Synthesis and antitumor activity of C-9 epimers of the tetrahydrofuran containing acetogenin 4-deoxyannoreticuin

Article abstract of DOI:10.1016/j.bmc.2008.08.030

A highly convergent synthesis of mono-tetrahydrofuran (THF) containing acetogenins, that is based on the cross-metathesis of THF and butenolide alkene precursors, was developed. This methodology was applied to the epimers of the C-9 alcohol of 4-deoxyannoreticuin, in an attempt to assign the configuration at this position in the naturally occurring material. Unfortunately, identification of one or the other epimeric structures with the natural product was not possible because of the closeness of the physical data for all three compounds. Both C-9 epimeric analogues showed similar cytotoxicity in the low micromolar range, against two human tumor cell lines PC-3 (prostate) and Jurkat (T-cell leukemia). This result contrasts to previous studies on closely related THF acetogenins, wherein configurational variation at analogous carbinol centers resulted in a significant effect on antitumor activity.

Full text of DOI:10.1016/j.bmc.2008.08.030

Bioorganic & Medicinal Chemistry 16 (2008) 8413–8418
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity of C-9 epimers of the tetrahydrofuran
containing acetogenin 4-deoxyannoreticuin
*
Feng Wang, Akira Kawamura, David R. Mootoo
Department of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10065, USA
The Graduate Center, CUNY, 365 5th Avenue, New York, NY 10016, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A highly convergent synthesis of mono-tetrahydrofuran (THF) containing acetogenins, that is based on
the cross-metathesis of THF and butenolide alkene precursors, was developed. This methodology was
applied to the epimers of the C-9 alcohol of 4-deoxyannoreticuin, in an attempt to assign the configura-
tion at this position in the naturally occurring material. Unfortunately, identification of one or the other
epimeric structures with the natural product was not possible because of the closeness of the physical
data for all three compounds. Both C-9 epimeric analogues showed similar cytotoxicity in the low micro-
molar range, against two human tumor cell lines PC-3 (prostate) and Jurkat (T-cell leukemia). This result
contrasts to previous studies on closely related THF acetogenins, wherein configurational variation at
analogous carbinol centers resulted in a significant effect on antitumor activity.
Received 18 June 2008
Revised 9 August 2008
Accepted 14 August 2008
Available online 19 August 2008
Keywords:
Cross metathesis
Tetrahydrofuran
Acetogenin
Ó 2008 Elsevier Ltd. All rights reserved.
Antitumor
1. Introduction
Against this backdrop we were attracted to 4-deoxyannoreti-
cuin, a relatively simple mono-THF acetogenin with low micromo-
The tetrahydrofuran (THF) containing acetogenin family of nat-
ural products, of which over 400 have been isolated, are known for
their cytotoxic, antitumoral, insecticidal, and immunosuppresive
activities.1 Three major subtypes, depending on the number and
connectivity of the THF rings, have been identified: mono-THF’s,
adjacently linked bis-THFs, and non-adjacently linked bis-THFs.
The cellular target is believed to be the reduced nicotinamide ade-
nine dinucleotide (NADH): ubiquinone oxidoreductase in complex
I, a membrane bound protein of the mitochondrial electron trans-
port system.2 Inhibition of electron transfer is believed to disrupt
ATP production, which may lead to cell death via a necrosis mech-
anism. More recent studies suggest that apoptotic pathways could
also be involved and may account for the high potency.3 The ubi-
quinone-linked NADH oxidase that is peculiar to the plasma mem-
brane of cancerous cells has also been suggested as a site of action,
and this may explain the activity of the acetogenins against multi-
drug resistant tumors.4 The generally high cytotoxicity of these
agents presents a challenge to their progress as clinical agents,
and in this vein less active analogues might be better suited for
lead development. Synthetic studies on the THF acetogenins are
relevant to bio-mechanistic investigations5 and have also been
valuable for structure determination, in particular with regard to
stereochemical assignment.6
lar level antitumor activity, because the stereochemistry was not
completely assigned.7 We were particularly interested in a syn-
thetic plan that would be practical for assigning an unambiguous
structure to 4-deoxyannoreticuin and in the longer term would
be applicable to other mono-THF acetogenin congeners. The gross
structure of 4-deoxyannoreticuin is represented as in 1 or 2 but the
configuration at C-9 was not determined (Scheme 1). The relative
stereochemistry of the C15–C20 portion was deduced as the
threo–trans–threo motif on the basis of the known 1H and 13C
NMR trends.8 The absolute stereochemistry of the THF core was as-
35
33
2
34
HO
() 11
9
O
1
20
15
O
O
OH
OH
OH
32CH3
1 C-9: R
2 C-9: S
Cross Metathesis
HO
O
9*
() 5
() 11
CH3
O
O
OH
3
(R)-4 or (S)-4
* Corresponding author. Contact Address: Department of Chemistry, Hunter
College, 695 Park Avenue, New York, 10065, USA. Tel.: +1 212 772 4356; fax: +1 212
772 5332.
