Design, synthesis, and biological evaluation of fluorinated analogues of salicylihalamide

Article abstract of DOI:10.1021/jm801265e

Salicylihalamide A (SA), a benzolactone enamide compound, possesses potent cytotoxicity against human tumor cell lines. SA is a selective inhibitor of mammalian vacuolar type H⁺-ATPase (V-ATPase), and is distinct from previously known V-ATPase

Full text of DOI:10.1021/jm801265e

798
J. Med. Chem. 2009, 52, 798–806
Design, Synthesis, and Biological Evaluation of Fluorinated Analogues of Salicylihalamide
Yoshinori Sugimoto,† Keiichi Konoki,† Michio Murata,† Masafumi Matsushita,‡ Hiroshi Kanazawa,‡ and Tohru Oishi*,†
Department of Chemistry, Graduate School of Science, Osaka UniVersity, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan, Department
of Biological Sciences, Graduate School of Science, Osaka UniVersity, Osaka, Japan
ReceiVed October 7, 2008
Salicylihalamide A (SA), a benzolactone enamide compound, possesses potent cytotoxicity against human
tumor cell lines. SA is a selective inhibitor of mammalian vacuolar type H+-ATPase (V-ATPase), and is
distinct from previously known V-ATPase inhibitors such as bafilomycins and concanamycins that do not
discriminate between mammalian and nonmammalian V-ATPases. Because of its potent antitumor activity
and structural simplicity, SA is a promising candidate for an anticancer drug. Although a number of
structure-activity relation studies using synthetic analogues have been reported, no fluorinated derivative
of SA has been evaluated even though selective addition of a fluorine atom into a therapeutic small molecule
candidate often enhances pharmacokinetic and physicochemical properties. We designed and synthesized
fluorinated analogues of SA and evaluated their V-ATPase inhibitory activities. Compared to the natural
product, the synthetic analogues were potent V-ATPase inhibitors, suggesting that these analogues are potential
drug candidates and potential molecular probes for mode-of-action studies using fluorine-based analytical
methods such as 19F-NMR spectroscopy.
Introduction
Development of antitumor drugs with limited side effects is
important for cancer patients undergoing chemotherapy. Al-
though combinatorial chemistry has greatly influenced the drug
discovery process,1 isolation of novel natural products remains
important for discovery of lead compounds possessing promising
biological profiles.2 Salicylihalamide A (SAa, 1a) was isolated
from the sponge Haliclona sp. (southwestern Australian coast)
by Boyd and co-workers in 1997 with the minor component
salicylihalamide B (SB, 1b),3 containing a labile enamide
Figure 1. Structures of salicylihalamide A and B.
linkage connecting a salicylic acid-derived macrolactone core
(Figure 1). Since the discovery of SA, a number of structurally
similar bioactive compounds that possess a benzolactone
enamide moiety have been isolated (Figure 2),4 e.g., lobatamides
A (2),5 apicularenes A (3),6 4 (CJ-12,950),7 and oximidines I
(5).8 SA elicited a unique differential cytotoxicity profile in the
human tumor NCI 60-cell line, with a mean panel GI50 value
of 15 nM, and melanoma cell lines showed the highest average
sensitivity (GI50 ) 7 nM).3 Furthermore, SA was a potent
inhibitor of vacuolar H+-ATPases (V-ATPases), with an IC50
value less than 1.0 nM against bovine brain V-ATPase.9 The
V-ATPases are ubiquitous proton-translocating pumps of eu-
* To whom correspondence should be addressed. Phone: +81-6-6850-
5775. Fax: +81-6-6850-5775. E-mail: oishi@chem.sci.osaka-u.ac.jp.
†
Department of Chemistry, Graduate School of Science, Osaka University.
Department of Biological Sciences, Graduate School of Science, Osaka
‡
University.
a Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; ATP,
adenosine 5′-triphosphate; BCA, bicinchoninic acid; CGM, chromaffin
granule membranes; CuTC; copper thiophene-2-carboxylate; dba, diben-
Figure 2. Structures of benzolactone enamide class compounds.
zylideneacetone; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; DEAD,
diethyl azodicarboxylate; DMA, N,N-dimethylacetamide; DMSO, dimethyl
sulfoxide; EDTA, ethylenediaminetetraacetic acid; FCCP, carbonyl cyanide
p-trifluoromethoxyphenylhydrazone; F-SA, 4-19F-salicylihalamide A; F-SB,
4-19F-salicylihalamide B; GI50, 50% growth inhibition; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high performance
liquid chromatography; IC50, 50% inhibition concentration; MOPS, 3-(N-
morpholino)propanesulfonic acid; NCI, National Cancer Institute; NMP,
N-methylpyrrolidone; NMR, nuclear magnetic resonance; PMB, p-meth-
oxybenzyl; SA, salicylihalamide A; SB, salicylihalamide B; Tf, trifluo-
romethanesulfonyl; TFP, 2-trifurylphosphine; THF, tetrahydrofuran; V-
ATPase, vacuolar type H+-ATPase.
karyotic cells located on the membranes of vacuoles, lysosomes,
and other components of the endomembrane system, which
regulate the pH of the compartments. Numerous physiological
processes depend on the activity of V-ATPases, which are
implicated as a contributing factor in multiple diseases including
osteoporosis and cancer.10 SA is distinct from previously known
V-ATPase inhibitors such as bafilomycins and concanamycins,
which do not discriminate between mammalian and nonmam-
10.1021/jm801265e CCC: $40.75
2009 American Chemical Society
Published on Web 12/31/2008

Fluorinated Analogues of Salicylihalamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 799
Scheme 1a
a Reagents and conditions: (a) Cl2CHCHCl2, 146 °C, 3 h, 12a:12b:12c:
10 ) 5:9:1:10; (b) Tf2O, pyridine, 0 °C, 1 h, 13 (16%, two steps), 14 (27%,
two steps); (c) n-Bu3SnCH2CHdCH2, [Pd2(dba)3]·CHCl3, TFP, LiCl, NMP,
rt, 18 h, 84%; (d) EtMgBr, CH2dCHCH2OH, THF, 0 °C to rt, 18 h, 81%;
(e) MeI, K2CO3, acetone, 24 h, 92%; (f) Pd(PPh3)4, morpholine, THF, 1 h,
85%.
Figure 3. Design and synthetic strategy of fluorinated salicylihala-
mides.
malian V-ATPases. SA binds to the transmembrane (V0) domain
of V-ATPase at a site different from bafilomycin A1 and
concanamycin A,11 while the precise mode-of-action of SA and
structure of the ligand-protein complex have not been elucidated.
Because of the potent antitumor activity and structural
simplicity compared with other V-ATPase inhibitors, SA is
expected to be a promising anticancer drug candidate.12
Although a number of synthetic studies and structure-activity
relation studies have been reported,13 no fluorinated derivatives
of SA have been evaluated, while examination of the role of
fluorine in medicinal chemistry reveals that selective addition
of fluorine into a therapeutic small molecule candidate can
enhance pharmacokinetic and physicochemical properties such
as metabolic stability and membrane permeability and can
increase binding affinity of drug candidates to the target
protein.14 Therefore, fluorinated SA may be a promising drug
candidate, as well as a possible molecular probe for elucidating
the mode-of-action when using fluorine-based analytical meth-
ods such as 19F-NMR spectroscopy. The properties of 19F
(nuclear spin of 1/2, high gyromagnetic ratio, 100% natural
abundance, and low background signals in biological systems)
make it useful for investigating biological systems.15 Here, we
report the design and synthesis of fluorinated salicylihalamides
and evaluation of the V-ATPase inhibitory activity.
at the final stage by cross-coupling using amide 8,13d and the
macrocyclic core was to be constructed by ring-closing me-
tathesis of the diene derived from salicylate derivative 7 and
alcohol 913g, by Mitsunobu esterification.20
For synthesis of building block 7, electrophilic fluorination
of the salicylate derivative 1021 was conducted as shown in
Scheme 1. Treatment of 10 with N-fluoro-5-(trifluoromethyl)-
pyridinium-2-sulfonate 1122 in 1,1,2,2-tetrachloroethane at 146
°C for 3 h afforded a mixture of monofluorinated 12a and 12b,
difluorinated 12c, and recovered 10 in ca. 5:9:1:10 ratio. The
mixture was separated by silica gel column chromatography as
a mixture of 12a and 10, and 12b and 12c, respectively. The
mixture of 12a and 10 was treated with Tf2O in pyridine to
afford triflate 13 (16%, 2 steps), which was separated from 14.23
According to De Brabander’s procedure,13a triflate 13g was
converted to carboxylic acid 7 via (i) Stille coupling of triflate
13 with allylstannane, (ii) transesterification of 15 with removal
of the acetonide by treatment with alkoxide generated from allyl
alcohol and ethylmagnesium bromide, (iii) protection of the
resulting phenol 16 as a methyl ether 17, and (iv) conversion
of the allyl ester to carboxylic acid 7 through the action of a
palladium catalyst.
Then, the coupling of the fragments was performed as shown
in Scheme 2. Condensation of carboxylic acid 7 with secondary
alcohol 9 under Mitsunobu esterification conditions using
DEAD/PPh3 afforded diene 18. Ring-closing metathesis of the
diene by the action of Grubbs catalyst24 resulted in the formation
of macrocycle 19 (E:Z ) 10:1). Removal of the PMB group
followed by Dess-Martin oxidation of the resulting alcohol 20
gave an aldehyde, which was converted to iodoolefin 21 by
Takai olefination25 under modified conditions reported by
Evans.26 Removal of the methyl group with BBr3 gave key
intermediate 22 for the coupling reaction to construct the
enamide moiety,27 which is the most critical step in the present
synthesis. Prior to the synthesis of fluorinated analogues,
coupling of counterpart 2313f with amide 8 giving natural SA
was examined. After considerable experimentation according
to Fu¨rstner’s procedure13f and modification by Panek,18a cou-
pling of iodoolefin 23 with amide 8 was achieved by treatment
Results and Discussion
Chemistry. The compounds, 4-19F-salicylihalamide A (F-
SA, 6a) and B (F-SB, 6b), were designed as target molecules
(Figure 3) because the salicylate moiety is a common structural
feature of natural benzoenamide class products, which is
considered to be a binding motif toward the target protein, and
electrophilic fluorination of phenol derivatives is a versatile
synthetic method.16 However, the utility of introduction of
fluorine at C4 position was uncertain because of its proximity
to the hydroxy group at C3, which might be involved in
hydrogen bonding with the target protein. Fluorinated analogues
were synthesized based on previous reports of the total synthesis
and synthetic studies of SA13 and benzolactone enamide class
natural products (Figure 3).17-19 The enamide side chain, the
most labile moiety of the molecule, was planned to be introduced

800 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Sugimoto et al.
