RE12 derivatives displaying Vaccinia H1-related phosphatase (VHR) inhibition in the presence of detergent and their anti-proliferative activity against HeLa cells

Article abstract of DOI:10.1016/j.bmc.2014.03.012

New derivatives of Vaccinia H1-related phosphatase (VHR) inhibitor RE12 (5) were designed by replacing the long straight alkyl chain with other hydrophobic functionalities containing two aromatic rings, with the aim of obtaining potent, cell-active inhibitors. We established a direct coupling reaction between tetronic acid derivative and thioimidate to prepare the RE derivatives 6a-6i efficiently. These compounds all showed VHR-inhibitory activity in the presence of 0.001% NP-40, whereas RE12 (5) was inactive under this condition, even at 100 μM. Further structure-activity studies focused on terminal substitution afforded trifluoromethyl derivative 6k (RE176) and nitro derivative 6l (RE177). The IC₅₀ value of 6l in the presence of NP-40 was almost equivalent to that of RE12 (5) in its absence. Compound 6k (RE176) potently inhibited proliferation of HeLa cells.

Full text of DOI:10.1016/j.bmc.2014.03.012

Accepted Manuscript
RE12 derivatives displaying Vaccinia H1-related phosphatase (VHR) inhibition
in the presence of detergent and their anti-proliferative activity against HeLa
cells
Frédéric Thuaud, Shuntaro Kojima, Go Hirai, Kana Oonuma, Ayako Tsuchiya,
Takako Uchida, Teruhisa Tsuchimoto, Mikiko Sodeoka
PII:
S0968-0896(14)00185-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.03.012
BMC 11459
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2014
7 March 2014
8 March 2014
Please cite this article as: Thuaud, F., Kojima, S., Hirai, G., Oonuma, K., Tsuchiya, A., Uchida, T., Tsuchimoto, T.,
Sodeoka, M., RE12 derivatives displaying Vaccinia H1-related phosphatase (VHR) inhibition in the presence of
detergent and their anti-proliferative activity against HeLa cells, Bioorganic & Medicinal Chemistry (2014), doi:
http://dx.doi.org/10.1016/j.bmc.2014.03.012
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Graphical Abstract
RE12 derivatives displaying Vaccinia H1-related
phosphatase (VHR) inhibition in the presence of
detergent and their anti-proliferative activity
against HeLa cells
Leave this area blank for abstract info.
Frédéric Thuauda, Shuntaro Kojimaa,b,c, Go Hiraia,d, Kana Oonumaa,d, Ayako Tsuchiyaa, Takako Uchidaa,e,
Teruhisa Tsuchimotob, and Mikiko Sodeokaa,c,d,e,f∗
aRIKEN, bMeiji Univ., cTokyo Medical and Dental Univ., dRIKEN CSRS, eSaitama Univ., fERATO-JST
O
X
O
O
OTBDPS
then TASF
NH
4
HO
O
O
NH
14
SMe
N
O
O
X
OH
O
4
O
OH
O
RE176: X = CF3 RE177: X = NO2
RE12

Bioorganic & Medicinal Chemistry
jo u rn al h ome p ag e : w ww .e lse vie r .c om
RE12 derivatives displaying Vaccinia H1-related phosphatase (VHR) inhibition in the
presence of detergent and their anti-proliferative activity against HeLa cells
Frédéric Thuauda, Shuntaro Kojimaa,b,c, Go Hiraia,d, Kana Oonumaa,d, Ayako Tsuchiyaa, Takako Uchidaa,e,
Teruhisa Tsuchimotob, and Mikiko Sodeokaa,c,d,e,f∗
aSynthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bDepartment of Applied Chemistry, School of Science and Technology, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
cTokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
dRIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
eGraduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
fERATO-JST, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
New derivatives of Vaccinia H1-related phosphatase (VHR) inhibitor RE12 (5) were designed
by replacing the long straight alkyl chain with other hydrophobic functionalities containing two
aromatic rings, with the aim of obtaining potent, cell-active inhibitors. We established a direct
coupling reaction between tetronic acid derivative and thioimidate to prepare the RE derivatives
6a~6i efficiently. These compounds all showed VHR-inhibitory activity in the presence of
0.001% NP-40, whereas RE12 (5) was inactive under this condition, even at 100 µM. Further
structure-activity studies focused on terminal substitution afforded trifluoromethyl derivative 6k
(RE176) and nitro derivative 6l (RE177). The IC50 value of 6l in the presence of NP-40 was
almost equivalent to that of RE12 (5) in its absence. Compound 6k (RE176) potently inhibited
proliferation of HeLa cells.
Received in revised form
Accepted
Available online
Keywords:
Protein Phosphatase
Inhibitor
Enamine
Thioimidate
Detergent
2009 Elsevier Ltd. All rights reserved.
1. Introduction
of VHR protein is not induced in response to activation of MAP
kinases, unlike many MKPs, it was reported that the VHR protein
Protein phosphorylation is one of the most important post-
translational modifications and has a key role in regulating cell
growth and intracellular signaling pathways.1 The activities of
protein kinases and protein phosphatases (PPases) dynamically
control the balance of phosphorylated and non-phosphorylated
proteins in cells. Consequently, protein kinase inhibitors have
been developed as therapeutic drug candidates.2 On the other
hand, PPases have been classified into protein serine/threonine
phosphatases3 (PPs) and protein tyrosine phosphatases (PTPs) on
the basis of structure and substrate specificity.4 Dual-specificity
protein phosphatases (DSPs), which possess a similar active-site
sequence and similar dephosphorylation mechanism to PTPs, can
hydrolyze phosphate ester on both a tyrosine residue and either a
serine or threonine residue located in the same protein.5 Vaccinia
H1-related phosphatase, VHR, is a member of the DSP family,
and dephosphorylates mitogen-activated protein kinases (MAP
kinases), extracellular signal-regulated kinases ERK1/2 and c-Jun
level changes during cell cycle progression. In cells lacking VHR,
cell cycle progression is blocked at the G1/S and G2/M
transitions.7 Moreover, protein levels of VHR were found to be
upregulated in several cervical cancer cell lines, including human
papillomavirus (HPV)-positive cell lines CaSki, HeLa, and SiHa,
as well as HPV-negative cell lines HT3 and C33.8 These findings
suggested that small-molecular inhibitors of VHR would be not
only novel drug candidates for cancer therapy, but also valuable
tools to investigate the roles of VHR in cervical cancer cells and
in cell cycle progression. However, creation of selective
inhibitors of VHR is a demanding task because of the structural
similarity of the active site residues among DSPs. Moreover, the
active site of VHR has a shallower pocket compared to that of
PTPs,9 and this contributes to the difficulty.
Nevertheless, several VHR inhibitors have been found by
screening of natural products or chemical libraries.10 Among
them, SA1 (1), which was reported by Tautz and co-workers, is
one of the most potent, having an IC50 value of 18 nM (Figure
1).11 Tautz et al. also reported that SA1 (1) and related molecule
N-terminal kinases JNK1/2.6 Dephosphorylation of these MAP
kinases by VHR, MAP kinase phosphatases (MKPs), or other
atypical DSPs results in their inactivation. Although expression
———
∗ Corresponding author. fax: +81-48-462-4666; e-mail: sodeoka@riken.jp

X
O
in the presence of detergent and at the cell level might be
improved by replacement of the long alkyl chain with other
hydrophobic substituents. Here we report the development of
advanced derivatives of the previously reported VHR inhibitors.
O
SO3H
N
N
(A) Hydrophobic Region 1
(B)
S
N
S
Tyr23
Leu25
SA1 (1): X = p-Cl
SA3 (2): X = o-F
F
Hydrophobic
Region 1
O
Tyr128
RE
Derivative
X
SA3 (2)
N
N
O
O
O
O
NH
O
Met69
N
S
R
O
R
O
SO3H
S
Hydrophobic
Region 2
OH
HO
RK-682 (3)
R = -(CH2)14CH3
OH
RE1 (4): X = H
RE12 (5): X = m-Me
Phe68
Hydrophobic Region 2
R = -(CH2)14CH3
(C)
m
o
p
o
Figure 1. Structures of VHR inhibitors (1~5).
