carbanionic character generated under these
conditions at the b-position of the enone system initiates the
cleavage of the oxirane ring at the allylic position.
Accordingly, treatment of 36 under these conditions led to
40% yield of the desired alcohol 38 accompanied by a
product of further reduction, diol 39 (33%).39 These
conditions were then used to reduce the enone system in
38 to give ketone 40 in 26% yield and diol 41 in 55%
yield.39
34
35
36
37
38
41
Acyclovir
VERO, Cercopithecus aethiops african green monkey kidney cell line
ATCC CCL 81.
a
Minimal toxic dose that detached 100% of the cell monolayer.
Ratio of the virus titer in the absence over virus titer in the presence of the
b
tested compound.
Maximal nontoxic dose that showed the highest viral reduction factor.
c
NA, no activity.
Data taken from Ref. 19.
d
cytotoxicity. The antiviral and cytotoxic activities of the
compounds and the positive control acyclovir against HSV-
2 were determined initially using a modified end-point
titration technique (EPPT)19 and are shown in Table 1. Most
of the compounds reduced the virus replication, though very
weakly. The examination of this preliminary assay indicates
the following: benzoates 21 and 27, analogues of SDB
lacking the carboxylic acid at C-4 and benzoate at C-6, have
no significant activity. Among the benzoates in the 8a-
scopadulane series, compounds 23–25, only 24 exhibited
some activity while 23 showed no activity at all. Among the
derivatives of enone 14, the compounds 27, 35 and 37 were
the only ones that showed slight activity. As can be seen in
Table 1, the highest antiviral activity was found in
compounds 24, 27 and 37 (a reduction factor of the virus
titer of 101.5). More accurate values of anti-HSV-2 activity
for compounds 24, 27 and 37 were obtained by the standard
plaque reduction assay and are given in Table 2. Therefore,
none of the new derivatives improved the highest inhibitory
values previously reported by us for other scopadulanes
derivatives, including alcohol 8 and enone 14. Comparison
of the data for 8 with derivatives 22 and 41 suggests that 6a
and 7b polar substituents in the scopadulane skeleton reduce
the activity. Furthermore, in the series of enone 14
derivatives the 6a-hydroxy enone 38 is much less active.
Finally, compounds 40 and 41 could be converted into SDB
analogues, lacking the carboxylic acid at C-4, using
standard functional group manipulation. In fact, diol 41
was oxidized selectively to ketone 42 in 70% yield using
pyridinium chlorochromate (PCC) adsorbed on alumina,40
which was then converted into its corresponding methoxy-
methyl (MOM) ether 43 (75%). Compound 43 is an
equivalent intermediate to those used by Overman and
co-workers in the synthesis of SDA and SDB.
The preparation of the scopadulane acid analogues (21–25,
27, and 34) and several C6-functionalized scopadulane
derivatives (35–38, and 41) enabled us to study the antiviral
activities of these compounds through biological testing.
Table 2. Anti-HSV-2 activity of scopadulane on vero cells determined by
plaque reduction assay
3. Antiviral evaluation
Compound
Antiviral activity (ED50) (mg/mL)
The antiviral activities of the scopadulcic acid analogues
(21–25, 27, and 34) including compounds 35–38, and 41
were evaluated against a common sexually transmitted
pathogen, Herpes simplex virus type 2 (HSV-2). These were
investigated at different concentrations depending on
24
27
37
6.7^0.7
5.4^0.4
9.3^0.6
VERO, Cercopithecus aethiops african green monkey kidney ATCC CCL
81.

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M. Arno et al. / Tetrahedron 59 (2003) 6455–6464
6460
In summary, as we described earlier,19 these results confirm
the importance of polar groups at C-13, as well as the
finding that the stereochemistry at C-8 is not critical for
antiviral activity, though in comparison with the anti-HSV-1
activity of SDB and analogues41 it is clear that the
carboxylic acid at C-4 and 6b polar substituents are
necessary for important antiviral properties.
(3H, m), 1.11, 1.08, 0.90 and 0.85 (3H each, each s, H-17,
H-18, H-19 and H-20); 13C NMR (75 MHz) dC 213.3 (s),
166.3 (s), 133.0 (d), 130.2 (s), 129.5£2 (d), 128.3£2 (d),
76.8 (d), 53.8 (s), 52.4 (s), 46.2 (d), 45.1 (t), 44.4 (d), 41.8
(t), 39.9 (t), 38.7 (s), 36.6 (t), 33.5 (q), 33.2 (s), 32.2 (t), 28.0
(t), 25.5 (t), 21.9 (q), 19.6 (q), 18.5 (t), 17.5 (q); HRMS (CI)
C27H36O3 requires 408.2664 (Mþ), found 408.2607.
