Total syntheses of (±)-ovalicin, C4(S*)-isomer, and its C5-analogs and anti-trypanosomal activities

Article abstract of DOI:10.1016/j.bmc.2008.03.009

Total syntheses of (±)-ovalicin, its C4(S^*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium aceta

Full text of DOI:10.1016/j.bmc.2008.03.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5232–5246
Total syntheses of ( )-ovalicin, C4(S*)-isomer, and its
C5-analogs and anti-trypanosomal activities
Duy H. Hua,a,* Huiping Zhao,a Srinivas K. Battina,a Kaiyan Lou,a Ana L. Jimenez,a
John Desper,a Elisabeth M. Perchellet,b Jean-Pierre H. Perchelletb and Peter K. Chiangc,d
aDepartment of Chemistry, Kansas State University, Manhattan, KS 66506-3701, USA
bDivision of Biology, Kansas State University, Manhattan, KS 66506, USA
cDepartment of Pathology and Sandler Center, QB3, University of California, San Francisco, CA 94143, USA
dPharmadyn Inc., 525 Del Rey Avenue, Sunnyvale, CA 94085, USA
Received 28 January 2008; revised 25 February 2008; accepted 3 March 2008
Available online 6 March 2008
This publication is dedicated to Professor E.J. Corey on the occasion of his 80th birthday.
Abstract—Total syntheses of ( )-ovalicin, its C4(S*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intra-
molecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate.
Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S*,5R*,6R*)-5-methoxy-6-(tert-butyldimethylsilyl-
oxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadi-
ene (27) followed by oxidation afforded ( )-ovalicin. The functional group manipulation afforded a number of regio- and
stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal
X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dic-
tated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evalu-
ated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor
cells in vitro. Compounds 46 and a precursor, (3S*,4R*,5R*,6R*)-5-methoxy-4-[(E)-(10,50-dimethylhexa-10,40-dienyl)]-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and eventually death if left untreated.1 Our interest
in finding anti-trypanosomal agents4,5 led us to study
the synthesis and bioevaluation of angiogenesis inhib-
itors, ovalicin and its analogs. Fumagillin, structurally
similar to ovalicin, has been shown to inhibit methio-
nine aminopeptidase 2 (MetAP2) leading to anti-angi-
ogenesis.4 Since MetAP2 presents in parasites,4
syntheses and studies of anti-trypanosomal activities
of ovalicin and its analogs were undertaken. Several
total syntheses of ovalicin have appeared starting from
functionalized six-membered ring compounds,6–11 but
an acyclic starting material is rare.12 Moreover, anti-
trypanosomal activity of ovalicin or its analogs has
not been reported previously. Herein, we report the
total syntheses of ( )-ovalicin, its C4(S*) isomer, and
C5-side chain isomer utilizing an intramolecular Heck
In search of small molecules that block the parasitic
growth of protozoan parasite Trypanosoma brucei
(T. brucei)1,2 and possess anti-angiogenic activity, we
studied the total synthesis of ( )-ovalicin (1)3 and its
analogs. The parasitic hemoflagellate T. brucei causes
human African Trypanosomiasis or sleeping sick-
ness.1,2 The disease affects over 55 million people in
Africa, and the transmission of parasite between mam-
malian hosts is largely through Glossina genus tsetse
fly bites. The parasite replicates in the blood, passes
the blood–brain barrier, and invades the central ner-
vous system resulting in loss of consciousness, coma,
Keywords: Total synthesis of ovalicin; Ovalicin analogs; Anti-trypan-
osomal activities; Anti-parasitic; Methionine aminopeptidase 2.
reaction to generate
a functionalized cyclohexene
intermediate from an acyclic alkenyl iodide, and their
anti-trypanosomal activities.
*
Corresponding author. Tel.: +1 785 532 6699; fax: +1 785 532
6666; e-mail: duy@ksu.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.009

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5233
2. Results and discussion
2.1. Synthesis of ( )-ovalicin
phenylphosphine, and potassium carbonate in
acetonitrile at 80 ꢁC,15 however under similar reaction
conditions, 4-methylene-2-cyclohexenol (9) was not de-
tected from the cyclization reaction of alcohol 8. By
using additives silver phosphate and proton sponge in
DMF, alcohol 8 was converted to 9 in a 61% yield
(Scheme 3). Under similar reaction conditions, silyl
ether 4 gave a 95% yield of cyclized product 3. Since sub-
sequent steps involved oxidant and reactive reagents, si-
lyl ether protected 316 was used in our synthesis. In the
epoxidation of 3, an oxidizing reagent such as MCPBA
does not provide satisfactory results, but methyl(trifluo-
romethyl)dioxirane (generated in situ)16,17 gave an 81%
yield of an inseparable mixture (1:1) of compounds 10
Our retrosynthetic analysis of ovalicin (1) is depicted in
Scheme 1 utilizing compound 2, a key intermediate re-
ported by Barton8 and others,10,12 because the stereo-
chemistry at C5 and 6 of compound 2 controls the
stereochemistry at C4 of 1 in the subsequent addition
reaction. Compound 2 is synthesized via an epoxidation
of the exo-methylene function of diene 3 followed by
dihydoxylation and functional group transformation.
The functionalization of diene 3 provides various stereo-
isomers at C4–C6 for bioevaluation and studies of the
stereochemical addition reactions at C4 of 2 and its ster-
eoisomers leading to 1 and its analogs, respectively.
Diene 3 is derived from an intramolecular Heck reaction
of alkenyl iodide 4, which is readily synthesized from 3-
butenylmagnesium bromide and cis-3-iodopropenal (7).
1
and 11 (determined from H NMR spectrum). Subse-
quent dihydroxylation of 10 and 11 with OsO4 and N-
methylmorpholine N-oxide (NMO) afforded diols 12
and 13 (85% yield), which can be partially separated
by silica gel column chromatography. A convenient sep-
aration method was found by selective mono-benzoyla-
tion of 12 and 13 (1:1) with benzoyl chloride and
triethylamine providing benzoate 14 (43% yield) and
unreacted diol 13 (49% recovery), which are readily sep-
arated. It appears that C4–axial–OH function of 12 is
more accessible than other hydroxyl functions of 12
and 13. Stereochemistry at C3-6 of 12 and 13 was deter-
mined from the subsequent conversion into the known
compound, 2, and 2D NOESY NMR spectroscopic
study of a subsequent intermediate (vide infra).
Alkenyl iodide 4 was readily synthesized from ethyl
propiolate (5) by a sequence of reactions depicted in
Scheme 2. Addition reaction of 5 with sodium iodide
in acetic acid13 followed by reduction of the ester func-
tion of resulting adduct 6 with Dibal-H in dichlorometh-
ane13,14 and addition with 3-butenylmagnesium bromide
afforded alcohol 8. The aldehyde intermediate, 7, from
the reduction of 6, is unstable and was used in the Grig-
nard reaction without purification. Silylation of alcohol
8 with tert-butyldimethylsilyl chloride gave silyl ether 4.
Compounds 13 and 14 possess the same stereochemistry
at C6 and C5, respectively, as that of 2, and they were
converted into 2 via two separated routes (Schemes 4
and 5). Oxidation of the hydroxyl function of 14 with
o-iodoxybenzoic acid (IBX) in DMSO18 followed by
Both alcohol 8 and silyl ether 4 were studied in the ring-
closing reactions. It has been reported that compounds
containing hydroxyl moiety underwent successful intra-
molecular Heck reaction with palladium acetate, tri-
O
1
O
I
O
3
2
1'
4
5
5
6
O
OMe
O
2'
MeO
OH
OSi-t-BuMe2
OSi-t-BuMe2
Me2t-Bu-SiO
1
4
3
2
Scheme 1. Retrosynthesis of ovalicin (1).
Dibal-H
CH2Cl2, -78oC
O
EtO2C
I
NaI, AcOH
(98% yield)
H
C
I
EtO2C
C
C
H
C
C
C
C
H
H
H
H
5
6
7
3-butenylmagnesium
bromide, THF, -78 ºC
-BuMe SiCl
t
2
imidazole, DMF
(76% overall yield
(98% yield)
OH
I
I
Me -Bu-SiO
2t
6
from
)
8
4
Scheme 2. Synthesis of 4.

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D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
Pd(OAc)2,
Ph3P, Ag3PO4
8
Proton sponge
DMF, 50oC
(61% yield)
OH
9
O
O
O
Pd(OAc)2,
Ph3P, Ag3PO4
H3C
CF3
+
4
EDTA, NaHCO3
oxone
Proton sponge
DMF, 50oC
(93% yield)
(82% yield; 1:1)
OSi-
11
t-BuMe2
OSi-t-BuMe2
OSi-t-BuMe2
10
3
O
O
O
O
1.1 eq PhCOCl,
Et3N, THF
OsO4, NMO
-BuOH, acetone
OCOPh
OH
OH
OH
OH
OH
t
+
+
H2O
OH
OH
t-BuMe2
(85% yield; 1:1)
OSi-
t
-BuMe2
OSi-t-BuMe2
OSi-
13
OSi-t-BuMe2
14
13
(49% yield)
(43% yield)
12
(separated)
Scheme 3. Synthesis of 14.
stereoselective reduction with diisobutylaluminum hy-
dride (Dibal-H) in THF at ꢀ78 ꢁC provided alcohol
16 as a single stereoisomer at C5. NMR spectra and
TLC appearances of stereoisomers 14 and 16 are differ-
ent. Methylation of alcohol 16 with trimethyloxonium
tetrafluoroborate and proton sponge followed by basic
hydrolysis and oxidation with IBX afforded ketone 2.
NMR spectral data of 2 are identical to those
reported.12
followed by removal of the silyl ether-protecting group
with tetra-n-butylammonium fluoride (TBAF) and oxi-
dation with IBX generated ketone 23 in excellent yield
(83% yield in three steps). Ketone 23 was stereoselective-
ly reduced with K-Selectride in THF to furnish alcohol
24 as a single stereoisomer. C6-S*-isomer of 24 was not
detected. Silylation of alcohol 24 with tert-butyldimeth-
ylsilyl chloride followed by basic hydrolysis of C4 ben-
zoyl function and oxidation with IBX gave ketone 2 in
a 93% overall yield (three steps).
The conversion of diol 13 to ketone 2 required a length-
ier route because monomethylation of the cis-dihydroxyl
functions and inversion and re-protection of C6 silyl
ether moiety are needed (Scheme 5). However, a regio-
isomer such as compound 20 is available for analog syn-
thesis (vide infra). As shown in the analog synthesis, the
stereochemistry at C6 (R*-configuration) of 2 is crucial
for the generation of the correct stereochemistry at C4
of ovalicin in the addition reaction with the alkenyllith-
ium (vide infra). Monomethylation of diol 13 with
Me3OÆBF4 and proton sponge led to an 81% yield of
methyl ether 19 and 20 in a ratio of 1.2:1 along with a
small amount (6% yield) of the dimethoxylated product.
Other methylation reactions were attempted such as the
uses of methyl iodide or methyl triflate and sodium hy-
dride or amine bases resulted in no methylation product.
Fortuitously, 19 and 20 are separable by silica gel col-
umn chromatography. Compound 20 was used in the
synthesis of analogs for bioevaluation (vide infra), and
its regiochemistry was verified by its 2D NOESY spec-
trum in which C4–OMe (d 3.42 ppm) shows NOE corre-
lation with C2–H (d 2.93 ppm) of the oxiran moiety.
Benzoylation of 19 with benzoyl chloride and pyridine
The synthesis of ovalicin 1 from 2 is similar to that re-
ported.8,10,12 Hence, the addition reaction of ketone 2
with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27)6 in
DME-toluene gave 28 as a single stereoisomer (Scheme
6). Compound 27 was generated in situ as described6
utilizing a Shapiro reaction from acetone 2,4,6-tri-
isopropylbenzenesulfonylhydrazone and 1-bromo-3-
methyl-2-butene. The C6-silyloxy and C5-methoxy of 2
shielded the si-face of the carbonyl function resulting
in the exclusive addition of alkenyllithium 27 from the
re-face. Removal of the silyl ether-protecting group with
TBAF followed by oxidation with IBX and epoxidation
with vanadyl acetylacetone and tert-butylhydroperoxide
afforded ( )-ovalicin (1) whose NMR data are identical
to those reported.8,12
2.2. Syntheses of C4(S*)-isomer 44 and C5-adduct 46
Although a number of total syntheses of ovalicin have
been reported,6–12 the synthesis of ovalicin analogs is
limited.11 Since a number of analogs are needed for
our bioevaluation and examination of the effect of C6-

