Studies directed toward the synthesis of the scabrosins: validation of a tandem enyne metathesis approach

Article abstract of DOI:10.1016/j.tet.2006.05.089

A synthetic approach to the scabrosin family of antibiotics using a ruthenium carbene-catalyzed tandem metathesis and?a Pd(II)-catalyzed cyclization is described. The chiral propargyl amino acid is furnished through enantioselective phase-transfer proparg

Full text of DOI:10.1016/j.tet.2006.05.089

Tetrahedron 62 (2006) 10528–10540
Studies directed toward the synthesis of the scabrosins:
validation of a tandem enyne metathesis approach
*
Mark D. Middleton, Brian P. Peppers and Steven T. Diver
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA
Received 20 January 2006; revised 6 May 2006; accepted 10 May 2006
Available online 17 August 2006
Abstract—A synthetic approach to the scabrosin family of antibiotics using a ruthenium carbene-catalyzed tandem metathesis and a
Pd(II)-catalyzed cyclization is described. The chiral propargyl amino acid is furnished through enantioselective phase-transfer propargylation.
The synthesis of the cyclohexadiene ring system is achieved through ring synthesis using tandem enyne metathesis, previously developed in
our lab. The complementary methods of methylene-free and 1,5-hexadiene-alkyne metatheses are compared. The indoline heterocycles are
€
formed using a two-step chloroacetoxylation (Backvall reaction) with subsequent nucleophilic attack by an amide nucleophile. The indoline
subunits were joined and cyclized to furnish the core diketopiperazine ring. The stereochemical assignment of intermediates is also discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
epoxide functionality. Scabrosin Awas isolated from a lichen
Xanthoparmelia scabrosa isolated on a coastal cliff face in
New South Wales, Australia; it features acetyl groups at
the flanks and is symmetrical. The same pentacyclic carbo-
cycle was isolated in 1998 by Williams et al. from a lichen
Usnea sp. found on a rotting tree in Ambewela, Sri Lanka;
these molecules, the ambewelamides, bear different acyl
The scabrosins are a group of epidithiadiketopiperazines
(edtdkp) symmetrically flanked by two highly functional-
ized cyclohexene rings (Scheme 1). Scabrosins 1–3 and
ambewelamides 2 and 4 share the same core pentacarbo-
cyclic framework with the symmetrical disposition of
O
6'
O
R2
3
2
O
E
N
4
S
O
5
D
B
S
O
N
C
O
6
1
R
A
O
O
1
R
2
R
Scabrosin A
1
2
3
4
CH3
CH3
C3H7
CH3
CH3
C3H7
Scabrosin B/Ambewelamide A
Scabrosin C
C3H7
Ambewelamide B
-(CH2)2CH3
O
OH
H
O
H
O
O
H
O
H
N
N
OH
O
O
N
H
S
S
S
AcO
OH
N
HO
H
S
H
S
S
S
OH
N
N
OH
HO
S
N
O
O
HO
N
O
O
Me
HO
gliotoxin
Scheme 1. Scabrosin/ambewelamide group of epidithiadiketopiperazines.
H
O
H
HO
aranotin
epicorazine A
rostratin A
€
Keywords: Enyne metathesis; Tandem metathesis; 1,3-Cyclohexadienes; Scabrosin; Epidithiadiketopiperazine; Grubbs’ catalyst; Hoveyda catalyst; Backvall
reaction.
* Corresponding author. Tel.: +1 716 645 6800x2201; fax: +1 716 645 6963; e-mail: diver@buffalo.edu
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.089

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10529
R
X
R
Ru cat.
R
(1)
+
cross enyne
ring-closing
metathesis
metathesis
(Z-only)
A X = RuL
C
n
B X = CH2
Mes
N
N Mes
Cl
Mes
N
N Mes
Cl
Ru CHPh
Ru
O
Cl
Cl
PCy3
5 (Grubbs gen-2)
6 (Hoveyda gen-2)
Scheme 2. Cyclohexadiene synthesis by tandem enyne metathesis.
substitution at the C6 and C60 positions.1 The best known
relative of ambewelamides is gliotoxin. Additional fungal
metabolites belonging to the edtdkp family have been iso-
lated including aranotin,2–4 epicorazine,5 the rostratins,6
and others. The central diketopiperazine ring is bridged by
a disulfide. This latter functionality spanning the diketo-
piperazine core (the C-ring) gives this family of natural
products their root name: epidithiadiketopiperazines. Of the
natural products in Scheme 1, only gliotoxin has been syn-
thesized despite an enormous amount of synthetic activity
directed toward the delicate introduction of the disulfide
moiety. Kishi and Fukuyama’s synthesis of gliotoxin stands
as a landmark achievement.7,8 In this report, we detail our
synthetic approach to the scabrosins relying on metal-
catalyzed transformation. In particular, ring building of the
A and E cyclohexene rings was accomplished through a
Grubbs’ ruthenium carbene-catalyzed tandem enyne meta-
thesis, developed in our labs (Eq. 1) and the nitrogen hetero-
an IC50 of 0.56 mM in the same assay, showing identical in-
hibition profile as found for ambewelamide.14,15 Because of
their identical core structure and similar biological profiles,
the ambewelamide/scabrosin family will be hereafter called
scabrosins.
Metathesis has become a powerful method for carbon–
carbon bond construction in synthesis.16–18 Enyne meta-
thesis is an adaptation of the parent reaction that provides
conjugated dienes as products. In particular, the cross, or
intermolecular, enyne metathesis19 offers a simple coupling
of unsaturated reactants to furnish diene products in a single
catalytic operation. The power of metathetic processes in
synthesis is amplified when metathesis reactions are used
in tandem. Our group developed the tandem diene–alkyne
metathesis as a cyclohexadiene ring synthesis as illustrated
above (Eq. 1). This methodology is attractive because it
generates a useful 2-substituted-1,3-cyclohexadiene from
simple alkynes and diene starting materials. There are few
methods for 1,3-cyclohexadiene synthesis and none that
offer a direct, catalytic synthesis achieved on mixing of
acyclic reactants. However, this tandem process, triggered by
intermolecular enyne metathesis, is not stereoselective. The
reaction between ruthenium carbene D and alkyne produces
E- and Z-vinyl carbene isomers E (Scheme 3). The lack of
Z-selectivity in intermolecular metathesis is a general prob-
lem in alkene metathesis research. Intermolecular enyne
metatheses can be more difficult than the intramolecular
ring-closing metathesis. The difficulty of the intermolecular
or ‘cross’ enyne metathesis is thought to arise due to the
slow catalyst turnover step E to F. The kinetics are known
for only a limited number of cross metatheses and the
rate-determining step can change depending on reactants
and catalyst employed.20
€
cycle formed by Pd(II)-catalyzed Backvall reaction. The
conjunction of the cyclohexadiene ring synthesis with the
Pd(II) chemistry for 1,4-difunctionalization validates our
approach and offers a powerful sequence of metal catalysis
for synthesis (Scheme 2).
The ambewelamide/scabrosins have potential as anticancer
agents. In general, the epidithiadiketopiperazine class of
fungal natural products display a range of biological activi-
ties. For instance, aranotin inhibits viral RNA polymer-
ase9,10 and gliotoxin is a reverse transcriptase inhibitor.11–13
However, the scabrosin/ambewelamide group displays
unique cytotoxicity and therefore has potential as anticancer
agents. This chemical biology is unique due to its dense
functionality, the presence of the epidisulfide and because
of the flanking epoxides. These factors combined are
thought to give scabrosins their unique and potent anticancer
activity.1 Recent studies suggest that the epoxides may not
be a major determinant in the unusual cytotoxicity observed
for these agents.14 Ambewelamide A is one of the best stud-
ied in the scabrosin family, with an acetyl and butanoyl
group at the flanks. Ambewelamide A is toxic to cancer
cell lines with an IC50 of 8.6 ng/mL (15 nM) for cytotoxicity
against the murine P388 leukemia cell line.1 Waring et al.
have also shown potent cytotoxicity against the MCF7
human breast cancer cell line with an IC50 of 1 nM.15 More-
over, tritiated thymidine incorporation was inhibited with an
IC50 of 0.5 mM in the P815 mastocytomia cell line (gliotoxin
gives IC50 of 2.9 mM in the same assay). Scabrosin A gives
OR1
R2
RuL
n
R
OR1
R1
+
L Ru
n
R
R
R
- L Ru
n
R2
R2
R
D
E
D
F
L Ru
n
OR1
R
R2
G
Scheme 3. The vinyl carbene intermediate in enyne metathesis.

