Convergent synthesis of fluorescence-labeled probes of Annonaceous acetogenins and visualization of their cell distribution

Article abstract of DOI:10.1016/j.bmc.2010.10.004

The convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, was accomplished by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with an alkyne tagged with fluorescent groups and another alkyne with an α,β-un

Full text of DOI:10.1016/j.bmc.2010.10.004

Bioorganic & Medicinal Chemistry 18 (2010) 8630–8641
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Convergent synthesis of fluorescence-labeled probes of Annonaceous
acetogenins and visualization of their cell distribution
Naoto Kojima a, , Takekuni Morioka a, Daisuke Urabe a, Masahiro Yano a, Yuki Suga a, Naoyoshi Maezaki b,
⇑
Ayako Ohashi-Kobayashi c, Yasuyuki Fujimoto c, Masatomo Maeda c, Takao Yamori d,
Takehiko Yoshimitsu a, Tetsuaki Tanaka a,
⇑
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tondabayashi, Osaka 584-8540, Japan
c School of Pharmacy, Iwate Medical University, Yahaba, Shiwa-gun, Iwate 028-3694, Japan
d Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, was
Received 17 August 2010
Revised 1 October 2010
Accepted 2 October 2010
Available online 16 October 2010
accomplished by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with an alkyne tagged
with fluorescent groups and another alkyne with an
a,b-unsaturated c-lactone. Assay for the growth
inhibitory activity against human cancer cell lines revealed that the probe with the fluorescent groups
at the end of the hydrocarbon chain may have the same mode of action as natural acetogenins. The
merged fluorescence of dansyl-labeled solamin and MitoTracker Red suggests that Annonaceous
acetogenins localize in the mitochondria.
Keywords:
Annonaceous acetogenins
Antitumor agents
Ó 2010 Elsevier Ltd. All rights reserved.
Fluorescence-labeled analogues
Convergent synthesis
Cell distribution
1. Introduction
NBD-NH-
O2N
=
H
N
Over 400 natural polyketides called Annonaceous acetogenins
have been isolated from Annona plants growing in tropical and
subtropical regions.1 Most acetogenins are white waxy derivatives
of long-chain fatty acids (C32 or C34) whose terminal carboxylic
acid is combined with a 2-propanol unit at the C-2 position to form
Me
N
N
=
O
R
O
O
8
11
O2
dansyl-NH-
HO
OH
solamin (1, R = n-C3H7)
NBD-labeled solamin (2, R = NBD-NH-)
dansyl-labeled solamin (3, R = dansyl-NH-)
a methyl-substituted a,b-unsaturated c-lactone (Fig. 1). One inter-
O
S
N
H
esting feature of their chemical structures is the single, adjacent, or
nonadjacent 2,5-disubstituted tetrahydrofuran (THF) system with
one or two flanking hydroxyl groups(s) at the center of a long
hydrocarbon chain. Much attention has also been paid to their
broad spectrum of biological activities, including immunosuppres-
sive, antimalarial, insecticidal, and probably the most impressive,
antitumor activity. Several strategies for the total synthesis of nat-
ural acetogenins2 and their analogues3 have been developed, moti-
vated by their unique structures and attractive biological activities.
It is generally accepted that the mode of action of acetogenins is
the inhibition of NADH-ubiquinone oxidoreductase (complex I) in
Me2N
Figure 1. Structures of
derivatives.
a mono-THF acetogenin, solamin, and its fluorescent
the mitochondria, thereby suppressing ATP production particularly
for cancer cells having high metabolic activity, and inducing
apoptosis.4–6 However, the structure–activity relationship for the
inhibition of complex I is not completely related to its cytotoxicity.
McLaughlin and co-workers suggested that as the mitochondrial
assay is cell-free, it does not take into consideration such factors
as membrane transport, intracellular transport, and metabolic
inactivation.7 Poupon and co-workers synthesized the first
⇑
Corresponding authors. Tel.: +81 6 6879 8212; fax: +81 6 6879 8214 (N.K.);
tel.: +81 6 6879 8210; fax: +81 6 6879 8214 (T.T.).
E-mail addresses: kojima@phs.osaka-u.ac.jp (N. Kojima), t-tanaka@phs.osaka-u.
ac.jp (T. Tanaka).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.004

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8631
fluorescence-labeled acetogenin, dansyl-labeled squamocin, in
which -lactone was replaced with a fluorescence label, and
in a sequential reaction: (1) selective pivaloylation of the primary
alcohol, (2) TBS protection of the secondary alcohol, (3) deprotec-
tion of the pivaloyl ester, and (4) oxidation with Dess–Martin peri-
odinane. The asymmetric alkynylation of aldehyde 9 with chiral
C4-unit 1018d under Carreira’s conditions26 with (1R,2S)-N-methyl-
ephedrine (NME) as the chiral ligand afforded propargyl alcohol
11a in high yield and selectivity. The stereochemistry was con-
firmed by comparison with the 1H NMR spectral data of corre-
sponding anti-adduct 11b synthesized with the antipode of NME.27
Hydrogenation of syn-adduct 11a using 10% Pd–C afforded sat-
urated triol 12, and subsequent selective sulfonylation of the pri-
mary alcohol with 2,4,6-triisopropylbenzenesulfonyl chloride
(TrisCl) furnished sulfonate 13 (Scheme 2). The formation of a
2,5-disubstituted THF ring in a domino reaction via epoxide 14
was achieved by treatment of 13 with K2CO3 in MeOH. Oxidation
of the primary alcohol of 15 was carried out with Dess–Martin
c
visualized its cell distribution.8 Yao and co-workers reported that
a fluorescence-labeled analogue, in which the THF ring moiety of
natural acetogenin was mimicked by ethylene glycol ether unit
replacements, showed different distributions in human normal
cells and cancer cells.9 Miyoshi and co-workers reported an
125I-labeled acetogenin analogue in which the (trifluoro-
methyl)phenyldiazirine group served as
a substitute for the
c-lactone moiety and a photoreactive group photo-cross-linked
to the ND1 subunit of bovine heart mitochondrial complex I with
high specificity.10 These reports prompted us to synthesize a novel
fluorescence-labeled acetogenin that retains all functionalities,11
because in living cells, the hydrophilic THF moiety in natural ace-
togenins may be located at the surface of the mitochondrial inner
membrane and the c-lactone moiety may interact with the enzyme
active site.12 Miyoshi and co-workers revealed that the hydrocar-
bon chain on the left side of the THF moiety is not essential for
the inhibitory activity against complex I.13 We planned the synthe-
sis of novel fluorescence-labeled acetogenins having a fluorescent
group at the left end of the THF moiety. After starting our synthetic
study, Sinha and co-workers reported the first synthesis of photo-
active asimicin with all functionalities, which has a benzophenone
group at the end of the hydrocarbon chain.14 However, the photo-
active probe was not evaluated.15 Herein, we describe the full
details of the first16 and second-generation17 syntheses of fluores-
cence-labeled acetogenins, the evaluation of their growth inhibi-
tory activities against human cancer cell lines, and their cell
distribution.
periodinane to give a-tetrahydrofuranyl aldehyde 16. The
introduction of a linker with a methyl ester at the end was
achieved by asymmetric alkynylation of aldehyde 16 with methyl
13-tetradecynoate 17 in 93% yield with high diastereoselectivity
(>97:3 dr).28,29
After protection of the secondary hydroxyl group of 18a as a TBS
ether, the aldol reaction of tris-TBS ether 19 with THP-protected
(S)-lactaldehyde30 gave an inseparable mixture of aldol product
20 (mixture of diastereomers) and the unreacted aldehyde
(Scheme 3). The selective deprotection of the THP group in 20 by
treatment with MgBr2 proceeded successfully, but the purification
of diol 21 was difficult because partial
during silica gel column chromatography. Therefore, we attempted
to promote -lactonization with CSA. However, although the depro-
tection of the terminal TBS group proceeded, the -lactonization
proceeded very slowly to give a mixture containing triol 22. Fortu-
nately, the -lactonization was completed by quenching the reac-
tion with NaHCO3, giving desired -lactone 23 in 41% yield in
three steps from 19. Construction of the ,b-unsaturated- -lactone
moiety was conducted at the late stage of the synthesis because the
methyl group on the
-lactone easily epimerized.31 Azidation of the
c-lactonization occurred
c
c
2. Results and discussion
c
2.1. First-generation synthesis of fluorescence-labeled solamin
c
a
c
We planned to synthesize fluorescence-labeled acetogenin with
our developed stereoselective synthesis of the THF moiety18,19 and
selected solamin,20 which has a comparatively simple structure
yet all the functionalities, as the lead compound. The 7-nitro-
benzo[c][1,2,5]oxadiazol-4-yl-amino (NBD-NH–) group was em-
ployed as a fluorescent tag due to its strong fluorescence with a
long wavelength, which is advantageous for the observation of cell
distribution.21
c
terminal alcohol in 23 via tosylation gave azide 25 in good yield.
After the reduction of the azide to the primary amine, the treatment
with NBDCl in the presence of Et3N in MeOH afforded NBD-labeled
OTBS
OH
The synthesis was started from Jacobsen’s kinetic optical reso-
b
a
TBSO
OH
OH
lution22 of known racemic epoxide 423 to give diol 5 with >98%
( )9
11a
OH
12
ee (Scheme 1).24,25 Diol 5 was converted into
a
-siloxyaldehyde 9
OH
OH
OTBS
OTBS
O
a
b
O
c
TBSO
c
TBSO
OPiv
TBSO
OH
TBSO
OTris
TBSO
TBSO
( )9
( )9
( )9
( )9
( )9
6
4
5
OH
13
OH
14
OTBS
OPiv
OTBS
OH
(Tris = triisopropylbenzenesulfonyl)
d
e
TBSO
TBSO
( )9
( )9
d
TBSO
7
8
( )8
TBSO
( )8
TBSO
CHO
O
O
Ph
O
Ph
OH
( )8
O
O
OTBS
16
15
OTBS
R
O
TBSO
( )9 CHO
TBSO
10
TBSO
OMe
17
( )9
CO2Me
( )10
( )9
f or g
9
O
11a: R = β-OH
11b: R = α-OH
O
e or f
TBSO
R
18a: R = β-OH
Scheme 1. Reagents and conditions: (a) (S,S)-(+)-N,N0-bis(3,5-di-tert-butylsalicy-
lidene)-1,2-cyclohexanediaminocobalt (III) acetate (0.5 mol%), ButOMe, H2O, 0 °C to
rt, 48% (>98% ee); (b) PivCl, pyridine, CH2Cl2, 0 °C to rt, 95%; (c) TBSCl, imidazole,
DMF, 0 °C to rt, 97%; (d) DIBAL-H, CH2Cl2, ꢀ78 °C, quantitative; (e) Dess–Martin
periodinane, pyridine, CH2Cl2, 0 °C, 91%; (f) Zn(OTf)2, Et3N, (1R,2S)-N-methylephe-
drine, toluene, rt, 91% (11a:11b = >97:3); (g) Zn(OTf)2, Et3N, (1S,2R)-N-methyl-
ephedrine, toluene, rt, 85% (11a:11b = 3:>97).
18b: R = α-OH
Scheme 2. Reagents and conditions: (a) H2 (3 atm), 10% Pd–C, EtOAc, rt, 94%;
(b) TrisCl, pyridine, CH2Cl2, 0 °C to rt, 78%; (c) K2CO3, MeOH, 0 °C to rt, 67%;
(d) Dess–Martin periodinane, pyridine, CH2Cl2, 0 °C to rt, 70%; (e) Zn(OTf)2, Et3N,
(1R,2S)-N-methylephedrine, toluene, rt, 93% (18a:18b = >97:3); (f) Zn(OTf)2, Et3N,
(1S,2R)-N-methylephedrine, toluene, rt, 68% (18a:18b = 3:>97).