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5235
O
O
O
OCOPh
OCOPh
OCOPh
OMe
IBX, DMSO
(95% yield)
Me3O•BF4
(86% yield)
Dibal-H
K2CO3
14
(85% yield)
(95% yield)
O
OH
OSi-t-BuMe2
16
OSi-t-BuMe2
OSi-t-BuMe2
15
17
O
OH
IBX, DMSO
(98% yield)
2
OMe
OSi-t-BuMe2
18
Scheme 4. Synthesis of 2.
O
O
OMe
OH
OH
Me3O•BF4
proton sponge
+
13
OMe
OSi-t-BuMe2
20
OSi-t-BuMe2
19
(37% yield)
(44% yield)
O
O
O
PhCOCl
pyridine
OCOPh
OCOPh
OCOPh
OMe
IBX, DMSO
(93% yield)
TBAF
(92% yield)
19
(97% yield)
OMe
OSi-t-BuMe2
21
OMe
OH
22
O
23
O
O
O
IBX
OCOPh
OMe
OCOPh
OMe
OH
K2CO3
(96% yield)
t-BuMe2SiCl
(98% yield)
K-Selectride
(93% yield)
DMSO
2
(99% yield)
OMe
OH
24
Scheme 5. Synthesis of 2 from 13.
OSi-t-BuMe2
25
OSi-t-BuMe2
26
stereochemistry onto the alkenyllithium addition reac-
tion, C4(S*)-isomer of ovalicin, 44, was synthesized
and characterized. C5-adduct, compound 46 was syn-
thesized and evaluated as well.
conditions, OsO4 and NMO, to give a 46% overall yield
of diols 34, 35, and 36 in a ratio of 5:4:1, which were sep-
arated by silica gel column chromatography. The rela-
tive stereochemistry of a major product, diol 34, was
established from a single-crystal X-ray analysis of its
C5-monosilyl ether derivative, 37, from the silylation
reaction with tert-butyldimethylsilyl chloride and trieth-
ylamine (Fig. 1). Stereochemistry of the other major
product, 35, was assigned based on NMR coupling con-
stant J values and the approach of osmium reagent pre-
dominantly from the opposite face of C6-benzoyloxy
group, similar to that of the formation of compound
34. Hence, C5–H of 35 shows a doublet of doublet at
d 4.01 ppm with J values of 7.6 Hz (axial–axial coupling)
Instead of the silyl ether-protecting group at C6 of 2, a
benzoyl-protecting group was used in this synthesis,
which provides benzoate analogs for bioevaluation (vide
infra). Benzoylation of alcohol 9 with benzoyl chloride
and triethylamine gave benzoate 31 (Scheme 7). Epoxi-
dation of 31 with MCPBA–NaF–KF in dichlorometh-
ane19 gave epoxides 32 and 33 in a 1:1 ratio, which
decomposed slowly on silica gel column. The crude mix-
ture of 32 and 33 was subjected to the dihydroxylation

5236
D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
Li
O
O
(27)
TBAF
IBX, DMSO
(92% yield)
2
OH
OH
DME, toluene
(99% yield)
OMe
ºC
-78
OMe
)
(75% yield
OSi-
t
-BuMe2
OH
29
28
O
VO(Acac) 2
-BuOOH
t
1
OH
benzene
(62% yield)
OMe
O
30
Scheme 6. Synthesis of ( )-ovalicin (1).
O
O
OsO4,N MO
MCPBA
NaF, KF
-BuOH, acetone
t
PhCOCl
+
9
H2O
(1:1)
(88% yield)
(46% overall yield;
5:4:1)
OCOPh
OCOPh
OCOPh
31
32
33
H
O
O
O
O
OH
OH
OH
OH
OH
O
Ph
6
+
+
4
HO
OH
O
OH
OCOPh
35
5
OCOPh
H
OCOPh
34
35
36
O
O
H
OH
O
H
OH
2
-BuMe SiCl
t
Et3N, D2MAP
H
O
Ph
34
Ph
H
O
6
OH
5
4
(53% yield)
6
4
OSi- -BuMe
H
t
2
O
O
5 HO
H
H
NOE
NOE
OCOPh
OH
37
36
36A
Scheme 7. Synthesis of 37.
and 3.2 Hz (axial–equatorial coupling). Stereochemistry
of the minor product, 36, was determined from a NMR
2D NOESY spectrum. The C4–H, d 3.82 ppm, of 36
exhibits NOE correlations with C2–Ha, d 3.15 ppm,
and C6–H, d 5.24 ppm. The stereoisomer of 36 or 36A
is not present. Compound 35 was used in bioevaluation
(vide infra) and not investigated further since it pos-
sesses the same stereochemistry at C6 as that of 2.
tetrafluoroborate afforded a 1.3:1 ratio of methyl ether
38 and 39, which were separated by silica gel column
chromatography (Scheme 8). Compound 39 was not
investigated further. Swern oxidation of 38 with trifluo-
roacetic anhydride and DMSO provided ketone 40,
which was subjected to the addition reaction with alke-
nyllithium 27. Adducts 41 and 42 were isolated in a ratio
of 1.5:1. Oxidation of alcohol 38 with IBX in DMSO at
25 ꢁC resulted in a poor yield of 40 (42% yield). It is
likely at 25 ꢁC, product 40 undergoes E1cb reaction un-
der IBX-DMSO reaction conditions, while the Swern
C4(S*)-isomer of ovalicin, 44, was synthesized from diol
34. Monomethylation of diol 34 with trimethyloxonium

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5237
Figure 1. An ORTEP drawing of the single-crystal X-ray analysis of
compound 37.
oxidation was carried out at low temperature (ꢀ78 to
0 ꢁC) in which elimination reaction does not occur.
Compound 42 likely derived from the cleavage of the
benzoyl ester function of 41 with 27 in situ. Basic hydro-
lysis of 41 with K2CO3 in methanol produced 42 (95%
yield). Oxidation of 42 with IBX-DMSO followed by
epoxidation with VO(Acac)2-t-BuOOH furnished
C4(S*)-isomer 44, of which the stereochemistry was af-
firmed from a single-crystal X-ray analysis (Fig. 2).
Hence, the carbonyl addition reaction of compound 40
with alkenyllithium 27 took place from the opposite face
of C6 benzoyloxy moiety and the same face of C5 meth-
Figure 2. An ORTEP drawing of the single-crystal X-ray analysis of
compound 44.
oxy group. The X-ray crystal structure reveals the axi-
ally oriented epoxy-alkenyl side chain, which is unusual.
C5-side chain analogs, such as compound 46, have not
been reported previously. For our bioevaluation, 46
O
O
Me3O•BF4
OH
OMe
OH
proton sponge
+
34
(80% yield; 1.3:1)
OMe
OCOPh
OCOPh
39
38
O
O
O
DMSO
O
27
(CF3CO)2O
+
OH
38
OH
DME, toluene
-78oC
(83% yield)
OMe
OMe
OMe
(66% yield;
1.5:1)
OH
OCOPh
OCOPh
42
41
40
K2CO3, MeOH
O
O
VO(Acac)2
-BuOOH
IBX, DMSO
(58% yield)
t
42
OH
OMe
OH
MeO
benzene
O
O
H
(65% yield)
O
43
44
Scheme 8. Synthesis of C4(S*)-isomer of ovalicin, 44.

5238
D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
O
OH
O
Li
H
OMe
Me2t-Bu-SiO
(27)
IBX, DMSO
(90% yield)
2
6
4
20
H
H
DME, toluene
-78oC
(72% yield)
O
OMe
NOE
2'
1'
NOE
OSi-
t
-BuMe2
NOE
45
46
Scheme 9. Synthesis of C5-analog 46.
was synthesized from alcohol 20 (Scheme 9) and evalu-
ated (vide infra). Hence, oxidation of 20 with IBX-
DMSO afforded ketone 45, which upon addition reac-
tion with alkenyllithium 27 gave a 72% yield of 46 as
a single stereoisomer. The stereochemistry was assigned
based on NMR 2D NOESY spectroscopy in which C4-
methoxy, d 3.29 ppm, shows NOE correlations with the
side chain C10-methyl, d 1.75 ppm, and C2–H, d
2.74 ppm. And, C6–H, d 4.27 ppm, of 46 exhibits
NOE correlations with C10-methyl and C20–H, d
5.75 ppm. Hence, the carbonyl addition reaction of
compound 45 with alkenyllithium 27 took place from
the opposite face of C6 silyloxyl moiety and the same
face of C4 methoxy group.
3. Conclusion
( )-Ovalicin, its C4(S*)-isomer, and C5-side chain ana-
log were synthesized via an intramolecular Heck reac-
tion utilizing a catalytic amount of palladium acetate.
Subsequent epoxidation, dihydroxylation, methylation,
and oxidation led to ketone 2, a reported key intermedi-
ate. The aforementioned functional group manipulation
afforded a number of regio- and stereoisomers, which al-
low the synthesis of analogs for bioevaluation. The ste-
reochemistry at C4 generated from the addition
reactions of alkenyllithium with ketones 2, 40, and 45
is dictated by C6-alkoxy functionality. Anti-trypanoso-
mal activities of various ovalicin analogs and synthetic
intermediates were evaluated, and C5-side chain analog,
46, shows the strongest activity. Compounds 46 and 28
may be used for the development of anti-parasitic drugs.
2.3. Bioevaluation
Various synthetic intermediates, compounds 28, 34, 35,
and 40, and ovalicin analogs, compounds 41, 43, and
46, were tested against T. brucei in vitro by following
our reported methods.20,21 C5-side chain compound,
46, possesses the most potent inhibitory activity
in vitro with an IC50 value of 0.28 0.07 lM
(Table 1). In comparison, ovalicin precursor 28, a C4-
side chain compound, is slightly less active with an
IC50 of 0.40 0.05. It is surprising to find that a struc-
turally simple diol analog, 35, has an IC50 of
0.72 0.15 and is 2.5-fold less active than 46.
Compounds 34 and 40, 41 and 43 are less active. The
anti-trypanosomal activities of compounds 28 and 46
are greater than those previously reported for 50-modi-
fied adenosine derivatives.21 Biological targets of this
class of compounds and efficacy in animal model will
be studied. The utilization of ovalicin analogs, such as
46, should be explored as anti-parasitic agents, as fum-
agillin and TNP-470,4 possessing a similar skeleton as
that of ovalicin, have been shown to inhibit methionine
aminopeptidase 2 resulting in anti-malaria and anti-
leishmaniasis.4
4. Experimental
4.1. General methods
Unless otherwise indicated, NMR spectra were obtained
at 400 MHz for 1H and 100 MHz for 13C in CDCl3, and
reported in ppm. High-resolution Mass spectra were ob-
tained from Maldi and ESI spectrometers. Maldi spectra
were taken using 2,5-dihydroxybenzoic acid as a matrix.
ESI spectra were acquired on a LCT Premier (Waters
Corp., Milford MA) time of flight mass spectrometer.
The instrument was operated at 10,000 resolution (W
mode) with dynamic range enhancement that attenuates
large intensity signals. The cone voltage was 60 eV.
Spectra were acquired at 16,666 Hz pusher frequency
covering the mass range 100–1200 l and accumulating
data for 2 s per cycle. Mass correction for exact mass
determinations was made automatically with the lock
mass feature in the MassLynx data system. A reference
compound in an auxiliary sprayer is sampled every third
cycle by toggling a ‘shutter’ between the analysis and
reference needles. The reference mass is used for a linear
mass correction of the analytical cycles. Samples are pre-
sented in Methanol Plus 0.1% formic acid as a 20 lL
loop injection using an auto injector (LC PAL, CTC
Analytics AG, Zwingen, Switzerland). Tetrahydrofuran
and diethyl ether were distilled over sodium and benzo-
phenone before use. Methylene chloride was distilled
over CaH2 and toluene and benzene were distilled over
LiAlH4.
Table 1. In vivo antitrypanosmal activity of ovalicin analogs and
synthetic intermediates
Compound
IC50 (lM)
Compound
IC50 (lM)
28
34
35
40
0.40 0.05
325 30
41
43
46
4.20 0.40
41.5 4.0
0.28 0.07
0.72 0.15
47.3 4.0

