Structural and synthetic investigations of tanikolide dimer, a SIRT2 selective inhibitor, and tanikolide seco-acid from the Madagascar marine cyanobacterium Lyngbya majuscula

Article abstract of DOI:10.1021/jo900578j

(Chemical Equation Presented) Tanikolide seco-acid 2 and tanikolide dimer 3, the latter a novel and selective SIRT2 inhibitor, were isolated from the Madagascar marine cyanobacterium Lyngbya majuscula. The structure of 2, isolated as the pure R enantiomer

Full text of DOI:10.1021/jo900578j

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Structural and Synthetic Investigations of Tanikolide Dimer, a SIRT2
Selective Inhibitor, and Tanikolide seco-Acid from the Madagascar
Marine Cyanobacterium Lyngbya majuscula
Marcelino Gutierrez,†,‡ Eric H. Andrianasolo,§,‡ Won Kyo Shin,^,‡ Douglas E. Goeger,§
ꢀ
Alexandre Yokochi, Jorg Schemies, Manfred Jung, Dennis France,
#
#
3
€
Susan Cornell-Kennon,3 Eun Lee,*,^ and William H. Gerwick*,†,§
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and The Skaggs
School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California
92037, §College of Pharmacy and Department of Chemical Engineering, Oregon State University, Corvallis,
Oregon 97331, ^Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul,
#
€
151-747, Korea, Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universitat Freiburg, Albertstrausse
25, 79104 Freiburg, Germany, and 3Novartis Institute for Biomedical Research, Cambridge,
Massachusetts 02139. ‡These authors contributed equally to the work (M.G., stereochemical analyses; E.H.
A., isolation and planar structure; W.K.S., synthesis).
wgerwick@ucsd.edu; eunlee@snu.ac.kr
Received April 3, 2009
Tanikolide seco-acid 2 and tanikolide dimer 3, the latter a novel and selective SIRT2 inhibitor, were
isolated from the Madagascar marine cyanobacterium Lyngbya majuscula. The structure of 2,
isolated as the pure R enantiomer, was elucidated by X-ray experiment in conjunction with NMR and
optical rotation data, whereas the depside molecular structure of 3 was initially thought to be a meso
compound as established by NMR, MS, and chiral HPLC analyses. Subsequent total synthesis of the
three tanikolide dimer stereoisomers 4, 5, and ent-5, followed by chiral GC-MS comparisons with
the natural product, showed it to be exclusively the R,R-isomer 5. Tanikolide dimer 3 (= 5) inhibited
SIRT2 with an IC50 = 176 nM in one assay format and 2.4 μM in another. Stereochemical
determination of symmetrical dimers such as compound 3 pose intriguing and subtle questions in
structure elucidation and, as shown in the current work, are perhaps best answered in conjunction
with total synthesis.
Introduction
hydroxymethyl groups, as well as two multicarbon chains
and exhibits potent antifungal activity against Candida
albicans (13 mm diameter zone inhibition using 100 μg/disk).
In addition, it shows strong toxicity in the brine shrimp
Artemia salina and schistosomiasis-carrying snail Biompha-
laria glabrata (LC50 =3.6 and 9.0 μg/mL, respectively)
biological assays. Owing to its juxtaposition of func-
tional groups, tanikolide (1) has been the target of numerous
enantioselective syntheses.2 As part of our ongoing search
for structurally and pharmacologically interesting substances
Tanikolide (1) is a biologically active δ-lactone that was
originally isolated from the lipid extract of a collection of
Lyngbya majuscula from Madagascar.1 Tanikolide possesses
a single chiral center, a quaternary carbon with hydroxyl and
*To whom correspondence should be addressed. Natural products isola-
tion and characterization. (W.H.G.) Tel: (858) 534-0578. Chemical synthesis.
(E.L.) Tel: þ82-2-880-6646.
(1) Singh, I. P.; Milligan, K. E.; Gerwick, W. H. J. Nat. Prod. 1999, 62,
1333–1335.
DOI: 10.1021/jo900578j
r
Published on Web 07/02/2009
J. Org. Chem. 2009, 74, 5267–5275 5267
2009 American Chemical Society

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from cyanobacteria,3-5 a detailed exploration of another
Malagasy L. majuscula collection was undertaken. Using a
human sirtuin type 2 (SIRT2) bioassay-guided approach, we
report here the isolation and structural elucidation of both
tanikolide seco-acid 2 and tanikolide dimer 3.
inhibitors of these proteins are of interest as potential
anticancer drugs. SIRT2 inhibitors have also emerged as
agents with potential utility in neuroprotection.12 Tanikolide
dimer 3 was found to be a potent inhibitor of SIRT2 (IC50=
176 nM in one assay format; 2.4 μM in another) as well as
active in a sodium channel blocking assay (54% inhibition at
5.2 μM). Conversely, the seco-acid 2 exhibited only moderate
cytotoxicity against the H-460 cancer cell line and was
inactive in both the SIRT2 and sodium channel blocking
assays.
Determination of the stereochemistry of symmetrical di-
meric compounds can be technically challenging because
such substances often possess subtle issues of chirality. For
example, our initial chiral analysis of tanikolide dimer 3
suggested it to be a meso compound (e.g., 4);13 however, total
chemical synthesis of the three stereoisomers of 3 and
comparison by chiral GC-MS showed it to be the R,R-
stereoisomer 5 and of high enantiomeric purity. Here, we
report the isolation of this new dilactone as a potent SIRT2
inhibitor, elucidation of its planar structure, and determina-
tion of its stereostructure through enantio-specific synthesis
and careful comparisons by chiral gas chromatography-
mass spectrometry.
SIRT2 is a NADþ-dependent cytoplasmic protein that is
co-localized with HDAC6 on microtubules. SIRT2 has been
shown to deacetylate R-tubulin and to control mitotic exit
from the cell cycle.6 Human SIRT2 proteins are closely
related in structure to the NAD-dependent deacetylases of
other species.7 The natural substrate for SIRT2 is believed
to be p53.8 Binding of p53 to DNA is activated by its acetyl-
ation in the C-terminal domain;9 thus, deacetylation of p53
by SIRT2 could be important in the regulation of cellular
responses to DNA-damaging agents.10 Because SIRT2 func-
tions to silence apoptotic responses mediated by p53,11
Results and Discussion
L. majuscula was collected from near Tanikely Island,
Madagascar, extracted with CH2Cl2/MeOH (2:1), and frac-
tionated by Si VLC. The resulting fractions were tested for
SIRT2 inhibitory activity and revealed that the 40% EtOAc/
hexanes eluting fraction was strongly active (IC50=2.5 μg/
mL). This material was subjected to further fractionation by
RP solid-phase extraction (SPE), and two fractions (D3 and
D4, see the Experimental Section) were found to be active
(81% and 75% inhibition at 10 μg/mL, respectively). Analy-
tical reversed-phase HPLC purification of D3 and D4 led to
the isolation of tanikolide dimer 3, which initially showed an
IC50 =176 nM to SIRT2. Compound 2 was isolated as a
crystalline substance from a VLC fraction eluting with 25%
MeOH in EtOAc and was inactive in the SIRT2 assay.
HR FABMS analyses of compound 2 gave an [MþNa]þ
ion at m/z 325.2366 for a formula of C17H34O4Na (one less
DBE than tanikolide). 13C NMR revealed the presence of a
carbonyl group, which on the basis of its chemical shift was
likely that of a carboxylic acid (δ 176.8). The C-5 quaternary
carbon shift (δ 74.4) was present in 2, indicating that hydro-
xyl, hydroxymethyl, and two alkyl groups were attached at
this position. Indeed, by the data presented above and
COSY, HSQC, and HMBC, four distinct sections of the
molecule were formulated: a hydroxy group, a hydroxy-
methyl group, a butanoic acid chain, and an undecanyl
chain, all of which were connected to the C-5 quaternary
carbon (Table 1). Because X-ray quality crystals of 2 were
deposited from a fraction eluting with 25% MeOH in
EtOAc, these structural features were confirmed via a
diffraction study (see the Supporting Information).
