Formal synthesis of (-)-englerin A and cytotoxicity studies of truncated englerins

Article abstract of DOI:10.1002/asia.201101021

An efficient formal synthesis of (-)-englerin A (1) is reported. The target molecule is a recently isolated guaiane sesquiterpene that possesses highly potent and selective activity against renal cancer cell-lines. Our enantioselective strategy involved the construction of the BC ring system of compound 1 through a Rh^II-catalyzed [4+3] cycloaddition reaction followed by subsequent attachment of the A ring through an intramolecular aldol condensation reaction. As such, this strategy allows the synthesis of truncated englerins. Evaluation of these analogues with the A498 renal cancer cell-line suggested that the A ring of englerin is crucial to its antiproliferative activity. Moreover, evaluation of these analogues led to the identification of potent growth-inhibitors of CEM cells with GI₅₀ values in the range 1-3 μM.

Full text of DOI:10.1002/asia.201101021

FULL PAPERS
DOI: 10.1002/asia.201101021
Formal Synthesis of (ꢀ)-Englerin A and Cytotoxicity Studies of Truncated
Englerins
Jing Xu, Eduardo J. E. Caro-Diaz, Ayse Batova, Steven D. E. Sullivan, and
Emmanuel A. Theodorakis*[a]
Dedicated to Professor K. C. Nicolaou on the occasion of his 65th birthday
Abstract: An efficient formal synthesis
of (ꢀ)-englerin A (1) is reported. The
target molecule is a recently isolated
guaiane sesquiterpene that possesses
highly potent and selective activity
against renal cancer cell-lines. Our
enantioselective strategy involved the
construction of the BC ring system of
compound 1 through a RhII-catalyzed
[4+3] cycloaddition reaction followed
by subsequent attachment of the A
ring through an intramolecular aldol
condensation reaction. As such, this
strategy allows the synthesis of truncat-
ed englerins. Evaluation of these ana-
logues with the A498 renal cancer cell-
line suggested that the A ring of eng-
lerin is crucial to its antiproliferative
activity. Moreover, evaluation of these
analogues led to the identification of
potent growth-inhibitors of CEM cells
with GI50 values in the range 1–3 mm.
Keywords: cancer · cycloaddition ·
leukemia · natural products · total
synthesis
Introduction
The need to identify new chemical motifs as potential drug-
leads has spurred the screening of plant extracts that are
used in traditional African, Ayurvedic (Indian), and Chinese
medicines.[1] In particular, South Africa has a remarkable
botanical diversity with over 30000 flowering species, from
which more than 3000 are used for medicinal purposes
throughout the country.[2] Among them, plants of the genus
Phyllanthus (Euphorbiaceae) are widely distributed and
have long been used in African folk medicine to treat
kidney and urinary tract infections.[3] With this in mind, the
Beutler laboratory has been screening extracts of the Tanza-
nian plant Plyllanthus engleri against renal cell carcinoma
(RCC) and has recently reported the isolation of two new
bioactive sesquiterpenes, named englerin A (1) and engler-
in B (2; Scheme 1).[4]
Scheme 1. Representative structures of natural and synthetic englerins.
clinical manifestations, and shows resistance to radiation;[6]
and 3) cannot be effectively treated with current chemother-
apeutic agents, thus leaving surgical procedures as the only
treatment option.[7]
Preliminary biological investigations[4] have shown that
compound 1 possesses very potent growth-inhibitory activity
(GI50 =1–87 nm) against RCC with approximately 1000-fold
tissue selectivity as compared to other carcinomas. These
findings are of particular significance because RCC: 1) is
among one of the ten leading cancer types in the US;[5] 2) is
characterized by a lack of early warning signs, has diverse
Biogenetically, the englerins belong to a family of guaiane
natural products in which a common bicyclic sesquiterpene
skeleton has undergone a sequence of oxygenation and oxo-
cyclization reactions.[8] Further decoration at the periphery
of this core produces the structures of compounds 1 and 2.
Owing to the combination of uncommon structural architec-
ture, potent and selective cytotoxicity against RCC, and
promising pharmacology, the englerins have received the at-
tention of the chemical community. Since their isolation in
2009, five total syntheses[9] and many strategies and stud-
ies[10] have been reported. These studies also led to the iden-
tification of synthetic englerin analogues, such as com-
pounds 3, 4, and 5, as products of various esterification reac-
tions of the common tricyclic core. Several recent reviews
have nicely summarized the current status of englerins.[11]
Our continuous interest in exploring natural products from
[a] Dr. J. Xu, E. J. E. Caro-Diaz, Dr. A. Batova, S. D. E. Sullivan,
Prof. Dr. E. A. Theodorakis
Department of Chemistry and Biochemistry
University of California, San Diego
9500 Gilman Drive, MC: 0358, La Jolla, CA 92093-0358 (USA)
Fax : (+1)858-822-0386
E-mail: etheodor@ucsd.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201101021.
1052
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 1052 – 1060

ethnomedicine as medicinal lead-compounds[12] has prompt-
ed us to design an enantioselective strategy toward the eng-
lerin family.[13] Herein, we describe in detail the results of
our efforts and the biological evaluation of structurally sim-
plified englerin analogues.
The transfer of chirality observed in this reaction can be ra-
tionalized by considering that the carbonyl moiety of panto-
lactone interacts with the Rh-carbenoid, as shown in
Scheme 3. This interaction blocked one of the two faces of
Results and Discussion
Retrosynthetic Analysis
Our retrosynthetic analysis is shown in Scheme 2. We antici-
pated that a sequence of reactions, including inversion of
ꢀ
the C6 stereocenter, hydrogenation of the C4 C5 alkene
(englerin numbering), and selective esterifications at the C6
and C9 hydroxy groups would form englerin (1) from diol 6.
Compound 6 could be derived from enone 7 by C3 deoxyge-
Scheme 3. Rationale of the stereochemical consequences of the Davies
Rh-catalyzed [4+3] cycloaddition reaction.
ꢀ
nation and a regioselective hydroboration of the C8 C9
double bond. We theorized that the cyclopentene moiety of
compound 7 could be constructed from an intramolecular
aldol condensation, whilst a-hydroxylation would produce
the C6 hydroxy moiety. Disconnection along these lines led
us to target the construction of bicyclic motif 8, which pos-
sessed the BC ring scaffold of englerins. We hypothesized
that compound 8 could be formed in its enantiomerically
pure form by exploring a RhII-catalyzed [4+3] cycloaddition
reaction between readily available furan 9, which represent-
ed the C ring of compound 1, and chiral pantolactone-de-
rived diazoester 10.
carbene 10, thereby leading to a selective si-face attack by
the 2-methyl-5-isopropyl-furan (9). The stereochemical out-
come was consistent with a tandem cyclopropanation/Cope
rearrangement (see intermediate [11]). Cyclopropanation of
achiral furan 9 was expected to occur regioselectively at the
ꢀ
less-substituted, and thus less-hindered, C9 C10 double
bond. The potential of this cycloaddition to construct bicy-
clic structure 8 was of great interest to our strategy, as it
provided efficient, rapid, and enantioselective access to the
englerin core. Notably, both starting materials 9[16] and
10[15a,17] could be readily prepared on a more than 50 gram
scale from commercially available materials in three steps.