(* acetogenin numbering)
E-mail address: dmootoo@hunter.cuny.edu (D.R. Mootoo).
Scheme 1. Retrosynthesis for 4-deoxyannoreticuin.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.030

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F. Wang et al. / Bioorg. Med. Chem. 16 (2008) 8413–8418
signed as 15R, 16R, 19R, 20R because this configuration was deter-
mined for the analogous segment in 2,4-trans-squamoxinone, a co-
isolate from the same plant material, and several other mono-THF
containing acetogenins.7 It was also assumed that the configura-
tion at C-34 is (S) by analogy with all the other acetogenins for
which this center has been determined.9 In order to establish the
configuration at C-9, we set as synthetic goals the C-9 epimeric
structures 1 and 2, with the plan of matching one or the other to
the natural product on the basis of its physical properties. Accord-
ingly, a modular synthesis in which a fixed THF segment 3 could be
paired with epimeric-C-9-hydroxy-butenolide components (R)-4
and (S)-4 (or appropriately protected alcohol derivatives), via an
olefin cross metathesis (CM), was conceived.10,11
reaction of methyl dec-9-enoate with mCPBA (Scheme 3). Treat-
ment of rac-7 under Jacobsen’s Hydrolytic Kinetic Resolution
(HKR) conditions using (R, R)-8, or its enantiomer gave either
(R)-7 or (S)-7 in 45% and 46% yields, respectively.12 Epoxides (R)-
7 and (S)-7 were next converted to the silyl ethers (R)-9 and (S)-
9, respectively, via a two-step epoxide opening-alcohol silylation
sequence. The ee of these products were deduced to be over 95%
by 1H NMR analysis of their Mosher ester derivatives ( Supple-
mentary Information).13 Silyl ethers (R)-9 and (S)-9 were individu-
ally partnered with aldehyde 10 and forwarded into a known aldol
reaction-elimination protocol to give butenolide alkenes (R)-11
and (S)-11, respectively, in 39% and 43% overall yield from (R)-9
and (S)-9.14 To compare the use of protected versus deprotected
alcohol butenolide partners in the CM and subsequent reactions
(vide infra), one of the silyl ethers (S)-11 was converted to the de-
rived alcohol (S)-4.
2. Results and discussion
The CM and subsequent steps were first evaluated for TBDPS-
protected butenolide (R)-11 (Scheme 4). The optimal CM condi-
tions were found to be methylene chloride as the solvent, a 1:2
molar ratio of 3:(R)-11, 10 mole% Grubbs II catalyst, at room tem-
perature for approximately 20 h. The desired product (R)-12 was
obtained in 73% isolated yield based on THF segment 3. The
homodimer of (R)-11 was also isolated in 20% yield together with
approximately 15% of unreacted (R)-11. Selective hydrogenation
of the isolated alkene in (R)-12 using RhCl(PPh3)3 as a catalyst pro-
vided an inseparable 7:1 mixture of the desired hydrogenated
product and the completely hydrogenated material in 70% com-
bined yield. Exposure of this mixture to methanolic HCl led to
9R-4-deoxyannoreticuin (1) that contained approximately 10% of
the corresponding over-reduced product. For the synthesis of 9S-
4-deoxyannoreticuin (2), the CM reaction was performed with
the desilylated butenolide (S)-4 thereby obviating the desilylation
step in a more advanced THF-butenolide synthetic intermediate.
Accordingly, application of the CM conditions to 3 and (S)-4 gave
The synthesis of the THF segment followed our earlier work on
the analogue of 3 that contained a hydrocarbon chain with two
carbons less.10b Thus, the copper (I)-mediated reaction of undecyl-
magnesium bromide with the known epoxide 5, followed by desi-
lylation of the product provided diol 3 (Scheme 2).
The epimeric butenolide segments (R)-4 and (S)-4, originated
from the racemic mixture of epoxided rac-7 are derived from the
1. C10H21MgBr,
CuBr, 80%
HO
( )11
CH3
O
O
OH
O
OTBDMS 2. TBAF, 89%
3
5
Scheme 2. Synthesis of THF segment.
1. MeOH,H
( )5
CO2Me
CO2Me
( )5
6
CO2H
O
2. mCPBA
(R/S)-7
78%
O
9
46%
( )5
(S)-7
(R, )-8
(S, S)-9
R
45%
O
OR
( )5
CO2Me
O
O
(R)-11 (9R); R= TBDPS; (2 eq.)
(S)-4 (9S); R= H; (2 eq.)
