Ti-catalyzed transannular cyclization of epoxygermacrolides. Synthesis of antifungal (+)-tuberiferine and (+)-dehydrobrachylaenolide

Article abstract of DOI:10.1016/j.tet.2008.08.114

We present a divergent strategy for the stereoselective synthesis of both eudesmanolides (+)-tuberiferine and (+)-brachylaenolide starting from the accessible germacrolide (+)-costunolide. The key steps of these syntheses are the Ti-catalyzed transannular cyclization of a 1,4-epoxygermacrolide in the presence or absence of water, respectively. The catalytic cycle operating in the presence of water probably involves the reduction of a tertiary radical by H-atom transfer from aquacomplex Cp₂Ti^III(OH₂)Cl. The catalytic cycle under anhydrous conditions presumably occurs through mixed disproportionation between a tertiary radical and Cp₂Ti^IIICl. Synthetic (+)-tuberiferine and (+)-brachylaenolide displayed an antifungal potency against Phycomyces blakesleeanus comparable or even higher than amphotericin B, the gold standard for antifungal therapy.

Full text of DOI:10.1016/j.tet.2008.08.114

Tetrahedron 64 (2008) 11938–11943
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Tetrahedron
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Ti-catalyzed transannular cyclization of epoxygermacrolides. Synthesis of
antifungal (þ)-tuberiferine and (þ)-dehydrobrachylaenolide
´
´
´
Jose Justicia, Luis Alvarez de Cienfuegos, Rosa E. Estevez, Miguel Paradas, Ana M. Lasanta,
*
*
Juan L. Oller, Antonio Rosales, Juan M. Cuerva , J. Enrique Oltra
Department of Organic Chemistry, University of Granada, Faculty of Sciences, Campus Fuentenueva s/n, E-18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2008
Accepted 19 August 2008
Available online 24 September 2008
We present a divergent strategy for the stereoselective synthesis of both eudesmanolides (þ)-tuber-
iferine and (þ)-brachylaenolide starting from the accessible germacrolide (þ)-costunolide. The key steps
of these syntheses are the Ti-catalyzed transannular cyclization of a 1,4-epoxygermacrolide in the
presence or absence of water, respectively. The catalytic cycle operating in the presence of water
probably involves the reduction of
a tertiary radical by H-atom transfer from aquacomplex
Cp2TiIII(OH2)Cl. The catalytic cycle under anhydrous conditions presumably occurs through mixed dis-
proportionation between a tertiary radical and Cp2TiIIICl. Synthetic (þ)-tuberiferine and (þ)-brachylae-
nolide displayed an antifungal potency against Phycomyces blakesleeanus comparable or even higher than
amphotericin B, the gold standard for antifungal therapy.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
two properties often associated with biological activity in small
molecules.15 Therefore, we decided to explore the transannular cy-
Since the pioneering work by RajanBabu and Nugent on TiIII-
promoted homolytic epoxide opening,1 biscyclopentadienyl tita-
nium chloride2 (Cp2TiCl) has become a formidable tool in organic
synthesis,3 especially after the catalytic version developed by Gan-
sa¨uer et al., who used 2,4,6-collidine hydrochloride as titanocene-
regenerating agent.4 Subsequently we developed an alternative,
non-protic titanocene-regenerating agent, the couple Me3SiCl/
2,4,6-collidine, which in situ presumably forms N-(trimethylsilyl)-
collidinium chloride, the actual titanocene-regenerating reagent.5
Ti-catalyzed homolytic epoxide opening has proven to be one of the
most convenient procedures to initiate radical cascade cyclizations,
which have been intensively exploited for the straightforward syn-
thesis of terpenoids, including monoterpenoids such as kar-
ahanaenone,6 sesquiterpenoids such as trans-4(11),8-daucadiene,6
clization of the 10-membered ring of germacrolides, sesquiterpene
lactones with a germacrane skeleton.16 We first assayed the cationic
cyclization of germacrolides, 1,10-epoxygermacrolides and 4,5-
epoxygermacrolides initiated by Brønsted and Lewis acids.14a,17
Thus, we obtained mixtures of isomeric eudesmanolides14a,17b
(sesquiterpene lactones with an eudesmane skeleton),16 oxidized
eudesmanolides17a or ring-contraction products,17c depending on
the nature of the substrate and the acid employed. We subsequently
began the study of radical cyclizations of 1,10-epoxygermacrolides
catalyzed by Cp2TiCl.5,14a Thus, we unexpectedly found that water
exerted a dramatic effect on the termination step of the radical cy-
clization process. In fact, whereas under anhydrous conditions an
eudesmanolide with an exocyclic double bond was selectively
obtained, in the presence of water the corresponding reduction
product was only formed.5,14a As at that time the reduction of a free
radical by hydrogen-atom transfer from water seemed counterin-
tuitive, the water effect observed was explained via trapping of
a tertiary radical by a Cp2TiCl species and subsequent hydrolysis of
the organometallic alkyl–TiIV intermediate formed.5 Nevertheless,
we have recently found that, possibly due to steric factors, tertiary
radicals are not trapped by Cp2TiCl under the conditions employ-
ed.14b Moreover, we have provided theoretical and experimental
evidence supporting the idea that free radicals can be effectively
reduced by hydrogen-atom transfer from the aquacomplex
Cp2Ti(OH2)Cl.14b Therefore, the catalytic cycle originally postulated5
should be modified as depicted in Scheme 1.