Scheme 2a
Figure 4. Change in pH of acidic organelles (lysosomes) of simian
renal cells (COS-7) induced by salicylihalamides: (A) control; (B)
salicyclihalamide A (SA, 1a), 1 µM; (C) SA; (D) salicyclihalamide B
(SB, 1b), 1 µM; (E) SB; (F) 4-19F-salicylihalamide A (F-SA, 6a), 1
µM; (G) F-SA; (H) 4-19F-salicylihalamide B (F-SB, 6b), 1 µM; (I)
F-SB; Change in pH was visualized by fluorescence microscopy using
LysoSensor Green. The panels B and C, D and E, F and G, and H and
I were the results of duplicate experiments. The fluorescent intensity
(arbitrary unit) is indicated in pseudocolor as shown in the color scale
on the top-right. Asterisks denote the nuclei of the cells. Scale bar, 20
µm.
a Reagents and conditions: (a) DEAD, PPh3, Et2O, rt, 36 min, 91%; (b)
(PCy3)2Cl2RudCHPh, CH2Cl2, rt, 21 min, 90%; (c) DDQ, CH2Cl2/H2O, 0
°C to rt, 53 min, 97%; (d) Dess-Martin periodinane, CH2Cl2, 0 °C to rt,
65 min; (e) CrCl2, CHI3, dioxane, THF, rt, 26 h 80% (two steps); (f) BBr3,
CH2Cl2, -78 °C, 1 h, 74%; (g) 8, CuTC, 2,2′-bipyridine, Rb2CO3, DMA,
90 °C, 60 min, 1a (23%), 1b (26%); (h) 8, CuTC, 2,2′-bipyridine, Rb2CO3,
DMA, 90 °C, 60 min, 6a (15%), 6b (22%).
with copper thiophene-2-carboxylate (CuTC)28 in the presence
of Rb2CO3 and 2,2′-bipyridine in N,N-dimethylacetamide (DMA)
at 90 °C for 60 min to afford a mixture of E,Z-isomers of the
enamide moiety in ca. 1:1 ratio. The mixture of enamide moiety
isomers was purified by HPLC to afford SA (1a, 23%) and SB
(1b, 26%), whose physical data were identical to reported
values.3 Note that the use of exhaustively degassed solvent
(DMA) is essential for a successful coupling reaction. Although
nearly equal amounts of isomers formed in contrast to the
previous report,13f,18a,27 it is advantageous for structure-activity
relation studies to obtain a significant amount of SB, which was
isolated as a minor compound. In an analogous sequence,
fluorinated analogues were synthesized and purified by HPLC
to afford F-SA (6a, 15%) and F-SB (6b, 22%).
Biological Results and Discussion. V-ATPase inhibitory
activity of fluorinated analogues F-SA (6a) and F-SB (6b), as
well as synthetic SA (1a) and SB (1b), were evaluated in
cultured simian renal cells (COS-7) and chromaffin granule
membranes prepared from porcine adrenal glands by monitoring
a pH change visualized by fluorescent dyes.
1. Imaging Assays Using Simian Renal Cells (COS-7). The
effect of salicylihalamides was examined on the luminal pH of
intracellular acidic compartments (lysosomes), which were
acidified prominently by the proton pumping activity of V-
ATPase. Simian renal cells (COS-7)29 were incubated with
salicylihalamides SA (1a) and SB (1b), and 4-19F-salicylihala-
mides, F-SA (6a) and F-SB (6b), at a concentration of 1 µM
for 5 h. Then, the cells were incubated with the pH indicator
LysoSensor Green (1 µM) for 1 min.30 As shown in Figure 4A,
lysosomes of intact cells emitted significant fluorescence,
Figure 5. Concentration-dependent inhibition of H+ transport activity
of V-ATPase (from porcine adrenal glands) by synthetic salicylihala-
mides.
indicating that these organelles were highly acidified. When the
cells were treated with SA (Figure 4B,C) or SB (Figure 4D,E),
fluorescence diminished, reflecting an increase in pH in these
organelles by inhibition of V-ATPase. To assess the viability,
the trypan blue dye exclusion test was also performed after the
drug treatment, and the results suggested that the viability was
not affected by the drug treatment at this time point (data not
shown). As expected, not only F-SA (Figure 4F,G) but also
F-SB (Figure 4H,I) also actively inhibited the proton pump.
2. Quantitative Assays Using Chromaffin Granule
Membranes. Although V-ATPase inhibitory activity is com-
monly evaluated by measuring ATP-dependent proton uptake
into isolated chromaffin granules from bovine adrenal glands,31
chromaffin granules from porcine adrenal glands were used in
this study due to short supply of fresh bovine tissue. Proton
uptake into the membrane vesicles was measured by monitoring
the fluorescence quenching of the 9-amino-6-chloro-2-meth-
oxyacridine (ACMA)31,32 employed as an indicator. The activity
of synthetic SA and SB, as well as fluorinated analogues F-SA
and F-SB, were evaluated (Figure 5), and the results are
summarized in Table 1. SA elicited potent inhibitory activity
with an IC50 value of approximately <1 (0.7) nM, comparable

Fluorinated Analogues of Salicylihalamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 801
Table 1. Inhibition of H+ Transport Activity of V-ATPase (from
recorded on an AB QSTAR Elite under ESI-TOF conditions.
Combustion elemental analyses were performed using Yanaco CHN
CORDER MT-3.
Porcine Adrenal Glands) by Synthetic Salicylihalamidesa
entry
compound
IC50/nM
8-Fluoro-5-trifluoromethanesulfonyloxy-2,2-dimethyl-4H-
benzo[d][1,3]dioxin-4-one (13). To the 300 mL two-necked flask
charged with N-fluoro-5-(trifluoromethyl)pyridinium-2-sulfonate 11
(3.98 g, 16.2 mmol) was added a solution of 5-hydroxy-2,2-
dimethyl-4H-benzo[d][1,3]dioxin-4-one 10 (3.73 g, 19.4 mmol) in
dry 1,1,2,2-tetrachloroethane (49 mL), and the resulting mixture
was refluxed for 4 h with light shielding. After cooling to room
temperature, the reaction mixture was diluted with dichloromethane
(90 mL), quenched with saturated aqueous Na2S2O3 at 0 °C, and
extracted with ether. The organic layer was washed with saturated
aqueous Na2S2O3, pH 7.0 phosphate buffer solution and brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (50:1
to 5:1 hexane/ethyl acetate) to afford a mixture of 12a and recovered
10 containing 1,1,2,2-tetrachloroethane, and a mixture of 12b and
12c. 12a: Rf ) 0.40 (10:1 hexane/ethyl acetate, developed in twice);
1H NMR (500 MHz, CDCl3) δ 9.99 (1H, br s), 7.26 (1H, dd, J )
9.7, 9.7 Hz), 5.98 (1H, dd, J ) 9.2, 3.4 Hz), 1.78 (6H, s). 12b: Rf
0.36 (10:1 hexane/ethyl acetate, developed in twice); 1H NMR (500
MHz, CDCl3) δ 10.3 (1H, br s), 7.24 (1H, dd, J ) 9.7, 9.7 Hz),
6.37 (1H, dd, J ) 8.6, 2.9 Hz), 1.73 (6H, s); 1H NMR (500 MHz,
CD2Cl2) δ 10.3 (1H, br s), 7.25 (1H, dd, J ) 10.9, 6.0 Hz), 6.39
(1H, dd, J ) 9.2, 3.4 Hz), 1.70 (6H, s). 8c: Rf 0.36 (hexane/ethyl
acetate ) 10/1, developed in twice); 1H NMR (500 MHz, CDCl3)
δ 9.98 (1H, br s), 7.18 (1H, dd, J ) 10.0, 10.0 Hz), 1.78 (6H, s);
1H NMR (500 MHz, CD2Cl2) δ 9.96 (1H, br s), 7.20 (1H, dd, J )
10.3, 10.3 Hz), 1.76 (6H, s).
1
2
3
4
1a (SA)
6a (F-SA)
1b (SB)
<1 (0.7)
2
30
10
6b (F-SB)
a IC50 values are the average of three experiments.
to reported values. As expected, F-SA acted as a potent inhibitor
of V-ATPase (IC50 ) 2 nM), but was slightly less potent than
SA. Since the V-ATPase inhibitory activity of SB has not been
reported (SB was isolated as minor compound: ∼0.5 mg), the
IC50 value of be approximately 30 nM was determined for the
first time. This value indicates that SB is 30 times less potent
than SA. Note that the activity of F-SB was comparable to that
of the corresponding natural product (IC50 ) 10 nM).
Conclusions
In conclusion, 4-19F-salicylihalamide A (F-SA) and B (F-
SB) were synthesized through electrophilic fluorination of the
salicylate derivative, ring-closing metathesis for the construction
of the macrocyclic core, and cross-coupling reaction for
installation of the enamide side chain. The V-ATPase inhibitory
activity of synthetic salicylihalamides was evaluated both in vivo
and in vitro using cultured simian renal cells (COS-7) and
chromaffin granule membranes prepared from porcine adrenal
glands, respectively. The F-SA was a potent V-ATPase inhibitor
(IC50 ) 2 nM) comparable to SA (<1 nM), suggesting that the
fluorinated analogue is a promising drug candidate and a
potential probe for mode-of-action studies using fluorine-based
analytical methods. In this study, the previously unknown IC50
value of SB was estimated to be 30 nM and that of F-SB (10
nM) was comparable to SB. Further studies on the antitumor
activity of fluorinated salicylihalamides and mode-of-action
studies based on 19F-NMR are currently in progress in our
laboratory.
To a solution of 12a and 10 in pyridine (18 mL), trifluo-
romethanesulfonic anhydride (11.0 mL, 2.09 mmol) was added
dropwise at 0 °C, and the mixture was stirred at 0 °C for 1 h. The
reaction mixture was quenched with saturated aqueous NaHCO3
at 0 °C and extracted with ether. The organic layer was washed
with saturated aqueous NH4Cl, water, and brine, dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography (30:1 to 10:1
hexane/ethyl acetate) to afford 13 (1.07 g, 3.11 mmol, 16%, two
steps) as a colorless solid which was separated from 14 (1.71 g,
5.25 mmol, 27%, two steps). 13: Rf ) 0.18 (10:1 hexane/ethyl
acetate); IR (film) 3092, 2986, 1749, 1632, 1497, 1419, 1403, 1395,
1381, 1319, 1301, 1286, 1264, 1229, 1219, 1182, 1141, 1068, 1033,
Experimental Section
1
979, 907, 856, 828, 649, 593 cm-1; H NMR (500 MHz, CDCl3)
Chemistry. General Procedures. All the solvents and chemicals
were used without further treatment unless otherwise stated. The
following solvents and chemicals used were purified by distillation
over the drying agents indicated in parentheses and were transferred
under argon atmosphere: THF, Et2O and 1,4-dioxane (Mg/ben-
zophenone), 1,1,2,2-tetrachloroethane, pyridine, triethylamine, and
morpholine (CaH2), dichloromethane (LiAlH4), allyl alcohol (Mg).
N-methylpyrrolidone (NMP) was dried over activated MS4A for 3
days and distilled. N,N′-dimethyl acetamide (DMA) was distilled
over di-n-butyltin dilaurilate, redistilled over CaH2 under reduced
pressure, and stocked in the presence of activated MS4A. Before
use, the DMA was degassed by freeze-thaw method over 10 times.
Analytical thin-layer chromatography (TLC) was performed using
E. Merck silica gel 60 F254 precoated plates (0.25-mm thickness).
For column chromatography, Kanto silica gel 60N (spherical,
neutral, 100-210 µm), and Merck silica gel 60 (40-63 µm, for
flash column chromatography) were used. Optical rotations were
recorded on a JASCO P-1010 polarimeter. IR spectra were recorded
on a JASCO FT-IR-300E Fourier transform infrared spectrometer.