Cl
m
o
p
o
Cl
SA3 (2) inhibited proliferation of cervical cancer cells such as
HeLa and CaSki. However, much higher concentrations of 1 or 2
compared to their IC50 values for VHR-inhibitory activity in vitro
were required (approximately 50% inhibition of proliferation of
HeLa cells by 20 µM 2). Development of 2 led to the first
crystallographic analysis of VHR protein bound with a small-
molecular inhibitor. Compounds 1 and 2 possess a sulfonic acid
moiety that interacts in a substrate (phosphate)-mimicking maner
with the active site of VHR. Despite this role of sulfonic acid, its
ionic nature at physiological pH may be unfavorable from the
viewpoint of cell permeability, resulting the poor activity at the
cell level. Other VHR inhibitors have similar problems. RK-68212
(3) and its derivatives,13 which possess a highly acidic 3-
acyltetronic acid structure, showed potent inhibitory activity
towards several PTPs and DSPs. However, again 3 was less
effective at the cell level.14 Thus, development of neutral
derivatives is expected to be a fruitful approach developing cell-
active VHR inhibitors.
O
m
p
O
C4
m
Direct
coupling
p
OH
N
O
+
H
O
O
N
Me
O
SMe
O
OTBDPS
Me
OH
New RE Derivatives
Thioimidate (7a~7i)
8
(6a~6i)
Figure 2. Design of new RE derivatives. (A) Molecular modeling of a
short alkyl chain analogue of RE1 (4) without the benzyl group on nitrogen
(ball-and-stick model) bound with VHR; (B) Schematic representation of the
reported binding model of 2 with VHR; (C) Structure and synthetic plan of
the new RE derivatives 6a~6i.
2. Results and Discussion
2.1. Design and Synthetic Plan of New RE derivatives
Our previous investigations revealed that transformation of the
3-acyltetronic acid core structure of RK-682 (3) to the polarized
but neutral enamine core structure (RE derivative) improved the
cell permeability with retention of the VHR-inhibitory activity.15
Namely, enamine derivatives RE1 (4) and RE12 (5) inhibited
VHR activity in vitro and arrested cell cycle progression of
NIH3T3 cells at the G1 phase. In addition, 4 and 5 inhibited
dephosphorylation of ERKs and JNK1 in serum-stimulated
NIH3T3 cells. In contrast, the parent compound 3 did not affect
either cell cycle progression or dephosphorylation of MAP
kinases. Moreover, 5 is highly selective for VHR, and it did not
inhibit other PTPs and DSPs, such as CDC25s and several MKPs.
However, there is still a room for improvement of the inhibitory
activity at the cell level, because the cell cycle-arresting activity
and inhibition of dephosphorylation of MAP kinases by 5 (50
µM) were only moderate. It is also noteworthy that the inhibitory
activity in in vitro enzyme assay was reduced in the presence of
detergent.16 Although the molecular mechanisms of the reduced
potency in the presence of detergent as well as in cells are not
clear,17-20 we speculate that the long hydrophobic alkyl chain in 4
or 5 might be a contributory factor. The long alkyl chain could
contribute to poor solubility in aqueous media and non-specific
hydrophobic interactions with unrelated proteins in cells.
However, simple replacement of the long alkyl of 4 with a
shorter one caused a drastic decrease of the VHR-inhibitory
potency, suggesting the importance of hydrophobic interaction
with VHR. Therefore, we envisioned that the inhibitory activity
We previously reported a binding model of RE derivatives
with VHR,15 which was constructed using Discovery Studio 1.1
and 1.2 (Accelrys, Figure 2A). The model with the Z-isomer of
the RE derivative suggested that the long hydrophobic alkyl
chain interacted with hydrophobic amino acid residues such as
Tyr 23 and Leu 25 (hydrophobic region 1). We expected that it
would be possible to replace a portion of the straight alkyl chain
of 5 with less lipophilic aromatic substituents while retaining
high affinity for VHR. The reported binding model constructed
based on the crystal structure of VHR-2 complex suggested that
the o-fluorobenzyl phenyl ether moiety of 2 could interact with
hydrophobic region 1 (Figure 2B). Since 1 was reported to show
more potent inhibition than 2, we decided to incorporate a
cholorobenzyl phenyl ether moiety at the end of the alkyl chain.
Another point to be considered was the length of the alkyl chain
serving as a linker between the enamine core structure and the
chlorobenzyl phenyl ether moiety. Eventually, we designed
compounds 6 with a C4 linker (Figure 2C), which appeared to be
suitable as judged from the binding model (Figure 2A). The
substitution pattern of the benzyl ether and chloride on the
terminal aromatic ring are expected to influence the interaction
with hydrophobic amino acids. Thus we envisioned the synthesis
and evaluation of all possible structural isomers of 6 (6a~6i). The
ClogP value of the new RE derivatives 6 (6.65) indicated a
moderate lipophilicity, lower than that of the parent compound 5
(ClogP of 5: 8.65). We anticipated that the replacement of the

Scheme 1. Synthesis of new RE derivatives. Reagents and conditions: a) H2SO4, MeOH, reflux (ortho: 2.5 h, 97%, meta: 1 h, 96%, para: 3.5 h, 89%); b)
DIBAL-H, THF, -78 °C (ortho: 3.5 h, 89%, meta: 1 h, quant., para: 2.5 h, quant.); c) (COCl)2, DMSO, CH2Cl2, -78 °C, 30 min, then Et3N, rt (ortho: 2.5 h, 95%,
meta: 20 min, 90%, para: 20 min 95%); d) MeOCOCH=PPh3, toluene, reflux (ortho: 10 min, 86%, meta: 20 min, 86%, para: 20 min, 95%); e) H2 (balloon),
Pd/C, EtOH, rt (ortho: 9 h, 98%, meta: 14 h, quant., para: 10 h, 99%); f) HBr, CH3COOH, reflux (ortho: 26 h, 66%, meta: 44 h, 76%, para: 3 h, 50%); g) m-
methylbenzylamine, EDC·HCl, DMAP, DMF, rt (for o-10: 7.5 h, 99%, for m-10: 11 h, 85%, for p-10: 8 h, quant.); h) o-chlorobenzyl bromide, m-chlorobenzyl
bromide, p-chlorobenzyl bromide, benzyl bromide, p-trifluoromethylbenzyl bromide, or p-nitrobenzyl bromide, NaH, THF:DMF (5:1), rt (11a: 10 min, 87%,
11b: 1.2 h, 75%, 11c: 5 min, 84%, 11d: 42 min, 44%, 11e: 1 h, 41%, 11f: 43 min, 52%, 11g: 15 min, 74%, 11h: 10 min, 84%, 11i: 15 min, 75%, 11j: 1 h, 74%,
11k: 10 min, 94%, 11l: 3 min, 85%); i) Lawesson’s reagent, THF, reflux (for 11a: 50 min, 93%, for 11b: 30 min, 91%, for 11c: 30 min, 87%, for 11d: 15 min,
quant., for 11e: 15 min, 87%, for 11f: 12 min, 91%, for 11g: 20 min, 93%, for 11h: 15 min, 98%, for 11i: 2 min, 87%, for 11j: 15 min, 96%, for 11k: 22 min,
97%, for 11l: 10 min, 99%); j) MeI, K2CO3, acetone, rt (7a: 15 h, 87%, 7b: 9 h, 78%, 7c: 12 h, 72%, 7d: 5 h, 84%, 7e: 7.5 h, 98%, 7f: 6.5 h, quant., 7g: 7 h, 88%,
7h: 6 h, 65%, 7i: 6 h, 77%, 7j: 19 h, 64%, 7k: 19 h, 82%, 7l: 22 h, 91%); k) 8, thioimidate 7 (1.4 equiv.), MS4A, CH2Cl2, rt, (12a: 30 h, 65%, 12b: 30 h, 53%,
12c: 30 h, 53%, 12d: 24 h, 87%, 12e: 48 h, 50%, 12f: 32 h, 67%, 12g: 30 h, 66%, 12h: 30 h, 73%, 12i: 31 h, 80%, 12j: 20 h, 59%, 12k: 20 h, 61%, 12l: 20 h,
55%); l) [(Me2N)3S]+[F2SiMe3]- (TASF), DMF, 0 °C (6a: 3.5 h, 61%, 6b: 3.5 h, 58%, 6c: 3.5 h, 62%, 6d: 5 h, 67%, 6e: 5 h, 61%, 6f: 5 h, 72%, 6g: 29 h, 70%, 6h:
4.5 h, 64%, 6i: 4.5 h, 70%, 6j: 6 h, 78%, 6k: 6 h, 72%, 6l: 6 h, 82%).
substituent would maintain the hydrophobic interaction with the
VHR surface and improve the inhibitory activity in the presence
of detergent.
m-methylbenzylamine was performed with EDC·HCl and DMAP
to give the corresponding amide with a free phenol moiety. Then,
alkylation with o-, m-, or p-chlorobenzyl bromide provided the 9
structural isomers of amides 11a~11i. Treatment of 11a~11i with
Lawesson’s reagent gave the corresponding thioamides, which
were converted into methyl thioimidates 7a~7i as mixtures of
geometric isomers.