4.1.2. 7b-Benzyloxyscopadulan-13a-ol (22). To a solution
of ketone 21 (8.3 mg, 0.020 mmol) in MeOH (0.8 mL) at
08C, NaBH4 (98%, 8 mg, 0.204 mmol) was added in one
portion. The reaction mixture was stirred for 50 min and
then diluted with diethyl ether. Usual workup followed by
column chromatography, using hexane–ethyl acetate (from
95:5 to 9:1) as eluent, yielded the a-alcohol 22 (5.8 mg,
70%) as a white solid: mp 176–1788C (from hexane–ethyl
acetate); [a]2D6¼þ30.2 (c 1.1, CHCl3); IR (NaCl) 3600–
3300, 1715, 1451, 1280 cm21; 1H NMR (300 MHz) d 8.05
(2H, m, OCOPh), 7.56 (1H, m, OCOPh), 7.44 (2H, m,
OCOPh), 4.96 (1H, ddd, J¼10.9, 10.9, 5.5 Hz, H-7), 3.39
(1H, dd, J¼10.9, 5.6 Hz, H-13), 1.07, 1.02, 0.86 and 0.82
(3H each, each s, H-17, H-18, H-19 and H-20); 13C NMR
(75 MHz) dC 166.3 (s), 132.8 (d), 130.6 (s), 129.5£2 (d),
128.3£2 (d), 77.0 (d), 76.0 (d), 53.2 (s), 46.0 (d), 44.6 (t),
44.1 (s), 42.2 (d), 42.0 (t), 38.4 (s), 34.2 (t), 33.5 (q), 33.2
(s), 32.3 (t), 30.6 (t), 28.0 (t), 25.9 (t), 23.1 (q), 22.0 (q), 18.6
(t), 17.6 (q); HRMS (CI) C27H38O3 requires 410.2812 (Mþ),
found 410.2846.
4. Experimental
4.1. General experimental details
Melting points were measured on a Kofler hot-stage
apparatus and are uncorrected. Optical rotations were
determined using a 5-cm path-length cell, using chloroform
as solvent (concentration expressed in g/100 mL). [a]D-
values are given in 1021 deg cm2 g21. IR spectra were taken
as KBr pellets, liquid films on NaCl plates or solutions in
chloroform. NMR spectra were recorded on 300 or
400 MHz spectrometers with tetramethylsilane as an
internal standard. All spectra were recorded in CDCl3 as
solvent. Complete assignments of 13C NMR multiplicities
were made on the basis of DEPT experiments. HMQC and
NOE experiments were used in some 1H NMR assignments.
J values are given in Hz. For all compounds, NMR
assignments are given with respect to the numbering
scheme shown in structure 1. Reactions were monitored
by thin-layer chromatography (TLC) using Merck silica gel
60 F-254 in 0.25 mm-thick plates. Compounds on TLC
plates were detected under UV light at 254 nm and
visualized by immersion in a 10% sulfuric acid solution
and heating on a hotplate. Purifications were performed by
flash chromatography on Merck silica gel (230–400 mesh).
All non-aqueous reactions were carried out in an argon
atmosphere in oven-dried glassware. Commercial reagent
grade solvents and chemicals were used as received unless
otherwise noted. THF was freshly distilled from sodium
benzophenone ketyl under argon atmosphere. Combined
organic extracts were washed with brine, dried over
anhydrous sodium sulphate, filtered and concentrated
under reduced pressure. Preparation and spectroscopic
data for compounds 14, and 17–20 can be found in Ref. 18.
4.1.3. 7a-Benzyloxy-8a-scopadulan-13-one (23). A sol-
ution of ketal–alcohol 20 (12.0 mg, 0.034 mmol) in dry
pyridine (1.6 mL) cooled in an ice-bath was treated with
freshly distilled benzoyl chloride (120 mL, 1.06 mmol).
After being stirred for 15 h at room temperature, the
reaction mixture was diluted with diethyl ether, washed with
5% aqueous HCl and brine, dried and concentrated. The
crude benzoate was dissolved in acetone/HCl 12 M (9:1,
2.0 mL) and stirred for 30 min. Then, the reaction mixture
was diluted with diethyl ether, washed with 10% aqueous
NaHCO3 and brine, dried and concentrated. The obtained
residue was purified by column chromatography, using
hexane–ethyl acetate (from 95:5 to 9:1) as eluent, yielded
the benzoate 23 (9.6 mg, 70%) as a colourless oil:
[a]2D5¼2102.8 (c 0.9, CHCl3); IR (NaCl) 1714, 1451,
1
1275 cm21; H NMR (300 MHz) d 8.02 (2H, m, OCOPh),
4.1.1. 7b-Benzyloxyscopadulan-13-one (21). A solution of
ketal-alcohol 19 (20.0 mg, 0.057 mmol) and 4-pyrrolidino-
pyridine (98%, 0.9 mg, 6 mmol) as a catalyst in dry Et3N
(0.6 mL) cooled in an ice-bath was treated with freshly
distilled benzoyl chloride (30 mL, 0.265 mmol) dropwise.