1H, 13C, and 19F-NMR spectra were recorded on a JEOL JNM-
GSX500 or JNM-ECA500 spectrometer. Chemical shifts of 1H and
13C NMR are reported in ppm from tetramethylsilane with reference
to internal residual solvent [1H NMR, CHCl3 (7.24), C6HD5 (7.15),
CHDCl2 (5.29); 13C NMR, CDCl3 (77.0), C6D6 (128)], chemical
shifts of 19F NMR are reported in ppm with reference to CF3COOH
(-76.5) in D2O. The following abbreviations are used to designate
the multiplicities: s ) singlet, d ) doublet, t ) triplet, q ) quartet,
and m ) multiplet. High resolution mass spectra (HRMS) were
δ 7.39 (1H, t, J ) 9.0 Hz), 6.95 (1H, dd, J ) 9.2, 3.6 Hz), 1.80
(6H, s); 13C NMR (125 MHz, CDCl3) δ 156.1 (d, JCF ) 4 Hz),
150.4 (d, 1JCF ) 254 Hz), 145.7 (d, JCF ) 15 Hz), 143.9 (d, JCF
)
1
4 Hz), 122.2 (d, JCF ) 19 Hz), 118.7 (d, JCF ) 312 Hz, -CF3),
110.0, 108.0, 25.5; 19F NMR (470 MHz, CDCl3) δ 132.4 (dd, J )
8.9, 4.0 Hz); HRMS (ESI-TOF) m/z calcd for C11H8F4NaO6S (M
+ Na+) 366.9875, found 366.9877.
8-Fluoro-5-(2-propenyl)-2,2-dimethyl-4H-benzo[d][1,3]dioxin-
4-one (15). Lithium chloride (395 mg, 9.33 mmol) in a Schlenk
flask was dried by being heated under reduced pressure. Tris-
(dibenzylideneacetone)dipalladium(0)-methylene chloride complex
(166 mg, 0.16 mmol), tris(2-furyl)phosphine (74.3 mg, 0.32 mmol),
and N-methylpyrrolidone (2.2 mL) were added to the flask, and a
solution of 13 (1.07 g, 3.11 mmol) in N-methylpyrrolidone (4.0
mL) was added and then stirred at room temperature for 12 min.
To the stirred solution, allyl tri-n-buthyltin (1.14 mL, 3.73 mmol)
was added dropwise at 0 °C. After being stirred at room temperature
for 8 h, the reaction mixture was quenched with saturated aqueous
KF at 0 °C, the resulting precipitates were removed filtration, and
the filtrate was extracted with ether. The organic layer was washed
with saturated aqueous KF, water, brine, dried over MgSO4, filtered,
and concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (20:1 to 10:1 hexane/
ethyl acetate) to afford 15 (620 mg, 2.62 mmol, 84%) as a pale
greenish-yellow oil. Rf ) 0.50 (5:1 hexane/ethyl acetate); IR (film)
2956, 2924, 1740, 1639, 1616, 1594, 1503, 1465, 1429, 1391, 1381,
1281, 1262, 1204, 1150, 1065, 1030, 997, 982, 911, 823 cm-1; 1H

802 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Sugimoto et al.
NMR (500 MHz, CDCl3) δ 7.24 (1H, dd. J ) 9.6, 8.6 Hz), 6.88
(1H, dd, J ) 8.6, 4.7 Hz), 5.98 (1H, ddt, J ) 16.9, 10.3, 6.5 Hz),
5.04 (1H, ddd, J ) 10.2, 2.9, 1.3 Hz), 5.01 (1H, ddd, J ) 17.1,
3.3, 1.7 Hz), 3.82 (2H, d, J ) 6.4 Hz), 1.73 (6H, s); 13C NMR
(500 MHz, CDCl3) δ 7.10 (2H, dd, J ) 11.2, 8.3 Hz), 6.92 (2H,
dd, J ) 8.6, 4.6 Hz), 5.90 (1H, ddt, J ) 17.0, 10.0, 6.3 Hz), 5.05
(1H, d, J ) 10.3 Hz), 5.04 (1H, d, J ) 17.2 Hz), 3.98 (3H, d, J )
1.7 Hz), 3.47 (2H, br d, J ) 6.9 Hz); IR (film) 3503, 2946, 2654,
2361, 1707, 1640, 1606, 1559, 1491, 1462, 1425, 1280, 1199, 1164,
1
(125 MHz, CDCl3) δ 159.2 (d, JCF ) 4 Hz), 149.7 (d, JCF ) 247
Hz), 145.1 (d, JCF ) 13 Hz), 140.1 (d, JCF ) 5 Hz), 136.4, 124.0
(d, JCF ) 6 Hz), 121.6 (d, JCF ) 17 Hz), 116.2, 113.6, 106.3, 37.7,
25.6; 19F NMR (470 MHz, CDCl3) δ -138.5 (dd, J ) 9.9, 4.5
Hz); HRMS (ESI-TOF) m/z calcd for C13H13F4NaO3 (M + Na+)
259.0746, found 259.0747.
1141, 1042, 994, 968, 921, 820, 768, 708, 668 cm-1
;
13C NMR
1
(125 MHz, CDCl3) δ171.7, 153.7 (d, JCF ) 246 Hz), 145.1 (d,
JCF ) 13 Hz), 136.0, 134.4 (d, JCF ) 4 Hz), 127.6, 125.1 (d, JCF
)
7 Hz), 118.6 (d, JCF ) 18 Hz), 116.7, 62.2 (d, JCF ) 6 Hz), 37.3;
19F NMR (470 MHz, CDCl3) δ -133.1 (br dq, J ) 10.8, 2.2 Hz);
HRMS (ESI-TOF) m/z calculated for C11H10FO3 (M-H-) 209.0613,
found 209.0639.
3-Fluoro-2-hydroxy-6-(2-propenyl)benzoic Acid 2-Propenyl
Ester (16). To a solution of 2-propene-1-ol (1.40 mL, 20.6 mmol)
in THF (1 mL), a solution of ethylmagnesium bromide in THF
(0.49 M, 21.1 mL, 10.3 mmol) was added dropwise at 0 °C, and
the resulting solution was stirred at room temperature for 20 min.
To the reaction mixture, a solution of 15 (610 mg, 2.58 mmol) in
THF (3 mL) was added dropwise at 0 °C. After being stirred at
room temperature for 17 h, the reaction mixture was diluted with
ether, quenched with aqueous NH4Cl at 0 °C, and extracted with
ether. The organic layer was washed with saturated aqueous NH4Cl,
water, and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (40:1 to 10:1 hexane/ethyl acetate) to afford 16
(494 mg, 2.09 mmol, 81%) as a pale greenish-yellow oil. Rf )
0.37 (20:1 hexane/ethyl acetate, developed in twice); IR (film) 3080,
2982, 1734, 1669, 1615, 1595, 1490, 1432, 1373, 1307, 1251, 1171,
3-Fluoro-2-methoxy-6-(2-propenyl)-benzoic acid (1S,3R,4S)-
3-(methoxymethoxy)-1-[2-[(4-methoxyphenyl)methoxy]ethyl]-4-
methyl-6-heptenyl Ester (18). To a stirred solution of triphe-
nylphosphine (254 mg, 0.97 mmol), 7 (0.86 mmol), and 9 (254
mg, 0.72 mmol) in ether (3.6 mL), DEAD (40% in toluene, 0.44
mL, 0.97 mmol) was added at 0 °C. After being stirred at room
temperature for 36 min, the reaction mixture was quenched with
saturated aqueous NH4Cl at 0 °C and extracted with ether. The
organic layer was washed with saturated aqueous NH4Cl, water,
and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (10:1 to 5:1 hexane/ethyl acetate) to afford 18 (368
27
mg, 0.68 mmol, 91%) as a pale greenish-yellow oil. [R]D +3.63
(c 0.32, CHCl3); Rf ) 0.50 (3:1 hexane/ethyl acetate); IR (film)
2935, 1727, 1640, 1613, 1514, 1490, 1458, 1442, 1421, 1362, 1275,
1248, 1197, 1173, 1155, 1139, 1097, 1039, 995, 975, 917, 820
1
1143, 983, 918, 822, 811, 797, 767 cm-1; H NMR (500 MHz,
CDCl3) δ 11.1 (1H, s), 7.14 (1H, dd, J ) 9.8, 8.7 Hz), 6.66 (1H,
dd, J ) 8.4, 5.0 Hz), 6.01 (1H, ddt, J ) 17.2, 10.5, 6.0 Hz), 5.92
(1H, ddt, J ) 17.0, 10.2, 6.3 Hz), 5.42 (1H, dd, J ) 17.0, 2.6 Hz),
5.34 (1H, d, J ) 10.5 Hz), 5.00 (1H, dd, J ) 10.2, 3.0 Hz), 4.92
(1H, dd, J ) 17.0, 3.4 Hz), 4.86 (2H, dt, J ) 6.0, 1.4 Hz), 3.64
(2H, br d, J ) 6.2 Hz); 13C NMR (125 MHz, CDCl3) δ 170.4 (d,
1
cm-1; H NMR (500 MHz, CDCl3) δ 7.25 (2H, d. J ) 8.6 Hz),
7.03 (1H, dd, J ) 11.2, 8.5 Hz), 6.87-6.82 (3H, m), 5.88 (1H,
ddt, J ) 17.0, 10.2, 6.4 Hz), 5.71 (1H, ddt, J ) 17.0, 10.0, 7.0
Hz), 5.42 (1H, dddd, J ) 6.2, 6.0, 6.0, 6.0, 6.0 Hz, H15), 5.05
(1H, dd, J ) 10.2, 3.0 Hz), 5.00 (1H, dd, J ) 17.0, 3.3 Hz), 4.94
(1H, dd, J ) 17.2, 3.2 Hz), 4.90 (1H, dd, J ) 10.0, 1.9 Hz), 4.68
(1H, d, J ) 6.9 Hz), 4.66 (1H, d, J ) 6.9 Hz), 4.45 (1H, d, J )
11.5 Hz), 4.41 (1H, d, J ) 11.5 Hz), 3.90 (3H, d, J ) 2.0 Hz,
-OMe), 3.78 (3H, s), 3.61 (1H, ddd, J ) 9.5, 8.4, 5.9 Hz, H17a),
3.60 (1H, dt, J ) 10.0, 3.6 Hz, H13), 3.55 (1H, ddd, J ) 9.5, 6.9,
6.9 Hz, H17b), 3.38 (3H, s), 3.30 (2H, br d, J ) 6.4 Hz), 3.30 (2H,
br d, J ) 6.4 Hz), 2.05 (1H, ddd, J ) 13.9, 6.6 Hz, H11a), 2.03
(1H, ddd, J ) 14.0, 6.0 6.0 Hz, H16a), 1.97 (1H, ddd, J ) 14.0,
6.9, 5.7 Hz, H16b), 1.93-1.85 (1H, m, H12), 1.80 (1H, ddd, J )
13.9, 8.6, 7.9 Hz, H11b), 1.73-1.69 (2H, m, H14), 0.87 (3H, d, J
) 6.7 Hz); 13C NMR (125 MHz, CDCl3) δ 166.6, 159.1, 153.4 (d,
1JCF ) 246 Hz), 144.3 (d, JCF ) 12 Hz), 137.1, 136.2, 133.3 (d,
JCF ) 5 Hz), 130.5, 129.6, 129.3, 124.5 (d, JCF ) 7 Hz), 117.7 (d,
JCF ) 17 Hz), 116.6, 115.9, 113.7, 96.7, 78.2, 72.7, 71.3, 66.4,
61.7 (d, JCF ) 7 Hz), 55.8, 55.3, 37.5, 36.7, 36.3, 35.3, 35.1, 13.7;
19F NMR (470 MHz, CDCl3) δ -133.6 (br ddq, J ) 10.8, 4.5, 1.8
Hz); HRMS (ESI-TOF) m/z calcd for C31H41FNaO7 (M + Na+)
567.2734, found 567.2731.