To synthesize 6a~6i efficiently, we planned to establish a new
synthetic route to RE derivatives with a variety of side chains. It
was reported that enamines conjugated with two carbonyl groups
could be constructed by mixing thioimidates with 1,3-dicarbonyl
compounds such as 1,3-cyclohexanedione derivatives or
Meldrum’s acid under basic conditions.21 Therefore we
envisioned that direct coupling reaction of thioimidates (7a~7i)
with the tetronic acid derivative 8 would provide the RE
derivatives with various side chains.
With the precursors of RE derivatives in hand, coupling
reactions of methyl thioimidates with tetronic acid derivative 8
protected with a t-butyldiphenylsilyl group (TBDPS)15 were
investigated. Several model studies indicated that the desired
enamine derivatives would be formed in moderate yields simply
by mixing tetronic acid and an almost equal amount of
thioimidate. Addition of silver nitrate or a catalytic amount of 4-
dimethylaminopyridine slightly increased the yield of enamine
derivative but 4 equivalents of thioimidate was necessary,
because decomposition of thioimidate occurred as a competitive
reaction. Instead, addition of molecular sieves 4A (MS4A)
slightly improved the chemical yields without the need for an
excess of thioimidate. Therefore we decided to use this condition.
All the synthesized thioimidates 7a~7i could be converted into
the corresponding enamine derivatives 12a~12i by treatment with
tetronic acid 8 in the presence of MS4A (800 wt%) in CH2Cl2 at
2.2. Synthesis of RE Derivatives (6a~6i)
Synthesis of the new RE derivatives was commenced from
commercially available o-, m-, or p-methoxyhydrocinnamic acids
(o-9, m-9, and p-9, scheme 1). Conversion to carboxylic acid (o-
10, m-10, and p-10) of methoxyhydrocinnamic acids 9 was
achieved via
a
conventional 6-step sequence: methyl
esterification, reduction to alcohol, oxidation to aldehyde, two-
carbon elongation by Wittig reaction, hydrogenation of double
bond, and de-methylation of methyl ester and methyl ether.
Condensation of the carboxylic acids (o-10, m-10, and p-10) with

room temperature. Finally, removal of the TBDPS group was
performed with [(Me2N)3S]+[F2SiMe3]- (TASF) to give the newly
designed RE derivatives 6a~6i.
synthetic procedure, and compounds 6k and 6l were synthesized
without difficulty (Scheme 1). Evaluation of their inhibitory
activity revealed that 6k and 6l were more potent VHR-inhibitors
than chlorine derivative 6c or non-substituted 6j (Table 1). In
paticular, polar nitro derivative 6l showed a comparable IC50
value even in the presence of NP-40 to that of RE12 (5) in the
absence of NP-40. These results strongly supported our
hypothesis that the terminal substituent contributes to the
interaction with VHR. Although the inhibitory potencies of 6k
and 6l were decreased at a higher concentration of NP-40
(0.01%), their activities at the cell level was expected to be
increased due to the improvement of their properties in solution.
2.3. Inhibitory Activity of RE Derivatives 5 and 6a~6i for VHR in
the presence of NP-40
Inhibitory activities of the synthesized compounds 6a~6i
towards VHR phosphatases were evaluated in vitro.15 As
described above, the inhibitory activity of RE derivatives towards
DSPs was decreased in the presence of NP-40. In fact, RE12 (5)
was inactive as a VHR inhibitor in the presence of 0.001% NP-40,
even at 100 µM (Table 1). In contrast, all the new compounds
showed VHR-inhibitory activity under the same condition, as
expected. These results strongly indicated that substitution of the
long hydrophobic methylene chain in 5 by aromatic groups was a
successful strategy to overcome the drawback of the original
RE12 (5). All structural isomers 6a~6i showed moderate
inhibition of VHR, but 6g having para-substitution of the internal
aromatic ring and ortho-substitution of the terminal aromatic ring
was less potent than the other derivatives. Among the synthesized
compounds 6a~6i, 6c showed the greatest inhibitory activity,
although its potency was still only moderate.
Table 1. Inhibition of VHR activity in the presence of NP-40
(0.001%) and anti-proliferative activity towards HeLa cells of
RE derivatives (6a~6l).
Inhibition of VHR
activity: IC50 values
(SD) [µM]
Anti-proliferative
activity for HeLa cells
IC50 Values (SD) [µM]
Compounds
Figure 3. Anti-proliferative activity of RE derivatives (6a~6l) at 20 µM
(black) and 30 µM (gray) against HeLa cells.
5a
5
1.6 (0.2)
>100
NDb
NDb
6a
6b
6c
6d
6e
6f
6g
6h
6i
18.8 (4.2)
16.1 (1.3)
14.0 (0.5)
17.2 (1.2)
18.3 (0.8)
14.7 (1.0)
28.8 (4.0)
18.6 (1.3)
19.1 (0.5)
8.2 (1.5)
7.7 (2.0)
4.6 (0.9)
2.5. Inhibitory activity for the proliferation on HeLa cells
NDb
Since VHR is overexpressed in cervical cancer cells, VHR
inhibitors might serve as anti-proliferative agents for these cell
lines. Indeed, Tautz and co-workers reported that VHR inhibitors
SA1 (1) and SA3 (2) inhibited the proliferation of HeLa and
CaSki cells. Therefore, we evaluated the inhibitory effect of all
the synthesized RE derivatives 5 and 6a~6l on proliferation of
HeLa cells. Unexpectedly, the original 5 and chloro derivatives
6a~6i did not show significant inhibition at 20 µM though they
showed moderate inhibition at 30 µM. The most potent VHR
inhibitor 6l showed weakest inhibition of HeLa cell proliferation
at 30 µM. In contrast, non-substituted 6j and trifluoromethyl
derivative 6k showed marked inhibition with IC50 values of 10.6
and 7.8 µM, respectively. These results indicated that
replacement of the long hydrophobic alkyl chain in 5 with an
aromatic substituent improved the inhibitory activity at the cell
level. Compound 6k (RE176) appears to be a good candidate as a
next-generation VHR inhibitor effective at the cell level.
NDb
NDb
NDb
NDb
NDb
NDb
NDb
6j
6k
6l
10.6 (0.2)
7.8 (0.5)
NDb
aThe IC50 value was determined in the absence of NP-40 (reported in
reference 15); bND: not determined.
2.4. Effect of terminal substituents on VHR-inhibitory activity
We next investigated the effect of a terminal chlorine
substituent on the inhibitory activity of 6c. First we synthesized
6j without any functionality on the terminal aromatic ring, using
the procedure described above (Scheme 1). Interestingly, the
VHR-inhibitory activity of 6j was higher than that of 6a~6c with
the same substitution pattern in the internal aromatic ring (Table
1). This result suggested that chlorine atom on terminal aromatic
ring in 6a~6c decreased the inhibitory activity. Hence, we further
examined compounds 6k (ClogP = 6.8) with a trifluoromethyl
group and 6l (ClogP = 5.7) with a nitro group at the para-
position of the terminal aromatic ring. Since these functionalities
are larger than the chlorine atom and have different electronic
and hydrophobic properties, we expected to obtain information
concerning the effect of terminal substituents. The
trifluoromethyl and nitro groups were compatible with the same
3. Conclusions
We synthesized a focused library of RE derivatives bearing
enamine conjugated with two carbonyl groups as a core structure.