isodrimenediol,7
3
b
-hydroxydihydroconfertifolin7 and (ꢀ)-
a-
ambrinol,8 diterpenoids such as 3 -hydroxymanool,9 rostratone,10
b
barekoxide,6 laukarlaool6 and sclareol oxide,11 meroterpenoids such
as zonarone and zonarol,12 and triterpenoids such as 3
b-hydroxy-
malabarica-14(26),17E,21-triene9 and achilleol A.13 In contrast, Ti-
catalyzed epoxide opening has been scarcely applied to achieve
radical transannular cyclizations.5,14
The transannular cyclization of medium-sized rings contributes
to the enhancement of molecular rigidity and structural complexity,
* Corresponding authors. Tel.: þ34 958248091; fax: þ34 958248437.
E-mail addresses: jmcuerva@ugr.es (J.M. Cuerva), joltra@ugr.es (J.E. Oltra).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.114

J. Justicia et al. / Tetrahedron 64 (2008) 11938–11943
11939
O
O
O
H
1
H
O
O
2
O
I
O
O
O
O
Cp2(Cl)TiO
H2O
+
O
O
O
2 Cp2TiCl
O
II
O
+
O
MnCl2
3
Cp2Ti(OH2)Cl
Scheme 2. Convergent retrosynthetic analysis from 1 and 2 to 3.
Cp2(Cl)TiO
Mn
Selective oxidation of 4 with m-chloroperbenzoic acid (MCPBA)
in the presence of pyridine provided an excellent yield (99%) of
epoxide 5.14a At this point we took advantage of the different be-
haviour of the Ti(III)-catalyzed cyclization of epoxygermacrolide 5
in the presence or absence of water. Thus, the key intermediate 6
was prepared in 72% yield by stirring epoxide 5 with a sub-
stoichiometric amount of Cp2TiCl2 (0.2 mmol), Mn dust (7 mmol),
H2O (5 mmol), 2,4,6-collidine (5 mmol) and 2,4,6-collidine hydro-
chloride (5 mmol) in THF. On the other hand, treatment of 5 with
Cp2TiCl2 (0.2 mmol), Mn (7 mmol), 2,4,6-collidine (7 mmol) and
trimethylsilyl chloride (4 mmol) in anhyd THF gave exocyclic al-
kene 7 (68% yield). Its physical properties, including optical rotation
H
O
H2O
+
2 Cp2TiCl2
III
O
+
Cp2Ti(OH)Cl
2
2
N
H
N
Cl
HO
and NMR data, matched those described for natural (þ)-11
b,13-
dihydroreynosin isolated from the plant Michelia compressa,24
supporting the usefulness of our procedure for the stereoselective
synthesis of natural eudesmanolides.
H
O
IV
O
It is obvious that transannular cyclization of 5 to 7, catalyzed by
Cp2TiCl under anhydrous conditions, cannot be explained by the
water-based catalytic cycle depicted in Scheme 1. We have recently
demonstrated that, under anhydrous conditions, tertiary radicals
are transformed into alkenes by a mixed disproportionation process
promoted by Cp2TiCl.14b In this scenario, anhydrous Ti-catalyzed
cyclization of 1,10-epoxygermacrolides (such as model compound I)
to eudesmanolides with an exocyclic double bond (such as model
VII) can be rationalized by the catalytic cycle depicted in Scheme 4.
Scheme 1. Revised catalytic cycle for TiIII-catalyzed transannular cyclization of
1,10-epoxygermacrolides in the presence of water.
Natural product synthesis constitutes one of the most de-
manding tests to prove the utility of novel synthetic methods.