10530
M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
Reaction conditions must be appropriate to kinetically drive
the reaction forward to nullify functional group coordination
(such as G) or chelative decomposition pathways. Func-
tional group chelation to the vinyl carbene intermediate
and catalyst decomposition occur by largely unstudied and
unknown pathways. Chelates have been suggested in order
to account for reduced efficiency in certain enyne metathe-
ses. Though plausible (see carbene 6), there is no experimen-
tal or kinetic evidence of their formation, and further studies
are needed to elucidate their relevance to catalytic efficiency.
For a typical intermolecular enyne metathesis, excess alkene
is used to drive the reaction. High alkene concentration may
also help propel catalysis forward relative to unwanted inter-
actions by functional groups (chelative traps are leading to
metal carbene decomposition).
The methylene-free conditions provide higher yields of
cyclohexadienes but the reactions are generally slower than
the metatheses of 1,5-hexadiene and alkynes.
For alkynes with certain functional groups, the tandem cross
metathesis using 1,5-hexadiene provides a complementary
ring synthesis to that of the methylene-free metathesis
conditions. The cross 1,5-hexadiene-alkyne metatheses are
faster in comparison, usually being done in minutes. We
have proposed that the 1,5-hexadiene-alkyne metatheses21
proceed quickly due to a fast vinyl carbene turnover step,
due to better binding of a 1-alkene and the high alkene
concentration used. These conditions are attractive for ring
synthesis where functional group interactions might pose
a problem or where low catalyst loadings are desired. In
contrast, the conditions of methylene-free metathesis were
designed to be slow, and in our original study,23 we specu-
lated that this might reduce functional group tolerance.
Going into the synthesis of the scabrosins, we wanted to
have two alternative procedures in hand to tackle potential
difficulties in the metathesis step.
Tandem enyne metathesis has evolved as a useful procedure
for ring synthesis. Our synthetic goal has been focused on cy-
clohexadiene synthesis. The one-step cyclohexadiene syn-
thesis was achieved in our original report.21 However, this
study also identified weaknesses. In particular, the lack of
stereoselection in the crossmetathesis step of the tandem pro-
cess presentedadifficultchallenge. Wedevelopedaworkable
solution to this problem using methylene-free conditions
(Scheme 4). These conditions proved effective for cyclohep-
tadiene (Eq. 2) and cyclohexadiene synthesis (Eq. 3). The cy-
cloheptadiene ring synthesis engendered a net two-carbon
ring expansion of cyclopentene by alkyne insertion, giving
H. This overall process is actually the result of a threefold
metathesis: ring-opening metathesis, cross metathesis, and
ring-closing metathesis. The cyclohexadiene synthesis was
accomplished from strain-free polybutadiene to form 7,
which was subsequently trapped in a thermal Diels–Alder
reaction with N-phenyl maleimide to provide 8 in good yield.
The fast cross metathesis with 1,5-hexadiene improves func-
tional group scope but this comes with reduced yield. The
yield is compromised by the triene by-product arising
from nonstereoselective cross metathesis (Eq. 4). Moreover,
the triene by-product J is difficult to separate from the
desired cyclohexadiene I. We developed a simple procedure
to separate the undesired triene from cyclohexadiene I. This
‘one-pot’ clean-up procedure transforms the triene J into
a polar, separable by-product K by alkene cross metathesis
(Scheme 5). The procedure can be executed with a variety
of alkenes in the second step of this sequential one-pot trans-
formation. To increase the polarity of the triene, we typically
R
Ru gen-2 (5 mol %)
CH2Cl2, reflux, 4 h
+
(2)
R
n
H (60-75%)
(4 equiv)
O
Ph
O
N
H
O
O
N(Ts)Boc
+
N
H
Ph
Ru gen-2 (5 mol %)
(3)
(2 equiv.)
CH2Cl2, 4 h
n
Boc(Ts)N
toluene, reflux
Boc
N
Ts
8 (58%)
(8 equiv)
Scheme 4. Methylene-free ring synthesis from polyalkenes.22,23
7
R1
R1
R1
Ru Hov-2 (5 mol %)
(4)
+
+
I
I
CO2H
(10 equiv)
HO2C
R1
R1
(3 equiv)
CH2Cl2, reflux
J
K
PMB
OTBS
N
OBz
OBn
Fmoc
CO2t-Bu
42%
Scheme 5. Tandem enyne metathesis and subsequent alkene cross metathesis.25
34%
32%
31%

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10531
used acrylic acid. The procedure was designed with alkene
cross selectivity in mind, using the Grubbs model for alkene
reactivity.24
effect the 1,4-N,O-difunctionalization of the 1,3-cyclohexa-
diene ring (Scheme 6). To make unsymmetrical ambewel-
amides, there are two possibilities. The cognate carboxylic
€
acid could be used in separate Backvall reactions to give
The procedure is remarkably efficient, including a cross
enyne metathesis, a ring-closing metathesis, and a cross
alkene metathesis, all under the same reaction conditions.
In the best cases, a single charge of catalyst was needed.
The theoretical yield of cyclohexadienes in these tandem
transformations is 50% (based on nonstereoselective cross
metathesis), and isolated yields are in the range of 35–
45%. Though this is not an ideal solution to the problem
of cyclohexadiene ring synthesis by tandem metathesis, the
clean-up procedure is attractive due to its simplicity and
practicality. The clean-up procedure can be used in cases
where functional groups interfere with the efficiency of
stereoselective, methylene-free enyne metathesis.
different acyl groups at C6 and C60 on the two indolines.
€
Alternatively, the product of Backvall cyclization can be
manipulated at the C6/C60 positions to install the desired
acyl groups. Our synthesis employs the latter approach.
€
The Backvall product M will be transformed to N and O
in two separate reactions. Indolines N and O are differen-
tially protected and ready for peptide coupling and cycliza-
tion to produce the pentacycle P.
The first goal in the synthesis was the development of a reli-
able synthetic route that would deliver significant quantities
of the metathesis precursor, chiral alkyne 12. After consider-
ing a number of possible methods for the synthesis of
propargylated amino acids, we decided to use the phase-
transfer-catalyzed alkylation approach (Scheme 7). The
phase-transfer-catalyzed alkylation of glycine imine ester
9 has been well studied by the groups of O’Donnell,26,27
Maruoka,28,29 Lygo,30 and others.31–33 We were attracted
to this approach because it is both operationally simple
and amenable to scale-up. We elected to use O-benzylated
cinchona catalyst 1330 due to the simplicity of the procedure,
the ready availability of phase-transfer catalyst 13 and the
low cost of starting materials.
2. Synthesis
Our approach to the ambewelamides is shown in Scheme 6.
Scabrosin is a symmetrical molecule, but we wanted to
develop a synthetic scheme that was applicable to the un-
symmmetrical ambewelamides (e.g., 2, 4). In these cases,
two different halves with differing acyl groups on the flank-
ing A and E rings would be synthesized and then joined. The
two metal-catalyzed reactions appear early in the synthesis
to make the indoline halves N and O. For this study, we
have chosen the desepoxycongener of scabrosin P as the
synthetic target in this study. First, the ring synthesis by
tandem enyne metathesis will be used to generate the cyclo-
hexadiene in M. The next metal-catalyzed reaction is the
High chemical and optical yields of chiral propargyl amino
acid 12 were obtained. Alkylation of 9 using a slight varia-
tion on the published conditions30 gave a good yield of the
propargylated glycine imine 10. Though 10 could be
purified and analyzed at this stage (e.g., for enantiomeric
excess), it proved sensitive to hydrolysis. The imine was
€
Pd(II)-promoted Backvall reaction, which will be used to
6'
OAc
6'
OAc
HO2C
N
Boc
N
H
N
H
H
N
RX
O
RO2C
RO2C
N
Fmoc
Fmoc
H
O
N
+
XR
Bäckvall
diene
functionalization
H
N
enyne tandem
metathesis
H
AcO
CO2Me
6
L
M
P
AcO
6
O
Scheme 6. Synthetic plan.
Br
N
Ph
N
Ph
tBuO2C
tBuO2C
tBuO2C
NH2
15% aq.
Citric acid,
THF,
50% aq. KOH,
CH2Cl2, Toluene
13 (5 mol %)
Ph
Ph
9
10
11 (73% 2 steps)
Fmoc-Cl, THF,
aq. NaHCO3
Br
N
H
N
tBuO2C
Fmoc
OBn
N
13
12 (78% >95% ee
after recrystallization)
Scheme 7. Synthesis of propargylated amino acid 12.