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TABLE 2. 1H and 13C NMR Assignments for Tanikolide Dimer 3a
TABLE 1. 1H and 13C NMR Assignments for Tanikolide seco-Acid 2a
position
δC
δH
position
δC
δH
HMBC
1/10
2/20
3/30
4/40
172.1
30.2
17.1
27.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
176.8
34.6
19.1
35.5
74.4
36.2
22.7
29.9
29.6
29.6
29.6
29.6
29.6
32.3
23.1
13.4
67.1
2.29 (t, J = 6.7 Hz)
1.67 (m)
1.49 (m)
2.51 (m)
1.92 (m)
1.78 (m)
1.95 (m)
3
2, 4
3
1, 3, 4
1, 5
3, 5
5/50
6/60
86.9
37.1
1.49 (m)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (br s)
1.29 (m)
0.91 (t, J = 6.9 Hz)
3.59 (d, J =14.1 Hz)
3.68 (d, J =14.1 Hz)
1.66 (m)
1.75 (m)
1.29 (m)
1.32 (m)
1.27 (br s)
1.27 (br s)
1.27 (br s)
1.27 (br s)
1.27 (br s)
1.27 (br s)
1.27 (br s)
1.30 (m)
0.86 (t, J=7.2 Hz)
3.59 (d, J=11.3 Hz)
3.68 (d, J=11.3 Hz)
7
6
4, 5, 17
7/70
23.9
8/80
9/90
30.4
30.2
30
10/100
11/110
12/120
13/130
14/140
15/150
16/160
17/170
30
29.9
29.7
32.3
23.1
14.1
67.9
15
14, 16
15
a 400 MHz 1H and 100 MHz for 13C NMR; CD3OD as solvent.
15, 14
4, 5, 6
a 400 MHz for 1H and 100 MHz for 13C NMR; CDCl3 as solvent.
The seco-acid 2 was found to be optically active, and only a
single enantiomer was observed in the X-ray study. Further,
when a sample of 2 was analyzed by chiral-phase HPLC, only
a single peak was observed. Because the R-enantiomer of
compound 2 has been chemically synthesized and its rotation
measured {[R]D -0.8 (c 1.0, CHCl3}, we conclude from the
negative rotation of natural tanikolide seco-acid {[R]25D -10
(c 0.87, CHCl3)} that it is also of R-configuration at C-5, the
same as in tanikolide (1) itself.14
The parent molecular ion for compound 3 was deduced by
various modes of ionization, including FABMS in various
matrices, ESIMS, and EIMS. LR FABMS displayed three
peaks at m/z 591.4 [M þ Na]þ, m/z 569.3 [M þ H]þ, and
m/z 551.2 [M - OH]þ. HR FABMS of the m/z 569.47804
[M þ H]þ peak showed a molecular formula of C34H65O6,
whereas HR EIMS of the m/z 551.4691 [M - OH]þ peak
FIGURE 1. Selected HMBC and 1H-1H COSY correlations for
tanikolide dimer 3.
1
Analyses of H-1H COSY and HMBC experiments of
3 led to the deduction that it was closely related to tanikolide
(1) (Figure 1).1 For example, the oxymethylene protons at
δ 3.68/3.59 (H2-17) showed HMBC correlations with car-
bons at δ 86.9 (C-5), 27.0 (C-4), and 37.1 (C-6). Additionally,
a pair of methylene protons at δ 2.51 (H2-2) showed HMBC
correlations with carbons at δ 172.1 (C-1), 17.1 (C-3), and
27.0 (C-4). 1H-1H COSY delineated a connected spin
system for the methylenes at C-2, C-3, and C-4. A methyl
group signal at δ 0.86 (H3-16) showed HMBC correlations
with two methylene carbons at δ23.1 (C-15) and 32.3 (C-14),
a sequence confirmed by 1H-1H COSY analysis and, hence,
was at the terminus of a long lipid tail. Based on the close
concordance in chemical shifts and coupling patterns be-
tween 3 and 1, and the MS analysis described above,
compound 3 was deduced to be a symmetrical dimer of
tanikolide (1).
Because tanikolide (1) and its seco-acid 2 had both been
isolated as optically active metabolites and their lone stereo-
centers determined to be R (Mosher ester for the former and
comparison with optical rotation for the latter), it was
surprising that dimer 3 initially gave no rotation at the
sodium D-line or any other wavelength examined [however,
remeasurement of the optical rotation of natural tanikolide
dimer on a more sensitive instrument subsequently gave an
[R]25D þ1.0 (c 1.2, CHCl3)]. This perplexing initial result was
further examined by acid hydrolysis of 3 and analysis by
chiral HPLC, first with diode array detection (DAD) and
then with ESI LCMS. In both cases, a 1:1 ratio of two peaks
showed a molecular formula of C34H63O5. However, the 13
C
NMR and DEPT spectra for 3 indicated the presence of only
17 carbon and 31 carbon-bound hydrogen atoms (Table 2).
These data combined with the MS information indicated
that only half of the signals were appearing in the NMR
spectra and suggested that 3 was a symmetrical dimer. The
three degrees of unsaturation implied by the molecular
formula were accounted for by the presence of two carbonyls
(δ 172.1) and one ring.
13C NMR showed the presence of an ester carbonyl
(δ 172.1; IR 1716 cm-1), a methylene carbon attached to
oxygen (δ 67.9), a quaternary carbon attached to oxygen
(δ 86.9), a methyl group (δ 14.5), and 13 high-field methy-
lene carbons. The 1H NMR of 3 showed a pair of mutually
coupled doublets (δ 3.68 and 3.59) for an isolated methylene
attached to oxygen and a two-proton multiplet (δ 2.51)
assigned to a methylene adjacent to a carbonyl group.
A series of multiplets in the range δ 1.29-1.95 accounted
for five methylene groups, and a broad singlet (δ 1.27)
contained an additional 14 degenerate protons. A ter-
minal methyl group was assigned to a three-proton triplet
at δ 0.86.
(14) We cannot explain the discrepancy in the magnitude in the optical
rotation of our crystalline natural sample of 2 from that of synthetic material,
except to note that the rotation of the natural material was run on an older,
less sensitive, and possibly inaccurate polarimeter (Perkin-Elmer 141).
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SCHEME 1. Synthesis of (R,R)-Tanikolide Dimer 5
SCHEME 2. Synthesis of (R,S)-Tanikolide Dimer 4
Moreover, we were unable to develop a convincing HPLC-
based method to conduct this analysis. Thus, we turned to a
chiral GC-MS approach as described below.
Natural tanikolide dimer 3 and the synthetic stereoisomers
(5R,50S)-tanikolide dimer 4, (5R,50R)-tanikolide dimer 5,
and (5S,50S)-tanikolide dimer ent-5 were analyzed by GC-
MS chromatography using a Cyclosil-B chiral column.
When (5R,50S)-tanikolide dimer 4 was injected, two peaks
were observed at 35.99 and 36.37 min, each having a mass
spectrum consistent with tanikolide (1) or its seco-acid 2
(e.g., each of these two compounds give the seco-acid follow-
ing injection into the GC). The presence of two peaks in the
chromatogram indicated that the dimer had undergone
thermal hydrolysis, probably in the GC injection port.
Consistent with this finding, when synthetic compounds
5 and ent-5 were injected separately, they each gave a single
peak, the former at 35.99 min and the latter at 36.37 min.
When natural tanikolide dimer 3 was analyzed by this
protocol, a single peak was observed at 35.99 min, thereby
indicating it was composed of two identical monomers, each
of R configuration.
We believe that the initial chiroptical and chiral HPLC
analyses, which suggested that natural tanikolide dimer was a
meso compound, were flawed for the following reasons. As
determined with the synthetic materials, the optical rotations of
both the R,R or S,S dimers are very weak such that an
essentially zero measurement was made with the small amount
of natural dimer material that we had originally isolated. This
was exacerbated by the use of an older polarimeter in these
initial measurements. More significantly, the conditions used
for the acid hydrolysis (6 N HCl at 105 ꢀC for 16 h) could very
well have led to racemization of the seco acid; retrospectively,
racemization of the tertiary alcohol under these vigorous
conditions could have been predicted.
was obtained which appeared to be due to the seco acid,
suggesting that 3 was a meso compound deriving from one
R-monomer and one S-monomer. To clarify this surprising
result, we turned to an integrated total chemical synthesis
approach of the three stereochemical possibilities for tani-
kolide dimer along with chiral GC-MS analysis. Remark-
ably, this approach gave quite different and unexpected
results as described below.
The known epoxy alcohol 6 served as the starting material
for the synthesis of (5R,50R)-tanikolide dimer 5. The tertiary
alcohol 7 was prepared from the benzyl ether derivative of 6
via reaction with vinylmagnesium bromide in the presence of
cuprous iodide. Radical-mediated reaction of 7 with thio-
phenol and then reaction with acryloyl chloride produced
acrylate 8. Cross-metathesis reaction of 7 and 8 in the
presence of the second-generation Grubbs catalyst15 in hot
dichloromethane led to the formation of the thioether 10.
Thermal elimination reaction of the sulfoxide derivative of
10 was carried out in hot toluene-pyridine, and the diene
acrylate 11 was prepared from the elimination product.