To the best of our knowledge, 2,5-disubstituted furans
have not previously been evaluated as substrates in the
RhII-catalyzed [4+3] cycloaddition. With this in mind, we in-
itially evaluated the regioselectivity of this reaction by using
achiral diazoester 14[18] as a model system (Scheme 4). The
synthesis of furan 9 is also shown in Scheme 4. Commercial-
ly available 2-methyl furan (12) was treated with nBuLi and
acetone, followed by dehydration (Ac2O/KOAc) to afford
Scheme 2. Retrosynthetic analysis of (ꢀ)-englerin A (1).
ACHTUNGTRENNUNG[4+3] Rhodium-Catalyzed Cycloaddition Reactions
Rhodium-triggered cyclization reactions have been widely
used in synthetic chemistry.[14] In 1996, Davies et al. reported
an elegant enantioselective RhII-catalyzed [4+3] cycloaddi-
tion between furans and chiral diazoesters.[15] Selection of
the appropriate pantolactone enantiomer as the chiral auxil-
iary allowed diastereocontrol of the oxy-bridged adduct.
Scheme 4. Reagents and conditions: a) nBuLi (0.94 equiv), Et2O, ꢀ208C,
reflux; then ꢀ208C, acetone (1.1 equiv), reflux; then Ac2O (1.5 equiv),
KOAc (0.63 equiv), 1108C; b) Pd/C (catalytic), H2 (1 bar), 1 h; c) [Rh2
AHCTUNGTERG(NUNN Ooct)4] (2 mol%), hexanes, reflux, 95%; d) DIBAL-H (2.5 equiv),
CH2Cl2, ꢀ788C; e) BF3·Et2O (1.5 equiv), CH2Cl2, ꢀ308C, 81%. DIBAL-
H=diisobutylaluminum hydride.
Chem. Asian J. 2012, 7, 1052 – 1060
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1053

Emmanuel A. Theodorakis et al.
FULL PAPERS
Table 1. Optimization of the key [4+3] cycloaddition reaction between
compounds 9 and 10.[a]
alkene 13. Regioselective hydrogenation of compound 13
proceeded smoothly by controlling the reaction time to
1 hour, and sequential distillation delivered the desired 2,5-
disubstituted furan (9). Notably, this three-step preparation
of compound 9 could be done on a more than 50 gram scale
and only one distillation was needed for purification. Heat-
ing a mixture of compound 9 and diazoester 14 to reflux in
n-hexane in the presence of catalytic amounts of rhodiu-
m(II) octanoate (2 mol%) afforded oxacyclic product (ꢁ)-
15 as the only regioisomer in excellent yield (95%). Cleav-
age of the ester auxiliary was more challenging than antici-
pated. In fact, the reported LiAlH4 reductive cleavage only
led to decomposition.[15a] However, treatment of ester 15
with DIBAL-H yielded labile b-hydroxy silyl enol ether 16
that, upon immediate treatment with stoichiometric
amounts of BF3·Et2O, underwent rearrangement[19] to afford
exocyclic enone (ꢁ)-17 in good yield (81%).
Furan 9 [equiv]
Catalyst (mol%)
[Rh2A(Ooct)4] (2)
[Rh2A(Ooct)4] (2)
[Rh2A(Ooct)4] (1)
[Rh2A(Ooct)4] (2)
[Rh2A(Ooct)4] (2)
[Rh2A(Ooct)4] (2)
[Rh2A(Ooct)4] (2)
[Rh2A(OAc)4] (2)
T [8C]
Yield [%] (d.r.)
10
2
2
2
2
2
5
5
G
N
reflux
reflux
reflux
ꢀ78
ꢀ40
0
91 (3:1)
90 (3:1)
73 (3:1)
n.r.
n.r.
n.r.
31 (ND)
n.r.
R
E
E
E
N
A
30
reflux
N
N
[a] n.r.=no reaction. ND=not determined.
compound 8 to produce optically active enone (ꢀ)-17, al-
though the yield was significantly lower (59%). This de-
crease in yield may have been due to the presence of the
two reactive carbonyl moieties in compound 8 that required
extended reaction times and excess DIBAL-H (7.5 equiv)
compared to the reduction of compound (ꢁ)-15 (2.5 equiv),
thereby leading to partial decomposition of the sensitive
silyl enol ether moiety.
Encouraged by these results, we synthesized chiral diazo-
ester 10 from (R)-pantolactone.[15a,17] The RhII-catalyzed
[4+3] cycloaddition of diazoester 10 with compound 9 pro-
ceeded efficiently with [Rh
(Ooct)4] to yield compound 8 in
Our attempts to install a hydroxy group onto the C6
carbon of compound (ꢀ)-17 by using Davis oxaziridine re-
agents[20] were unsuccessful. However, Rubottom oxida-
tion[21] provided the desired hydroxy enone 19 in good yield
(36%; 87% brsm), although with inverse stereochemistry at
the englerin C6 hydroxy moiety. The absolute stereochemis-
try of hydroxy enone 19 was established by single-crystal X-
ray analysis,[22] which simultaneously confirmed the absolute
stereochemistry of oxy-bridged ester 8. We were pleased to
observe that the Rubottom oxidation proceeded regioselec-
tively at the more-electron-rich TMS enol ether without af-
fecting any other alkenes in this molecule. The stereochemi-
cal outcome of this reaction was also satisfactory because it
proceeded exclusively from the top face of the intermediate
TMS-enolate [18], thus suggesting that this face was less
hindered. We predicted that this selectivity would allow us
to have complete substrate-control during the subsequent
steps and invert the C6 stereochemistry at a later stage in
our synthesis.
good yield (90%), albeit in moderate diastereoselectivity
(d.r. 3:1, calculated by using 1H NMR spectroscopy;
Scheme 5). Under the same conditions, the use of [Rh2
ACHTUNGTRENNUNG(OAc)2] only led to decomposition of the starting materials.
Moreover, attempts to increase the d.r. by performing this
reaction at lower temperatures or by using lower catalyst
loading led to a significant decrease in the yield without any
significant enhancement in the diastereoselectivity. Table 1
shows our efforts to optimize this cycloaddition reaction.
The diastereomeric mixture of compound 8 was separated
by column chromatography on silica gel. Our previously es-
tablished reductive-cleavage conditions were applied to
Formation of the A Ring Using Olefin Metathesis
The next stage of the synthesis involved constructing the tri-
cyclic core of the englerin from compound 19. We envi-
sioned accomplishing this cyclization by the means of
a Grubbs ring-closing metathesis (RCM)[23] of precursor 21
(Scheme 6), which, after oxidation of the C6 hydroxy group,
would reveal a conjugate system for the 1,4-addition of
a methyl nucleophile at the C4 site. To this end, a Lewis-
acid-promoted conjugate allylation with allyltrimethylsilane
afforded the corresponding ketone (20) in moderate yield
(68%) as a single diastereomer. However, the subsequent
olefination of the C5 position was unexpectedly challenging.