10532
M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
isolated after the asymmetric propargylation, and the crude
material taken through to hydrolysis with aqueous citric acid
in THF. In this way, amine 11 was obtained in 73% isolated
yield over the two steps. For stereochemical assignment,
imine 10 was analyzed directly. The only absolute configura-
tion data available on chiral propargyl amino acid derivatives
comes from alkyne 10. The optical rotation of 10 was deter-
mined on an analytically pure sample (column chromato-
graphy), which established the absolute configuration as S,
based on the literature precedence29–31,34 for the identical
compound. The amine was then protected with (9-fluorenyl-
methyl)chloroformate under Schotten–Baumann conditions
to give 12.27 At this point, the enantiopurity of the material
was established by HPLC using a chiral stationary phase.
The material derived directly from 10 had 89% ee, assigned
as the S-configuration on the basis of the absolute configura-
tion correlation of imine 10 above. With a single recrystalli-
zation, the enantiomeric excess was improved to greater that
95% ee, with a chemical yield of 78% (after a single recrys-
tallization). The sequence outlined in Scheme 7 proved
amenable to scale-up and delivered up to 40 g batches of 12.
basic aqueous wash followed by column chromatography,
which yielded 14 in 38% overall yield. Initially we ran
the two metathesis reactions with 5 mol % each of Grubbs’
second-generation carbene catalyst. However, on scale-up
this was optimized to a lower loading of 3.5 mol % each,
or 7% overall. Lowering the catalyst loading further was
unsuccessful: for example, the first cross metathesis stalled
at 3 mol % catalyst loading.
Kulkarni and Diver have recently developed the methylene-
free metathesis conditions, which deliver good yields of
1,3-cyclohexadienes.23 Our group is interested in catalyti-
cally efficient tandem processes and we typically do not
screen reaction conditions employing catalyst loadings
above 5 mol % Grubbs carbene complex. In this instance, we
were interested in comparatively evaluating the methylene-
free method with the clean-up procedure above, which
gave modest yields. When alkyne 17 was treated with the
second-generation Grubbs carbene complex and polybuta-
diene, only trace conversion to the cyclohexadiene was
detected at high (20 mol %) carbene catalyst loadings.25
Some conversion was obtained by treating the reaction mix-
ture with successive portions of catalyst, but the loading was
too high for practical use in the early stages of total synthesis.
The next challenge was the synthesis of cyclohexadiene 14.
The successive alkene metathesis ‘clean-up’ procedure of-
fered an expedient solution to cyclohexadiene synthesis for
the scabrosin synthesis. When 12 was treated with 5 equiv
of 1,5-hexadiene and Ru gen-2, a 1:1.2 mixture of diene
14 and triene 15 was produced (Scheme 8). The clean-up
procedure was then applied. Without isolation or solvent ex-
change, the mixture of 14 and 15 was directly treated with
a second portion of Ru gen-2 and acrylic acid. Continued re-
flux in dichloromethane solvent effected the alkene cross
metathesis, which occurred with high chemoselectivity on
the terminal alkene of 15. The new mixture of 14 and alkene
cross metathesis product 16 could be separated. Separation
was readily achieved through extractive work-up using a
Cyclohexadiene 14 can be accessed by methylene-free
enyne metathesis using 1,5-COD as the alkene. In these
runs, higher catalyst loadings (10–20 mol %) were required
in small-scale reactions.35 The high catalyst loadings are
needed because the free N–H is considered problematic
for cross enyne metathesis.36 Full conversion of alkyne
12 was achieved with 4 equiv COD at 14 mol % total
loading of Ru gen-2 complex (Eq. 5).37 The cyclodiene
was obtained in 68% isolated yield after treatment of
the crude reaction with DMSO (the Georg protocol).38 Typi-
cally the reaction requires the addition of a fresh portion of
Fmoc
NH
H
Ru gen-2 (14 mol %)a
tBuO
2C
tBuO2C
N
NHTs
Fmoc
(5)
1,5-cyclooctadiene
(4 equiv, high dilution)
(syringe pump)
17
CH2Cl2, reflux, 6h
12
14 (68%)
(a) Conditions: 10 mol % Ru gen-2 initial catalyst charge. Additional 4 mol %
Ru gen-2 was added after 2 h.
H
N
Fmoc
NH
tBuO2C
Fmoc
tBuO2C
Fmoc
NH
tBuO2C
Ru gen-2 (3.5 mol %)
Ru gen-2 (3.5 mol %)
14 (38%)
CO2H
(3 equiv)
CH2Cl2, reflux
14
(9 equiv)
CH2Cl2, reflux
Fmoc
NH
H
N
tBuO2C
tBuO2C
12
Fmoc
CO2H
separable mixture
15
16
Inseparable mixture
Scheme 8. Clean-up procedure applied to synthesis of 14.

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10533
catalyst. Currently, the best results are obtained with an ini-
tial charge of 10 mol % Ru gen-2 followed by addition of
additional 4 mol % halfway through the 4 h syringe pump
addition. We do not fully understand the nature of catalyst
decomposition. Further experiments to optimize the reaction
conditions are underway.39
19 and 22 and the oxidation product 29 did not vary signif-
icantly. Diene 14 could be undergoing air oxidation or could
be undergoing activation by Pd(II) with a b-hydride elimina-
tion competing with nucleophilic attack (Scheme 10).
BÄCKVALL and ANDERSON, 1990
H
AcO
(i)
(6)
With cyclohexadiene 14 accessible by two different
approaches, we proceeded to investigate cyclization to the
indoline. Ideally, direct transformation of 14 into indoline
N
NHR
H
R
R (82%, >93% trans)
Q
€
18 would be desired through use of the Backvall reac-
tion40–43 (top path, Scheme 9). Ultimately, we found that
an indirect two-step sequence via 19 was necessary to obtain
desired indoline 18.
THIS WORK
AcO
(i)
(7)
(8)
N
NHTs
H
21
Ts
reaction fails
20
Pd(II), LiOAc
AcO
CO2t-Bu
NHFmoc
trans-selective
(i) conditions: Pd(OAc)2 (5 mol %), benzoquinone (2 equiv), LiOAc,
acetone/HOAc (4:1 v/v)
CO2tBu
1,4-amido
N
H
H
acetoxylation
Fmoc
BÄCKVALL, et al., 1985
Pd(OAc)2 (5 mol %)
14
18
AcO
Cl
Pd(II), LiOAc, LiCl
cis-selective
1,4-chloro
HOAc-pentanes
LiCl, LiOAc
intramolecular
backside attack
benzoquinone
55%; 97% cis
acetoxylation
AcO
CO2t-Bu
NHFmoc
(2 equiv)
€
Scheme 10. Cyclization to indoline based on Backvall reactions (Eqs. 6 and
Cl
19
8 from Backvall42,44).
€
Scheme 9. Direct and indirect paths to indoline 18.
Deprotection of the fluorenylmethyloxycarbonyl (Fmoc)
group was accomplished over two discrete treatments with
base. Initial experiments using 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU) in acetonitrile on the mixture of diaste-
reomers 19/22 gave Fmoc deprotection with the formation of
up to 30% of 26 and 28. The formation of cyclic carbamates
was surprising. This is unusual, seldom observed probably
because most carbamic acids formed in situ during Fmoc de-
protection do not have a cyclization path. During the depro-
tection of 19/22 with DBU in acetonitrile, the intermediate
carbamic acids gave cyclization by substitution reaction on
the allylic chloride providing the cyclic carbamates 26 and
28. Other solvents such as tetrahydrofuran, dichloro-
methane, dichloroethane, and benzene all result in the for-
mation of 26 and 28 in decreasing amounts, respectively.
Each diastereomer 19/22 gave cleaner deprotection in
toluene without the formation of the cyclic carbamates.
Treatment of 19 or 22 with DBU in toluene at room temper-
ature afforded the corresponding primary amino acids in
97% and 94% yields, respectively. The addition of DBU
too quickly similarly produced trace amounts of 26 and 28
in toluene. Last, when the primary amine was cyclized in
the same pot as the Fmoc deprotection, there were several
products formed including a nonpolar adduct S between
the primary amine and fulvene. The fulvene is formed as
a by-product of Fmoc protecting group removal. For in-
stance it is known that the primary amines can add into the
fulvene to form the adduct.8 To optimize the yield of cycli-
zation, it proved necessary to remove the nonpolar fulvene
before the allylic chloride 27 was subjected to the more forc-
ing conditions required for the cyclization.
€
Backvall’s original paper describing the cyclization showed
that the amidoacetoxylation42 proceeds efficiently using
diene Q (Eq. 6). Our earlier efforts to effect an analogous
cyclization on the constitutional isomer 20 failed. The
tethered amine must have enough flexibility to attack the
h4-diene–Pd(II) complex; in diene 20 there is too much
ring strain for the out-of-plane deformation needed for
C–N bond formation to produce 21. To overcome this diffi-
culty, we imagined that a two-step sequence could be used.
€
Backvall has also described Pd(II)-catalysis of a stereoselec-
tive syn-chloroacetoxylation44–46 (Eq. 8). Interestingly, the
reaction is completely regioselective. Since the direct amido-
acetoxylation in Eq. 7 failed, we examined the two-step
approach described in Scheme 9 above. The syn-selective
chloroacetoxylation would be followed by a base-catalyzed
intramolecular displacement by an amide nucleophile. This
process results in the desired, net 1,4-trans amidoacetoxyla-
tion (lower pathway in Scheme 9).
€
The synthesis of the indoline relied on the Backvall chloro-
acetoxylation–cyclization two-step sequence. Exposing 14
to syn-1,4-chloroacetoxyation (LiCl and LiOAc in THF–
acetic acid) to Pd(II) under the oxidative conditions (2 equiv
benzoquinone) yielded functionalized alkene product 19 and
a diastereomer 22. The diastereomers arise from nondiscri-
minant complexation to the Pd(II). The a-chiral center is
too remote to have any effect on facial selectivity. As a result,
both diastereomers were produced in equal yields. During
purification of 19 and 22, phenylalanine 29 was isolated in
17% yield. It is presumed that this oxidation product arises
directly from the 1,3-cyclohexadiene 14. Several observa-
tions support this rationalization. Under the extended reac-
tion time, the ratio of the 1,4-acetoxychlorination products
The cyclization of 19 and 22 had to be conducted under
different conditions. Treatment of 22 with 10 equiv of
1,1,3,3-tetramethylguanidine in refluxing toluene for 15 h