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5239
4.1.1. (Z)-1-Iodo-1,6-heptadien-3-ol (8). To a three-neck
flask equipped with a dropping funnel and a reflux con-
denser under argon were added 1.6 g (67 mmol) of mag-
nesium turnings and 30 mL THF, and the mixture was
stirred vigorously at 70 ꢁC. To it, 3.0 g (22 mmol) of 4-
bromo-1-butene was added dropwise through a drop-
ping funnel and the resulting mixture was stirred at
75 ꢁC for 2 h, cooled to 25 ꢁC to give the Grignard re-
agent, which was maintained under argon. To a solution
of (Z)-3-iodopropenal (7, generated from 16.5 mmol of
6)14 in 40 mL of dichloromethane at ꢀ78 ꢁC under ar-
gon, was added the above 3-butenylmagnesium bromide
via a cannula. The solution was stirred at ꢀ78 ꢁC for
1 h, warmed to 25 ꢁC, diluted with 100 mL of water,
and extracted three times with diethyl ether (50 mL
each). The combined organic layer was washed with
aqueous NaHCO3 and brine, dried (anhydrous
Na2SO4), concentrated, and column-chromatographed
on silica gel using a gradient mixture of hexane and
diethyl ether as eluant to give 2.98 g (76% overall yield;
2.35–2.25 (m, 1H), 1.95–1.85 (m, 1H), 1.65–1.55 (m,
1H), 0.82 (s, 9 H), 0.01 (s, 3H), 0.01 (s, 3H); 13C
NMR d 142.6, 133.9, 130.2, 111.9, 67.1, 32.9, 27.9,
26.1, 18.4, ꢀ4.3.
4.1.4. 4-Methylene-2-cyclohexen-1-ol (9). A solution of
1.0 g (4.2 mmol) of 8, 94 mg (0.42 mmol) of Pd(OAc)2,
0.22 g (0.84 mmol) of Ph3P, 1.8 g (4.2 mmol) of Ag3PO4,
and 1.98 g (0.24 mmol) of proton sponge in 60 mL of
DMF was stirred at 50 ꢁC under argon for 10 h. The
solution was cooled to 25 ꢁC, diluted with water
(200 mL), and extracted three times with dichlorometh-
ane (50 mL each). The combined organic layer was
washed with water, and brine, dried (MgSO4), concen-
trated, and column-chromatographed on silica gel using
a gradient mixture of hexane and diethyl ether as eluant
1
to give 0.28 g (61% yield) of 9: H NMR (CDCl3)d 6.21
(d, J = 9 Hz, 1H), 5.81 (d, J = 9 Hz, 1H), 4.88 (s, 2H),
4.40–4.30 (m, 1H), 2.60–2.20 (m, 2H), 2.10–1.60 (m,
2H), 1.56 (bs, 1H); 13C NMR (CDCl3) d 142.1, 132.2,
131.4, 112.9, 66.1, 32.4, 27.3; HRMS calcd for C7H11O
(M+H+) 111.0804, found 111.0811.
1
based on 6) of 8 a colorless liquid. H NMR d 6.35 (d,
J = 8 Hz, 1H), 6.27 (t, J = 8 Hz, 1H), 5.90–5.70 (m,
1H), 5.07 (dd, J = 18, 4 Hz, 1H), 4.98 (dd, J = 12,
4 Hz, 1H), 4.43 (q, J = 8 Hz, 1H), 2.25–2.05 (m, 2H),
1.80–1.50 (m, 31H); 13C NMR d 143.4, 138.2, 115.4,
82.7, 74.2, 35.1, 29.5; HRMS calcd for C7H12IO
(M+H+) 238.9927, found 238.9931.
Silylation of 9 with tert-butyldimethylsilyl chloride,
imidazole, and DMAP in dichloromethane gave a 97%
yield of 3.
4.1.5. (3R*,6R*) and (3R*,6S*)-6-(tert-Butyldimethylsilyl-
oxy)-1-oxaspiro[2.5]oct-4-ene (10 and 11). A three-
necked round-bottom flask was equipped with a
dry-ice reflux condenser and a stir bar and maintained
under argon. To it, were added 10.5 g (46.8 mmol) of
4.1.2. (Z)-1-Iodo-3-(tert-butyldimethylsilyloxy)-1,6-hept-
adiene (4). To a cold (0 ꢁC) solution of 5.6 g (23 mmol)
of alcohol 8 in 40 mL of DMF under argon, were added
4.8 g (71 mmol) of imidazole, 2.0 g (16.4 mmol) of 4-
dimethylaminopyridine (DMAP), and 4.6 g (31 mmol)
of tert-butyldimethylsilyl chloride. The reaction solution
was stirred at 25 ꢁC for 12 h, diluted with 100 mL of
water, and extracted twice with diethyl ether (50 mL
each). The organic layer was washed with 1 N HCl
(30 mL), aqueous NaHCO3, and brine, dried (anhy-
drous Na2SO4), concentrated, and column-chromato-
graphed on silica gel using a gradient mixture of
hexane and diethyl ether as eluant to give 8.1 g (98%
yield) of 4. 1H NMR d 6.19 (s, 2H), 5.90–5.75 (m,
1H), 5.03 (d, J = 18 Hz, 1H), 4.92 (d, J = 12 Hz, 1H),
4.31 (q, J = 6 Hz, 1H), 2.25–2.05 (m, 2H), 1.70–1.50
(m, 2H), 0.88 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H); 13C
NMR d 144.8, 138.6, 114.9, 80.07, 75.2, 36.21, 29.4,
26.06, 18.3, ꢀ3.98. HRMS calcd for C13H26IOSi
(M+H+) 353.0792, found 353.0799.
3
and 125 mL of EDTA (0.4 mM)/CH3CN/THF
(9:8.8:2.2), and the solution was cooled to 0 ꢁC. Dry
ice and acetone were added to the condenser to maintain
at ꢀ78 ꢁC. To the above solution, were added 19.7 g
(0.23 mol) of sodium bicarbonate and 2.53 mL
(26.9 mmol) of 1,1,1-trifluoroacetone. Oxone (14.4 g,
23.4 mmol) was added to the solution over 20 min in
portions. The resulting mixture was stirred at 0 ꢁC for
1 h, added 2.53 mL (26.9 mmol) of 1,1,1-trifluoroace-
tone, and stirred 0 ꢁC for 6 h. The reaction was moni-
tored by TLC, and a small amount of 3 remained.
Prolonged reaction time led to the decomposition of
the products. The mixture was diluted with CH2Cl2
and aqueous Na2SO4, and the organic layer was sepa-
rated, washed with brine, dried (anhydrous Na2SO4),
concentrated, and column-chromatographed on silica
gel using a 1:1 mixture of hexane and diethyl ether as
eluant. The silica gel was pretreated with hexane con-
taining 1% of triethylamine by sonication for 1 h. The
chromatography provided 9.10 g (82% yield) of a mix-
ture of epoxides 10 and 11 (1:1) and 0.95 g (9% recovery)
of 3. The ratio of the two isomeric products was deter-
mined from their 1H NMR spectrum. 1H NMR (of
4.1.3. 3-Methylene-6-(tert-butyldimethylsilyloxy)-1-cyclo-
hexene (3). A mixture of 0.42 g (1.18 mmol) of 4,
27 mg (0.12 mmol) of Pd(OAc)2, 63 mg (0.24 mmol) of
PPh3, 0.49 g (1.18 mmol) of Ag3PO4, and 0.51 g
(2.36 mmol) of proton sponge was dried under vacuum
and maintained under argon. To it was added 5 mL of
DMF, and the solution was stirred at 50 ꢁC for 8 h, di-
luted with water, and extracted with diethyl ether. The
combined organic layer was washed with water, and
brine, dried (MgSO4), concentrated, and column-chro-
matographed on silica gel using hexane as an eluant
to give 0.25 g (93% yield) of 3:16 1H NMR d 6.12 (d,
J = 8 Hz, 1H), 5.71 (d, J = 8 Hz, 1H), 4.80 (d,
J = 8 Hz, 2H), 4.40–4.30 (m, 1H), 2.55–2.45 (m, 1H),
one isomer)
d 6.0 (d, J = 10 Hz, 1H), 5.22 (t,
J = 10 Hz, 1H), 4.35–4.25 (m, 1H), 2.84 (d, J = 5 Hz,
1H), 2.76 (d, J = 5 Hz, 1H), 1.95–1.85 (m, 2H), 1.80–
1.60 (m, 2H), 0.89 (s, 9H), 0.03 (s, 3H), 0.029 (s, 3H);
1H NMR (of another isomer) d 6.00 (d, J = 10 Hz,
1H), 5.22 (t, J = 10 Hz, 1H), 4.35–4.25 (m, 1H), 2.84
(d, J = 5 Hz, 1H), 2.76 (d, J = 5 Hz, 1H), 1.95–1.85
(m, 2H), 1.80–1.60 (m, 2H), 0.89 (s, 9H), 0.03 (s, 3H),