Therefore, once we were confident about the possibility of con-
trolling the termination step of Ti-catalyzed transannular cycliza-
tions of epoxygermacrolides, by means of the water effect observed,
we decided taking advantage of this phenomenon for the
straightforward synthesis of potentially antifungal (vide infra)
eudesmanolides scarce in nature. As target molecules we chose
(þ)-tuberiferine (1), a metabolite from the plant Sonchus tuberifer,18
and (þ)-dehydrobrachylaenolide (2), isolated from the roots of
Brachylaena transvaalensis.19
3
H2/Pd
O
2. Results and discussion
4 (93%)
O
MCPBA, Py
CH2Cl2
To the best of our knowledge, synthetic procedures previously
reported for the preparation of tuberiferin (1)20 and dehydro-
brachylaenolide (2)21 are restricted to chemical transformations of
other eudesmanolides. In contrast, our retrosynthetic analysis for
both products (Scheme 2) converged to the germacrolide (þ)-cos-
tunolide (3), which can be obtained in (multi)gram quantities from
the commercially available extract Costus Resinoid.22
O
Cp2TiCl2 (cat)
Cp2TiCl2 (cat)
Mn, THF/H2O
2,4,6-collidine·HCl
HO
Mn, THF anhyd
O
2,4,6-collidine, Me3SiCl
HO
O
5 (99%)
Therefore, the synthesis began with the selective reduction of
the conjugated double bond of 3 to avoid potential complications
with a reactive Michael-acceptor system. This double bond can
easily be restored at the end of the synthetic sequence. Thus, cat-
alytic hydrogenation of 3 by our previously described procedure22
took place regio- and stereoselectively, giving a 93% yield of
H
H
O
H
O
6 (72%)
7 (68%)
O
O
(þ)-11
b
,13-dihydrocostunolide23 (4) (Scheme 3).
Scheme 3. TiIII-catalyzed synthesis of intermediates 6 and 7.

11940
J. Justicia et al. / Tetrahedron 64 (2008) 11938–11943
O
I
a) TsNHNH2
b) NaH
DMP
6
(50 %)
(95 %)
H
H
O
Cp2(Cl)TiO
O
O
2 Cp2TiCl
9
8
O
O
O
a) KHMDS
PhSeCl
b) H2O2, Py
SeO2
O
II
(70 %)
(58 %)
H
HO
O
HO
O
+
H
10
H
11
O
MnCl2
Cp2TiCl
O
Cp2(Cl)TiO
Mn
DMP
(90 %)
DBU
1
(92 %)
H
12
O
O
O
V
+
O
Scheme 5. Synthesis of (þ)-tuberiferine (1).
2 Cp2TiCl2
Cp2Ti(H)Cl
(47%) without apparent isomerization of the exocyclic double bond.
Allylic oxidation of the doubly activated C-3 position of 14 yielded
allylic alcohol 15 (75%). Subsequently, the modified Grieco’s pro-
tocol provided a moderate 50% yield of conjugated lactone 16. This
lactone is the C-3 epimer of (þ)-brachylaenolide isolated from B.
transvaalensis.19 In natural brachylaenolide H-3 resonates at
4.72 ppm whereas H-3 of 16 appears at 4.43 ppm. Finally, oxidation
of alcohol 16 with DMP yielded the second target molecule,
(þ)-dehydrobrachylaenolide (2). Physical and spectroscopic data of
synthetic 2, including optical rotation, were in agreement with
those described for the natural product.19 Thus, we completed the
synthesis of 2 from 3 in nine steps and an 8% overall yield.
2
Me3SiH + 2
N
N
SiMe3
Cl
Me3SiO
H2O
HO
O
O
O
VI
VII
O
Scheme 4. Hypothetical catalytic cycle for TiIII-catalyzed transannular cyclization of
1,10-epoxygermacrolides under anhydrous conditions.
Divergent stereoselective synthesis of both eudesmanolides
(þ)-tuberiferine (1) and (þ)-dehydrobrachylaenolide (2) from
germacrolide 3 highlights the synthetic utility of Ti-catalyzed rad-
ical transannular cyclizations of 1,10-epoxygermacrolides.
Moreover, the high regioselectivity observed for the formation of
exocyclic alkene 7 suggest that hydrogen-atom abstraction by the
bulky Cp2TiCl species from the methyl group of a tertiary radical
such as II is much faster than from the methylene or the methyne
3. Antifungal activity of (D)-tuberiferine (1) and (D)-
dehydrobrachylaenolide (2)
groups located at the a-positions of the carbon-centred free radical.
At the moment, we do not know what are the factors responsible for
these different reaction rates.
In the last decades, a considerable increase of fungal infections
has been observed worldwide, with special incidence of opportu-
nistic infections in inmunodeficient patients and individuals infec-
ted by multidrug-resistant fungal strains.26 Therefore, the
development of novel, more effectiveantifungal drugs is desirable.26
In this context, Oltra et al. observed that sesquiterpene lactones
require a relatively low polarity and, at least, one Michael-acceptor
system to display antifungal activity.27 As both sesquiterpene lac-
tones 1 and 2 fitted these requirements, we decided to check their
Intermediate 6 was subsequently transformed into (þ)-tuber-
iferine (1) in a sequence of seven steps (Scheme 5). Oxidation of
secondary alcohol 6 with Dess–Martin periodinane (DMP) yielded
the corresponding ketone 8 (95%). At this point, we chose the
Shapiro elimination of the tosylhydrazone derived from 8 to form
the D1,2 double bond characteristic of tuberiferine. In this way, we
obtained the desired alkene 9 in 50% yield (two steps). Subsequent
allylic oxidation of 9 provided a 70% yield of pseudo-equatorial
alcohol 10. The C-11–C-13 double bond was then restored in a one-
pot reaction applying a slight modification of the selenium-based,
two-step protocol described by Grieco and Nishizawa23 Thus, we
obtained a 58% yield of conjugated lactone 11.25 Oxidation of al-
cohol 11 with DMP provided conjugated ketone 12 (90% yield).