Ring-forming metathesis reaction of 11 in the presence of
Hoveyda-Grubbs catalyst 1216 proceeded smoothly, and
(5R,50R)-tanikolide dimer 5 was obtained in high yield upon
hydrogenation-hydrogenolysis (Scheme 1). The synthesis of
(5S,50S)-tanilkolide dimer ent-5 was carried out in the same
manner using the enantiomeric starting material.
The synthesis of (5R,50S)-tanikolide dimer 4 also started
with ent-6. Cross metathesis reaction between ent-7 and 8
produced a new thioether 13, which was converted into the
diene acrylate 14 via oxidation-elimination and acrylate
formation. Ring-forming metathesis reaction of 14 and
hydrogenation-hydrogenolysis led to the formation of
(5R,50S)-tanikolide dimer 4 in high yield (Scheme 2).
Pure compounds 2 and 3 (= 5) were tested in the SIRT2
inhibitory assay as well as a mammalian cell sodium channel
blocking assay.17 Compound 3 (=5) was initially found to
have an IC50 value of 176 nM, against SIRT2 whereas 2 was
inactive at the highest concentration tested (50 μM). Com-
paring these data with those reported for sirtinol and
8,9-dihydroxybenzo[4,5]furo[3,2-c]chromen-6-one (IC50 va-
lues of 38 and 45 μM respectively)18 indicates that 3 (= 5) is
When the NMR spectra of compounds 4 and 5 were
compared, they looked very similar, and it was impractical to
distinguish them on this basis (Supporting Information).
(17) Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama,
Y.; Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M.
J. AOAC Int. 1995, 78, 521–527.
(15) Sanford, M. S.; Love, J. A.; Grubbs, R. H. J. Am. Chem. Soc. 2001,
123, 6543–6554.
(16) Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am.
Chem. Soc. 2000, 122, 8168–8179.
(18) Grozinger, C. M.; Chao, E. D.; Blackwell, H. E.; Moazed, D.;
Schreiber, S. L. J. Biol. Chem. 2001, 276, 38837–38843.
5270 J. Org. Chem. Vol. 74, No. 15, 2009

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TABLE 3. Inhibitory Properties of Synthetic Tanikolide Dimers to SIRT1
and SIRT2
not important to this drug-protein interaction. That
the seco-acid 2 was inactive despite its containing two
hydroxyl groups suggests that either the free carboxyl
functionality interferes with binding of the drug or that the
entire dimeric structure is sterically required for effective
inhibition.
It seems likely that the biosynthesis of tanikolide (1), its
seco-acid 2, and its dimer 3 proceeds via a PKS biosynthetic
pathway. However, the occurrence of a branching carbon
atom at C-5(50) in these metabolites, a site on the polyketide
backbone logically deriving from C-1 of acetate, suggests the
involvement of an HMGCoA synthase-like enzyme as has
recently been deduced in curacin A and jamaicamide A
biosynthesis.23,24 With clarification of the configuration of
this stereocenter in tanikolide dimer 3 from combined syn-
thetic/chiral GCMS analysis, it appears that the condensa-
tion of acetate with a β-carbonyl functionality occurs with
the same chiral preference for both monomers present in the
dimer.
compd
SIRT1 IC50 þ SE (μM)
28.8 ( 4.3
SIRT2 IC50 þ SE (μM)
2.4 ( 0.2
(R,S)-tanikolide
dimer (4)
(R,R)-tanikolide
dimer (5)
(S,S)-tanikolide
dimer (ent-5)
36.4 ( 8.1
34.5 ( 3.5
3.3 ( 0.2
3.1 ( 0.3
200-300-fold more active than either of these agents. In the
sodium channel blocking assay, compound 3 induced a 54%
inhibition at 5.2 μM, while 2 was again inactive at the highest
concentration tested (10 μM). However, cytotoxicity assays
revealed that 2 was moderately toxic at 9.9 μM to the human
lung H-460 cell line, while 3 did not have any activity at
10 μM. This observation is consistent with the limited
cytotoxicity observed with other selective SIRT2 inhibitors19
and suggests that tanikolide dimer may have more potential
for use as a neuroprotectant.
Synthetic production of an additional supply of the nat-
ural (5R,50R)-tanikolide dimer 5 as well as the stereoisomers
(5S,50S)-tanilkolide dimer ent-5 and (5R,50S)-tanikolide di-
mer 4 allowed for a further exploration of the SIRT1 and
SIRT2 inhibitory properties of this compound class. Un-
fortunately, the original assay used for measuring these
enzyme inhibitory properties was no longer available, but
this problem was overcome using another assay system.20,21
Surprisingly, all three stereoisomers (4, 5, ent-5) showed
similar potencies in their inhibition of SIRT1 (20-36 μM)
or SIRT2 (2.4-3.3 μM) but were overall approximately
10-fold more potent to the SIRT2 isoform (Table 3). The
decreased potency of natural tanikolide 3 (= 5) in this
second assay system relative to its initial assay may be due
to small differences in the way the assays were run, the
preparation of the enzymes, the substrates, duration of
incubation, or a combination of factors. None of these three
isomers showed any activity at 25 μM to a rat liver HDAC
preparation composed of a mixture of HDACs,20,21 and
thus, the SIRT inhibitory properties of these tanikolide
dimers is not a general and nonspecific property.
A study on novel inhibitors of SIRT2 reported that a
phenol or hydroxyl group is important for inhibitory activity
based on the structure of the putative SIRT2 active site.22
These polar functional groups together with a hydrophobic
moiety and hydrogen-bonding features are suggested to
form an active SIRT2 pharmacophore. In addition, it is
believed that SIRT2 inhibitors must sterically block the
opening of a narrow channel near the putative active site,
adjacent to Ile169 and Asp170, and continuing throughout
the length of the enzyme.22 Interestingly, the functional
groups and overall dimeric structure of 3 meet these require-
ments; however, the results with the synthetic stereoisomers
of tanikolide dimer (4, 5, and ent-5) indicate that chirality is
Conclusions
In summary, the structure elucidation of tanikolide dimer
3 (= 5) raised a number of intriguing configurational and
biosynthetic questions for further study, and these were
effectively answered by the combined approach of synthesis
of the three candidate stereoisomers and chiral GC-MS
analysis. It was intriguing that all three stereoisomers had
equivalent activity in the SIRT2 assay, suggesting an ac-
hiral interaction between inhibitor and enzyme. Neverthe-
less, given tanikolide dimer’s potent biological effects to
SIRT2, expanding a deeper understanding of its struc-
tural subtleties and mechanism of formation is important
to developing its lead compound and biotechnological
potential.
Experimental Section
Collection, Extraction, and Isolation Procedures. L. majuscula
was collected by hand using scuba in April 2000 near Tanikely
Island, Nosy-Be Madagascar (voucher specimen available from
WHG as collection no. MNT-26/Apr/00-02). The alga was
stored at -20 ꢀC in 70% EtOH until workup. A total of 944 g
(dry wt) of the alga was extracted three times with CH2Cl2-
MeOH (2:1) to yield 8.73 g of crude extract. A portion of this
(1.5 g) was fractionated using vacuum-liquid chromatography
(VLC) on Si gel by a stepwise gradient of hexanes-EtOAc and
EtOAc-MeOH to give nine fractions (A through I). Fraction D
(96.7 mg, 40% EtOAc in hexanes) showed inhibitory activity
against SIRT2 and was subjected to further fractionation by
solid-phase extraction (NP SPE) to yield five fractions (D1-D5).
Fraction D3 and D4 gave 80% and 75% inhibition at 10 μg/mL,
respectively, in the SIRT2 assay. Analytical reversed-phase
HPLC (Phenomenex Sphereclone ODS, 250 ꢀ 10.0 mm, 5 μm,
2.5 mL/min, RI detection, MeOH/H2O, 95:5) purification of
D3 and D4 led to the isolation of 15 mg of 3 (tR=8 min) as a
colorless oil. Fraction H from the initial Si gel chromatography
(25% MeOH-EtOAc) directly deposited crystals of compound
2 in several milligram yield, and these were used in X-ray
crystallographic and other experiments without any further
purification.
(19) Neugebauer, R. C.; Uchiechowska, U.; Meier, R.; Hruby, H.;
Valkov, V.; Verdin, E.; Sippl, W.; Jung, M. J. Med. Chem. 2008, 51, 1203–
1213.
(20) Heltweg, B.; Trapp, J.; Jung, M. Methods 2005, 36, 332–337.
(21) Uciechowska, U.; Schemies, J.; Neugebauer, R. C.; Huda, E.-M.;
Schmitt, M. L.; Meier, R.; Verdin, E.; Jung, M.; Sippl, W. ChemMedChem.