1
JCF ) 2 Hz), 151.0 (d, JCF ) 12 Hz), 150.4 (d, JCF ) 243 Hz),
137.7 (d, JCF ) 5 Hz), 137.3, 131.0, 121.2 (d, JCF ) 6 Hz), 121.2
(d, JCF ) 6 Hz), 120.1 (d, JCF ) 13 Hz), 120.0, 115.6, 66.8, 39.7;
19F NMR (470 MHz, CDCl3) δ -138.8 (dd, J ) 9.9, 4.5 Hz);
HRMS (ESI-TOF) m/z calcd for C13H13F4NaO3 (M + Na+)
259.0746, found 259.0759.
3-Fluoro-2-methoxy-6-(2-propenyl)benzoic Acid 2-Propenyl
Ester (17). To a solution of 16 (481 mg, 2.04 mol) in acetone (4.0
mL), potassium carbonate (366 mg, 2.65 mmol), and methyl iodide
(1.27 mL, 20.4 mmol) were added. After being stirred at room
temperature for 23 h, the reaction mixture was concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (20:1 hexane/ethyl acetate) to afford 17 (471 mg,
1.88 mmol, 93%) as a pale greenish-yellow oil. Rf ) 0.65 (20:1
hexane/ethyl acetate); IR (film) 2982, 2944, 1734, 1640, 1606, 1490,
1458, 1421, 1359, 1277, 1195, 1160, 1134, 1105, 1043, 986, 922,
1
820, 734 cm-1; H NMR (500 MHz, CDCl3) δ 7.05 (1H, dd, J )
11.2, 8.5 Hz), 6.86 (1H, dd, J ) 8.5, 4.5 Hz), 5.99 (1H, ddt, J )
17.2, 10.5, 5.9 Hz), 5.85 (1H, ddt, J ) 16.9, 10.2, 6.6 Hz), 5.41
(1H, ddd, J ) 17.2, 2.9, 1.4 Hz), 5.28 (1H, ddd, J ) 10.3, 2.4, 1.3
Hz), 5.04 (1H, ddd, J ) 10.2, 3.0, 1.3 Hz), 5.02 (1H, ddd, J )
16.9, 3.4, 1.7 Hz), 4.79 (2H, dd, J ) 5.9, 1.4 Hz), 3.92 (3H, d, J
) 1.9 Hz, -OMe), 3.32 (2H, br d, J ) 6.7 Hz); 13C NMR (125
(3S,5R,6S,8E)-3,4,5,6,7,10-Hexahydro-13-fluoro-14-methoxy-
5-(methoxymethoxy)-3-[2-[(4-methoxyphenyl)methoxy]ethyl]-6-
methyl-1H-2-benzoxacyclododecin-1-one (19). To a flask charged
with dichloromethane (18 mL) under vigorous stirring, a solution
of 18 (358 mg, 0.66 mmol) in dichloromethane (67 mL) and
(Cy3P)2Cl2RudCHPh (41 mg, 0.0495 mmol) in dichloromethane
(63 mL) were added simultaneously at room temperature over 26
min, and the resulting solution was stirred at room temperature for
21 min. The reaction was quenched with triethylamine (3.2 mL),
exposed to air for 13 h with vigorous stirring, and concentrated
under reduced pressure. The residue was purified by flash silica
gel column chromatography (10:1 to 5:1 hexane/ethyl acetate) to
afford 19 (306 mg, 0.59 mmol, 90%) as a pale-gray oil (10:1
mixture of E:Z isomers at C9). [R]D26 -44.5 (c 0.21, CHCl3); Rf )
0.28 (3:1 hexane/ethyl acetate); IR (film) 2954, 2933, 1725, 1613,
1587, 1514, 1489, 1456, 1417, 1359, 1275, 1248, 1207, 1173, 1155,
1142, 1099, 1067, 1040, 975, 917, 822 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.25 (2H, d. J ) 8.6 Hz), 6.97 (1H, dd, J ) 11.3, 8.3
Hz), 6.85 (2H, d. J ) 8.6 Hz), 6.78 (1H, dd, J ) 8.4, 4.5 Hz),
5.50-5.40 (2H, m, H15 and H10), 5.29 (1H, ddt, J ) 15.2, 9.5,
1
MHz, CDCl3) δ 166.5 (d, JCF ) 4 Hz), 153.6 (d, JCF ) 246 Hz),
144.7 (d, JCF ) 12 Hz), 136.0, 133.7 (d, JCF ) 5 Hz), 131.7, 129.1,
124.7 (d, JCF ) 7 Hz), 119.1, 117.9 (d, JCF ) 19 Hz), 116.5, 66.1,
62.0 (d, JCF ) 5 Hz), 37.0; 19F NMR (470 MHz, CDCl3) δ -133.5
(br ddq, J ) 11.1, 4.5, 1.8 Hz); HRMS (ESI-TOF) m/z calcd for
C14H15FNaO3 (M + Na+) 273.0903, found 273.0929.
3-Fluoro-2-methoxy-6-(2-propenyl)benzoic Acid (7). To a flask
charged with tetrakis(triphenylphosphine)palladium (8.3 mg, 7.2
µmol), a solution of 17 (18.0 mg, 71.9 µmol) in THF (2.3 mL),
followed by morpholine (62.7 µL, 0.72 mmol), was added dropwise.
After being stirred at room tempeature for 1 h, the reaction mixture
was concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (1:1 hexane/ethyl acetate,
containing 0.05% HCOOH) to afford 7 (12.9 mg, 61.2 µmol, 85%)
1
as a colorless oil. Rf ) 0.44 (1:1 hexane/ethyl acetate); H NMR

Fluorinated Analogues of Salicylihalamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 803
2.2 Hz, H9), 4.84 (1H, d, J ) 6.9 Hz), 4.76 (1H, d, J ) 6.9 Hz),
4.45 (2H, s), 4.06 (1H, dd, J ) 9.5, 3.7 Hz, H13), 3.82 (3H, d, J
) 1.9 Hz), 3.78 (3H, s), 3.63 (1H, br dd, J ) 16.4, 10.0 Hz, H8a),
3.61 (2H, t,dd, J ) 6.7, 6.7 Hz, H17), 3.41 (3H, s), 3.28 (1H, ddt,
J ) 16.4, 4.6, 2.6 Hz, H8b), 2.28 (1H, br ddd, J ) 14.0, 5.7, 3.3
Hz, H11a), 2.17-2.06 (1H, m, H12), 2.01 (1H, dddd, J ) 14.2,
8.3, 6.0, 6.0 Hz, H16a), 1.90 (1H, dddd, J ) 14.2, 7.2, 7.2, 4.6 Hz,
H16b), 1.72 (1H, dd, J ) 15.6, 8.9 Hz, H14a), 1.68 (1H, dd, J )
14.1, 11.6 Hz, H11b), 1.46 (1H, dd, J ) 15.5, 9.5, H14b), 0.84
(3H, d, J ) 6.9); 13C NMR (125 MHz, CDCl3) δ 166.9, 159.1,
153.6x (d, 1JCF ) 246 Hz), 144.7 (JCF ) 12 Hz), 134.1, 131.5 (JCF
) 2 Hz), 130.6, 130.2, 129.1, 128.3, 125.5 (JCF ) 7 Hz), 117.1
(JCF ) 19 Hz), 113.7, 96.8, 79.3, 72.8, 72.6, 66.3, 61.6 (JCF ) 7
Hz), 55.6, 55.3, 37.7, 37.3, 36.4, 35.5, 34.0, 13.4; 19F NMR (470
MHz, CDCl3) δ -133.0 (br ddq, J ) 11.7, 4.5, 1.8 Hz); HRMS
(ESI-TOF) m/z calcd for C29H37FNaO7 (M + Na+) 539.2421, found
539.2426.
(32 mL), and the suspension was poured into a mixture of ether
(12 mL) and water (36 mL) at 0 °C. The organic layer was
separated, washed with saturated aqueous Na2S2O3, saturated
aqueous NaHCO3, water, and brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (15:1 to 5:1 hexane/
ethyl acetate) to afford 21 (221 mg, 0.43 mmol, 80%) as a pale-
27
yellow oil (3.3:1 mixture of E:Z isomers at C17). [R]D -84.5 (c
0.21, CHCl3); Rf ) 0.49 (3:1 hexane/ethyl acetate); IR (film) 2955,
2932, 1727, 1606, 1489, 1456, 1426, 1417, 1382, 1347, 1273, 1230,
1196, 1155, 1139, 1102, 1042, 975, 917, 821, 811 cm-1; 1H NMR
(500 MHz, CDCl3) δ 6.99 (1H, dd, J ) 11.5, 8.5 Hz), 6.78 (1H,
dd, J ) 8.5, 4.4 Hz), 6.69 (1H, ddd, J ) 14.4, 8.2, 6.2 Hz, H17),
6.15 (1H, d, J ) 14.5 Hz, H18), 5.45 (1H, dddd, J ) 14.8, 11.4,
2.7, 2.7 Hz, H10), 5.33 (1H, ddd, J ) 8.3, 8.3, 4.6 Hz, H15), 5.28
(1H,dddd, J ) 15.2, 9.6, 2.1, 2.1 Hz, H9), 4.85 (1H, d, J ) 6.7
Hz), 4.78 (1H, d, J ) 6.7 Hz), 4.05 (1H, dd, J ) 9.4, 3.7 Hz,
H13), 3.96 (3H, d, J ) 2.0 Hz), 3.62 (1H, dd, J ) 16.3, 9.5 Hz,
H8a), 3.43 (3H, s), 3.28 (1H, dddd, J ) 16.4, 3.9, 2.4, 2.4 Hz,
H8b), 2.45 (1H, dddd, J ) 14.6, 8.0, 6.3, 1.5 Hz, H16a), 2.33 (1H,
dddd, J ) 14.9, 8.2, 4.6, 0.9 Hz, H16b), 2.29 (1H, br d, J ) 14.2
Hz, H11a), 2.17-2.07 (1H, m, H12), 1.68 (dd, J ) 15.1, 8.6 Hz,
H14a), 1.67 (1H, ddd, J ) 14.2, 11.5, 11.5 Hz, H11b), 1.40 (1H,
dd, J ) 15.4, 9.5 Hz, H14b), 0.84 (3H, d, J ) 6.9 Hz); 13C NMR
(3S,5R,6S,8E)-3,4,5,6,7,10-Hexahydro-3-(2-hydroxyethyl)-13-
fluoro-14-methoxy-5-(methoxymethoxy)-6-methyl-1H-2-benzox-
acyclododecin-1-one (20). To a solution of 19 (296 mg, 0.57 mmol)
and water (0.57 mL) in dichloromethane (11 mL), 2,3-dichloro-
5,6-dicyano-p-benzoquinone (182 mg, 0,80 mmol) was added at 0
°C. After being stirred at room temperature for 53 min, the reaction
mixture was quenched with saturated aqueous NaHCO3 and
saturated aqueous Na2S2O3 at 0 °C, and extracted with ether. The
organic layer was washed with saturated aqueous Na2S2O3, water,
and brine, dried over Na2SO4, filtrated, and concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (3:1 to 1:1 hexane/ethyl acetate) to afford 20 (220
mg, 0.55 mmol, 97%) as a pale bright-yellow oil (10:1 mixture of
1
(125 MHz, CDCl3) δ 166.9 (d, JCF ) 4 Hz), 153.6 (d, JCF ) 246
Hz), 144.8 (d, JCF ) 12 Hz), 141.71, 136.9, 134.1 (d, JCF ) 5 Hz),
131.5, 128.3, 125.4 (d, JCF ) 7 Hz), 117.2 (d, JCF ) 19 Hz), 97.1,
79.4, 77.6, 73.8, 61.9 (d, JCF ) 7 Hz), 55.7, 42.4, 37.6, 37.2, 35.1,
34.1, 13.3; 19F NMR (470 MHz, CDCl3) δ -132.7 (ddq, J ) 11.3,
4.5, 2.4 Hz); HRMS (ESI-TOF) m/z calcd for C22H28FINaO5 (M
+ Na+) 541.0863, found 541.0881.