10534
M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
yielded 82% of 23. In contrast, the 2,5-trans-pyrrolidine sub-
structure found in indoline 25 (2,9-anti-configuration based
on scabrosin numbering) makes it more strained than 23. As
a result, indoline 25 was found to be much harder to syn-
thesize. Treatment of 24 with 1,1,3,3-tetramethylguanidine
under identical conditions resulted in trace conversion to
indoline 25. After screening several bases, DBU in aceto-
nitrile at 50 ꢀC for 36 h yielded the desired indoline 25 in
58% isolated yield. Access to both diastereomers of the
indolines proved advantageous for the assignment of relative
stereochemistry. The stereochemistry of 23 was established
directly by NOE experiments. Irradiation of the resonance
at d 3.41 (C9 proton) gave an NOE of the resonances at
d 3.73 (C2 proton, scabrosin numbering) and d 1.70. Irradi-
ation of the proton at d 3.73 ppm showed enhancement in
the resonances at d 3.41 (C9 proton) and d 2.85 (C3 proton).
This was expected for a 2,5-cis-disubstituted pyrrolidine,47
and led to the assignment of the indoline relative stereo-
chemistry shown for 23 (Scheme 11). For diastereomer 25,
the analogous NOE between d 3.77 (C2 proton) and d 3.64
(C9 proton) of 25 was not observed, suggestive of trans-
orientation. The two observations taken together lead to an
assignment of the 2,5-trans-pyrrolidine substructure in 25.
Ultimately this conclusion was corroborated by crystal
structure of the pentacycle (vide supra).
coupling. A second portion of amino acid 25 was deprotected
and converted (di-tert-butyl dicarbonate in aqueous sodium
carbonate) to Boc-protected carboxylic acid 31 in 75% yield.
Indoline half 31 features the carboxylic acid to be activated in
the peptide coupling step. Next, the two pieces were joined
through conventional peptide coupling. Conventional pep-
tide coupling agents used for difficult couplings gave poor re-
sults. Both BOP reagent and PyBroP were attempted butgave
low conversion to dipeptide 33. The hindered nature of the
secondary amine in 32 hampered the efficiency of coupling.
Activation of the carboxylic acid 31 with bis(oxazolidinyl)-
phosphoryl chloride (BOP-Cl)48,49 gave an improved yield,
providing amide 33, isolated in 60% yield. Amide 33 ap-
peared as a mixture of amide and carbamate rotamers in
the NMR. The final cyclization required intramolecular ami-
nolysis of the methyl ester and was accomplished by depro-
tection of the N-Boc group with TFA in CH2Cl2. The excess
TFA was then removed and the secondary ammonium salt
neutralized with triethylamine, refluxed in CH2Cl2 overnight
to furnish the pentacycle 34 in 49% yield.
The structure of 34 was confirmed by single crystal X-ray
structure (Fig. 1). The pentacyclic diketopiperazine 34 was
crystallized from ethyl acetate and hexanes to provide white
colorless crystals, mp 259–261 ꢀC. Importantly, the struc-
ture determination corroborated the diastereomeric assign-
ment of the indolines based on observed NOE’s (Scheme
Completion of the synthesis of the pentacyclic core of scab-
rosin is summarized in Scheme 12. Indoline 25 was divided
into two portions and manipulated separately for eventual
union to form the diketopiperazine ring. The tert-butyl ester
was deprotected with trifluoroacetic acid, then esterified with
trimethylsilyldiazomethane in 1:1 v/v CH2Cl2–methanol to
produce methyl ester 32 in 84% yield. This amino ester has
a free amine in the proline ring ready for peptide bond
€
11 describing the Backvall chloroacetoxylation–cyclization
sequence). Since the molecule 34 does not contain any heavy
atoms and since it crystallized in a centrosymmetric space
group, the structure determination was not used to corrobo-
rate absolute configuration. The structure determination of
34 illustrates relative configuration. Furthermore, the crystal
structure showed disorder in the ester moieties, with the
CO2t-Bu
AcO
AcO
CO2t-Bu
NHFmoc
5% Pd(OAc)2, AcOH
Acetone, LiCl, LiOAc
benzoquinone
NHFmoc
14
Cl
19 (24%)
+
AcO
3
CO2t-Bu
NHFmoc
1) DBU, tol, rt, 1 h, 94%
2
CO2t-Bu
9
N
H
2) 1,1,3,3-tetramethylguanidine (10 eq)
tol, reflux, 82%
H
Cl
8
H
DBU, tol.,
rt, 1 h
noe
22 (24%)
23 (77%, two steps)
AcO
AcO
2 CO2t-Bu
CO2t-Bu
NH2
DBU, MeCN
9
N
H
50 ºC, 36 h
58%
H
H
Cl
24 (97%)
25 (56%, two steps)
CO2t-Bu
CO2t-Bu
CO2t-Bu
NH2
AcO
AcO
H
AcO
H
N
N
Cl
O
H
O
O
H
26
O
28
27
H
N
AcO
CO2t-Bu
NHFmoc
CO2t-Bu
Cl
S
29 (17%)
Scheme 11. Synthesis of diastereomeric indolines 23 and 25.