2008, 3, 1965–1976.
(23) Chang, Z.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P. M.;
Jia, J.; Sherman, D. H.; Gerwick, W. H. J. Nat. Prod. 2004, 67, 1356–1367.
(24) Edwards, D. J.; Marquez, B.; Nogle, L. M.; McPhail, K.; Goeger,
D.; Roberts, M. A.; Gerwick, W. H. Chem. Biol. 2004, 11, 817–833.
€
(22) Tervo, A. J.; Kyrylenko, S.; Niskanen, P.; Salminen, A.; Leppanen,
J.; Nyronen, T. H.; Jarvinen, T.; Poso, A. J. Med. Chem. 2004, 47, 6292–6298.
€
€
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X-ray Crystallography of Tanikolide seco-Acid 2. The crystal-
line sample of 2 obtained from the fraction eluting with 25:75
MeOH/EtOAc was used without further preparation. Determi-
nation of the crystallographic parameters, data collection, and
structure solution and refinement were done as described else-
where,25 with the following details.
SIRT1 and SIRT2 enzymes were overexpressed and their activ-
ities assayed as described in Uciechowska et al.21
Sodium Channel Blocking Assay. Chemicals were evaluated
for their capacity to either activate or block sodium channels
using the following modifications to the cell-based bioassay of
Manger et al.17 Twenty-four hours prior to chemical testing,
mouse neuroblastoma (neuro-2a) cells were seeded in 96-well
plates at 6.0 ꢀ 104 cells/well in a volume of 200 μL. Test
chemicals dissolved in DMSO were serially diluted in medium
without fetal bovine serum and added at 10 μL/well. DMSO was
less than 0.5% final concentration. Plates to evaluate sodium
channel activating activity received 20 μL/well of either a
mixture of 3 mM ouabain and 0.3 mM veratridine (Sigma
Chemical Co.) in 5 mM HCl or 5 mM HCl in addition to the
test chemical. Plates were incubated for 18 h and results com-
pared to similarly treated solvent controls with 10 μL of medium
added in lieu of the test chemical. The sodium channel activator
brevetoxin PbTx-3 (Calbiochem) was used as the positive con-
trol and added at 10 ng/well in 10 μL of medium. Sodium
channel blocking activity was assessed in a similar manner
except that ouabain and veratridine were 5.0 and 0.5 mM,
respectively, and the sodium channel blocker saxitoxin (Calbio-
chem) was used as the positive control. Plates were incubated for
approximately 22 h.
A well-shaped crystal of dimensions 0.30 ꢀ 0.10 ꢀ 0.02 mm
was selected and mounted on the tip of a thin glass fiber using a
dab of Paratone.25 Crystal quality evaluation and preliminary
indexing were performed from four images of 10ꢀ rotation about
ω, each separated from the others by 50ꢀ. Proving to be a
satisfactory crystal, a full set of 160 frames of 5ꢀ rotation about
ω was collected. The frames were integrated using the deter-
mined unit cell, using the program TwinSolve as included in
Rigaku/MSC’s software package CrystalClear to yield a redun-
dant data set of 8951 reflections. Correction for the effects of
absorption anisotropy was carried out by means of multiscans
as programmed in TwinSolve.26 Finally, a data set consisting of
2294 unique reflections in the range (0-6, -9-9, -24-24) was
generated with an R(merge) of 0.1640. The reported unit cell was
refined using all 2048 reflections with intensities greater than
10 times their esd’s in the range 2.25ꢀ < θ < 64.64ꢀ.
The structure was solved using direct methods as pro-
grammed in SHELXS-9026 and refined using the program
SHELXL-97.27 Although all hydrogen atoms could be clearly
identified from the Fourier map, in order to preserve a favorable
data-to-parameter ratio the hydrogen atoms were placed in
geometrically idealized positions. The hydrogen atoms were
given a displacement parameter equal to 1.5 times (methyl
group) or 1.2 times (all other hydrogens) the equivalent isotropic
displacement parameter of the atom to which it was attached.
During the final cycle of least-squares refinement, all non-
hydrogen atoms were refined with anisotropic displacement
parameters. The refined value of the absolute structure para-
meter (Flack parameter)28 of 0.5(7) indicates that no clear
indication of the absolute structure of the molecule can be
derived from the diffraction experiment alone. An ORTEP29
of the final model for compound 2 is given in the Supporting
Information, with displacement ellipsoids drawn at the 50%
probability level.
Cytotoxicity Assay. Cytotoxicity was measured in NCI-H460
lung tumor cells and neuro-2a cells using the method of Alley
et al.30 with cell viability being determined by MTT reduction.17
Cells were seeded in 96-well plates at 6000 cells/well in 180 μL.
Twenty-four hours later, the test chemical dissolved in DMSO
and diluted into medium without fetal bovine serum was added
at 20 μL/well DMSO was less than 0.5% final concentration.
After 48 h, the medium was removed and cell viability deter-
mined.
Tanikolide seco-acid 2:. [R]25 -10 (c 0.87, CHCl3); no UV
D
absorbance observed; IR νmax (neat) 3425, 3410, 2850, 2360,
1715, 1250, 1050, 939 cm-1; 1H NMR (CD3OD, 400 MHz) and
13C NMR (CD3OD, 100 MHz), see Table 1; LR FABMS (nba)
obsd m/z (rel int) 325.3 (100) [M þ Na]þ; HR FABMS obsd
[M þ Na]þ m/z 325.23668 for C17H34O4Na (-1.2 mmu).
Natural tanikolide dimer 3:. initial measurement [R]25 0 -
D
Initial SIRT2 Inhibitory Assay. The assay was run in 96-well
format in a final volume of 50 μL. All reaction components were
prepared in buffer A. Blank wells contained no NAD. The
reaction was run for 2 h at 37 ꢀC and stopped with 50 μL of
stop buffer. Development was allowed to proceed for 20 min,
and then the plate was read with a fluorescence plate reader
(excitation at 360 nm, emission at 460 nm). Final assay con-
centrations contained 50 μM fluor de Lys substrate (purchased
from Biomol and used according to the manufacturer’s instruc-
tions at http://www.biomol.com/Online_Catalog/Online_Cata-
log/Products/Product_Detail/38/?categoryId=226&produc-
tId=749&mid=75), 270 nM SIRT2 (12.5 μg/50 μL), 1 mM
NAD, ( inhibitor, and adjusted to a 50 μL volume with buffer
A. Buffer A was composed of 25 mM Tris-HCl, pH 8.0, 137
mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. Stop buffer was
composed of 2.7 mg/mL trypsin in buffer A.
(c 1.0, CHCl3, Perkin-Elmer 141); second measurement
[R]25 þ1.0 (c 1.2, CHCl3, JASCO P-2000); UV (CHCl3) λmax
D
202 nm (log ε = 2); IR νmax (neat) 3420, 3402, 2853, 2360, 1716,
1
1248, 1049, 940 cm-1; H NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz), see Table 2; LR FABMS (otg matrix)
obsd m/z (rel int) 591.4 (5) [M þ Na]þ, 569.3 (35) [M þ H]þ,
267.1 (100); LR FABMS (nba matrix) obs m/z (rel int) 591.4 (7)
[M þ Na]þ, 569.3 (14) [M þ H]þ, 551.2 (20) [M - OH]þ, 533.2
(10) [M - 2OH - H]þ, 267.1 (100); LR TOF MS ESþ obsd m/z
(rel int) 607.4 (5) [M þ K]þ, 591.5 (5) [M þ Na]þ, m/z 569.5 (10)
[M þ H]þ, m/z 551.4 (60) [M - OH]þ, and m/z 533.4 (20) [M -
2OH - H]þ; HR FABMS obs [M þ H]þ m/z 569.47804 for
C34H65O6 (0.1 mmu); HR EI MS m/z 551.4691 [M - OH]þ and
533.4567 [M - 2OH - H]þ for C34H63O5 (-0.5 mmu) and
C34H61O4 (2.8 mmu), respectively.
Initial Chiral HPLC Analysis of Tanikolide Dimer 3 and Its
Acid Hydrolysate. Chiral LCMS analyses of 3 employed a
Chirobiotic T (4.6 ꢀ 100 mm, 10 μm) column and yielded a
single peak (tR =3.0 min) using a gradient elution begin-
ning with 70:30 MeOH/H2O and ending in pure MeOH.
Chiral LCMS analyses on the same column of the hydrolysate
of 2 (6 N HCl at 105 ꢀC for 16 h) yielded two peaks (tR =
4.73 and 5.22 min) using an isocratic elution (40:60 H2O/
EtOH).