(R)-7
1. 3-butenylMgBr,
1. 3-butenylMgBr,
CuBr
75%
75%
CuBr
2. TBDPSCl
2. TBDPSCl
Mes
N
N
Mes
Ph
3
Cl
Cl
( )5
OTBDPS
(S)-9
( )5
CO2Me
Ru
PCy3
CO2Me
CH2Cl2
20h, rt
OTBDPS
(R)-9
10 mol%
1, LDA, 10
1, LDA, 10
2. MeOH,T sOH,
2-propanol
2. MeOH,T sOH,
2-propanol
HO
( )5
CH3
43%
O
9
39%
3. MsCl, Et3N
3. MsCl, Et3N
O
O
OH
OR
(
)-12 (9R); R= TBDPS; 73% based on 3
(S)-13 (9S); R= H; 57% based a
R
O
O
3
( )5
( )5
O
O
OR
OR
AcCl
For reaction of(R)-12: For reaction of(S)-13:
(R)-11 R= TBDPS
(S)-11 R= TBDPS
MeOH
80%
(i) RhCl, H2 (70%)
(ii) MeOH, AcCl( 72%)
TsNHNH2,N aOAc (90%)
(S)-4 R= H
HO
9
O
H
N
H
OH
2 N
Co
OTHP
( )5
O
O
OH
OH
t-Bu
O
O
t-Bu
OAc
CHO
CH3
t-Bu
(R, R)-8t-Bu
1 9 R -4 -deoxyannoreticuin
10
2 9 S -4 -deoxyannoreticuin
Scheme 3. Synthesis of butenolide segments.
Scheme 4. Cross metathesis and final products.

F. Wang et al. / Bioorg. Med. Chem. 16 (2008) 8413–8418
8415
(S)-13 in 57% isolated yield based on 3. Given the difficulty in con-
trolling the RhCl(PPh3)3-catalyzed hydrogenation of (R)-12, the dii-
mide reduction of (S)-13 was explored and found to be superior,
giving 9S-4-deoxyannoreticuin (2) in 90% yield with no evidence
of the over-reduced product.
ium sheets and flash column chromatography (FCC) was per-
formed using Kieselgel 60 (32–63 mesh, Scientific Adsorbents).
Elution for FCC usually employed a stepwise solvent polarity gradi-
ent, correlated with TLC mobility. Chromatograms were observed
under UV (short and long wavelength) light, and/or were visual-
ized by heating plates that were dipped in a solution of ammonium
(VI) molybdate tetrahydrate (12.5 g) and cerium (IV) sulfate tetra-
hydrate (5.0 g) in 10% aqueous sulfuric acid (500 mL). Optical rota-
In the absence of an actual sample of 4-deoxyannoreticuin we
attempted to assign the configuration at C-9 in the natural product
by comparison of the NMR data for synthetic 1 and 2 with the re-
ported listings for natural 4-deoxyannoreticuin.7 All three sets of
data were essentially identical, which corroborated the gross struc-
ture assigned to the natural product, but at the same time made
assignment of the C-9 configuration impossible. This scenario
was not completely surprising given the close similarity (albeit
noticeably different) of the NMR data for the analogous epimers
of corossolin, the C-10 regioisomer of 4-deoxyannoreticuin.15,16
Similar observations have been made in a recent more rigorous
investigation on acetogenin diastereomers that vary with respect
to the configuration at remote stereogenic centers.6e Examination
of optical rotations were also inconclusive because the values ob-
tained for 1 and 2 were appreciably different from that reported
tions ([a]D were recorded using a Rudolph Autopol III polarimeter,
which has a thermally jacketed 10 cm cell (path length of 1 dm),
and are given in units of 10ꢀ1 deg cm2 g at 589 nm (sodium D-line).
NMR spectra were recorded using either Varian Unity Plus 500 or
Bruker Ultra Shield instruments (1H and 13C; 500 and 125 MHz,
respectively). Spectra were recorded in CDCl3 solutions with resid-
ual CHCl3 as internal standard (dH 7.27 and dC 77.0 ppm). Chemical
shifts are quoted in ppm relative to tetramethysilane (dH 0.00), and
coupling constants (J) are given in Hertz (Hz). First-order approxi-
mations are employed throughout. High-resolution mass spec-
trometry was performed on an Ultima Micromass Q-Tof
instrument at the Mass Spectrometry Laboratory of the University
of Illinois, Urbana-Champaign.