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10535
AcO
AcO
AcO
TFA,
CH2Cl2
CO2tBu
CO2H
N
H
N
H
H
H
H
H
coupling agent
yield 33
30
25
trace
BOP/ Et3N, CH2Cl2
PyBroP/i-Pr2NEt, CH2Cl2 24%
BOP-Cl/Et3N, CH2Cl2
1) TFA, CH2Cl2
2) TMSCHN2
CH2Cl2
Boc2O,
THF,
60%
aq Na2CO3
MeOH
AcO
+
coupling conditions
CO2Me
CO2H
N
H
N
H
Boc
H
H
H
32 (84%, 2 steps)
31 (75%, 2 steps)
O
O
H
H
H
H
N
OAc
OAc
N
N
H
1) TFA, CH2Cl2
N
AcO
H
AcO
2) Et3N, CH2Cl2
Boc
CO2Me
H
H
O
34 (49%, 2 steps)
33
Scheme 12. Completion of scabrosin pentacycle synthesis.
Figure 1. ORTEP drawing of pentacycle 34. The major conformer (63%) found in the unit cell is shown. Thermal ellipsoids are drawn at the 50% probability
level. The numbering on the right half is the same as scabrosin numbering. The crystallographic numbering C10–C18 corresponds to C10–C90 (scabrosin
numbering).
major (63% population) isomer shown. Both structures have
the same relative stereochemistry. A further discussion is
provided in Section 3.
ether were dried by passage through alumina and toluene
was dried and deoxygenated using columns of alumina and
Q5. Ruthenium [1,3-bis-(2,4,6-trimethylphenyl)-2-imidazo-
lidinylidine]dichloro(phenylmethylene)(tricyclohexylphos-
phine) (Grubbs’ second-generation catalyst) was obtained
from Materia, Inc. (Pasadena, CA) or purchased from
Aldrich Chemical Co. 1,5-Hexadiene was purified by distil-
lation from sodium metal. All other chemicals were pur-
chased from Aldrich Chemical Co. and used as received.
Column chromatography was carried out on Merck silica
In conclusion, we have achieved a stereocontrolled synthesis
of the scabrosin pentacycle using tandem diene–alkyne me-
tathesis conjoined with the Pd(II)-promoted chloroacetoxy-
lation–base-catalyzed cyclization. The combination of the
two metal-catalyzed reactions demonstrates the value and
versatility of the metathesis chemistry when linked with
1,4-difunctionalization. The cyclohexadiene ring synthesis
can be achieved under either 1,5-hexadiene-alkyne cross
metathesis or under methylene-free conditions using cyclo-
1
gel 60 (230–400 mesh). H NMR spectra were recorded
at 300, 400, or 500 MHz and 13C NMR spectra at either
75 or 125 MHz in the indicated solvent. H NMR spectra
1
€
octadiene as the alkene. The Backvall 1,4-N,O-difunctional-
were referenced on the TMS signal for CDCl3. The
13C NMR spectra were referenced at 77 ppm for CDCl3.
Enantiomeric excesses were determined by HPLC using
a Chiralcel OD-H column (4.6 mmꢁ250 mm, 5 mm particle
size) using UV detection. Proton and carbon NMR data can
be found in Supplementary data file.
ization chemistry of the 1,3-cyclohexadiene results in the
formation of the indoline ring system. The synthetic ap-
proach is amenable to the synthesis of unsymmetrical epidi-
thiadiketopiperazines of this family of natural products.
Further studies directed toward the stereospecific sulfuriza-
tion of the diketopiperazine ring are in progress.
3.1.1. 2-(Benzhydrylidene-amino)-pent-4-ynoic acid tert-
butyl ester (10). In a 5 L Erlenmeyer flask equipped with
magnetic stirbar, tert-butyl N-(diphenylmethylene)glycinate
9 (53 g, 0.18 mol) was dissolved in toluene (1.3 L) and
CH2Cl2 (0.54 L). The solution was treated sequentially with
catalyst 13 (5.9 g, 9.0 mmol), propargyl bromide (80% in
toluene, 24 mL, 0.22 mol), and 50% aqueous potassium
hydroxide (0.36 L, 3.2 mol). The mixture was stirred
vigorously for 14 h at room temperature, the phases were
separated and the aqueous layer was extracted with Et2O
3. Experimental
3.1. General information
Reactions were conducted under argon atmosphere unless
otherwise noted. Solvents were dried and degassed under
argon by a solvent purification system and drawn immedi-
ately prior to use. Dichloromethane, tetrahydrofuran, and

10536
M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
(2ꢁ300 mL). The organic layers were combined, dried
(Na2SO4), filtered, and concentrated in vacuo (rotary evapo-
rator). The residue was then passed though a short plug of
silica gel, eluted with 20% ethyl acetate–hexanes, and con-
centrated to give crude imine 10 (57 g, 95%), which was
used directly in the subsequent hydrolysis step.
ethyl acetate–hexanes) to afford a white solid, which was
recrystallized (ethyl acetate–hexanes) to give 12 as white
needles (39.2 g, 76%): mp 68–70 ꢀC; Rf¼0.53 (1:3 ethyl
1
acetate–hexanes); H NMR (500 MHz, CDCl3) d 7.79 (d,
J¼8.0 Hz, 2H), 7.64 (d, J¼7.5 Hz, 2H), 7.4 (dd, J¼7.5,
7.5 Hz, 2H), 7.34 (dd, J¼8.0, 7.5 Hz, 2H), 5.5 (d,
J¼8.0 Hz, 1H), 4.48–4.38 (m, 3H), 4.27 (dd, J¼7.0 Hz,
1H), 2.81–2.78 (m, 2H), 2.09 (br s, 1H), 1.53 (s, 9H); 13C
NMR (125 MHz, CDCl3) d 169.3, 155.6, 143.9, 143.8,
141.3, 127.7, 127.1, 125.2, 120, 82.8, 78.6, 71.6, 67.2,
52.6, 47.1, 28.0, 23.0; FTIR (thin film, cmꢂ1) 3298, 2977,
1721, 1507, 1450, 1349, 1223, 1156; high-resolution MS
(EI+) calcd for C24H25O4N (M+): 391.1778, found:
391.1776; enantiomeric excess determination by HPLC
(0.6 mL/min, gradient elution 15% 2-propanol–hexane to
40% 2-propanol–hexane over 40 min, tR¼15.1, 1.6% (R),
26.0, 98.4% (S) min) indicated 97% ee of the S-enantiomer;
[a]2D5 +31.6 (c 1.0, CHCl3).
An analytical sample of 10 was obtained in a separate small-
scale reaction, conducted analogous to the procedure above
on 10 mmol scale. After the usual work-up, the residue was
further purified by flash column chromatography on silica
gel (gradient elution with 1:30 ethyl acetate–hexane to 1:9
ethyl acetate–hexane) to give 10 (2.38 g, 71%) as a green
oil: Rf¼0.39 (1:9 ethyl acetate–hexanes); 1H NMR
(300 MHz, CDCl3) d 7.67–7.64 (m, 2H), 7.45–7.25 (m,
8H), 4.19–4.15 (m, 1H), 2.83–2.71 (m, 2H), 1.94 (m, 1H);
13C NMR (75 MHz, CDCl3) d 171.4, 169.6, 139.7, 136.3,
130.4, 129.0, 128.7, 128.4, 128.3, 128.1, 81.6, 81.3, 70.1,
64.8, 28.1, 23.4; FTIR (thin film, cmꢂ1) 3297, 2978, 1732,
1624, 1446, 1368, 1285, 1152, 696; high-resolution MS
(ESI+) calcd for C22H24O2N1 (M++H): 334.1802, found:
334.1809; [a]2D5 ꢂ96.8 (c 1.0, CHCl3). The negative sign
of optical rotation established the S-configuration in agree-
ment with the literature assignment.31,34
3.1.4. (2S)-tert-Butyl 3-cyclohexa-1,5-dienyl-2-(9H-fluo-
ren-9-ylmethoxycarbonylamino)-propionate (14).
3.1.4.1. Method A: 1,5-cyclooctadiene. A 50 mL air-
free Schlenk tube was charged with dichloromethane
(5 mL) and 1,5-cycloctadiene (188 mL, 1.5 mmol). Argon
was bubbled through this solution for 10 min. Ru gen-2
(21.2 mg, 0.025 mmol, 10 mol %) was then added to the
room temperature solution and subsequently placed into
a 55 ꢀC oil bath. To this solution was added 12 (100 mg,
0.256 mmol) in 2 mL dichloromethane over 4 h via gas-tight
syringe (addition rate 0.5 mL/h).50 After 2 h of addition, an
additional 4 mol % of Ru gen-2 (8.7 mg, 0.010 mmol,
4 mol %) was added to the reaction. After 2 h, the addition
was complete and the flask was heated for another 6 h.
The reaction was allowed to cool to room temperature, con-
centrated in vacuo (rotary evaporator) diluted with 5 mL
of dichloromethane and 182 mL of dimethyl sulfoxide
(2.56 mmol, 1000 mol %), and stirred for 12 h. The solution
was then concentrated in vacuo (rotary evaporator) and the
residue purified by column chromatography on silica gel,
eluting with 1:7 ethyl acetate–hexanes to obtain 75 mg of
14 (68%) as a pale yellow oil: Rf¼0.27 (1:5 ethyl acetate–
hexanes); 1H NMR (500 MHz, CDCl3) d 7.76 (d,
J¼7.5 Hz, 2H), 7.60 (m, 2H), 7.40 (t, J¼7.0 Hz, 2H), 7.31
(t, J¼7.5 Hz, 2H), 5.83 (m, 2H), 5.55 (s, 1H), 5.27 (d,
J¼8.0 Hz, 1H), 4.41 (m, 1H), 4.34 (m, 2H), 4.23 (m, 1H),
2.48 (m, 2H), 2.10 (m, 4H), 1.47 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 171.1, 155.5, 143.9, 141.3, 130.9,
127.7, 127.3, 127.0, 126.6, 125.2, 124.4, 120.0, 82.1, 66.1,
3.1.2. 2-Amino-pent-4-ynoic acid tert-butyl ester (11). A
2 L rb flask equipped with magnetic stirbar and rubber sep-
tum was charged with imine 10 (57 g, 0.17 mol) and 650 mL
THF. To the stirred solution was added 15% aqueous citric
acid (350 mL) and stirring was continued for 12 h. The mix-
ture was diluted with 1 M HCl (200 mL), extracted with
Et2O (3ꢁ350 mL), and the combined organics were subse-
quently washed with water (2ꢁ300 mL). The combined
aqueous layers were basified to pH 11 by the addition of
K2CO3. The aqueous layer was then extracted with ethyl
acetate (3ꢁ400 mL) and all of the organic layers were
then combined, dried (Na2SO4), filtered, and concentrated
in vacuo (rotary evaporator). The residue was purified by
flash column chromatography on silica gel (elution with
3:1 ethyl acetate–hexanes) to give 11 (22.3 g, 77%) as a clear
oil: Rf¼0.32 (3:1 ethyl acetate–hexanes); 1H NMR
(300 MHz, CDCl3) d 3.51 (dd, J¼9.0, 9.0 Hz, 1H), 2.61–
2.58 (m, 2H), 2.06–2.05 (m, 1H), 1.67 (br s, 1H), 1.48 (s,
12H); 13C NMR (75 MHz, CDCl3) d 173.1, 81.6, 79.8,
71.0, 53.6, 28.0, 25.0; FTIR (thin film, cmꢂ1) 2979, 2362,
1730, 1394, 1368, 1251, 1221, 1153; high-resolution MS
(ESI+) calcd for C9H15O2N1Na (M++Na): 192.0995, found:
192.0994; [a]2D5 ꢂ23.8 (c 1.0, CHCl3).
53.6, 47.2, 38.5, 28.0, 22.3, 22.1; FTIR (thin film, cmꢂ1
)
3.1.3. (2S)-tert-Butyl 2-(9H-fluoren-9-ylmethoxycarbo-
nylamino)-pent-4-ynoate (12). A 2 L rb flask containing
magnetic stirbar and rubber septum was charged with amine
11 (22.3 g, 0.132 mol) in THF (500 mL) to which 9-(fluor-
enylmethoxycarbonyl)chloride (38.0 g, 0.145 mol) was
added. A solution of 10% aqueous Na2CO3 (500 mL) was
added and the mixture was stirred for 16 h. After this time,
the mixture was diluted with ethyl acetate (400 mL) and
the layers were separated. The aqueous layer was extracted
with ethyl acetate (2ꢁ400 mL) and the organic layers
were combined, dried (MgSO4), filtered, and concentrated
in vacuo (rotary evaporator). The residue was subsequently
purified by flash column chromatography on silica gel
(gradient elution with 1:20 ethyl acetate–hexanes to 1:5
3326, 2933, 1718, 1507, 1450, 1367, 1224, 1155, 1050;
high-resolution ESI molecular ion calcd for C28H31O4N+Na
468.2145, found: 468.2152; [a]2D5 +10.0 (c 1.6, CHCl3).
3.1.4.2. Method B: preparative scale using 1,5-hexa-
diene. Into a 250 mL rb flask equipped with a condenser,
magnetic stirbar, and rubber septum was added alkyne 12
(15.6 g, 40 mmol) and 1,5-hexadiene (24 mL, 200 mmol),
dissolved in CH2Cl2 (80 mL). The Grubbs’ catalyst Ru
gen-2 (1.2 g, 1.4 mmol, 3.5 mol %) was added and the solu-
tion was then brought immediately to reflux by immersion in
a 50 ꢀC oil bath. Heating was maintained for 5 h, the mixture
was subsequently cooled and concentrated to a volume of
10 mL (rotary evaporator). The residue was passed though