8632
N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
MeO
( )9
O
OMe
OTHP
Me
a
( )9
TBSO
b
c, d
TBSO
( )8
18a
( )8
TBSO
O
O
OTBS
OH
O
19
TBSO
OTBS
20
Me
Me
HO
( )9
HO
( )9
MeO
( )9
O
OH
Me
HO
R
e
PO
f
( )8
( )8
O
O
( )8
O
O
O
O
OH
O
TBSO
OTBS
23
TBSO
OTBS
TBSO
OTBS
24: R = OTs
25: R = N3
21: P = TBS
g
22: P = H
H
Me
H
N
HO
AcO
( )11
Me
O
N
( )8
h, i
( )8
j
k
O
( )11
OTBS
O2N
O
N
O2N
O
N
TBSO
TBSO
O
N
O
N
OTBS
O
O
27
26
H
N
Me
( )8
l
O
2
( )11
OTBS
O2N
O
N
TBSO
N
O
O
28
Scheme 3. Reagents and conditions: (a) TBSCl, imidazole, DMF, 0 °C to rt, 96%; (b) THP-protected-(S)-lactaldehyde, LDA, THF, ꢀ78 °C; (c) MgBr2, Et2O, rt; (d) CSA,
MeOH–CH2Cl2 (1:1), 0 °C; (e) NaHCO3, 41% from 19; (f) TsCl, pyridine, CH2Cl2, 0 °C to rt, 85%; (g) NaN3, DMSO, rt, 83%; (h) H2 (3 atm), 10% Pd–C, MeOH, rt; (i) NBDCl, Et3N,
MeOH, 0 °C to rt, 95% in two steps; (j) Ac2O, pyridine, rt, 77%; (k) DBU, THF, rt, 93%; (l) 48% aq HF, MeCN–THF (5:3), rt, 91%.
derivative 26 in 97% yield in two steps. The synthesis of fluores-
cent solamin 2 was completed via acetylation of the secondary
alcohol, DBU-promoted b-elimination, and deprotection with HF in
MeCN–THF.
To confirm that the bioactivity of fluorescence-labeled solamin
2 is similar to that of natural solamin 1, we would need a sample of
1. The total synthesis of solamin 1 was easily accomplished with
our strategy from our previously synthesized aldehyde 2918d over
nine steps (Scheme 4).32
With fluorescence-labeled solamin 2 and synthetic solamin 1 in
hand, we attempted to visualize cell distribution with 2. Unfortu-
nately, we could not observe in detail the cell distribution of 2 be-
cause the quenching of the fluorescence of 2 was very fast. We had
to prepare a new analogue with a different fluorescent tag from the
NBD group, but the present synthetic route was long and the total
yield was low because the synthetic route was linear. These
problems prompted us to develop a novel approach to the fluores-
cence-labeled acetogenins.
2.2. Second-generation synthesis of fluorescence-labeled
solamin
Scheme 5 outlines the second-generation synthesis of fluores-
cence-labeled acetogenins. Fluorescence-labeled acetogenins are
divided into three fragments retrosynthetically: fluorescent frag-
ment 35, THF fragment 36, and c-lactone fragment 37. This synthe-
sis should provide ready access to diverse fluorescent derivatives
that have other fluorescent groups or long hydrocarbon chains as
linker.
The preparation of THF fragment 36 started from the asymmet-
ric alkynylation of 2,3-O-isopropylidene-D
-glyceraldehyde 3833
with alkyne 10 (Scheme 6). Asymmetric alkynylation of 38 with
10 proceeded with high stereoselectivity to give propargyl alcohol
39a.34,35 Hydrogenation of the triple bond accompanied by depro-
tection of the benzylidene acetal with 10% Pd–C afforded saturated
triol 40 in 84% yield. After selective sulfonylation of the primary
alcohol with TrisCl, treatment of resulting sulfonate 41 with
( )9
CO2Me
a or b
c, d
-C12H25
-C12H25
n
n
n-C12H25
TBSO
( )11
CHO
CO2Me
O
O
O
TBSO
R
TBSO
OTBS
31
30a :R = β-OH
29
30b: R = α-OH
Me
Me
RO
e-g
i
n-C12H25
O
n-C12H25
O
( )11
( )11
OTBS
O
O
RO
OR
TBSO
O
O
32: R = H
34: R = TBS
1: R = H
h
j
33: R = Ac
Scheme 4. Reagents and conditions: (a) 17, Zn(OTf)2, Et3N, (1R,2S)-N-methylephedrine, toluene, rt, 85% (30a:30b = >97:3); (b) 17, Zn(OTf)2, Et3N, (1S,2R)-N-methylephedrine,
toluene, rt, 91% (30a:30b = 3:>97); (c) H2 (1 atm), 10% Pd–C, EtOAc, rt; (d) TBSCl, imidazole, DMF, 0 °C to rt, 69% from 30a; (e) THP-protected-(S)-lactaldehyde, LDA, THF,
ꢀ78 °C; (f) MgBr2, Et2O, rt; (g) NaHCO3, 49% from 31; (h) Ac2O, pyridine, rt, 90%; (i) DBU, CH2Cl2, rt, quantitative; (j) 48% aq HF, MeCN–THF (5:3), rt, 91%.

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8633
Me
Me
Me
a
fluorescent acetogenins
(FG = fluorescent group)
I
+
FG
O
( )9
O
O
( )n
( )11
( )9
O
PhS
PhS
HO
OH
O
O
O
O
O
46
47
37
Me
Scheme 8. Reagents and conditions: (a) LDA, THF–HMPA (2:1), ꢀ78 °C to rt, 97%.
CHO
FG
+
+
O
( )n-1
( )9
PhS
O
alcohol of 48a as TBDPS ether, followed by hydrolysis of acetonide,
gave diol 50 in high yield. Oxidative cleavage of the 1,2-diol moiety
O
fluorescent fragment (35) THF fragment (36) γ-lactone fragment (37)
in 50 afforded
a-tetrahydrofuranyl aldehyde 51 in 99% yield. The
Scheme 5. Retrosynthesis of second-generation approach of fluorescent
assembly of aldehyde 51 and fluorescent fragment 45 was per-
formed by asymmetric alkynylation and high diastereoselectivity
(91:9 dr) was achieved.41 Unexpectedly, the reduction of diyne
52a by catalytic hydrogenation over Pd–C, or the diimide reduction
(TsNHNH2, AcONa) was difficult to perform and gave a complex
mixture of olefinic products that were obtained by partial reduc-
tion. Fortunately, hydrogenation with Wilkinson’s catalyst under
3 atm pressure of hydrogen in benzene gave desired saturated
alcohol 53 in 73% yield. Oxidation of the sulfide, followed by ther-
acetogenins
Ph
Ph
O
O
O
a or b
O
O
+
O
O
O
CHO
O
OH
38
10
39a: R = β-OH
39b: R = α-OH
mal elimination of the resulting sulfoxide, afforded
rated- -lactone 54. The synthesis of dansyl-labeled solamin 3
was completed via deprotection of TBDPS ether with 48% HF in
a,b-unsatu-
c
OH
d
O
OH
c
O
O
OH
OTris
MeCN.42
OH
OH
41
40
2.3. Growth inhibitory activity of fluorescence-labeled solamins
against human cancer cell lines
To examine the influence of the introduction of fluorescent
groups at the end of the hydrocarbon chain on the biological activ-
ity, we used COMPARE analysis.43,44 It is well established in a panel
of 39 human cancer cell lines (JFCR39) that a pair of compounds
potentially share the same mode of action when they indicate
fingerprints closely resembling each other (r >0.75; r = Pearson
correlation coefficient). Synthesized fluorescence-labeled solamins
2 and 3 and solamin 1 were tested for in vitro antiproliferative
activity against JFCR39. Figure 2 shows the growth inhibitory
profiles across the JFCR39 (fingerprints) of the three compounds.
We noted that the either fingerprint of the fluorescent probes 2
or 3 was very similar to that of solamin 1 although 2 and 3 showed
stronger growth inhibition against some cancer cell lines than sol-
amin 1. The COMPARE analysis revealed that the fingerprints of
fluorescence-labeled solamins 2 and 3 significantly correlated with
that of solamin 1 (2 versus 1: r = 0.864, p <0.01; 3 vs 1: r = 0.857, p
<0.01), suggesting that fluorescent probes 2 and 3 may have the
same mode of action as natural solamin 1.
f
O
CHO
e
O
O
O
O
OH
O
42
36
Scheme 6. Reagents and conditions: (a) Zn(OTf)2, Et3N, (1R,2S)-N-methylephe-
drine, toluene, rt, 93% (39a:39b = >97:3); (b) Zn(OTf)2, Et3N, (1S,2R)-N-methyl-
ephedrine, toluene, rt, 96% (39a:39b = 3:>97); (c) H2 (3 atm), 10% Pd–C, EtOAc, rt,
84%; (d) TrisCl, pyridine, CH2Cl2, 0 °C to rt, 79%; (e) K2CO3, MeOH, 0 °C to rt, 98%; (f)
SO3ꢁpyridine, DMSO, i-Pr2NEt, CH2Cl2, 0 °C to rt, 89%.
K2CO3 in MeOH gave threo/trans-THF alcohol 42 in good yield.
DMSO oxidation of 42 with SO3ꢁpyridine complex gave
a-tetrahy-
drofuranyl aldehyde 36 in high yield.36
Fluorescent fragment 45 was synthesized by the coupling reac-
tion of dansyl chloride with 8-nonyn-1-amine 44, which was pre-
pared by the azidation of 9-iodo-1-nonyne 43 followed by
reduction under Staudinger conditions (Scheme 7).
c
-Lactone fragment 37 was easily prepared by
a-alkylation of
known -sulfenyl-
a
c
-lactone 4737 with 12-iodo-1-dodecyne 4638
2.4. Cell distribution of fluorescence-labeled solamin
under basic conditions in an almost quantitative yield (Scheme 8).
With all the fragments in hand, we examined the assembly of
THF fragment 36 and c-lactone fragment 37 because we thought
it best that the expensive fluorescent fragment 45 be introduced
at the late stage of the synthesis, and this synthesis should provide
ready access to diverse fluorescent derivatives that have other fluo-
To identify where in the cell solamin and its derivatives might
localize, dansyl-labeled solamin 3 was introduced into CHO-K1
cells and localization was examined by confocal laser scanning
microscopy. MitoTracker Red and ER-Tracker Red were used as
organelle markers. To visualize the localization of the probes in
lysosome, CHO-K1 cells in which GFP-tagged hTAPL (human trans-
porter associated with antigen processing-like) was expressed sta-
bly were used. It is known that hTAPL localizes in lysosomal
rescent groups (Scheme 9). Asymmetric alkynylation of a-tetrahy-
drofuranyl aldehyde 36 with alkyne 37 proceeded with high
diastereoselectivity (95:5 dr).39,40 Protection of the secondary
membranes.45 The observation of cells treated with 10
lM of fluo-
rescence-labeled solamin 3 for 5 h and stained with MitoTracker
Red revealed the localization of dansyl-labeled solamin 3 in the
mitochondria, as confirmed by the overlapping patterns of fluores-
cence (green and red). This result confirms the previous proposal
that Annonaceous acetogenins would act as an inhibitor of complex
I in living cells. Moreover, the fluorescence derived from ER-Tracker
Red partially co-localized with the signal of 3, indicating that part
H
N
b, c
NMe2
R
( )5
S
( )5
O2
43: R = I
a
45
44: R = N3
Scheme 7. Reagents and conditions: (a) NaN3, DMSO, rt, 98%; (b) PPh3, Et2O–H2O
(17:1), 0 °C; (c) dansyl chloride, i-Pr2NEt, CH2Cl2, 0 °C to rt, 96% in two steps.
of dansyl-labeled solamin
3 localized in the endoplasmic