Finally, base-catalyzed epimerization of the methyl group at C-4 led
to the target molecule (þ)-tuberiferine (1) (92%). Physical and
spectroscopic data, including optical rotation, of synthetic 1 were in
agreement with those described for the natural product.18 Thus, we
completed the synthesis of 1 from 3 in 10 steps and an 11% overall
yield.
O
1
a) TsNHNH2
DMP
b) NaH
7
3
(89 %)
(47 %)
H
H
O
O
O
14
13
O
O
O
O
a) KHMDS
PhSeCl
b) H2O2, Py
SeO2
(75 %)
(50 %)
HO
HO
H
15
H
16
The synthesis of (þ)-dehydrobrachylaenolide (2), from the key
intermediate 7 (Scheme 6), was performed using a synthetic se-
quence closely related to that employed for the synthesis of 1. Thus,
DMP-based oxidation of 7 gave an 89% yield of ketone 13. In-
terestingly, the Shapiro reaction of 13 gave the expected diene 14
O
DMP
2
(84 %)
Scheme 6. Synthesis of (þ)-dehydrobrachylaenolide (2).

J. Justicia et al. / Tetrahedron 64 (2008) 11938–11943
11941
potential antifungal properties. For this purpose, we chose Phyco-
myces blakesleeanus, a filamentous fungus the metabolites of which
were previously analyzed in our laboratory.28 Phycomyces was in-
cubated in minimal medium29 (control experiment) and in minimal
media supplemented with different concentrations of (þ)-tuber-
iferine (1), (þ)-brachylaenolide (2) and a reference antifungal drug,
amphotericin B, which is considered the ‘gold standard’ for anti-
fungal therapy.26 At the end of the incubation period (7 days), my-
celia were filtered off from culture broths, lyophilized and weighted.
Comparison of the biomass weights of mycelia from cultures con-
taining antifungal products with that of the control experiment
allowed the determination of the concentration required to achieve
a 50% of fungal growth inhibition (GI50). Thus, we determined the
(ddd, J¼15.6, 11.7, 5.5 Hz, 1H), 2.37 (m, 1H), 2.25 (dt, J¼15.0, 5.3 Hz,
1H), 2.10–1.75 (m, 5H), 1.60–1.40 (m, 4H), 1.26 (s, 3H), 1.21 (d,
J¼7.7 Hz, 3H), 1.21 (d, J¼5.4 Hz, 3H). 13C NMR (75 MHz, CDCl3,
DEPT):
d 214.2 (C), 179.2 (C), 79.4 (CH), 52.9 (CH), 49.9 (CH), 41.6
(CH), 36.9 (C), 34.9 (CH2), 34.4 (CH2), 32.5 (CH2), 26.9 (CH), 22.7
(CH2), 20.3 (CH3), 15.4 (CH3), 12.5 (CH3). HRMS (FABMS): m/z
273.1460 [MþNa]þ, calcd for C15H22O3Na: 273.1466.
5.3. Synthesis of alkene 9
A sample of p-toluenesulfonyl hydrazide (290 mg, 1.85 mmol)
was added to a solution of 8 (420 mg, 1.68 mmol) in MeOH (10 mL).
The mixture was stirred under reflux for 30 min and at 25 ꢁC for
14 h. The solvent was then removed and the residue dissolved in t-
BuOMe and washed with brine. The organic layer was dried with
anhyd Na2SO4, and the solvent removed to give the hydrazone in-
termediate. To this intermediate (130 mg, 0.36 mmol) dissolved in
anhyd toluene (28 mL), NaH (260 mg, 10.8 mmol) was added under
an argon atmosphere. The mixture was stirred at 90 ꢁC for 2 h, di-
luted with t-BuOMe (50 mL) and washed with brine. The organic
layer was dried over anhyd Na2SO4, the solvent was removed and
the residue was submitted to flash chromatography (t-BuOMe/
hexane 1:4) to give alkene 9 (50 mg, 50% after two steps). IR (film)
following values: GI50¼12
m
g/mL for amphotericin B, GI50¼12
mg/mL
for (þ)-tuberiferine and GI50¼6
m
g/mL for (þ)-brachylaenolide.
Despite of a more in-depth antifungal analysis is obviously needed;
the above values indicate that, at least for P. blakesleeanus, lactones 1
and 2 have an antifungal potency comparable or even higher than
amphotericin B.