Secondary SIRT1 and SIRT2 Inhibitory Assays. These sec-
ondary assays for inhibition to the sirtuins and HDACs fol-
lowed the procedures outlined in Heltweg et al.20 except that the
(25) Bourland, T. C.; Carter, R. G.; Yokochi, A. F. T. Org. Biomol.
Chem. 2004, 2, 1315–1329.
(26) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467.
(27) Sheldrick, G. M. In Crystallographic Computing 6; Flack, H. D.,
Parkanyi, L., Simon, K., Eds.; Oxford University Press: Oxford, 1993.
(28) Flack, H. D. Acta Crystallogr. 1983, A39, 876–881.
(29) (a) ORTEP-III: Burnett, M. N.; Johnson, C. K. Report ORNL-
6895, Oak Ridge National Laboratory, Oak Ridge, TN, 1996. (b) ORTEP3
for Windows: Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
(30) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski,
M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R.
Cancer Res. 1988, 48, 589–601.
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Chiral GC-MS Analysis of Natural Tanikolide Dimer 3,
(5R,50S)-Tanikolide Dimer 4, (5R,50R)-Tanikolide Dimer 5, and
(5S,50S)-Tanikolide Dimer ent-5. Natural tanikolide dimer 3 and
the synthetic standards 4, 5, and ent-5 were analyzed by GC-
MS using a Cyclohexil-B chiral column. All of the samples were
analyzed using a ramp from 80 to 240 ꢀC at 7 ꢀC/min. Natural
tanikolide dimer 3 gave a single peak at 35.99 min with the
following mass spectrum (the dimer thermally hydrolyzes in the
GC-injection inlet to its component “monomers”): EIMS m/z
253 (38), 225 (34), 129 (70), 97 (52), 57 (57), 55 (100). Synthetic
(R,S)-tanikolide dimer 4 gave two peaks at 35.99 min [EIMS m/z
253 (52), 225 (25), 129 (68), 97 (42), 57 (69), 54 (100)] and 36.37
min [EIMS m/z 253 (47), 225 (42), 129 (59), 97 (38), 71 (48), 57
(62), 54 (100)], while synthetic (R,R)-tanikolide dimer 5 gave a
single peak at 35.99 min [EIMS m/z 253 (63), 225 (32), 153 (12),
129 (73), 97 (59), 57 (68), 55 (100)], and synthetic (S,S)-taniko-
lide dimer ent-5 gave a single peak at 36.37 min [EIMS m/z 253
(58), 225 (38), 129 (67), 83 (42), 71 (58), 57 (77), 55 (100)].
Formation of the Benzyl Ether of Epoxide 6. NaH (60% in oil,
87 mg, 2.18 mmol) was added to a solution of epoxy alcohol 6 in
THF (10 mL) at 0 ꢀC. After the mixture was stirred 30 min,
benzyl bromide (0.17 mL, 1.42 mmol) and TBAI (80 mg, 0.22
mmol) were added, and the reaction mixture was stirred at room
temperature for 2 h. After dilution with Et2O (10 mL), the
reaction mixture was cooled to 0 ꢀC and treated with satd
NH4Cl solution (10 mL). The aqueous phase was extracted with
Et2O (10 mL ꢀ 2), and the combined organic extracts were
washed with brine (15 mL ꢀ 2), dried over MgSO4, filtered,
and concentrated. The residue was purified by flash column
chromatography (hexanes-EtOAc, 20:1) to give the benzyl
ether of epoxide 6 (328 mg, 95%): Rf 0.51 (hexanes-EtOAc,
heated to 80 ꢀC and stirred for 12 h. The mixture was cooled to
room temperature. After dilution with Et2O (3 mL), the solution
was washed successively with 5% NaOH solution (5 mL) and
brine (5 mL ꢀ 2), dried over Na2SO4, filtered, and concentrated.
The residue was purified by flash column chromatography
(hexanes-EtOAc, 15:1) to give the sulfide derivative of 7 (104
mg, quant): Rf 0.30 (hexanes-EtOAc, 8:1); [R]25D -1.1 (c 1.50,
CHCl3); IR (neat) νmax =3469, 3063, 3030, 2925, 2857, 1950,
1872, 1582, 1459, 1370, 1299, 1204, 1099, 1021, 739, 696 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.10-7.43 (m, 10 H), 4.52 (s, 2
H), 3.29 (s, 2 H), 2.80-3.00 (m, 2 H), 2.18 (s, 1 H), 1.57-1.74 (m,
4 H), 1.38-1.50 (m, 2 H), 1.13-1.35 (m, 18 H), 0.88 (t, J=6.8
Hz, 3 H); 13C NMR (125 MHz, CDCl3) δ 138.4, 136.9, 129.2,
129.1, 128.7, 128.0, 127.9, 126.0, 75.7, 74.0, 73.7, 36.8, 35.8, 34.4,
32.2, 30.5, 29.9, 29.9, 29.8, 29.6, 23.7, 23.5, 23.0, 14.4; MS m/z
(FAB, relative intensity) 456 (Mþ, 3), 439 (13), 347 (3), 331 (6),
239 (5), 225 (15), 123 (10), 91 (100), 55 (8), 43 (9), 29 (2); HRMS
(FAB) calcd for C29H44O2S (Mþ) 456.3062, found 456.3048.
Acrylate 8. Ethylmagnesium bromide (1 M solution in THF,
5.3 mL, 5.3 mmol) was added dropwise to a solution of the
sulfide derivative of 7 (1.2 g, 2.65 mmol) in THF (27 mL) at
room temperature. After being stirred for 20 min, acryloyl
chloride (0.75 mL, 9.3 mmol) was added to this mixture at room
temperature. After being stirred for 2 h, the reaction mixture was
diluted with Et2O (20 mL) and quenched with satd NaHCO3
solution (20 mL). The aqueous phase was extracted with Et2O
(30 mL ꢀ 1), and the combined organic extracts were washed
with brine (30 mL x 2), dried over MgSO4, filtered, and
concentrated. The residue was purified by flash column chro-
matography (hexanes-EtOAc, 10:1) to give acrylate 8 (1.1 g,
79%): Rf 0.27 (hexanes-EtOAc, 8:1); [R]25 -1.6 (c 0.25,
D
8:1); [R]25 þ2.3 (c 0.55, CHCl3); IR (neat) νmax=3034, 2925,
CHCl3); IR (neat) νmax =3030, 2925, 2857, 1945, 1722, 1628,
1583, 1459, 1405, 1285, 1198, 1105, 980, 810, 740, 476 cm-1; 1H
NMR (500 MHz, CDCl3) δ 7.08-7.39 (m, 10 H), 6.28 (d, J=
17.24 Hz, 1 H), 6.02 (dd, J=17.2, 10.3 Hz, 1 H), 5.73 (d, J=10.3
Hz, 1 H), 4.48 (s, 2 H), 3.66 (s, 2 H), 2.89 (t, J = 7.3 Hz, 2 H), 2.05
(m, 2 H), 1.78-1.94 (m, 2 H), 1.55-1.67 (m, 2 H), 1.11-1.37 (m,
18 H), 0.88 (t, J = 6.8 Hz, 3 H); 13C NMR (125 MHz, CDCl3) δ
165.4, 138.5, 136.8, 130.2, 129.9, 129.2, 129.1, 128.6, 127.8,
127.8, 126.0, 86.1, 73.5, 71.2, 34.0, 34.0, 33.3, 32.2, 30.1, 29.9,
29.9, 29.9, 29.8, 29.6, 23.3, 23.2, 22.9, 14.4; MS m/z (FAB,
relative intensity) 510 (Mþ, 4), 439 (83), 403 (6), 347 (9), 331 (29),
239 (16), 199 (4), 136 (76), 123 (55), 91 (100), 55 (87); HRMS
(FAB) calcd for C32H46O3S (Mþ) 510.3168, found 510.3150.