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10537
a short plug of silica gel (eluting with 20% ethyl acetate–
hexanes) and concentrated. The crude mixture was then dis-
solved in CH2Cl2 (80 mL) and acrylic acid (11.0 mL,
160 mmol) was then added. The Grubbs’ catalyst Ru gen-
2 (1.2 g, 1.4 mmol, 3.5 mol %) was then added and the solu-
tion was then brought immediately to reflux by immersion in
a 50 ꢀC oil bath. Heating was maintained for 12 h, the mix-
ture was subsequently cooled to room temperature, diluted
with CH2Cl2 (200 mL), and washed with saturated aqueous
NaHCO3 (2ꢁ250 mL). The organic layers were combined,
dried (MgSO4), filtered, and the solvent removed in vacuo
(rotary evaporator). Purification was accomplished by flash
column chromatography on silica gel (elution with 1:5 ethyl
acetate–hexanes) afforded 14 (6.8 g, 38%), as a brown oil.
Spectral data matched that reported for 14 above (using
COD), though 17–20% butadiene was present by proton
NMR. This by-product proved difficult to remove, so the
diene was carried through to the next step.
155.5 (143.8, 143.7, rotamers), 141.8, 137.2, 129.2, 127.7,
127.0, 125.0, 120.0, 82.7, 69.1, 67.0, 55.8, 53.0, 47.1,
37.7, 30.2, 28.0, 23.2, 21.1; FTIR (thin film, cmꢂ1) 3339,
2977, 1733, 1520, 1450, 1369, 1237, 1154, 1033; high-reso-
lution ESI molecular ion calcd for C30H34O4ClN+Na
562.1967, found: 562.1982; [a]2D5 ꢂ4.4 (c 2.00, CHCl3).
Compound 22: mp¼52–54 ꢀC. Rf¼0.15 (1:5 ethyl acetate–
hexanes); 1H NMR (500 MHz, CDCl3) d 7.76 (d, J¼
12.0 Hz, 1H), 7.58 (d, J¼11.5 Hz, 1H), 7.40 (t, J¼
12.0 Hz, 1H), 7.32 (t, J¼12.0 Hz, 1H), 5.61 (s, 1H), 5.29
(d, J¼13.0 Hz, 1H), 5.20 (m, 1H), 4.66 (s, 1H), 4.31 (m,
4H), 2.94 (m, 1H), 2.10 (m, 6H), 1.48 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 170.8, 170.4, 155.9, 143.8, 141.3,
137.4, 129.1, 127.7, 127.0, 125.0, 120.0, 82.7, 69.1, 67.1,
55.7, 52.3, 47.1, 38.4, 30.1, 28.0, 23.3, 21.1; FTIR (thin
film, cmꢂ1) 3338, 2978, 1734, 1525, 1450, 1369, 1240,
1156; high-resolution ESI molecular ion calcd for
C30H34O4ClN+Na 562.1967, found: 562.1978; [a]D25
+18.5 (c 2.00, CHCl3).
3.1.5. Preparation of 19, 22, and 29. To a stirred solution of
Pd(OAc)2 (149 mg, 0.664 mmol), LiCl (111 mg, 2.6 mmol),
LiOAc–2H2O (667 mg, 6.64 mmol), and benzoquinone
(2.87 g, 26.6 mmol) in glacial acetic acid (3.9 mL) and
acetone (37 mL) was added two solutions via syringe
pump over 12 h. Solution 1 was cyclohexadiene 14 (5.9 g,
13.2 mmol) in acetone (5.7 mL). Solution 2 was LiCl
(1.0 g, 23.9 mmol) in glacial acetic acid (5.7 mL). After
15 h, the reaction was concentrated in vacuo (rotary evapora-
tor), diluted with ethyl acetate (300 mL), transferred to a sep-
aratory funnel, and washed with 1 M NaOH(aq) (3ꢁ200 mL).
The aqueous layers were then combined and back-extracted
with ethyl acetate (3ꢁ100 mL), the organic layers were then
combined, dried (MgSO4), concentrated in vacuo (rotary
evaporator) to give a viscous black sludge. The black residue
was dissolved in a minimal amount of chloroform and passed
through a short column of silica gel (3.7 cmꢁ12 cm) eluted
with 1:10 ethyl acetate–hexanes ramping up to 1:5 ethyl ace-
tate–hexanes to remove palladium and other polar by-prod-
ucts (this operation removes most of 29 from 19 and 22).
The desired fractions (containing 29 Rf¼0.27 in 1:5 ethyl
acetate–hexanes) were collected and concentrated into a vis-
cous yellow oil. This oil was further purified by column chro-
matography on silica gel (3.7 cmꢁ30 cm), eluting with 1:10
ethyl acetate–hexanes ramping polarity to 1:6 ethyl acetate–
hexanes giving 1.7 g of 19 (24%), 1.7 g of 22 (24%), and
1.0 g of 29 (17%), each obtained as viscous pale yellow
oils. If 19 and 22 are dissolved in a minimal amount of ether
(at rt) and diluted with five volumes of pentane and concen-
trated, a pale yellow solid is obtained in quantitative yield.
The pale yellow product is clean enough to be taken on to
the next reaction. If another column is performed it is possi-
ble to obtain 19 and 22 as a clear oil or white powder as
described above. The 19, 22, and 29 are stable at room tem-
perature in a vial in regular lab light for months.
1
Compound 29: Rf¼0.27 (1:5 ethyl acetate–hexanes); H
NMR (500 MHz, CDCl3) d 7.77 (d, J¼7.5 Hz, 2H), 7.57 (t,
J¼7.0 Hz, 2H), 7.40 (t, J¼7.5 Hz, 2H), 7.28 (m, 5H), 7.15
(d, J¼7.0 Hz, 2H), 5.28 (d, J¼7.5 Hz, 1H), 4.55 (m, 1H),
4.44 (dd, J¼11.0, 7.5 Hz, 1H), 4.32 (dd, J¼11.0, 7.5 Hz,
1H), 4.21 (t, J¼7.0 Hz, 1H), 3.10 (d, J¼5.5 Hz, 2H), 1.42
(s, 9H); 13C NMR (125 MHz, CDCl3) d 170.5, 155.4,
143.7, 141.1, 136.0, 129.3, 128.2, 127.5, (126.8, 127.7 roto-
mers), 124.9, 119.8, 82.0, 66.7, 55.0, 47.0, 38.2, 27.8; FTIR
(thin film, cmꢂ1) 3334, 2979, 1723, 1511, 1368, 1253; high-
resolution ESI molecular ion calcd for C28H29O4N+Na
466.1989, found: 466.1988; [a]2D5 +19.6 (c 2.00, CHCl3).
3.1.6. Preparation of 24. A 100 mL Schlenk tube was
charged with 19 (780 mg, 1.44 mmol) and 74 mL of toluene.
To this stirring solution was added dropwise 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (208 mL, 1.44 mmol) over 2 min,
then stirred for 1 h. The color changed from yellow to
a pink/red on addition of DBU. The reaction was concen-
trated in vacuo (rotary evaporator) to a volume of w1 mL
and purified by column chromatography on silica gel, elut-
ing with 1:1 ethyl acetate–hexane ramping polarity to
100% ethyl acetate (once the fulvene has been eluted, the
solvent polarity is increased from 1:1 to 4:1 ethyl acetate–
hexane and then to 100% ethyl acetate after 24 starts to
elute), to yield 436 mg of 24 (96%) as a pale yellow oil:
1
Rf¼0.17 (4:1 ethyl acetate–hexanes); H NMR (400 MHz,
CDCl3) d 5.60 (s, 1H), 5.82 (t, J¼7.0 Hz, 1H), 4.57 (t,
J¼3.5 Hz, 1H), 3.5 (t, J¼7.0 Hz, 1H), 2.45 (m, 2H), 2.04
(m, 7H), 1.81 (s, 2H), 1.42 (s, 9H); 13C NMR (125 MHz,
CDCl3) d 174.2, 170.7, 138.4, 128.4, 81.5, 69.2, 56.5,
54.0, 40.2, 30.3, 28.0, 23.2, 21.2; FTIR (thin film, cmꢂ1
)
2919, 2850, 1734, 1369, 1241, 1154, 1026; high-resolution
ESI molecular ion calcd for C15H24O4NCl+H 318.1467,
found: 318.1456; [a]2D5 ꢂ16.4 (c 3.70, CHCl3).
Compound 19: mp¼51–54 ꢀC. Rf¼0.11 (1:5 ethyl acetate–
hexanes); 1H NMR (500 MHz, CDCl3) d 7.76 (d,
J¼7.5 Hz, 2H), 7.60 (d, J¼7.0 Hz, 2H), 7.40 (t, J¼7.5 Hz,
2H), 7.32 (t, J¼7.5 Hz, 2H), 5.63 (s, 1H), 5.34 (d,
J¼8.0 Hz, 1H), 5.30 (m, 1H), 4.52 (s, 1H), 4.38 (m, 3H),
4.23 (t, J¼7.0 Hz, 1H), 2.76 (dd, J¼14.5, 5.5 Hz, 1H),
2.49 (dd, J¼14.5, 8.0 Hz, 1H), 2.19 (m, 1H), 2.01 (m, 6H),
1.47 (s, 9H); 13C NMR (125 MHz, CDCl3) d 170.9, 170.5,
3.1.7. Preparation of 27. A 100 mL Schlenk tube was
charged with 22 (511 mg, 0.927 mmol) and 46 mL of
toluene. To this stirring solution was added dropwise 1,8-
diazabicyclo (5.4.0) undec-7-ene (130 mL, 0.927 mmol) over
2 min and then stirred for 1 h. The color changed from
yellow to a pink/red on addition of DBU. The reaction was