27
E:Z isomers at C9). [R]D -37.8 (c 0.26, CHCl3); Rf ) 0.29 (1:1
hexane/ethyl acetate); IR (film) 3441, 2954, 1725, 1605, 1489, 1457,
1417, 1382, 1348, 1275, 1231, 1197, 1155, 1142, 1102, 1041, 975,
917, 822, 717 cm-1; 1H NMR (500 MHz, CDCl3) δ 6.98 (1H, dd,
J ) 11.5, 8.5 Hz), 6.80 (1H, dd, J ) 8.4, 4.5 Hz), 5.48 (1H, dddd,
J ) 8.5, 8.5, 4.7, 4.7 Hz, H15), 5.46 (1H, ddt, J ) 15.2, 11.5, 2.6
Hz, H10), 5.30 (1H, ddt, J ) 15.2, 9.6, 2.2 Hz, H9), 4.85 (1H, d,
J ) 6.8 Hz), 4.80 (1H, d, J ) 6.8 Hz), 4.08 (1H, dd, J ) 9.4, 3.4
Hz, H13), 3.92 (3H, d, J ) 2.0 Hz), 3.82 (1H, ddd, J ) 11.6, 7.7,
4.4 Hz, H17a), 3.75 (1H, ddd, J ) 11.2, 6.0, 5.3 Hz, H17b), 3.63
(1H, dd, J ) 16.5, 9.6 Hz, H8a), 3.43 (3H, s), 3.29 (1H, dddd, J )
16.5, 4.2, 2.4, 2.4 Hz, H8b), 2.29 (1H, ddd, J ) 14.4, 5.6, 3.2 Hz,
H11a), 2.18-2.07 (1H, m, H12), 1.94 (1H, dddd, J ) 14.5, 9.5,
5.9, 4.4 Hz, H16a), 1.84 (1H, dddd, J ) 14.4, 7.7, 4.7, 4.7 Hz,
H16b), 1.75 (1H, dd, J ) 15.6, 8.2 Hz, H14a), 1.69 (1H, ddd, J )
14.3, 11.6, 11.6 Hz, H11b), 1.46 (1H, dd, J ) 15.6, 9.3 Hz, H14b),
0.85 (3H, d, J ) 6.7 Hz); 13C NMR (125 MHz, CDCl3) δ 167.1 (d,
(3S,5R,6S,8E)-3,4,5,6,7,10-Hexahydro-13-fluoro-3-(3-iodo-2-
propenyl)-14-hydroxy-5-(methoxymethoxy)-6-methyl-1H-2-ben-
zoxacyclododecin-1-one (22). To a vigorously stirred solution of
21 (211 mg, 0.41 mmol) in dichloromethane (45 mL), a solution
of tribromoborane in dichloromethane (1.0 M, 1.06 mL, 1.06 mmol)
was added at -78 °C. After being stirred at -78 °C for 65 min,
the reaction mixture was allowed to warm up to room temperature,
then quenched with ice-cold water (70 mL) at -5 °C and extracted
with dichloromethane. The organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash silica gel column
chromatography (5:1 to 3:1 hexane/ethyl acetate) to afford 22 (138
mg, 0.30 mmol, 76%) as a colorless powder (4:1 mixture of E:Z
27
isomers at C17). [R]D +19.4 (c 0.69, CHCl3); Rf ) 0.27 (3:1
hexane/ethyl acetate, developed in twice); IR (film) 3469, 2956,
2359, 1703, 1612, 1491, 1425, 1356, 1291, 1274, 1168, 1141, 1028,
968, 820, 767, 669, 623 cm-1; 1H NMR (500 MHz, CDCl3) δ 10.1
(1H, br s, -OH), 7.10 (1H, dd, J ) 8.5 Hz), 6.63 (1H, dd, J ) 8.3,
5.0 Hz), 6.57 (1H, ddd, J ) 14.3, 7.4, 7.4 Hz, H17), 6.18 (1H,
ddd, J ) 14.3, 1.3, 1.3 Hz, H18), 5.52 (1H, dddd, J ) 10.6, 5.9,
5.9, 1.0 Hz, H15), 5.40 (1H, ddd, J ) 15.5, 4.6, 4.6 Hz, H9), 5.12
(1H, br ddd, J )15.2, 9.3, 5.3 Hz, H10), 3.69 (1H, dd, J ) 8.7, 3.4
Hz, H13), 3.59 (1H, br dd, J ) 16.7, 5.1 Hz, H8a), 3.40 (1H, ddd,
J ) 16.8, 2.0, 2.0 Hz, H8b), 2.47-2.41 (2H, m, H16), 2.33 (1H,
br dddd, J ) 13.5, 6.7, 1.8, 1.8 Hz, H11a), 1.93 (1H, dd, J ) 15.0,
10.6 Hz, H14a), 1.84-1.92 (2H, m, H12), 1.79 (1H, ddd, J ) 13.6,
9.2, 9.2 Hz, H11b), 1.33 (1H, ddd, J ) 15.1, 8.8, 1.0 Hz, H14b),
0.90 (3H, d, J ) 6.9 Hz); 13C NMR (125 MHz, CDCl3) δ 169.7,
1
JCF ) 2 Hz), 153.5 (d, JCF ) 247 Hz), 144.3 (d, JCF ) 12 Hz),
134.1 (d, JCF ) 3 Hz), 131.6, 129.9, 128.2, 125.7 (d, JCF ) 7 Hz),
117.3 (d, JCF ) 19 Hz), 97.0, 73.5, 61.9 (d, JCF ) 7 Hz), 59.4,
55.6, 38.9, 37.6, 37.3, 35.5, 34.1, 13.4; 19F NMR (470 MHz, CDCl3)
δ -132.8 (ddq, J ) 11.7, 4.5, 1.8 Hz); HRMS (ESI-TOF) m/z calcd
for C21H29FNaO6 (M + Na+) 419.1846, found 419.1861.
(3S,5R,6S,8E)-3,4,5,6,7,10-Hexahydro-13-fluoro-3-(3-iodo-2-
propenyl)-14-methoxy-5-(methoxymethoxy)-6-methyl-1H-2-ben-
zoxacyclododecin-1-one (21). To a solution of 20 (211 mg, 0.53
mmol) in dichloromethane (11 mL) Dess-Martin periodinane (339
mg, 0,80 mmol) was added at 0 °C. After being stirred at room
temperature for 73 min, the reaction mixture was quenched with
saturated aqueous NaHCO3 and saturated aqueous Na2S2O3 at 0
°C, and extracted with ether. The organic layer was washed with
saturated aqueous Na2S2O3, water, and brine, dried over Na2SO4,
filtrated, and concentrated under reduced pressure to afford aldehyde
as a pale bright-yellow oil, which was used in the next reaction
without further purification. Chromium chloride(II) (782 mg, 6.36
mmol) was dried by being heated with flame under reduced pressure
for 15min. To a suspension of the chromium chloride(II) in THF
(4.6 mL), a solution of the aldehyde and iodoform (835 mg, 2.12
mmol) in dioxane (27.6 mL) was added at 0 °C to give a reddish-
brown suspension. After being stirred at room temperature for 26 h
with light shielding, the reaction mixture was diluted with ether
1
150.5 (d, JCF ) 241 Hz), 149.4, 140.7, 136.8 (d, JCF ) 5 Hz),
135.9, 132.0, 127.4, 122.3 (d, JCF ) 7 Hz), 119.0 (d, JCF ) 17
Hz), 78.4, 73.7, 70.5, 41.4, 38.5, 38.2, 37.4, 35.2, 13.6; 19F NMR
(470 MHz, CDCl3) δ -139.1 (br s); HRMS (ESI-TOF) m/z calcd
for C19H22FINaO4 (M + Na+) 483.0445, found 483.0457.
Salicylihalamide A (1a) and B (1b). N,N′-dimethyl acetamide
(DMA) used in this reaction was degassed by freeze-thaw method
over 10 times. To the 20 mL Schlenk flask dried by heating with
flame, under reduced pressure for 10 min Rb2CO3 (139 mg, 0.60
mmol) was added under argon atmosphere and dried by a drier
under reduced pressure for 15 min and charged with argon gas after
cooling to room temperature. To this flask, copper(I) thiophene

804 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Sugimoto et al.
carboxylate (19 mg, 0.10 mmol), a solution of (2Z,4Z)-2,4-
heptadienamide 8 (75 mg, 0.60 mmol) and 2,2′-bipyridine (20 mg,
0.12 mmol) in DMA (0.9 mL), which was dried over MS4A, and
then the resulting mixture was immediately degassed until no
bubbles were found. After being stirred for 30 min at room
temperature with light shielding, a solution of 23 (44 mg, 0.10
mmol) in DMA (0.6 mL), which was dried over MS4A, was added
and the resulting solution was immediately degassed until no
bubbles were found. After being stirred for 1 h at 90 °C with light
shielding, the reaction solution was diluted with ether at room
temperatur, and was quenched with pH 7.0 phosphate buffer at 0
°C. The organic layer was separated, washed with pH 7.0 phosphate
buffer, water and brine, and dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (hexane/ethyl acetate )
3/1 to 1/3) to afford the roughly purified substance (58.3 mg), which
was purified by the preparative HPLC to afford salicylihalamide
A (1a) (10.1 mg, 0.023 mol, 23%) and salicylihalamide B (1b)
(11.4 mg, 0.026 mmol, 26%) as a colorless wax, respectively. HPLC
conditions: column: COSMOSIL 5C18-MS-II (NACALAI TESQUE,
INC.), 10 mm × 250 mm; detection: λ ) 280 nm; flow rate: 2.0
mL/min; elution with a gradient solvent system: MeOH/H2O ) 7/3,
0-100 min: MeOH/H2O ) 7/3 f MeOH, 100-120 min: MeOH,
120-140 min; The tR of salicylihalamide A (1a) and salicylihala-
mide B (1b) were 64.2 and 81.0 min, respectively.
