Total synthesis of 27-hydroxy-bullatacin and its C-15 epimer, and studies on their inhibitory effect on bovine heart mitochondrial complex I functions

Article abstract of DOI:10.1016/j.tet.2007.11.089

The total synthesis of 27-hydroxybullatacin and its C-15 epimer has been achieved using rhenium(VII) oxides-mediated and Co(modp)₂-catalyzed oxidative cyclization (OC), diastereoselective alkynylation, Brown's enantioselective allylboration, an

Full text of DOI:10.1016/j.tet.2007.11.089

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 1603e1611
www.elsevier.com/locate/tet
Total synthesis of 27-hydroxy-bullatacin and its C-15 epimer,
and studies on their inhibitory effect on bovine heart
mitochondrial complex I functions
*
Zhiyong Chen, Subhash C. Sinha
The Skaggs Institute for Chemical Biology and the Department of Molecular Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 12 September 2007; received in revised form 15 November 2007; accepted 15 November 2007
Available online 21 December 2007
Abstract
The total synthesis of 27-hydroxybullatacin and its C-15 epimer has been achieved using rhenium(VII) oxides-mediated and Co(modp)2-
catalyzed oxidative cyclization (OC), diastereoselective alkynylation, Brown’s enantioselective allylboration, and Grubbs’ cross metathesis
as the key reactions. The inhibitory effect of these compounds on the complex I function, as determined by using bovine submitochondrial
particles, was in low nanomolar range.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
can be considered potential drug candidates for the treatment
of cancers. Our interest in such a compound is further aug-
mented because of our ongoing program on the development
of ‘smart cytotoxins’ for selective cancer therapy. Presumably,
a smart acetogenin would target cancer cells preferentially
over normal cells. Thus, to obtain the target molecule for
the biological studies and to validate the proposed structure,
we embarked on the total synthesis of 1a.
Previously, we prepared a number of adjacent bis-THF ace-
togenins, including asimicin, bullatacin, squamotacin, triloba-
cin, trilobin, and uvaricin using a combination of modular,
convergent and/or bidirectional approaches.5,6 While most
methods were highly stereoselective, and the target com-
pounds and selected asimicin-type non-natural bis-THF aceto-
genins were obtained with very high diastereoselectivities,
they often required many synthetic steps. Therefore, we sought
to develop highly versatile methods that might not only rapidly
afford the naturally occurring asimicin-type natural bis-THF
acetogenins, but also their analogs. Here, we describe the ini-
tial findings of our study toward the stated goal, including
a successful synthesis of 27-hydroxybullatacin 1a and its
C-15 epimer, 1b, and validation of the proposed structure
for the natural product. The key reactions used in the synthesis
27-Hydroxybullatacin, 1a,1 is an adjacent asimicin-type
bis-THF acetogenin, isolated from the leaves extract of
Annona glabra L., family Annonaceae.2,3 The absolute struc-
ture of 1a was determined on the basis of detailed NMR spec-
troscopy studies of the parent molecule and its Mosher’s
esters. Evident from its nomenclature, 27-hydroxy-bullatacin
shares a framework identical to bullatacin, 2,4 possessing the
threo-trans-threo-trans-erythro bis-THF system, though the
structure of the former remains to be confirmed. Both bullata-
cin and 27-hydroxybullatacin are extremely potent anticancer
compounds with reported activities of approximately 105
times higher than adriamycin against several cancer cell lines,
including human prostate adenocarcinoma (PC-3) and pancre-
atic carcinoma (PACA-2). 27-Hydroxybullatacin is also very
potent against the human kidney carcinoma (A-498) cell
line. Therefore, annonaceous acetogenins and an asimicin-
type bis-THF acetogenin (such as 1a and 2) in particular,
* Corresponding author. Tel.: þ1 858 784 8512; fax: þ1 858 784 8732.
E-mail address: subhash@scripps.edu (S.C. Sinha).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.089

1604
Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
of 1a and 1b included the rhenium(VII) oxides-mediated
trans oxidative cyclization (OC),7 the Co(modp)2-catalyzed
Mukaiyama’s trans-OC,8 Ti-alkyne mediated diastereoselec-
tive alkynylation,6b the Brown’s allylboration,9 and Grubbs’
cross metathesis10 reactions. We also provide a report on the
effect of 1a and 1b on the bovine mitochondrial enzyme func-
tions as compared to 2.
stereoisomeric bis-THF acetogenins can be prepared by an
asymmetric alkylation (alkynylation or allylation) of a single
bis-THF aldehyde V. In other words, only 16 stereoisomeric
V’s will be required to prepare 64 stereoisomeric asimicins
or their alkene precursors, I’s. In contrast, our previous
methods required a minimum of 36 bis-THF lactones to pro-
duce 64 stereoisomeric bis-THF alkene precursors analogous
to I’s.12 Furthermore, the cross metathesis approach eliminates
any need of additional reactions on alkenes, such as the Wittig
salt formation or an iodoalkene for the Pd coupling reaction.5
The synthesis of 1a and 1b started with the readily avail-
able deca-1,9-dien-5-yne, 3. The diene 3 was mono-dihy-
droxylated with AD-b giving the corresponding monodiol in
67% yield and 86% ee. The alkyne function in the diol was
reduced to trans-alkene using Na/NH3, and the primary alco-
hol was selectively protected as benzoate ester giving com-
pound 4. Compound 4 was oxidatively cyclized using
TFAOReO3 affording the trans-monoTHF compound 5. The
latter product was then oxidatively cyclized using Co(modp)2-
catalyzed Mukaiyama’s OC reaction affording the bifunctional
intermediate 6. An attempted second oxidative cyclization of
5 using TFAOReO3 afforded a bis-THF compound that was
different from 6, and later identified as the corresponding cis
analog. For the stereoselective addition of the alkyl and
butenyl side chains on the two sides of 6, we decided to use
diastereoselective and enantioselective processes, respectively.