After being stirred 3 h at room temperature, the reaction
mixture was diluted with diethyl ether. Workup as usual
gave the crude ketal-benzoate, which was hydrolyzed with a
9:1 mixture of acetone/12 M HCl (1.2 mL). After being
stirred for 30 min at room temperature, the mixture was
diluted with diethyl ether and washed with 10% aqueous
NaHCO3 solution, dried and concentrated. The residue was
purified by column chromatography, using hexane–ethyl
acetate (from 95:5 to 9:1) as eluent, to give the benzoate 21
(15.2 mg, 65%) as a colourless oil: [a]2D5¼þ64.0 (c 1.2,
CHCl3); IR (NaCl) 1710, 1275 cm21; 1H NMR (300 MHz)
d 8.03 (2H, m, OCOPh), 7.57 (1H, m, OCOPh), 7.44 (2H,
m, OCOPh), 5.10 (1H, ddd, J¼10.9, 10.9, 5.4 Hz, H-7),
2.56 (1H, dd, J¼15.5, 6.0 Hz), 2.43 (1H, m), 2.23–2.07
7.56 (1H, m, OCOPh), 7.44 (2H, m, OCOPh), 5.29 (1H, m,
H-7), 2.63 (1H, dd, J¼16.7, 5.9 Hz), 1.15, 1.09, 0.97 and
0.83 (3H each, each s, H-17, H-18, H-19 and H-20); 13C
NMR (75 MHz) dC 214.4 (s), 166.3 (s), 132.9 (d), 130.3 (s),
129.5£2 (d), 128.3£2 (d), 75.4 (d), 52.8 (s), 51.2 (s), 45.1
(d), 42.9 (d), 42.0 (t), 40.7 (t), 40.2 (t), 38.2 (s), 35.6 (t), 35.5
(t), 34.0 (t), 33.9 (s), 33.1 (q), 27.8 (t), 22.3 (q), 21.2 (q),
20.1 (q), 18.9 (t); HRMS (CI) C27H36O3 requires 408.2664
(Mþ), found 408.2622.
4.1.4. 7a-Benzyloxy-8a-scopadulan-13b-ol (24) and 7a-
Benzyloxy-8a-scopadulan-13a-ol (25). To a solution of
ketone 23 (12.7 mg, 0.031 mmol) in MeOH (1.2 mL) and
THF (0.3 mL) at 08C, NaBH4 (98%, 12 mg, 0.31 mmol) was
added in one portion. The reaction mixture was stirred for
50 min and then diluted with diethyl ether. Usual workup
followed by column chromatography, using hexane–ethyl
acetate (from 9:1 to 8:2) as eluent, gave the b-alcohol 24

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M. Arno et al. / Tetrahedron 59 (2003) 6455–6464
6461
(7.9 mg, 62%) followed by the a-alcohol 25 (2.8 mg, 22%)
as colourless oils. For compound 24: [a]2D6¼23.5 (c 1.1,
CHCl3); IR (NaCl) 3600–3370, 1699, 1451, 1283 cm21; 1H
NMR (300 MHz) d 8.05 (2H, m, OCOPh), 7.55 (1H, m,
OCOPh), 7.44 (2H, m, OCOPh), 5.90 (1H, ddd, J¼9.0, 6.8,
1.3 Hz, H-7), 3.46 (1H, br s, Wh/2¼7 Hz, H-13), 1.07, 0.95,
0.87£2 (3H each, each s, H-17, H-18, H-19 and H-20); 13C
NMR (75 MHz) dC 167.1 (s), 132.8 (d), 130.9 (s), 129.6£2
(d), 128.3£2 (d), 75.5 (d), 74.9 (d), 50.8 (s), 45.6 (d), 45.1
(s), 41.0 (t), 40.0 (d), 38.8 (s), 38.2 (t), 36.0 (t), 34.3 (t), 33.9
(q), 33.2 (s), 31.2 (t), 29.7 (t), 26.4 (t), 24.4 (q), 22.4 (q),
20.2 (t), 17.6 (q); HRMS (EI) C27H38O3 requires 410.2812
(Mþ), found 410.2821. For compound 25: [a]2D6¼231.1 (c
0.4, CHCl3); IR (NaCl) 3550–3350, 1712, 1596, 1452,
each, each s, H-17, H-18, H-19 and H-20); 13C NMR
(75 MHz) dC 204.9 (s), 163.2 (s), 133.0 (d), 130.3 (d), 129.8
(s), 129.5£2 (d), 128.5£2 (d), 72.7 (d), 58.2 (s), 50.7 (s),
45.0 (t), 42.5 (d), 41.9 (t), 37.3 (s), 33.8 (t), 33.4 (q), 33.1 (s),
32.9 (t), 29.7 (t), 29.6 (t), 22.4 (q), 21.1 (q), 20.1 (q), 18.3 (t);
HRMS (EI) C27H34O3 requires 406.2508 (Mþ), found
406.2511. Due to sample size one quaternary carbon signal
is not observed.