5240
D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
0.029 (s, 3H); 13C NMR (two isomers) d 139.4, 138.2,
130.0, 128.8, 66.8, 65.9, 55.3, 54.9, 31.7, 30.9, 27.8,
26.0, 18.3, ꢀ4.4, ꢀ4.48. HRMS calcd for C13H25O2Si
(M+H+) 241.1624, found 241.1631.
(br s, 2H), 1.95–1.87 (m, 1H), 1.75–1.60 (m, 3H), 0.91
(s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13C NMR (CDCl3)
d 74.8, 70.36, 70.01, 59.6, 51.1, 28.3, 26.3, 25.8, 17.9,
ꢀ4.7, ꢀ4.8; HRMS calcd for C13H27O4Si (M+H+)
275.1679, found 275.1676.
4.1.6. (3S*,4S*,5R*,6R*)-6-(tert-Butyldimethylsilyloxy)-
1-oxaspiro[2.5]octane-4,5-diol (12) and (3S*,4R*,5S*,6S*)-
6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octane-4,5-
diol (13). To a solution of 9.0 g (37.4 mmol) of a mixture
of epoxides 10 and 11 in 665 mL of acetone–water (3:1),
was added 90 mL of tert-butanol. To it, were added
13.1 g (0.112 mol) of N-methylmorpholine-N-oxide
(NMO) and 0.50 g (1.97 mmol) of osmium tetroxide,
and the solution was stirred at 25 ꢁC for 36 h. The solu-
tion was distilled under vacuum to remove acetone, di-
luted with water, and extracted twice with CH2Cl2.
The organic layer was washed with brine, dried (anhy-
drous Na2SO4), concentrated, and column-chromato-
graphed on silica gel using a mixture of hexane and
diethyl ether (1:1) as eluant to give 8.71 g (85% yield)
of diols 12 and 13 in 1:1 ratio. The ratio of the two iso-
meric products was determined from their 1H NMR
spectrum. These two isomers can be separated partially
by column chromatography at this stage. Based on
NMR spectra of pure diol 13 obtained in the following
reaction (vide infra), NMR spectral data of diol 12 are
deduced (by subtracting signals of 13). Compound 12:
1H NMR d 3.42–3.92 (m, 1H), 3.86 (dd, J = 7.6,
3.2 Hz, 1H), 3.88–3.66 (m, 1H), 2.84 (d, J = 5 Hz, 1H),
2.45 (d, J = 5 Hz, 1H), 1.90–1.80 (m, 2H), 1.60–1.40
(m, 2H), 0.91 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13C
NMR d 75.8, 71.4, 71.2, 59.0, 49.7, 28.8, 26.9, 26.0,
14.4, ꢀ4.3, ꢀ4.5; HRMS calcd for C13H27O4Si
(M+H+) 275.1679, found 275.1676.
4.1.8. (3S*,4R*,6R*)-4-Benzoyloxy-6-(tert-butyldimethyl-
silyloxy)-1-oxaspiro[2.5]-5-octanone (15). To 33 mg
(0.087 mmol) of alcohol 14 under argon, was added a
solution of 97 mg (0.35 mmol) of IBX in 2 mL of
DMSO, and the solution was stirred at 25 ꢁC for 12 h.
The solution was diluted with diethyl ether, washed with
water, and brine, dried (MgSO4), concentrated, and
column-chromatographed on silica gel using a gradient
mixture of hexane and diethyl ether as eluant to give
31 mg (95% yield) of 15. 1H NMR d 8.12–8.07 (m,
2H), 7.64–7.60 (m, 1H), 7.50–7.46 (m, 2H), 6.24 (s,
1H), 4.37 (t, J = 3.4 Hz, 1H), 3.04 (d, J = 4.4 Hz,
1H), 2.76 (d, J = 4.4 Hz, 1H), 2.15 ꢁ 2.03 (m, 2H),
1.55–1.51 (m, 1H), 1.47–1.43 (m, 1H); 13C NMR d
201.0, 165.2, 133.7, 130.2, 129.3, 128.7, 75.5, 72.7,
61.9, 50.1, 31.2, 26.7, 25.9, 18.3, ꢀ4.7, ꢀ5.0; HRMS
calcd for C20H28O5SiNa (M+Na+) 399.1598, found
399.1605.
4.1.9. (3S*,4S*,5S*,6R*)-4-Benzoyloxy-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]-5-octanol (16). To a cold
(ꢀ78 ꢁC) solution of 11 mg (0.03 mmol) of 15 in 0.5 mL
of THF under argon, was added 46 lL (0.046 mmol) of
diisobutylaluminum hydride (1 M in toluene). The solu-
tion was stirred at ꢀ78 ꢁC for 8 h, added 30 lL of acetic
acid, and diluted with diethyl ether. The organic layer
was washed with water, and brine, dried (MgSO4), con-
centrated, and column-chromatographed on silica gel
using a gradient mixture of hexane and diethyl ether
1
4.1.7. (3S*,4S*,5R*,6R*)-4-Benzoyloxy-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]-5-octanol (14) and (3S*,
4R*,5S*,6S*)-6-(tert-butyldimethylsilyloxy)-1-oxaspiro-
to give 9.6 mg (85% yield) of 16. H NMR d 8.09–8.05
(m, 2H), 7.58–7.54 (m, 1H), 7.47–7.43 (m, 2H), 5.54
(d, J = 10 Hz, 1H), 4.29–4.25 (m, 1H), 3.90 (td, J = 10,
3 Hz, 1H), 2.87 (d, J = 4.8 Hz, 1H), 2.67 (d,
J = 4.4 Hz), 2.22–2.18 (m, 2H), 1.90–1.86 (m, 2H), 0.97
(s, 9H), 0.15 (s, 3H), 0.14 (s, 3H); 13C NMR d 165.6,
133.5, 130.1, 130.0, 128.7, 77.4, 73.7, 71.7, 71.5,
50.6, 28.5, 26.8, 26.0, 18.3, ꢀ4.8, ꢀ4.9; HRMS calcd
for C20H30O5SiNa (M+Na+) 401.1760, found
401.1752.
[2.5]octane-4,5-diol (13). To
a solution of 6.7 g
(24.5 mmol) of mixture of 12 and 13 (1:1) in 150 mL
THF under argon at 0 ꢁC, were added 10.2 mL
(73 mmol) of triethylamine and 3.1 mL (27 mmol) of
benzoyl chloride. The solution was stirred at 0 ꢁC for
1 h and 25 ꢁC for 12 h, diluted with dichloromethane,
and washed with 1 N HCl. The organic layer was
washed with aqueous NaHCO3, and brine, dried (anhy-
drous Na2SO4), concentrated, and column-chromato-
graphed on silica gel using a mixture of hexane and
ethyl acetate (1:1) as eluant to give 3.97 g (43% yield)
of 14 and 3.3 g (49% recovery) of 13.
4.1.10. (3S*,4S*,5S*,6R*)-4-Benzoyloxy-5-methoxy-6-
(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]-octane (17).
A solution of 27 mg (68 lmol) of 16, 35 mg (0.16 mmol)
of proton sponge, and 12 mg (82 lmol) of Me3OBF4 in 2
mL of dichloromethane under argon was stirred at 0 ꢁC
for 10 h, diluted with diethyl ether, washed with water,
and brine, dried (anhydrous Na2SO4), concentrated,
and column-chromatographed on silica gel using a gra-
dient mixture of hexane and diethyl ether as eluant to
give 23 mg (86% yield) of 17: 1H NMR d 8.10–8.06
(m, 2H), 7.59–7.55 (m, 1H) 7.49–7.45 (m, 2H), 5.59 (d,
J = 9 Hz, 1H), 4.34–4.30 (m, 1H), 3.50 (dd, J = 9,
3 Hz, 1H), 3.41 (s, 3H), 2.76 (d, J = 4.4 Hz, 1H), 2.65
(d, J = 4.4 Hz, 1H), 2.32–2.28 (m, 1H), 1.88–1.84 (m,
2H), 1.44–1.40 (m, 1H), 0.92 (s, 9H), 0.11 (s, 3H), 0.08
(s, 3H); 13C NMR d 166.1, 133.3, 130.1, 130.0, 128.6,
82.7, 70.9, 68.6, 59.1, 58.9, 51.1, 28.9, 27.0, 26.0, 18.4,
1
Compound 14: H NMR d 8.07 (d, J = 7 Hz, 2H), 7.58
(t, J = 7 Hz, 1H), 7.49 (t, J = 7 Hz, 2H), 5.49 (d,
J = 3 Hz, 1H), 4.20–4.10 (m, 1H), 4.00–3.95 (m, 1H),
2.82 (d, J = 5 Hz, 1H), 2.61 (d, J = 5 Hz, 1H), 2.25–
2.05 (m, 2H), 2.05 (s, 1H), 1.75–1.55 (m, 2H), 0.94 (s,
9 H), 0.13 (s, 3H), 0.12 (s, 3H); 13C NMR d 152.5,
141.5, 133.5, 130.0, 128.7, 74.5, 71.2, 70.6, 58.8, 49.6,
27.1, 26.8, 25.9, 18.2, ꢀ4.5, ꢀ4.7. HRMS calcd for
C20H30O5SiNa (M+Na+) 401.1760, found 401.1754.
1
Diol 13: H NMR d 4.02–3.95 (m, 1H), 3.84–3.80 (m,
2H), 2.94 (d, J = 5 Hz, 1H), 2.69 (d, J = 5 Hz), 2.50