Thus, compound 6 was oxidized to aldehyde 7 using a Swern
oxidation, and reacted with the readily available alkyne 814
erythro
trans
threo
threo
trans
HO
OH
15
HO
4
O
O
HO
O
O
C7H15
1a: 27-Hydroxybullatacin (15R)
1b: 15-epi-27-Hydroxybullatacin (15S)
erythro
threo
trans
threo
OH
trans
HO
HO
4
O
O
C10H21
2: Bullatacin
O
O
2. Results and discussion
Scheme 1 describes our retrosynthesis of 27-hydroxy-
bullatacin, 1a, involving a cross metathesis of alkene I with
II to produce an alkene precursor of compound 1. Whereas,
compound II could be prepared analogous to one described
earlier for its homologs,11,12 compound I could be prepared
by asymmetric allylation of aldehyde III. The latter product
should be prepared by an asymmetric alkynylation of the pre-
viously reported bifunctional aldehyde V.6b,13 Alternatively,
we could develop a modified process to prepare aldehyde V
starting with dieneyne, 3. In a similar manner, compound 1b
could be prepared from III via Ib. The salient features of
the current approach include the versatility and a compara-
tively less number of steps to produce the bis-THF fragments,
Is’, and quick assembly of the I’s to the butenolide fragment
II by the Grubbs’ cross metathesis reaction. Thus, four
using BuLi and ClTi(O-iPr)3 affording the alkynylated
6b
product with 9:1 diastereoselectivity, in favor of the desired
erythro configuration. The major product was separated out
from the undesired diastereomer, and the hydroxy function
in the former was protected as a TBS ether giving compound
9. The latter product was hydrogenated using 10% PdeC cata-
lyst under hydrogen atmosphere (balloon) and the benzoate
group was removed by basic hydrolysis affording alcohol
10. The latter was oxidized and the resulting aldehyde 11
was allylated using (þ)-Ipc2B-allyl yielding alcohols 12a
and 12b in 83% combined yield and 4:1 diastereoselectivity.
Using (ꢀ)-Ipc2B-allyl, aldehyde 11 was converted to 12a
and 12b in 73% combined yield and 1:10 diastereoselectivity.
Pure products 12a and 12b were obtained by flash
chromatography.
HO
OH
15
HO
4
O
O
HO
O
1a (15R)
1b (15S)
O
C7H15
Separately, alkene 13 was synthesized from butyrolactone
15 and iodide 16, as shown in Scheme 2B, using the process
described earlier for similar compounds.11 Iodide 16 was
prepared from 1,10-undecadiene in four steps, including a
Sharpless asymmetric dihydroxylation of the diene with
AD-b to afford (R)-1,2-dihydroxyundec-10-ene, monotosyla-
tion at the primary alcohol, TBS protection of the secondary
alcohol, and substitution of the tosylate function with an
iodide. Alkylation of 15 with iodide 16 was achieved using
LDAeHMPA, and the reaction product 17 was oxidized to
its sulfoxide and then heated in toluene giving 13. The latter
compound was then cross-metathesized with compounds 12a
and 12b in the presence of Grubbs’ catalyst (RuCl2[imid-
H2-Mes2][CHPh]PCy3), and the resulting products 14a and
RO
OH
15
RO
4
+
O
O
RO
II
O
O
Ia (15R)
Ib (15S)
C7H15
RO
C7H15
O
O
RO
OR
IV
O
O
O
O
RO
III
V
C7H15
Scheme 1. Retrosynthesis of 27-hydroxybullatacin, 1a, and its 15-epimer, 1b.

Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
1605
1. AD- , tert-BuOH-H2O
A
TFAOReO3, TFAA, CH2Cl2,
0 °C, then Aq. NaHCO3, H2O2
2. Na, NH3, THF
3. BzCl, Et3N, CH2Cl2
55%
80%
HO
O
HO
OBz
OBz
3
5
4
Oxalyl chloride, DMSO,
CH2Cl2, -78 °C, then iPr2EtN
1. BuLi, Ti(OiPr)3Cl, Et2O, -78 °C
2. TBSOTf, 2,6-lutidine, CH2Cl2, 0 ºC
Co(modp)2, TBHP,
iPrOH, RT
TBSO
67%
90%
O
O
O
O
HO
OBz
O
OBz
6
7
C7H15
8
1.H2, Pd/C, EtOAc, RT
2. Aq. LiOH, MeOH, THF, RT
Oxalyl chloride, DMSO,
CH2Cl2, -78 °C, then iPr2EtN
TBSO
24
TBSO
24
O
O
O
O
O
67%
OH
OBz
TBSO
9, 75%
10, 75%
TBSO
C7H15
C7H15
TBSO
TBSO
(+)- or (-)-Ipc2B-allyl, Et2O, -78 °C
Grubbs' catalyst, CH2Cl2, RT
CHO
73-83%
TBSO
O
O
O
OH
6
TBSO
TBSO
11
O
12a (R)
12b (S)
O
C7H15
TBSO
C7H15
13
OH
15
TBSO
1. TsNHNH2, AcONa, DME, H2O, reflux
2. 5%HF, CH2Cl2, CH3CN, 0 °C
O
O
1a, 1b
84-92%
TBSO
O
O
14a (15R), 66%
14b (15S), 58%
C7H15
1. m-CPBA, CH2Cl2, 0 °C
2. toluene, 90 °C
B
PhS
O
SPh
LDA, THF, HMPA, -78 °C to RT
13
6
90%
I
O
15
6
TBSO
O
TBSO
O
16
17 (51%)
Scheme 2. Stereoselective synthesis of 27-hydroxybullatacin, 1a, and 15-epi-27-hydroxybullatacin, 1b.
14b were hydrogenated and deprotected to yield 1a and 1b.15
The 1H and 13C NMR spectral data of synthetic compound 1a
was identical to the reported data for the natural 27-
hydroxybullatacin.
heart submitochondrial particles (SMPs) in the presence and
absence of these compounds, as described in the literature.17
Briefly, compounds at various concentrations were added to
15 mg of SMPs/ml containing 0.25 M sucrose, 1 mM MgCl2,
and 50 mM phosphate buffer (pH 7.5) and mixed. After
5 min equilibration of SMPs with inhibitors, the reaction
was started using 150 mM NADH, and progress of reaction
was determined spectrometrically using a UV instrument at
340 nm. Under these conditions, all three compounds showed
comparable activities in the low nanomolar range. Thus, the
IC50 value for bullatacin was determined to be 3.3 nM, and
those for 27-hydroxybullatacin and its C-15 epimer were re-
corded as 10 and 5 nM, respectively. Obviously, hydroxylation
of bullatacin at C-27 caused three-fold reduction in its binding
to the mitochondrial enzymes, and that was partially compen-
sated when C-15 was epimerized in 15-epi-27-
hydroxybullatacin.
2.1. Effect of 27-hydroxybullatacin and its C-15 epimer
on the mitochondrial enzymes
Annonaceous acetogenins are among the most potent inhib-
itors of the mitochondrial enzymes, in particular complex I.16
Though the exact mechanism of action still remains elusive,
studies using the photoreactive acetogenins and competition
assays with a variety of complex I inhibitors, including pieri-
cidin A and rotenone, have revealed that they share the com-
mon binding domain and that ND1 subunit constructs the
inhibitor binding site.17 Because both bullatacin and 27-
hydroxybullatacin are known to possess comparable cytotoxic-
ity against several human cancer cell lines, we decided to
investigate their effect on the mitochondrial enzyme functions
and compare. The mitochondrial activity of 1a, 1b, and 2 was
determined by measuring the catalytic activity of the bovine
In conclusion, the synthesis of 27-hydroxybullatacin and its
C-15 epimer has been achieved using a bidirectional approach
in 15 linear steps (approximately 5.6% yield) with compound
3 and 27 total number of steps from the commercially

1606
Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
available materials. The key steps used in the synthesis of the
target compounds included rhenium(VII) oxides-mediated and
the Co(modp)2-catalyzed OC reactions to form the THF rings,
Ti-alkyne mediated diastereoselective alkynylation, the asym-
metric allylboration, and Grubbs’ cross metathesis reactions.