4.1.7. 13,13-Ethylenedioxy-8(14)-scopadulen-7-one (29).
To a mixture of alkene 17 (20 mg, 0.060 mmol) and oven-
dried celite (77 mg) under Argon, benzene (0.73 mL),
pyridine
(29 mL,
0.363 mmol),
PDC
(114 mg,
0.303 mmol) and t-BuOOH (80% in t-BuOH, 59 mL,
0.303 mmol) were sequentially added. After being stirred
for 32 h the reaction mixture was directly chromatographed
on silica gel, using hexane–ethyl acetate (from 95:5 to 9:1)
as eluent, to afford the enone 29 (10.4 mg, 50%) as an
amorphous white solid: [a]2D5¼240.0 (c 0.4); IR (NaCl)
1685, 1558, 1118 cm21; 1H NMR (300 MHz) d 6.51 (1H, s,
H-14), 4.15–4.05 (3H, m), 3.85 (1H, m), 2.51 (1H, d0d,
J¼19.4, 6.3 Hz, H-6), 2.36 (1H, dd, J¼19.4, 12.5 Hz, H-6 ),
1.08, 0.96, 0.92 and 0.85 (3H each, each s, H-17, H-18, H-19
and H-20); 13C NMR (75 MHz) dC 200.6 (s), 143.3 (s),
131.9 (d), 110.9 (s), 65.7 (t), 65.3 (t), 55.2 (s), 46.1 (s), 44.0
(d), 41.8 (t), 41.7 (t), 37.1 (t), 36.4 (s), 33.2 (s), 32.7 (q), 31.7
(t), 31.7 (t), 31.3 (t), 21.5 (q), 19.8 (q), 18.9 (q), 18.3 (t);
HRMS (EI) C22H32O3 requires 344.2351 (Mþ), found
344.2344. Deketalization of 29: enone 29 (6 mg,
0.017 mmol) was dissolved in a 9:1 mixture of acetone–
HCl (0.6 mL) and stirred for 30 min at room temperature.
After this time the reaction mixture was then diluted with
diethyl ether, washed with 10% aqueous NaHCO3 and brine.
Workup (Na2SO4) and flash chromatography, using 9:1
hexane–ethyl acetate as eluent, gave endione 34 (4.2 mg,
80%). Data: see below.
1
1278 cm21; H NMR (300 MHz) d 8.04 (2H, m, OCOPh),
7.57 (1H, m, OCOPh), 7.44 (2H, m, OCOPh), 5.22 (1H,
ddd, J¼9.0, 6.1, 1.1 Hz, H-7), 3.62 (1H, dd, J¼10.2, 5.5 Hz,
H-13), 1.05, 0.94, 0.84, 0.80 (3H each, each s, H-17, H-18,
H-19 and H-20); 13C NMR (75 MHz) dC 166.7 (s), 132.8
(d), 130.7 (s), 129.5£2 (d), 128.3£2 (d), 74.9 (d), 73.9 (d),
49.9 (s), 46.0 (d), 45.2 (s), 44.8 (t), 41.3 (d), 40.8 (t), 38.6
(s), 35.8 (t), 33.8 (q), 33.2 (s), 32.3 (t), 31.3 (t), 28.0 (t), 26.1
(t), 23.6 (q), 22.3 (q), 20.2 (t), 17.7 (q): HRMS (EI)
C27H38O3 requires 410.2812 (Mþ), found 410.2825.
4.1.5. 7a-Hydroxy-8(14)-scopadulen-13-one (26). Solid
85% m-chloroperoxybenzoic acid (6.5 mg, 0.032 mmol)
was added to a stirred solution of dienol acetate 18 (6.8 mg,
0.020 mmol) in 95% ethanol (0.16 mL) at 08C. The reaction
mixture was stirred for 6 h at room temperature, treated with
a solution of sodium thiosulphate (10 mg) and sodium
hydrogen carbonate (5 mg) in the minimum amount of
water and the stirring was continued for a further hour. The
solution was poured into cold water and extracted with
dichloromethane. The extracts were washed with sodium
hydrogen carbonate and brine. Workup as usual followed by
column chromatography, using hexane–ethyl acetate (6:4)
as eluent, yielded the alcohol 26 (4.2 mg, 67%) as a white
solid: [a]2D0¼þ15.3 (c 0.2, CHCl3); IR (NaCl) 3600–3100,
1705, 1666, 1596, 1461 cm21; 1H NMR (300 MHz) d 5.96
(1H, s, H-14), 4.50 (1H, dd, J¼3.4, 2.9 Hz, H-7), 1.17, 0.93
and 0.90£2 (3H each, each s, H-17, H-18, H-19 and H-20);
13C NMR (75 MHz) dC 205.3 (s), 169.5 (s), 127.8 (d), 70.8
(d), 58.0 (s), 45.0 (t), 42.0 (t), 41.2 (d), 37.5 (s), 34.1 (t), 33.4
(q), 33.1 (s), 32.9 (t), 30.4 (t), 29.7 (t), 22.6 (q), 21.1 (q),
20.1 (q), 18.3 (t); HRMS (EI) C20H30O2 requires 302.2246
(Mþ), found 302.2253. Due to sample size one quaternary
carbon signal is not observed.