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5241
ꢀ4.4, ꢀ4.9; HRMS calcd for C21H32O5SiNa (M+Na+)
Compound 20 (less polar isomer): 1H NMR d 4.00–3.90
(m, 1H), 3.85–3.75 (m, 1H), 3.42 (s, 3H), 3.35 (d,
J = 4 Hz, 1H), 2.93 (d, J = 5 Hz, 1H), 2.68 (d,
J = 5 Hz, 1H), 2.69 (d, J = 5 Hz, 1H), 1.95–1.85 (m,
1H), 1.70–1.60 (m, 3H), 0.91 (s, 9H), 0.11 (s, 3H),
0.089 (s, 3H); 13C NMR d 80.4, 74.3, 70.6, 58.6, 58.0,
51.9, 28.7, 26.6, 26.0, 18.2, ꢀ4.4, ꢀ4.6; HRMS calcd
for C14H29O4Si (M+H+) 289.1830, found 289.1826.
415.1917, found 415.1906.
4.1.11.
(3S*,4S*,5S*,6R*)-5-Methoxy-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (18). A solu-
tion of 23 mg (59 lmol) of 17 and 81 mg (0.59 mmol)
of potassium carbonate in 2 mL of methanol was stirred
at 0 ꢁC for 8 h, diluted with diethyl ether, washed with
water, and brine, dried (MgSO4), concentrated, and col-
umn-chromatographed on silica gel using a mixture of
hexane and diethyl ether (2:1) as eluant to give 16 mg
4.1.14. (3S*,4R*,5S*,6S*)-4,5-Dimethoxy-6-(tert-butyldi-
1
methylsilyloxy)-1-oxaspiro[2.5]octane. H NMR d 4.08–
1
(95% yield) of 18: H NMR d 4.34–4.30 (m, 1H), 4.10
3.98 (m, 1H), 3.45 (s, 3H), 3.41 (s, 3H), 3.39–3.35 (m,
1H), 3.35–3.30 (m, 1H), 2.95 (d, J = 5 Hz, 1H), 2.71
(d, J = 5 Hz, 1H), 1.90–1.80 (m, 1H), 1.70–1.50 (m,
3H), 0.91 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H); 13C NMR
d 83.9, 79.7, 69.1, 59.1, 58.6, 58.3, 52.3, 29.3, 26.5,
26.0, 18.3, ꢀ4.54, ꢀ4.59; HRMS calcd for C15H31O4Si
(M+H+) 303.1992, found 303.1997.
(dd, J = 9, 6 Hz, 1H), 3.44 (s, 3H), 3.13 (d, J = 4.7 Hz,
1H), 3.07 (dd, J = 9, 2.2 Hz, 1H), 2.62 (d, J = 4.7, 1H),
2.37–2.33 (m, 1H), 2.29–2.25 (m, 1H), 1.99 (d, J = 6.4,
1H), 1.80 ꢁ 1.68 (m, 1H), 1.25–1.21 (m, 1H), 0.91 (s,
9H), 0.11 (s, 3H), 0.08 (s, 3H); 13C NMR d 85.5, 67.6,
66.9, 60.2, 57.8, 50.1, 29.0, 26.5, 26.0, 18.3, ꢀ4.5, ꢀ4.8;
HRMS calcd for C14H29O4Si (M+H+) 289.1830, found
289.1837.
4.1.15. (3S*,4R*,5S*,6S*)-4-Benzoyloxy-5-methoxy-6-
(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]-octane (21).
To a solution of 0.123 g (0.43 mmol) of 19 in 2 mL of
pyridine under argon, was added 0.11 mL (0.86 mmol)
of benzoyl chloride. After stirring at 40 ꢁC for 15 min,
the solution was diluted with diethyl ether, washed with
aqueous NH4Cl, aqueous NaHCO3, water, and brine,
dried (MgSO4), concentrated, and column-chromato-
graphed on silica gel using a mixture of hexane and
diethyl ether (4:1) as eluant to give 0.164 g (97% yield)
of 21: 1H NMR d 8.02 (d, J = 7.0 Hz, 2H), 7.55 (t,
J = 7.0 Hz, 1H), 7.45 (t, J = 7 Hz, 2H), 5.34 (d,
J = 2.9 Hz, 1H), 4.09 (td, J = 6.6, 3.3 Hz, 1H), 3.50
(dd, J = 6.6, 2.9 Hz, 1H), 3.42 (s, 3H), 3.04 (d, J = 5.1
Hz, 1H), 2.68 (d, J = 5.1 Hz, 1H), 2.00–1.90 (m, 1H),
1.82–1.60 (m, 3H), 0.93 (s, 9H), 0.12 (s, 3H), 0.11 (s,
3H); 13C NMR d 165.7, 133.3, 130.3, 129.9, 128.6,
83.4, 72.1, 69.5, 59.0, 57.7, 52.1, 29.3, 27.0, 26.0, 18.3,
ꢀ4.5, ꢀ4.7; HRMS calcd for C21H32O5SiNa (M+Na+)
415.1917, found 415.1920.
4.1.12. (3S*,5R*,6R*)-5-Methoxy-6-(tert-butyldimethylsi-
lyloxy)-1-oxaspiro[2.5]octan-4-one (2). A solution of
41 mg (0.14 mmol) of 18 and 0.16 g (0.56 mmol) of
IBX in 5 mL of DMSO under argon was stirred at
25 ꢁC for 12 h, diluted with diethyl ether, washed with
water, and brine, dried (MgSO4), and concentrated to
give 39.9 mg (98% yield) of 2.12 This material was used
in the next step without purification. Its proton and car-
bon-13 NMR spectral data are similar to those re-
ported.12 1H NMR d 4.46–4.42 (m, 1H), 3.98 (d,
J = 2.6 Hz, 1H), 3.44 (s, 3H), 3.29 (d, J = 4.7 Hz, 1H),
2.77 (d, J = 4.7 Hz, 1H), 2.52–2.48 (m, 1H), 2.10–2.0
(m, 2H), 1.58–1.54 (m, 1 H), 0.92 (s, 9H), 0.08 (s, 3H),
0.07 (s, 3H); 13C NMR d 202.48, 87.5, 72.3, 60.7, 58.5,
51.4, 28.9, 27.1, 25.9, 18.3, ꢀ4.4, ꢀ5.0; HRMS
calcd for C14H26O4SiNa (M+Na+) 309.1498, found
309.1499.
4.1.13. (3S*,4R*,5S*,6S*)-5-Methoxy-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]octan-4-o1 (19), (3S*,4R*,
5S*,6S*)-4-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxa-
spiro[2.5]octan-5-o1 (20), and (3S*,4R*,5S*,6S*)-4,5-
dimethoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]-
octane. A solution of 0.27 g (0.97 mmol) of diol 13,
0.50 g (2.3 mmol) of proton sponge, and 0.17 g
(1.2 mmol) of Me3OÆBF4 in 5 mL of dichloromethane
was stirred at 0 ꢁC for 10 h under argon, diluted with
diethyl ether, washed with water, and brine, dried (anhy-
drous Na2SO4), concentrated, and column-chromato-
graphed using a mixture of hexane and ethyl acetate
(3:1) as eluant to give 0.123 g (44% yield) of 19, 0.103
g (37% yield) of 20 and 18 mg (6% yield) of the dimethy-
lated by-product.
4.1.16. (3S*,4R*,5S*,6S*)-4-Benzoyloxy-5-methoxy-1-
oxaspiro[2.5]-6-octanol (22). To a cold (0 ꢁC) solution of
0.15 g (0.39 mmol) of 21 in 3 mL of THF under argon,
was added 0.78 mL (0.78 mmol) of tetra-n-butylammo-
nium fluoride in THF (1 M solution). The solution
was stirred at 0 ꢁC for 4 h, diluted with diethyl ether,
washed with water, and brine, dried (MgSO4), concen-
trated, and column-chromatographed on silica gel using
a gradient mixture of hexane and diethyl ether as eluant
1
to give 99 mg (92% yield) of 22: H NMR d 8.02 (d,
J = 7 Hz, 2H), 7.55 (t, J = 7 Hz, 1H), 7.45 (t, J = 7 Hz,
2H), 5.09 (d, J = 3 Hz, 1H), 4.18–4.08 (m, 1H), 3.43 (s,
3H), 3.39 (d, J = 3 Hz, 1H), 2.96 (d, J = 4.4 Hz, 1H),
2.73 (d, J = 4.4 Hz, 1H), 2.35–2.25 (m, 1H), 2.22–2.12
(m, 1H), 1.90–1.70 (m, 1H), 1.40–1.20 (m, 1H); 13C
NMR d165.6, 133.6, 130.0, 129.8, 128.7, 83.9, 72.1,
68.7, 57.6, 57.5, 53.2, 28.2, 26.7; HRMS calcd for
C15H19O5 (M+H+) 279.1227, found 279.1233.
1
Compound 19 (more polar isomer): H NMR d 4.03–
3.99 (m, 1H), 3.84–3.80 (m, 1H), 3.45 (s, 3H, OMe),
3.38 (dd, J = 6.2, 3.0 Hz, 1H), 2.91 (d, J = 4.8 Hz, 1H),
2.67 (d, J = 4.8 Hz, 1H), 2.32 (br s, 1H), 1.92–1.88 (m,
1H), 1.70 ꢁ 1.60 (m, 3H), 0.91 (s, 9H), 0.10 (s, 3H),
0.09 (s, 3H); 13C NMR d 84.5, 70.3, 68.6, 59.2, 58.8,
51.5, 29.1, 26.2, 25.9, 18.3, ꢀ4.6; HRMS calcd for
C14H29O4Si (M+H+) 289.1830, found 289.1841.
4.1.17. (3S*,4R*,5S*)-4-Benzoyloxy-5-methoxy-1-oxaspi-
ro[2.5]-6-octanone (23). To a solution of 71 mg
(0.25 mmol) of 22 in 2 mL of DMSO under argon,
was added 0.29 g (1.0 mmol) of IBX, and the solution

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D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
was stirred at 25 ꢁC for 12 h, diluted with diethyl ether,
washed with water, and brine, dried (MgSO4), concen-
trated, and column-chromatographed on silica gel using
a gradient mixture of hexane and diethyl ether as eluant
J = 8 Hz, 1H), 3.44 (s, 3H), 3.42–3.38 (m, 1H), 3.31–
3.27 (m, 1H), 2.83 (d, J = 4.4 Hz, 1H), 2.74 (d,
J = 4.4 Hz, 1H), 2.59–2.40 (m, 1H), 1.90–1.70 (m, 2H),
1.15–1.02 (m, 1H), 0.92 (s, 9H), 0.15 (s, 3H), 0.12 (s,
3H); 13C NMR d 79.0, 74.0, 70.5, 59.9, 56.7, 53.2,
29.0, 25.9, 22.3, 18.2, ꢀ4.8, ꢀ4.9; HRMS calcd for
C14H29O4Si (M+H+) 289.1830, found 289.1824.
1
to give 66 mg (93% yield) of 23: H NMR d 7.80–7.96
(m, 2H), 7.56–7.52 (m, 1H), 7.45–7.41 (m, 2H), 5.25
(dd, J = 3.7, 1.8 Hz, 1H), 4.33 (dd, J = 3.7, 1.1 Hz,
1H), 3.48 (s, 3H), 3.21 (d, J = 4.4 Hz, 1H), 2.93 (dd,
J = 4.4 Hz, 1H), 2.80–2.76 (m, 1H), 2.66–2.62 (m, 2H),
1.65–1.50 (m, 1H); 13C NMR d 205.1, 165.4, 133.8,
130.1, 129.3, 128.7, 83.1, 58.5, 57.2, 54.5, 37.1, 29.9,
28.7; HRMS calcd for C15H17O5 (M+H+) 277.1071,
found 277.1079.
Oxidation of 26 with IBX in DMSO gave a 99% yield of
2, whose NMR spectra are identical to those derived
from 18 (vide supra).
4.1.21. (3S*,4R*,5R*,6R*)-5-Methoxy-4-[(E)-(10,50-dim-
ethylhexa-10,40-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-
oxaspiro[2.5]octan-4-ol (28). Alkenyllithium 27 was pre-
pared by following the reported procedure.6,8,10,12 To a
cold (ꢀ78 ꢁC) solution of 21 mg (73 lmol) of 2 in
1 mL of toluene under argon, was added 1.22 mL
(0.146 mmol) of 27, and the solution was stirred at
ꢀ78 ꢁC for 2 h, warmed to 0 ꢁC, diluted with diethyl
ether, washed with water, and brine, dried (MgSO4),
concentrated, and column-chromatographed on silica
gel using a mixture of hexane and diethyl ether (2:1) as
4.1.18.
(3S*,4R*,5S*,6R*)-4-Benzoyloxy-5-methoxy-1-
oxaspiro[2.5]-6-octanol (24). To a cold (ꢀ78 ꢁC) solution
of 33 mg (0.12 mmol) of 23 in 0.5 mL of THF under ar-
gon, was added 0.14 mL (0.14 mmol) of K-Selectride (1
M solution in THF), and the solution was stirred at
ꢀ78 ꢁC for 2 h, diluted with diethyl ether, washed with
water, and brine, dried (MgSO4), concentrated, and col-
umn-chromatographed on silica gel using a mixture of
hexane and ethyl acetate (3:1) as eluant to give 31 mg
1
1
(93% yield) of 24: H NMR d 8.10–8.06 (m, 2H), 7.65–
eluant to give 22.1 mg (75% yield) of 288,12: H NMR
7.61 (m, 1H), 7.48–7.44 (m, 2H), 5.18 (d, J = 3.3 Hz,
1H), 4.20–4.10 (m, 1H), 3.71 (t, J = 3.3 Hz, 1H), 3.50
(s, 3H), 3.02 (d, J = 4.7 Hz, 1H), 2.71 (d, J = 4.7 Hz,
1H), 2.48 (d, J = 5 Hz, 1H), 2.24–2.16 (m, 1H), 2.05–
1.90 (m, 2H), 1.50–1.30 (m, 1H); 13C NMR d 165.7,
133.5, 130.0, 129.8, 128.8, 79.9, 72.5, 68.1, 58.8, 57.7,
52.4, 27.8, 24.8; HRMS calcd for C15H19O5 (M+H+)
279.1227, found 279.1235.
d 5.71 (t, J = 7 Hz, 1H), 5.20–5.13 (m, 1H), 4.86 (br s,
1H), 4.50–4.40 (m, 1H), 3.51 (d, J = 2.6 Hz, 1H), 3.45
(s, 3H), 2.81 (d, J = 5.1 Hz, 1H), 2.80–2.70 (m, 2H),
2.60–2.45 (m, 1H), 2.42 (d, J = 5.1 Hz, 1H), 1.9 ꢁ 1.75
(m, 2H), 1.67 (s, 6H), 1.61 (s, 3 H), 1.22–1.18 (m, 1H),
0.92 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H); 13C NMR d
132.7, 131.7, 127.8, 123.0, 80.6, 79.3, 68.6, 62.2, 57.8,
50.7, 28.7, 27.3, 25.9, 25.8, 25.5, 18.1, 18.0, 14.2, ꢀ4.7,
þ
ꢀ4.8; HRMS calcd for C22H44O4SiN (M þ NH4
)
4.1.19. (3S*,4R*,5S*,6R*)-4-Benzoyloxy-5-methoxy-6-
(tert–butyldimethylsilyloxy)-1-oxaspiro[2.5]-octane (25).
To a cold (0 ꢁC) solution of 40 mg (0.14 mmol) of 24
in 2 mL of DMF under argon, were added 29 mg
(0.42 mmol) of imidazole, 2 mg of DMAP, and 33 mg
(0.22 mmol) of tert-butyldimethylsilyl chloride. The
solution was stirred at 0 ꢁC for 8 h, diluted with diethyl
ether, washed with water, and brine, dried (MgSO4),
concentrated, and column-chromatographed on silica
gel using a mixture of hexane and diethyl ether (4:1) as
eluant to give 55 mg (98% yield) of 25: 1H NMR d
8.06–8.02 (m, 2H), 7.59–7.55 (m, 1H), 7.46–7.42 (m,
2H), 5.24 (d, J = 2.6 Hz, 1H), 4.02–3.92 (m, 1H), 3.74
(t, J = 2.6 Hz, 1H), 3.54 (s, 3H), 3.10 (d, J = 5.1 Hz,
1H), 2.65 (d, J = 5.1 Hz, 1H), 2.10–1.90 (m, 1H), 1.82–
1.60 (m, 3H), 0.92 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H);
13C NMR d 152.6, 133.4, 130.1, 130.0, 128.6, 82.6,
71.6, 71.2, 61.0, 57.6, 51.3, 28.4, 27.9, 26.0, 18.4,
ꢀ4.48, ꢀ4.53; HRMS calcd for C21H32O5SiNa
(M+Na+) 415.1917, found 415.1916.
414.3040, found 414.3036.
4.1.22. (3S*,4R*,5R*,6R*)-5-Methoxy-4-[(E)-(10,50-dim-
ethylhexa-10,40-dienyl)]-1-oxaspiro[2.5]-octane-4,6-diol
(29).12 To a cold (0 ꢁC) solution of 12 mg (0.03 mmol)
of 28 in 1 mL of THF under argon, was added 60 lL
(0.06 mmol) of tetra-n-butylammonium fluoride (1 M
solution in THF), and the solution was stirred at 0 ꢁC
for 6 h, diluted with diethyl ether, washed with water,
and brine, dried (MgSO4), concentrated, and column-
chromatographed on silica gel using a gradient mixture
of hexane and diethyl ether to give 8.4 mg (99% yield) of
2912: 1H NMR d 5.65 (t, J = 7.0 Hz, 1H), 5.10 (th,
J = 7.0, 1.0 Hz, 1H), 4.40–4.30 (m, 1H), 3.61 (d,
J = 3.3 Hz, 1H), 3.49 (s, 3H), 3.26 (bs, 1H), 3.15 (br d,
J = 7 Hz, 1H), 2.79 (d, J = 5.1 Hz, 1H), 2.75 (t,
J = 7 Hz, 2H), 2.45 (d, J = 5.1 Hz, 1H), 2.50–2.35 (m,
1H), 2.10–2.00 (m, 1H), 1.90–1.80 (m, 1H), 1.68 (s,
6H), 1.62 (s, 3H), 1.30–1.20 (m, 1H); 13C NMR d
133.9, 132.3, 127.5, 122.6, 80.0, 79.9, 67.0, 61.5, 57.8,
50.4, 27.8, 27.3, 25.9, 25.2, 18.0, 14.3; HRMS calcd for
C16H30NO4 ðM þ NH4þÞ 300.2169, found 300.2171.
4.1.20. (3S*,4R*,5S*,6R*)-5-Methoxy-6-(tert-butyldi-
methylsilyloxy)-1-oxaspiro[2.5]octan-4-o1 (26). A cold
(0 ꢁC) solution of 18 mg (45 lmol) of 25 and 63 mg of
potassium carbonate in 2 mL of methanol was stirred
for 8 h, diluted with diethyl ether, washed with water,
and brine, dried (MgSO4), concentrated, and column-
chromatographed on silica gel using a mixture of hexane
and diethyl ether (2:1) as eluant to give 13 mg (96%
4.1.23. (3S*,4R*,5S*)-4-Hydroxy-5-methoxy-4-[(E)-(10,
50-dimethylhexa-10,40-dienyl)]-1-oxaspiro[2.5]octan-6-one
(30). A solution of 4.7 mg (17 lmol) of 29 and 19 mg
(67 lmol) of IBX in 0.5 mL of DMSO under argon
was stirred at 25 ꢁC for 12 h, diluted with diethyl ether,
washed with water, and brine, dried (MgSO4), concen-
trated, and column-chromatographed using a mixture
1
yield) of 26: H NMR d 4.32–4.26 (m, 1H), 4.20 (d,