Using bovine submitochondrial particles, both 27-hydroxybul-
latacin and its 15-epimer were shown to discern low nanomo-
lar inhibitory effect, comparable to bullatacin, on complex I
functions.
m), 3.63 (1H, m), 3.45 (1H, m), 2.89 (2H, m), 2.85 (1H, m),
2.56 (1H, m), 2.27 (4H, m), 1.60 (2H, m).
Freshly cut pieces of Na metal (900 mg) were added in por-
tions to liquid NH3 (30 ml) in a round bottom flask at low
temperature (ꢀ78 ꢁC). After 10 min, a solution of the above-
described diol (1.1 g, 6.5 mmol) in dry THF (10 ml) was
added, and the mixture was stirred at this temperature for
4 h. The reaction mixture was quenched using solid NH4Cl
(2 g) and the cooling bath was removed. After ammonia has
evaporated, the mixture was diluted with water and extracted
using EtOAc. The organic layer was washed with brine, dried
over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified over silica gel (using
CH2Cl2eCH3OH) to yield the corresponding trans olefin
product, (9R)-dec-1,5-diene-9,10-diol (1.1 g, 100% yield).
1H NMR: d 5.83 (1H, m), 5.47 (2H, m), 4.98 (2H, m), 3.73
(1H, m), 3.64 (1H, m), 3.44 (1H, m), 2.21 (8H, m), 1.50
(2H, m); 13C NMR: d 138.32, 130.48, 129.84, 114.62, 71.80,
66.74, 33.68, 32.81, 31.89, 28.63.
3. Experimental section
3.1. General procedures
All reactions were carried out under an inert argon atmo-
sphere with dry solvents under anhydrous conditions unless
otherwise stated. Tetrahydrofuran (THF) was distilled over
potassium, diethyl ether over sodium and benzophenone, tolu-
ene over lithium aluminum hydride, and dichloromethane and
triethyl amine over calcium hydride. Other solvents and re-
agents were purchased at the highest commercial quality and
used without further purification, unless otherwise stated. Re-
actions were monitored by thin layer chromatography (TLC)
carried out on 0.25 mm E. Merck silica gel plates (60F-254)
using UV light (lmax¼254) for visualization and an acidic
mixture of p-anisaldehyde, phosphomolybdic acid, ceric am-
monium molybdate or basic aqueous potassium permanganate
(KMnO4) and heat as the developing agents. E. Merck silica
gel (60, particle size 0.043e0.063 mm) was used for flash col-
umn chromatography. NMR spectra were recorded on Bruker
DRX-500 or Varian Inova-400 instruments and calibrated
using residual undeuterated solvent as an internal reference.
The following abbreviations (or combinations thereof) were
used to explain the multiplicities: s¼singlet, d¼doublet,
t¼triplet, q¼quartet, m¼multiplet, br¼broad. Low resolution
mass spectra were recorded on Micromat-LCT. High-resolu-
tion mass spectra (HRMS) were recorded on Agilent LC/
MSD TOF time-of-flight mass spectrometer by electrospray
ionization time-of-flight reflectron experiments.
Benzoyl chloride (0.37 ml, 3.1 mmol) was added dropwise
to a solution of the above-described diol product (450 mg,
2.6 mmol) and Et3N (0.56 ml, 4 mmol) in CH2Cl2 (20 ml).
The solution was stirred overnight at 0 ꢁC, then diluted with
CH2Cl2, washed sequentially with saturated NaHCO3 solution
and brine, and evaporated. The residue was purified by flash
chromatography (using hexaneeEtOAc) to afford the mono
1
protected compound 4 (600 mg, 82%). H NMR: d 8.06 (2H,
d, J¼8.0 Hz), 7.56 (1H, t, J¼7.0 Hz), 7.44 (2H, t, J¼7 Hz), 5.81
(1H, m), 5.47 (2H, m), 4.97 (2H, m), 4.37 (1H, m), 4.23 (1H,
m), 3.99 (1H, m), 2.19 (6H, m), 1.62 (2H, m); 13C NMR: d
166.70, 138.31, 133.12, 133.12, 130.61, 129.82, 129.61,
129.59, 128.37, 114.58, 69.49, 69.04, 33.64, 33.09, 31.87,
28.45.
3.3. ((2R,5R)-5-((R)-1-Hydroxypent-4-enyl)tetrahydro-
furan-2-yl)methyl benzoate 5
TFAOReO3 was prepared in situ as described in literature.18
In a general process, Re2O7 (484 mg, 1 mmol) was transferred
into a flask, and THF (10 ml) and TFAA (170 ml, 1.2 mmol)
were added sequentially. The mixture was stirred at room tem-
perature for 1 h. The flask was cooled using an ice cold bath
and solvents were removed in vacuo. The residue was washed
twice using cold pentane and used directly for the OC reac-
tion. Cold CH2Cl2 (10 ml) was added to the above prepared
TFAOReO3 and the mixture was cooled to 0 ꢁC. A cold solu-
tion of TFAA (170 ml, 1.2 mmol) and compound 4 (140 mg,
0.5 mmol) in CH2Cl2 (5 ml) was added dropwise sequentially.
After the reaction was stirred at 0 ꢁC to room temperature for
4 h, the reaction mixture was worked up using a saturated
solution of NaHCO3 (5 ml) and 30% H2O2 (0.5 ml), then
extraction with EtOAc. The organic layer was washed with
saturated NaHSO3 solution followed by brine, dried over
Na2SO4, and solvents were removed. The resulting residue
was purified over silica gel using hexaneseEtOAc as the elut-
ing solvents affording the corresponding trans-mono OC prod-
uct, 5. 1H NMR: d 7.98 (2H, d, J¼7.0 Hz), 7.54 (1H, t,
3.2. (R,E)-2-Hydroxydeca-5,9-dienyl benzoate 4
Compound 3 (20 g, 150 mmol) was added to the well-
stirred solution of potassium carbonate (61.5 g, 450 mmol),
potassium ferricyanide (147 g, 45 mmol), DHQD(PHAL)
(1.17 g, 1.5 mmol), and K2OsO4 (111 mg, 0.3 mmol) in 500 ml
H2O and 1000 ml tert-butanol at 0 ꢁC, the reaction was stirred
at 0 ꢁC overnight, and then quenched with sodium sulfite
(Caution! Sodium sulfite should be added to reaction mixture
in portions and slowly to avoid any spill due to effervescence).
The mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine, dried over anhy-
drous Na2SO4, and concentrated under reduced pressure.
