Design and synthesis of 24-fluorinated bafilomycin analogue as an NMR probe with potent inhibitory activity to vacuolar-type ATPase

Article abstract of DOI:10.1246/cl.131099

A fluorine-labeled bafilomycin analogue was designed and convergently synthesized from three segments via the Stille coupling, macrolactonization, and diastereoselective aldol reaction. The V-ATPase inhibitory activity of the analogue was comparable to that of the natural product, indicating its utility as a potential molecular probe for investigating the inhibition mechanism of bafilomycin by NMR.

Full text of DOI:10.1246/cl.131099

CL-131099
Received: November 25, 2013 | Accepted: December 16, 2013 | Web Released: December 20, 2013
Design and Synthesis of 24-Fluorinated Bafilomycin Analogue as an NMR Probe
with Potent Inhibitory Activity to Vacuolar-type ATPase
Hajime Shibata,1,2 Hiroshi Tsuchikawa,1 Nobuaki Matsumori,1 Michio Murata,*1,2 and Takeo Usui3
1Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043
2ERATO, Lipid Active Structure Project, Japan Science and Technology Agency,
1-1 Machikaneyama, Toyonaka, Osaka 560-0043
3Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572
(E-mail: murata@chem.sci.osaka-u.ac.jp)
A fluorine-labeled bafilomycin analogue was designed
and convergently synthesized from three segments via the Stille
coupling, macrolactonization, and diastereoselective aldol
reaction. The V-ATPase inhibitory activity of the analogue was
comparable to that of the natural product, indicating its utility
as a potential molecular probe for investigating the inhibition
mechanism of bafilomycin by NMR.
such a complicated membrane-bound system. We aimed to solve
this problem using solid-state NMR techniques, especially
REDOR5 known as a powerful tool for interatomic distance
measurements. For solid-state NMR measurements, labeled
compounds with NMR-sensitive nucleus are essential; among
several elements and isotopes, 19F is often regarded as the most
appropriate nucleus, despite its higher perturbations in biology,
because of its characteristic properties such as a nuclear spin of
1/2, high gyromagnetic ratio, and low background signals in
biological samples.6 We have demonstrated that REDOR
provides important evidence for elucidating the structure of a
channel complex of 19F-labeled amphotericin B in lipid
bilayers.7 In this study we designed and synthesized a novel
19F-labeled Baf analogue that has potent inhibitory activity
toward V-ATPase and could be used for investigating the
molecular interactions between Baf and the protein.
As a target molecule, 24,24-didesmethyl-24-F3-Baf (24-F-
Baf, 2), where the isopropyl unit of 1 was replaced by a
trifluoromethyl group, was designed in consideration of the
following points. (i) To minimize perturbations on bioactivity
brought by fluorine substitution, the tetrahydropyran (THP)
portion was selected based upon previous structure-activity
relationship studies.8 (ii) Fluorine substitution for hydroxy
groups in the THP ring was avoided because the destabilization
of products was expected. (iii) A CF3 group could be easily
introduced and equivalently replace the i-Pr group in terms of
the bulkiness.9
Vacuolar-type ATPase (V-ATPase) is a ubiquitous proton
pump that occurs in the endomembrane systems of all eukaryotic
cells and in the plasma membranes of diverse animal cells.1 This
protein family has various functions including the regulation of
intracellular or intraorganellar pH and the facilitation of trans-
port processes across the membrane. In recent years, it has
become more evident that the malfunction of V-ATPase is
correlated with an increasing number of age-related diseases
such as osteoporosis, renal tubular acidosis, and cancer. There-
fore, the inhibitors of V-ATPase play a pivotal role in under-
standing the molecular pathology of these diseases and in
developing corresponding molecular-targeting drugs. Since
bafilomycin A1 (Baf, 1) (Figure 1) was first isolated in 1983
from Streptomyces griseus ssp. sulphurus,2 the antibiotic has
long been regarded as a representative V-ATPase inhibitor.3 The
potent and specific activity of Baf has accelerated mechanism-
of-action studies, resulting in the elucidation of its putative
binding site to be the transmembrane Vo domain of V-ATPase.4
However, the precise location of the binding site remains
unclear. The largest problem lies in the structural analysis of
The efficient synthesis of 2 was planned by using the
diastereoselective aldol reaction of a novel CF3-labeled C18-
C24 segment 3 and the known C1-C17 macrocyclic core,10b
which could be constructed via the Stille coupling and macro-
lactonization from C1-C11 segment 4 and C12-C17 segment 5
by following previous synthetic studies of 1.10
Macrolactonization
MeO
24
R
1
O
HO
Synthesis of 3 commenced with PMB protection of the
known Wienreb amide 6 prepared from 1,3-propanediol in 4
steps,11 followed by DIBAL reduction to afford 7 (Scheme 1).