4-19F-Salicylihalamide A (6a) and B (6b). N,N′-dimethyl
acetamide (DMA) used in this reaction was degassed by freeze-thaw
method over 10 times. Rb2CO3 (139 mg, 0.60 mmol) in a Schlenk
flask was dried by heating with a drier under reduced pressure for
15 min. After cooling to room temperature, copper(I) thiophen-
ecarboxylate (19 mg, 0.10 mmol) was added in glove box. A
solution of (2Z,4Z)-2,4-heptadienamide 8 (75 mg, 0.60 mmol) and
2,2′-bipyridine (20 mg, 0.12 mmol) in DMA (0.7 mL), which was
dried over MS4A, was added and the resulting mixture was
immediately degassed over and over again until no bubbles were
found. After being stirred for 30 min at room temperature with
light shielding, a solution of of 22 (46 mg, 0.10 mmol) in DMA
(0.7 mL), which was dried over MS4A, was added and the resulting
solution was immediately degassed over and over again until no
bubbles were found. After being stirred for 1 h at 90 °C with light
shielding, the reaction mixture was diluted with ether at room
temperature and was quenched with pH 7.0 phosphate buffer at 0
°C. The organic layer was separated, washed with pH 7.0 phosphate
buffer, water and brine, and dried over Na2SO4, filtrered, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (3:1 to 1:3 hexane/ethyl
acetate) to afford the roughly purified substance (29 mg), which
was purified by the preparative HPLC to afford 4-19F-salicylihala-
mide A (6a) (7.1 mg, 0.015 mol, 15%) and 4-19F-salicylihalamide
B (6b) (10.1 mg, 0.022 mmol, 22%) as a colorless wax, respectively.
HPLC conditions: column: COSMOSIL 5C18-MSII, 10 mm × 250
mm; detection: 280 nm; flow rate: 2.0 mL/min, eluent: 70:30
MeOH/H2O, 100 min, 70:30 f 100:0, 20 min, 100:0, 30 min. The
retention times (tR) of 6a and 6b were 63.4 and 73.4 min,
19
1a. [R]D ) -32.6 (c 0.169, MeOH); Rf 0.25 (hexane/ethyl
acetate ) 1/1); IR (film) 3285, 2964, 1700, 1653, 1589, 1523, 1464,
1
1293, 1248, 1216, 1123, 1066, 971 cm-1; H NMR (500 MHz,
C6D6) δ 11.5 (1H, br, C3-OH), 7.93 (1H, br dd, J ) 11.4, 11.4 Hz,
H22), 7.12 (1H, dd, J ) 14.3, 10.9 Hz, H18), 6.99-6.94 (2H, m,
H4 and H5), 6.62 (1H, dd, J ) 11.6, 11.6 Hz, H21), 6.50 (1H, br
d, -NH), 6.48 (1H, dd, J ) 6.6, 2.3 Hz, H6), 5.62 (1H, ddddd, J
) 10.8, 7.7, 7.7, 1.3, 1.3 Hz, H23), 5.54(1H, ddd, J ) 10.0, 6.6,
6.6 Hz, H15), 5.28 (1H, ddd, J ) 15.5, 4.3, 4.3 Hz, H9), 5.10 (1H,
br d, J ) 10.9 Hz, H20), 5.05 (1H, br ddd, J ) 14.6, 6.9, 6.9 Hz,
H10), 4.77 (1H, br ddd, J ) 14.3, 7.2, 7.2 Hz, H17), 3.62 (1H, br
dd, J ) 16.5, 5.3 Hz, H8a), 3.44 (1H, dd, J ) 8.3, 2.9 Hz, H13),
3.32 (1H, br d, J ) 16.0 Hz, H8b), 2.17 (1H, ddd, J ) 14.0, 7.2,
7.2 Hz, H16a), 2.12 (1H, ddd, J ) 12.3, 5.7, 5.7 Hz, H11a), 2.05
(1H, ddd, J ) 14.0, 6.9, 6.9 Hz, H16b), 1.97 (2H, tdd, J ) 7.6,
7.6, 1.4 Hz, H24), 1.66 (1H, dd, J ) 15.1, 10.8 Hz, H14a), 1.62
(1H, dd, J ) 13.5, 8.9 Hz, H11b), 1.56-1.45 (1H, m, H12), 1.27
(1H, dd, J ) 14.9, 8.6 Hz, H14b), 0.82 (3H, d, J ) 6.9 Hz, H26),
0.77 (3H, t, J ) 7.7 Hz, H25); 13C NMR (125 MHz, C6D6) δ 171.2,
170.0, 163.0, 162.9, 142.6, 141.7, 136.9, 134.0, 132.7, 127.1, 126.0,
124.9, 123.6, 119.7, 117.1, 114.7, 107.0, 75.3, 70.4, 39.3, 38.6,
37.6, 36.4, 35.5, 20.8, 14.0, 13.9; HRMS (ESI-TOF) m/z calcd for
C26H33NNaO5 (M + Na+) 462.2256, found 462.2253.
28
respectively. 6a: [R]D -50.0 (c 0.213, MeOH); Rf ) 0.34 (1:1
hexane/ethyl acetate); λmax (MeOH) 282 nm (ε ) 26200); IR (film)
3281, 2963, 2931, 1691, 1656, 1611, 1492, 1461, 1288, 1248, 1218,
1138, 1081, 1027, 971, 820, 678 cm-1; 1H NMR (500 MHz, C6D6)
δ 10.5 (1H, br, C3-OH), 7.91 (1H, br dd, J ) 11.5, 11.5 Hz, H22),
7.08 (1H, dd, J ) 14.3, 10.6 Hz, H18), 6.72 (1H, dd, J ) 9.7, 8.3
Hz, H5), 6.61 (1H, ddd, J ) 11.5, 11.5, 0.9 Hz, H21), 6.56 (1H, br
s, -NH), 6.19 (1H, dd, J ) 8.3, 4.6 Hz, H6), 5.63 (1H, ddddd, J
) 10.9, 7.7, 7.7, 1.3, 1.3 Hz, H23), 5.53 (1H, m, H15), 5.23 (1H,
ddd, J ) 15.5, 4.7, 4.7 Hz, H9), 5.10 (1H, br d, J ) 11.5 Hz,
H20), 5.13-5.03 (1H, m, H10), 5.05 (1H, br ddd, J ) 14.6, 6.9,
6.9 Hz, H10), 4.85 (1H, ddd, J ) 14.3, 7.2, 7.2 Hz, H17), 3.68
(1H, br d, J ) 6.0 Hz, H13), 3.37 (2H, m, H8), 2.21 (1H, ddd, J
) 14.3, 7.4, 7.4 Hz, H16a), 2.12 (1H, m, H11a), 2.08 (1H, ddd, J
) 13.6, 6.0, 6.0 Hz, H16b), 1.97 (2H, tdd, J ) 7.6, 7.6, 1.4 Hz,
H24), 1.66 (1H, dd, J ) 15.0, 9.9 Hz, H14a), 1.65-1.53 (2H, m,
H11b and H12), 1.31 (1H, dd, J ) 14.6, 9.2 Hz, H14b), 0.83 (3H,
d, J ) 6.9 Hz, H26), 0.77 (3H, t, J ) 7.6 Hz, H25); 13C NMR (125
MHz, C6D6) δ 169.7 164.2, 160.5, 151.5 (d, 1JCF ) 240 Hz), 141.9,
137.2, 135.8, 130.8, 129.5, 126.0, 124.9, 121.7, 119.7, 117.5, 108.8,
76.0, 71.1, 38.3, 37.6, 36.7, 36.2, 20.8, 14.0, 13.7 (1 of the sp2
carbons were obserded); 19F NMR (470 MHz, C6D6) δ -138.6 (s);
HRMS (ESI-TOF) m/z calculated for C26H33FNO5 (M + H+)
458.2343, found 458.2341. 6b: [R]D28 -71.0 (c 0.301, MeOH); Rf
) 0.34 (hexane/ethyl acetate ) 1/1); λmax (MeOH) 282 nm (ε )
22,100); IR (film) 3357, 2964, 2931, 1707, 1654, 1590, 1491, 1274,
1248, 1207, 1170, 1137, 1032, 973, 678 cm-1; 1H NMR (500 MHz,
C6D6) δ 7.90 (1H, dd, J ) 11.5, 11.4 Hz, H22), 7.71 (1H, br s,
-NH), 7.26 (1H, dd, J ) 9.9 Hz, H18), 6.72 (1H, dd, J ) 9.7, 9.7
Hz, H5), 6.67 (1H, dd, J ) 11.7, 11.7 Hz, H21), 6.17 (1H, dd, J )
8.2, 4.6 Hz, H6), 5.63 (1H, br ddd, J ) 10.9, 7.9, 7.9 Hz, H23),
5.51 (1H, d, J ) 10.9 Hz, H20), 5.36 (1H, m, H15), 5.20-5.08
(2H, m, H9 and H10), 4.65 (1H, ddd, J ) 8.3, 8.3, 8.0 Hz, H17),
3.60 (1H, m, H13), 3.34 (1H, br d, J ) 15.7 Hz, H8a), 3.30 (1H,
br d, J ) 15.7 Hz, H8b), 2.17 (1H, ddd, J ) 14.0, 7.2, 7.2 Hz,
H16a), 2.11-2.01 (1H, m, H11a), 2.06 (1H, br d, J ) 14.5 Hz,
H16b), 1.97 (2H, tdd, J ) 7.6, 7.6, 1.4 Hz, H24), 1.76 (1H, dd, J
) 14.9, 10.0 Hz, H14a), 1.72-1.65 (1H, m, H11b), 1.65-1.55 (1H,
m, H12), 1.30 (1H, dd, J ) 14.9, 8.9 Hz, H14b), 0.85 (3H, d, J )
6.9 Hz, H26), 0.78 (3H, t, J ) 7.7 Hz, H25); 13C NMR (125 MHz,
C6D6) δ 170.0, 164.0, 151.2 (d, 1JCF ) 246 Hz), 142.0, 137.4, 136.7,
131.1, 128.9, 124.9 (d, JCF ) 12 Hz), 122.2 (d, JCF ) 5 Hz), 119.6,
20
1b. [R]D ) -90.4 (c 0.025, MeOH); Rf 0.25 (hexane/ethyl
acetate ) 1/1); IR (film) 3286, 2960, 2924, 1695, 1653, 1587, 1507,
1
1464, 1294, 1247, 1211, 1119, 1032, 966 cm-1; H NMR (500
MHz, C6D6) δ 11.8 (1H, br, C3-OH), 7.97 (1H, br dd, J ) 11.4,
11.4 Hz, H22), 7.57 (1H, br d, J ) 9.2 Hz, -NH), 7.31 (1H, dd, J
) 10.0, 10.0 Hz, H18), 6.98-6.94 (2H, m, H4 and H5), 6.62 (1H,
ddd, J ) 11.4, 11.4, 0.9 Hz, H21), 6.45 (1H, dd,6.3, 2.6 Hz, H6),
5.26-5.12 (2H, m, H15 and H10), 6.48 (1H, dd, J ) 6.6, 2.3 Hz,
H6), 5.62 (1H, ddddd, J ) 10.8, 7.7, 7.7, 1.3, 1.3 Hz, H23), 5.44
(1H, d, J ) 11.5 Hz, H20), 5.25-5.12 (2H, m, H15 and H10),
5.07 (1H, ddd, J ) 15.3, 7.2, 7.2 Hz, H9), 4.48 (1H, ddd, J ) 8.0,
8.0, 8.0 Hz, H17), 3.56 (1H, br dd, J ) 16.5, 5.3 Hz, H8a), 3.24
(1H, br d, J ) 16.9 Hz, H8b), 3.21 (1H, br d, J ) 9.7 Hz, H13),
2.11-1.99 (1H, m, H11a), 2.04 (1H, ddd, J ) 14.0, 7.4, 7.4 Hz,
H16a), 1.94 (2H, tdd, J ) 7.6, 7.6, 1.4 Hz, H24), 1.84 (1H, ddd, J
) 14.3, 7.7, 7.7 Hz, H16b), 1.77-1.68 (1H, m, H11b), 1.72 (1H,
dd, J ) 14.7, 10.7 Hz, H14a), 1.45 (1H, br q, J ) 6.8 Hz, H12),
1.19 (1H, dd, J ) 15.2, 8.6 Hz, H14b), 0.82 (3H, d, J ) 6.9 Hz,
H26), 0.77 (3H, t, J ) 7.4 Hz, H25); 13C NMR (125 MHz, C6D6)
δ 172.0, 162.9, 141.9, 137.3, 134.6, 132.8, 126.8, 125.4, 124.9,
123.7, 119.5, 117.2, 103.2, 76.1, 70.9, 39.4, 38.4, 38.0, 36.2, 31.4,
20.8, 14.0, 13.8; HRMS (ESI-TOF) m/z calcd for C26H33NNaO5
(M + Na+) 462.2256, found 462.2245.