8634
N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
Me
Me
Me
O
O
O
a or b
c
d
( )9
PhS
( )9
PhS
( )9
PhS
36
HO
O
O
O
O
O
O
O
O
HO
OTBDPS
50
O
R
O
OTBDPS
49
48a: R = β-OH
48b: R = α-OH
Me
Me
e
f or g
H
N
h
O
O
O
Me2N
( )9
PhS
( )9
PhS
( )6
S
O2
OHC
O
O
O
O
OTBDPS
OTBDPS
R
52a: R = α-OH
52b: R = β-OH
51
Me
Me
i, j
H
H
N
Me2N
Me2N
N
O
O
S
( )6
( )9
OTBDPS PhS
S
( )6
( )9
O
O2
O2
HO
HO
OR
O
O
53
54: R = TBDPS
dansyl-labeled solamin (3): R = H
k
Scheme 9. Reagents and conditions: (a) 37, Zn(OTf)2, i-Pr2NEt, (1R,2S)-N-methylephedrine, toluene, rt, 74% (48a:48b = 95:5); (b) 37, Zn(OTf)2, i-Pr2NEt, (1S,2R)-N-
methylephedrine, toluene, rt, 66% (48a:48b = 4:96); (c) TBDPSCl, imidazole, DMF, 0 °C to rt, quantitative; (d) Dowex 50 W, THF–MeOH (1:1), rt to 60 °C, 96%; (e) NaIO4,
THF–H2O (4:1), rt, 99%; (f) 45, Zn(OTf)2, i-Pr2NEt, (1R,2S)-N-methylephedrine, toluene, rt, 66% (52a:52b = 91:9); (g) 45, Zn(OTf)2, i-Pr2NEt, (1S,2R)-N-methylephedrine,
toluene, rt, 81% (52a:52b = 4:96); (h) H2 (3 atm), (Ph3P)3RhCl, benzene, rt, 73%; (i) mCPBA, CH2Cl2, 0 °C; (j) toluene, reflux, 68% in two steps; (k) 48% HF aq, MeCN, rt, 71%.
Figure 2. Growth inhibitory profiles (fingerprints) of fluorescence-labeled solamins 2 and 3 and solamin 1 across the JFCR39. Inhibition of cell growth was assessed by the
change in total cellular protein following 48 h of treatment with a given test compound, and was measured using the sulforhodamine B colorimetric assay. Fingerprints were
produced by computer processing of the 50% growth inhibition (GI50) values. The logarithm of the GI50 value for each cell line is indicated. In the plot, columns to the right of
zero indicate sensitivity of the cell line to the compound, and columns to the left of zero indicate resistance to the compound. The x-axis represents the logarithm of difference
between the mean of GI50 values for the 39 cell lines and the GI50 value for each cell line. One scale represents one logarithm difference. MG-MID = mean of logarithm of GI50
values of all cell lines tested. CNS = central nervous system.
reticulum. However, localization in the lysosome was not
observed. McLaughlin and co-workers reported that natural
acetogenins showed selective inhibitory activity against cancer
cells but not normal cells.46 Wu, Yao and co-workers also described
the selectivity of their analogues having the ethylene glycol moiety
instead of bis-THF rings for cancer but not normal cells.9 They sug-
gested that a special mechanism is involved at the level of the can-
cer cell membrane. On the other hand, our fluorescence-labeled
analogue 3 easily penetrated CHO-K1 cells, which are ‘normal
cells’. Interestingly, however, no toxicity was observed when cells
treated with 10 lM of 3 were incubated for 24 h. This result
suggests the existence of different mechanism from membrane
recognitions that would account for the selective action of
Annonaceous acetogenins, though the detail is unclear because no
study of localization of our probes in cancer cells has yet been
conducted.

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8635
Figure 3. Visualization of dansyl-labeled solamin 3 by fluorescence microscopy. CHO-K1 cells were pretreated with 10
l
M dansyl-labeled solamin 3 for 5 h before the cells
were stained with organelle markers. (A) CHO-K1 cells labeled with dansyl-labeled solamin 3 were stained with MitoTracker Red and then observed under a confocal
microscope. The signal of MitoTracker Red (second from left) merged (third from left) with that of 3 (left) in living cells. (B) CHO-K1 cells labeled with dansyl-labeled solamin
3 were stained with ER-Tracker Red and then observed under a confocal microscope. The signal of ER-Tracker Red (second from left) merged (third from left) with that of 3
(left) in living cells. (C) CHO-K1 cells expressing GFP-fusion proteins labeled with dansyl-labeled solamin 3 and then observed under a confocal microscope. The signal derived
from hTAPL-GFP (second from left, red pseudo-color) merged (third from left) with that of 3 (left) in living cells. Bar indicates 20 lm. DIC = Differential interference contrast.
are listed. Mass spectra were obtained with a JEOL JMS-600H and
JEOL JMS-700 mass spectrometer. Column chromatography was
carried out by using Kanto Chemical Silica Gel 60 N (spherical, neu-
3. Conclusion
We have accomplished the convergent synthesis of fluorescence-
labeled solamin, an antitumor Annonaceous acetogenin, by two
asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with
two kinds of alkynes as the key steps. Examination of their growth
inhibitory activity against cancer cells revealed that the introduc-
tion of fluorescent groups at the end of the hydrocarbon chain of
acetogenins did not interfere with the mode of action of the probes.
It was observed that dansyl-labeled solamin 3 localized in the
mitochondria. Our convergent synthesis is a useful tool for the elu-
cidation of the mechanism of action of Annonaceous acetogenins.
New results of biological studies will be reported in due course.
tral, 63–210
lm) and flash column chromatography was carried
out by using Merck Silica Gel 60 (0.040–0.063
lm). All air- or mois-
ture-sensitive reactions were carried out in flame-dried glassware
under an atmosphere of Ar or N2. All solvents were dried and
distilled according to standard procedures. All organic extracts
were dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure with rotary evaporator.
4.1.1. (1R)-1-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-3-[(2RS,4S)-
2-phenyl-1,3-dioxolan-4-yl]-2-propyn-1-ol (39a)
A flask was charged with Zn(OTf)2 (7.52 g, 20.7 mmol). Vacuum
(7 mmHg) was applied and the flask was heated to 120 °C for 10 h.
After the flask was cooled to rt, the vacuum was released. (1R,2S)-
N-Methylephedrine (3.71 g, 20.7 mmol), toluene (10 mL), and Et3N
(3.13 mL, 22.4 mmol) were added to the flask with stirring at rt.
4. Experimental section
4.1. Chemistry
Melting points are uncorrected. Optical rotations were measured
by using a JASCO DIP-360 digital polarimeter or a JASCO P-1020
digital polarimeter. 1H NMR spectra were recorded in CDCl3 solu-
tion with a JEOL JNM-GX-500 spectrometer (500 MHz). 13C NMR
spectra were recorded in CDCl3 solution with a JEOL JNM-AL300
spectrometer (75 MHz) or a JEOL JNM-GX-500 spectrometer
(125 MHz). All signals are expressed as d values in ppm downfield
from the internal standard tetramethylsilane. The following abbre-
viations are used: broad = br, singlet = s, doublet = d, triplet = t,
quartet = q, quintet = qn, sextet = sext, septet = sep and multi-
plet = m. IR absorption spectra (FT = diffuse reflectance spectros-
copy) were recorded with KBr powder by using a Horiba FT-210
IR spectrophotometer, or as neat films on NaCl plates by using a
After 2.5 h,
a solution of 10 (3.27 g, 18.8 mmol) in toluene
(5.7 mL) was added to the mixture at the same temperature. After
15 min, 38 (4.05 g, 31.1 mmol) in toluene (5 mL) was added to the
reaction mixture and the whole mixture was stirred for 6 h at rt.
The reaction was quenched with saturated NH4Cl and the mixture
was extracted with EtOAc. The combined organic layers were
washed with brine prior to drying and solvent evaporation.
Purification by column chromatography over silica gel with
n-hexane/EtOAc (2:1?1:1?2:3) as eluent yielded 39a (5.32 g,
93%, 39a:39b = >97:3) as a colorless oil. ½a D26
ꢂ
+54.5 (c 3.19, CHCl3);
1H NMR (500 MHz, CDCl3) d: 1.38 (s, 1.5H), 1.39 (s, 1.5H), 1.46 (s,
1.5H), 1.48 (s, 1.5H), 2.45 (d, 0.5H J = 4.9 Hz), 2.50 (d, 0.5H,
J = 4.9 Hz), 3.86 (dd, 0.5H, J = 7.9, 4.9 Hz), 3.93 (dd, 0.5H, J = 7.9,
4.9 Hz), 4.00 (dd, 0.5H, J = 7.9, 6.7 Hz), 4.06 (dd, 0.5H, J = 7.9,
Shimazu FTIR-8400S and only noteworthy absorptions (in cmꢀ1
)