Among them, two potent VHR inhibitors 6j and 6k effective at
the cell level were found. Further structure-activity relationship
studies and investigation of the biological activity of compound
6k (RE176) at the cell level are under way.
4. Experimental
4.1. General
NMR spectra were recorded on a JEOL ECS400 or ECP500
spectrometer, or a Varian NMR System 500 spectrometer,
1
operating at 400 or 500 MHz for H-NMR and 100.4 or 125.8

MHz for 13C-NMR. Chemical shifts were reported in the scale
relative to CHCl3 as an internal reference. IR spectra were
measured on a Thermo Scientific Nicolet iS5 FT-IR with ZnSe
iD3 ATR. EI-MS were taken on a JEOL JMS-HX/HX110. ESI-
To a solution of 13a (103 mg, 346 µmol) in THF (1.44 mL)
and DMF (288 µL), o-chlorobenzyl bromide (67.4 µL, 519 µmol)
and sodium hydride (NaH: 20.8 mg, 519 µmol) were added at 0
°C. The mixture was stirred for 10 min at room temperature, then
water and ethyl acetate were added at 0 °C. The organic layer
was separated and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were dried over anhydrous
magnesium sulfate and concentrated in vacuo. Further
purification was carried out by silica gel column chromatography
(eluent: hexane/ethyl acetate = 3) to give 11a (126.8 mg, 87%) as
a colorless oil. IR (neat): 3292, 3062, 3022, 2941, 2862, 1645,
MS were taken on
a
JEOL JMS-T100LC. Column
chromatography was performed with silica gel 60 (40-50 µm)
purchased from KANTO CHEMICAL CO., INC. In general,
reactions were carried out under a nitrogen atmosphere, unless
otherwise noted. Tetrahydrofuran (THF), diethyl ether,
dichloromethane (CH2Cl2), N,N-dimethylformamide (DMF),
toluene, and methanol were purified by a Glass Contour Solvent
Dispensing System.
1
1550, 1493, 1454, 1240, 1059, 1047, 1034 cm-1; H-NMR (400
MHz, CDCl3) δ 1.60-1.78 (m, 4H), 2.22 (t, J = 7.6 Hz, 2H), 2.33
(s, 3H), 2.73 (brt, J = 7.2 Hz, 2H), 4.37 (d, J = 5.6 Hz, 2H), 5.15
(s, 2H), 5.63 (br, 1H), 6.91 (m, 2H), 7.03-7.09 (m, 3H), 7.15-7.31
(m, 5H), 7.39 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.55 (brd, J = 7.6 Hz,
1H); 13C-NMR (100 MHz, CDCl3) δ 21.3, 25.5, 29.5, 29.9, 36.5,
43.4, 67.0, 111.6, 120.9, 124.7, 126.9, 127.0, 128.1, 128.4, 128.5
(2C), 128.8, 129.2, 130.0, 130.9, 132.3, 135.0, 138.2, 138.3,
156.1, 172.8; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNO2Na, 421.1809; found, 421.1807.
4.2. Synthesis of RE derivatives
4.2.1. Representative procedure for transformation of 10 to 13
To a solution of o-10 (915 mg, 4.71 mmol) in DMF (15.7
mL), 3-methylbenzylamine (701 µL, 5.65 mmol) and 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC:
1.20 g, 6.26 mmol) and N,N-dimethyl-4-aminopyridine (DMAP:
153 mg, 1.25 mmol) were added at 0 °C. The mixture was stirred
for 7.5 h at room temperature, then water and ethyl acetate were
added at 0 °C. The organic layer was separated and washed with
water and brine. The combined aqueous layers were extracted
with ethyl acetate. The combined organic layers were dried over
magnesium sulfate and concentrated in vacuo. Further
purification was carried out by silica gel column chromatography
(eluent: hexane/ethyl acetate = 1/1) to give o-13 (1.38 g, 99%) as
a pale yellow oil. IR (neat) 3296, 2943, 1643, 1608, 1593, 1535,
11b (103 mg, 75%): IR (neat) 3294, 3062, 2924, 2860, 1643,
1
1549, 1493, 1454, 1433, 1240, 1115, 1051, 1030, 872 cm-1; H-
NMR (400 MHz, CDCl3) δ 1.62-1.77 (m, 4H), 2.22 (t, J = 8.0 Hz,
2H), 2.32 (s, 3H), 2.70 (t, J = 6.8 Hz, 2H), 4.37 (d, J = 5.6 Hz,
2H), 5.02 (s, 2H), 5.60 (br, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.91
(brt, J = 7.2 Hz, 1H), 7.03-7.09 (m, 3H), 7.13-7.17 (m, 2H), 7.21
(t, J = 7.6 Hz, 1H), 7.26-7.31 (m, 3H), 7.41 (brs, 1H); 13C-NMR
(100 MHz, CDCl3) δ 21.3, 25.5, 29.5, 29.8, 36.5, 43.4, 68.9,
111.5, 120.9, 124.7, 125.0, 126.9 (3C), 127.8, 128.1, 128.5 (2C),
129.8, 130.0, 130.9, 134.3, 138.2, 139.4, 156.1, 172.8; HRMS-
ESI (m/z): [M+Na+] calcd for C26H28ClNO2Na, 421.1809; found,
421.1808.
1
1456, 1240 cm-1; H-NMR (400 MHz, CDCl3) δ 1.65-1.78 (m,
11c (129 mg, 84%): IR (neat) 3296, 3064, 3024, 2925, 2860,
1
1643, 1547, 1495, 1454, 1240, 1115, 1090, 1014, 810 cm-1; H-
4H), 2.30 (t, J = 6.8 Hz, 2H), 2.33 (s, 3H), 2.65 (brt, J = 7.2 Hz,
2H), 4.40 (d, J = 5.2 Hz, 2H), 5.74 (br, 1H), 5.98 (s, 1H), 6.78
(dd, J = 0.8 Hz, 7.6 Hz, 1H), 6.82 (td, J = 7.6 Hz, 0.8 Hz, 1H),
7.05-7.10 (m, 5H), 7.22 (brt, J = 7.6 Hz, 1H); 13C-NMR (100
MHz, CDCl3) δ 21.3, 25.4, 29.3, 29.5, 36.2, 43.6, 115.6, 120.0,
124.8, 127.0, 128.2, 128.4, 128.6 (2C), 130.0, 137.9, 138.4, 154.3,
173.7; HRMS-ESI (m/z): [M+Na+] calcd for C19H23NO2Na,
297.1729; found, 297.1728.
NMR (400 MHz, CDCl3) δ 1.62-1.75 (m, 4H), 2.20 (t, J = 7.2 Hz,
2H), 2.32 (s, 3H), 2.68 (t, J = 7.2 Hz, 2H), 4.37 (d, J = 5.2 Hz,
2H), 5.01 (s, 2H), 5.58 (br, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.90 (td,
J = 7.6 Hz, 1.2 Hz, 1H), 7.03-7.10 (m, 3H), 7.13-7.17 (m, 2H),
7.21 (t, J = 7.6 Hz, 1H), 7.34 (s, 4H); 13C-NMR (100 MHz,
CDCl3) δ 21.3, 25.5, 29.5, 29.8, 36.6, 43.5, 69.1, 111.6, 120.9,
124.8, 127.0, 128.2, 128.4 (2C), 128.6 (2C), 128.7 (2C), 130.0,
130.9, 133.5, 135.9, 138.2, 138.4, 156.2, Signal of a carbon
(C=O) was weak; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNO2Na, 421.1809; found, 421.1804.
m-13 (65.3 mg, 85%): IR (neat) 3282, 2931, 1645, 1587, 1533,
1
1455, 1275, 1240, 1157 cm-1; H-NMR (400 MHz, CDCl3) δ
1.58-1.76 (m, 4H), 2.22 (t, J = 6.8 Hz, 2H), 2.33 (s, 3H), 2.58 (t,
J = 7.2 Hz, 2H), 4.39 (d, J = 5.6 Hz, 2H), 5.61 (br, 1H), 5.69 (br,
1H), 6.64-6.68 (m, 2H), 6.71 (brd, J = 7.6 Hz, 1H), 7.04-7.14 (m,
4H), 7.22 (t, J = 7.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ
21.3, 25.2, 30.6, 35.3, 36.5, 43.7, 112.9, 115.4, 120.2, 124.8,
128.3, 128.6 (2C), 129.4, 137.8, 138.4, 143.7, 156.3, 173.4;
HRMS-ESI (m/z): [M+Na+] calcd for C19H23NO2Na, 297.1729;
found, 297.1729.