The residue was purified over silica gel (using CH2Cl2e
CH3OH) to yield the corresponding monodihydroxylated
product, (9R)-dec-1-en-5-yne-9,10-diol (17 g, 67% yield,
1
86% ee). H NMR: d 5.83 (1H, m), 5.03 (2H, m), 3.84 (1H,

Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
1607
J¼7.5 Hz), 7.41 (2H, t, J¼7.0 Hz), 5.78 (1H, m), 5.04 (1H, dd,
J¼17.0, 1.0 Hz), 4.98 (1H, d, J¼10.0 Hz), 4.56 (1H, m), 4.35
(2H, m), 4.26 (1H, m), 4.10 (1H, m), 3.04 (3H, s), 2.28e2.06
(4H, m), 1.78 (1H, m), 1.66 (3H, m); 13C NMR d 166.08,
136.81, 133.01, 129.75, 129.37, 128.29, 115.63, 85.38,
80.19, 76.71, 66.36, 38.71, 30.41, 28.90, 28.59, 28.33.
diethyl ether at 0 ꢁC under argon atmosphere. After 30 min,
the reaction mixture was cooled to ꢀ78 ꢁC and ClTi(O-iPr)3
(1 M in toluene, 2.75 ml, 2.75 mmol) was added. The solution
was further stirred for 30 min, and aldehyde 6 (300 mg,
1.0 mmol) in 5 ml diethyl ether was added through a canula.
Then the reaction was warmed to room temperature slowly.
After 16 h the mixture was quenched with 10% tartaric acid
solution and stirred for 0.5 h, and extracted with ethyl acetate.
The organic layers were washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue
(10:1 dr in favor of the erythro isomer) was purified by flash
chromatography on silica gel to afford the pure alkynylated
product of 7 (480 mg, 86%) in the form of a colorless oil.
1H NMR: d 8.05 (2H, d, J¼8.0 Hz), 7.56 (1H, t, J¼7.5 Hz),
7.44 (2H, t, J¼8.0 Hz), 4.57 (1H, s), 4.38 (4H, m), 4.18
(1H, m), 4.06e3.97 (2H, m), 2.35 (1H, d, J¼4.5 Hz), 2.13
(1H, m), 2.04 (2H, m), 1.82e1.59 (6H, m), 1.39 (2H, m),
1.27 (9H, m), 0.89 (9H, s), 0.88 (3H, t, J¼7.0 Hz), 0.11
(3H, s), 0.092 (3H, s); 13C NMR: d 165.21, 133.62, 132.95,
129.66, 128.31, 83.32, 82.35, 81.94, 81.26, 66.78, 64.06,
62.92, 38.58, 31.79, 29.19, 28.66, 28.54, 28.40, 26.01,
25.78, 25.19, 22.63, 18.21, 14.08, ꢀ4.50, ꢀ5.03. MS (ESI):
m/z: 573 [MþH]þ, 595 [MþNa]þ.
3.4. ((2R,20R,5R,50R)-50-(Hydroxymethyl)octahydro-2,20-
bifuran-5-yl)methyl benzoate 6
TBHP (0.1 ml, 0.5e0.6 mmol, 5e6 M in decane) was
added dropwise to a well-stirred solution of 5 (150 mg,
0.5 mmol) and Co(modp)2 (41 mg, 0.076 mmol) in 10 ml iso-
propanol and 0.05 ml water under 1 atm oxygen. The solution
was warmed up to 50 ꢁC and stirred for 24 h. Solvents were
evaporated and the residue was purified by flash chromatogra-
phy to give 6 (72 mg, 46%) and recovered starting material
1
(36 mg). [a]2D0 ꢀ8.3 (c 2.25, CHCl3); H NMR: d 8.04 (2H,
d, J¼7.5 Hz), 7.55 (1H, t, J¼7.5 Hz), 7.42 (2H, t, J¼
7.5 Hz), 4.40 (1H, m), 4.36 (2H, m), 4.16 (1H, m), 4.01
(1H, dd, J¼14.0, 6.5 Hz), 3.94 (1H, dd, J¼14.0, 7.0 Hz),
3.71 (1H, dd, J¼11.5, 2.5 Hz), 3.51 (1H, dd, J¼11.5,
3.5 Hz), 2.13 (1H, m), 2.07e1.95 (3H, m), 1.82e1.66 (4H,
m); 13C NMR: d 166.51, 132.94, 130.06, 129.65, 128.29,
82.37, 82.14, 80.03, 77.25, 66.78, 64.53, 28.77,
28.53, 28.41, 27.38. MS (ESI): m/z: 307 [MþH]þ, 329
[MþNa]þ.
TBSOTf (230 ml, 1.0 mmol) was added to a solution of the
above-described hydrogenated product (480 mg, 0.86 mmol)
and 2,6-lutidine (140 ml, 1.2 mmol) in CH2Cl2 at 0 ꢁC. The
mixture was stirred at this temperature for 2 h and worked
up using saturated NaHCO3 solution and CH2Cl2. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure. The resultant residue was purified over silica gel
3.5. ((2R,20R,5R,50R)-50-Formyloctahydro-2,20-bifuran-
5-yl)methyl benzoate 7
1
affording the corresponding TBS ether (605 mg, 100%). H
DMSO (0.4 ml, 5 mmol) in 2 ml dichloromethane was
added to a pre-cooled solution of oxalyl chloride (2 ml,
4 mmol, 2 M in dichloromethane) in 10 ml dichloromethane
at ꢀ78 ꢁC. After 20 min, the bis-THF 6 (600 mg, 2 mmol)
in 2 ml dichloromethane was added dropwise, and the reaction
was stirred for 30 min at this temperature. Diisopropylethyl-
amine (1.74 ml, 10 mmol) was added into the above solution,
the reaction was warmed to room temperature slowly,
quenched by saturated ammonium chloride solution, and ex-
tracted with dichloromethane. The organic layers were com-
bined and washed with brine, dried over sodium sulfate, and
concentrated under reduced pressure. The residue was purified
by flash chromatography to yield aldehyde 7 (500 mg, 85%).
1H NMR: d 9.69 (1H, d, J¼1.5 Hz), 8.03 (2H, d, J¼7.5 Hz),
7.54 (1H, t, J¼7.5 Hz), 7.42 (2H, t, J¼7.5 Hz), 4.34 (3H,
m), 4.04 (2H, m), 2.23e1.92 (5H, m), 1.82e1.70 (3H,
m); 13C NMR: d 202.67, 166.43, 132.92, 129.59, 128.32,
128.27, 83.28, 83.17, 81.67, 77.32, 66.70, 28.47, 28.38,
27.90, 27.29.