Fluorinated Analogues of Salicylihalamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 805
118.3 (d, JCF ) 17 Hz), 105.2, 76.2, 71.3, 38.5, 38.3, 37.8, 36.3,
31.9, 20.8, 14.0, 13.6 (3 of the sp2 carbons were obserded); 19F
NMR (470 MHz, C6D6) δ -138.4 (s); HRMS (ESI-TOF) m/z calcd
for C26H32FNNaO5 (M + Na+) 480.2162, found 480.2162.
diluted into 1.8 mL of the MOPS solution (20 mM MOPS, pH 7.5
with NaOH, 25% glycerol, 5 mM Na2ATP, 5 mM monothioglyc-
erol, and 10 µg/mL of the protease inhibitors). The resulting solution
contained 4-8 mg of the membrane proteins including V-ATPase,
judging from both the quantitative determination of the total protein
concentration by the BCA assay, and the Western blotting with
the antibody against subunit b on the V1 domain of V-ATPase.
The prepared CGM was suspended at the protein concentration of
5 mg/mL in the MOPS solution and kept frozen at -80 °C.
Assay of Synthetic Salicylihalamides in the Inhibitory
Activity against Chromaffin Granule Membrane V-ATPase. The
measurement of fluorescence quenching of 9-amino-6-chloro-2-
methoxyacridine (ACMA) as an indicator for proton uptake into
the membrane vesicles was employed.31 To a 2 mL of the reaction
medium for proton pump assay, chromaffin granule membranes
from porcine adrenal glands containing 50 µg of protein, 4.0 µL
of 1.0 M ACMA, and 0.67 µL of valinomycin were added, and the
resulting solution was incubated at 37 °C for 10 min. A solution
of the synthesized salicylihalamide in DMSO (prepared to give 0.1%
final concentration of DMSO) was added and incubated at 37 °C
for 3 min, and then 10 µL of 0.1 M MgATP was added and
incubated for 2 min. The proton gradients made by the V-ATPases
in the membranes were canceled by addition of 3.4 µL of 1 mM
FCCP. The fluorescence quenching in the presence of MgATP was
monitored by a spectrofluorometer with an excitation wavelength
of 412 nm and emission at 480 nm. The initial rate of quenching
was estimated as the slope of the quenching trace performed on
sets of 20 points (20 s of quenching trace) and is expressed as the
rate of quenching/min. The proton transport activity is expressed
as the percentage versus DMSO-matched controls with standard
deviation from triplicate experiments. IC50 values were obtained
graphically by plotting the percent activity of proton transport versus
the log values of the inhibitor concentrations.
Biology. Materials. All the chemical used in this study were
commercially available special grade ones without further treatment.
Water was purified with a Millipore Simpli Laboratory system
(Millipore Inc., Bedford, MA) and used within a few hours. The
following reagents used in this protocol were as follows: SME
buffer (0.3 M sucrose, 5 mM EDTA, 10 mM MOPS and pH 7.5
with NaOH), pepstatin A (10 mg/mL in ethanol), leupeptin (10
mg/mL in ethanol), reaction medium for proton pump assay (0.3
M sucrose, 40 mM KCl, 5 mM tricine, and pH 8.0 with NaOH),
9-amino-6-chloro-2-methoxyacridine (ACMA) (0.1 M in ethanol),
valinomycin (1 mg/mL in ethanol), MgATP (0.1 M aq and pH 7.5
with NaOH), and carbonyl cyanide p-trifluoromethoxyphenylhy-
drazone (FCCP) (1 mM in ethanol), Hanks’ balanced salt solutions
(HBSS) (137 mM NaCl, 5.3 mM KCl, 1.3 mM CaCl2, 0.82 mM
MgSO4, 0.34 mM Na2HPO4, 4.2 mM KH2PO4, 4.2 mM NaHCO3,
5.6 mM glucose, 15 mM HEPES (4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid) pH 7.4 with NaOH).
Assay of Synthesized Salicylihalamides Using Simian
Kidney Cell. Simian (Cercopithecus aethiops) kidney COS-7 cells
were maintained in Dulbecco’s modified Eagle medium containing
10% fetal calf serum at 37 °C in 5% CO2/95% air with a CO2
incubator.28 The cells were seeded on poly-L-lysine coated cover-
slips. After overnight culture, the synthesized salicylihalamide was
added into the culture media to give a final concentration of 1 µM,
and incubated at 37 °C for 5 h. Then, the cells were loaded with
pH indicator LysoSensor Green DND-189 (Invitrogen, Molecular
probes)29 at a final concentration of 1 µM. After 1 min, an excess
of the indicator was washed out with HBSS. The cells were
observed using a fluorescence microscope, and images were
acquired using a cooled CCD camera. For tryptan blue exclusion
test, the intact or drug-treated cells were soaked in phosphate
buffered saline containing 0.3% trypan blue. The cells were
observed with bright-field and phase-contrast microscope.
Preparation of Chromaffin Granule Membranes (CGM)
from Porcine Adrenal Glands. Preparation of chromaffin granule
membranes (CGM) from porcine adrenal glands was operated
according to refs 30, 31 in which the preparation of CGM from
bovine adrenal glands was described. The following manipulation
was carried out at 4 °C unless otherwise noted. Porcine adrenal
glands are obtained from the local meat distributor (IKEDA meat-
packing company Co., Ltd., Kyoto, Japan). Then 30-35 adrenal
medullae were separated from the cortex by pinching the connecting
tissue with pointed forceps. The isolated adrenal medullae were
kept at 4 °C in 200 mL of freshly prepared SME buffer containing
5 µg/mL of pepstatin A and leupeptin. The medullae were briefly
homogenized in a Waring blender and rehomogenized in the
Polytron homogenizer on ice bath. The mixture was filtered through
two layers of gauze, and the filtrate was kept on ice. The pink-
colored filtrate was centrifuged at 2000g for 10 min. The pellet
was discarded and the supernatant was centrifuged at 10000g for
20 min. The obtained pellet was gently suspended in ca. 25 mL of
SME containing the protease inhibitors. The suspension was applied
on top of sucrose layers in polyallomer tubes. The bottom layer of
3.0 mL buffer contained 1.8 M sucrose, and the top layer of 3.6
mL buffer contained 1.2 M sucrose. The protease inhibitors were
added to the each layer just before use. About 4.2 mL of the
suspension was applied on the layers in six tubes and centrifuged
at 72000g for 17 h. The supernatant was removed and the pale-
pink pellet containing the chromaffin granules was suspended in a
small amount of SME containing the protease inhibitors and
homogenized with a glass homogenizer. The suspension was poured
into 300 mL of the hypotonic solution (10 mM MOPS, pH 7.5
with NaOH, 1.25 mM Na2ATP, and 1 µg/mL of the protease
inhibitors). After vigorous stirring for 1 min, the solution was
centrifuged at 3000g for 10 min. The pellet was discarded and the
supernatant was centrifuged at 200000g for 60 min. The chromaffin
granule membranes were obtained as a brownish-pink pellet and
Acknowledgment. We thank Prof. Yoshinori Moriyama,
Okayama University, for helpful advice on the isolation of
chromaffin granule membranes. This work was supported by a
Grant-in-Aid for Scientific Research on Priority Areas (no.
16073211) from MEXT, Japan.
References
(1) (a) Lee, A.; Breitenbucher, J. G. The impact of combinatorial chemistry
on drug discovery. Curr. Opin. Drug DiscoVery DeV. 2003, 6, 494–
508. (b) Sehgal, A. Drug discovery and development using chemical
genomics. Curr. Opin. Drug DiscoVery DeV. 2002, 5, 526–531.
(2) (a) Galm, U.; Shen, B. Natural product drug discovery: The times
have never been better. Chem. Biol. 2007, 14, 1098–1104. (b) Lam,
K. S. New aspects of natural products in drug discovery. Trends
Microbiol. 2007, 15, 279–289.
(3) (a) Erickson, K. L.; Beutler, J. A.; Cardellina, J. H., II; Boyd, M. R.
Salicylihalamide A and B, novel cytotoxic macrolides from the marine
sponge Haliclona sp. J. Org. Chem. 1997, 62, 8188–8192. (b)
Erickson, K. L.; Beutler, J. A.; Cardellina, J. H., II; Boyd, M. R.
Salicylihalamides A and B, Novel Cytotoxic Macrolides from the
Marine Sponge Haliclona sp. [Erratum]. J. Org. Chem. 2001, 66, 1532.
(4) Yet, L. Chemistry and biology of salicylihalamide A and related
compounds. Chem. ReV. 2003, 103, 4283–4306.
(5) (a) McKee, T. C.; Galinis, D. L.; Pannell, L. K.; Cardellina, J. H., II;
Laakso, J.; Ireland, C. M.; Murray, L.; Capon, R. J.; Boyd, M. R. The
lobatamides, novel cytotoxic macrolides from southwestern pacific
tunicates. J. Org. Chem. 1998, 63, 7805–7810. (b) Suzumura, K.;
Takahashi, I.; Matsumoto, H.; Nagai, K.; Setiawan, B.; Rantiatmodjo,
R. M.; Suzuki, K.; Nagano, N. Structural elucidation of YM-75518,
a novel antifungal antibiotic isolated from Pseudomonas sp. Q38009.
Tetrahedron Lett. 1997, 38, 7573–7576.
(6) (a) Jansen, R.; Kunze, B.; Reihenbach, H.; Ho¨fle, G. Antibiotics from
gliding bacteria, LXXXVI. Apicularen A and B, cytotoxic 10-
membered lactones with a novel mechanism of action from Chon-
dromyces species (mycobacteria): isolation, structure elucidation, and
biosynthesis. Eur. J. Org. Chem. 2000, 913–919. (b) Kunze, B.; Jansen,
R.; Sasse, F.; Ho¨fle, G.; Reichenbach, H. Apicularens A and B, new
cytostatic macrolides from Chondromyces species (Myxobacteria):
production, physicochemical and biological properties. J. Antibiot.
1998, 51, 1075–1080.

806 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Sugimoto et al.
(7) Dekker, K. A.; Aiello, R. J.; Hirai, H.; Inagaki, T.; Sakakibara, T.;
Suzuki, Y.; Thompson, J. F.; Yamauchi, Y.; Kojima, N. Novel lactone
compounds from Mortierella Verticillata that induce the human low
density lipoprotein receptor gene: fermentation, isolation, structural
elucidation and biological activities. J. Antibiot. 1998, 51, 14–20.
(8) Kim, J. W.; Shin-ya, K.; Furihata, K.; Hayakawa, Y.; Seto, H.
Oximidines I and II: Novel antitumor macrolides from Pseudomonas
sp. J. Org. Chem. 1999, 64, 153–155.