After attempting many different methods, including the
Wittig reaction, Peterson olefination,[24] Petasis,[25] and the
Tebbe olefination,[26] only the TiCl4-assisted Nysted olefina-
tion[27] gave low yields (15–36%) of trialkene 21. Despite
Scheme 5. Reagents and conditions: a) compound
9 (2.0 equiv), [Rh2
(Ooct)4] (2 mol%), hexanes, reflux, 95% for compound (ꢁ)-16, 90% for
compound (ꢀ)-16 (d.r. 3:1); b) DIBAL-H (7.5 equiv), CH2Cl2, ꢀ788C,
then BF3·Et2O (1.5 equiv), CH2Cl2, ꢀ308C, 59%; c) LDA (4.6 equiv),
TMSCl (4.6 equiv), ꢀ788C to 08C; d) mCPBA (1.1 equiv), NaHCO3,
CH2Cl2, 08C, then (COOH)2 (4.6 equiv), 36%; 87% brsm. brsm=based
on recovered starting material.
1054
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 1052 – 1060

Formal Synthesis of (ꢀ)-Englerin A
Scheme 7. Reagents and conditions: a) propanal (4 equiv), compound 26
(20 mol%), Et3N (1.2 equiv), 808C, 75%; b) KOH (5 equiv), EtOH, RT,
76%.
Motivated by this result, we converted compound 19 into
C6 silyl ether 29 and treated this product with propanal
under the previously established Stetter conditions. This re-
action proceeded efficiently under basic conditions to fur-
nish diketone 30 as a single diastereomer in good yield
(75% over two steps). However, the application of the pre-
viously successful KOH condensation procedure only result-
ed in deprotection of the TBS ether, with no further reac-
tion. This result prompted an extensive investigation on this
intramolecular condensation reaction, with different C6 pro-
tecting groups (H, MOM, TES, etc.), various bases (tBuOK,
KOH, NaOMe, LDA, etc.), and several reaction conditions
tested. Many failed attempts at providing the tricyclic motif
confirmed that this reaction was unexpectedly difficult.
Eventually, the treatment of diketone 30 with NaHMDS af-
forded the corresponding aldol addition product, which un-
derwent a sequential dehydration process (NaOMe/MeOH,
heat) to produce the key tricyclic core (7) in acceptable
yield (36%; 43% brsm). NaBH4 reduction of enone 7 yield-
ed allylic alcohol 31 (99%) as a single diastereomer that,
without further purification, was protected as a benzyl ether
to furnish compound 32 in good yield (71%). Treatment of
compound 32 with BH3·THF followed by oxidation with
H2O2 afforded alcohol 33 (60%) regio- and stereoselec-
tively. Silylation of compound 33 followed by deprotection
of the C3 benzyl ether yielded compound 35 via compound
34 in 99% overall yield (Scheme 8). Our efforts to hydro-
genate the tetrasubstituted alkene of compound 35 by using
different catalysts and H2 pressure were unsuccessful, pre-
sumably owing to the steric hindrance of the double bond
(C4=C5). This result prompted us to deoxygenate the C3 hy-
droxy group under Barton–McCombie conditions[30] (40%
yield). In our efforts to improve this transformation, we dis-
covered that the dehydration of compound 35 with Burgess
reagent,[31] followed by standard hydrogenation, significantly
improved the yield to 90% over two steps. Deprotection of
di-TBS ether 36 with TBAF gave poor results; however, we
were pleased to find that a microwave-accelerated reaction
under similar conditions (3 equiv TBAF, THF, 808C) made
diol 6 in a quick and high-yielding conversion (45 min,
93%).
Scheme 6. Reagents and conditions: a) TiCl4 (1.2 equiv), allyltrimethylsi-
lane (3 equiv), CH2Cl2, ꢀ788C, 68%; b) Nysted reagent (10 equiv), TiCl4
(10 equiv), NaHCO3 (7 equiv), ꢀ788C, 36%; c) Grubbs second-genera-
tion catalyst (5 mol%), CH2Cl2, RT, 99%; d) NMO (3 equiv), TPAP
(0.1 equiv), CH2Cl2, RT, 72%; e) various methyl cuprate reagents;
f) TiCl4 (1.1 equiv), trimethyl(2-methylallyl)silane (5 equiv), CH2Cl2,
ꢀ788C, 71%. NMO=N-methylmorpholine-N-oxide, TPAP=tetrapropy-
lammonium perruthenate.
the inefficient formation of compound 21, we attempted the
RCM. We were pleased to see that this reaction proceeded
smoothly in almost quantitative yield to give the allylic alco-
hol, which, after TPAP/NMO oxidation,[28] formed a,b-unsa-
turated ketone 22. Unfortunately, all efforts to add methyl
nucleophiles to compound 22 by conjugate addition were
unsuccessful. To overcome this issue, we sought to perform
the RCM on substrate 24, which contained the challenging
C11 methyl group. Consequently, we performed the allyla-
tion of compound 19 with methylallyltrimethyl silane.
Whilst we were successful in producing compound 24, olefi-
nation of this precursor at the C5 position gave irreproduci-
ble results, which prompted us to abandon this approach
and seek an alternative strategy for the formation of the 5-
membered ring from compound 19.
Formation of the 5-Membered Ring (A Ring) from an
Intramolecular Aldol Condensation Reaction and
Completion of the Synthesis
Based on these results (see above), we pursued an alterna-
tive strategy for the construction of the tricyclic core of eng-
lerin that was based on an intramolecular aldol condensa-
tion reaction. Initially, we explored the feasibility of this re-
action by using non-hydroxylated enone 17 as a model
system. To this end, the treatment of propanal with thiazoli-
um salt 26 and enone 17 under Stetter conditions[29] pro-
duced diketone 27 (Scheme 7). To our delight, the intramo-
lecular aldol reaction of compound 27 proceeded under
mild conditions (KOH/EtOH, RT, 24 h) to afford compound
28 in good yield (76%).
To access the natural product from diol 6, we would need
to achieve stereoselective saturation of the tetrasubstituted
bond, followed by esterification of the C9 hydroxy group, in-
version of C6 stereocenter by oxidation/reduction sequence,
Chem. Asian J. 2012, 7, 1052 – 1060
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1055

Emmanuel A. Theodorakis et al.
FULL PAPERS
pound 19, which represents the BC ring system of englerins.
In other words, this approach could provide information on
the biological significance of the A ring of englerin. With
this in mind, we synthesized truncated englerins 37, 38, and
39 (Scheme 9). To further expand such SAR studies, we de-
Scheme 9. Reagents and conditions: for compound 37: Ac2O (1.5 equiv),
pyridine, 608C, 95%; for compound 38: benzoic acid (2.0 equiv), 2,4,6-tri-
chlorobenzoyl chloride (2.5 equiv), Et3N (3.0 equiv), 4-DMAP (catalytic),
toluene, 90%; for compound 39: cinnamic acid (2.0 equiv), 2,4,6,-trichlor-
obenzoyl chloride (2.5 equiv), Et3N (3.0 equiv), 4-DMAP (catalytic), tol-
uene, 88%. 4-DMAP=4-dimethylaminopyridine.