NMR: d 8.05 (2H, d, J¼7.5 Hz), 7.55 (1H, t, J¼7.5 Hz),
7.43 (2H, t, J¼7.5 Hz), 4.55 (1H, m), 4.34 (4H, m), 4.14
(1H, m), 3.98 (2H, m), 2.11 (2H, m), 1.98 (2H, m), 1.75 (2H,
m), 1.61 (4H, m), 1.38 (2H, m), 1.25 (8H, m), 0.89 (9H, s),
0.87 (9H, s), 0.88 (3H, t, J¼7.5 Hz), 0.12 (3H, s), 0.097
(6H, s), 0.080 (3H, s); 13C NMR: d 166.53, 132.88, 130.18,
129.67, 128.28, 82.83, 82.67, 81.97, 77.24, 66.93,
65.62, 62.94, 38.61, 31.78, 31.57, 29.22, 29.19, 28.28,
25.77, 25.59, 25.19, 22.62, 21.03, 18.21, 18.17, 14.08,
ꢀ4.50, ꢀ4.71, ꢀ4.99, ꢀ5.06. MS (ESI): m/z: 687 [M+H]+,
709 [M+Na]+.
3.7. ((2R,20R,5R,50R)-50-(1S,4S)-1,4-Di(tert-butyl-
dimethylsilyloxy)undecyl)octahydro-2,20-bifuran-
5-yl)-methanol 10
The TBS-protected compound 9 (605 mg, 0.86 mmol) and
PdeC (10% w/w, 60 mg) in EtOAc (30 ml) was stirred under
H2 atmosphere using balloon affording the desired hydroge-
nated product (600 mg, 99%). 1H NMR: d 8.04 (2H, dd,
J¼8.5, 1.5 Hz), 7.54 (1H, t, J¼7.5 Hz), 7.42 (2H, t, J¼
8.0 Hz), 4.34 (3H, m), 3.99 (1H, m), 3.90 (2H, m), 3.77 (1H,
m), 3.58 (1H, m), 2.11 (1H, m), 2.01 (1H, m), 1.92e1.67 (6H,
m), 1.54 (1H, m), 1.45 (1H, m), 1.39e1.23 (16H, m) 0.89 (9H,
s), 0.86 (9H, s), 0.87 (3H, t, J¼6.5 Hz), 0.056 (3H, s), 0.047
(3H, s), 0.023 (3H, s), 0.015 (3H, s); 13C NMR: d 166.50,
3.6. ((2R,20R,5R,50R)-50-((1S,4S)-1,4-di(tert-Butyldimethyl-
silyloxy)undec-2-ynyloctahydro-2,20-bifuran-5-yl)methyl
benzoate 9
BuLi (2.5 M in hexanes, 1.1 ml, 2.75 mmol) was added
dropwise to a solution of 8 (730 mg, 2.72 mmol) in 40 ml

1608
Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
132.86, 130.18, 129.05, 128.25, 82.30, 81.86, 81.57, 77.25,
73.85, 72.53, 66.96, 37.12, 32.77, 31.80, 30.45, 29.74,
29.27, 28.62, 28.54, 28.23, 25.97, 25.89, 25.84, 25.62,
25.29, 22.62, 18.13, 18.09, 14.07, ꢀ4.29, ꢀ4.43, ꢀ4.46,
ꢀ4.48. MS (ESI): m/z: 691 [MþH]þ, 713 [MþNa]þ.
(160 mg, 0.5 mmol) in 10 ml Et2O at 0e25 ꢁC, 1 h) aldehyde
11 (60 mg, 0.1 mmol in 5 ml Et2O) was added at ꢀ78 ꢁC.
The reaction was stirred overnight, and quenched with
MeOH and treated with Et3N (0.5 ml) and 30% H2O2
(2 mmol, 0.15 ml). Standard work-up and purification of the
resulting crude product by chromatography on silica gel gave
the homoallylic alcohols 12b and 12a in a ratio of 10:1. Simi-
larly aldehyde 10 reacted with (þ)-B-allyldiisopino-
campheylborane affording 12a and 12b in a ratio of 4:1. Physi-
The above-described hydrogenated product (600 mg,
0.9 mmol) was hydrolyzed using 2 M aqueous LiOH
(2.5 ml) in a mixture of THFeMeOH (1:1, 10 ml) at room
temperature (overnight). The desired product 10 (520 mg,
99%) was obtained after usual work-up (using ether and wa-
ter), and purification over silica gel. [a]2D0 1.5 (c 2.95,
cal data of compound 12a. [a]2D0 þ7.0 (c 0.97, CHCl3); H
1
NMR: d 5.88 (1H, m), 5.08 (2H, m), 3.93e3.82 (4H, m),
3.78 (1H, m), 3.58 (1H, m), 3.49 (1H, m), 2.47 (1H, br),
2.24 (2H, m), 1.99e1.82 (6H, m), 1.71e1.62 (5H, m),
1.55e1.26 (13H, m), 0.88 (18H, s), 0.87 (3H, t, J¼7.5 Hz),
0.063 (3H, s), 0.052 (3H, s), 0.022 (6H, s); 13C NMR:
d 134.88, 116.99, 82.36, 81.97, 81.92, 81.69, 73.83, 73.25,
72.53, 38.32, 37.13, 32.82, 31.82, 30.42, 29.76, 29.29,
28.71, 28.33, 25.98, 25.91, 25.73, 25.73, 25.31, 22.64,
18.14, 18.12, 14.09, ꢀ4.28, ꢀ4.42, ꢀ4.44, ꢀ4.46. MS (ESI):
m/z: 627 [MþH]þ, 649 [MþNa]þ. Physical data of compound
1
CHCl3); H NMR: d 4.10 (1H, m), 3.93e3.83 (3H, m), 3.79
(1H, m), 3.68 (1H, dd, J¼11.5, 3.0 Hz), 3.58 (1H, m), 3.47
(1H, dd, J¼11.5, 5.5 Hz), 1.97e1.82 (6H, m), 1.73e1.53
(5H, m), 1.46e1.23 (13H, m), 0.88 (9H, s), 0.87 (9H, s),
0.88 (3H, t, J¼6.5 Hz), 0.052 (3H, s), 0.041 (3H, s), 0.023
(3H, s), 0.020 (3H, s); 13C NMR: d 82.31, 82.00, 81.86,
79.70, 73.84, 72.54, 64.67, 37.14, 32.82, 31.82, 30.49,
29.77, 29.29, 28.67, 27.43, 25.98, 25.92, 25.64, 25.33,
22.65, 18.15, 18.12, 14.10, ꢀ4.29, ꢀ4.41, ꢀ4.43, ꢀ4.46.
MS (ESI): m/z: 587 [MþH]þ, 609 [MþNa]þ.