(9) Boyd, M. R.; Farina, C.; Belfiore, P.; Gagliardi, S.; Kim, J. W.;
Hayakawa, Y.; Beutler, J. A.; McKee, T. C.; Bowman, B. J.; Bowman,
E. J. Discovery of a novel antitumor benzolactone enamide class that
selectively inhibits mammalian vacuolar-type (H+)-ATPases. J. Phar-
macol. Exp. Ther. 2001, 297, 114–120.
(10) Sun-Wada, G.-H.; Wada, Y.; Futai, M. Diverse and essential roles of
mammalian vacuolar-type proton pump ATPase: toward the physi-
ological understanding of inside acidic compartments. Biochim.
Biophys. Acta 2004, 1658, 106–114.
(11) Xie, X.-S.; Padron, D.; Liao, X.; Wang, J.; Roth, M. G.; De Brabander,
J. K. Salicylihalamide A inhibits the V0 sector of the V-ATPase
through a mechanism distinct from bafilomycin A1. J. Biol. Chem.
2004, 279, 19755–19763.
assignment of the salicylate antitumor macrolide lobatamide C. J. Am.
Chem. Soc. 2002, 124, 5650–5651. (c) Shen, R.; Lin, C. T.; Bowman,
E. J.; Bowman, B. J.; Porco, J. A., Jr. Synthesis and V-ATPase
inhibition of simplified lobatamide analogues. Org. Lett. 2002, 4, 3103–
3106.
(18) Apicularene: (a) Su, Q.; Panek, J. S. Total synthesis of (-)-apicularen.
J. Am. Chem. Soc. 2004, 126, 2425–2430. (b) Nicolaou, K. C.; Kim,
D. W.; Baati, R.; O’Brate, A.; Giannakakou, P. Total synthesis and
biological evaluation of (-)apicularen A and analogues thereof.
Chem.sEur. J. 2003, 9, 6177–6191. (c) Nicolaou, K. C.; Kim, D. W.;
Baati, R. Stereocontrolled total synthesis of apicularen A and its ∆17,18
Z isomer. Angew. Chem., Int. Ed. 2002, 41, 3701–3704. (d) Bhatta-
charjee, A.; Seguil, O. R.; De Brabander, J. K. Total synthesis and
biological evaluation of apicularen A and synthetic analogs. Tetra-
hedron Lett. 2001, 42, 1217–1220.
(19) Oximidine: (a) Wang, X.; Bowman, E. J.; Bowman, B. J.; Porco, J. A.,
Jr. Total synthesis of the salicylate enamide macrolide oximidine III:
application of relay ring-closing metathesis. Angew. Chem., Int. Ed.
2004, 43, 3601–3605. (b) Wang, X.; Porco, J. A., Jr. Total synthesis
of the salicylate enamide macrolide oximidine II. J. Am. Chem. Soc.
2003, 125, 6040–6041.
(12) Bowman, E. J.; Bowman, B. J. V-ATPases as drug targets. J. Bioenerg.
Biomembr. 2005, 37, 431–435.
(20) Mitsunobu, O. The use of diethyl azodicarboxylate and triphenylphos-
phine in synthesis and transformation of natural products. Synthesis
1981, 1–28.
(13) (a) Wu, Y.; Esser, L.; De Brabander, J. K. Revision of the absolute
configuration of salicylihalamide A through asymmetric total synthesis.
Angew. Chem., Int. Ed. 2000, 39, 4308–4310. (b) Wu, Y.; Seguil,
O. R.; De Brabander, J. K. Synthesis and initial structure-activity
relationships of modified salicylihalamides. Org. Lett. 2000, 2, 4241–
4244. (c) Smith, A. B., III; Zheng, J. A first generation total synthesis
of (+)-salicylihalamide A. Synlett 2001, 1019–1023. (d) Labrecque,
D.; Charron, S.; Rej, R.; Blais, C.; Lamothe, S. Enantioselective total
synthesis of salicylihalamides A and B. Tetrahedron Lett. 2001, 42,
2645–2648. (e) Snider, B. B.; Song, F. Total Synthesis of (-)-
Salicylihalamide A. Org. Lett. 2001, 3, 1817–1820. (f) Fu¨rstner, A.;
Dierkes, T.; Thiel, O. R.; Blanda, G. Total synthesis of (-)-
salicylihalamide. Chem. Eur. J. 2001, 7, 5286–5298. (g) Wu, Y.; Liao,
X.; Wang, R.; Xie, X.-S.; De Brabander, J. K. Total synthesis and
initial structure-function analysis of the potent V-ATPase inhibitors
salicylihalamide A and related compounds. J. Am. Chem. Soc. 2002,
124, 3245–3253. (h) Smith, A. B., III; Zheng, J. Total synthesis of
(-)-salicylihalamide A and related congeners. Tetrahedron 2002, 58,
6455–6471. (i) Yang, K. L.; Haack, T.; Blackman, B.; Diederich,
W. E.; Roy, S.; Pusuluri, S.; Georg, G. I. Enantiospecific formal total
syntheses of (-)-salicylihalamides A and B from D-glucose and
L-rhamnose. Org. Lett. 2003, 5, 4007–4009. (j) Yadav, J. S.; Srihari,
P. Total synthesis of (-)-salicylihalamides A and B. Tetrahedron:
Asymmetry 2004, 15, 81–89. (k) Lebreton, S.; Xie, X.-S.; Ferguson,
D.; De Brabander, J. K. Ring-closing metathesis: a powerful tool for
the synthesis of simplified salicylihalamide-based V-ATPase inhibitors.
Tetrahedron 2004, 60, 9635–9647. (l) Herb, C.; Bayer, A.; Maier,
M. E. Total synthesis of salicylihalamides A and B. Chem. Eur. J.
2004, 10, 5649–5660.
(14) (a) Isanbor, C.; O’Hagan, D. Fluorine in medicinal chemistry: A review
of anti-cancer agents. J. Fluorine Chem. 2006, 127, 303–319. (b)
Be´gue´, J.-P.; Bonnet-Delpon, D. Recent advances (1995-2005) in
fluorinated pharmaceuticals based on natural products. J. Fluorine
Chem. 2006, 127, 992–1012. (c) Shah, P.; Westwell, A. D. The role
of fluorine in medicinal chemistry. J. Enzyme Inhib. Med. Chem. 2007,
22, 527–540.
(15) (a) Watts, A. Solid-state NMR in drug design and discovery for
membrane-embedded targets. Nat. ReV. Drug DiscoVery 2005, 4, 555–
568. (b) Ulrich, A. S. Solid state 19F NMR methods for studying
biomembranes. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46, 1–21.
(c) Yu, J.-X.; Kodibagkar, V. D.; Cui, W.; Mason, R. P. 19F: a versatile
reporter for non-invasive physiology and pharmacology using magnetic
resonance. Current Med. Chem. 2005, 12, 819–848. (d) Dalvit, C.
Ligand- and substrate-based 19F NMR screening: principles and
applications to drug discovery. Prog. Nucl. Magn. Reson. Spectrosc.
2007, 51, 243–271.
(21) Hadfield, A.; Schweitzer, H.; Trova, M. P.; Green, K. Practical, large-
scale synthesis of 2,2-dimethyl-5-hydroxy-4-oxobenzo-1,3-dioxin.
Synth. Commun. 1994, 24, 1025–1028.
(22) Umemoto, T.; Tomizawa, G. Highly selective fluorinating agents: A
counteranion-bound N-fluoropyridinium salt system. J. Org. Chem.
1995, 60, 6563–6570.
(23) Fu¨rstner, A.; Konetzki, I. Synthesis of 2-hydroxy-6-([(16R)-ꢀ-D-
mannopyranosyloxy]heptadecyl) benzoic acid, a fungal metabolite with
GABAA ion channel receptor inhibiting properties. Tetrahedron 1996,
52, 15071–15078.
(24) Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. A series of well-
defined metathesis catalysts-synthesis of [RuCI2(dCHR′)(PR3)2] and its
reactions. Angew. Chem., Int. Ed. 1995, 34, 2039–2041.
(25) Takai, K.; Nitta, K.; Utimoto, K. Simple and selective method for
RCHO f (E)-RCHdCHX conversion by means of a CHX3-CrCl2
system. J. Am. Chem. Soc. 1986, 108, 7408–7410.
(26) Evans, D. A.; Black, W. C. Total synthesis of (+)-A83543A [(+)-
lepicidin A]. J. Am. Chem. Soc. 1993, 115, 4497–4513.
(27) (a) Shen, R.; Porco, J. A., Jr. Synthesis of enamides related to the
salicylate antitumor macrolides using copper-mediated vinylic substitu-
tion. Org. Lett. 2000, 2, 1333–1336. (b) Coleman, R. S.; Liu, P.-H.
Divergent and stereocontrolled synthesis of the enamides side chains
of oximidines I/II/III, salicylihalamides A/B, lobatamides A/D, and
CJ-12,950. Org. Lett. 2004, 6, 577–580.
(28) (a) Zhang, S.; Zhang, D.; Liebeskind, L. S. Ambient temperature,
Ullmann-like reductive coupling of aryl, heteroaryl, and alkenyl
halides. J. Org. Chem. 1997, 62, 2312–2313. (b) Allred, G. D.;
Liebeskind, L. S. Copper-mediated cross-coupling of organostannanes
with organic iodides at or below room temperature. J. Am. Chem. Soc.
1996, 118, 2748–2749.
(29) Matsushita, M.; Tanaka, S.; Nakamura, N.; Inoue, H.; Kanazawa, H.
A novel kinesin-like protein, KIF1Bb3 is involved in the movement
of lysosomes to the cell periphery in non-neuronal cells. Traffic 2004,
5, 140–151.
(30) Lyubimov, A. V.; Carr, S. N.; Brown, A. P.; Art, J. J.; Crowell, J. A.;
Levine, B. S. Evaluation of hydrogen ion concentrations in prostates
from rats and dogs using fluorescent confocal microscopy. J. Photo-
chem. Photobiol., B 2005, 80, 225–234.
(31) Nelson, N.; Cidon, S.; Moriyama, Y. Methods in Enzymology Fleischer,
S. Fleischer, B., Eds.; Academic Press: New York, 1988; Vol. 157,
pp 619-633.
(32) (a) Galkin, M. A.; Ishmukhametov, R. R.; Vik, S. B. A functionally
inactive, cold-stabilized form of the Escherichia coli F1F0 ATP
synthase. Biochim. Biophys. Acta 2006, 1757, 206–214. (b) McCarty,
R. E. The decay of the ATPase activity of light plus thiol-activated
thylakoid membranes in the dark. J. Bioenerg. Biomembr. 2006, 38,
67–74. (c) Camarasa, C.; Prieto, S.; Ros, R.; Salmon, J.-M.; Barre, P.
Evidence for a selective and electroneutral K+/H+-exchange in
Saccharomyces cereVisiae using plasma membrane vesicles. Yeast
1996, 12, 1301–1313.
(16) Lal, G. S.; Pez, G. P.; Syvret, R. G. Electrophilic NF fluorinating
agents. Chem. ReV. 1996, 96, 1737–1755.
(17) Lobatamide: (a) Shen, R.; Lin, C. T.; Bowman, E. J.; Bowman, B. J.;
Porco, J. A., Jr. Lobatamide C: total synthesis, stereochemical
assignment, preparation of simplified analogues, and V-ATPase
inhibition studies. J. Am. Chem. Soc. 2003, 125, 7889–7901. (b) Shen,
R.; Lin, C. T.; Porco, J. A., Jr. Total synthesis and stereochemical
JM801265E