signed a new approach towards compound 46, which repre-
sented the nor-A-ring of englerin A. The synthesis of com-
pound 46 (Scheme 10) was inspired by Wenderꢁs pioneering
[5+2] cycloaddition reactions.[32] It is worth pointing out that
this reaction has also been applied to the synthesis of engler-
in by the Nicolaou group.[9d] First, 2-methyl-5-furfural (40)
was alkylated under Grignard conditions and the resulting
alcohol was converted into hemiacetal 41 in 75% overall
yield. Treatment of compound 41 with excess acrylonitrile
Scheme 8. Reagents and conditions: a) TBSOTf (2 equiv), NEt3
(5 equiv), CH2Cl2, 08C to RT; b) propanal (4 equiv), compound 26
(20 mol%), Et3N, 80 8C, 75% over 2 steps; c) NaHMDS (5 equiv), THF,
08C; d) NaOMe (1.3 equiv), MeOH, 658C, 36%; 43% brsm for 2 steps;
e) NaBH4 (5 equiv), MeOH, RT; f) NaH (4 equiv), BnBr (8 equiv), DMF,
608C, 71% for 2 steps; g) BH3·THF (3 equiv), THF, RT then 3m NaOH/
30% H2O2, 60%; h) TBSOTf (2 equiv), Et3N (4 equiv), CH2Cl2, RT,
97%; i) H2 (1 atm), Pd(OH)2 (catalytic), MeOH, RT, 99%; j) Burgess re-
agent (5 equiv), toluene, 808C, 90%; k) H2 (1 atm), Pd/C (catalytic),
MeOH, RT, 99%; l) TBAF (3 equiv), THF, 808C, microwave, 45 min,
93%. NaHMDS=sodium bis(trimethylsilyl)amide, Bn=benzyl, TBS=
tert-butyldimethylsilyl, TBAF=tetrabutylammonium fluoride.
and finally esterification in the presence of cinnamic acid.
An alternative synthesis of compound (+)-6 was reported
by Ma and co-workers.[9b] Our synthetic route to compound
(+)-6 provides another new and facile method for the con-
struction of englerin A and related compounds. More signifi-
cantly, the Rh-catalyzed [4+3]-annulation/intramolecular-
aldol-condensation sequence presents a very useful, practi-
cal, and general method for the preparation of the guaiane
sesquiterpene core that is present in englerin-related com-
pounds.
Scheme 10. Reagents and conditions: a) iPr2MgCl (1.5 equiv), Et2O,
ꢀ108C; b) mCPBA (1.0 equiv), CH2Cl2, 75% over two steps; c) iPr2NEt
(1.2 equiv), MsCl (1.2 equiv), acrylonitrile (80 equiv), 1008C, microwave,
45%; d) NaBH4 (1.0 equiv), CeCl3·H2O (1 equiv), MeOH, 08C, 98%;
e) H2, Pd/C (catalytic), EtOH, 99%; f) LDA (2 equiv), THF, ꢀ788C, then
O2, then SnCl2·HCl (30 equiv), 08C, 40%; g) cinnamic acid (2.0 equiv),
2,4,6,-trichlorobenzoyl chloride (2.5 equiv), Et3N (3.0 equiv), 4-DMAP
(catalytic), toluene, 95%; h) NaBH4 (1.0 equiv), MeOH, 08C, 100%;
i) Ac2O (1.5 equiv), Et3N (4.0 equiv), 4-DMAP (0.5 equiv), CH2Cl2, RT,
80%; j) LiHMDS (1.3 equiv), Im2SO2 (1.3 equiv), THF, 08C to RT, 99%;
k) cesium hydroxy acetate (5 equiv), [18]crown-6 (5 equiv), toluene,
1108C, 74%. mCPBA=meta-chloroperbenzoic acid, MsCl=methanesul-
fonyl chloride, LDA=lithium diisopropylamide.
Synthesis of Truncated Englerin Analogues
The intriguing bioactivity of englerin has prompted several
groups to perform structure–activity relationship (SAR)
studies[9f,10e] that have focused exclusively on modification of
the C6 and C9 side-chains. An advantage of our strategy is
that it can efficiently and stereoselectively produce com-
1056
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 1052 – 1060

Formal Synthesis of (ꢀ)-Englerin A
(iPr2NEt/MsCl, 1008C, 14 h) gave the desired bicyclic prod-
uct (42) in 32% yield. Moreover, this reaction was accelerat-
ed under microwave conditions to afford an improved yield
(iPr2NEt/MsCl, 1508C, 4 h, 45%). To the best of our knowl-
edge, this is the first example of microwave acceleration of
this type of [5+2] cycloaddition reaction. Luche reduction[33]
followed by hydrogenation provided compound 43[22] in
almost quantitative yield. The transformation from com-
pound 43 into compound 44 was accomplished in a three-
step sequence that included: 1) oxidative cleavage of the cy-
anide group to the corresponding ketone; 2) esterification
with cinnamic acid under Yamaguchi conditions;[34] and
3) hydride reduction of the C9 carbonyl moiety. The last two
steps were performed according to a literature procedure[9b]
to furnish compound 46. Moreover, acetate analogue 45 was
readily prepared from acetylation of compound 44.
Figure 2. Dose-dependent inhibition of cell-proliferation by selected eng-
lerin analogues in T-ALL cells.
gate electrophile with bionucleophiles.[35] In contrast, engler-
in A and analogues that contained an additional ring, such
as compound 7, had little or no cytotoxicity at concentra-
tions as high as 20 mm. These results suggested that the
single-ring analogues can target leukemia cells effectively
and the addition of structural complexity may result in
a loss of cytotoxicity towards leukemia cells.
Cytotoxicity Studies of Truncated Englerins
The cytotoxicity of the truncated englerins was evaluated in
both A498 renal cancer cells and CEM T-cell acute lympho-
3
blastic leukemia (T-ALL) cells by using a H-thymidine-in-
corporation assay. In our initial study, we compared the
growth-inhibitory activity of englerin A to that of truncated
englerins 44 and 46. We found that englerin A inhibited the
growth of A498 renal cancer cells with a GI50 of 45 nm,
which is in agreement with previous results (Fig-
ure 1).[4,9d,f,10e] However, compounds 44 and 46 did not have
any effect on the growth of A498 cells, even at concentra-
tions of 100 nm or greater (Figure 1). These results suggest
that the A ring is essential for the growth-inhibitory activity
of englerin A in renal cancer cells.
Table 2. Inhibition of cell-proliferation by (ꢀ)-englerin A and its ana-
logues in T-ALL cells.[a]
Compound
% of Control at 20 mM
GI50 [mM]
(ꢀ)-englerin A
81.4
74.6
75.4
88.7
98.7
0.2(ꢁ0)
ND
0.4
N
>20
>20
>20
>20
>20
3.3
44
45
46
7
17
(ꢁ)-17
1.8
2.4
19
37
38
39
0.3
0.2
0.1(ꢁ0)
U
ND
ND
ND
[a] ND=not determined.
Conclusions
We have accomplished an efficient and enantioselective
formal synthesis of englerin A (1), a potent and selective
growth inhibitor of renal cancer cells. Our synthetic ap-
proach to intermediate 6 proceeded in 15 steps from readily
available compounds 9 and 10 in 5% overall yield. Key to
our strategy was the enantioselective formation of the BC
ring of compound 1 from a RhII-induced enantioselective
[4+3] cycloaddition reaction, followed by the construction
of the A ring from an intramolecular aldol condensation re-
action. Inspired by this sequence, we also synthesized
a small family of truncated englerins and evaluated their
growth-inhibitory activities against certain renal cancer and
leukemia cell-lines. These studies suggested that the A ring
of englerin A plays an important role in its bioactivity and
Figure 1. Effect of englerin A and analogues 44 and 46 on the prolifera-
tion of A498 renal cancer cells.