Next, diastereoselective trifluoromethylation was attempted by
the exposure of the aldehyde 7 with TMSCF3 in the presence of
TBAF, which resulted in formation of undesired 23R-epimer 8
as a major product. Since the chelation-controlled conditions
using the β-hydroxy group of 7 was not successful, we attempted
to invert the C23 configuration of 8. After several trials,12 the
Dess-Martin oxidation of 8 followed by treatment with L-
Selectride was found to give the desired epimer 9 preferentially
in 4.2:1 ratio.13 After removal of the PMB group, the resulting
diol was protected as a cyclic silyl ether to furnish 10.
Hydrogenolysis of the benzyl group followed by the Dess-
Martin oxidation afforded an aldehyde, which was then treated
O
OH
17
O
11
18
HO
12
OH
OMe
Stille coupling
Aldol reaction
1: R= i-Pr (bafilomycin A1, Baf)
2: R= CF3 (24,24-didesmethyl-24-F3-Baf, 24-F-Baf)
OMe
t-Bu
t-Bu
1
Si
OH
O
O
O
MeO2C
OH
24
17
18
F3C
11
SnBu3
MMTrO
I
12
OMe
C18-C24 segment 3
C12-C17 segment 5
C1-C11 segment 4
Figure 1. Structure of bafilomycin and its fluorinated ana-
logue.
474 | Chem. Lett. 2014, 43, 474–476 | doi:10.1246/cl.131099
© 2014 The Chemical Society of Japan

with the Grignard reagent and the produced alcohol was
oxidized to give 3 in 80% yield for 4 steps.
Next, preparation of the C1-C11 segment 4 was carried out
via carbon elongations from the known hydroxyketone 1114 as
shown in Scheme 2. Enantiopure alcohol 11 was silylated with
TBSOTf and 2,6-lutidine, and the reduction of 9-ketone
followed by one-pot treatment with K2CO3 furnished the diol.
Then oxidative cleavage and NaBH4 reduction resulted in the
formation of 12. Then, the primary alcohol 12 was converted to
alkyne 1315 via tosylation, addition of lithium acetylide and
removal of the PMB group. Then the resultant alcohol 13 was
converted to 4, chiefly following a report by Marshall;10f,16 after
three carbon elongation of the allylic alcohol moiety in 3 steps,
the formation of the vinyl iodide was performed under Negishi’s
conditions17 to produce 14. The TEMPO oxidation of the allylic
alcohol to aldehyde and the subsequent Horner-Wadsworth-
Emmons reaction proceeded in a Z-selective manner, which was
followed by removal of the TBS group to afford the C1-C11
segment 4 successfully.
O
OH
O
OPMB
a,b
c
MeO
N
OBn
OBn
6
7
OH OPMB
OH OPMB
f,g
F3C 23
OBn
F3C 23
OBn
Next, the coupling reaction between 4 and separately
prepared C12-C17 segment 518 and construction of the macro-
cyclic structure were performed following the previous proto-
col.10 The Stille coupling of segments 4 and 5 under the
conditions reported by Marshall et al.10f efficiently gave the E,E-
diene product in 80% yield. Then, saponification of methyl
ester10b afforded the corresponding seco acid and the following
macrolactonization was examined under Yamaguchi condi-
tions10e,19 to furnish the desired macrolactone 15 in moderate
yield.