4.1.8. 13,13-Ethylenedioxy-7-scopadulen-14a-ol (32). To
a stirred mixture of SeO2 (1.7 mg, 0.015 mmol) in DCM
(0.25 mL) and t-BuOOH (80% in t-butylperoxide; 11.0 mL,
0.09 mmol) during 20 min at room temperature, the ketal 17
(10 mg, 0.03 mmol) in DCM (0.5 mL) was added. After
being stirred for 1 h the solvent was removed and 1 mL of
MeOH and 2 mg of NaBH4 were added. After 5 min 1 mL
of water was added and the resulting mixture was diluted
with ethyl acetate, washed with brine and workup as usual.
The residue thus obtained was chromatographed on silica
gel, using 8:2 hexane–ethyl acetate as eluent, to give the
alcohol 32 (10.0 mg, 95%) as a colourless oil: [a]2D3¼277.5
(c 0.8); IR (NaCl) 3600–3400, 1456, 1118 cm21; 1H NMR
(300 MHz) d5.72 (1H, dd, J¼5.3, 2.2 Hz, H-7), 4.18–3.85
(4H, m), 3.86 (1H, s, H-14), 1.01, 0.92£2 and 0.84 (3H each,
each s, H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC
142.2 (s), 126.7 (d), 111.8 (s), 74.9 (d), 66.1 (t), 65.4 (t),
54.0 (s), 45.8 (s), 44.5 (d), 42.3 (t), 40.7 (t), 37.1 (s), 33.5
(q), 32.9 (s), 32.5 (t), 32.2 (t), 31.2 (t), 24.6 (t), 22.9 (q), 20.3
(q), 18.7 (t), 18.5 (q); HRMS (EI) C22H34O3 requires
346.2508 (Mþ), found 346.2505.
4.1.6. 7a-Benzyloxy-8(14)-scopadulen-13-one (27). To a
solution of alcohol 26 (3.0 mg, 9.8 mmol) and 4-pyrro-
lidinopyridine (98%, 0.02 mg, 0.13 mmol) as a catalyst in
dry Et3N (0.2 mL) cooled in an ice-bath, freshly distilled
benzoyl chloride (10 mL, 0.088 mmol) was added dropwise.
After being stirred 2 h at room temperature, the reaction
mixture was diluted with diethyl ether. Workup as usual
followed by column chromatography, using hexane–ethyl
acetate (from 98:2 to 9:1) as eluent, yielded the benzoate 27
(2.8 mg, 70%) as a colourless oil: [a]2D0¼þ29.0 (c 0.2,
CHCl3); IR (NaCl) 1713, 1677, 1596, 1264, 1107 cm21; 1H
NMR (300 MHz) d 7.94 (2H, m, –OCOPh), 7.56 (1H, m,
–OCOPh), 7.44 (2H, m, –OCOPh), 6.14 (1H, br s, H-14),
5.98 (1H, dd, J¼3.2, 3.2 Hz), 1.20, 1.01, 0.94 and 0.90 (3H
4.1.9. 8(14)-Scopadulen-7,13-dione (34). A mixture of
dienolacetate 18 (6.5 mg, 0.02 mmol), celite (25 mg) in
benzene (0.25 mL) and pyridine (7 mL, 0.08 mmol) was
treated with PDC (32 mg, 0.08 mmol) and t-BuOOH (5.5 M

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6462
in nonane, 15 mL, 0.08 mmol). After being stirred for 8 h
the reaction mixture was directly purified by flash
chromatography, using 95:5 hexane–ethyl acetate as eluent,
to give endione 34 (3.7 mg, 63%) as a yellow oil:
EtOH (0.2 mL) was added dropwise. After being stirred for
1 h 30 min, 0.1 mL of water were added and stirring
continued for 30 min more, then the reaction mixture was
filtered through celite. After concentration the obtained
residue was purified by chromatography, using 7:3 hexane–
ethyl acetate as eluent, to give 1.0 mg of recovered starting
material followed by the epoxyalcohol 37 (3.0 mg, 60%) as
a white solid: mp 108–1108C (hexane–ethyl acetate);
[a]2D5¼230.0 (c 0.8); IR (NaCl) 1681, 1458 cm21
;
1H
NMR (300 MHz) d 6.64 (1H, s, H-14), 2.62 (1H, d0d,
J¼19.0, 7.0 Hz, H-6), 2.51 (1H, dd, J¼19.0, 12.5 Hz, H-6 ),
1.24, 1.00, 0.96 and 0.89 (3H each, each s, H-17, H-18, H-19
and H-20); 13C NMR (75 MHz) dC 204.5 (s), 200.1 (s),
158.0 (s), 128.3 (d), 57.4 (s), 52.2 (s), 44.9 (t), 44.0 (d), 41.5
(t), 37.0 (t), 36.6 (s), 33.3 (s), 32.9 (t), 32.6 (q), 31.6 (t), 31.2
(t), 21.5 (q), 20.1 (q), 19.5 (q), 18.1 (t); HRMS (EI)
C20H28O2 requires 300.2089 (Mþ), found 300.2086.