11d (36.3 mg, 44%, impure): IR (neat) 3298, 3062, 3032,
2929, 2860, 1645, 1608, 1583, 1549, 1489, 1444, 1259, 1157,
1
1036 cm-1; H-NMR (400 MHz, CDCl3) δ 1.61-1.79 (m, 4H),
2.22 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.62 (t, J = 7.2 Hz, 2H),
4.39 (d, J = 5.2 Hz, 2H), 5.15 (s, 2H), 5.62 (br, 1H), 6.78-6.81 (m,
3H), 7.05-7.09 (m, 3H), 7.17-7.23 (m, 2H), 7.25-7.31 (m, 2H),
7.38-7.41 (m, 1H), 7.55-7.57 (m, 1H); 13C-NMR (100 MHz,
CDCl3) δ 21.3, 25.3, 30.9, 35.6, 36.5, 43.5, 66.9, 111.8, 115.1,
121.3, 124.8, 126.9, 128.2, 128.5 (2C), 128.8, 128.9, 129.27,
129.30, 132.5, 134.8, 138.2, 138.3, 143.9, 158.5, 172.6; HRMS-
ESI (m/z): [M+Na+] calcd for C26H28ClNO2Na, 421.1809; found,
421.1807.
p-13 (905 mg, quant.): IR (neat) 3290,2925, 1641, 1612, 1541,
1514, 1446, 1230 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.57-1.73
(m, 4H), 2.30 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.54 (t, J = 7.6 Hz,
2H), 4.39 (d, J = 5.6 Hz, 2H), 5.69 (br, 1H), 5.74 (br, 1H), 6.74
(d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 7.04-7.10 (m, 3H),
7.22 (t, J = 7.2 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 21.2,
25.2, 31.1, 34.6, 36.4, 43.5, 115.2 (2C), 124.7, 128.1, 128.4,
128.5, 129.1 (2C), 133.2, 137.7, 138.2, 154.5, 173.7; HRMS-ESI
(m/z): [M+Na+] calcd for C19H23NO2Na, 297.1729; found,
297.1728.
11e (26.6 mg, 41%): IR (neat) 3294, 3057, 3026, 2925, 2860,
1645, 1583, 1549, 1487, 1448, 1431, 1257, 1157, 1032, 876 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.61-1.75 (m, 4H), 2.22 (t, J = 7.6
Hz, 2H), 2.33 (s, 3H), 2.61 (t, J = 7.2 Hz, 2H), 4.39 (d, J = 5.2
Hz, 2H), 5.01 (s, 2H), 5.62 (br, 1H), 6.76-6.80 (m, 3H), 7.01-7.10
(m, 3H), 7.17-7.23 (m, 2H), 7.30 (m, 3H), 7.44 (br, 1H); 13C-
NMR (100 MHz, CDCl3) δ 21.3, 25.3, 30.9, 35.6, 36.5, 43.5,
4.2.2. Representative procedure for transformation of 13 to 11

69.0, 111.8, 115.1, 131.3, 124.8, 125.3, 127.4, 128.0, 128.2,
128.6 (2C), 129.3, 129.8, 134.4, 138.2, 138.4, 139.2, 143.9, 158.5,
172.6; HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNO2Na,
421.1809; found, 421.1810.
J = 5.6 Hz, 2H), 5.12 (s, 2H), 5.62 (br, 1H), 6.85 (d, J = 8.0 Hz,
1H), 6.92 (t, J = 7.6 Hz, 1H), 7.03-7.09 (m, 3H), 7.13-7.17 (m,
2H), 7.21 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.64 (d, J
= 8.4 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 21.3, 25.5, 29.5,
29.9, 36.6, 43.5, 68.9, 111.5, 121.0, 124.1 (q, J = 277.4 Hz),
124.8, 125.5 (q, J = 3.9 Hz, 2C), 126.99 (2C), 127.02, 128.2,
128.6 (2C), 129.9 (q, J = 32.8 Hz), 130.1, 130.9, 138.2, 138.4,
141.5, 156.1, 172.8; 19F-NMR (376Hz, CDCl3) δ -62.4 HRMS-EI
(m/z): [M+] calcd for C27H28F3NO2, 455.2072; found, 455.2072.
11f (31.5 mg, 52%, impure): IR (neat) 3290, 3068, 3030, 2924,
2860, 1643, 1601, 1583, 1543, 1491, 1448, 1255, 1155, 1093,
1
1030, 1014, 810 cm-1; H-NMR (400 MHz, CDCl3) δ 1.58-1.78
(m, 4H), 2.22 (t, J = 6.8 Hz, 2H), 2.33 (s, 3H), 2.60 (t, J = 7.2 Hz,
2H), 4.39 (d, J = 5.6 Hz, 2H), 5.01 (s, 2H), 5.64 (br, 1H), 6.74-
6.82 (m, 3H), 7.03-7.09 (m, 3H), 7.16-7.23 (m, 2H), 7.32-7.40
(m, 4H); 13C-NMR (100 MHz, CDCl3) δ 21.3, 25.3, 30.9, 35.7,
36.6, 43.6, 69.1, 111.8, 115.1, 121.3, 124.8, 128.2, 128.6 (2C),
128.7 (2C), 128.8 (2C), 129.3, 133.6, 135.6, 138.2, 138.4, 143.9,
158.6, 172.6; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNO2Na, 421.1809; found, 421.1803.
11l (74.3 mg, 85%): IR (neat) 3303, 3072, 2924, 2860, 1645,
1
1601, 1520, 1493, 1452, 1346, 1240, 1111, 1053, 854 cm-1; H-
NMR (400 MHz, CDCl3) δ 1.64-1.78 (m, 4H), 2.23 (t, J = 7.6
Hz, 2H), 2.32 (s, 3H), 2.72 (t, J = 7.6 Hz, 2H), 4.38 (d, J = 5.6
Hz, 2H), 5.16 (s, 2H), 5.63 (br, 1H), 6.82 (d, J = 8.4 Hz, 1H),
6.93 (td, J = 7.6 Hz, 1.2 Hz, 1H), 7.03-7.09 (m, 3H), 7.13-7.22
(m, 3H), 7.59 (d, J = 8.8 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H); 13C-
NMR (100 MHz, CDCl3) δ 21.3, 25.5, 29.5, 29.9, 36.6, 43.5,
68.5, 111.4, 121.3, 123.8 (2C), 124.2, 124.8, 127.1, 127.3, 127.4
(2C), 128.2, 128.6 (2C), 130.2, 130.8, 138.2, 138.4, 144.8, 147.4,
155.8, Signal of a carbon (C=O) was weak; HRMS-EI (m/z):
[M+] calcd for C26H28N2O4, 432.2049; found, 432.2055.
11g (101 mg, 74%): IR (neat) 3292, 3057, 3032, 2931, 2858,
1
1639, 1610, 1547, 1512, 1442, 1242, 1036 cm-1; H-NMR (400
MHz, CDCl3) δ 1.57-1.74 (m, 4H), 2.22 (t, J = 7.6 Hz, 2H), 2.33
(s, 3H), 2.58 (t, J = 7.2 Hz, 3H), 4.39 (d, J = 5.6 Hz, 2H), 5.14 (s,
2H), 5.64 (br, 1H), 6.90 (d, J = 8.8 Hz, 2H), 7.05-7.09 (m, 5H),
7.22 (t, J = 7.2 Hz, 1H), 7.25-7.31 (m, 2H), 7.40 (dd, J = 1.6 Hz,
7.2 Hz, 1H), 7.56 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 21.3,
25.2, 31.2, 34.7, 36.4, 43.4, 67.1, 114.6 (2C), 124.7, 126.8, 128.1,
128.5 (2C), 128.7, 128.8, 129.2 (3C), 132.4, 134.76, 134.83,
138.2 (2C), 156.6, 172.6; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNO2Na, 421.1809; found, 421.1805.