1
12b. [a]2D0 þ7.7 (c 0.57, CHCl3); H NMR: d 5.85 (1H, m),
5.11 (2H, m), 3.94e3.78 (6H, m), 3.58 (1H, m), 2.20 (2H,
m), 1.99e1.81 (7H, m), 1.69e1.51 (5H, m), 1.48e1.21 (12,
m), 0.88 (21H, s and m), 0.061 (3H, s), 0.052 (3H, s), 0.023
(3H, s), 0.018 (3H, s); 13C NMR: d 134.77, 117.24,
82.29 (2), 81.97, 81.84, 73.85, 72.53, 70.97, 32.83, 31.82,
30.48, 29.76, 29.66, 29.29, 28.63, 28.57, 25.98, 25.92,
25.59, 25.31, 25.18, 22.64, 18.14, 18.11, 14.09, ꢀ4.30,
ꢀ4.42, ꢀ4.43, ꢀ4.46. MS (ESI): m/z: 627 [MþH]þ, 649
[MþNa]þ.
3.8. (2R,20R,5R,50R)-50-((1S,4S)-1,4-Di(tert-butyl-
dimethylsilyloxy)undecyl)octahydro-2,20-bifuran-5-
carbaldehyde 11
DMSO (340 ml, 4.4 mmol) in 2 ml dichloromethane was
added to a pre-cooled solution of oxalyl chloride (1 ml,
2 mmol, 2 M in dichloromethane) in 10 ml dichloromethane
at ꢀ78 ꢁC. After 20 min, compound 10 (260 mg, 0.44 mmol)
in 2 ml dichloromethane was added dropwise, and the reaction
was stirred for further 30 min at this temperature. Diisopropyl-
ethylamine (1.74 ml, 10 mmol) was added into the above
solution, the reaction was warmed to room temperature slowly,
quenched by saturated ammonium chloride solution, extracted
with dichloromethane. The organic layers were combined and
washed with brine, dried over sodium sulfate, and concen-
trated under reduced pressure. The residue was purified by
flash chromatography to yield aldehyde 11 (240 mg, 92%).
1H NMR: d 9.68 (1H, d, J¼2.0 Hz), 4.04e3.86 (4H, m),
3.79e3.74 (1H, m), 3.59 (1H, m), 1.97e1.62 (10H, m),
1.60e1.26 (14H, m), 0.87 (18H, s), 0.88 (3H, t, J¼6.5 Hz),
0.068 (3H, s), 0.054 (3H, s), 0.065 (6H, s); 13C NMR:
d 203.10, 83.30, 83.07, 82.50, 82.34, 82.16, 73.81, 72.59,
72.54, 37.15, 32.82, 31.84, 30.50, 29.78, 29.31, 28.68, 27.96,
27.44, 26.00, 25.98, 25.94, 25.85, 25.75, 25.33, 22.66, 18.15,
18.13, 14.10, ꢀ4.21, ꢀ4.27, ꢀ4.27, ꢀ4.42.
3.10. (S)-3-((R)-2-(tert-Butyldimethylsilyloxy)undec-10-
en-yl)-5-methylfuran-2(5H)-one 13
3.10.1. Preparation of iodide 16
To a well-stirred solution of potassium carbonate (8.2 g,
60 mmol), potassium ferricyanide (19.6 g, 60 mmol) in
200 ml tert-butanol and 100 ml water was added (DHQD)-
PHAL (156 mg, 0.2 mmol), K2OsO4 (14.8 mg, 0.02 mmol)
and 1,10-undecadiene (3.8 g, 25 mmol) at 0 ꢁC. After the
solution was stirred for 24 h at the same temperature, it was
quenched with sodium sulfite (Caution! Sodium sulfite should
be added to reaction mixture in portions and slowly to avoid
any spill due to effervescence), diluted with water and ex-
tracted with ethyl acetate. Solvent was removed under reduced
pressure, and the residue was purified by flash chromatography
to give (10R)-undece-1-en-10,11-diol (2.2 g, 60% yield, 85%
1
ee) in the form of a solid. H NMR: d 5.82 (1H, m), 5.06
3.9. (S)-1-((2R,20R,5R,50R)-50-((1S,4S)-1,4-Di(tert-butyl-
dimethylsilyloxy)undecyl)octahydro-2,20-bifuran-5-yl)-
but-3-en-1-ol 12a and (R)-1-((2R,20R,5R,50R)-50-
((1S,4S)-1,4-Di(tert-butyldimethylsilyloxy)undecyl)octa-
hydro-2,20-bifuran-5-yl)but-3-en-1-ol 12b
(1H, dd, J¼17.5, 2.0 Hz), 4.95 (1H, d, J¼10.5 Hz), 3.70 (1H,
s), 3.65 (1H, d, J¼11.0 Hz), 3.43 (1H, t, J¼9.5 Hz), 2.15 (1H,
s), 2.03 (3H, m), 1.43e1.25 (12H, m); 13C NMR: 139.14,
114.15, 72.30, 66.83, 33.76, 33.18, 29.56, 29.36, 29.02, 28.87,
25.51.
TsCl (2.37 g, 12 mmol) was added in portions to a solution
of the above-described diol (1.86 g, 10 mmol) and triethyl
amine (2.8 ml, 20 mmol), in dry dichloromethane at 0 ꢁC.
The mixture was stirred at room temperature for 24 h, and
To a solution of (ꢀ)-B-allyldiisopinocampheylborane (pre-
pared from 0.5 ml allylmagnesium bromide, 0.5 mmol, 1 M so-
lution in Et2O, and (þ)-B-methoxydiisopinocampheylborane

Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
1609
then diluted with dichloromethane, washed using saturated
sodium bicarbonate and brine. The organic layer was dried
over anhydrous Na2SO4 and concentrated, and the residue
was purified by flash chromatography to give the correspond-
ing monotosylate (2.7 g, 91% yield) of the primary alcohol
136.63, 130.39, 129.55, 128.92, 114.11, 73.25, 69.44,
60.31, 55.28, 41.18, 39.49, 37.94, 33.74, 29.67, 29.53, 28.95,
28.85, 25.96, 24.37, 21.29, 20.99, 17.98, 14.16, ꢀ3.85.
3.10.3. Conversion of 17 to 13
1
function. H NMR: d 7.78 (2H, d, J¼8.0 Hz), 7.33 (2H, d,
Compound 17 (610 mg, 1.2 mmol) was dissolved in 10 ml
dichloromethane and treated with m-CPBA (200 mg,
1.2 mmol) at 0 ꢁC. After the reaction solution was stirred for
10 min, it was diluted with 20 ml dichloromethane and washed
with saturated NaHCO3. The organic layer was separated,
dried, and concentrated. The residue was re-dissolved into
10 ml toluene, and then the solution was heated at 90 ꢁC for
2 h, cooled down and concentrated and purified by flash chro-
matography to afford the desired compound 13 (410 mg,
J¼8.0 Hz), 5.78 (1H, m), 4.96 (1H, J¼17.5, 2.0 Hz), 4.90
(1H, d, J¼10.5 Hz), 4.00 (1H, dd, J¼10.0, 3.0 Hz), 3.86 (1H,
dd, J¼10.0, 7.0 Hz), 3.80 (1H, s), 2.43 (3H, s), 2.33 (1H, s),
2.02 (2H, m), 1.34 (4H, m), 1.23 (8H, m); 13C NMR: d
144.92, 138.99, 132.61, 129.84, 127.85, 114.08, 73.89,
69.30, 60.31, 33.65, 32.56, 29.27, 29.18, 28.88, 28.75,
25.09, 21.54.