[a]2D5¼266.7 (c 0.3); IR (NaCl) 3500–3300, 1381 cm21
;
1H NMR (300 MHz) d5.80 (1H, d, J¼1.9 Hz, H-14), 4.20
(1H, br s, H-13), 3.42 (1H, d, J¼4.1 Hz, H-7), 3.22 (1H, dd,
J¼4.1, 2.3 Hz, H-6), 1.13, 1.07, 1.03 and 0.93 (3H each,
each s, H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC
141.6 (s), 132.8 (d), 77.7 (d), 55.6 (d), 54.4 (d), 53.3 (s), 48.6
(d), 43.5 (s), 43.4 (t), 41.3 (t), 37.6 (s), 33.2 (s), 32.9 (q),
31.8 (t), 31.0 (t), 28.8 (t), 24.7 (q), 22.7 (q), 19.4 (q), 18.4 (t);
HRMS (EI) C20H30O2 requires 302.2246 (Mþ), found
302.2251.
4.1.10. 6,8(14)-Scopaduladien-13-one (35). To a solution
of enone 14 (67.5 mg, 0.236 mmol) in benzene (2.7 mL) at
reflux was added DDQ (202 mg, 0.89 mmol) and reflux
continued for 24 h. The reaction mixture was then cooled
and directly chromatographed on silica gel, using 9:1
hexane–ethyl acetate as eluent, to give the dienone 35
(59.8 mg, 89%) as a colourless oil which solidified on
standing: mp 104–1058C (from hexane); [a]2D3¼2119.7 (c
4.1.13. 6a-Hydroxy-8(14)-scopadulen-13-one (38) and
7-scopadulen-6a,13a-diol (39). To a solution of lithium
(12 mg, 1.72 mmol) in NH3 (7 mL) at 2358C under argon,
epoxide 36 (22 mg, 0.073 mmol) in THF (1.2 mL) was
added. After being stirred for 30 min, solid NH4Cl was
slowly added and the excess of NH3 was evaporated under a
stream of Argon. The reaction mixture was then diluted with
diethyl ether and water (2 mL), washed with brine, dried and
concentrated. The white solid residue was chromatographed
on silica gel using hexane–ethyl acetate (from 7:3 to 5:5)
eluent, to furnish alcohol 38 (9.0 mg, 41%) and the diol 39
(7.4 mg, 33%) as white solids. Compound 38: mp 71–738C
(hexanes–ethyl acetate); [a]2D5¼þ72.7 (c 0.9); IR (NaCl)
1
1.3); IR (NaCl) 3033, 3009, 1665, 1616, 1191 cm21; H
NMR (300 MHz) d 6.23 (1H, dd, J¼9.9, 2.3 Hz), 6.18 (1H,
dd, J¼9.9, 1.6 Hz), 5.68 (1H, s, H-14), 2.20 (1H, m, H-16),
2.04 (1H, br s, H-5), 1.22, 0.99 and 0.95£2 (3H each, each s,
H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC 205.3
(s), 163.0 (s), 138.4 (d), 127.2 (d), 122.5 (d), 56.6 (s), 51.0
(s), 50.0 (d), 43.1 (t), 41.1 (t), 38.1 (s), 33.1 (t), 33.0 (q), 32.8
(s), 31.2 (t), 29.1 (t), 22.7 (q), 20.3 (q), 18.4 (q), 18.2 (t);
HRMS (EI) C20H28O requires 284.2140 (Mþ), found
284.2144.