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5243
1
of hexane and diethyl ether (2:1) to give 4.3 mg (92%
yield) of 30:12 1H NMR d 5.64 (t, J = 7 Hz, 1H), 5.10
(bt, J = 7 Hz, 1H), 4.26 (s, 1H), 3.50 (s, 3H), 2.85 (d,
J = 4.9 Hz, 1H), 2.80–2.62 (m, 4H), 2.61 (d,
J = 4.9 Hz, 1H), 2.53–2.42 (m, 2H), 1.70 (s, 6H), 1.62
(s, 3H), 1.60–1.48 (m, 1H); 13C NMR d 207.7, 133.8,
132.6, 127.9, 122.3, 85.9, 83.1, 61.1, 59.7, 51.2, 37.1,
30.5, 27.2, 25.9, 18.0, 14.6; HRMS calcd for
C16H28NO4 (M þ NH4þ) 298.2018, found 298.2020.
2.86–2.96 (m, 2H), 1.83–2.34 (m, 2H); H NMR d (for
another isomer) 8.08–8.04 (m, 2H), 7.59–7.55 (m, 1H),
7.47–7.43 (m, 2H), 6.16 (d, J = 11 Hz, 1H), 5.64–5.56
(m, 1H), 5.50 (t, J = 11 Hz, 1H), 2.95 (d, J = 5 Hz,
1H), 2.86 (d, J = 5 Hz, 1H), 2.86–2.96 (m, 2H), 1.83–
2.34 (m, 2H); 13C NMR (two isomers) d 166.3, 166.2,
133.6, 133.3, 133.29, 133.2, 130.5, 133.0, 132.8, 130.4,
129.9, 129.8, 128.6, 128.59, 68.3, 67.7, 55.2, 55.19,
55.1, 55.09, 27.4, 27.39, 27.3, 27.29; HRMS calcd for
C14H15O3 (M+H+) 231.1016, found 231.1022.
4.1.24. ( )-Ovalicin (1).8,10,12 To a cold (5 ꢁC) solution
of 4.0 mg (14 lmol) of 30 and 1.1 mg (4.3 lmol) of
VO(Acac)2 in 0.2 mL of benzene, was added 4.0 lL
(28 lmol) of t-BuOOH. The resulting brown solution
was stirred at 25 ꢁC for 1.5 h and subjected to column-
chromatographic separation using a mixture of hexane
and diethyl ether (2:1) as eluant to give 2.6 mg (62%
yield) of ( )-ovalicin:12 1H NMR d 5.18 (t, J = 6.6 Hz,
1H), 4.23 (s, 1H), 3.57 (s, 3 H), 3.14 (s, 1H), 3.10 (d,
J = 4.2 Hz, 1H), 2.90 (t, J = 6.3 Hz, 1H), 2.73 (d,
J = 4.2 Hz, 1H), 2.72–2.62 (m, 2H), 2.51–2.47 (m, 1H),
2.45–2.39 (m, 1H), 2.18–2.12 (m, 1H), 1.75 (s, 3H),
1.66 (s, 3H), 1.45–1.41 (m, 1H), 1.37 (s, 3H); 13C
NMR d 206.8, 135.7, 118.2, 86.3, 78.8, 60.7, 60.5, 59.5,
57.0, 51.5, 36.9, 30.5, 27.2, 25.9, 18.2, 14.6; HRMS calcd
for C16H24O5Na (M+Na+) 319.1522, found 319.1515.
4.1.27. (3S*,4R*,5S*,6S*)-6-(Benzoyloxy)-1-oxaspiro[2.5]-
octane-4,5-diol (34), (3S*,4S*,5R*,6R*)-6-(benzoyloxy)-1-
oxaspiro[2.5]octane-4,5-diol (35), and (3S*,4S*,5R*,6S*)-
6-(benzoyloxy)-1-oxaspiro[2.5]octane-4,5-diol (36). To a
solution of 1.00 g (4.3 mmol) of epoxides 32 and 33
(1:1) in 80 mL of acetone and water (3:1) and 3.5 mL
of tert-butanol, were added 1.64 g (14.0 mmol) of
NMO and 0.24 mg (0.93 mmol) of OsO4. The solution
was stirred at 25 ꢁC for 12 h, and acetone was removed
by vacuum distillation. The remaining solution was ex-
tracted with diethyl ether, and the organic layer was
washed with water, and brine, dried (anhydrous
Na2SO4), concentrated, and column-chromatographed
on silica gel using a gradient mixture of hexane and
diethyl ether as eluant to give 0.28 g (25% yield) of 34,
0.23g (20% yield) of 35, and 57 mg (5% yield) of 36. Ste-
reochemistry of 36 was determined from a NMR 2D
NOESY experiment.
4.1.25. 3-Methylene-6-(benzoyloxy)cyclohexene (31). To
a cold (0 ꢁC) solution of 0.64 g (5.8 mmol) of alcohol 9
and 4.8 mL (35 mmol) of triethylamine under argon,
was added 2.6 mL (15 mmol) of benzoyl chloride, and
the solution was slowly allowed to warm to 25 ꢁC and
stirred for 6 h, diluted with diethyl ether, washed with
1 N HCl, aqueous sodium bicarbonate, and brine, dried
(anhydrous Na2SO4), concentrated, and column-chro-
matographed on silica gel using a mixture of hexane
and diethyl ether (3:1) as eluant to give 1.09 g (88%
Compound 34: 1H NMR d 8.08–8.04 (m, 2H), 7.61–7.57
(m, 1H), 7.48–7.44 (m, 2H), 5.35 (td, J = 7.6, 3.6 Hz,
1H), 4.11 (dd, J = 7.6, 3.6 Hz, 1H), 3.73 (d, J = 3.6 Hz,
1H), 3.21 (s, 1H), 2.94 (d, J = 4.8 Hz, 1H), 2.76 (d,
J = 4.8 Hz, 1H), 2.2–2.0 (m, 2H), 1.9–1.6 (m, 2H); 13C
NMR d 166.6, 133.3, 129.8, 129.7, 128.4, 73.0, 72.8,
72.6, 58.7, 51.7, 26.0, 25.7; HRMS calcd for C14H17O5
(M+H+) 265.1076, found 265.1081.
1
yield) of 31: H NMR d 8.14–8.10 (m, 2H), 7.57–7.53
(m, 1H), 7.45–7.41 (m, 2H), 6.32 (d, J = 9.6 Hz, 1H),
5.90 (dd, J = 9.6, 3.6 Hz, 1H), 5.61 (q, J = 3.6 Hz, 1H),
4.95 (s, 2H), 2.70–2.50 (m, 1H), 2.47–2.43 (m, 1H),
2.21–2.11 (m, 1H), 1.02–1.92 (m, 1H); 13C NMR d
166.3, 141.5, 133.3, 133.0, 130.2, 129.7, 128.4, 127.5,
113.9, 68.7, 28.5, 26.9; HRMS calcd for C14H15O2
(M+H+) 215.1067, found 215.1070.
Compound 35: 1H NMR d 8.07–8.03 (m, 2H), 7.61–7.57
(m, 1H), 7.48–7.44 (m, 2H), 5.41 (td, J = 7.6, 4 Hz, 1H),
4.00 (dd, J = 7.6, 3.2 Hz, 1H), 3.86 (d, J = 3.2 Hz, 1H),
2.88 (d, J = 5 Hz, 1H), 2.65 (d, J = 5 Hz, 1H), 2.3–1.7
(m, 4H); 13C NMR (CDCl3) d 166.5, 133.5, 130.0,
129.9, 128.7, 73.6, 73.1, 72.8, 51.9, 50.6, 26.0, 24.6;
HRMS calcd for C14H17O5 (M+H+) 265.1076, found
265.1069.
4.1.26. (3R*,8R*) and (3R*,8S*)-6-(Benzoyloxy)-1-oxa-
spiro[2.5]oct-4-ene (32 and 33). To a cold (0 ꢁC) solution
of 1.0 g (4.7 mmol) of diene 31 in 25 mL of dichloro-
methane under argon, were added 0.33 g (7.9 mmol) of
sodium fluoride, 0.19 g (3.3 mmol) of potassium fluo-
ride, and 1.2 g (7.0 mmol) of meta-chloroperbenzoic
acid (MCPBA). The mixture was stirred at 25 ꢁC for
4 h, diluted with diethyl ether, washed with aqueous so-
dium thiosulfate, aqueous sodium bicarbonate, and
brine, dried (anhydrous Na2SO4), and concentrated to
give 1.0 g (93% yield) of a mixture of 32 and 33 (1:1;
based on integration in 1H NMR spectrum), which
was used in the following step without further purifica-
1
Compound 36: H NMR (CDCl3) d 8.07 (d, J = 7 Hz,
2H), 7.58 (t, J = 7 Hz, 1H), 7.46 (t, J = 7 Hz, 2H), 5.23
(dt, J = 9.2, 4 Hz, 1H, C6H), 4.38 (br s, 1H), 3.83 (d,
J = 2.5 Hz, 1H), 3.07 (d, J = 5 Hz, 1H, C2H), 2.71 (d,
J = 5 Hz, 1H, C2H), 2.27–2.17 (m, 1H), 2.25–1.75 (m,
1H), 1.60–1.56 (m, 2H); 13C NMR (CDCl3) d 166.3,
133.5, 130.3, 130.0, 128.7, 72.9, 71.3, 71.1, 58.9, 51.1,
26.7, 24.7; HRMS calcd for C14H17O5 (M+H+)
265.1076, found 265.1080.
4.1.28. (3S*,4R*,5S*,6S*)-6-Benzoyloxy-4-hydroxy-5-
(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]-octane (37).
To a cold (0 ꢁC) solution of 0.26 g (0.98 mmol) of diol
34 and 25 mg (0.20 mmol) of DMAP in 10 mL dichloro-
methane under argon, were added 0.28 mL (2.0 mmol)
1
tion. H NMR d (for one isomer) 8.08–8.04 (m, 2H),
7.59–7.55 (m, 1H), 7.47–7.43 (m, 2H), 6.18 (d,
J = 11 Hz, 1H), 5.66–5.60 (m, 1H), 5.50 (t, J = 11 Hz,
1H), 2.90 (d, J = 5 Hz, 1H), 2.88 (d, J = 5 Hz, 1H),