We then evaluated the antiproliferative activity of engler-
in analogues in CEM cells, a cell-line in which englerin A
has little activity (GI50 =20.4 mm).[4] Of all of the analogues
tested, compounds 17, (ꢁ)-17, and 19 had significant cyto-
toxicity, with GI50 values of 3.3, 1.8, and 2.4 mm, respectively
(Figure 2, Table 2). It is likely that these cytotoxicities were
due to the exocyclic enone moiety, which acted as a conju-
Chem. Asian J. 2012, 7, 1052 – 1060
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1057

Emmanuel A. Theodorakis et al.
FULL PAPERS
THF (800 mL) at ꢀ788C. The reaction mixture was stirred for 15 min,
and then a solution of compound 17 (5.2 g, 27.0 mmol) in dry THF
(500 mL) was quickly added dropwise to the reaction mixture. The tem-
perature was raised from ꢀ788C to 08C over 45 min and stirred for 1 h at
08C. The reaction mixture was cooled to ꢀ788C and TMSCl (15.8 mL,
124.2 mmol) was added dropwise. The temperature was raised from
ꢀ788C to 08C over 45 min and stirred for 1 h at 08C, at which time TLC
analysis showed no remaining starting material. The reaction mixture was
diluted with hexanes (650 mL), quenched with 5% NaHCO3 solution
(500 mL), washed with brine (300 mL), dried over Na2SO4, filtered, and
concentrated under vacuum. To a solution of the crude enol-ether prod-
uct in CH2Cl2 (250 mL) was added a solution of NaHCO3 (250 mL, 10%)
in a single portion at 08C. Then, a solution of mCPBA (5.2 g, 30.0 mmol)
in CH2Cl2 (60 mL) was added slowly under vigorous stirring. The reac-
tion was closely monitored by TLC. When trace amounts of enol ether
(<5%) were observed, the reaction was further diluted in CH2Cl2
(200 mL), quenched with saturated NaHSO3 solution (300 mL), allowed
to warm to RT, extracted with CH2Cl2 (2ꢂ200 mL), washed with brine
(300 mL), dried over Na2SO4, filtered, and concentrated under vacuum to
tissue-selectivity. Interestingly, compounds (ꢀ)-17, (ꢁ)-17,
and (ꢀ)-19 have shown significant growth-inhibitory activity
against CEM cell-lines at low micromolar concentrations
(GI50 =1–3 mm). Consequently, these compounds may repre-
sent new lead structures for the development of small-mole-
cule therapeutics against leukemia.
Experimental Section
ACHTUNGTRENNUNG(1R,5S)-(R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-3-[(tert-
butyldimethylsilyl)oxy]-5-isopropyl-1-methyl-8-oxabicycloACTHNUTRGNE[UNG 3.2.1]octa-2,6-
diene-2-carboxylate (8)
A solution of compound 10 (11.2 g, 31.6 mmol) in dry hexanes (750 mL)
was added dropwise over 5.5 h to a refluxing solution of compound 9
(7.85 g, 8.8 mL, 63.2 mmol) and rhodium(II) octanoate dimer (492 mg,
0.63 mmol) in anhydrous hexanes (750 mL). The reaction mixture was
stirred for an additional 30 min, after which TLC analysis showed no re-
maining starting material. The reaction mixture was allowed to cool to
RT, filtered through a silica plug, and concentrated under vacuum.
Column chromatography on silica gel (hexanes/EtOAc, 100:1 to 9:1 slow
gradient) afforded 8.1 g of bicyclic ester 8 as a thick colorless oil (57%).
approximately 250 mL. To this solution was added
a solution of
(COOH)2 (15.6 g, 124.2 mmol) in MeOH (150 mL). After stirring for
30 min at RT, TLC analysis showed no remaining epoxide product. The
reaction mixture was slowly quenched with saturated K2CO3 solution
until neutral pH was achieved and then extracted with CH2Cl2 (2ꢂ
200 mL). The combined organic layers were washed with brine (300 mL),
dried over Na2SO4, filtered, and concentrated under vacuum. Purification
by column chromatography on silica gel (hexanes/EtOAc, 100:1 to 4:1)
afforded compound 17 (2.7 g) and a-hydroxy ketone 19 as a crystalline
solid (2.2 g, 36%; 87% brsm). Recrystallization from hexanes afforded
crystals suitable for X-ray diffraction. ½aꢂ2D3 =+101.00 (c=1.3, CHCl3);
1H NMR (500 MHz, CDCl3): d=6.08 (dd, J=5.8, 0.9 Hz, 1H), 6.00 (d,
J=5.9 Hz, 1H), 5.97 (s, 1H), 5.28 (s, 1H), 3.81 (d, J=6.4 Hz, 1H), 2.37–
2.28 (m, 1H), 1.62 (s, 3H), 1.05 (d, J=6.9 Hz, 3H), 0.94 ppm (d, J=
6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=199.0, 146.9, 140.2, 130.1,
116.5, 93.4, 85.5, 73.3, 28.4, 19.4, 17.7, 17.0 ppm; HRMS (ESI): m/z calcd
for C12H16O3Na: 231.0992 [M+Na]+; found: 231.0993.
1
½aꢂ2D3 =+36.40 (c=1.0, CHCl3); H NMR (500 MHz, CDCl3): d=6.38 (dd,
J=5.7, 0.5 Hz, 1H), 5.72 (d, J=5.7 Hz, 1H), 5.40 (s, 1H), 4.04 (q, J=
8.9 Hz, 2H), 2.41 (d, J=17.4 Hz, 1H), 1.95–1.83 (m, 2H), 1.62 (s, 3H),
1.24 (s, 3H), 1.17 (s, 3H), 1.00 (d, J=6.9 Hz, 3H), 0.95 (d, J=6.8 Hz,
3H), 0.92 (s, 9H), 0.19 (s, 3H), 0.18 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=172.4, 164.2, 157.1, 141.6, 126.8, 117.9, 88.5, 82.8, 76.3, 75.0,
40.2, 37.1, 34.3, 25.8, 23.3, 21.5, 20.6, 18.4, 17.2, 17.1, ꢀ3.4, ꢀ3.5 ppm;
HRMS (FAB): m/z calcd for C24H38O6SiNa: 473.2330 [M+Na]+; found:
473.2331.