1
3600–3200, 1656, 1193 cm21; H NMR (300 MHz) d5.78
4.1.11. 6a,7a-Epoxy-8(14)-scopadulen-13-one (36). To a
refluxing mixture of dienone 35 (57 mg, 0.20 mmol) in
DCM (6 mL) and a buffer solution (pH¼8) of Na2HPO4/
KH2PO4 (6 mL), MCPBA (ca. 85%, 96 mg, 0.48 mmol)
was added in four portions over 48 h. The reaction was
carried out at 408C in the dark, and after two days the
mixture was then cooled, diluted with diethyl ether, washed
with Na2S2O3, 10% aqueous NaHCO3 and brine. The
resulting organic extract was dried (Na2SO4) and concen-
trated to afford a residue which was chromatographed on
silica gel, using 8:2 hexane–ethyl acetate as eluent, to give
4.0 mg of recovered starting material followed by the
epoxyenone 36 (30.7 mg, 51%) as a colourless oil which
solidified on standing: [a]2D2¼267.8 (c 1.8); IR (NaCl)
1712, 1680, 1458 cm21; 1H NMR (400 MHz) d6.27 (1H, s,
H-14), 3.58 (1H, d, J¼3.8 Hz, H-7), 3.38 (1H, dd, J¼3.8,
2.4 Hz, H-6), 1.50 (1H, br s, H-5), 1.20 (3H, s, H-17), 1.11
(3H, s, H-18), 1.08 (3H, s, H-19), 1.02 (3H, s, H-20); 13C
NMR (75 MHz) dC 203.9 (s), 162.2 (s), 131.0 (d), 56.3 (s),
56.1 (d), 53.3 (d), 51.2 (s), 48.6 (d), 44.7 (t), 41.0 (t), 37.9
(s), 33.8 (t), 33.2 (s), 32.9 (q), 30.8 (t), 30.0 (t), 22.7 (q), 20.3
(q), 20.1 (q), 18.1 (t); HRMS (EI) C20H28O2 requires
300.2089 (Mþ), found 300.2086.
(1H, dd, J¼1.7, 1.6 Hz, H-14), 4.10 (1H, m, H-6), 3.02 (1H,
ddd, J¼17.9, 5.8, 1.6 Hz, H-7), 2.52 (1H, ddd, J¼17.9, 6.6,
1.7 Hz, H-70), 1.20, 1.12, 1.07 and 0.99 (3H each, each s,
H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC 204.3
(s), 169.7 (s), 124.6 (d), 67.5 (d), 58.1 (s), 54.3 (d), 51.0 (s),
45.0 (t), 42.8 (t), 40.3 (t), 37.8 (s), 35.3 (q), 34.6 (t), 33.8 (s),
33.3 (t), 29.8 (t), 22.8 (q), 21.8 (q), 20.3 (q), 18.2 (t); HRMS
(EI) C20H30O2 requires 302.2246 (Mþ), found 302.2253.
Compound 39: 1H NMR (300 MHz) d 5.17 (1H, br s, H-7),
4.18 (1H, br d, J¼8.7 Hz, H-6), 3.47 (1H, dd, J¼10.4,
6.8 Hz, H-13), 1.13, 1.09, 1.06 and 0.96 (3H each, each s,
H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC 142.2
(s), 122.3 (d), 76.0 (d), 69.7 (d), 54.6 (s), 52.4 (d), 44.0 (t),
43.5 (s), 42.4 (t), 38.5 (s), 37.8 (t), 36.6 (q), 33.9 (t), 33.1 (s),
31.6 (t), 29.1 (t), 23.5 (q), 23.5 (q), 19.7 (q), 18.6 (t); HRMS
(EI) C20H32O2 requires 304.2402 (Mþ), found 304.2410.
4.1.14. 6a-Hydroxy-13-scopadulanone (40) and 6a,13a-
scopadulandiol (41). To a solution of lithium (10 mg,
1.44 mmol) in NH3 (4 mL) at 2358C under Argon, enone 38
(8.8 mg, 0.029 mmol) in THF (1.0 mL) was slowly added
dropwise. After being stirred for 8 min, the reaction mixture
was cooled to 2788C and solid NH4Cl was slowly added.
The excess of NH3 was evaporated under a stream of Argon
while warming to room temperature. The reaction mixture
was then diluted with diethyl ether and water (1 mL),
washed with brine, dried and concentrated. The white solid
residue was chromatographed on silica gel using hexane–
ethyl acetate (from 7:3 to 5:5) eluent, to furnish alcohol 40
(2.3 mg, 26%) as a colourless oil and the diol 41 (4.9 mg,
4.1.12. 6a,7a-Epoxy-8(14)-scopadulen-13a-ol (37).