for the natural product, ([a]
= +19o, +25o and +6.8o for 1, 2, and
D
natural 4-deoxyannoreticuin, respectively). Parenthetically, the
optical rotations for 1 and 2 were essentially identical to the values
observed for the respective C-10 epimers of corossolin.15
4.1.1. (2R,5R,10R,100R)-2-[10-Hydroxy-20-propenyl]-5-[100-
hydroxytridecyl]-tetrahydrofuran (3)
In the case of corossolin15, the cytotoxic activities of the C-10
epimers were significantly different. Therefore, we next attempted
to identify 1 or 2 with the natural product by comparing their cyto-
toxicity against PC-3 (human prostate) with the expectation that
the activity of one or the other would more closely match the
reported data for the natural product. The GI50 values for 1 and 2
In a 50 mL round-bottomed flask equipped with magnetic stir-
ring bar were placed CuBr (36.3 mg, 0.253 mmol) and anhydrous
THF (10 mL). Pre-prepared C11H23MgBr (11.5 mL, ca. 0.4 M in
THF) was added dropwise at 0 oC, and then epoxide 510b (120 mg,
0.422 mmol) was introduced. The reaction mixture was stirred at
0 °C for 3 h, then poured into ice-cold saturated aqueous NH4Cl,
and extracted with ether. The organic phase was washed with
brine, dried (Na2SO4), filtered, and evaporated under reduced pres-
sure. The residue was purified by FCC (2–5% EtOAc:petroleum
ether) to give the derived alcohol (160 mg, 86%): Rf = 0.45 (5%
EtOAc: petroleum ether); 1H NMR (CDCl3) d 5.81 (m, 1H), 5.24–
5.28 (dd, 2H, J = 17.1 Hz, 10.6 Hz), 4.10 (t, 1H), 3.88–3.89 (q, 1H),
3.75–3.76 (q, 1H), 3.35 (m, 1H), 2.31 (d, 1H, J = 4.1 Hz), 1.86–1.89
(m, 2H), 1.52–1.70 (m, 2H), 1.36 (m, 1H), 1.36–1.38 (m, 2H), 1.23
(s, br, 19H), 0.88 (s, br, 9H), 0.85 (t, J = 6.7 Hz, 3H), 0.05 (s, 3H),
0.03 (s, 3H); 13C NMR (CDCl3) d 137.6, 115.7, 82.7, 82.4, 76.0,
74.0, 33.6, 31.9, 29.7 (two signals), 29.6 (three signals), 29.3,
28.4, 27.8, 25.8, 25.6, 22.7, 18.3, 14.1, 1.0, ꢀ4.6, ꢀ4.8; HRMS
(FAB) calcd for C26H53O3Si (M+H+) 441.3764, found 441.3744.
To a solution of the product from the previous step (64 mg,
0.145 mmol) in THF (10 mL) was added Bu4NF (1.45 mL, 1 M in
THF) at 0ꢀC. The reaction mixture was stirred at rt until TLC indi-
cated complete disappearence of the starting material, at which
time the mixture was diluted with water and extracted with
EtOAc. The organic phase was dried (Na2SO4), filtered, and evapo-
rated under reduced pressure. The residue was purified by FCC to
give 3 (40 mg, 85%): white powder; Rf = 0.3 (25% EtOAc: petroleum
ether); 1H NMR (CDCl3) d 5.78 (m, 1H), 5.18–5.36 (dd, 2H, J = 17.5,
10.5 Hz), 3.93 (m, 1H), 3.80–3.88 (m, 2H), 3.39 (m, 1H), 2.45 (b,
1H), 2.25 (b, 1H), 1.94–1.98 (m, 2H), 1.68–1.73 (m, 2H), 1.40–
1.41 (m, 1H), 1.29–1.38 (m, 3H), 1.24 (s, br, 18H), 0.86 (t, J = 6.8
Hz, 3H); 13C NMR (CDCl3) d 136.7, 117.1, 82.9, 82.2, 75.5, 74.0,
33.6, 31.9, 29.7 (three signals), 29.6 (three signals), 29.3, 28.5,
28.4, 25.6, 22.7, 14.1; HRMS (FAB) calcd for C20H38O3Na (M+Na+)
349.2719, found 349.2708.
were found to be approximately 15 and 10
lg/mL in a 3-day MTS
assay. The ED50 reported for the natural sample is 2.7
lg/mL in a
7-day MTT assay.7 Thus, preliminary biological evaluation was also
not structurally insightful. Epimers 1 and 2 also showed similar
activity against Jurkat cells (human T-cell leukemia), with GI50
values in the range of 5–10 lg/mL. These data suggest that the
notion that a change in the configuration at such remote carbinol
centers leads to significant difference in cytotoxic activity may
hold only for certain cell lines and is not general.
3. Conclusion
In conclusion we have developed a highly convergent synthesis
for mono-THF containing acetogenins that is based on the CM cou-
pling of THF and butenolide components. This methodology was
applied to the epimeric analogues of the natural product 4-deoxy-
annoreticuin, in an attempt to resolve the unassigned configura-
tion at C-9. Unfortunately, identification of one or the other
epimeric structures with the natural product was not possible be-
cause of the closeness of the physical data for all three compounds,
illustrating the limitation of this strategy for the assignment of ste-
reochemistry at remote stereogenic centers. In contrast to observa-
tions made for closely related THF acetogenins, the cytotoxicity of
the epimeric analogues of 4- deoxyannoreticuin was found to be
very similar.