4.2.3. Representative procedure for transformation of 11 to 14
11h (99.3 mg, 84%): IR (neat) 3284, 3066, 3032, 2927, 2858,
1
1641, 1543, 1510, 1460, 1431, 1236, 1028, 825 cm-1; H-NMR
(400 MHz, CDCl3) δ 1.58-1.72 (m, 4H), 2.20 (t, J = 7.6 Hz, 2H),
2.31 (s, 3H), 2.55 (t, J = 6.8 Hz, 2H), 4.37 (d, J = 5.6 Hz, 2H),
4.99 (s, 2H), 5.60 (br, 1H), 6.84 (d, J = 8.8 Hz, 2H), 7.02-7.09
(m, 5H), 7.20 (brt, J = 7.2 Hz, 1H), 7.28 (m, 3H), 7.42 (br, 1H);
13C-NMR (100 MHz, CDCl3) δ 21.3, 25.2, 31.2, 34.7, 36.4, 43.4,
69.1, 114.6 (2C), 124.7, 125.2, 127.3, 127.9, 128.1, 128.5 (2C),
129.3 (2C), 129.7, 134.3, 134.8, 138.2, 138.3, 139.2, 156.6,
172.6; HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNO2Na,
421.1809; found, 421.1808.
To a solution of 11a (118 mg, 280 µmol) in THF, Lawesson’s
reagent (226 mg, 559 µmol) was added. The mixture was
refluxed for 50 min and concentrated in vacuo. Further
purification was carried out by silica gel column chromatography
(eluent: hexane/ethyl acetate = 10/1) to give 14a as a pale yellow
oil. IR (neat) 3352, 3246, 3059, 3024, 2924, 2860, 1523, 1493,
11i (83.1 mg, 75%): IR (neat) 3290, 3037, 2937, 2920, 2852,
1
1635, 1545, 1512, 1379, 1238, 1176, 1016, 876, 810 cm-1; H-
NMR (400 MHz, CDCl3) δ 1.59-1.72 (m, 4H), 2.20 (t, J = 7.6 Hz,
2H), 2.32 (s, 3H), 2.56 (t, J = 6.8 Hz, 2H), 4.38 (d, J = 6.0 Hz,
2H), 4.98 (s, 2H), 5.61 (br, 1H), 6.84 (d, J = 8.8 Hz, 2H), 7.02-
7.09 (m, 5H), 7.20 (t, J = 7.2 Hz, 1H), 7.34 (s, 4H); 13C-NMR
(100 MHz, CDCl3) δ 21.3, 25.3, 31.2, 34.7, 36.5, 43.5, 69.2,
114.6 (2C), 124.8, 128.2, 128.5 (2C), 128.6 (2C), 128.7 (2C),
129.3 (2C), 133.6, 134.7, 135.7, 138.2, 138.3, 156.6, 172.6;
HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNO2Na, 421.1809;
found, 421.1805.
1
1452, 1402, 1238, 1107, 1057, 1034 cm-1; H-NMR (400 MHz,
CDCl3) δ 1.69 (m, 2H), 1.87 (m, 2H), 2.34 (s, 3H), 2.68 (t, J =
7.2 Hz, 2H), 2.72 (t, J = 7.2 Hz, 2H), 4.74 (d, J = 5.2 Hz, 2H),
5.12 (s, 2H), 6.87-6.92 (m, 2H), 7.07-7.18 (m, 5H), 7.23 (d, J =
7.2 Hz, 2H), 7.23 (br, 1H), 7.29 (td, J = 7.2 Hz, 1.6 Hz, 1H), 7.38
(dd, J = 1.6 Hz, 7.2 Hz, 1H), 7.53 (dd, J = 1.6 Hz, 7.2 Hz, 1H);
13C-NMR (100 MHz, CDCl3) δ 21.3, 29.0, 29.1, 29.8, 46.8, 50.2,
67.1, 111.7, 120.9, 125.2, 126.9, 127.0, 128.4, 128.7, 128.75,
128.80, 128.9, 129.2, 130.0, 130.8, 132.3, 134.9, 136.0, 138.6,
156.1, 205.3; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNOSNa, 437.1580; found, 437.1576.
11j (56.2 mg, 74%): IR (neat) 3282, 3062, 3035, 2937, 2862,
1
1645, 1545, 1493, 1452, 1240, 1115, 1026 cm-1; H-NMR (400
MHz, CDCl3) δ 1.62-1.75 (m, 4H), 2.19 (t, J = 7.6 Hz, 2H), 2.32
(s, 3H), 2.69 (t, J = 6.8 Hz, 2H), 4.35 (d, J = 5.6 Hz, 2H), 5.05
(s, 2H), 5.52 (br, 1H), 6.87-6.92 (m, 2H), 7.01-7.09 (m, 3H),
7.13-7.23 (m, 3H), 7.28-7.31 (m, 1H), 7.35-7.42 (m, 4H); 13C-
NMR (100 MHz, CDCl3) δ 21.3, 25.4, 29.5, 29.8, 36.6, 43.5,
69.8, 111.6, 120.7, 124.8, 127.0, 127.1 (2C), 127.7, 128.2, 128.50
(2C), 128.53 (2C), 130.0, 130.9, 137.4, 138.26, 138.34, 156.5,
Signal of a carbon (C=O) was weak; HRMS-EI (m/z): [M+] calcd
for C26H29NO2, 387.2198; found, 387.2195.
14b (96.3 mg, 91%, pale green oil): IR (neat) 3357, 3221,
3030, 2927, 2860, 1599, 1523, 1493, 1452, 1433, 1331, 1292,
1240, 1107, 1051, 960 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.67
(m, 2H), 1.86 (m, 2H), 2.34 (s, 3H), 2.68 (t, J = 8.3 Hz, 2H), 2.69
(t, J = 8.3 Hz, 2H), 4.74 (d, J = 5.2 Hz, 2H), 5.00 (s, 2H), 6.84 (d,
J = 7.4 Hz, 1H), 6.90 (t, J = 7.4 Hz, 1H), 7.07-7.17 (m, 5H), 7.22
(br, 1H), 7.23-7.32 (m, 4H), 7.39 (br, 1H); 13C-NMR (100 MHz,
CDCl3) δ 21.3, 29.1 (2C), 29.8, 46.8, 50.3, 69.0, 111.7, 121.0,
125.0, 125.3, 127.02, 127.05, 127.9, 128.77, 128.80, 129.0, 129.9,
130.1, 130.9, 134.3, 136.0, 138.6, 139.4, 156.1, 205.3; HRMS-
11k (80.8 mg, 94%): IR (neat) 3296, 3070, 3032, 2927, 2860,
1643, 1547, 1493, 1454, 1325, 1240, 1163, 1124, 1066, 1018,
823 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.63-1.77 (m, 4H), 2.22
(t, J = 7.2 Hz, 2H), 2.32 (s, 3H), 2.71 (t, J = 6.8 Hz, 2H), 4.38 (d,

ESI (m/z): [M+Na+] calcd for C26H28ClNOSNa, 437.1580; found,
437.1575.
J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 4.77 (d, J = 5.1 Hz,
2H), 5.00 (s, 2H), 6.86 (d, J = 8.3 Hz, 2H), 7.07 (d, J = 8.3 Hz,
2H), 7.08-7.16 (m, 5H), 7.20 (br, 1H), 7.25 (t J = 7.4 Hz, 1H),
7.30 (br, 3H), 7.43 (br, 1H); 13C-NMR (100 MHz, CDCl3) δ 21.3,
28.9, 30.7, 34.6, 46.8, 50.2, 69.1, 114.6 (2C), 125.2 (2C), 127.3,
127.9, 128.7, 128.8, 129.0, 129.2 (2C), 129.7, 134.3, 134.7, 135.9,
138.6, 139.2, 156.6, 205.1; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNOSNa, 437.1580; found, 437.1571.