Ester 10. Grubbs’ second-generation catalyst (8 mg, 0.0063
mmol) was added to a solution of homoallylic alcohol 7 (90 mg,
0.25 mmol) and acrylate 8 (195 mg, 0.37 mmol) in CH2Cl2 (4
mL) at room temperature. The reaction mixture was heated
under reflux for 5 h, and the solvent was evaporated. The residue
was purified by flash column chromatography (hexanes-
EtOAc, 8:1) to give ester 10 (164 mg, 77%): Rf 0.5 (hexanes-
EtOAc, 4:1); [R]25D -1.5 (c 1.00, CHCl3); IR (neat) νmax=3471,
2925, 2854, 1719, 1652, 1457, 1381, 1270, 1175, 1101, 739, 699
D
2856, 1459, 1371, 1208, 1103, 1022, 953, 903, 815 cm-1 1H
;
NMR (500 MHz, CDCl3) δ 7.18-7.43 (m, 5 H), 4.58 and 4.53
(ABq, J = 12.0 Hz, 2 H), 3.60 and 3.46 (ABq, J=11.0 Hz, 2 H),
2.70 and 2.64 (ABq, J=4.8 Hz, 2 H), 1.72-1.88 (m, 1 H), 1.47-
1.64 (m, 1 H), 1.14-1.43 (m, 18 H), 0.88 (t, J=6.7 Hz, 3 H); 13
C
NMR (125 MHz, CDCl3) δ 138.3, 128.6, 127.9, 127.9, 73.5, 72.1,
58.9, 50.5, 32.2, 32.2, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 24.9, 22.9,
14.4; MS m/z (FAB, relative intensity) 319 (Mþ þ 1, 13), 289 (2),
227 (11), 181 (17), 154 (22), 137 (24), 107 (39), 91 (100), 55 (30),
43 (32), 29 (9); HRMS (FAB) calcd for C21H35O2 (Mþþ1)
319.2637, found 319.2641.
Homoallylic Alcohol 7. Vinylmagnesium bromide (1 M solu-
tion in THF, 1.5 mL, 1.5 mmol) was added slowly to a suspen-
sion of CuI (44 mg, 0.2 mmol) in THF (6 mL) at -20 ꢀC. After
being stirred for 30 min, a solution of the benzyl ether of epoxide
6 (320 mg, 1.0 mmol) in THF (3 mL) was added, and the reaction
mixture was stirred at -20 ꢀC for 2 h. After dilution with Et2O
(10 mL), the reaction mixture was warmed to 0 ꢀC and treated
with satd NH4Cl solution (10 mL). The aqueous phase was
extracted with Et2O (15 mL ꢀ 2), and the combined organic
extracts were washed with brine (15 mLꢀ2), dried over MgSO4,
filtered, and concentrated. The residue was purified by flash
column chromatography (hexanes-EtOAc, 20:1) to give homo-
allylic alcohol 7 (309 mg, 89%): Rf 0.40 (hexanes-EtOAc, 8:1);
[R]25D þ2.4 (c 2.00, CHCl3); IR (neat) νmax=3457, 3071, 3031,
2925, 2857, 1817, 1639, 1459, 1371, 1208, 1101, 1002, 915, 800,
740 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.23-7.41 (m, 5 H),
5.74-5.87 (m, 1 H), 5.08 (d, J=12.5 Hz, 2 H), 4.54 (s, 2 H), 3.34
and 3.31 (ABq, J=9.0 Hz, 2 H), 2.25-2.38 (m, 2 H), 2.22 (s, 1
H), 1.42-1.52 (m, 2 H), 1.18-1.36 (m, 18 H), 0.88 (t, J=6.8 Hz,
3 H); 13C NMR (125 MHz, CDCl3) δ 138.5, 134.1, 128.6, 127.9,
127.9, 118.4, 75.5, 73.4, 73.7, 41.6, 36.9, 32.2, 30.5, 29.9, 29.9,
29.9, 29.8, 29.6, 23.4, 22.9, 14.4.
cm-1 1H NMR (500 MHz, CDCl3) δ 7.12-7.40 (m, 15 H),
;
6.81-6.92 (m, 1 H), 5.75 (d, J=15.4 Hz, 1 H), 4.54 (s, 2 H), 4.51
(s, 2 H), 3.66 (s, 2 H), 3.32 (s, 2 H), 2.88 (t, J=7.1 Hz, 2 H), 2.13-
2.25 (m, 2 H), 1.80-1.94 (m, 4 H), 1.55-1.69 (m, 4 H), 1.48 (d,
J=4.7 Hz, 2 H), 1.10-1.40 (m, 34 H), 0.88 (t, J=6.5 Hz, 6 H);
13C NMR (125 MHz, CDCl3) δ 165.5, 144.1, 138.5, 138.2, 136.9,
129.2, 129.1, 128.7, 128.6, 128.0, 127.9, 127.8, 127.8, 125.9,
125.4, 85.8, 75.4, 74.0, 73.7, 73.6, 71.4, 40.1, 37.2, 34.1, 34.1,
33.4, 32.2, 30.4, 30.2, 29.9, 29.9, 29.9, 29.9, 29.8, 29.8, 29.6, 29.6,
23.4, 23.4, 23.3, 22.9, 14.4.
Ester 13. Grubbs’ second-generation catalyst (10 mg, 0.0078
mmol) was added to a solution of homoallylic alcohol ent-7 (110
mg, 0.31 mmol) and acrylate 8 (246 mg, 0.46 mmol) in CH2Cl2
(5 mL) at room temperature. The reaction mixture was heated
under reflux for 5 h, and the solvent was evaporated. The residue
Sulfide Derivative of 7. AIBN (7.5 mg, 0.05 mmol) was added
to a solution of homoallylic alcohol 7 (81 mg, 0.23 mmol) in
thiophenol (1.2 mL) at room temperature. The mixture was
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was purified by flash column chromatography (hexanes-
EtOAc, 8:1) to give ester 13 (161 mg, 76%): Rf 0.5 (hexanes-
EtOAc, 4:1); [R]25D þ1.6 (c 0.60, CHCl3); IR (neat) νmax=2925,
relative intensity) 772 (Mþ, 1), 699 (1), 665 (1), 593 (3), 503 (1),
355 (3), 329 (8), 328 (2), 265 (4), 235 (3), 91 (100); HRMS (FAB)
calcd. for C50H76O6 (Mþ) 772.5642, found 772.5646.
2853, 2349, 1713, 1650, 1456, 1272, 1100, 738, 696 cm-1; H
Diene Product of 13. m-CPBA (50 mg, 0.21 mmol) in CH2Cl2
(5 mL) was added dropwise to a solution of 13 (270 mg, 0.33
mmol) in CH2Cl2 (30 mL) at -78 ꢀC. After 10 min, the reaction
mixture was quenched with satd NaHCO3 solution (30 mL) at -
78 ꢀC and warmed to room temperature. The aqueous phase was
extracted with CH2Cl2 (50 mL ꢀ 1), and the combined organic
extracts were washed with brine (50 mL ꢀ 2), dried over MgSO4,
filtered, and concentrated. The crude sulfoxide was dissolved in
freshly prepared toluene (30 mL) and pyridine (30 mL), and then
the mixture was heated under reflux for 7 d. This mixture was
concentrated and flash column chromatography (hexanes-
EtOAc, 15:1) gave the diene product of 13 (220 mg, 94%): Rf
1
NMR (500 MHz, CDCl3) δ 7.13-7.33 (m, 15 H), 6.82-6.87 (m,
1 H), 5.76 (d, J=15.4 Hz, 1 H), 4.52 (s, 2 H), 4.48 (s, 2 H), 3.65 (s,
2 H), 3.30 (s, 2 H), 2.89 (t, J=7.3 Hz, 2 H), 2.40 (d, J=7.3 Hz, 2
H), 2.03-2.06 (m, 2 H), 1.80-1.91 (m, 2 H),1.57-1.61 (m, 2 H),
1.45-1.47 (m, 2 H), 1.18-1.31 (m, 36 H), 0.88 (t, J=7.0 Hz, 6
H); 13C NMR (125 MHz, CDCl3) δ 165.5, 144.1, 138.5, 138.2,
136.9, 129.2, 129.1, 128.7, 128.6, 128.0, 127.9, 127.8, 127.8,
125.9, 125.4, 85.7, 75.4, 74.0, 73.7, 73.5, 71.4, 40.1, 37.2, 34.1,
34.1, 33.4, 32.2, 30.4, 30.2, 29.9, 29.9, 29.9, 29.9, 29.8, 29.8, 29.6,
29.6, 23.4, 23.4, 23.2, 22.9, 14.4.