4. Experimental
4.1. Chemistry
4.1.2. Methyl 8-(oxiran-2-yl)octanoate (7)
Solvents were purified by standard procedures or used from
commercial sources as appropriate. Petroleum ether refers to the
fraction of petroleum ether boiling between 40 and 60 oC. Unless
otherwise stated, thin layer chromatography (TLC) was done on
0.25 mm thick precoated silica gel 60 (HF-254, Whatman) alumin-
To a solution of methyl dec-9-enoate (1.40 g, 7.60 mmol) in
CH2Cl2 (25 mL) was added mCPBA (4.26 g, 19 mmol, 77% purity)
at room temperature. The mixture was stirred for 1 h and
quenched by adding saturated aqueous NaHCO3 and saturated
aqueous Na2S2O3, and extracted with EtOAc. The organic phase

8416
F. Wang et al. / Bioorg. Med. Chem. 16 (2008) 8413–8418
was dried (Na2SO4), filtered and evaporated under reduced pres-
sure. The residue was purified by FCC to give 7 (1.31 g, 86.4%) as
a yellow oil; Rf = 0.70 (10% EtOAc: petroleum ether); 1H NMR
(CDCl3) d 3.69 (s, 3H), 2.92 (m, 1H), 2.75–2.77 (m, 1H), 2.47–2.48
(m, 1H), 2.32 (t, J = 7.5 Hz, 2H), 1.60–1.62 (m, 2H), 1.34–1.55 (m,
10H); 13C NMR (CDCl3) d 174.2, 52.3, 51.4, 47.1, 34.1, 32.4, 29.2,
29.1, 29.0, 25.9, 24.9.
to afford the aldol product as clear syrup (400 mg, 70%) based on
consumed (R)-9.
To the solution of product from the previous step (400 mg,
0.613 mmol) in a mixture of MeOH (10 mL) and 2-propanol
(1 mL) was added TsOH (12 mg, 0.06 mmol) at 0 °C. The reaction
mixture was stirred at room temperature for 20 h, then concen-
trated under reduced pressure. The residue was purified by FCC
to afford the derived lactone as a yellow oil (227 mg, 69%).
To a solution of the material from the previous step (227 mg,
0.42 mmol) in CH2Cl2 (10 mL) was added Et3N (0.3 mL, 2.1 mmol)
and MsCl (0.07 mL, 0.84 mmol) at 0 °C. After stirring at room tem-
perature for 14 h, the reaction mixture was diluted with saturated
aqueous NH4Cl and extracted with ether. The organic phase was
dried (Na2SO4) and concentrated under reduced pressure. The res-
idue was purified by FCC to afford (R)-11 (176 mg, 81%) as a clear
oil. Rf = 0.75 (15% EtOAc: petroleum ether); 1H NMR (CDCl3) d 7.66–
7.68 (m, 4H), 7.35–7.41 (m, 6H), 6.94 (d, J = 1.5 Hz, 1H), 5.69–5.72
(m, 1H), 4.97–4.99 (m, 1H), 4.88–4.94 (m, 2H), 3.70–3.74 (m, 1H),
2.20–2.24 (m, 2H), 1.90–1.91 (m, 2H), 1.38–1.47 (m, 11H), 1.14–
1.1.23 (m, 6H), 1.05 (s, 9H); 13C NMR (CDCl3) d 173.8, 148.8,
138.9, 135.9, 134.8, 134.7, 134.3, 129.4 (two signals), 127.4,
114.3, 73.1, 36.3, 35.8, 33.8, 29.3, 29.1, 27.3, 27.1, 25.1, 24.7,
4.1.3. (R)-Methyl 8-(oxiran-2-yl)octanoate [(R)-7]
A solution of (R/S)-7 (1.83 g, 9.17 mmol) in DME (10 mL) was
treated with (R,R)-salen-Co(OAc) (182 mg, 0.275 mmol) and dis-
tilled water (0.091 mL, 5.06 mmol). The mixture was stirred at
room temperature for 72 h, then purified by FCC to give (R)-7
(677 mg, 37%) and the derived diol (765 mg, 38%). The TLC and
NMR data for (R)-7 are as listed for (R/S)-7.
4.1.4. (S)-Methyl 8-(oxiran-2-yl)octanoate [(S)-7]
(S)-7 (4.2 g, 46%) was obtained from (R/S)-7 (9.2 g. 46 mmol)
using (S,S)-8, following the procedure described for (R)-7. The
TLC and NMR data for (S)-7 are as listed for (R/S)-7.
4.1.5. (S)-methyl 9-tert-butyldiphenylsilyloxytetradec-13-
enoate [(S)-9]
24.2, 19.4, 19.2; HRMS (FAB) calcd for
519.3294, found 519.3282.