A
ACHTUNGTREN[NUNG 3.2.1]oct-6-en-3-
AHCTUNGTRENNUNG
To a solution of compound 8 (20.7 g, 45.9 mmol) in dry CH2Cl2 (459 mL)
was added DIBAL-H (344.3 mmol, 344.3 mL, 1.0m in heptanes) dropwise
quickly via an addition funnel at ꢀ788C. After the addition had been
completed, the reaction was stirred for 15 min, after which TLC analysis
showed no remaining starting material. The reaction mixture was
quenched with saturated Rochelle salt solution (300 mL), allowed to
warm to RT, and stirred for 1 h. The reaction mixture was filtered
through Celite and washed with CH2Cl2 until TLC analysis showed no re-
maining crude product in the filter cake. The filtered mixture was sepa-
rated, extracted with CH2Cl2 (2ꢂ150 mL), washed with brine (300 mL),
dried over Na2SO4, filtered, and concentrated to 500 mL of CH2Cl2 under
reduced pressure. The solution was flushed with argon and treated direct-
ly with BF3·Et2O (68.9 mmol, 8.7 mL) dropwise at ꢀ308C. After 5 min,
TLC analysis showed no remaining starting material. The reaction mix-
ture was further diluted with CH2Cl2 (250 mL), quenched with saturated
NaHCO3 solution (350 mL), and extracted with CH2Cl2 (2ꢂ200 mL). The
combined organic layers were washed with brine (300 mL), dried over
Na2SO4, filtered, and concentrated under vacuum. Purification by column
chromatography on silica gel (hexanes/EtOAc, 100:1 to 9:1) afforded
5.2 g of ketone 17 as a yellow oil (59%). ½aꢂ2D3 =+103.01 (c=1.0, CHCl3);
1H NMR (500 MHz, CDCl3): d=6.05 (d, J=5.8 Hz, 1H), 5.96 (s, 1H),
5.92 (d, J=5.7 Hz, 1H), 5.24 (s, 1H), 2.56 (d, J=17.7 Hz, 1H), 2.46 (d,
J=17.7 Hz, 1H), 2.00–1.89 (m, 1H), 1.61 (s, 3H), 0.99 (d, J=4.9 Hz,
3H), 0.98 ppm (d, J=4.7 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
197.8, 147.3, 136.3, 133.4, 115.3, 89.8, 84.9, 45.9, 33.7, 19.8, 17.3, 17.3 ppm;
HRMS (FAB): m/z calcd for C12H16O2Na: 215.1043 [M+Na]+; found:
215.1045.
AHCTUNGTRENNUNG
To a solution of compound 19 (5 mg, 0.024 mmol) in pyridine (0.25 mL)
was added 4-DMAP (0.6 mg, 0.005 mmol) and Ac2O (0.011 mL,
0.12 mmol). The mixture was heated at 608C for 3 h. The mixture was al-
lowed to cool to RT, quenched with saturated NaHCO3, and extracted
three times with EtOAc. The combined organic layers were dried over
MgSO4, concentrated under vacuum, and the residue was purified by
silica gel preparatory thin layer chromatography (hexanes/EtOAc, 20:1)
to yield compound 37 as
a
white foam (5.7 mg, 95%). 1H NMR
(400 MHz, CDCl3): d=6.16 (d, J=5.9 Hz, 1H), 6.02 (d, J=5.9 Hz, 1H),
6.00 (s, 1H), 5.30 (s, 1H), 5.27 (s, 1H), 2.16 (s, 3H), 2.16 (m, 1H), 1.65 (s,
3H), 0.95 (d, J=6.9 Hz, 3H), 0.92 ppm (d, J=6.9 Hz, 3H); 13C NMR
(126 MHz, CDCl3): d=194.1, 170.0, 146.6, 141.2, 129.3, 116.9, 92.6, 85.7,
71.7, 29.9, 28.8, 19.5, 17.4, 17.1 ppm; HRMS (ESI): m/z calcd for
C14H18O4Na: 273.1097 [M+Na]+; found: 273.1098.
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
NEt3 (15 mL, 0.11 mol) and 2,4,6-trichlorobenzoyl chloride (9 mL,
0.075 mol) were added successively to a stirring mixture of benzoic acid
(7.3 mg, 0.06 mol) and compound 19 (6.5 mg, 0.026 mol) in dry toluene
(0.55 mL). The reaction mixture was stirred for 10 min, then a catalytic
amount of 4-DMAP (1 crystal) was added. After stirring for 16 h at RT,
the mixture was diluted with EtOAc and the organic phase was washed
successively with aqueous HCl solution (1m), saturated sodium bicarbon-
ate solution, and brine. The organic phase was dried over MgSO4 and
concentrated under vacuum after filtration. The residue was purified by
column chromatography on silica gel (hexanes/EtOAc, 20:1) to afford
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
benzoic keto-ester 38 (9.1 mg, 90%) as
a
colorless oil. 1H NMR
n-Butyllithium (77.6 mL, 124.2 mmol, 1.6m in hexanes was added drop-
wise to a solution of dry diisopropylamine (19 mL, 135.0 mmol) in dry
(400 MHz, CDCl3): d=8.10 (m, 2H), 7.57 (m, 1H), 7.24 (m, 2H), 6.21
(dd, J=5.8, 0.9 Hz, 1H), 6.09 (d, J=5.8 Hz, 1H), 6.02 (s, 1H), 5.51 (s,
1058
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 1052 – 1060

Formal Synthesis of (ꢀ)-Englerin A
1H), 5.32 (s, 1H), 2.25 (m, 1H), 1.69 (s, 3H), 0.96 (d, J=6.9 Hz, 3H),
0.93 ppm (d, J=6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3): d=193.8,
165.5, 146.7, 141.1, 133.4, 130.1, 129.4, 128.4, 92.8, 85.6, 72.0, 28.9, 19.6,
17.4, 17.1 ppm; HRMS (ESI): m/z calcd for C19H20O4Na: 335.1254
[M+Na]+; found: 335.1256.
[1] a) A. Gurib-Fakim, Mol. Aspects. Med. 2006, 27, 1–93; b) D. F. R. P.
Burslem, N. C. Garwood, S. C. Thomas, Science 2001, 291, 606–607;
c) G. Cragg, D. J. Newmann, Pure Appl. Chem. 2005, 77, 7–24; d) T.
Eisner, Issues Sci. Technol. 1989, 6, 31–34; e) S. P. Rout, K. A.
Choudary, D. M. Kar, L. Das, A. Jain, Int. J. Pharm. Pharmaceut.
Sci. 2009, 1, 1–23.
ACHTUNGTRENNUNG
[2] a) B. E. Van Wyk, B. Van Oudtshoorn, N. Gericke in Medicinal
plants of South Africa, Briza, Pretoria, 1997, pp. 1–304; b) C. Fabri-
cius, E. Koch, H. Magome, S. Turner in Rights, resources and rural
development: community-based natural resource management in
Southern Africa, Earthscan, 2004, pp. 98; c) R. A. Street, W. A.
Stirk, J. Van Staden, J. Ethnopharmacol. 2008, 119, 705–710.
[3] a) J. B. Calixto, A. R. S. Santos, V. C. Filho, R. A. Yunes, Med. Res.
Rev. 1998, 18, 225–258; b) G. Bagalkotkar, S. R. Sagineedu, M. S.
Saad, J. Stanslas, J. Pharm. Pharmacol. 2006, 58, 1559–1570.
[4] a) R. Ratnayake, D. Covell, T. T. Ransom, K. R. Gustafson, J. A.