To the above-described tosylate product (2.7 g, 7.88 mmol)
and 2,6-lutidine (1.4 ml, 11.8 mmol) in 30 ml dichloro-
methane was added TBSOTf (2.2 ml, 9.46 mmol) at ꢀ78 ꢁC.
The reaction was stirred for 1 h at this temperature, quenched
with water, and extracted with dichloromethane. The organic
layer was dried and concentrated. The residue was purified
by flash chromatography to give the corresponding TBS ether
(the precursor of iodide 16) (3.3 g, 92% yield). 1H NMR:
d 7.78 (2H, d, J¼8.0 Hz), 7.34 (2H, d, J¼8 Hz), 5.80 (1H,
m), 4.97 (1H, m), 3.85 (3H, m), 3.80 (1H, s), 2.45 (3H, s),
2.02 (2H, s), 1.53 (6H, m), 1.39 (4H, m), 1.26 (8H, m), 0.83
(9H, s), 0.02 (3H, s), 0.005 (3H, s); 13C NMR: d 144.87,
139.12, 132.60, 129.78, 127.96, 114.17, 73.19, 69.97, 34.05,
33.76, 29.52, 29.29, 28.99, 28.87, 25.74, 25.70, 24.73,
21.62, 18.02, ꢀ4.56, ꢀ4.81.
1
90%). H NMR: d 7.11 (1H, s), 5.80 (1H, m), 4.96 (3H, m),
3.94 (1H, m), 2.40 (2H, d, J¼5.0 Hz), 2.02 (2H, m),
1.41e1.22 (16H, m), 0.86 (9H, s), 0.038 (3H, s), 0.010 (3H,
s); 13C NMR: d 173.94, 151.40, 139.11, 130.80, 114.07,
77.39, 70.13, 36.90, 33.73, 32.71, 29.59, 29.36, 28.98,
28.84, 25.82, 25.07, 18.92, 17.99, ꢀ4.50.
3.11. 4,24,27-Tri-(tert-butyldimethylsilyl)-12,13-
dehydro-27-hydroxybullatacin 14a and 4,24,27-tri-(tert-
butyl-dimethylsilyl)-12,13-dehydro-15-epi-27-hydroxy-
bullatacin 14b
The homoallylic alcohol compound 12a (13 mg, 0.02 mmol)
and butenolide 13 (31 mg, 0.08 mmol) in 2 ml dichloromethane
were added to the solution of the Grubbs’ catalyst (RuCl2[imid-
H2-Mes2][CHPh]PCy3, 4.0 mg, 0.004 mmol) in 2 ml dichloro-
methane at reflux over 12 h by syringe pump. The solution was
concentrated by reduced pressure after further refluxing for an
additional hour. Column chromatography on silica gel afforded
A mixture of the above-described TBS-protected tosylate
compound (2.0 g, 4.4 mmol), sodium bicarbonate (440 mg,
5.0 mmol), and sodium iodide (7.7 g, 50 mmol) in acetone
(40 ml) was refluxed for 24 h. Solvents were removed in vacuo
and the residue was purified to give iodide 16 (1.7 g, 94%
1
1
yield). H MNR: d 5.81 (1H, m), 4.92e5.02 (2H, m), 3.54
compound 14a (11 mg, 66%). H NMR: d 7.12 (1H, s), 5.48
(1H, m), 3.18 (2H, d, J¼5.0 Hz), 2.04 (2H, m), 1.60 (1H,
m), 1.53 (1H, m), 1.38 (2H, m), 1.30 (8H, m), 0.91 (9H, s),
0.10 (3H, s), 0.07 (3H, s); 13C NMR: d 139.06, 114.15,
71.42, 36.87, 33.77, 29.46, 29.36, 29.01, 28.88, 25.83,
24.88, 18.05, 13.98, ꢀ4.36, ꢀ4.56.
(1H, qd, J1¼1.0 Hz, J2¼5.5 Hz), 4.99 (1H, m), 3.96e3.78 (6H,
m), 3.59 (1H, m), 3.43 (1H, m), 2.42 (2H, m), 2.18 (2H, m),
2.04e1.88 (8H, m), 1.67e1.63 (4H, m), 1.56 (6H, m), 1.45e
1.26 (20H, m), 1.41 (3H, d, J¼7 Hz), 0.88 (27H, s), 0.87 (3H,
t, J¼7.5 Hz), 0.066 (3H, s), 0.054 (3H, s), 0.050 (3H, s), 0.023
(9H, s); 13C NMR: d 177.95, 151.43, 133.28, 125.88, 82.35,
80.01, 81.84, 81.68, 77.44, 73.83, 73.69, 72.54, 70.17, 37.14,
37.08, 36.95, 32.84, 32.74, 32.63, 31.82, 30.41, 29.77, 29.68,
29.45, 29.30, 29.16, 28.71, 28.32, 25.99, 25.92, 25.87,
25.70, 25.33, 25.14, 22.64, 18.97, 18.15, 18.12, 18.04, 14.09,
ꢀ4.26, ꢀ4.41, ꢀ4.44. MS (ESI): m/z: 979 [MþH]þ, 1001
[MþNa]þ.
3.10.2. Alkylation of lactone 15 to produce 17
A solution of N,N-diisopropylamine (424 ml, 3.0 mmol) in
15 ml THF was treated with n-BuLi (1.25 ml, 3.125 mmol,
2.5 M in THF) at 0 ꢁC for 15 min and then cooled to
ꢀ78 ꢁC. Lactone 15 (1.0 g, 2.4 mmol) in 5 ml THF was
added dropwise via syringe and the mixture was stirred at
0 ꢁC for 30 min. Iodide 16 (500 mg, 2.4 mmol) in 1.3 ml
HMPA and 2 ml THF was added to the above solution
and the mixture was stirred for 24 h. The solution was di-
luted with ethyl acetate and washed with saturated NH4Cl
and brine. The organic solution was dried, filtered, concen-
trated by rotary evaporation, and purified by flash column
chromatography to afford compound 17 (620 mg, 51%).