C
33H47O3Si (M+H+)
To a suspension of CuBr (258 mg, 1.8 mmol) in THF (15 mL) at
0ꢀC was added 3-butenylmagnesium bromide (18 mL, 0.5 M in
THF, 9.0 mmol) for over 5 min. A solution of (S)-7 (600 mg,
3.00 mmol) in anhydrous THF (3 mL) was then introduced, drop-
wise over 5 min. The reaction mixture was stirred at 0ꢀC for
10 min at which time saturated aqueous NH4Cl was added and
the mixture was extracted with ether. The organic phase was
washed with brine, dried (Na2SO4), filtered, and evaporated under
reduced pressure. The residue was purified by FCC to give the de-
rived alcohol: Rf = 0.50 (20% EtOAc: petroleum ether).
A solution of the product from the previous step (358 mg,
1.4 mmol), TBDPSCl (769 mg, 2.8 mmol), and imidazole (477 mg,
7.00 mmol) in anhydrous CH2Cl2 (20 mL) was stirred under nitro-
gen at room temperature for 2 h. The mixture was quenched with
brine and extracted with ether (3 ꢁ 50 mL). The organic phase was
dried (Na2SO4), filtered, and evaporated under reduced pressure.
The residue was purified by FCC to afford 9 (600 mg, 87%) as a pale
yellow oil. Rf = 0.50 (5% EtOAc: petroleum ether); 1H NMR (CDCl3) d
7.69–7.70 (m, 4H), 7.37–7.44 (m, 6H), 5.71–5.74 (m, 1H), 4.91–4.96
(m, 2H), 3.75 (m, 1H), 3.70 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H), 1.92–1.93
(m, 2H), 1.59–1.60 (m, 2H), 1.40–1.45 (m, 6H), 1.14–1.23 (m, 8H),
1.08 (s, 9H); 13C NMR (CDCl3) d 174.5, 139.2, 136.2, 135.1, 135.0,
129.6, 127.6, 114.5, 77.0, 73.3, 51.6, 36.5, 36.0, 34.3, 34.0, 29.7,
29.3 (two signals), 27.3, 25.1, 25.0, 24.4, 19.6.
4.1.8. Butenolide (S)-11
Colorless oil; Rf = 0.70 (15% EtOAc: petroleum ether); 1H NMR
(CDCl3) d 7.69–7.70 (m, 4H), 7.37–7.45 (m, 6H), 6.96 (d, J = 1.4 Hz,
1H), 5.71–5.76 (m, 1H), 5.00–5.01 (m, 1H), 4.91–4.96 (m, 2H),
3.74–3.76 (m, 1H), 2.23–2.26 (t, J = 8.1 Hz, 2H), 1.92–1.93 (m,
2H), 1.25–1.49 (m, 11H), 1.16–1.23 (m, 6H), 1.08 (s, 9H); 13C
NMR (CDCl3) d 173.8, 148.7, 138.9, 135.9, 134.8, 134.7, 134.4,
129.4, 127.4, 114.3, 73.1, 36.3, 35.8, 33.8, 29.3, 29.1, 27.3, 27.1,
25.1, 24.7, 24.2, 19.4, 19.2; HRMS (FAB) calcd for C33H47O3Si
(M+H+) 519.3294, found 519.3293.
4.1.9. Butenolide (S)-4
AcCl (5%) in MeOH (2.1 mL) was added at room temperature to
a solution of (S)-11 in CH2Cl2 (5 mL). The mixture was stirred for
16 h, then diluted with CH2Cl2 and washed with saturated aqueous
NaHCO3. The organic phase was dried (Na2SO4) and concentrated
under reduced pressure. FCC of the residue afforded (S)-4 (50 mg,
82%): clear oil; Rf = 0.45 (30% EtOAc: petroleum ether); 1H NMR
(CDCl3) d 7.00 s, 1H), 5.81–5.86 (m, 1H), 4.97–5.06 (m, 3H), 3.61–
3.62 (m, 1H), 2.27–2.31 (m, 2H), 2.02–2.11 (m, 2H), 1.37–1.58
(m, 18H); 13C NMR (CDCl3) d 173.8, 148.8, 138.7, 134.3, 114.6,
71.8, 37.4, 36.9, 33.7, 29.3, 29.1, 27.4, 25.5, 24.9, 19.2; HRMS
(FAB) calcd for C17H29O3 (M+H+) 281.2117, found 281.2111.
4.1.6. (R)-methyl 9-tert-butyldiphenylsilyloxytetradec-13-
enoate [(R)-9]
4.1.10. THF-butenolide (R)-12
(R)-9 (600 mg, 75%) was obtained from (R)-7 (324 mg.
1.62 mmol) following the two-step reaction sequence described
for (S)-9. The TLC and NMR data for (R)-9 was as listed for (S)-9.