4. Conclusions
We have developed a divergent procedure for the stereo-
selective synthesis of the eudesmanolides (þ)-tuberiferine (1) and
(þ)-brachylaenolide (2), which are scarce in nature, starting from
the accessible germacrolide (þ)-costunolide (3). The key steps of
these syntheses were the Ti-catalyzed transannular cyclizations of
1,4-epoxygermacrolide 5 in the presence or absence of water, re-
spectively. The catalytic cycle operating in the presence of water
probably involves the reduction of a tertiary radical by H-atom
transfer from aquacomplex Cp2TiIII(OH2)Cl. The catalytic cycle un-
der anhydrous conditions presumably occurs through mixed dis-
proportionation between a tertiary radical and Cp2TiIIICl. Synthetic
(þ)-tuberiferine (1) and (þ)-brachylaenolide (2) displayed an an-
tifungal potency against P. blakesleeanus comparable or even higher
than amphotericin B.
nmax 2936,1768 cmꢀ1. 1H NMR (300 MHz, CDCl3):
d 5.04 (s, 2H), 4.01
(dd, J¼11.5, 9.7 Hz, 1H), 2.40–1.20 (m, 10H), 1.19 (d, J¼6.9 Hz, 3H),
1.08 (s, 3H), 1.04 (d, J¼6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3, DEPT):
d
179.8 (C), 136.9 (CH), 123.4 (CH), 80.2 (CH), 54.1 (CH), 49.2 (CH),
41.9 (CH), 41.2 (CH2), 36.9 (C), 32.4 (CH2), 25.5 (CH), 23.4 (CH2), 22.2
(CH3), 17.2 (CH3), 12.6 (CH3). HRMS (FABMS): m/z 257.1516
[MþNa]þ, calcd for C15H22O2Na: 257.1517.
5.4. Synthesis of alcohol 10
Under an argon atmosphere, SeO2 (72 mg, 0.63 mmol) was
added to a solution of 9 (50 mg, 0.21 mmol) in anhyd dioxane
(10 mL). The mixture was stirred at 100 ꢁC for 3 h, diluted with t-
BuOMe and washed with brine. The organic layer was dried over
anhyd Na2SO4 and the solvent removed. The residue was submitted
to flash chromatography (t-BuOMe/hexane 1:1) to provide alcohol
10 (35 mg, 70%). IR film nmax 3418, 1768 cmꢀ1. 1H NMR (300 MHz,
At the moment, we are engaged in the study of Ti-catalyzed
transannular cyclizations of 4,5-epoxygermacrolides and in a more
in-depth study of the antifungal activity of lactones 1 and 2.
5. Experimental section
5.1. General details
CDCl3):
d
5.70 (d, J¼10 Hz, 1H), 5.63 (dd, J¼10.0, 4.0 Hz, 1H), 3.98
(dd, J¼11.1, 10.0 Hz, 1H), 3.85 (d, J¼3.8 Hz, 1H), 2.30 (m, 2H), 2.18 (br
s, 1H(OH)), 2.01 (dd, J¼11.7, 4.4 Hz, 1H), 1.81 (m, 1H), 1.70–1.20 (m,
4H), 1.12 (d, J¼6.8 Hz, 3H), 1.01 (s, 3H), 0.97 (d, J¼7.7 Hz, 3H). 13C
For all reactions employing titanocene, solvents and additives
were thoroughly deoxygenated prior to use. The numbering used
corresponds to the germacrane and eudesmane sesquiterpene
systems and not to the IUPAC nomenclature.16 (þ)-Costunolide (3)
can be obtained in (multi)gram quantities from the commercially
available extract Costus Resinoid (Pierre Chauvet S.A., Seillans,
France) as described elsewhere.17b We followed previously de-
NMR (75 MHz, CDCl3, DEPT): d 179.9 (C),140.7 (CH),124.8 (CH), 79.6
(CH), 69.9 (CH), 54.0 (CH), 44.5 (CH), 41.8 (CH), 40.4 (CH2), 37.1 (C),
35.7 (CH), 23.2 (CH2), 21.8 (CH3), 14.8 (CH3), 12.6 (CH3). HRMS
(FABMS): m/z 273.1463 [MþNa]þ, calcd for C15H22O3Na: 273.1466.