Since macrolactone 15 was successfully obtained, connec-
tion with 3 by diastereoselective aldol reaction established by
Evans et al.10a was next attempted. First, the secondary alcohol
of 15 was protected as a diethylisopropylsilyl ether, which was
necessary for the following reactions.10b The deprotection of
the primary alcohol with PPTS followed by the Swern oxidation
provided 16, which was treated with 3 under the reported
conditions.10a,10b As a result, the diastereoselective aldol reaction
proceeded smoothly to afford the desired β-hydroxyketone 17 in
8
9: 23S-isomer
: 23R-isomer
d,e
t-Bu
Si
t-Bu
O
t-Bu
t-Bu
Si
O
O
O
O
h-k
F3C
F3C
OBn
10
C18-C24 segment 3
Scheme 1. Synthesis of the C18-C24 segment 3. Reagents and
conditions: a) PMBOCNHCCl3, Sc(OTf)3, toluene, rt, 70%; b)
DIBAL, CH2Cl2, ¹78 °C, 80%; c) TMSCF3, TBAF, THF, rt,
49% for 8, 27% for 9; d) Dess-Martin periodinane, NaHCO3,
CH2Cl2, rt, 92%; e) L-Selectride, THF, ¹78 °C, 68% for 9, 16%
for 8; f) DDQ, CH2Cl2, H2O, rt, then AcOH, THF, 40 °C, 79%;
g) (t-Bu)2Si(OTf)2, 2,6-lutidine, DMF, rt, 89%; h) Pd/C, H2,
EtOAc, rt, 98%; i) Dess-Martin periodinane, NaHCO3, CH2Cl2,
rt, 94%; j) EtMgBr, THF, rt, 93%; k) Dess-Martin periodinane,
NaHCO3, CH2Cl2, rt, 90%.
OMe
OH
HO
OTBS
PMBO
OTBS
OH
HO
OTBS
PMBO
OH
MeO2C
7
1
a-d
e-g
h-k
l-n
O
11
11
9
I
I
OBz
11
12
13
14
C1-C11 segment 4
OMe
OMe
OH
O
ODEIPS
O
OH
17
o-q
SnBu3
r-t
+
MMTrO
4
O
O
12
O
OMe
C12-C17 segment 5
MMTrO
16
OMe
15
OMe
O
OMe
OMe
MeO
O
t-Bu
O
t-Bu
O
7
ODEIPS
CF3
OH
Si
t-Bu
t-Bu
O
Si
u
v,w
O
O
O
OH
O
O
OH O
+
16
F3C
18
F3C
17
HO
OH
OMe
C18-C24 segment 3
2
24-F-Baf ( )
17
Scheme 2. Synthesis of fluorinated bafilomycin derivative 2. Reagents and conditions: a) TBSOTf, 2,6-lutidine, ¹78 °C; b) NaBH4,
THF, MeOH, rt, then K2CO3, rt, 79% (2 steps); c) NaIO4, CH2Cl2, pH 7.0 buffer, rt; d) NaBH4, THF, MeOH, rt, 91% (2 steps); e) TsCl,
Py, rt, 99%; f) LiCCH¢H2NCH2CH2NH2, DMSO, rt, 64%; g) DDQ, CH2Cl2, pH 7.0 buffer, 94%; h) (COCl)2, DMSO, Et3N, CH2Cl2,
¹78 °C; i) Ph3P=C(Me)CO2Et, toluene, 100 °C; j) DIBAL, CH2Cl2, ¹78 °C, 83% (3 steps); k) Cp2ZrCl2, AlMe3, ClCH2CH2Cl, 60 °C
then I2, THF, ¹30 °C, 66%; l) TEMPO, TBACl, NCS, CH2Cl2, pH 8.6 buffer, rt, 88%; m) (i-PrO)2P(O)CH(OMe)CO2Me, KHMDS,
18-crown-6 ether, THF, rt, 49%; n) TBAF, THF, rt, 84%; o) [Pd2(dba)3]¢CHCl3, Ph3As, LiCl, NMP, rt, 85%; p) KOH, dioxane, 80 °C;
q) 2,4,6-trichlorobenzoyl chloride, i-Pr2NEt, toluene, rt, then diluted with toluene, DMAP, rt, 51% (2 steps); r) DEIPSOTf, 2,6-lutidine,
CH2Cl2, 0 °C, 85%; s) PPTS, THF, MeOH, rt, 78%; t) (COCl)2, DMSO, Et3N, CH2Cl2, ¹78 °C; u) PhBCl2, i-Pr2NEt, CH2Cl2, ¹78 °C,
32% (2 steps); v) 18% HF¢Py, THF, rt; w) TsOH¢H2O, CH2Cl2, MeCN, H2O, rt, then HPLC purification, 36% (2 steps).