Diene Product of 10. m-CPBA (50 mg, 0.21 mmol) in CH2Cl2
(5 mL) was added dropwise to a solution of 10 (190 mg, 0.22
mmol) in CH2Cl2 (25 mL) at -78 ꢀC. After 10 min, the reaction
mixture was quenched with satd NaHCO3 solution (20 mL) at -
78 ꢀC and then warmed to room temperature. The aqueous
phase was extracted with CH2Cl2 (30 mL ꢀ 1), and the combined
organic extracts were washed with brine (30 mL ꢀ 2), dried over
MgSO4, filtered, and concentrated. This crude sulfoxide was
dissolved in freshly prepared toluene (13 mL) and pyridine (13
mL), and the solution was heated under reflux for 72 h. This
mixture was concentrated, and the residue was purified by flash
column chromatography (hexanes-EtOAc, 15:1) to give the
diene product of 10 (131 mg, 83%): Rf 0.45 (hexanes-EtOAc,
4:1); [R]25D -4.1 (c 1.00, CHCl3); IR (neat) νmax=3460, 3067,
3031, 2925, 2854, 2100, 1714, 1648, 1495, 1455, 1271, 1102, 735,
0.45 (hexanes-EtOAc, 4:1); [R]25 þ3.0 (c 0.35, CHCl3); IR
D
(neat) νmax=3469, 2925, 2854, 1715, 1650, 1457, 1270, 1102, 991,
917, 737, 698 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.26-7.35
(m, 10 H), 6.83-6.88 (m, 1 H), 5.79 (d, J = 16.2 Hz, 1 H), 5.71
(dd, J=20.7, 9.9 Hz, 1 H), 5.04-5.09 (m, 2 H), 4.53 (s, 4 H), 3.71
and 3.65 (ABq, J=9.7 Hz, 2 H), 3.31 (s, 2 H), 2.73 and 2.66
(ABX, JAB=16.8, JAX=7.4, JBX=7.5 Hz, 2 H), 2.41 (d, J=7.7
Hz, 2 H), 1.88-1.90 (m, 2 H), 1.45-1.48 (m, 2 H), 1.25-1.31 (m,
36 H), 0.88 (t, J=6.8 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ
165.5, 144.0, 138.6, 138.2, 133.2, 128.7, 128.6, 128.1, 127.9,
127.8, 127.8, 125.5, 118.7, 85.4, 75.5, 74.1, 73.8, 73.6, 71.3,
40.1, 38.7, 37.2, 33.8, 32.2, 30.4, 30.2, 29.9, 29.9, 29.9, 29.9,
29.9, 29.8, 29.8, 29.6, 23.4, 23.2, 22.9, 14.4; MS m/z (FAB,
relative intensity) 719 (Mþ þ 1, 1), 611 (1), 519 (1), 463 (1), 373
(2), 329 (16), 265 (2), 221 (5), 181 (12), 131 (7), 91 (100); HRMS
(FAB) calcd for C47H75O5 (Mþþ1) 719.5615, found 719.5613.
Triene 14. Acryloyl chloride (0.1 mL, 2.85 mmol) was added
to a solution of the diene product of 13 (40 mg, 0.057 mmol) and
DIPEA (0.9 mL) in CH2Cl2 (0.9 mL) at 0 ꢀC. Acryloyl chloride
(0.04 mL) was added two more times every 2 h at 0 ꢀC. After 6 h,
the reaction mixture was quenched with satd NH4Cl solution (2
mL). The aqueous phase was extracted with CH2Cl2 (5 mL ꢀ 1),
and the combined organic extracts were washed with brine
(3 mLꢀ2), dried over MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography (hex-
anes-EtOAc, 10:1) to give triene 14 (28 mg, 62%): Rf 0.5
(hexanes-EtOAc, 4:1); [R]25D þ0.98 (c 1.00, CHCl3); IR (neat)
1
cm-1; H NMR (500 MHz, CDCl3) δ 7.21-7.41 (m, 10 H),
6.80-6.92 (m, 1 H), 5.79 (d, J = 15.8 Hz, 1 H), 5.71 (dd, J=17.1,
9.7 Hz, 1 H), 4.98-5.15 (m, 2 H), 4.51 (s, 4 H), 3.71 and 3.65
(ABq, J = 9.6 Hz, 2 H), 3.31 (s, 2 H), 2.73 and 2.65 (ABX, JAB=
13.9, JAX = 7.3, JBX = 7.3 Hz, 2 H), 2.41 (d, J = 7.3 Hz, 2 H),
1.83-1.95 (m, 2 H), 1.42-1.53 (m, 2 H), 1.25 (m, 36 H), 0.88 (t,
J=6.8 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ 165.5, 144.0,
138.5, 138.2, 133.2, 128.7, 128.5, 128.0, 127.9, 127.8, 127.8,
125.5, 118.7, 85.4, 75.4, 74.0, 73.7, 73.5, 71.3, 40.1, 38.6, 37.2,
33.8, 32.2, 30.4, 30.2, 29.9, 29.9, 29.9, 29.9, 29.9, 29.9, 29.8, 29.8,
29.6, 29.6, 23.4, 23.2, 22.9, 14.4; MS m/z (FAB, relative inten-
sity) 718 (Mþ, 2), 644 (2), 643 (1), 548 (3), 458 (5), 284 (3), 258 (8),
156 (2), 121 (3), 91 (100), 55 (87); HRMS (FAB) calcd for
C47H74O5 (Mþ) 718.5536, found 718.5532.
ν
max = 3066, 3032, 2925, 2854, 1722, 1653, 1457, 1402, 1273,
1
Triene 11. Acryloyl chloride (0.1 mL, 2.85 mmol) was added
to a solution of the diene product of 10 (205 mg, 0.285 mmol) in
CH2Cl2 (2.8 mL) and DIPEA (2.8 mL) at 0 ꢀC. Acryloyl chloride
(0.1 mL) was added two more times every 2 h at 0 ꢀC. After 6 h,
the reaction mixture was quenched with satd NH4Cl solution
(5 mL). The aqueous phase was extracted with CH2Cl2 (5 mL ꢀ
1), and the combined organic extracts were washed with brine
(5 mL ꢀ 2), dried over MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography (hex-
anes-EtOAc, 10:1) to give triene 11 (70 mg, 51%): Rf 0.5
(hexanes-EtOAc, 4:1); [R]25D -2.73 (c 0.25, CHCl3); IR (neat)
1197, 1114, 985, 917, 809, 738, 698, 609 cm-1; H NMR (500
MHz, CDCl3) δ 7.26-7.34 (m, 10 H), 6.77-6.83 (m, 1 H), 6.32
and 6.28 (ABq, J=1.4 Hz, 1 H), 6.02 (dd, J=17.2, 10.3 Hz, 1 H),
5.81 (d, J = 15.4 Hz, 1 H), 5.75 and 5.73 (ABq, J=1.5 Hz, 1 H),
5.65-5.71 (m, 1 H), 5.03-5.08 (m, 2 H), 4.51 (s, 4 H), 3.70 (d,
J=10.0 Hz, 2 H), 3.63 (dd, J=11.2, 2.5 Hz, 2 H), 2.86 and 2.80
(ABX, JAB=17.5, JAX=7.1, JBX=7.7 Hz, 2 H), 2.72 and 2.64
(ABX, JAB=16.8, JAX=7.3, JBX=7.5 Hz, 2 H), 1.86-1.95 (m, 4
H), 1.10-1.37 (m, 36 H), 0.88 (t, J=6.9 Hz, 6 H); 13C NMR (125
MHz, CDCl3) δ 165.5, 165.3, 142.9, 138.6, 138.3, 133.2, 130.5,
129.7, 128.6, 128.6, 127.9, 127.9, 126.0, 118.7, 85.5, 85.3, 73.6,
73.6, 71.3, 71.2, 38.6, 37.2, 34.1, 33.8, 32.2, 30.2, 30.1, 29.9, 29.9,
29.8, 29.8, 29.6, 29.6, 23.2, 23.2, 23.0, 14.4; MS m/z (FAB,
relative intensity) 773 (Mþ þ 1, 1), 699 (1), 665 (1), 593 (1), 463
(1), 373 (1), 329 (5), 265 (4), 219 (2), 154 (10), 136 (11), 91 (100);
HRMS (FAB) calcd for C50H77O6 (Mþ þ 1) 773.5720, found
773.5706.