Beutler, Org. Lett. 2009, 11, 57–60; b) J. A. Beutler, R. Ratnayake,
D. Covell, T. R. Johnson, WO 2009/088854.
ACHTUNGTRENNUNG
NEt3 (11 mL, 0.08 mol) and 2,4,6-trichlorobenzoyl chloride (7 mL,
0.07 mol) were added successively to a stirring mixture of cinnamic acid
(8 mg, 0.053 mol) and compound 19 (5.5 mg, 0.026 mol) in dry toluene
(0.5 mL). The reaction mixture was stirred for 10 min, then a catalytic
amount of 4-DMAP (1 crystal) was added. After stirring for 16 h at RT,
the reaction was diluted with EtOAc and the organic phase was washed
successively with aqueous HCl solution (1m), saturated sodium bicarbon-
ate solution, and brine. The organic phase was dried over MgSO4 and
concentrated under vacuum after filtration. The residue was purified by
column chromatography on silica gel (hexanes/EtOAc, 20:1) to afford
compound 39 (7.8 mg, 88%) as a colorless oil. 1H NMR (500 MHz,
CDCl3): d=7.70 (d, J=15.9 Hz, 1H), 7.52 (m, 2H), 7.39 (m, 3H), 6.52
(d, J=16.0 Hz, 1H), 6.19 (d, J=5.8 Hz, 1H), 6.06 (d, J=5.8 Hz, 1H),
6.03 (s, 1H), 5.42 (s, 1H), 5.33 (s, 1H), 2.22 (m, 1H), 1.68 (s, 3H),
0.96 ppm (t, J=6.9 Hz, 6H); 13C NMR (126 MHz, CDCl3): d=194.1,
166.0, 146.5, 141.2, 134.3, 130.7, 129.4, 129.0, 128.4, 117.2, 116.9, 92.8,
85.7, 71.7, 28.8, 19.6, 17.5, 17.1 ppm; HRMS (ESI): m/z calcd for
C21H22O4Na: 361.1416 [M+Na]+; found: 361.1417.
[5] A. Jemal, R. Siegel, J. Xu, E. Ward, CA Cancer J. Clin. 2010, 60,
277–300.
[6] a) A. J. Schrader, R. Hofmann, Anticancer Drugs 2008, 19, 235–245;
b) J. M. G. Larkin, E. L. S. Kipps, C. J. Powell, C. Swanton, Ther.
Adv. Med. Oncol. 2009, 1, 15–27; c) H. T. Cohen, F. J. McGovern,
N. Engl. J. Med. 2005, 353, 2477–2490; d) R. J. Motzer, N. H.
Bander, D. M. Nanus, N. Engl. J. Med. 1996, 335, 865–875.
[7] E. C. Nelson, C. P. Evans, P. N. Lara Jr., Cancer Treat. Rev. 2007, 33,
299–313.
G
ACHTUNGTREN[NUNG 3.2.1]oct-3-ene-6-
[8] G.-P. Peng, G. Tian, X.-F. Huang, F.-C. Lou, Phytochemistry 2003,
63, 877–881.
[9] a) M. Willot, L. Radtke, D. Kçnning, R. Frçhlich, V. H. Gessner, C.
Strohmann, M. Christmann, Angew. Chem. 2009, 121, 9269–9272;
Angew. Chem. Int. Ed. 2009, 48, 9105–9108; b) Q. Zhou, X. Chen,
D. Ma, Angew. Chem. 2010, 122, 3591–3594; Angew. Chem. Int. Ed.
2010, 49, 3513–3516; c) K. Molawi, N. Delpont, A. M. Echavarren,
Angew. Chem. 2010, 122, 3595–3597; Angew. Chem. Int. Ed. 2010,
49, 3517–3519; d) K. C. Nicolaou, Q. Kang, S. Y. Ng, D. Y.-K. Chen,
J. Am. Chem. Soc. 2010, 132, 8219–8222; e) Z.-W. Li, M. Nakashige,
W. J. Chain, J. Am. Chem. Soc. 2011, 133, 6553–6556; f) L. R.
Radtke, M. Willot, H. Sun, S. Ziegler, S. Sauerland, C. Strohmann,
R. Frçhlich, P. Habenberger, H. Waldmann, M. Christmann, Angew.
Chem. 2011, 123, 4084–4088; Angew. Chem. Int. Ed. 2011, 50, 3998–
4002.
[10] a) B.-F. Sun, C.-L. Wang, R. Ding, J.-Y. Xu, G.-Q. Lin, Tetrahedron
Lett. 2011, 52, 2155–2158; b) C.-L. Wang, B.-F. Sun, S.-G. Chen, R.
Ding, G.-Q. Lin, J.-Y. Xu, Y.-J. Shang, Synlett 2012, 263–266;
c) D. B. Ushakov, V. Navickas, M. Strçbele, C. Maichle-Mçssmer, F.
Sasse, M. E. Maier, Org. Lett. 2011, 13, 2090–2093; d) V. Navickas,
D. B. Ushakov, M. E. Maier, M. Strobele, H. J. Meyer, Org. Lett.
2010, 12, 3418–3421; e) K. P. Chan, D. Y.-K. Chen, ChemMedChem
2011, 6, 420–423.
To a solution of compound 41 (1.6 g, 11.0 mmol) in acrylonitrile (60 mL)
was added diisopropylethylamine (2.3 mL, 13 mmol), followed by metha-
nesulfonyl chloride (1.0 mL, 13 mmol). The resulting solution was heated
in a microwave at 1508C for 4 h. After being allowed to cool to RT, the
reaction mixture was filtered through a plug of Celite and the filtrate was
concentrated in vacuo. Column chromatography on silica gel (EtOAc/
hexanes, 20%) provided cycloadduct 42 (880 mg, 45%) as a colorless oil.
1H NMR (500 MHz, CDCl3): d=6.93 (d, J=9.7 Hz, 1H), 6.01 (d, J=
9.8 Hz, 1H), 3.06 (dd, J=9.2, 2.9 Hz, 1H), 2.52 (dd, J=14.3, 2.9 Hz, 1H),
2.30 (p, J=7.0 Hz, 1H), 2.19 (dd, J=14.3, 9.3 Hz, 1H), 1.74 (s, 3H), 1.08
(d, J=6.9 Hz, 3H), 1.04 ppm (d, J=6.9 Hz, 3H); 13C NMR (126 MHz,
CDCl3): d=196.7, 151.8, 128.1, 118.8, 100.0, 90.4, 37.4, 35.2, 30.0, 21.6,
17.6, 16.5 ppm; HRMS (ESI): m/z calcd for C12H15NNaO2: 228.0995
[M+Na]+; found: 228.0996.
3H-Thymidine-Incorporation Assay
CEM cells were plated at 10–20ꢂ103 cells/well and A498 cells at
3500 cells/well in 96-well plates in Roswell Park Memorial Institute
medium (RPMI) that was supplemented with 10% fetal bovine serum,
2 mm glutamine, and 100 umLꢀ1 penicillin/streptomycin (complete
medium). Englerin A or its analogues were added to the cells at increas-
ing concentrations and 0.1% DMSO was added to the control cells. The
cells were incubated for 48 h and then pulsed with 3H-thymidine for 6 h.
The incorporation of 3H-thymidine was determined by a scintillation
counter (Beckman Coulter Inc., Fullerton, CA) after the cells had been
washed and deposited onto glass microfiber filters using a cell-harvester
M-24 (Brandel, Gaithersbur, MD).
[11] a) M. Willot, M. Christmann, Nat. Chem. 2010, 2, 519–520; b) W. J.
Chain, Synlett 2011, 2605–2608; c) R. H. Pouwer, J.-A. Richard, C.-
C. Tseng, D. Y.-K. Chen, Chem. Asian J. 2012, 7, 22–35; d) Y.-Y.
Lu, H.-Q. Yao, B.-F. Sun, Chin. J. Org. Chem. DOI: 10.6023/
cjoc1110141.
[12] For selected publications on this topic, see: a) T.-T. Ling, B. A.