Chem. Lett. 2014, 43, 474–476 | doi:10.1246/cl.131099
© 2014 The Chemical Society of Japan | 475

32% yield over 2 steps with 20:1 diastereomer ratio.20 This
result clearly revealed that the CF3 moiety had no adverse effect
on the high stereoselectivity reported in the syntheses of 1.10a,10b
Finally, stepwise removal of silyl groups was achieved using
HF¢Py, followed by TsOH¢H2O, to afford 24-F-Baf (2) in 36%
yield after HPLC purification.
Biochemistry 2008, 47, 13463.
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The V-ATPase inhibitory activity of 24-F-Baf (2) was
evaluated by two methods (See Supporting Information for
details).21,22 First, the effect on the acidification of intracellular
acidic organelles by V-ATPase in rat 3Y1 fibroblasts was
examined. Treatment with 100 nM 2 caused the complete
disappearance of red fluorescence in acridine orange staining,
indicating that 2 strongly inhibited V-ATPase in the cells. Next,
to evaluate the inhibition of V-ATPase directly and quantita-
tively, we tested the effect of 2 on V-ATPases obtained from the
purified vacuole membrane of budding yeast by measuring the
liberated inorganic phosphate from ATP. The V-ATPase activity
was inhibited by 2 in a dose-dependent manner and its IC50
value (2.5 nM) was comparable with that of natural bafilomycin
1 (2.3 nM). These results demonstrated that our rational design
led to the preparation of the first fluorine-labeled analogue of
bafilomycin with potent ATPase inhibitory activity.
10 a) D. A. Evans, M. A. Calter, Tetrahedron Lett. 1993, 34,
6871. b) K. Toshima, T. Jyojima, H. Yamaguchi, Y. Noguchi,
T. Yoshida, H. Murase, M. Nakata, S. Matsumura, J. Org.
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In summary, the fluorine-labeled Baf analogue (24-F-Baf, 2)
was properly designed and convergently synthesized from three
key segments. 24-F-Baf (2) was shown to possess potent
V-ATPase inhibition activity comparable with that of 1 and
expected to be a potential molecular probe for elucidating the
inhibition mechanism of bafilomycin using solid-state NMR and
other spectroscopic techniques.
12 Inversion of the secondary alcohol of 8 by Mitsunobu
conditions did not proceed due to the large steric hindrance.
1,3-anti-Selective reductions of 18 derived from 8 led to the
low diastereoselectivity or the epimerization at the C22
position bacause of the undesired chelation of a CF3 group to
the reagent and/or its high electron-withdrawing property.
O
OH
OH OH
22
F3C 23
(A) Me4NBH(OAc)3
22
23
F3C
OBn
OBn
(B) SmI2, PhCHO
then K2CO3
We thank Dr. N. Inazumi (Osaka University) for his help in
NMR measurements. This work was supported in part by Grants
for Excellent Graduate Schools, MEXT, Japan. H.S. expresses
his special thanks for the supports from Global COE Programs
of Osaka University.
18
19
13 The C23 configuration of 9 was determined by NOE
experiments of its benzylideneacetal derivative. See
Supporting Information for details.
14 I. Paterson, S. B. Blakey, C. J. Cowden, Tetrahedron Lett.
2002, 43, 6005.
References and Notes
15 Another preparation of the alcohol 13 was reported. See
refs 10c and 10f.
16 Roush and co-workers have also reported the synthesis of
C1-C11 segment 4 from alkyne 13 via allylic alcohol 14.
See ref 10c.
17 E.-i. Negishi, D. E. Van Horn, T. Yoshida, J. Am. Chem. Soc.
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20 The stereochemistry of the major product 17 was determined
by analogy with the known derivatives in refs 8b and 10b,
See Supporting Information for details.
21 S. Kazami, M. Muroi, M. Kawatani, T. Kubota, T. Usui, J.
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22 Supporting Information is available electronically on the
CSJ-Journal Web site, http://www.csj.jp/journals/chem-lett/
index.html.
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476 | Chem. Lett. 2014, 43, 474–476 | doi:10.1246/cl.131099
© 2014 The Chemical Society of Japan