νmax = 3460, 3067, 3031, 2925, 2854, 2100, 1714, 1648, 1495,
1
1455, 1271, 1102, 735 cm-1; H NMR (500 MHz, CDCl3) δ
7.21-7.37 (m, 10 H), 6.74-6.85 (m, 1 H), 6.31 and 6.28 (ABq,
J = 1.5 Hz, 1 H), 6.02 (dd, J = 17.2, 10.3 Hz, 1 H), 5.81 (d, J =
15.4 Hz, 1 H), 5.75 and 5.72 (ABq, J = 1.4 Hz, 1 H), 5.65-5.72
(m, 1 H), 4.97-5.12 (m, 2 H), 4.51 (s, 4 H), 3.71 and 3.65 (ABq,
J=9.6 Hz, 4 H), 2.86 and 2.80 (ABX, JAB = 14.2, JAX=7.5,
Diene 12. Hoveyda-Grubbs’ second-generation catalyst (8
mg, 0.00625 mmol) was added to a solution of triene 11 (48 mg,
0.062 mmol) in benzene (6 mL) at room temperature. The
reaction mixture was warmed to 70 ꢀC. After 2 h, the solvent
was evaporated. The residue was purified by flash column
chromatography (hexanes-EtOAc, 8:1) to give diene 12 (36
JBX=7.7 Hz, 2 H), 2.72 and 2.64 (ABX, JAB = 13.8, JAX=7.2,
JBX=7.5 Hz, 2 H), 1.82-2.00 (m, 4 H), 1.10-1.37 (m, 36 H),
0.88 (t, J=6.9 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ 165.5,
165.3, 142.9, 138.6, 138.3, 133.2, 130.5, 129.7, 128.6, 128.5,
127.9, 127.8, 127.8, 126.0, 118.7, 85.4, 85.3, 73.6, 73.5, 71.3,
71.2, 38.6, 37.2, 34.1, 33.8, 32.2, 30.6, 30.2, 30.0, 29.9, 29.9, 29.9,
29.8, 29.7, 29.6, 29.6, 23.2, 23.2, 22.9, 14.4; MS m/z (FAB,
mg, 77%): Rf 0.5 (hexanes-EtOAc, 4:1); [R]25 -0.45 (c 1.15,
D
CHCl3); IR (neat) νmax =3031, 2924, 2854, 1723, 1458, 1383,
5274 J. Org. Chem. Vol. 74, No. 15, 2009

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Gutierrez et al.
JOCArticle
1252, 1113, 1031, 957, 812, 739, 699 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.26-7.36 (m, 10 H), 6.70-6.74 (m, 2 H), 5.97 (td, J=
7.2, 4.0 Hz, 2 H), 4.54 (d, J=2.6 Hz, 4 H), 3.58 and 3.46 (ABq,
J=9.7 Hz, 4 H), 2.35-2.70 (m, 4 H), 1.68-1.81 (m, 4 H), 1.22-
1.32 (m, 36 H), 0.88 (t, J=9.9 Hz, 6 H); 13C NMR (125 MHz,
CDCl3) δ 163.6, 143.8, 138.1, 128.7, 128.0, 127.9, 120.8, 83.5,
73.8, 72.9, 37.2, 32.1, 30.1, 29.9, 29.8, 29.8, 29.7, 29.6, 23.4, 22.9,
14.3; MS m/z (FAB, relative intensity) 745 (Mþ þ 1, 3), 655 (1),
598 (1), 508 (1), 463 (1), 395 (2), 373 (19), 307 (2), 289 (1), 281 (2),
263 (2), 181 (2), 154 (14), 137 (12), 91 (100); HRMS (FAB) calcd
for C48H73O6 (Mþ þ 1) 745.5407, found 745.5422.
546 (1), 508 (1), 463 (1), 395 (6), 373 (13), 355 (2), 281 (2), 265 (7),
251 (5), 181 (3), 154 (5), 136 (5), 91 (100); HRMS (FAB) calcd for
C48H73O6 (Mþ þ 1) 745.5407, found 745.5412.
Diolide 4. Pd/C was added to a solution of the initial olefin
metathesis product of 14 (19 mg, 0.026 mmol) in hexane (2 mL)
under H2. After 3 h, the mixture was filtered and concentrated to
give diolide 4 (14 mg, quant): Rf 0.3 (hexanes-EtOAc, 1:1);
[R]25D þ0.00 (c 0.90, CHCl3); IR (neat) νmax=3444, 2923, 2850,
1
1716, 1467, 1385, 1252, 1173, 1103, 1051, 928, 723 cm-1; H
NMR (500 MHz, CDCl3) δ 3.66 and 3.55 (ABX, JAB =14.2,
JAX=6.6, JBX=6.6 Hz, 4 H), 2.47-2.50 (m, 4 H), 1.87-1.92 (m,
6 H), 1.71-1.79 (m, 4 H), 1.59 - 1.65 (m, 2 H), 1.12-1.42 (m, 36
H), 0.88 (t, J=7.0 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ
171.9, 86.7, 67.8, 36.9, 32.1, 30.2, 30.0, 29.8, 29.8, 29.8, 29.7,
29.6, 26.8, 23.7, 22.9, 16.9, 14.3; MS m/z (FAB, relative inten-
sity) 569 (Mþ þ 1, 25), 537 (1), 421 (2), 391 (2), 341 (1), 285 (48),
267 (100), 253 (12), 225 (8), 154 (51), 137 (64); HRMS (FAB)
calcd for C34H65O6 (Mþ þ 1) 569.4781, found 569.4766.
Diolide 5. Pd/C was added to a solution of diene 12 (46 mg,
0.062 mmol) in hexane (2 mL) under H2. After 3 h, the mixture
was filtered and concentrated to give diolide 5 (34 mg, quant): Rf
0.3 (hexanes-EtOAc, 1:1); [R]25 þ2.9 (c 0.25, CHCl3); IR
D
(neat) νmax =3384, 2925, 2855, 1734, 1660, 1586, 1496, 1459,
1382, 1243, 1088, 1052, 755, 722, 700 cm-1; 1H NMR (500 MHz,
CDCl3) δ 3.66 and 3.56 (ABX, JAB=14.3, JAX=6.8, JBX=6.8
Hz, 4 H), 2.47-2.51 (m, 4 H), 1.84-1.94 (m, 6 H), 1.71-1.77 (m,
4 H), 1.59-1.65 (m, 2 H), 1.12-1.42 (m, 36 H), 0.88 (t, J=7.0
Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ 171.9, 86.8, 67.8, 36.8,
32.2, 30.2, 30.0, 29.9, 29.8, 29.8, 29.7, 29.6, 26.8, 23.7, 22.9, 16.9,
14.4; MS m/z (FAB, relative intensity) 591 (Mþ þ Na, 7), 551
(18), 533 (10), 391 (5), 339 (3), 267 (85), 154 (100), 136 (78), 91
(56), 89 (51); HRMS (FAB) calcd for C34H64O6 (Mþ þ Na)
591.4601, found 591.4604.
Acknowledgment. We thank the Malagasy government
for permission to make sample collections. We also thank
the Chemistry NMR Facilities at OSU and J. Morre and the
EHS center at OSU for mass spectral data acquisition.
The National Institute of Health (CA 52955 and NS
053398) and the Fulbright program (E.H.A.) provided fi-
nancial support. This research was also supported by a
grant from the Marine Biotechnology Program funded by
Ministry of Land, Transport and Maritime Affairs, Republic
of Korea. We thank M. Johnson and T. Suyama for help
with construction of the manuscript and the Supporting
Information.
Initial olefin metathesis product of triene 14: Hoveyda-
Grubbs’ second-generation catalyst (6 mg, 0.0048 mmol) was
added to a solution of triene 14 (37 mg, 0.048 mmol) in benzene
(5 mL) at room temperature. The reaction mixture was warmed
to 70 ꢀC. After 2 h, the solvent was evaporated. The residue was
purified by flash column chromatography (hexanes-EtOAc,
8:1) to give the diene product from metathesis of 14 (34 mg,
96%): Rf 0.5 (hexanes-EtOAc, 4:1); [R]D þ0.16 (c 2.00, CHCl3);
IR (neat) νmax=3031, 2925, 2854, 1723, 1458, 1383, 1252, 1113,
1031, 957, 812, 739, 699 cm-1; 1H NMR (500 MHz, CDCl3) δ
7.26-7.36 (m, 10 H), 6.70-6.74 (m, 2 H), 5.97 (td, J=7.2, 4.0
Hz, 2 H), 4.54 (d, J=2.6 Hz, 4 H), 3.57 and 3.46 (ABq, J=9.7 Hz,
4 H), 2.35-2.70 (m, 4 H), 1.68-1.81 (m, 4 H), 1.22-1.32 (m, 36
H), 0.88 (t, J=7.0 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ
163.6, 143.8, 138.0, 128.6, 128.0, 127.9, 120.7, 83.4, 73.8, 72.8,
37.2, 32.1, 30.1, 29.8, 29.8, 29.8, 29.7, 29.6, 23.3, 22.9, 14.3; MS
m/z (FAB, relative intensity) 745 (Mþ þ 1, 2), 668 (1), 626 (1),
Supporting Information Available: 1H NMR, 13C NMR,
and X-ray crystal structure data including ORTEP drawing and
CIF for compound 2 and 1H NMR, 13C NMR, 1H-1H COSY,
HSQC, HMBC, and mass spectrometric data for compound 3;
1H and 13C NMR spectra for synthetic intermediates 6-14 and
products 4 and 5; chiral GC-MS analysis of compounds 3-5
and ent-5. Inhibition dose-response curves for compounds 4, 5,
and ent-5 to SIRT1 and SIRT2. This material is available free of
charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 15, 2009 5275