Kramer, M. A. Palladino, E. A. Theodorakis, Org. Lett. 2000, 2,
2073–2076; b) T.-T. Ling, C. Chowdhury, B. A. Kramer, B. G. Vong,
M. A. Palladino, E. A. Theodorakis, J. Org. Chem. 2001, 66, 8843–
8853; c) E. J. Tisdale, I. Slobodov, E. A. Theodorakis, Proc. Natl.
Acad. Sci. USA 2004, 101, 12030–12035; d) E. J. Tisdale, I. Slobo-
dov, E. A. Theodorakis, Org. Biomol. Chem. 2003, 1, 4418–4422;
e) J. Xu, L. Trzoss, W. K. Chang, E. A. Theodorakis, Angew. Chem.
2011, 123, 3756–3760; Angew. Chem. Int. Ed. 2011, 50, 3672–3676;
f) L. Trzoss, J. Xu, M. H. Lacoske, W. C. Mobley, E. A. Theodorakis,
Org. Lett. 2011, 13, 4554–4557; g) O. Chantarasriwong, A. Batova,
W. Chavasiri, E. A. Theodorakis, Chem. Eur. J. 2010, 16, 9944–9962.
[13] J. Xu, E. J. E. Caro-Diaz, E. A. Theodorakis, Org. Lett. 2010, 12,
3708–3711.
Acknowledgements
We gratefully acknowledge the National Institutes of Health (NIH) for
financial support of this work through Grant No. R01 GM081484. We
thank the National Science Foundation for instrumentation grants
CHE9709183 and CHE0741968. We also thank Dr. Anthony Mrse
(UCSD NMR Facility), Dr. Yongxuan Su (UCSD MS Facility), and Dr.
Arnold L. Rheingold and Dr. Curtis E. Moore (UCSD X-ray Facility).
Chem. Asian J. 2012, 7, 1052 – 1060
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1059

Emmanuel A. Theodorakis et al.
FULL PAPERS
[14] N. Jeong, J. E. Robinson, P. A. Wender, G. G. Gamber, T. J. Wil-
liams, H. M. L. Davies, A. M. Walji in Modern Rhodium-Catalyzed
Organic Reactions (Ed.: P. A. Evans), Wiley-VCH, Weinheim, 2005,
Chap. 11–14, pp. 215–340.
[15] a) H. M. L. Davies, G. Ahmed, M. R. Churchill, J. Am. Chem. Soc.
1996, 118, 10774–10782; b) for a recent application of this strategy
on natural product synthesis, see also: K. L. Jackson, J. A. Hender-
son, H. Motoyoshi, A. J. Phillips, Angew. Chem. 2009, 121, 2382–
2386; Angew. Chem. Int. Ed. 2009, 48, 2346–2350.
[16] P. Weyerstahl, J. Brendel, Liebigs Ann. Chem. 1988, 1015–1016.
[17] a) R.-D. Shan, S. E. Howlett, E. E. Knaus, J. Med. Chem. 2002, 45,
955–961; b) R. J. Clemens, J. A. Hyatt, J. Org. Chem. 1985, 50,
2431–2435; c) for detailed experimental procedure, see the Support-
ing Information.
[26] F. N. Tebbe, G. W. Parshall, G. S. Reddy, J. Am. Chem. Soc. 1978,
100, 3611–3613.
[27] L. N. Nysted, US Patent 3865848.
[28] a) W. P. Griffith, S. V. Ley, G. P. Whitcombe, A. D. White, J. Chem.
Soc. Chem. Commun. 1987, 1625–1627; b) S. V. Ley, J. Norman,
W. P. Griffith, S. P. Marsden, Synthesis 1994, 639–666.
[29] a) H. Stetter, Angew. Chem. 1976, 88, 695–704; Angew. Chem. Int.
Ed. Engl. 1976, 15, 639–647; b) H. Stetter, H. Kuhlmann, W. Haese,
Org. Synth. 1987, 65, 26–31; H. Stetter, H. Kuhlmann, W. Haese,
Org. Synth. 1993, 8, 620–624.
[30] D. H. R. Barton, S. W. McCombie, J. Chem. Soc. Perkin Trans.
1 1975, 1574–1585.
[31] G. M. Atkins, E. M. Burgess, J. Am. Chem. Soc. 1968, 90, 4744–
4745.
[32] a) P. A. Wender, J. L. MascareÇas, J. Org. Chem. 1991, 56, 6267–
6269; b) P. A. Wender, J. L. MascareÇas, Tetrahedron Lett. 1992, 33,
2115–2118; c) D. R. Williams, J. W. Benbow, J. G. McNutt, E. E.
Allen, J. Org. Chem. 1995, 60, 833–843; d) K. A. Marshall, A. K.
Mapp, C. H. Heathcock, J. Org. Chem. 1996, 61, 9135–9145.
[33] A. L. Gemal, J. L. Luche, J. Am. Chem. Soc. 1981, 103, 5454–5459.
[34] J. Inanaga, K. Hirata, H. Saeki, T. Katsuki, M. Yamaguchi, Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
[35] For selected examples of natural products that exhibit potent bioac-
tivities due to enone functionalities in their structures, see: a) B.
Siedle, A. J. Garcꢃa-PiÇeres, R. Murillo, J. Schulte-Mçnting, V.
Castro, P. Rꢄngeler, C. A. Klaas, F. B. Da Costa, W. Kisiel, I. Mer-
fort, J. Med. Chem. 2004, 47, 6042–6054; b) B. H. B. Kwok, B. Koh,
M. I. Ndubuisi, M. Elofsson, C. M. Crews, Chem. Biol. 2001, 8, 759–
766; c) S. A. Rizvi, D. S. Courson, V. A. keller, R. S. Rock, S. A.
Kozmin, Proc. Natl. Acad. Sci. USA 2008, 105, 4088–4092.
[18] See the Supporting Information.
[19] J.-M. Poirier, L. Hennequin, Tetrahedron 1989, 45, 4191–4202.
[20] a) F. A. Davis, S. Chattopadhyay, J. C. Towson, S. Lal, T. Reddy, J.
Org. Chem. 1988, 53, 2087–2089; b) L. C. Vishwakarma, O. D.
Stringer, F. A. Davis, Org. Synth. 1993, 8, 546; c) F. A. Davis, B.-C.
Chen, Chem. Rev. 1992, 92, 919–934.
[21] G. M. Rubottom, M. A. Vazquez, D. R. Pelegrina, Tetrahedron Lett.
1974, 15, 4319–4322.
[22] CCDC 782508 (19) and CCDC 857553 (43) contain the supplemen-
tary crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
[23] a) G. C. Vougioukalakis, R. H. Grubbs, Chem. Rev. 2010, 110, 1746–
1787 and references herein; b) R. H. Grubbs, Handbook of Metathe-
sis, Wiley-VCH, Weinheim, 2003; c) M. Scholl, S. Ding, C. W. Lee,
R. H. Grubbs, Org. Lett. 1999, 1, 953–956.
[24] D. J. Peterson, J. Org. Chem. 1968, 33, 780–784.
Received: December 16, 2011
[25] N. A. Petasis, E. I. Bzowej, J. Am. Chem. Soc. 1990, 112, 6392–6394.
Published online: March 13, 2012
1060
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 1052 – 1060