5.5. Synthesis of conjugated lactone 11
scribed procedures for the preparation of (þ)-11
b
,13-dihy-
A solution of compound 10 (30 mg, 0.12 mmol) in anhyd THF
(3 mL) was cooled until ꢀ78 ꢁC under argon. KHMDS (192 mg,
0.48 mmol) was added and the mixture was stirred at ꢀ78 ꢁC for
30 min. Then, PhSeCl (93 mg, 0.42 mmol) was added and the mix-
ture was stirred at ꢀ78 ꢁC for 2 h. The mixture was diluted with t-
BuOMe and washed with brine. The organic layer was dried over
anhyd Na2SO4 and the solvent removed under vacuum. The residue
was dissolved in CH2Cl2 (6 mL) and pyridine (1 mL) and H2O2 (1 mL,
30% w/w) were added. The mixture was heated under reflux for
10 min, diluted with CH2Cl2 and washed with a 10% HCl solution
(10 mL) and brine. The organic layer was dried over anhyd Na2SO4,
the solvent was removed and the residue was purified by flash
chromatography (t-BuOMe/hexane 3:7) to afford 11 (17 mg, 58%). IR
drocostunolide22 (4), (þ)-1,10-epoxy-11
b
,13-dihydrocostunolide14a
(5), and the key intermediates 6 and 7.5
5.2. Synthesis of ketone 8
Water (100 mL) and Dess–Martin periodinane (DMP) (1.84 g,
4.3 mmol) were added to a solution of compound 6 (900 mg,
3.6 mmol) in CH2Cl2 (125 mL). The mixture was stirred at rt for 1 h,
diluted with CH2Cl2 and washed with a 1:1 mixture (50 mL) of
saturated solutions of NaHCO3 and Na2S2O3, and with brine. The
organic layer was dried over anhyd Na2SO4 and the solvent re-
moved. The residue was submitted to flash chromatography (t-
BuOMe) to provide ketone 8 (85 mg, 95%). IR (film) nmax 1767,
(film) nmax 3423, 1766 cmꢀ1. 1H NMR (300 MHz, CDCl3):
J¼3.1 Hz, 1H), 5.74 (d, J¼10.0 Hz, 1H), 5.67 (dd, J¼10.0, 4.2 Hz, 1H),
d 6.04 (d,
1703 cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
4.04 (t, J¼10.3 Hz,1H), 2.73

11942
J. Justicia et al. / Tetrahedron 64 (2008) 11938–11943
5.37 (d, J¼3.1 Hz, 1H), 3.98 (t, J¼11.0 Hz, 1H), 3.89 (d, J¼4.0 Hz, 1H),
2.55 (tq, J¼11.1, 3.2 Hz,1H), 2.36 (m,1H), 2.16 (dd, J¼11.7, 4.4 Hz,1H),
2.03 (dq, J¼13.3, 3.6 Hz, 1H), 1.53 (m, 3H), 1.03 (s, 3H), 1.01 (d,
dried with anhyd Na2SO4 and the solvent was removed to give the
corresponding hydrazone intermediate. NaH (260 mg, 10.8 mmol)
was added to this intermediate (130 mg, 0.36 mmol) in anhyd tol-
uene(28 mL) underan argon atmosphere. Themixturewas stirred at
90 ꢁC for 2 h, diluted with t-BuOMe (50 mL) and washed with brine.
The organic layer was dried over anhyd Na2SO4, the solvent was
removed and the residue was purified by flash chromatography (t-
BuOMe/hexane 1:4) toprovidediene 14 (47 mg, 47% aftertwosteps).
J¼7.8 Hz, 3H).13C NMR (75 MHz, CDCl3, DEPT):
d 171.0(C),140.6(CH),
139.9 (C),124.8 (CH),116.6 (CH2), 79.9 (CH), 69.9 (CH), 51.4 (CH), 45.0
(CH), 40.1 (CH2), 37.3 (C), 35.6 (CH), 21.8 (CH2), 21.6 (CH3),14.9 (CH3).
HRMS (FABMS): m/z 271.1313 [MþNa]þ, calcd for C15H20O3Na:
271.1310.
IR film nmax 1770 cmꢀ1.1H NMR (300 MHz, CDCl3):
d 5.52 (s, 2H), 5.02
5.6. Synthesis of conjugated ketone 12
(br s, 1H), 4.89 (s, 1H), 4.07 (dd, J¼10.9, 10.4 Hz, 1H), 2.94–2.69 (m,
2H), 2.39 (d, J¼11.7 Hz, 1H), 2.34 (dq, J¼13.6, 6.9 Hz, 1H), 1.87–1.43
(m, 5H),1.22 (d, J¼6.8 Hz, 3H), 0.80 (s, 3H). 13C NMR (75 MHz, CDCl3,
DMP (103 mg, 0.24 mmol) was added to a solution of 11 (50 mg,
0.20 mmol) in CH2Cl2/H2O (10 mL/10
mL). The mixture was stirred
DEPT): d 179.4 (C), 141.4 (C), 137.2 (CH), 123.9 (CH), 109.4 (CH2), 79.3
at rt for 1.5 h, diluted with CH2Cl2 and washed with a 1:1 mixture
(20 mL) of saturated solutions of NaHCO3 and Na2S2O3, and with
brine. The organic layer was dried over anhyd Na2SO4 and the
solvent removed. The residue was purified by flash chromatogra-
phy (t-BuOMe/hexane 3:2) to afford ketone 12 (45 mg, 90%). IR film
(CH), 52.8 (CH), 52.3 (CH), 41.2 (CH), 39.4 (C), 37.5 (CH2), 34.8 (CH2),
23.2 (CH2), 20.8 (CH3), 12.5 (CH3). HRMS (FABMS): m/z 255.1358
[MþNa]þ, calcd for C15H20O2Na: 255.1361.