1H NMR: d 7.57 (2H, m), 7.35 (3H, m), 5.80 (1H, m), 4.94
(2H, m), 4.52 (1H, m), 4.25 (1H, m), 3.02 (1H, m), 2.00
(3H, m), 1.85 (1H, m), 1.46e1.22 (17H, m), 0.90 (9H, s),
0.16 (3H, s), 0.12 (3H, s); 13C NMR: d 177.48, 139.09,
Similarly, compound 12b was reacted with 13 affording
1
14b in 58% yield. H NMR: d 7.12 (1H, s), 5.51 (1H, m),
5.41 (1H, m), 4.99 (1H, m), 3.97e3.81 (7H, m), 3.59 (1H,
m), 2.42 (1H, d, J¼5.5 Hz), 2.14 (2H, m), 2.02e1.80 (10H,
m), 1.68e1.52 (6H, m), 1.41 (3H, d, J¼6.5 Hz), 1.48e1.25
(24H, m), 0.88 (30H, s and m), 0.062 (3H, s), 0.051 (3H, s),
0.023 (6H, s), 0.020 (6H, s); 13C NMR: d 174.85, 151.43,
133.65, 125.70, 82.28, 81.95, 77.43, 73.85, 72.52, 71.42,
70.17, 37.12, 36.94, 36.19, 32.85, 32.73, 32.61, 31.82, 30.47,
29.76, 29.68, 29.42, 29.35, 29.29, 29.13, 29.09, 28.64, 28.56,
25.99, 25.92, 25.87, 25.53, 25.32, 25.28, 25.13, 22.65, 18.96,

1610
Z. Chen, S.C. Sinha / Tetrahedron 64 (2008) 1603e1611
18.15, 18.14, 14.09, ꢀ4.30, ꢀ4.41, ꢀ4.46. MS (ESI): m/z: 979
[MþH]þ, 1001 [MþNa]þ.
14.11. HRMS (ESI-TOF, MþHþ) calcd for C37H66O8
639.483, found 639.4813.
3.12. 27-Hydroxybullatacin 1a and 15-epi-
hydroxybullatacin 1b
3.13. Determination of the effect of 27-hydroxybullatacin
and its C-15 epimer on the mitochondrial enzymes’
inhibitors
A solution of NaOAc (100 mg, 1.2 mmol) in water (2 ml)
was added to a stirred solution of metathesis product (14a,
11 mg, 0.011 mmol) and p-toluenesulfonyl-hydrazine (186 mg,
1 mmol) in dimethoxyethane (2 ml) at reflux over 5 h. The
mixture was then cooled to room temperature, poured into
water, and extracted with dichloromethane. The combined
organic layers were dried and concentrated under reduced
pressure. Purification by column chromatography on silica gel
(hexaneeEtOAc 6:1) yielded the corresponding hydrogenated
Bovine heart SMPs were provided by our collaborator,
Takao Yagi, at The Scripps Research Institute, who prepared
them using a sonication medium containing 0.25 M sucrose,
1 mM succinate, 1.5 mM ATP, 10 mM MgCl2, 10 mM
MnCl2, and 10 mM TriseHCl (pH 7.4) and stored in a buffer
containing 0.25 M sucrose and 10 mM TriseHCl (pH 7.4) at
ꢀ84 ꢁC. A solution of 27-hydroxy-bullatacin or its C-15
epimer at various concentrations between 1 and 20 nM were
added to 15 mg of SMPs in 500 ml volume containing
0.25 M sucrose, 1 mM MgCl2, and 50 mM phosphate buffer
(pH 7.5), and mixed. After 5 min equilibration of SMP with
inhibitors, the reaction was started using 150 mM NADH and
progress of reaction was determined spectrometrically using
a UV instrument at 340 nm. Minimum concentration of the
compounds required to inhibit the oxidation of NADH by
50% was recorded as their IC50 value.
1
product (10 mg, 90%). H NMR: d 7.11 (1H, d, J¼1.5 Hz),
5.01 (1H, m), 3.96e3.77 (6H, m), 3.58 (1H, m), 3.38 (1H, m),
2.42 (2H, m), 1.98e1.82 (5H, m), 1.71e1.60 (4H, m), 1.60e
1.25 (35H, m), 1.41 (3H, d, J¼6.5 Hz), 0.88 (27H, s), 0.87 (3H,
t, J¼7.5 Hz), 0.065 (3H, s), 0.054 (3H, s), 0.052 (3H, s), 0.023
(9H, s). MS (ESI): m/z: 981 [MþH]þ, 1003 [MþNa]þ.
The above-described hydrogenated product (10 mg,
0.01 mmol) was stirred with 2 ml 5% HF in MeCN and 2 ml
THF at room temperature for 12 h, the reaction mixture was
partitioned between dichloromethane and brine. The organic
layer was separated and the aqueous layer was extracted
with dichloromethane. The combined organic layers were
washed with brine prior to drying and solvent evaporation.
Purification by column chromatography on silica gel yielded
1a (6.0 mg, 91%) as a colorless waxy solid. [a]2D0 þ5.8 (c
1.04, CHCl3); (lit.1 þ11.0); 1H NMR: d 7.18 (1H, d, J¼
1.0 Hz), 5.05 (1H, m), 3.93 (2H, m), 3.85 (4H, m), 3.65
(1H, m), 3.40 (1H, m), 2.52 (1H, m), 2.40 (1H, m), 2.34
(1H, m), 2.02e1.84 (6H, m), 1.74e1.39 (10H, m), 1.43 (3H,
d, J¼7.0 Hz), 1.36e1.16 (27H, m), 0.88 (3H, t, J¼7.5 Hz);
13C NMR: d 174.60, 151.78, 131.23, 83.23, 82.81, 82.54,
82.22, 77.98, 74.10, 71.84, 71.53, 70.02, 37.49, 37.44,
34.04, 33.37, 31.94, 31.85, 29.71 (2), 29.67 (2), 29.52,
29.37, 29.30, 22.71, 22.67, 19.14, 14.11. HRMS (ESI-TOF,
MþHþ) calcd for C37H66O8 639.483, found 639.4813.
Similarly, compound 14b was hydrogenated affording the
precursor of 1b. 1H NMR: d 7.12 (1H, s), 5.00 (1H, m),
3.96e3.80 (7H, m), 3.59 (1H, m), 2.42 (2H, d, J¼5.0 Hz),
2.13e1.84 (10H, m), 1.64e1.26 (36H, m), 1.41 (3H, d,
J¼6.5 Hz), 0.88 (30H, m), 0.050 (9H, s), 0.023 (9H, s). MS
(ESI): m/z: 981 [MþH]þ, 1003 [MþNa]þ.
Acknowledgements
We are thankful to Drs. Z.-Z. Huang and Eiko Nakamaru-
Ogiso for determining binding of the synthetic compounds.
This work was supported by The Skaggs Institute for Chemi-
cal Biology.
References and notes
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1
CHCl3); H NMR: d 7.18 (1H, d, J¼1.5 Hz), 5.05 (1H, qd,
J¼13.5, 1.0 Hz), 3.95e3.83 (9H, m), 3.64 (1H, m), 2.54e
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