Grubb’s II catalyst (6 mg, 0.007 mmol) in CH2Cl2 (1 mL) was
injected, at rt, into a degassed solution of alcohol 3 (23 mg,
0.07 mmol) and (R)-11 (74 mg, 0.14 mmol), in CH2Cl2 (2 mL). The
reaction mixture was stirred for 20 h at rt, then quenched by addi-
4.1.7. Butenolide (R)-11
tion of DMSO (50 lL) and concentrated in vacuo. FCC of the residue
To a solution of diisopropylamine (1.2 mL, 8.5 mmol) in anhy-
drous THF (15 mL) at ꢀ78 °C was added BuLi (2.4 mL, 2.5 M in hex-
ane, 6 mmol). The mixture was warmed to 0 °C and stirred at this
temperature for 10 min, then cooled to ꢀ78 °C, at which time a
solution of (R)-9 (600 mg, 1.21 mmol) in anhydrous THF (6 mL)
was added. The reaction mixture was stirred at ꢀ78 °C for 1 h
and a solution of aldehyde 1014 (574 mg, 3.63 mmol) in anhydrous
THF (6 mL) was slowly introduced. After 30 min, the reaction mix-
ture was quenched with saturated aqueous NH4Cl and extracted
with ether. The organic extract was dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by FCC
afforded (R)-12 (42 mg, 73% based on 3), recovered (R)-11 (ca.
15%), and the homodimer (R)-11 (ca. 20%). For (R)-12: clear oil;
Rf = 0.50 (30% EtOAc: petroleum ether); 1H NMR (CDCl3) d 7.65–
7.67 (m, 4H), 7.34–7.42 (m, 6H), 6.94 (s, 1H), 5.65–5.68 (m, 1H),
5.32–5.34 (m, 1H), 4.97–4.99 (m, 1H), 3.81–3.85 (m, 3H), 3.70–
3.73 (m, 1H), 3.41 (m, 1H), 2.43–2.44 (m, 1H), 2.20–2.21 (m, 3H),
1.88–1.89 (m, 4H), 1.20–1.47 (m, 41H), 1.04 (s, 9H), 0.89 (t,
J = 3.2 Hz, 3H); 13C NMR (CDCl3) d 173.8, 149.5, 149.3, 149.1,
148.7, 135.9, 134.7, 134.5, 134.3, 129.4, 128.3, 127.4, 123.1, 123.0
122.7, 82.7, 82.6, 75.6, 74.0, 73.0, 33.6, 31.9, 29.7 (three signals),
29,6 (three signals), 29.3 (two signals), 29.1, 28.4 (two signals),

F. Wang et al. / Bioorg. Med. Chem. 16 (2008) 8413–8418
8417
27.3, 27.1, 25.1, 22.7, 19.2, 14.1; HRMS calcd for C51H80O6SiNa
4.2. Cytotoxicity assay
(M+Na+) 839.5622, found, 839.5613.
Cells were cultured in RPMI medium supplemented with 10%
fetal bovine serum and penicillin-streptomycin-fungizone mixture
4.1.11. THF-butenolide (S)-13
Grubb’s II catalyst (9.3 mg, 0.01 mmol) in CH2Cl2 (1 mL) was in-
(100 U/mL, 100 lg/mL, and 0.25 lg mL, respectively) and main-
jected, at rt, into a degassed solution of alcohol
3
(36 mg,
tained in a 37 °C humidified 5% CO2 incubator. On the day before
0.11 mmol), and (S)-4 (50 mg, 0.178 mmol), in CH2Cl2 (2 mL). The
the drug treatment, cells were plated onto each well of 96-well
reaction mixture was stirred for 20 h at rt, then quenched by addi-
plate at 2000 cells/well (200 ll of the medium per well). After
tion of DMSO (50
l
L) and concentrated in vacuo. FCC of the residue
24 h, cells were treated with different concentrations of com-
pounds and incubated for 72 h. After the incubation, cell growth
was evaluated using a CellTiter 96 AQueous One Solution Cell Pro-
liferation Assay (Promega). UV absorption (490 nm) of each well
was quantified by SpectraMax Plus 384 microplate reader (Molec-
ular Devices). The data are provided as Supplementary
Information.
provided (S)-13 (36 mg, 57% based on 3), and the homodimer of
(S)-4 (ca. 27%). For (S)-13: clear oil; Rf = 0.50 (80% EtOAc: petro-
leum ether); 1H NMR (CDCl3) d 7.01 (s, 1H), 5.76–5.82 (m, 1H),
5.41–5.46 (m, 1H), 5.02–5.03 (m, 1H), 3.85–3.92 (m, 3H), 3.61 (s,
br, 1H), 3.42–3.44 (m, 1H), 2.29 (t, J = 7.5 Hz, 2H), 2.10 (m, 2H),
1.97–2.01 (m, 2H), 1.28–1.70 (m, 44H), 0.90 (t, J = 6.6 Hz, 3H);
13C NMR (CDCl3) d 173.9, 148.9, 134.3, 128.6, 82.8, 82.5, 76.8,
76.7, 75.5, 74.0, 71.7, 37.4, 36.9, 32.3, 31.9, 29.7 (five signals),
29.6, 29.4, 29.3, 29.2, 27.4, 25.6, 25.5, 25.2, 22.7, 19.2, 14.2; HRMS
(FAB) calcd for C35H63O6 (M+H+) 579.4614, found 579.4625.