5.10. Synthesis of alcohol 15
nmax 1768, 1668 cmꢀ1 1H NMR (300 MHz, CDCl3):
. d 6.78 (d,
J¼10.0 Hz, 1H), 6.10 (d, J¼3.2 Hz, 1H), 5.92 (d, J¼10.1 Hz, 1H), 5.43
(d, J¼3.0 Hz, 1H), 4.03 (t, J¼11.1 Hz, 1H), 2.86 (quint, J¼7.5 Hz, 1H),
2.54 (tq, J¼14.0, 3.1 Hz, 1H), 2.43 (dd, J¼11.6, 6.4 Hz, 1H), 2.12 (m,
1H), 1.73–1.55 (m, 3H), 1.29 (d, J¼7.8 Hz, 3H), 1.23 (s, 3H). 13C NMR
To a solution of 14 (80 mg, 0.34 mmol) in CH2Cl2 (10 mL), SeO2
(20 mg, 0.17 mmol) and t-BuOOH (0.28 mL, 6 M in decane) were
added. The mixture was stirred at 25 ꢁC for 4 h, diluted with CH2Cl2
(20 mL) and washed with brine. The organic layer was dried over
anhyd Na2SO4, the solvent was removed and the residue was puri-
fied by flash chromatography (t-BuOMe/hexane 1:1) to give alcohol
15 (56 mg, 75%). IR film nmax 3421, 1768 cmꢀ1. 1H NMR (300 MHz,
(75 MHz, CDCl3, DEPT):
d 201.3 (C), 170.3 (C), 158.2 (CH), 139.0 (C),
127.1 (CH), 117.4 (CH2), 79.0 (CH), 50.5 (CH), 48.6 (CH), 40.2 (CH),
38.8 (CH2), 38.2 (C), 22.7 (CH3), 21.4 (CH2), 13.7 (CH3). HRMS
(FABMS): m/z 269.1156 [MþNa]þ, calcd for C15H18O3Na: 269.1153.
CDCl3):
d
5.72 (d, J¼9.8 Hz, 1H), 5.66 (dd, J¼9.8, 3.5 Hz, 1H), 5.27 (s,
1H), 5.08 (s,1H), 4.38 (d, J¼3.3 Hz,1H), 4.05 (t, J¼10.5 Hz,1H), 2.66 (d,
J¼11.0 Hz, 1H), 2.33 (dq, J¼13.5, 6.8 Hz, 1H), 1.98 (s, 1H(OH)), 1.88–
1.50 (m, 5H), 1.20 (d, J¼6.8 Hz, 3H), 0.83 (s, 3H). 13C NMR (75 MHz,
5.7. Synthesis of (D)-tuberiferine (1)
DBU (0.24 mL) was added to a solution of 12 (40 mg, 0.16 mmol)
in anhyd toluene (8 mL) under an argon atmosphere. The mixture
was stirred at 60 ꢁC for 1.5 h and at 25 ꢁC for 10 h. The mixture was
diluted with CH2Cl2 and washed with a 10% HCl solution (20 mL) and
brine. The organic layer was dried over anhyd Na2SO4, the solvent
was removed and the residue was purified by flash chromatography
(t-BuOMe/hexane 3:2) to afford 1 (37 mg, 92%). Spectroscopic
properties, including IR and NMR data, and optical rotation were in
agreementwiththose previouslydescribedfor(þ)-tuberiferine.18,20c
CDCl3, DEPT): d179.3 (C),145.1 (C),141.5 (CH),125.4(CH),115.6 (CH2),
78.7 (CH), 69.1 (CH), 52.7 (CH), 48.2 (CH), 41.2 (CH), 40.0 (C), 36.9
(CH2), 23.0 (CH2), 19.5 (CH3), 12.5 (CH3). HRMS (FABMS): m/z
271.1304 [MþNa]þ, calcd for C15H20O3Na: 271.1310.