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D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
of Et3N and 0.23 g (1.5 mmol) of t-butyldimethylsilyl
chloride. The solution was stirred at 25 ꢁC for 6 h, di-
luted with aqueous NH4Cl, and extracted with ethyl ace-
tate. The organic layer was washed with 1 N HCl,
aqueous NaHCO3, and brine, dried (anhydrous
Na2SO4), concentrated, and column-chromatographed
on silica gel using a mixture of hexane and diethyl ether
(2:1) as eluant to give 0.196 g (53% yield) of 37: 1H
NMR d 8.05 (d, J = 7 Hz, 2H), 7.58 (t, J = 7 Hz, 1H),
7.45 (t, J = 7 Hz, 2H), 5.36 (td, J = 8, 4.4 Hz, 1H),
3.93 (td, J = 8, 3.2 Hz, 1H), 3.69 (d, J = 3.2 Hz, 1H),
2.91 (d, J = 4.8 Hz, 1H), 2.75 (d, J = 4.8 Hz, 1H), 2.34
(s, 1H), 2.18–2.12 (m, 1H), 2.12–2.08 (m, 1H), 1.95–
1.85 (m, 1H), 1.55–1.49 (m, 1H), 0.83 (s, 9H), 0.10 (s,
3H), 0.02 (s, 3H); 13C NMR (CDCl3) d 165.9, 133.2,
130.0, 129.8, 128.5, 73.6, 73.4, 72.9, 58.7, 51.8, 26.3,
25.8, 25.7, 18.0, ꢀ4.6, ꢀ4.7; HRMS calcd for
C20H30O5SiNa (M+Na+) 401.1760, found 401.1758.
trifluoroacetic anhydride, and the solution was stirred
for 0.5 h. To it, a solution of 16 mg (58 lmol) of 38 in
1 mL of dichloromethane was added, and the solution
was stirred at ꢀ78 ꢁC for 1 h, warmed to 0 ꢁC, diluted
with diethyl ether, washed with aqueous NH4Cl, and
brine, dried (anhydrous Na2SO4), concentrated, and col-
umn-chromatographed on silica gel using a mixture of
hexane and ethyl acetate (1:1) as eluant to give 13 mg
(83% yield) of compound 40: 1H NMR d 7.99 (d,
J = 8 Hz, 2H), 7.58 (t, J = 8 Hz 1H), 7.45 (t, J = 8 Hz,
2H), 5.5 (td, J = 6, 3 Hz, 1H), 3.88 (d, J = 6 Hz, 1H),
3.41 (s, 3H), 3.01 (d, J = 6 Hz, 1H), 2.9 (d, J = 6 Hz,
1H), 2.50–2.38 (m, 2H), 2.28–2.18 (m, 1H), 1.82–1.74
(m, 1H); 13C NMR d 203, 165, 137, 133.5, 129.7,
128.6, 83.9, 72.7, 59.0, 54.04, 29.9, 27.3, 24.4; HRMS
calcd for C15H17O5 (M+H+) 277.1071, found 277.1077.
4.1.31. (3S*,4S*,5R*,6S*)-6-Benzoyloxy-5-methoxy-4-[(E)-
(10,50-dimethylhexa-10,40-dienyl)]-1-oxaspiro[2.5]octan-4-
ol (41) and (3S*,4S*,5R*,6S*)-5-methoxy-4-[(E)-(10,50-
dimethylhexa-10,40-dienyl)]-1-oxaspiro[2.5]-4,6-octanediol
(42). To a cold (ꢀ78 ꢁC) solution of 49 mg (0.18 mmol) of
ketone 40 in 0.5 mL of diethyl ether and 0.5 mL of toluene
under argon, was added 2.7 mL (0.36 mmol) of alkenyl-
lithium 27, and the solution was stirred for 1 h, and slowly
warmed to 25 ꢁC. The reaction solution was diluted with
aqueous NH4Cl and extracted with CH2Cl2. The organic
layer was washed with brine, dried (anhydrous Na2SO4),
concentrated, and column-chromatographed on silica gel
using a gradient mixture of hexane and ethyl acetate as
eluant to give 28 mg (40% yield) of 41 and 18 mg (26%
yield) of 42.
Crystallization of 37 in diethyl ether gave white crystals,
mp 121–123 ꢁC, whose structure was determined by a
single-crystal X-ray analysis (Fig. 1).22
4.1.29.
(3S*,4R*,5S*,6S*)-6-Benzoyloxy-4-hydroxy-5-
methoxy-1-oxaspiro[2.5]octane (38) and (3S*,4R*,
5S*,6S*)-6-benzoyloxy-5-hydroxy-4-methoxy-1-oxaspi-
ro[2.5]octane (39). To a cold (0 ꢁC) solution of 0.19 g
(0.74 mmol) of diol 34 in 3 mL of dichloromethane un-
der argon, were added 0.31 g (1.5 mmol) of proton
sponge and 0.11 g (0.73 mmol) of Me3OBF4, and the
solution was stirred at 0 ꢁC for 7 h. To it, 20 mg
(0.14 mmol) of Me3OBF4 was added, and the solution
was stirred at 0 ꢁC for 1.5 h, diluted with diethyl ether,
washed with 1 N HCl, aqueous sodium dicarbonate,
and brine, dried (MgSO4), concentrated, and column-
chromatographed on silica gel using a mixture of hexane
and ethyl acetate (3:2) as eluant to give 93 mg (46%
yield) of compound 38 and 69 mg (34% yield) of 39.
1
Compound 41: H NMR d 8.08 (d, J = 9 Hz, 2H), 7.59
(t, J = 9 Hz, 1H), 7.56 (t, J = 9 Hz, 2H), 6.13 (t,
J = 7 Hz, 1H), 5.58 (td, J = 8, 4 Hz, 1H), 5.14 (t,
J = 7 Hz, 1H), 3.60 (s, 3H), 3.56 (d, J = 8 Hz, 1H), 3.3
(d, J = 5 Hz, 1H), 2.82 (d, J = 5 Hz, 1H), 2.82–2.74
(m, 2H), 2.25–2.15 (m, 1H), 1.714 (s, 3H), 1.712 (s,
3H), 1.65 (s, 3H); HRMS calcd for C23H34NO5
ðM þ NHþ4 Þ 404.2431, found 404.2435.
Compound 42: 1H NMR d 5.90 (t, J = 7 Hz, 1H, C20H),
5.11 (t, J = 7 Hz, 1H, C40H), 3.89 (td, J = 10, 4 Hz, 1H),
3.68 (s, 3H), 3.16 (d, J = 10 Hz, 1H), 3.14 (d, J = 5 Hz,
1H), 2.74 (d, J = 5 Hz, 1H), 2.68 (bs, 1H), 2.27 (s,
1H), 2.07–1.97 (m, 2H), 1.72 (s, 3H), 1.69 (s, 3H), 1.63
(s, 3H), 1.38–1.25 (m, 1H), 1.25–1.15 (m, 1H);13C
NMR d 132.8, 132.5, 131.1, 122.5, 93.5, 70.8, 62.7,
62.4, 55.8, 51.3, 28.9, 28.0, 27.5, 25.9, 18.1, 13.7; HRMS
calcd for C16H30NO4 ðM þ NHþ4 Þ 300.2169, found
300.2173.
Compound 38: 1H NMR d 8.08–8.04 (m, 2H), 7.61–7.57
(m, 1H), 7.49–7.45 (m, 2H), 5.43 (td, J = 8, 4 Hz, 1H),
3.77 (dd, J = 8, 4 Hz, 1H), 3.68 (dd, J = 7, 4 Hz, 1H),
3.49 (s, 3H), 2.93 (d, J = 4.8 Hz, 1H), 2.76 (d,
J = 4.8 Hz, 1H), 2.51 (d, J = 7 Hz, 1H), 2.15–2.09 (m,
2H), 1.85–1.79 (m, 1H), 1.60–1.52 (m, 1H); 13C NMR
d 166.0, 133.2, 129.9, 129.8, 128.6, 81.8, 71.3, 71.3,
58.8, 58.7, 51.9, 26.1, 26.09; HRMS calcd for
C15H19O5 (M+H+) 279.1227, found 279.1223.
Compound 39: 1H NMR d 8.09–8.05 (m, 2H), 7.58–7.54
(m, 1H), 7.47–7.43 (m, 2H), 5.32 (td, J = 9, 4 Hz, 1H),
3.99 (td, J = 9, 4 Hz, 1H), 3.46 (s, 3H), 3.18 (d,
J = 4 Hz, 1H), 2.92 (d, J = 4.8 Hz, 1H), 2.81 (d,
J = 4.8 Hz, 1H), 2.51 (br s, 1H), 2.20–2.14 (m, 2H),
1.90–1.80 (m, 1H), 1.43–1.37 (m, 1H); 13C NMR d
166.6, 133.2, 130.0, 129.4, 128.6, 83.0, 73.3, 72.8, 58.4,
57.2, 52.7, 26.3, 26.2; HRMS calcd for C15H19O5
(M+H+) 279.1227, found 279.1236.
Treatment of 41 with 5 equiv of K2CO3 in methanol
afforded a 95% yield of compound 42.
4.1.32. (3S*,4S*,5S*)-4-Hydroxy-5-methoxy-4-[(E)-(10,
50-dimethylhexa-10,40-dienyl)]-1-oxaspiro[2.5]-6-octanone
(43). To a solution of 7 mg (25 lmol) of 42 in 1 mL
DMSO under argon was added 14 mg (50 lmol) of
IBX, and the solution was stirred at 25 ꢁC for 5 h, di-
luted with aqueous NH4Cl, and extracted with diethyl
ether. The organic layer was washed with brine, dried
4.1.30. (3S*,5R*,6S*)-6-(Benzoyloxy)-5-methoxy-1-oxa-
spiro[2.5]-4-octanone (40). To a cold (ꢀ78 ꢁC) solution
of 14 mg (0.17 mmol) of DMSO in 1.5 mL of dichloro-
methane under argon was added 30 mg (0.15 mmol) of