8636
N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
6.7 Hz), 4.10–4.15 (m, 0.5H), 4.18 (dd, 0.5H, J = 6.7, 4.9 Hz), 4.19
(dd, 0.5H, J = 7.9, 6.7 Hz), 4.20–4.25 (m, 0.5H), 4.22 (dd, 0.5H,
J = 6.7, 4.9 Hz), 4.34 (ddd, 0.5H, J = 6.7, 4.9, 1.8 Hz), 4.38 (dd,
J = 7.9, 6.7 Hz, 0.5H), 4.39 (ddd, 0.5H, J = 6.7, 4.9, 1.8 Hz), 4.90
(ddd, 0.5H, J = 6.7, 4.9, 1.8 Hz), 4.94 (ddd, 0.5H, J = 6.7, 4.9,
1.8 Hz), 5.88 (s, 0.5H), 5.96 (s, 0.5H), 7.38–7.55 (m, 5H); 13C NMR
(75 MHz, CDCl3) d: 25.0 (0.5C), 25.1 (0.5C), 26.6, 63.8, 65.5 (0.5C),
65.89 (0.5C), 65.91 (0.5C), 65.93 (0.5C), 70.6 (0.5C), 70.9 (0.5C),
78.27 (0.5C), 78.30 (0.5C), 83.06 (0.5C), 83.12 (0.5C), 83.3 (0.5C),
83.9 (0.5C), 103.6 (0.5C), 104.8 (0.5C), 110.28 (0.5C), 110.32
(0.5C), 126.4, 126.7, 128.19, 128.24, 129.36 (0.5C), 129.41 (0.5C),
136.3 (0.5C, Ar), 136.8 (0.5C, Ar); IR (KBr) cmꢀ1: 3428, 2989; MS
(FAB) m/z: 305 [M+H]+; HRMS (FAB) calcd for C17H21O5:
305.1389; found: 305.1391 [M+H]+.
ane–EtOAc); ½a 2D8
ꢂ
+6.4 (c 1.57, CHCl3); 1H NMR (500 MHz, CDCl3)
d: 1.26 (d, 18H, J = 6.1 Hz), 1.36 (s, 3H), 1.43 (s, 3H), 1.46–1.80
(m, 4H), 2.48 (br s, 1H), 2.75 (br s, 1H), 2.92 (sept, 1H, J = 6.1 Hz),
3.47–3.56 (m, 1H), 3.72 (dd, 1H, J = 8.5, 6.1 Hz), 3.89–4.06 (m,
4H), 4.02 (dd, 1H, J = 8.5, 7.3 Hz), 4.13 (sep, 2H, J = 6.1 Hz), 7.19
(s, 2H); 13C NMR (75 MHz, CDCl3) d: 23.5 (2C), 24.66 (2C), 24.68
(2C), 25.2, 26.6, 29.1, 29.2, 29.6 (2C), 34.2, 66.0, 69.3, 72.3, 72.6,
78.9, 109.5, 123.8 (2C), 128.9, 150.8 (2C), 153.9; IR (KBr) cmꢀ1
;
3473, 2958, 1601, 1566; MS (FAB) m/z: 487 [M+H]+; HRMS (FAB)
calcd for C25H43O7S: 487.2729; found: 487.2722 [M+H]+.
4.1.5. (2R,5R)-2-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-5-
hydroxymethyltetrahydrofuran (42)
K2CO3 (9.66 g, 69.9 mmol) was added to a solution of 41 (3.40 g,
6.99 mmol) in MeOH (300 mL) with stirring at 0 °C. After 30 min at
the same temperature, the mixture was stirred at rt for 39 h. Water
was added to the reaction mixture and MeOH was removed under
the reduced pressure. The mixture was extracted with EtOAc. The
combined organic layers were washed with brine prior to drying
and solvent evaporation. Purification by column chromatography
over silica gel with n-hexane/EtOAc (1:1?1:2) as eluent yielded
4.1.2. (1S)-1-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-3-[(2RS,4S)-
2-phenyl-1,3-dioxolan-4-yl]-2-propyn-1-ol (39b)
The procedure was same as that used for preparation of 39a.
Colorless oil; ½a D25
ꢂ
+59.7 (c 0.46, CHCl3); 1H NMR (500 MHz, CDCl3)
d: 1.37 (s, 1.5H), 1.39 (s, 1.5H), 1.46 (s, 1.5H), 1.48 (s, 1.5H),
2.32–2.46 (br, 1H), 4.00 (t, 0.5H, J = 8.5 Hz), 4.01 (t, 0.5H,
J = 8.5 Hz), 4.02–4.09 (m, 1H), 4.09 (m, 1H), 4.19 (dd, 0.5H, J = 7.3,
6.1 Hz), 4.22 (td, 0.5H, J = 6.1, 3.7 Hz), 4.27 (td, 0.5H, J = 6.1,
3.7 Hz), 4.37 (dd, 0.5H, J = 8.5, 7.3 Hz), 4.48–4.52 (m, 0.5H), 4.54
(ddd, 0.5H, J = 4.9, 3.7, 1.2 Hz), 4.91 (ddd, 0.5H, J = 7.3, 4.9,
2.4 Hz), 4.95 (ddd, 0.5H, J = 8.5, 7.3, 1.2 Hz), 5.87 (s, 0.5H), 5.97
(s, 0.5H), 7.35–7.55 (m, 5H); 13C NMR (125 MHz, CDCl3) d: 25.1,
26.4, 62.6 (0.5C), 62.7 (0.5C), 65.2 (0.5C), 65.3 (0.5C), 65.7 (0.5C),
66.1 (0.5C), 70.8 (0.5C), 71.1 (0.5C), 77.6, 83.3 (0.5C), 83.4 (0.5C),
83.79 (0.5C), 83.81 (0.5C), 103.8 (0.5C), 105.0 (0.5C), 110.16
(0.5C), 110.21 (0.5C), 126.6, 126.8, 128.3, 128.4, 129.47 (0.5C),
129.53 (0.5C), 136.4 (0.5C), 137.0 (0.5C); IR (neat) cmꢀ1: 3439,
2988; MS (FAB) m/z: 305 [M+H]+; HRMS (FAB) calcd for
42 (1.39 g, 98%) as a colorless oil. ½a D26
ꢂ
+2.6 (c 0.94, CHCl3); 1H
NMR (500 MHz, CDCl3) d: 1.38 (s, 3H), 1.44 (s, 3H), 1.60–2.05 (m,
4H), 2.20 (br s, 1H), 3.52 (dd, 1H, J = 11.5, 4.8 Hz), 3.66 (t, 1H,
J = 6.7 Hz), 3.72 (dd, 1H, J = 11.5, 3.0 Hz), 3.92–4.03 (m, 2H),
4.03–4.13 (m, 1H), 4.16 (m, 1H); 13C NMR (75 MHz, CDCl3) d:
25.5, 26.5, 27.3, 28.3, 64.4, 65.9, 78.8, 80.2 (2C), 109.8; IR (KBr)
cmꢀ1 3477, 2983; MS (FAB) m/z: 203 [M+H]+; HRMS (FAB) calcd
for C10H19O4: 203.1283; found: 203.1285 [M+H]+.
4.1.6. (2R,5R)-2-Formyl-5-[(4R)-2,2-dimethyl-1,3-dioxolan-4-
yl]tetrahydrofuran (36)
i-Pr2NEt (2.00 mL, 11.8 mmol) and DMSO (0.601 mL,
7.85 mmol) were added to a solution of 42 (397 mg, 1.96 mmol)
in CH2Cl2 (2 mL) with stirring at 0 °C. After 10 min at the same
temperature, SO3ꢁpy (625 mg, 3.93 mmol) was added to the mix-
ture and the mixture was stirred for 30 min at the same tempera-
ture. After 1 h at rt, brine was added to the reaction mixture and
the mixture was extracted with EtOAc. The combined organic lay-
ers were washed with brine prior to drying and solvent evapora-
tion. Purification by column chromatography over silica gel with
n-hexane/EtOAc (2:1) as eluent yielded 36 (350 mg, 89%) as a col-
orless oil. The aldehyde was unstable and therefore used immedi-
ately in the next reaction.
C
17H21O5: 305.1389; found: 305.1366 [M+H]+.
4.1.3. (2S,5R)-5-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-1,2,5-
pentanetriol (40)
A solution of 39a (1.60 g, 5.26 mmol) in EtOAc (50 mL) was
hydrogenated on 10% Pd–C (80 mg) with stirring at rt for 31 h un-
der 3 atm pressure of hydrogen. The Pd–C was filtered off and the
filtrate was concentrated under reduced pressure. Purification by
column chromatography over silica gel with CHCl3/MeOH (10:1)
as eluent yielded 40 (974 mg, 84%) as a colorless solid. ½a D28
ꢂ
+7.1
(c 3.97, CHCl3); 1H NMR (500 MHz, CDCl3) d: 1.36 (s, 3H), 1.44 (s,
3H), 1.45–1.79 (m, 4H), 3.25 (br s, 1H), 3.44 (dd, 1H, J = 11.5,
6.7 Hz), 3.50–3.65 (m, 2H), 3.72 (m, 2H), 3.80–4.30 (m, 4H); 13C
NMR (75 MHz, CDCl3) d: 24.9, 26.2, 28.8, 28.9, 65.5, 65.9, 71.6,
72.0, 78.6, 109.0; IR (KBr) cmꢀ1: 3384, 2938; MS (FAB) m/z: 221
[M+H]+; HRMS (FAB) calcd for C10H21O5: 221.1389; found:
221.1387 [M+H]+.
4.1.7. 9-Azidonon-1-yne (44)
NaN3 (1.28 g, 19.7 mmol) was added to a solution of 43 (2.46 g,
9.84 mmol) in DMSO (49 mL) with stirring at rt. After 3 h, water
was added to the reaction mixture and the mixture was extracted
with EtOAc. The combined organic layers were washed with brine
prior to drying and solvent evaporation. Purification by column
chromatography over silica gel with n-hexane/EtOAc (10:1) as elu-
ent yielded 44 (1.60 g, 98%) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d: 1.30–1.64 (m, 10H), 1.95 (t, 1H, J = 2.4 Hz), 2.19 (td, 2H,
J = 7.3, 2.4 Hz), 3.26 (t, 2H, J = 7.3 Hz); 13C NMR (75 MHz, CDCl3)
d: 18.2, 26.4, 28.1, 28.35, 28.44, 28.6, 51.2, 68.1, 84.3; IR (neat)
cmꢀ1: 3306, 2936, 2097; elemental Anal. Calcd for C9H15N3: C,
65.42; H, 9.15; N, 25.43. Found: C, 65.15; H. 9.21; N, 25.11.
4.1.4. (2S,5R)-5-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-2,5-dihy-
droxypentyl 2,4,6-triisopropylbenzene sulfonate (41)
2,4,6-Triisopropylbenzenesulfonyl chloride (206 mg, 0.681
mmol) was added to a solution of 40 (50.0 mg, 0.227 mmol) in
pyridine (0.9 mL) and CH2Cl2 (1.4 mL) with stirring at 0 °C. After
stirred for 15 min at the same temperature, the mixture was
stirred at rt for 38 h. Water was added to the reaction mixture
and the mixture was extracted with EtOAc. The combined organic
layers were washed with water and brine prior to drying. Concen-
tration followed by azeotropic removal of pyridine with toluene
was repeated three times. Purification by column chromatography
over silica gel with n-hexane/EtOAc (3:1) as eluent yielded 41
(87.3 mg, 79%) as a colorless solid. Mp 108.3–109.4 °C (n-hex-
4.1.8. 5-(Dimetylamino)-N-(non-8-ynyl)naphthalene-1-
sulfonamide (45)
PPh3 (1.12 g, 4.28 mmol) was added to a solution of 44 (707 mg,
4.28 mmol) in Et2O (2.7 mL) with stirring at 0 °C. After 2 h, water
was added to the reaction mixture and the whole was stirred for