5.11. Synthesis of a,b-unsaturated lactone 16
Compound 15 (90 mg, 0.36 mmol) was dissolved in anhyd THF
(9 mL) under an argon atmosphere and the solution was cooled at
ꢀ78 ꢁC. KHMDS (504 mg, 2.52 mmol) was added to this solution
and the mixture was stirred at ꢀ78 ꢁC for 30 min. Then, PhSeCl
(242 mg, 1.26 mmol) was added and the mixture was stirred at
ꢀ78 ꢁC for 2 h. The mixture was diluted with t-BuOMe and washed
with brine. The organic layer was dried over anhyd Na2SO4 and the
solvent removed under vacuum. The residue was dissolved in
CH2Cl2 (12 mL), and pyridine (1 mL) and H2O2 (1 mL, 30% w/w)
were added. The mixture was heated under reflux for 10 min, di-
luted with CH2Cl2 and washed with a 10% HCl solution (10 mL) and
brine. The organic layer was dried over anhyd Na2SO4, the solvent
was removed and the residue was purified by flash chromatogra-
phy (t-BuOMe/hexane 3:7) to afford 16 (45 mg, 50%). IR film nmax
5.8. Synthesis of ketone 13
A sample of DMP (920 mg, 2.15 mmol) was added to a solution of
7 (450 mg,1.8 mmol) in CH2Cl2/H2O (70 mL/50 mL). The mixture was
stirred at rt for 1 h, diluted with CH2Cl2 and washed with a 1:1
mixture (30 mL) of saturated solutions of NaHCO3 and Na2S2O3, and
with brine. The organic layer was dried over anhyd Na2SO4 and the
solvent removed. The residue was submitted to flash chromatogra-
phy (t-BuOMe) to give ketone 13 (400 mg, 89%). IR film nmax 2936,
1782, 1708 cmꢀ1. 1H NMR (400 MHz, CDCl3):
d 5.21 (s, 1H), 5.08 (s,
1H), 4.12 (t, J¼10.5 Hz,1H), 2.74–2.57 (m, 2H), 2.46–2.30 (m, 4H),1.95
(dq, J¼12.9, 3.0 Hz, 1H), 1.85 (dt, J¼14.4, 3.2 Hz, 1H), 1.75 (td, J¼12.9,
4.2 Hz, 1H), 1.60 (m, 1H), 1.47 (td, J¼12.6, 3.8 Hz, 1H), 1.24 (d,
3402, 1763 cmꢀ1
.
1H NMR (300 MHz, CDCl3):
d
6.09 (d, J¼2.4 Hz,
1H), 5.76 (d, J¼9.9 Hz, 1H), 5.70 (dd, J¼9.9, 3.2 Hz, 1H), 5.41 (d,
J¼2.3 Hz, 1H), 5.32 (s, 1H), 5.15 (s, 1H), 4.43 (d, J¼3.2 Hz, 1H), 4.05 (t,
J¼10.9 Hz, 1H), 2.81 (d, J¼11.1 Hz, 1H), 2.62 (m, 1H), 2.08 (m, 1H),
1.75–1.60 (m, 3H), 0.84 (s, 3H). 13C NMR (75 MHz, CDCl3, DEPT):
J¼6.9 Hz, 3H), 1.12 (s, 3H). 13C NMR (CDCl3, 100 MHz, DEPT):
d 212.0
(C), 178.9 (C), 140.8 (C), 112.6 (CH2), 78.6 (CH), 52.2 (CH), 51.8 (CH),
50.2 (C), 41.0 (CH), 37.6 (CH2), 34.0 (CH2), 31.5 (CH2), 22.5 (CH2), 18.1
(CH3), 12.4 (CH3). HRMS (FABMS): m/z 271.1307 [MþNa]þ, calcd for
C15H20O3Na: 271.1310.
d
171.6 (C), 144.8 (C), 141.4 (CH), 139.1 (C), 125.4 (CH), 117.1 (CH2),
114.0 (CH2), 78.9 (CH), 69.2 (CH), 50.0 (CH), 48.6 (CH), 40.2 (C), 36.7
(CH2), 21.4 (CH2), 19.5 (CH3). HRMS (FABMS) m/z 269.1153
[MþNa]þ, calcd for C15H18O3Na: 269.1153.
5.9. Synthesis of diene 14
A sample of p-toluenesulfonyl hydrazide (193 mg, 1.23 mmol)
was added to a solution of 13 (280 mg,1.12 mmol) in MeOH (10 mL).
The mixture was stirred under reflux for 30 min and, subsequently,
at 25 ꢁC for 14 h. The solvent was removed and the residue was
dissolved in t-BuOMe and washed with brine. The organic layer was
5.12. Synthesis of (D)-dehydrobrachylaenolide (2)
Water (10 mL) and DMP (72 mg, 0.16 mmol) were added to a so-
lution of compound 16 (30 mg, 0.12 mmol) in CH2Cl2 (10 mL). The
mixture was stirred at rt for 1.5 h, diluted with CH2Cl2 and washed

J. Justicia et al. / Tetrahedron 64 (2008) 11938–11943
11943
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Na2S2O3, and with brine. The organic layer was dried over anhyd
Na2SO4 and the solvent removed. The residue was submitted to flash
chromatography (t-BuOMe/hexane 1:1) to give 2 (25 mg, 84%).
Optical rotation,1H, and 13C NMR data of 2 matched those previously
described.19,21
´
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filamentous fungus P. blakesleeanus28b were inoculated
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wards, THF solutions (1 mL) with different concentrations of
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