10538
M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
concentrated in vacuo (rotary evaporator) to a volume of
w1 mL and purified by column chromatography on silica
gel, eluting with 1:1 ethyl acetate–hexanes ramping polarity
to 100% to ethyl acetate (once the fulvene been eluted, the
solvent polarity is changed from 1:1 to 4:1 ethyl acetate–
hexanes and then to straight ethyl acetate after 27 starts to
elute), to yield 286 mg of 27 (94%) as a pale yellow oil:
with 1:1 ethyl acetate–hexane ramping polarity to 3:1 ethyl
acetate–hexane, to yield 110 mg of 23 (82%) as a pale
1
yellow oil: Rf¼0.22 (4:1 ethyl acetate–hexanes); H NMR
(500 MHz, CDCl3) d 5.43 (m, 2H), 3.73 (dd, J¼6.5,
2.5 Hz, 1H), 3.41 (d, J¼8.0 Hz, 1H), 2.85 (ddd, J¼17.0,
9.5, 2.0 Hz, 1H), 2.43 (ddd, J¼17.0, 6.0, 2.0 Hz, 1H), 2.24
(m, 2H), 2.10 (s, 3H), 1.7 (s, 1H), 1.45 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 172.9, 170.8, 146.5, 117.4, 81.4,
71.1, 58.6, 58.3, 35.4, 28.9, 27.9, 27.4, 21.2; FTIR (thin
film, cmꢂ1) 2977, 2939, 2867, 1733, 1369, 1244, 1155,
1020; high-resolution ESI molecular ion calcd for
C15H23O4N+H 282.1700, found: 282.1706; [a]2D5 ꢂ118.2
(c 1.43, CHCl3).
1
Rf¼0.10 (4:1 ethyl acetate–hexanes); H NMR (500 MHz,
CDCl3) d 5.63 (s, 1H), 5.34 (t, J¼7.5 Hz, 1H), 4.56 (t,
J¼3.5 Hz, 1H), 3.53 (dd, J¼9.5, 4.5 Hz, 1H), 2.76 (dd,
J¼14.5, 4.5 Hz, 1H), 2.20 (m, 2H), 2.03 (m, 6H), 1.54 (s,
2H), 1.47 (s, 9H); 13C NMR (125 MHz, CDCl3) d 174.3,
170.6, 138.2, 128.3, 81.4, 69.2, 56.0, 53.0, 39.4, 30.3,
28.0, 23.3, 21.2; FTIR (thin film, cmꢂ1) 3380, 2977, 1733,
1369, 1243, 1157, 1027; high-resolution ESI molecular ion
calcd for C15H24O4NCl+H 318.1467, found: 318.1466;
[a]2D5 +18.0 (c 2.00, CHCl3).
3.1.11. 5-Acetoxy-2,3,5,6,7,7a-hexahydro-1H-indole-2-
carboxylic acid methyl ester (32). Into a 10 mL rb flask
containing magnetic stirbar and rubber septum was placed
indoline 25 (30 mg, 0.11 mmol) dissolved in CH2Cl2
(1.25 mL) at room temperature. To the stirred solution was
added trifluoroacetic acid (1.25 mL), stirred at ambient tem-
perature for 12 h then concentrated in vacuo and placed
under high vacuum for 2 h. The residue was dissolved in
1:1 v/v CH2Cl2–MeOH (1 mL of each) and (trimethylsily)-
diazomethane (2.0 M in Et2O, 205 mL) was added dropwise
by microliter syringe until gas evolution ceased and the
solution remained yellow. The mixture was stirred for 8 h,
concentrated in vacuo (rotary evaporator) and the residue
was subsequently purified by flash column chromatography
on silica gel (gradient elution with 3:1 ethyl acetate–hexane
to 100% ethyl acetate) to provide 32 (14 mg, 84%), as
3.1.8. Preparation of 26 and 28. Substituting toluene for
acetonitrile in the procedure above will result in the forma-
tion of 20–30% 26 and 28 as a clear oil (Rf¼0.30,
Rf¼0.32, respectively, 4:1 ethyl acetate–hexanes). The use
of tetrahydrofuran, dichloromethane, dichloroethane or
benzene all resulted in the formation of 26 and 28 in decreas-
ing amounts, respectively. The addition of DBU too quickly
will also produce trace amounts of 26 and 28 in toluene.
Work-up is the same as for 24 and 27.
Compound 28: Rf¼0.32 (4:1 ethyl acetate–hexanes); 1H
NMR (500 MHz, CDCl3) d 5.83 (m, 2H), 5.26 (d,
J¼4.0 Hz, 1H), 4.72 (t, J¼4.0 Hz, 1H), 4.08 (ddd, J¼12.5,
8.5, 4.5 Hz, 1H), 2.78 (m, 2H), 2.1 (m, 5H), 1.73 (m, 1H),
1.48 (s, 9H); 13C NMR (125 MHz, CDCl3) d 170.4, 168.9,
157.7, 136.5, 127.3, 83.4, 73.6, 66.7, 53.6, 37.8, 27.9,
25.8, 24.1, 21.1; FTIR (thin film, cmꢂ1) 3314, 2967, 1728,
1376, 1244, 1155, 1014; high-resolution ESI molecular ion
calcd for C16H23O6N+Na 348.1418, found: 348.1414;
[a]2D5 ꢂ118.0 (c 0.50, CHCl3).
1
a brown oil: Rf¼0.15 (3:1 ethyl acetate–hexane); H NMR
(500 MHz, CDCl3) d 5.49 (s, 1H), 5.43–5.42 (m, 1H), 3.91
(dd, J¼9.0, 5.0 Hz, 1H), 3.74 (s, 3H), 3.64–3.62 (m, 1H),
2.84 (ddd, J¼16.5, 9.0, 1.5 Hz, 1H), 2.70 (ddd, J¼16.5,
4.5, 2.0 Hz, 1H), 2.49 (br s, 1H), 2.23–2.13 (m, 2H), 2.05
(s, 3H), 1.56–1.48 (m, 1H), 1.39–1.31 (m, 1H); 13C NMR
(125 MHz, CDCl3) d 175.6, 171.0, 146.0, 118.1, 71.0,
57.4, 56.4, 52.3, 33.9, 29.9, 27.1, 21.4; FTIR (thin film,
cmꢂ1) 2950, 1734, 1441, 1373, 1243, 1138; high-resolution
MS (ESI+) calcd for C12H18O4N1 (M++H): 240.1230, found:
240.1235; [a]2D5 +43.6 (c 0.5, CHCl3).
3.1.9. Preparation of 25. To a 100 mL rb flask was added 24
(252 mg, 0.795 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene
(112 mL, 0.795 mmol) and 40 mL of acetonitrile. This solu-
tion was heated to 50 ꢀC for 36 h. After 36 h, the reaction
was concentrated in vacuo (rotary evaporator) and purified
by column chromatography on silica gel, eluting with 1:1
ramping polarity to 3:1 ethyl acetate–hexane, to yield
130 mg of 25 (58%) as a pale yellow oil: Rf¼0.28 (4:1 ethyl
3.1.12. 5-Acetoxy-2,3,5,6,7,7a-hexahydro-indole-1,2-di-
carboxylic acid 1-tert-butyl ester (31). Into a 10 mL rb flask
containing magnetic stirbar and rubber septum was placed in-
doline 25 (45 mg, 0.16 mmol) dissolved in CH2Cl2 (2 mL) at
room temperature. To the stirred solution was added tri-
fluoroacetic acid (2 mL), stirred at ambient temperature for
8 h, then concentrated in vacuo (rotary evaporator) and
placed under high vacuum for 2 h. The residue was then dis-
solved in a mixture of THF (4 mL) and 10% aqueous Na2CO3
(4 mL). To the stirred solution was added di-tert-butyl dicar-
bonate (Boc2O, 42 mg, 0.19 mmol), the mixture was stirred
at ambient temperature for 11 h, acidified to pH 3 with 1 M
aq HCl and then extracted with ethyl acetate (3ꢁ15 mL).
The combined organic layers were then dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was sub-
sequently purified by flash column chromatography on silica
gel (gradient elution with 5% MeOH–CH2Cl2 to 10%
MeOH–CH2Cl2) to afford 31 (50 mg, 96%) as a yellow
1
acetate–hexanes); H NMR (500 MHz, CDCl3) d 5.48 (s,
1H), 5.43 (m, 1H), 3.77 (m, 1H), 3.64 (m, 1H), 2.84 (m,
1H), 2.63 (m, 1H), 2.16 (m, 2H), 2.03 (s, 1H), 1.77 (s, 1H),
1.47 (m, 11H); 13C NMR (125 MHz, CDCl3) d 174.5,
170.9, 146.4, 118.0, 81.5, 71.1, 58.1, 56.3, 30.1, 28.0, 27.1,
21.3; FTIR (thin film, cmꢂ1) 3343, 2935, 1731, 1370, 1242,
1156; high-resolution ESI molecular ion calcd for
C15H23O4N+H 282.1700, found: 282.1704; [a]2D5 +79.3 (c
1.20, CHCl3).
3.1.10. Preparation of 23. To a 50 mL rb flask was added
27 (149 mg, 0.47 mmol), 1,1,3,3-tetramethylguanidine
(112 mL, 0.795 mmol) and 24 mL of toluene. This solution
was gently refluxed for 15 h. After this time, the reaction
was filtered, concentrated in vacuo (rotary evaporator), and
purified by column chromatography on silica gel, eluting
1
gum: Rf¼0.18 (5% MeOH–CH2Cl2); H NMR (500 MHz,
CDCl3) d 5.56 (s, 1H), 5.41–5.34 (m, 1H), 4.28–4.18
(m, 2H), 2.87–2.81 (m, 1H), 2.65–2.49 (m, 1H), 2.09–2.22