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8637
3 h at the same temperature. HCl (3 N) was added to the mixture,
the mixture was washed with Et2O. After the aqueous layer was
basified using NaOH, the mixture was extracted with Et2O, and
the solvent was evaporated. To a solution of the residue in CH2Cl2
(11 mL) was added i-Pr2NEt (0.853 mL, 5.02 mmol). After stirred
for 10 min at the same temperature, 5-dimetylaminonaphthal-
enesulfonyl chloride (1.36 g, 5.02 mmol) was added to the reaction
mixture and the whole was stirred for 3.5 h. After solvent evapora-
tion, purification by column chromatography over silica gel with n-
hexane/EtOAc (10:1) as eluent yielded 5 (1.53 g, 96% in two steps)
as a pale green solid. Mp 58.1–59.1 °C (n-hexane–EtOAc); 1H NMR
(500 MHz, CDCl3) d: 1.06–1.44 (m, 10H), 1.92 (t, 1H, J = 2.4 Hz),
2.11 (td, 2H, J = 7.3, 2.4 Hz), 2.88 (q, 2H, J = 6.7 Hz), 2.89 (s, 6H),
4.56 (br t, 1H, J = 6.7 Hz), 7.19 (d, 1H, J = 7.3 Hz), 7.53 (t, 1H,
J = 7.9 Hz), 7.57 (dd, 1H, J = 8.5, 7.9 Hz), 8.25 (d, 1H, J = 7.3 Hz),
8.28 (d, 1H, J = 8.5 Hz), 8.54 (d, 1H, J = 7.9 Hz); 13C NMR (75 MHz,
CDCl3) d: 18.2, 26.1, 28.1, 28.29, 28.31, 29.32, 43.1, 45.4 (2C),
68.1, 84.5, 115.1, 118.7, 123.1, 128.3, 129.6 (2C), 129.8, 130.3,
134.7, 151.9; IR (neat) cmꢀ1 : 3295, 2936, 2114, 1587; MS (FAB)
m/z: 395 [M+Na]+; HRMS (FAB) m/z: calcd for C21H28N2NaO2S:
395.1769; found: 395.1758 [M+Na]+.
(236 mg, 74%, 48a:48b = 95:5) as a colorless oil. ½a D26
ꢂ
ꢀ13.8 (c
1.50, CHCl3); 1H NMR (500 MHz, CDCl3) d: 1.18 (d, 3H, J = 7.3 Hz),
1.25–1.79 (m, 19H), 1.38 (s, 3H), 1.44 (s, 3H), 1.79–1.86 (m, 1H),
1.95–2.08 (m, 1H), 1.97 (dd, 1H, J = 13.4, 7.3 Hz), 2.10–2.16 (m,
1H), 2.20 (td, 2H, J = 7.3, 2.4 Hz), 2.33 (dd, 0.15H, J = 13.4, 4.9 Hz),
2.52 (dd, 0.85H, J = 13.4, 7.3 Hz), 2.53 (br s, 1H), 3.70 (t, 1H,
J = 7.3 Hz), 4.01 (t, 1H, J = 7.3 Hz), 4.00–4.05 (m, 1H), 4.07–4.14
(m, 2H), 4.21–4.25 (m, 1H), 4.49 (m, 0.85H), 4.61 (m, 0.15H),
7.33–7.43 (m, 3H), 7.51–7.57 (m, 2H); 13C NMR (75 MHz, CDCl3)
(Major isomer) d: 18.7, 21.4, 24.6, 25.5, 26.4, 27.86, 27.88, 28.4,
28.7, 28.9, 29.2, 29.3 (2C), 29.5, 36.4, 40.0, 56.2, 65.5, 65.8, 73.2,
77.7, 78.3, 79.9, 83.1, 86.5, 109.7, 128.9 (2C), 129.6, 130.3, 136.7
(2C), 177.1; IR (neat) cmꢀ1: 3462, 2928, 2231, 1763, 1458, 1439;
MS (FAB) m/z: 573 [M+H]+; HRMS (FAB) m/z: calcd for
C
33H49O6S: 573.3250; found: 573.3223 [M+H]+.
4.1.11. (3RS,5S)-3-{(13S)-13-Hydroxy-13-[(2R,5R)-5-[(1R)-2,2-
dimethyl-1,3-dioxolan-1-yl]-5-hydroxy-methyltetrahydro-
furan-2-yl]tridec-11-ynyl}-5-methyl-3-(phenylsulfenyl)tetra-
hydrofuran-2-one (48b)
The procedure was same as that used for preparation of 48a.
Colorless oil; ½a D26
ꢂ
ꢀ7.2 (c 0.95, CHCl3); 1H NMR (500 MHz, CDCl3)
4.1.9. (3RS,5S)-5-methy-3-phenylsulfenyl-3-(dodec-11-ynyl)-
tetrahydrofuran-2-one (37)
d: 1.18 (d, 3H, J = 6.7 Hz), 1.25–1.82 (m, 19H), 1.38 (s, 3H), 1.44 (s,
3H), 1.97 (dd, 1H, J = 14.0, 6.7 Hz), 2.00–2.09 (m, 3H), 2.19 (td, 2H,
J = 7.3, 1.8 Hz), 2.33 (dd, 0.15H, J = 13.4, 5.5 Hz), 2.34 (d, 1H,
J = 4.9 Hz), 2.52 (dd, 0.85H, J = 14.0, 6.7 Hz), 3.66 (dd, 1H, J = 7.3,
6.7 Hz), 4.01 (dd, 1H, J = 7.3, 6.7 Hz), 4.06 (q, 1H, J = 6.7 Hz),
4.08–4.14 (m, 1H), 4.19 (td, 1H, J = 7.3, 3.1 Hz), 4.49 (sext, 0.85H,
J = 6.7 Hz), 4.52–4.56 (m, 1H), 4.57–4.64 (m, 0.15H), 7.32–7.44
(m, 3H), 7.50–7.58 (m, 2H); 13C NMR (75 MHz, CDCl3) (Major iso-
mer) d: 18.6, 21.4, 24.5, 25.4, 25.8, 26.4, 28.0, 28.4, 28.7, 28.9,
29.2, 29.3 (2C), 29.4, 36.4, 40.0, 56.2, 63.9, 65.7, 73.1, 77.5, 78.6,
81.1, 82.3, 86.5, 109.6, 128.9 (2C), 129.6, 130.3, 136.7 (2C), 177.0;
IR (neat) cmꢀ1 : 3460, 2928, 1765; MS (FAB) m/z: 573 [M+H]+;
n-BuLi (1.58 M in n-hexane, 8.65 mL, 13.7 mmol) was added to
a solution of i-Pr2NH (1.92 mL, 13.7 mmol) in THF (5.6 mL) with
stirring at ꢀ78 °C and the mixture was stirred for 30 min. A
solution of 47 (3.03 g, 14.5 mmol) in THF (5.6 mL) was added to
the mixture. After 1.5 h, a solution of 46 (2.66 g, 9.10 mmol) in
THF/HMPA (1:2.7, 9.3 mL) was added to the mixture and the whole
was stirred for 5 min at the same temperature. After stirred for
16 h at rt, the reaction was quenched with saturated NH4Cl and
the mixture was extracted with EtOAc. The combined organic
layers were washed with brine prior to drying and solvent evapo-
ration. Purification by column chromatography over silica gel with
n-hexane/EtOAc (10:1) as eluent yielded 37 (3.28 g, 97%) as a yel-
HRMS (FAB) m/z: calcd for
C33H49O6S: 573.3250; found:
573.3261 [M+H]+.
low oil. ½a 2D5
ꢂ
ꢀ33.4 (c 1.11, CHCl3); 1H NMR (500 MHz, CDCl3) d:
1.18 (d, 3H, J = 6.1 Hz), 1.21–1.81 (m, 18H), 1.94 (t, 1H,
J = 2.4 Hz), 1.96 (dd, 1H, J = 14.6, 7.3 Hz), 2.18 (td, 2H, J = 7.3,
2.4 Hz), 2.33 (dd, 0.15H, J = 13.4, 4.9 Hz), 2.52 (dd, 0.85H, J = 14.6,
7.3 Hz), 4.49 (qt, 0.85H, J = 7.3, 6.1 Hz), 4.60 (qt, 0.15H, J = 13.4,
4.6 Hz), 7.31–7.44 (m, 3H), 7.49–7.58 (m, 2H); 13C NMR (75 MHz,
CDCl3) (Major isomer) d: 18.2, 21.3, 24.4, 28.2, 28.5, 28.8, 29.1,
29.2 (2C), 29.3, 36.3, 39.9, 56.1, 68.0, 73.0, 84.5, 128.8 (2C),
129.5, 130.3, 136.6 (2C), 176.9; IR (neat) cmꢀ1: 3304, 2928, 2116,
1763; MS (FAB) m/z: 373 [M+H]+; HRMS (FAB) m/z: calcd for
4.1.12. (3RS,5S)-3-{(13R)-13-tert-Butyldiphenylthylsilyoxy-13-
[(2R,5S)-5-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]tetrahydro-
furan-2-yl]tridec-11-ynyl}-5-methyl-3-(phenylsulfenyl)tetra-
hydrofuran-2-one (49)
Imidazole (164 mg, 2.41 mmol) was added to the solution of 48a
(627 mg, 1.10 mmol) in DMF (11 mL) at 0 °C. After stirred for
10 min, TBDPSCl (0.569 mL, 2.19 mmol) was added to the reaction
mixture and the whole was stirred for 10 min at the same temper-
ature. After stirred for 12 h at rt, water was added to the mixture.
The mixture was extracted with EtOAc. The combined organic layers
were washed with brine prior to drying and solvent evaporation.
Purification by column chromatography over silica gel with n-hex-
ane/EtOAc (7:1) as eluent yielded 49 (888 mg, quant.) as a colorless
C
23H33O2S: 373.2201; found: 373.2202 [M+H]+.
4.1.10. (3RS,5S)-3-{(13R)-13-Hydroxy-13-[(2R,5R)-5-[(1R)-2,2-
dimethyl-1,3-dioxolan-1-yl]-5-hydroxy-methyltetrahydro-
furan-2-yl]tridec-11-ynyl}-5-methyl-3-(phenylsulfenyl)tetra-
hydrofuran-2-one (48a)
oil. ½a 2D5
ꢂ
ꢀ38.9 (c 1.11, CHCl3); 1H NMR (500 MHz, CDCl3) d: 1.06 (s,
9H), 1.18 (d, 3H, J = 6.1 Hz), 1.22–1.82 (m, 19H), 1.35 (s, 3H), 1.39 (s,
3H), 1.94–2.03 (m, 1H), 1.96 (dd, 1H, J = 14.0, 7.3 Hz), 2.05 (t, 2H,
J = 7.3 Hz), 2.08–2.18 (m, 2H), 2.33 (dd, 0.15H, J = 13.4, 4.9 Hz),
2.51 (dd, 0.85H, J = 14.0, 7.3 Hz), 3.66 (dd, 1H, J = 7.3, 6.7 Hz), 3.96
(dd, 1H, J = 7.3, 6.7 Hz), 3.98–4.05 (m, 2H), 4.13 (q, 1H, J = 6.1 Hz),
4.44–4.52 (m, 1.85H), 4.57–4.64 (m, 0.15H), 7.30–7.43 (m, 9H),
7.51–7.57 (m, 2H), 7.65–7.76 (m, 4H); 13C NMR (75 MHz, CDCl3)
(Major isomer) d: 18.6, 19.3, 21.4, 24.6, 25.5, 26.4, 26.8 (3C), 27.1,
27.9, 28.3, 28.7, 29.0, 29.3, 29.4 (2C), 29.5, 36.4, 40.0, 56.2, 65.8,
66.5, 73.2, 78.4, 78.9, 80.0, 82.0, 86.4, 109.5, 127.2 (2C), 127.4
(2C), 128.9 (2C), 129.4, 129.6 (2C), 130.4, 133.4, 133.7, 135.8 (2C),
135.9 (2C), 136.8 (2C), 177.1; IR (neat) cmꢀ1 : 2930, 2238, 1767;
MS (FAB) m/z: 833 [M+H]+; HRMS (FAB) m/z: calcd for C49H66NaO6S-
Si: 833.4247; found: 833.4240 [M+H]+.
A
flask was charged with Zn(OTf)2 (447 mg, 1.23 mmol).
Vacuum (8 mmHg) was applied and the flask was heated to
120 °C for 9 h. After the flask was cooled to rt, the vacuum was re-
leased. (1R,2S)-N-methylephedrine (240 mg, 1.34 mmol), toluene
(0.8 mL), and i-Pr2NEt (0.228 mL, 1.34 mmol) were added to the
flask with stirring at rt. After 2.5 h, a solution of 37 (417 mg,
1.12 mmol) in toluene (0.8 mL) was added to the mixture at the
same temperature. After 15 min, 36 (112 mg, 0.559 mmol) in tolu-
ene (0.9 mL) was added to the reaction mixture and the whole
mixture was stirred for 5 h at rt. The reaction was quenched with
saturated NH4Cl and the mixture was extracted with EtOAc. The
combined organic layers were washed with brine prior to drying
and solvent evaporation. Purification by column chromatography
over silica gel with n-hexane/EtOAc (2:1) as eluent yielded 48a