14c (117 mg, 87%, pale brown oil): IR (neat) 3356, 3232,
3033, 2924, 2860, 1601, 1493, 1452, 1406, 1331, 1238, 1105,
1
1053, 1014, 810 cm-1; H-NMR (400 MHz, CDCl3) δ 1.65 (m,
2H), 1.84 (m, 2H), 2.34 (s, 3H), 2.64 (t, J = 8.3 Hz, 2H), 2.68 (t,
J = 8.3 Hz, 2H), 4.74 (d, J = 5.2 Hz, 2H), 4.99 (s, 2H), 6.84 (d, J
= 7.8 Hz, 1H), 6.90 (t, J = 7.8 Hz, 1H), 7.07-7.17 (m, 5H), 7.20
(br, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.33 (br, 4H); 13C-NMR (100
MHz, CDCl3) δ 21.3, 29.0, 29.1, 29.7, 46.8, 50.2, 69.1, 111.7,
120.9, 125.2, 127.0, 128.4 (2C), 128.6 (2C), 128.7, 128.8, 129.0,
130.0, 130.8, 133.5, 135.7, 136.0, 138.6, 156.1, 205.3; HRMS-
ESI (m/z): [M+Na+] calcd for C26H28ClNOSNa, 437.1580; found,
437.1575.
14i (71.1 mg, 87%, white amorphous solid): IR (neat) 3213,
3051, 2929, 2854, 1599, 1510, 1460, 1396, 1335, 1298, 1246,
1174, 1105, 1014, 962, 812 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.63 (m, 2H), 1.84 (m, 2H), 2.35 (s, 3H), 2.58 (t, J = 8.0 Hz, 2H),
2.67 (t, J = 8.0 Hz, 2H), 4.77 (d, J = 4.6 Hz, 2H), 4.99 (s, 2H),
6.85 (d, J = 8.3 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 7.08-7.16 (m,
3H), 7.21 (br, 1H), 7.25 (t J = 7.4 Hz, 1H), 7.35 (s, 4H); 13C-
NMR (100 MHz, CDCl3) δ 21.3, 28.9, 30.7, 34.6, 46.8, 50.3,
69.1, 114.6 (2C), 125.3, 128.6 (2C), 128.66 (2C), 128.76, 128.81
129.0, 129.2 (2C), 133.5, 134.6, 135.6, 135.9, 138.6, 156.6,
205.1; HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNOSNa,
437.1580; found, 437.1578.
14d (20.1 mg, quant., white oil): IR (neat) 3344, 3230, 3033,
2931, 2858, 1583, 1525, 1489, 1444, 1402, 1331, 1257, 1157,
1
1059, 1045, 1034 cm-1; H-NMR (400 MHz, CDCl3) δ 1.67 (m,
2H), 1.85 (m, 2H), 2.34 (s, 3H), 2.62 (t, J = 7.2 Hz, 2H), 2.67 (t,
J = 7.7 Hz, 2H), 4.77 (d, J = 5.3 Hz, 2H), 5.15 (s, 2H), 6.77-6.81
(m, 3H), 7.09-7.31 (m, 7H), 7.24 (br, 1H), 7.39 (m, 1H), 7.56 (m,
1H); 13C-NMR (100 MHz, CDCl3) δ 21.3, 29.0, 30.5, 35.6, 47.0,
50.4, 67.0, 111.9, 115.2, 121.3, 125.4, 126.9, 128.8, 128.87,
128.91 (2C), 129.1, 129.3, 129.4, 132.6, 134.8, 136.0, 138.8,
143.8, 158.6, 205.1; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNOSNa, 437.1580; found, 437.1578.
14j (54.9 mg, 96%): IR (neat) 3363, 3230, 3033, 2924, 2860,
1601, 1523, 1493, 1452, 1397, 1333, 1290, 1240, 1188, 1134,
1
1109, 1051, 1026 cm-1; H-NMR (400 MHz, CDCl3) δ 1.67 (m,
2H), 1.84 (m, 2H), 2.34 (s, 3H), 2.65 (dd, J = 7.8 Hz, 7.4 Hz,
2H), 2.70 (dd, J = 7.8 Hz, 7.4 Hz, 2H), 4.72 (d, J = 5.1 Hz, 2H),
5.02 (s, 2H), 6.86-6.91 (m, 2H), 7.05-7.19 (m, 4H), 7.17 (br, 1H),
7.22-7.31 (m, 2H), 7.34-7.41 (m, 4H); 13C-NMR (100 MHz,
CDCl3) δ 21.3, 29.0, 29.1, 29.7, 46.8, 50.3, 70.0, 111.8, 120.8,
125.3, 127.0, 127.1 (2C), 127.8, 128.5 (2C), 128.77, 128.79,
129.0, 130.0, 130.9, 136.0, 137.3, 138.6, 156.4, 205.4; HRMS-EI
(m/z): [M+] calcd for C26H29NOS,403.1970; found, 403.1968.
14e (23.1 mg, 87%, white oil): IR (neat) 3348, 3236, 3033,
2925, 2860, 1583, 1525, 1489, 1448, 1433, 1404, 1331, 1259,
1157, 1095, 1078, 1043, 877 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.66 (m, 2H), 1.84 (m, 2H), 2.34 (s, 3H), 2.61 (t, J = 7.2 Hz, 2H),
2.67 (t, J = 7.2 Hz, 2H), 4.77 (d, J = 5.1 Hz, 2H), 5.01 (s, 2H),
6.74-6.79 (m, 3H), 7.08-7.31 (m, 8H), 7.24 (br, 1H), 7.43 (m,
1H); 13C-NMR (100 MHz, CDCl3) δ 21.3, 28.9, 30.4, 35.6, 46.9,
50.4, 69.0, 111.9, 115.1, 121.3, 125.4 (2C), 127.4, 128.0, 128.86,
128.91, 129.1, 129.4, 129.8, 134.4, 136.0, 138.8, 139.2, 143.8,
158.5, 205.1; HRMS-ESI (m/z): [M+Na+] calcd for
C26H28ClNOSNa, 437.1580; found, 437.1577.
14k (77.8 mg, 97%): IR (neat) 3240, 3028, 2931, 2862, 1601,
1523, 1493, 1454, 1404, 1325, 1242, 1165, 1124, 1066, 1018,
825 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.68 (m, 2H), 1.87 (m,
2H), 2.34 (s, 3H), 2.67 (dd, J = 7.8 Hz, 7.4 Hz, 2H), 2.72 (dd, J
= 7.8 Hz, 7.4 Hz, 2H), 4.74 (d, J = 5.1 Hz, 2H), 5.10 (s, 2H),
6.87 (dd, J = 8.7 Hz, 0.9 Hz, 1H), 6.91 (td, J = 7.4 Hz, 0.9 Hz,
1H), 7.06-7.26 (m, 6H), 7.21 (br, 1H), 7.52 (d, J = 7.8 Hz, 2H),
7.64 (d, J = 7.8 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 21.3,
29.08, 29.14, 29.8, 46.9, 50.3, 69.0, 111.6, 121.1, 124.0 (q, J =
272.6 Hz), 125.3, 125.5 (q, J = 3.9 Hz, 2C), 127.0 (2C), 127.1,
128.79, 128.84, 129.0, 129.9 (q, J = 32.7 Hz), 130.1, 130.8, 136.0,
138.7, 141.4, 156.0, 205.3; 19F-NMR (376Hz, CDCl3) δ -62.4;
HRMS-EI (m/z): [M+] calcd for C27H28F3NOS, 471.1844; found,
471.1836.
14f (11.5 mg, 91%, colorless oil): IR (neat) 3236, 3041, 2927,
2856, 1601, 1493, 1448, 1408, 1333, 1259, 1157, 1093, 1045,
1016, 960, 810 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.66 (m, 2H),
1.84 (m, 2H), 2.34 (s, 3H), 2.61 (t, J = 7.6 Hz, 2H), 2.67 (t, J =
7.6 Hz, 2H), 4.77 (d, J = 5.1 Hz, 2H), 5.01 (s, 2H), 6.75-6.79 (m,
3H), 7.08-7.27 (m, 5H), 7.24 (br, 1H), 7.35 (s, 4H); 13C-NMR
(100 MHz, CDCl3) δ 21.3, 28.9, 30.5, 35.6, 46.9, 50.4, 69.1,
111.9, 115.1, 121.3, 125.4, 128.7 (2C), 128.8 (2C), 128.87,
128.92, 129.1, 129.3, 133.7, 135.6, 136.0, 138.8, 143.8, 158.6,
205.1; HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNOSNa,
437.1580; found, 437.1571.