Acknowledgments
This investigation was supported by Grant R01 GM57865 from
the National Institutes of Health (NIH). ‘Research Centers in Minor-
ity Institutions’ Award RR-03037 from the National Center for Re-
search Resources of the NIH, which supports the infrastructure and
instrumentation of the Chemistry Department at Hunter College, is
also acknowledged.
4.1.12. (9R)-4-deoxyannoreticuin (1)
Chlorotris(triphenylphosphine)-rhodium (I) (7.1 mg, 0.0077
mmol) was added to a degassed solution of (R)-12 (23 mg,
0.028 lmol) in a mixture of benzene–EtOH (2 mL, 50% v/v). The
mixture was stirred under an atmosphere of hydrogen for 12 h,
at which time the solvent was removed under reduced pressure.
FCC of the residue gave an inseparable mixture of the 13,14-dihy-
dro- and tetrahydro derivatives of (R)-12 (16 mg, 70%): Rf = 0.50
(30% EtOAc: petroleum ether).
A mixture of 5% AcCl in MeOH (0.5 mL) was added at room tem-
perature to a solution of the material obtained in the previous step
(10 mg, 12.2 lmol) in CH2Cl2 (1 mL). The mixture was stirred at
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.08.030.
References and notes
this temperature for 3 h, diluted with CH2Cl2, and washed with a
saturated aqueous NaHCO3. The organic layer was dried (Na2SO4),
filtered, and concentrated under reduced pressure. FCC of the res-
idue afforded a mixture of 1 and the completely hydrogenated
product (7.2 mg, 72%) in an approximate 7:1 ratio: Rf = 0.35 (60%
EtOAc: petroleum ether). Repeated FCC provided a sample of 1 that
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contained less than 10% of the completely reduced material as a
22
white waxy solid; [
a]
+19 (c 2.00, CH2Cl2); 1H NMR (CDCl3) d
D
6.96 (s, 1H), 4.96–4.98 (m, 1H), 3.76–3.80 (m, 2H), 3.56 (s, br,
1H), 3.36–3.41 (m, 2H), 2.25–2.27 (m, 4H, CH2, 2ꢁ OH, D2O ex.),
1.96–1.97 (m, 2H), 1.24–1.52 (m, 48H), 0.87 (t, J = 6.8 Hz, 3H);
13C NMR (CDCl3) d 173.8, 148.9, 134.3, 82.7, 77.4, 74.0 (two sig-
nals), 71.9, 37.4, 33.5, 33.4, 31.9, 29.7 (three signals), 29.6 (three
signals), 29.3, 29.1, 28.7, 27.3, 25.6 (three signals), 25.5, 25.2,
22.7, 19.2, 14.1; HRMS (FAB) calcd for C35H65O6 (M+H) 581.4781,
found 581.4794.
4.1.13. (9S)-4-deoxyannoreticuin (2)
A solution of sodium acetate (280 mg, 3.41 mmol) in water
(5 mL) was added via a syringe pump, for over 4 h, to a mixture
of (S)-13 (24 mg, 0.041 mmol), p-toluenesulfonyl hydrazide
(511 mg, 2.74 mmol), and DME (4 mL) at reflux. After cooling to
rt, the reaction mixture was poured into water and extracted with
EtOAc. The combined organic extract was washed with 2 M HCl,
water, and brine, dried (Na2SO4), filtered, concentrated under re-
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duced pressure and purified by FCC to give 2 as a white waxy solid
22
(21.5 mg, 90%). Rf = 0.50 (80% EtOAc: petroleum ether); [
a]
+25
D
(c 4.00, CH2Cl2); 1H NMR (CDCl3) d 6.96 (s, 1H), 4.95–4.99 (m, 1H),
3.76–3.80 (m, 2H), 3.56 (s, br, 1H), 3.36–3.40 (m, 2H), 2.23–2.24
(m, 2H), 1.95–1.96 (m, 2H), 1.66 (m, 2H), 1.23–1.53 (m, 48H),
0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) d 173.8, 148.9, 134.3,
82.7, 82.6, 77.4, 74.0, 71.9, 37.4, 33.5, 33.4, 31.9, 29.7, 29.6, 29.3,
29.1, 28.7, 27.4, 25.6, 25.5, (two signals), 22.7, 19.2, 14.1; HRMS
calcd for C35H65O6 (M+H) 581.4781, found 581.4789.
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8418
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a
remote
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