8638
N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
4.1.13. (3RS,5S)-3-{(13R)-13-tert-Butyldiphenylsilyloxy-13-
[(2R,5R)-5-[(1R)-1,2-dihydroxyethan-1-yl]tetrahydrofuran-2-
yl]tridec-11-ynyl}-5-methyl-3-(phenylsulfenyl)tetrahydro-
furan-2-one (50)
2.86 (q, 2H, J = 6.1 Hz), 2.87 (s, 6H), 3.94 (q, 1H, J = 6.1 Hz), 4.05–
4.16 (m, 2H), 4.31–4.37 (m, 1H), 4.42–4.52 (m, 0.85H), 4.54–4.63
(m, 0.15H), 4.74–4.90 (m, 1H), 7.17 (d, 1H, J = 7.3 Hz), 7.30–7.40
(m, 9H), 7.48–7.58 (m, 4H), 7.69 (d, 2H, J = 7.3 Hz), 7.75 (d, 2H,
J = 7.3 Hz), 8.24 (d, 1H, J = 7.3 Hz), 8.30 (d, 1H, J = 8.5 Hz), 8.53 (d,
1H, J = 8.5 Hz); 13C NMR (75 MHz, CDCl3) (Major isomer) d: 18.5,
18.6, 19.3, 21.4, 24.6, 26.1, 26.8 (3C), 27.5, 27.9, 28.1, 28.2, 28.3,
28.7, 29.0, 29.26, 29.29, 29.35, 29.38, 29.5, 29.6, 36.4, 39.9, 43.1,
45.3 (2C), 56.2, 65.6, 66.8, 73.2, 77.8, 78.6, 82.5, 82.9, 86.1, 86.4,
115.0, 118.6, 123.1, 127.2 (2C), 127.4 (2C), 128.3, 128.9 (2C),
129.4, 129.5 (2C), 129.56, 129.62, 129.7, 130.2, 130.3, 133.5 (2C),
134.7, 135.7 (2C), 136.0 (2C), 136.7 (2C), 151.8, 177.1; IR (neat)
cmꢀ1 : 3526, 3310, 2930, 2239, 1761; MS (FAB) m/z: 1111
[M+H]+; HRMS (FAB) m/z: calcd for C66H87N2O7S2Si: 1111.5724;
found: 1111.5709 [M+H]+.
Dowex 50 W (418 mg) was added to a solution of 49 (424 mg,
0.523 mmol) in THF/MeOH (1:1, 12 mL) at rt. After stirred for
10 h at 60 °C, the catalyst was filtered off and the filtrate was con-
centrated under reduced pressure. Purification by column chroma-
tography over silica gel with n-hexane/EtOAc (2:1) as eluent
yielded 50 (386 mg, 96%) as a colorless solid. ½a D26
ꢀ36.8 (c 1.03,
ꢂ
CHCl3); 1H NMR (500 MHz, CDCl3) d: 1.07 (s, 9H), 1.17 (d, 3H,
J = 6.1 Hz), 1.18–1.83 (m, 19H), 1.85–1.99 (m, 2H), 1.96 (dd, 1H,
J = 13.4, 6.1 Hz), 2.01–2.13 (m, 1H), 2.05 (t, 2H, J = 7.3 Hz), 2.33
(dd, 0.15H, J = 13.4, 6.1 Hz), 2.45 (br s, 1H), 2.51 (dd, 0.85H,
J = 13.4, 7.3 Hz), 2.54 (br, 1H), 3.44–3.51 (m, 1H), 3.54–3.67 (m,
2H), 3.87 (dt, 1H, J = 8.5, 4.9 Hz), 4.11 (q, 1H, J = 6.1 Hz), 4.32 (d,
1H, J = 6.1 Hz), 4.44–4.51 (m, 0.85H), 4.56–4.63 (m, 0.15H), 7.32–
7.43 (m, 9H), 7.51–7.56 (m, 2H), 7.71 (d, 2H, J = 7.3 Hz), 7.75 (d,
2H, J = 7.3 Hz); 13C NMR (75 MHz, CDCl3) (Major isomer) d: 18.5,
19.3, 21.4, 24.6, 26.8 (3C), 27.9 (2C), 28.3, 28.7, 29.0, 29.3, 29.4
(2C), 29.5, 36.4, 40.0, 56.2, 64.6, 66.9, 73.0, 73.2, 78.7, 80.7, 82.6,
86.6, 127.2 (2C), 127.4 (2C), 128.9 (2C), 129.5, 129.57, 129.63,
130.3, 133.51, 133.54, 135.7 (2C), 136.0 (2C), 136.7 (2C), 177.1;
IR (neat) cmꢀ1 : 3458, 2930, 1767; MS (FAB) m/z: 771 [M+H]+;
4.1.16. 5-Dimethylamino-N-[(10S)-10-hydroxy-10-((2R,5R)-5-
{(1R)-1-tert-butyldiphenylsilyloxy-13-[(3RS,5S)-2-oxo-5-methyl-
3-(phenylsulfenyl)tetrahydrofuran-3-yl]tridec-2-ynyl}tetra-
hyrofran-2-yl)dec-8-ynyl]naphthalene-1-sulfonamide (52b)
The procedure was same as that used for preparation of 52a.
Pale green amorphous solid; ½a D26
ꢂ
ꢀ21.2 (c 0.95, CHCl3); 1H NMR
(500 MHz, CDCl3) d: 1.00–1.82 (m, 28H), 1.07 (s, 9H), 1.16 (d, 3H,
J = 6.1 Hz), 1.87–2.20 (m, 4H), 1.96 (dd, 1H, J = 14.6, 7.3 Hz), 2.06
(t, 2H, J = 7.3 Hz), 2.10 (t, 2H, J = 7.3 Hz), 2.33 (dd, 0.15H, J = 13.4,
6.1 Hz), 2.39 (br, 1H), 2.51 (dd, 0.85H, J = 13.4, 7.3 Hz), 2.87 (q,
2H, J = 6.1 Hz), 2.90 (s, 6H), 4.01 (td, 1H, J = 7.3, 2.4 Hz), 4.19 (m,
1H), 4.31–4.39 (m, 2H), 4.44–4.51 (m, 0.85H), 4.55–4.62 (m,
0.15H), 5.05 (br t, 1H, J = 6.1 Hz), 7.20 (d, 1H, J = 7.3 Hz), 7.29–
7.43 (m, 9H), 7.47–7.59 (m, 4H), 7.70 (d, 2H, J = 7.3 Hz), 7.75 (d,
2H, J = 7.3 Hz), 8.24 (d, 1H, J = 7.3 Hz), 8.35 (d, 1H, J = 8.5 Hz),
8.57 (d, 1H, J = 7.3 Hz); 13C NMR (75 MHz, CDCl3) (Major isomer)
d: 18.46, 18.53, 19.2, 21.3, 24.5, 26.0 (2C), 26.8 (3C), 27.5, 28.1,
28.16, 28.23, 28.3, 28.7, 28.9, 29.2, 29.25, 29.3 (2C), 29.4, 36.3,
39.9, 43.0, 45.4 (2C), 56.2, 64.1, 66.7, 73.1, 77.7, 78.7, 82.1, 83.0,
86.1, 86.4, 115.2, 119.1, 123.2, 127.2 (2C), 127.3 (2C), 128.1,
128.9 (2C), 129.36, 129.41 (2C), 129.5 (2C), 129.6, 130.0, 130.3,
133.45, 133.53, 134.8, 135.7 (2C), 135.9 (2C), 136.7 (2C), 151.3,
177.1; IR (neat) cmꢀ1: 3509, 3298, 2930, 1761; MS (FAB) m/z:
1111 [M+H]+; HRMS (FAB) m/z: calcd for C66H87N2O7S2Si:
1111.5724; found: 1111.5723 [M+H]+.
HRMS (FAB) m/z: calcd for
C46H63O6SSi: 771.4115; found:
771.4107 [M+H]+.
4.1.14. (3RS,5S)-3-{(13R)-13-tert-Butyldiphenylsilyloxy-13-
[(2R,5R)-5-formyltetrahydrofuran-2-yl]tridec-11-ynyl}-5-
methyl-3-(phenylsulfenyl)tetrahydrofuran-2-one (51)
NaIO4 (193 mg, 0.902 mmol) was added to a solution of 50
(580 mg, 0.752 mmol) in THF/H2O (4:1, 10 mL) at rt. After stirred
for 2 h, NaIO4 (48.3 mg, 0.226 mmol) was added to the mixture
and the whole was stirred for 2 h. Water was added to the reaction
mixture and the mixture was extracted with CHCl3. The combined
organic layers were washed with brine prior to drying and solvent
evaporation. Purification by column chromatography over silica gel
with n-hexane/EtOAc (3:1?2:1) as eluent yielded 51 (550 mg,
99%) as a colorless oil. The aldehyde was unstable and therefore
used immediately in the next reaction.
4.1.15. 5-Dimethylamino-N-[(10R)-10-hydroxy-10-((2R,5R)-5-
{(1R)-1-tert-butyldiphenylsilyloxy-13-[(3RS,5S)-2-oxo-5-methyl-
3-(phenylsulfenyl)tetrahydrofuran-3-yl]tridec-2-ynyl}tetra-
hyrofran-2-yl)dec-8-ynyl]naphthalene-1-sulfonamide (52a)
A flask was charged with Zn(OTf)2 (125 mg, 0.343 mmol). Vac-
uum (8 mmHg) was applied and the flask was heated to 120 °C
for 10 h. After the flask was cooled to rt, the vacuum was released.
(1R,2S)-N-methylephedrine (67.1 mg, 0.375 mmol), toluene
(0.2 mL), and i-Pr2NEt (0.106 mL, 0.624 mmol) were added to the
flask with stirring at rt. After 2.5 h, a solution of 45 (116 mg,
0.312 mmol) in toluene (0.3 mL) was added to the mixture at the
same temperature. After 15 min, 51 (77.3 mg, 0.104 mmol) in tol-
uene (0.7 mL) was added to the reaction mixture and the whole
mixture was stirred for 4 h at rt. The reaction was quenched with
saturated NH4Cl and the mixture was extracted with EtOAc. The
combined organic layers were washed with brine prior to drying
and solvent evaporation. Purification by flash column chromatog-
raphy over silica gel with n-hexane/Et2O (3:1) as eluent yielded
52a (76.2 mg, 66%, 52a:52b = 91:9) as a pale green amorphous so-
4.1.17. 5-Dimethylamino-N-[(10R)-10-hydroxy-10-((2R,5R)-5-
{(1R)-1-tert-butyldiphenylsilyloxy-13-[(3RS,5S)-2-oxo-5-methyl-
3-(phenylsulfenyl)tetrahydrofuran-3-yl] tridecyl}tetrahyrofran-
2-yl)decanyl]naphthalene-1-sulfonamide (53)
A solution of 52a (74.0 mg, 0.0666 mmol) in benzene (1.5 mL)
was hydrogenated on Rh(PPh3)3Cl (43.0 mg, 0.0466 mmol) for
67.5 h with stirring at rt under 3 atm pressure of hydrogen.
The catalyst was filtered off and the filtrate was concentrated un-
der reduced pressure. Purification by column chromatography over
flash silica gel with n-hexane/Et2O (3:1) as eluent yielded 53
(54.7 mg, 73%) as a pale green oil. ½a D24
ꢂ
ꢀ7.3 (c 0.45, CHCl3); 1H
NMR (500 MHz, CDCl3) d: 1.02 (s, 9H), 1.02–1.82 m, 43H), 1.18
(d, 3H, J = 6.1 Hz), 1.82–1.90 (m, 1H), 1.97 (dd, 1H, J = 13.4,
6.1 Hz), 2.33 (dd, 1H, J = 13.4, 6.1 Hz), 2.51 (dd, 1H, J = 13.4,
7.3 Hz), 2.87 (q, 2H, J = 6.1 Hz), 2.89 (s, 6H), 3.03–3.12 (m, 1H),
3.43 (dt, 1H, J = 7.3, 6.1 Hz), 3.45–3.54 (m, 1H), 3.92 (dt, 1H,
J = 8.5, 7.3 Hz), 4.42–4.62 (m, 1H), 4.56 (t, 1H, J = 6.1 Hz), 7.19 (d,
1H, J = 7.3 Hz), 7.29–7.43 (m, 9H), 7.45–7.60 (m, 4H), 7.70 (d, 2H,
J = 8.5 Hz), 7.74 (d, 2H, J = 7.3 Hz), 8.25 (d, 1H, J = 7.3 Hz), 8.28 (d,
1H, J = 8.5 Hz), 8.54 (d, 1H, J = 8.5 Hz); 13C NMR (75 MHz, CDCl3)
(Major isomer) d: 19.6, 21.5, 24.7, 24.9, 25.5, 26.4, 27.1 (3C),
28.7, 28.9, 29.0, 29.3, 29.4 (2C), 29.46 (2C), 29.48 (2C), 29.6 (4C),
29.7, 33.2, 33.4, 36.5, 40.0, 43.3, 45.4 (2C), 56.3, 73.2, 74.0, 76.6,
lid. ½a 2D4
ꢂ
ꢀ25.1 (c 1.04, CHCl3); 1H NMR (500 MHz, CDCl3) d: 1.04–
1.64 (m, 28H), 1.07 (s, 9H), 1.16 (d, 3H, J = 6.1 Hz), 1.66–1.82 (m,
1H), 1.91–2.14 (m, 3H), 1.96 (dd, 1H, J = 14.6, 7.3 Hz), 2.09 (td,
2H, J = 7.3, 2.4 Hz), 2.10 (td, 2H, J = 7.3, 2.4 Hz), 2.33 (dd, 0.15H,
J = 13.4, 4.9 Hz), 2.42 (br, 1H), 2.51 (dd, 0.85H, J = 14.6, 7.3 Hz),