M. D. Middleton et al. / Tetrahedron 62 (2006) 10528–10540
10539
(m, 1H), 2.04 (s, 3H), 1.48–1.22 (m, 12H); 13C NMR
(125 MHz, CDCl3) d 178.8, 171.1, 154.9, 153.8, 142.8,
142.3, 120.3, 120.2, 81.1, 81.0, 70.4, 70.2, 67.7, 67.5, 58.6,
58.4, 57.1, 56.7, 34.3, 33.8, 29.7, 29.5, 29.0, 28.4, 28.3,
28.2, 26.9, 26.7, 23.8, 21.3; FTIR (thin film, cmꢂ1) 2978,
1733, 1698, 1478, 1418, 1368, 1243, 1145; high-resolution
MS (ESI+) calcd for C16H23O6NNa (M++Na): 348.1418,
found: 348.1418; [a]2D5 ꢂ23.2 (c 1.0, CHCl3).
on the ambewelamides and for advice regarding the methyl-
ene-free metathesis. We also thank Dr. Cara L. Nygren for
refinement of the crystal structure data, Professor Philip
Coppens (UB) for use of his X-ray diffraction equipment,
and Materia, Inc. for samples of the Grubbs second-genera-
tion complex.
Supplementary data
3.1.13. Amide 33. Into a 10 mL rb flask containing magnetic
stirbar and rubber septum was placed acid 31 (18 mg,
0.55 mmol) and amino ester 32 (13 mg, 0.055 mmol), dis-
solved in CH2Cl2 (1 mL). To this solution was added succes-
sively BOP-Cl (21 mg 0.083 mmol) then Et3N (11 mg,
0.11 mmol). The mixture was then stirred at room tempera-
ture for 18 h, water (5 mL) was added, and the mixture was
extracted with CH2Cl2 (3ꢁ5 mL). The combined organic
layers were then dried (MgSO4), filtered, and concentrated
in vacuo. The residue was subsequently purified by flash col-
umn chromatography on silica gel (gradient elution with 1:2
ethyl acetate–hexanes to 2:1 ethyl acetate–hexanes) to pro-
vide 33 (18 mg, 60%) as a yellow gum: Rf¼0.47 (3:1 ethyl
acetate–hexanes); The NMR spectra showed a mixture of
amide and carbamate rotamers that did not coalesce on heat-
ing in the NMR probe. The compound was therefore par-
tially characterized and taken on to the cyclization step.
High-resolution MS (ESI+) calcd for C28H38O9N2Na
(M++Na): 569.2470, found: 569.2462.
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.tet.2006.05.089.
References and notes
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hexanes); 1H NMR (500 MHz, CDCl3) d 5.73 (s, 1H), 5.43
(m, 1H), 4.40–4.24 (m, 2H), 2.86–2.80 (m, 3H), 2.30–2.25
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39. While this manuscript was under review, we found that lower
catalyst loadings can be used at higher concentrations of