Tellurium powder (9 mg, 0.068 mmol) and NaBH4 (5 mg,
0.132 mmol) were added to degasified EtOH (0.2 mL) and
the resulting solution was heated at 808C with vigorous
stirring. After 1 h, the pale purple suspension was cooled to
08C, and a solution of epoxide 36 (5 mg, 0.017 mmol) in

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6463
55%) as white solid. Compound 40: [a]2D5¼þ55.0 (c 0.4);
1H NMR (300 MHz) d 3.94 (1H, ddd, J¼10.8, 10.8, 5.0 Hz,
H-6), 1.15, 1.07, 1.04 and 1.00 (3H each, each s, H-17,
H-18, H-19 and H-20); 13C NMR (75 MHz) dC 214.0 (s),
69.7 (d), 53.3 (d), 52.5 (s), 52.4 (s), 45.1 (t), 43.8 (t), 43.1 (t),
41.3 (t), 40.1 (s), 39.4 (d), 36.9 (q), 36.6 (t), 33.6 (t), 33.3 (s),
24.4 (t), 22.5 (q), 19.8 (q), 18.6 (t), 18.5 (q); HRMS (EI)
C20H32O2 requires 304.2402 (Mþ), found 304.2408.
Compound 41: 1H NMR (300 MHz) d 3.90 (1H, ddd,
J¼11.1, 11.1, 5.6 Hz, H-6), 3.40 (1H, dd, J¼10.6, 5.6 Hz,
H-13), 1.12, 1.02, 1.01 and 0.96 (3H each, each s, H-17,
H-18, H-19 and H-20); 13C NMR (75 MHz) dC 76.1
(d), 70.1 (d), 53.3 (d), 51.8 (s), 44.3 (t), 44.2 (s), 43.9 (t),
41.4 (t), 40.0 (s), 37.6 (t), 37.0 (d), 37.0 (q), 33.6 (t), 33.3
(s), 30.5 (t), 24.7 (t), 23.3 (q), 22.5 (q), 18.7 (t), 18.6 (q);
HRMS (EI) C20H34O2 requires 306.2559 (Mþ), found
306.2564.
the virus suspension by quadruplicated for a period of
48 h.
2. Plaque reduction assay. Confluent monolayer VERO
cells were grown in 24-well flat-bottomed plates. 500 mL
of two-fold dilutions of the compound in MM were
added 1 h before viral infection. Treated cells were
inoculated with 100 mL of approximately 100 PFU
(plaque forming units) of virus; after 1 h 400 mL of
MM with 2% carboxymethylcellulose was added and
finally incubated again at 378C in humidified 5% CO2
atmosphere for 72 h. At least two assays were carried out
in duplicated with four concentrations on separate
occasion and reproducible results were obtained. The
ED50 concentration is the dilution tested that 50%
eliminated macroscopic plaque formation without overt
toxicity to cell monolayers.
4.1.15. 13a-Hydroxy-6-scopadulanone (42) and 13a-
methoxymethyloxy-6-scopadulanone (43). To a solution
of diol 41 (4 mg, 0.013 mmol) in DCM (1.0 mL) at 08C,
PCC over alumina (35 mg; 0.8 mmol CrO3/g) was added.
After 2 h the reaction mixture was allowed to warm to room
temperature and stirred for a further 1 h 30 min. Then, the
reaction mixture was directly filtered on silica using fresh
DCM and concentrated to give a white solid residue. This
residue was chromatographed on silica using 7:3 hexane–
ethyl acetate as eluent to afford the ketone 42 (2.8 mg, 70%)
as a white solid: 1H NMR (300 MHz) d3.44 (1H, dd,
J¼10.4, 5.7 Hz, H-13), 1.21, 1.05, 0.93, and 0.93 (3H each,
each s, H-17, H-18, H-19 and H-20); 13C NMR (75 MHz) dC
211.1 (s), 75.7 (d), 60.9 (d), 52.3 (s), 46.8 (t), 44.5 (s), 43.7
(t), 42.7 (t), 42.5 (s), 39.2 (d), 37.6 (t), 33.0 (q), 32.1 (t), 32.1
(s), 30.6 (t), 25.6 (t), 23.2 (q), 21.8 (q), 19.9 (q), 18.2 (t).
Compound 42 (2.7 mg, 0.009 mmol) in DCM (0.3 mL) at
08C under Argon was treated with iPr2NEt (15 mL,
0.072 mmol) and MOMCl (4 mL, 0.054 mmol). The reac-
tion mixture was allowed to warm to room temperature and
stirred overnight. After being stirred for 14 h, the reaction
mixture was diluted with diethyl ether, washed with 5%
aqueous HCl, 10% NaHCO3, and brine. Workup as usual of
the resulting organic extract gave a residue which was
purified by flash chromatography with 8:2 hexane–ethyl
acetate as eluent to furnish methoxy methyl ether 43
Acknowledgements
Financial support from the Conselleria de Educacion y
Ciencia de la Generalidad Valenciana and Programa
Iberoamericano de Ciencia y Tecnologia para el desarrollo
(CYTED) is gratefully acknowledged.
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