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8639
82.2, 82.3, 115.1, 118.6, 123.2, 127.1 (2C), 127.3 (2C), 128.3, 129.0
4.2. Biology
(2C), 129.2, 129.3, 129.6, 129.65, 129.68, 129.8, 130.3, 130.4, 133.9,
134.6, 135.2, 135.7 (2C), 136.2 (2C), 136.8 (2C), 152.0, 177.2; IR
(neat) cmꢀ1 : 3547, 3296, 2926, 1765; MS (FAB) m/z: 1196
[M+H]+; HRMS (FAB) m/z: calcd for C66H95N2O7S2Si: 1119.6350;
found: 1119.6305 [M+H]+.
4.2.1. Determination of cell growth inhibition profiles
(fingerprint) and COMPARE analysis
This experiment was carried out at the Cancer Chemo therapy
Center, Japanese Foundation for Cancer Research. The screening pa-
nel consisted of the following 39 human cancer cell lines (JFCR39):
breast cancer HBC-4, BSY-1, HBC-5, MCF-7, and MDA-MB-231; brain
cancer U251, SF-268, SF-295, SF-539, SNB-75, and SNB-78; colon
cancer HCC2998, KM-12, HT-29, HCT-15, and HCT-116; lung cancer
NCI-H23, NCIH-226, NCI-H522, NCI-H460, A549, DMS273, and
DMS114; melanoma LOX-IMVI; ovarian cancer OVCAR-3, OVCAR-4,
OVCAR-5, OVCAR-8, and SK-OV-3; renal cancer RXF-631L and
ACHN; stomach cancer St-4, MKN1, MKN7, MKN28, MKN45, and
MKN74; and prostate cancer DU-145 and PC-3. Inhibition of cell
growth was assessed by the change in total cellular protein follow-
ing 48 h of treatment with a given test compound, and was mea-
sured using the sulforhodamine B colorimetric assay. The molar
concentration of a test compound required for 50% growth inhibi-
tion (GI50) of cells was calculated as reported previously. A detailed
method is described elsewhere.43 A graphic representation (termed
fingerprint) of the differential growth inhibition of each compound
for the cells in the JFCR39 panel was plotted based on a calculation
employing a set of GI50 values. We used COMPARE analysis to com-
pare two compounds for the similarity of their mode of action based
on their fingerprints. COMPARE analysis was performed by calculat-
ing the Pearson correlation coefficient (r) between the GI50 mean
graphs of the compounds X and Y using the following formula:
r = ((xi ꢀ xm) (yi ꢀ ym))/((xi ꢀ xm)2 (yi ꢀ ym)2)1/2, where xi and yi are
Log GI50 of the two compounds, respectively, for each cell line, and
xm and ym are the mean values of xi and yi, respectively
(n = 39).43,44 The Pearson correlationcoefficients were used to deter-
mine the degree of similarity. The greater the coefficient is, the high-
er the similarity between X and Y.
4.1.18. 5-Dimethylamino-N-[(10R)-10-hydroxy-10-((2R,5R)-5-
{(1R)-1-tert-butyldiphenylsilyloxy-13-[(5S)-2-oxo-5-methyl-
2,5-dihydrofuran-3-yl)tridecyl]tetrahyrofran-2-yl}decanyl]-
naphtalene-1-sulfonamide (54)
mCPBA (75% in water, 6.8 mg, 0.0294 mmol) was added to a
solution of 53 (24.5 mg, 0.0219 mmol) in CH2Cl2 (1.0 mL) with stir-
red at 0 °C. After stirred for 15 min, the reaction was quenched
with satd. NaHCO3 and the mixture was extracted with CH2Cl2.
The combined organic layers were washed with brine prior to dry-
ing and solvent evaporation. The residue was dissolved in toluene
(1.0 mL), and the mixture was stirred for 20 min under reflux. Puri-
fication by flash column chromatography over silica gel with n-
hexane/EtOAc (3:1) as eluent yielded 54 (15.1 mg, 68% in two
steps) as a pale green oil. ½a D23
ꢂ
+13.6 (c 1.35, CHCl3); 1H NMR
(500 MHz, CDCl3) d: 1.00–1.89 (m, 44H), 1.02 (s, 9H), 1.40 (d, 3H,
J = 7.3 Hz), 2.26 (t, 2H, J = 7.3 Hz), 2.87 (q, 2H, J = 6.1 Hz), 2.89 (s,
6H), 3.05–3.13 (m, 1H), 3.43 (q, 1H, J = 7.3 Hz), 3.47–3.54 (m,
1H), 3.92 (q, 1H, J = 7.3 Hz), 4.64 (t, 1H, J = 6.1 Hz), 4.99 (q, 1H,
J = 7.3 Hz), 6.98 (s, 1H), 7.18 (d, 1H, J = 7.3 Hz), 7.31–7.43 (m, 6H),
7.52 (dd, 1H, J = 8.5, 7.3 Hz), 7.56 (t, 1H, J = 8.5, 7.3 Hz), 7.70 (d,
2H, J = 7.3 Hz), 7.74 (d, 2H, J = 7.3 Hz), 8.25 (d, 1H, J = 7.3 Hz),
8.29 (d, 1H, J = 8.5 Hz), 8.54 (d, 1H, J = 8.5 Hz); 13C NMR (75 MHz,
CDCl3) d: 19.2, 19.6, 24.9, 25.1, 25.5, 26.4, 27.1 (3C), 27.4, 28.7,
28.9, 29.0, 29.27, 29.30, 29.4, 29.47, 29.49, 29.52, 29.56 (2C),
29.58, 29.60, 29.67, 29.70, 33.2, 33.4, 43.3, 45.4 (2C), 74.0, 77.4,
76.6, 82.2, 82.3, 115.1, 118.6, 123.2, 127.1 (2C), 127.3 (2C), 128.3,
129.2, 129.3, 129.59, 129.65, 129.8, 130.3, 133.9, 134.3, 134.6,
135.2, 135.7 (2C), 136.2 (2C), 148.9, 152.0, 173.9; IR (neat) cmꢀ1
: 3565, 3291, 2926, 1755; MS (FAB) m/z: 1009 [M+H]+; HRMS
(FAB) m/z: calcd for C60H89N2O7SSi: 1009.6160; found: 1009.6153
[M+H]+.
4.2.2. Fluorescence imaging study
CHO-K1 cells were inoculated in 35 mm glass-bottomed dishes
(Matsunami, Japan) and cultured for two days in Ham’s F12 medium
(Sigma, USA) with 7% fetal calf serum at 37 °C. To accomplish double
staining with dansyl-labeled solamin 3 and MitoTracker Red, cells
4.1.19. 5-Dimethylamino-N-[(10R)-10-hydroxy-10-((2R,5R)-5-
{(1R)-1-hydroxy-13-[(5S)-2-oxo-5-methyl-2,5-dihydrofuran-3-
yl)tridecyl]tetrahyrofran-2-yl}decanyl]naphthalene-1-sulfon-
amide (3)
were first treated with 10 lM dansyl-labeled solamin 3 in the med-
ium for 5 h. Then, the dishes were washed five times with fresh med-
ium. Thereafter, the cells were further incubated for 30 min in the
presence of 50 nM MitoTracker Red CMX-Ros (Molecular Probes,
Two drops of 48% aq HF were added to a solution of 54 (25.0 mg,
0.0248 mmol) in MeCN (1.0 mL) with stirring at rt. After stirred
for 41 h, the reaction was quenched with satd. NaHCO3, and the
mixture was extracted with CH2Cl2, The combined organic layers
were washed with brine prior to drying and solvent evaporation.
Purification by flash column chromatography over silica gel with
CHCl3/EtOAc (4:1) as eluent yielded 3 (13.6 mg, 71%) as a pale
USA) in Ham’s F12 medium or for 40 min in the presence of 1 lM
ER-Tracker Red (Molecular Probes) in Hank’s balanced salt solution.
After washing with fresh medium, fluorescence images were ob-
tained with a confocal microscope (FV1000-IX81; Olympus, Japan)
with 405 nm (dansyl-labeled solamin 3) and 559 nm (MitoTracker
and ER-Tracker) excitation lines. For the simultaneous detection of
dansyl-labeled solamin 3 and lysosomes, a CHO-K1 sub-line stably
expressing GFP-tagged hTAPL45 was cultured and stained with dan-
syl-labeled solamin 3 as above, and GFP fluorescence was observed
with the 473 nm excitation line. For each experiment, samples were
run in duplicate, and any fields were similarly stained. Representa-
tive images are displayed in Figure 3. No toxicity of compound 3
against CHO-K1 cells was confirmed because desquamated and sus-
pended cells were not observed by microscope observation in dishes
which were incubated for 24 h after the distribution study by a
microscopy.
green powder. Mp 58.9–60.2 °C; ½a D25
ꢂ
+17.3 (c 0.60, CHCl3); 1H
NMR (500 MHz, CDCl3) d: 1.09–1.58 (m, 38H), 1.40 (d, 3H,
J = 7.3 Hz,), 1.60–1.78 (m, 2H), 1.94–2.05 (m, 2H), 2.26 (t, 2H,
J = 7.3 Hz), 2.40 (br, 2H), 2.87 (q, 2H, J = 6.1 Hz), 2.89 (s, 6H),
3.35–3.44 (m, 2H), 3.75–3.84 (m, 2H), 4.62 (t, 1H, J = 6.1 Hz), 5.00
(q, 1H, J = 7.3 Hz), 6.99 (s, 1H), 7.19 (d, 1H, J = 7.3 Hz), 7.53 (dd,
1H, J = 8.5, 7.3 Hz), 7.57 (dd, 1H, J = 8.5, 7.3 Hz), 8.25 (d, 1H,
J = 7.3 Hz), 8.28 (d, 1H, J = 8.5 Hz), 8.53 (d, 1H, J = 8.5 Hz); 13C
NMR (75 MHz, CDCl3) d: 19.2, 25.1, 25.5, 25.6, 26.3, 27.4, 28.7
(2C), 28.9, 29.1, 29.2, 29.27, 29.30, 29.46, 29.48, 29.53, 29.6 (4C),
29.7, 33.38, 33.43, 43.3, 45.4 (2C), 74.00, 74.02, 77.4, 82.58,
82.61, 115.1, 118.6, 123.2, 128.3, 129.6, 129.7, 129.8, 130.3,
134.3, 134.7, 148.9, 152.0, 174.0; IR (neat) cmꢀ1 : 3503, 3310,
2926, 1751; MS (FAB) m/z: 771 [M+H]+; HRMS (FAB) m/z: calcd
for C44H71N2O7S: 771.4982; found: 771.4985 [M+H]+.
Acknowledgments
In vitro antiproliferative activities of fluorescence-labeled sola-
mins and natural solamin against human cancer cell lines were
examined by the Screening Committee of New Anticancer Agents