D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
5245
(anhydrous Na2SO4), concentrated, and column-chro-
matographed on silica gel using a mixture of hexane
and diethyl ether (1:1) as eluant to give 4 mg (58% yield)
of 43: 1H NMR d 5.76 (t, J = 7 Hz, 1H), 5.02 (t,
J = 7 Hz, 1H), 4.01 (s, 1H), 3.58 (s, 3H), 3.43 (d,
J = 5 Hz, 1H), 2.91 (d, J = 5 Hz, 1H), 2.75–2.67 (m,
2H), 2.54–2.46 (m, 1H), 2.42 (s, 1H), 2.25–2.15 (m,
2H), 1.67 (s, 3H, CH3), 1.63 (s, 3H), 1.59 (s, 3H), 1.45
(ddd, J = 14, 7, 2 Hz, 1H); 13C NMR d 209.2, 132.5,
132.3, 131.4, 121.7, 91.0, 79.2, 61.5, 61.1, 52.2, 36.8,
27.5, 27.2, 25.8, 18.1, 12.5; HRMS calcd for
C16H28NO4 ðM þ NHþ4 Þ 298.2018, found 298.2015.
chromatographed on silica gel using a mixture of hexane
and diethyl ether (2:1) as eluant to give 25 mg (72%
yield) of 46: HNMR d 5.75 (td, J = 7.0, 1.1 Hz, 1H,
1
C20H), 5.18–5.08 (m, 1H, C40H), 4.28 (dd, J = 10,
5 Hz, 1H, C6H), 3.29 (s, 3H, OMe), 2.75 (d,J = 4.8
Hz, 1H, C2H), 2.78–2.70 (m, 2H, C30H), 2.73 (s, 1H,
C4H), 2.62 (d, J = 4.8 Hz, 1H, C2H), 2.28 (s, 1H,
OH), 2.26 ꢁ 1.84 (m, 3H), 1.76 (s, 3H, CH3C@), 1.67
(s, 3H, CH3C@), 1.62 (s, 3H, CH3C@), 1.24–1.16 (m,
1H), 0.84 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H); 13C NMR
d 138.1, 131.5, 126.1, 123.2, 88.5, 78.4, 70.1, 59.2, 56.4,
50.5, 28.0, 27.4, 26.6, 25.9, 25.87, 18.2, 17.9, 14.4,
ꢀ3.11, ꢀ4.76; HRMS calcd for C22H44O4SiN
(M þ NH4þ) 414.3040, found 414.3031.
4.1.33. (3S*,4S*,5S*,10S*,20R)-4-Hydroxy-5-methoxy-4-
[20-methyl-30-(300-methylbut-200-enyl)oxiran-20-yl-1-
oxaspiro[2.5]-6-octanone (44). To a cold (0 ꢁC) solution
of 3.5 mg (13 lmol) of 43 in 0.5 mL of toluene under ar-
gon, were added 0.3 mL (2.5 lmol) of vanadyl aceto-
acetonate (19 mM solution in toluene) and 0.15 mL
(20 lmol) of tert-butylhydroperoxide in toluene
(0.1 mL of t-BuOOH in 10 mL of toluene). The solution
was stirred at 0 ꢁC for 3 h, subjected to a silica gel col-
umn, and eluted with a mixture of hexane and ethyl ace-
The stereochemistry was determined from a 2D NOESY
NMR spectrum of 46.
4.2. Culturing of parasites
The bloodstream form of Trypanosoma brucei 427 strain
was maintained under the standard cell culture condi-
tions (37 ꢁC, 5% CO2). The parasites were grown in
complete HMI-9 medium containing 10% FBS, 10%
Serum Plus and 1 · Penicillin/Streptomycin.20,21
1
tate (1:1) to give 2.4 mg (65% yield) of 44: H NMR d
5.10 (t, J = 8 Hz, 1H), 3.85 (s, 1H), 3.62 (t, J = 6 Hz,
1H), 3.51 (s, 3H), 3.38 (d, J = 5 Hz, 1H), 2.95 (d,
J = 5 Hz, 1H), 2.74–2.66 (m, 1H), 2.62 (s, 1H, OH),
2.58–2.48 (m, 2H), 2.34–2.26 (m, 1H), 2.18–2.08 (m,
1H), 1.714 (s, 3H), 1.711 (s, 3H), 1.32 (s, 3H), 1.57–
1.50 (m, 1H); 13C NMR (CDCl3) d 212.5, 130.9, 128.0,
88.4, 86.5, 61.0, 60.4, 59.5, 53.0, 51.8, 36.5, 29.9, 27.7,
25.6, 22.2, 13.4; HRMS calcd for C16H24O5Na
(M+Na+) 319.1522, found 319.1518.
4.3. Luciferase assay20,21
Luciferase assay was used to measure ATP-biolumines-
cence in T. brucei cultured in 96-well plates at 37 ꢁC for
48 h. Parasites were diluted to 1.0 · 105 cells/mL in com-
plete HMI-9 medium. One hundred microliters (100 lL)
of the diluted parasites were aliquoted into sterile 96-
well flat white opaque culture plates (Greiner). Each
compound was serially diluted from 10 lM to 0.1 lM
in DMSO and then mixed in the appropriate wells con-
taining parasites. The treated parasites were then incu-
bated for 48 h at 37 ꢁC with 5% CO2 before
monitoring viability. To measure the viability of the par-
asites after treatment with each compound, the parasites
were lysed in the wells by adding 100 lL of CellTiter-
GloTM (Promega). After lysis, the ATP-bioluminescence
of the 96-well plates was measured with a SpectraFluor
Plus multidetection plate reader (Tecan).
Crystallization of the material from diethyl ether gave
white crystals, mp 116–118 ꢁC, which was used in a sin-
gle-crystal X-ray analysis (Fig. 2).22
4.1.34. (3S*,4S*,6S*)-4-Methoxy-6-(tert-butyldimethylsi-
lyloxy)-1-oxaspiro[2.5]octan-5-one (45). To a solution of
31 mg (0.11 mmol) of 20 in 1 mL of DMSO under ar-
gon, was added 0.18 g (0.65 mmol) of IBX, and the solu-
tion was stirred at 25 ꢁC for 12 h, diluted with diethyl
ether, washed with water, and brine, dried (MgSO4),
concentrated, and column-chromatographed on silica
gel using a mixture of hexane and diethyl ether (2:1) as
eluant to give 28 mg (90% yield) of 45: 1H NMR d
4.34 (dd, J = 5.4, 3.4 Hz, 1H), 4.25 (s, 1H), 3.43 (s,
3H), 3.02 (d, J = 5.2 Hz, 1H), 2.57 (d, J = 5.2 Hz, 1H),
2.40–2.32 (m, 1H), 2.02–1.94 (m, 1H), 1.82–1.74 (m,
1H), 1.70–1.62 (m,1H), 0.93 (s, 9H), 0.10 (s, 6H); 13C
NMR d 206.0, 83.2, 75.1, 61.6, 59.5, 49.9, 30.4, 26.7,
25.9, 18.3, ꢀ4.67, ꢀ4.90; HRMS calcd for C14H26O4Si-
Na (M+Na+) 309.1498, found 309.1506.
Acknowledgments
D.H.H. gratefully acknowledges financial support from
the National Science Foundation (CHE-0555341 and
NSF43529), National Institutes of Health (National
Institute of Aging, R01AG025500), American Heart
Association, Heartland Affiliate (0750115Z), and Amer-
ican Chemical Society PRF (40345-AC). We thank Dr.
James H. McKerrow at University of California in
San Francisco for advice and support.
4.1.35. (3S*,4S*,5R*,6S*)-4-Methoxy-5-[(E)-(10, 50-dim-
ethylhexa-10,40-dienyl)–6-(tert-butyldimethylsilyloxy)-1-
oxaspiro[2.5]octan-5-ol (46). To a cold (ꢀ78 ꢁC) solution
of 25 mg (87 lmol) of 45 in 1 mL of toluene under ar-
gon, was added 1.5 mL (0.17 mmol) of alkenyllithium
27. The solution was stirred at ꢀ78 ꢁC for 2 h, warmed
to 0 ꢁC, diluted with diethyl ether, washed with water,
and brine, dried (MgSO4), concentrated, and column-
References and notes
1. Horn, D. Curr. Mol. Med. 2004, 4, 563.
2. Welburn, S. C.; Maudlin, I. Parasitol. Today 1999, 15,
399.

5246
D. H. Hua et al. / Bioorg. Med. Chem. 16 (2008) 5232–5246
3. Bollinger, P.; Sigg, H.-P.; Weber, H.-P. Helv. Chim. Acta
1973, 56, 819.
14. Marek, I.; Meyer, C.; Normant, J.-F. Org. Syn. 1997, 74,
194.
4. Zhang, P.; Nicholson, D. E.; Bujnicki, J. M.; Su, X.;
Brendle, J. J.; Ferdig, M.; Kyle, D. E.; Milhous, W. K.;
Chiang, P. K. J. Biomed. Sci. 2002, 9, 34.
5. Hua, D. H.; Tamura, M.; Egi, M.; Werbovetz, K.; Delfin,
D.; Salem, M.; Chiang, P. K. Bioorg. Med. Chem. 2003,
11, 4357.
6. Corey, E. J.; Dittami, J. P. J. Am. Chem. Soc. 1985, 107,
256.
7. Corey, E. J.; Guzman-Perez, A.; Noe, M. C. J. Am. Chem.
Soc. 1994, 116, 12109.
8. Barton, D. H. R.; Bath, S.; Billington, D. C.; Gero, S. D.;
Quiclet-Sire, B.; Samadi, M. J. Chem. Soc., Perkin Trans.
1 1995, 1551.
9. Barco, A.; Benetti, S.; De Risi, C.; Marchetti, P.; Pollini,
G. P.; Zanirato, V. Tetrahedron: Asymmetry 1998, 9, 2857.
10. Takahashi, S.; Hishinuma, N.; Koshino, H.; Nakata, T. J.
Org. Chem. 2005, 70, 10162.
15. Korbe, S.; de Meijere, A.; Labahn, T. Helv. Chim. Acta
2002, 85, 3161.
16. Compound 3 was synthesized previously via a different
route from (+)-limonene oxide. Evarts, J. B.; Fuchs, P. L.
Tetrahedron Lett. 2001, 42, 3673.
17. Yang, D.; Wong, M.-K.; Yip, Y.-C. J. Org. Chem. 1995,
60, 3887.
18. Nicolaou, K. C.; Montagnon, T.; Baran, P. S.; Zhong, Y.
L. J. Am. Chem. Soc. 2002, 124, 2245.
19. Urones, J. G.; Marcos, I. S.; Perez, B. G.; Lithgow, A. M.;
Gomez, D. D. P. M.; Basabe, P.; Garrido, N. M.
Tetrahedron 1995, 51, 1845.
20. Rapp, M.; Haubrich, T. A.; Perrault, J.; Mackey, Z. B.;
McKerrow, J. H.; Chiang, P. K.; Wnuk, S. F. J. Med.
Chem. 2006, 49, 2096.
21. Chiang, P. K.; Bujnicki, J. M.; Su, X. Z.; Lanar, D. E.
Curr. Mol. Med. 2006, 6, 309.
11. Yamaguchi, J.; Toyoshima, M.; Shoji, M.; Kakeya, H.;
Osada, H.; Hayashi, Y. Angew. Chem. Int. Ed. 2006, 45, 789.
12. Tiefenbacher, K.; Arion, V. B.; Mulzer, J. Angew. Chem.
Int. Ed. 2007, 46, 2690.
13. Marek, I.; Alexakis, A.; Normant, J. F. Tetrahedron Lett.
1991, 32, 5329.
22. The authors have deposited atomic coordinates for
compounds 37 and 44 with the Cambridge Crystallo-
graphic Data Centre. The coordinates can be obtained
on request, from the Director, Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge, CB2
1EZ, UK.