8640
N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
Tominaga, H.; Maezaki, N.; Yanai, M.; Kojima, N.; Urabe, D.; Ueki, R.; Tanaka, T.
Eur. J. Org. Chem. 2006, 1422; (d) Maezaki, N.; Tominaga, H.; Kojima, N.; Yanai,
M.; Urabe, D.; Ueki, R.; Tanaka, T.; Yamori, T. Chem. Eur. J. 2005, 11, 6237; (e)
Maezaki, N.; Tominaga, H.; Kojima, N.; Yanai, M.; Urabe, D.; Tanaka, T. Chem.
Commun. 2004, 406.
supported by a Grant-in-Aid for Scientific Research on Priority Area
‘Cancer’ from The Ministry of Education, Culture, Sports, Science
and Technology, Japan. We acknowledge financial support in the
form of a Grant-in-Aid for Young Scientists (B) [No. 21790113], a
Grant-in-Aid for Scientific Research (B) [No. 22390021], and a
Grant-in Aid for Scientific Research on Priority Area ‘Creation
of Biologically Functional Molecules’ [No. 18032048] from The
Ministry of Education, Culture, Sports, Science and Technology,
Japan.
20. (a) Nakanishi, Y.; Chang, F.-R.; Liaw, C.-C.; Wu, Y.-C.; Bastow, K. F.; Lee, K.-H. J.
Med. Chem. 2003, 46, 3185; (b) Sinha, S. C.; Keinan, E. J. Am. Chem. Soc. 1993,
115, 4891; (c) Myint, S. H.; Cortes, D.; Laurens, A.; Hocquemiller, R.; Leboeuf,
M.; Cavé, A.; Cotte, J.; Quéro, A.-M. Phytochemistry 1991, 30, 3335.
21. Imai, K.; Watanabe, Y. Anal. Chim. Acta 1981, 130, 377.
22. Schaus, S. E.; Brånalt, J.; Jacobsen, E. N. J. Org. Chem. 1998, 63, 4876.
23. Vyvyan, J. R.; Meyer, J. A.; Meyer, K. D. J. Org. Chem. 2003, 68, 9144.
24. The stereochemistry of diol 5 was confirmed by the modified Mosher method.
The enantiomeric excess was determined by comparison of the 1H NMR
spectroscopic data of the (R)- and (S)-MTPA esters of 5.
Supplementary data
Δδ =
–0.02
Δδ = –0.08
Supplementary data (experimental procedures and character-
ization data of novel compounds not listed in the Experimental
Section) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2010.10.004.
O
Δδ = +0.05
( )7
OMTPA
H
H
Δδ = δS − δR
(Hz)
O
OTBS
1H NMR (500 MHz, CDCl3)
Δδ = –0.04
25. The diol was synthesized by Sinha using asymmetric epoxidation; see: Sinha, S.
C.; Sinha-Bagchi, A.; Keinan, E. J. Org. Chem. 1993, 58, 7789.
References and notes
26. (a) El-Sayed, E.; Anand, N. K.; Carreira, E. M. Org. Lett. 2001, 3, 3017; (b) Anand,
N. K.; Carreira, E. M. J. Am. Chem. Soc. 2001, 123, 9687; (c) Sasaki, H.; Boyall, D.;
Carreira, E. M. Helv. Chim. Acta 2001, 84, 964; (d) Boyall, D.; López, F.; Sasaki, H.;
Frantz, D.; Carreira, E. M. Org. Lett. 2000, 2, 4233; (e) Frantz, D. E.; Fässler, R. C.;
Tomooka, S.; Carreira, E. M. Acc. Chem. Res. 2000, 33, 373; (f) Frantz, D. E.;
Fässler, R.; Carreira, E. M. J. Am. Chem. Soc. 2000, 122, 1806.
1. Recent review of Annonaceous acetogenins: (a) McLaughlin, J. L. J. Nat. Prod.
2008, 71, 1311; (b) Bermejo, A.; Figadère, B.; Zafra-Polo, M.-C.; Barrachina, I.;
Estornell, E.; Cortes, D. Nat. Prod. Rep. 2005, 22, 269.
2. Recent review of total synthesis of Annonaceous acetogenins: Li, N.; Shi, Z.;
Tang, Y.; Chen, J.; Li, X. Beilstein J. Org. Chem. 2008, 4, 48.
3. Recent review of synthesis of acetogenin analogues: Kojima, N.; Tanaka, T.
Molecules 2009, 14, 3621.
4. (a) Morré, R. D.; de Cabo, J.; Farley, C.; Oberlies, N. H.; McLaughlin, J. L. Life Sci.
1995, 56, 343; (b) Wolvetang, E. J.; Johnson, K. L.; Krauer, K. S.; Ralph, J.;
Linnane, A. W. FEBS Lett. 1994, 339, 40.
5. Recently, Nagle and Zhou reported that Annonaceous acetogenins inhibited
hypoxia-inducible factor-1 (HIF-1) mediated hypoxic signaling pathways in
tumor cells: Coothankandaswamy, V.; Liu, Y.; Mao, S.-C.; Morgan, J. B.; Mahdi,
F.; Jekabsons, M. B.; Nagle, D. G.; Zhou, Y.-D. J. Nat. Prod. 2010, 73, 956.
6. Höglinger and co-workers have described that complex I inhibitor including
acetogenins may contribute to the pathogenesis of neurodegenerative
disorders: Höllerhage, M.; Matusch, A.; Champy, P.; Lombès, A.; Ruberg, M.;
Oertel, W. H.; Höglinger, G. U. Exp. Neurol. 2009, 220, 133.
7. Oberlies, N. H.; Chang, C.; McLaughlin, J. L. J. Med. Chem. 1997, 40, 2102.
8. Derbré, S.; Roué, G.; Poupon, E.; Susin, S. A.; Hocquemiller, R. ChemBioChem
2005, 6, 979.
9. Liu, H.-X.; Huang, G.-R.; Zhang, H.-M.; Jiang, S.; Wu, J.-R.; Yao, Z.-J.
ChemBioChem 2007, 8, 172.
27. In a comparison of the related compounds synthesized in our laboratory, the
hydroxyl proton of the anti-adduct appears at a higher field than that of the
syn-adduct. On the other hand, the signals of methine protons at both C2 and
C3 positions showed a greater downfield shift in the anti-adduct than in the
syn-adduct. For details, see Ref. 18d.
28. The stereochemistry of 18a was determined by comparison with the 13C
NMR data of Fujimoto’s model compounds after reduction of the triple bond
and deprotection of the TBS group with HF. For details, see Supplementary
data.
29. Fujimoto, Y.; Murasaki, C.; Shimada, H.; Nishioka, S.; Kakinuma, K.; Singh, S.;
Singh, M.; Gupta, Y.; Sahai, M. Chem. Pharm. Bull. 1994, 42, 1175.
30. (a) Yao, Z.-J.; Wu, Y.-L. J. Org. Chem. 1995, 60, 1170; (b) Yao, Z.-L.; Wu, Y.-L.
Tetrahedron Lett. 1994, 35, 157.
31. Duret, P.; Figadère, B.; Hocquemiller, R.; Cavé, A. Tetrahedron Lett. 1997, 38, 8849.
32. For the total synthesis of solamin, see: (a) Makabe, H.; Kuwabara, A.; Hattori,
Y.; Konno, H. Heterocycles 2009, 78, 2369; (b) Raghavan, S.; Subramanian, S. G.;
Tony, K. A. Tetrahedron Lett. 2008, 49, 1601; (c) Prestat, G.; Baylon, C.;
Heck, M.-P.; Grasa, G. A.; Nolan, S. P.; Mioskowski, C. J. Org. Chem. 2004, 69,
5770; (d) Kuriyama, W.; Ishigami, K.; Kitahara, T. Heterocycles 1999, 50, 981;
(e) Makabe, H.; Tanaka, A.; Oritani, T. J. Chem. Soc., Perkin Trans. 1 1994, 1975;
(f) Trost, B. M.; Shi, Z. J. Am. Chem. Soc. 1994, 116, 7459.
10. (a) Sekiguchi, K.; Murai, M.; Miyoshi, H. Biochim. Biophys. Acta 2009, 1787,
1106; (b) Murai, M.; Ishihara, A.; Nishioka, T.; Yagi, T.; Miyoshi, H. Biochemistry
2007, 46, 6409.
11. After the publication of our first-generation synthesis of fluorescence-labeled
acetogenins, Poupon and co-workers reported the semisynthesis of
fluorescence-labeled squamocin having all functionalities, but their
analogues have a bulky fluorescent group near the THF moiety: Derbré, S.;
Gil, S.; Taverna, M.; Boursier, C.; Nicolas, V.; Demey-Thomas, E.; Vinh, J.; Susin,
S. A.; Hocquemiller, R.; Poupon, E. Bioorg. Med. Chem. Lett. 2008, 18, 5741.
12. (a) Kuwabara, K.; Takada, M.; Iwata, J.; Tatsumoto, K.; Sakamoto, K.; Iwamura,
H.; Miyoshi, H. Eur. J. Biochem. 2000, 267, 2538. and references cited therein;
(b) Shimada, H.; Grutzner, J. B.; Kozlowski, J. F.; McLaughlin, J. L. Biochemistry
1998, 37, 854; (c) Shimada, H.; Kozlowski, J. F.; McLaughlin, J. L. Pharmacol. Res.
1998, 37, 357.
33. Jackson, D. Y. Synth. Commun. 1988, 18, 337.
34. The stereochemistry of propargyl alcohol 39a was confirmed by the modified
Mosher method. The enantiomeric excess was determined by comparison of
the 1H NMR spectroscopic data of 39a and 39b.
Δδ = +0.006
Δδ = +0.026
Ph
Ph
Δδ =
Δδ = –0.023
–0.013
O
O
O
O
O
O
O
O
13. Motoyama, T.; Yabunaka, H.; Miyoshi, H. Bioorg. Med. Chem. Lett. 2002, 12,
2089.
14. Han, H.; Sinha, M. K.; D’Souza, L. J.; Keinan, E.; Sinha, S. C. Chem. Eur. J. 2004, 10,
2149.
OMTPA
Δδ = +0.063
major isomer
OMTPA
Δδ =
+0.109
minor isomer
15. During the preparation of this manuscript, Nakamaru-Ogiso and Sinha et al.
reported that the ND2 subunit in the mitochondria is labeled by their asimicin
analogue, which has a photoaffinity tag at the end of the left side of the THF
moiety: Nakamaru-Ogiso, E.; Han, H.; Matsuno-Yagi, A.; Keinan, E.; Sinha, S. C.;
Yagi, T.; Ohnishi, T. FEBS Lett. 2010, 584, 883.
1H NMR (500 MHz, C6D6)
Δδ = δS − δR
(Hz)
35. The stereochemistry at C4 position in compound 39a was also determined by
transformation to known bis-acetal I (Goto, A.; Fujiwara, K.; Kawai, A.; Kawai,
H.; Suzuki, T. Org. Lett. 2007, 9, 5373.) via acetalization of 40.
16. Maezaki, N.; Urabe, D.; Yano, M.; Tominaga, H.; Morioka, T.; Kojima, N.; Tanaka,
T. Heterocycles 2007, 73, 159.
17. Kojima, N.; Morioka, T.; Yano, M.; Suga, Y.; Maezaki, N.; Tanaka, T. Heterocycles
2009, 79, 387.
Me2C(OMe)2
O
OH
O
O
PPTS
O
O
OH
O
CH2Cl2, 0 ºC
87%
18. (a) Maezaki, N.; Kojima, N.; Tanaka, T. Synlett 2006, 993; (b) Kojima, N.
Yakugaku Zasshi 2004, 124, 673; (c) Kojima, N.; Maezaki, N.; Tominaga, H.;
Yanai, M.; Urabe, D.; Tanaka, T. Chem. Eur. J. 2004, 10, 672; (d) Kojima, N.;
Maezaki, N.; Tominaga, H.; Asai, M.; Yanai, M.; Tanaka, T. Chem. Eur. J. 2003, 9,
4980; (e) Maezaki, N.; Kojima, N.; Tominaga, H.; Yanai, M.; Tanaka, T. Org. Lett.
2003, 5, 1411; (f) Maezaki, N.; Kojima, N.; Asai, M.; Tominaga, H.; Tanaka, T.
Org. Lett. 2002, 4, 2977.
19. (a) Kojima, N.; Hayashi, H.; Suzuki, S.; Tominaga, H.; Maezaki, N.; Tanaka, T.;
Yamori, T. Bioorg. Med. Chem. Lett. 2008, 18, 6451; (b) Kojima, N.; Fushimi, T.;
Maezaki, N.; Tanaka, T.; Yamori, T. Bioorg. Med. Chem. Lett. 2008, 18, 1637; (c)
OH
40
OH
I
36. Oxidation of alcohol 42 with Dess–Martin periodinane gave aldehyde 36 in 49%
yield.
37. White, J. D.; Somers, T. C.; Reddy, G. N. J. Org. Chem. 1992, 57, 4991.
38. Valicenti, A. J.; Pusch, F. J.; Holman, R. T. Lipids 1985, 20, 234.
39. The stereoselectivity was determined by comparison of the 1H NMR spectra
data of acetyl derivatives IIa–b.

N. Kojima et al. / Bioorg. Med. Chem. 18 (2010) 8630–8641
8641
42. The stereochemistry around the THF moiety of
3
was determined by
Ac2O
comparison with the 13C NMR data of Fujimoto’s model compounds. For
details, see Supplementary data.
pyridine
O
O
O
O
43. (a) Dan, S.; Okamura, M.; Seki, M.; Yamazaki, K.; Sugita, H.; Okui, M.;
Mukai, Y.; Nishimura, H.; Asaka, R.; Nomura, K.; Ishikawa, Y.; Yamori, T.
Cancer Res. 2010, 70, 4982; (b) Yaguchi, S.; Fukui, Y.; Koshimizu, I.;
Yoshimi, H.; Matsuno, T.; Gouda, H.; Hirono, S.; Yamazaki, K.; Yamori, T. J.
Natl. Cancer Inst. 2006, 98, 545; (c) Yamori, T.; Matsunaga, A.; Saito, S.;
Yamazaki, K.; Komi, A.; Ishizu, K.; Mita, I.; Edatsugi, H.; Matsuba, Y.;
Takezawa, K.; Nakanishi, O.; Kohno, H.; Nakajima, Y.; Komatsu, H.; Andoh,
T.; Tsuruo, T. Cancer Res. 1999, 59, 4042.
44. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.; Rubinstein,
L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989, 81, 1088.
45. Kamakura, A.; Fujimoto, Y.; Motohashi, Y.; Ohashi, K.; Ohashi-Kobayashi, A.;
Maeda, M. Biochem. Biophys. Res. Commun. 2008, 377, 847.
R
R'
R
R'
O
rt
O
48a: R = OH, R' = H
48b: R = H, R' = OH
IIa: R = OAc, R' = H, 92% (95:5 dr)
IIb: R = H, R' = OAc, quant. (96:4 dr)
40. The stereochemistry of 48a–b was confirmed by comparison of their 1H and
13C NMR spectra with those of model compounds that we have synthesized
previously; see Ref. 18d.
41. The stereoselectivity was determined by comparison of the 1H NMR spectra
data of acetyl derivatives IIIa–b.
Ac2O
pyridine
46. Oberlies, N. H.; Jones, J. L.; Corbett, T. H.; Fotopoulos, S. S.; McLaughlin, J. L.
Cancer Lett. 1995, 96, 55.
O
O
rt
R
R'
R R'
OTBDPS
OTBDPS
IIIa: R = H, R' = OAc, quant. (91:9 dr)
IIIb: R = OAc, R' = H, quant. (96:4 dr)
52a: R = H, R' = OH
52b: R = OH, R' = H