14l (74.2 mg, 99%): IR (neat) 3342, 3226, 3024, 2924, 2858,
1601, 1520, 1493, 1452, 1404, 1344, 1240, 1109, 1053, 839 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.67 (m, 2H), 1.86 (m, 2H), 2.32
(s, 3H), 2.66 (dd, J = 7.8 Hz, 7.4 Hz, 2H), 2.71 (dd, J = 7.8 Hz,
7.4 Hz, 2H), 4.73 (d, J = 4.6 Hz, 2H), 5.13 (s, 2H), 6.80 (d, J =
8.3 Hz, 1H), 6.91 (td, J = 7.8 Hz, 0.9 Hz, 1H), 7.04-7.16 (m, 5H),
7.21 (d, J = 7.8 Hz, 1H), 7.22 (br, 1H), 7.56 (d, J = 8.7 Hz, 2H),
8.23 (dt, J = 8.7 Hz, 2H), J = 1.6 Hz, 2H); 13C-NMR (100 MHz,
CDCl3) δ 21.3, 29.1, 29.2, 29.9, 46.9, 50.3, 68.6, 111.5, 121.3,
123.8 (2C), 125.3, 127.1, 127.3 (2C), 128.80, 128.85, 129.0,
130.2, 130.8, 136.0, 138.7, 144.7, 147.4, 155.8, 205.2; HRMS-EI
(m/z): [M+] calcd for C26H28N2O3S, 448.1821; found, 448.1822.
14g (93.6 mg, 93%, pale yellow amorphous solid): IR (neat)
3348, 3232, 3028, 2922, 2856, 1610, 1510, 1442, 1402, 1238,
1
1176, 1034 cm-1; H-NMR (400 MHz, CDCl3) δ 1.64 (m, 2H),
1.84 (m, 2H), 2.354 (s, 3H), 2.59 (t, J = 8.0 Hz, 2H), 2.68 (t, J =
8.0 Hz, 2H), 4.77 (d, J = 4.6 Hz, 2H), 5.13 (s, 2H), 6.89 (d, J =
8.7 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H), 7.11-7.16 (m, 3H), 7.22 (br,
1H), 7.23-7.31 (m, 3H), 7.40 (m, 1H), 7.56 (m, 1H); 13C-NMR
(100 MHz, CDCl3) δ 21.3, 28.9, 30.8, 34.7, 46.9, 50.3, 67.1,
114.7 (2C), 125.3, 126.9, 128.7, 128.78, 128.83 (2C), 129.0,
129.3 (3C), 132.4, 134.7, 134.8, 136.0, 138.7, 156.6, 205.1;
HRMS-ESI (m/z): [M+Na+] calcd for C26H28ClNOSNa,
437.1580; found, 437.1571.
4.2.4. Representative procedure for transformation of 14 to 7
To a solution of 14a (112 mg, 256 µmol) in dry acetone (1.23
mL), potassium carbonate (70.9 mg, 513 µmol) and methyl
iodide (48.0 µL, 769 µmol) were added. The mixture was stirred
for 13.5 h, and further methyl iodide (32.0 µL, 514 µmol) was
added. Stirring was continued for 1 h, and the reaction mixture
14h (98.2 mg, 98%, pale yellow amorphous solid): IR (neat)
3359, 3226, 3030, 2924, 2856, 1608, 1510, 1458, 1433, 1402,
1
1331, 1298, 1236, 1176, 1105, 1036, 958 cm-1; H-NMR (400
MHz, CDCl3) δ 1.63 (m, 2H), 1.84 (m, 2H), 2.35 (s, 3H), 2.58 (t,

was filtered through Celite. Further purification was carried out
by GPC (eluent: chloroform) to give 7a (100 mg, 87%, 1:1
isomeric mixture) as a pale green oil. IR (neat) 3062, 3026, 2926,
2860, 1624, 1604, 1493, 1452, 1238, 1126, 1090, 1057, 1047,
7e (18.4 mg, 98%, colorless oil): IR (neat) 3028, 2927, 2858,
1608, 1583, 1487, 1450, 1431, 1259, 1157, 1095, 1080, 1043,
877 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.59-1.78 (m, 4H), 2.34
(s, 3H), 2.35 (s, 1.5H), 2.45 (s, 1.5H), 2.47-2.49 (m, 1H), 2.58-
2.67 (m, 3H), 4.48 (s, 1H), 4.58 (s, 1H), 5.01 (s, 1H), 5.02 (s, 1H),
6.76-6.83 (m, 3H), 7.05-7.25 (m, 5H), 7.30 (m, 3H), 7.45 (brs,
1H); 13C-NMR (100 MHz, CDCl3) δ 12.7, 14.3, 21.4, 21.5, 26.8,
27.0, 30.7, 30.9, 34.1, 35.5, 35.7, 37.4, 54.5, 56.0, 69.0 (2C),
111.8, 111.9, 115.1 (2C), 121.4 (2C), 124.3, 124.8, 125.4 (2C),
127.2, 127.3, 127.4 (2C), 128.0 (2C), 128.19, 128.21, 128.57
(2C), 129.3, 129.4, 129.8 (2C), 134.4 (2C), 137.8, 137.9, 139.21,
139.24, 140.0, 140.5, 143.8, 144.1, 158.5 (2C), 165.0, 168.1;
HRMS-ESI (m/z): [M+H+] calcd for C27H31ClNOS, 452.1809;
found, 452.1806.
1
1034 cm-1; H-NMR (400 MHz, CDCl3) δ 1.63-1.82 (m, 4H),
2.34 (s, 3H), 2.35 (s, 1.5H), 2.44 (s, 1.5H), 2.47 (dd, J = 8.3 Hz,
6.9 Hz, 1H), 2.63 (brdd, J = 7.4 Hz, 5.5 Hz, 1H), 2.73 (brdd, J =
8.3 Hz, 6.9 Hz, 1H), 2.76 (brdd, J = 7.4 Hz, 6.9 Hz, 1H), 4.49 (s,
1H), 4.58 (s, 1H), 5.16 (s, 1H), 5.17 (s, 1H), 6.88-6.95 (m, 2H),
7.05 (brd, J = 7.4 Hz, 1H), 7.12-7.32 (m, 7H), 7.40 (m, 1H), 7.57
(m, 1H); 13C-NMR (100 MHz, CDCl3) δ 12.7, 14.3, 21.42, 21.45,
27.1, 27.5, 29.4, 29.6, 30.0, 30.1, 34.2, 37.4, 54.4, 55.7, 67.0 (2C),
111.60, 111.64, 120.9 (2C), 124.3, 124.9, 126.92 (2C), 126.99,
127.1, 127.2, 127.4, 128.0, 128.17, 128.21, 128.3, 128.4, 128.6,
128.74, 128.77, 129.25, 129.28, 129.94, 129.99, 130.7, 131.0,
132.4 (2C), 135.06, 135.10, 137.8, 137.9, 140.40 (2C), 156.2
(2C), Signals of carbons (SC=N) were weak; HRMS-ESI (m/z):
[M+H+] calcd for C27H31ClNOS, 452.1809; found, 452.1807.
7f (11.9 mg, quant., colorless oil): IR (neat) 3022, 2925, 2860,
1626, 1612, 1493, 1450, 1259, 1157, 1095, 1041, 1016, 810 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.59-1.75 (m, 4H), 2.33 (s, 3H),
2.34 (s, 1.5H), 2.45 (s, 1.5H), 2.44-2.49 (m, 1H), 2.57-2.65 (m,
3H), 4.48 (s, 1H), 4.58 (s, 1H), 5.00 (s, 1H), 5.01 (s, 1H), 6.75-
6.81 (m, 3H), 7.02-7.24 (m, 5H), 7.35 (s, 4H); 13C-NMR (100
MHz, CDCl3) δ 12.7, 14.3, 21.4, 21.5, 26.8, 27.1, 30.7, 30.9, 34.1,
35.5, 35.7, 37.5, 54.5, 56.0, 69.1 (2C), 111.8, 111.9, 115.1 (2C),
121.3 (2C), 124.3, 124.8, 127.25, 127.33, 128.0, 128.20, 128.22,
128.6, 128.7 (4C), 128.8 (4C), 129.3, 129.4, 133.6, 133.7, 135.6,
135.7, 137.85, 137.89, 140.0, 140.5, 143.8, 144.1, 158.57, 158.59,
165.0, 168.1; HRMS-ESI (m/z): [M+H+] calcd for C27H31ClNOS,
452.1809; found, 452.1811.
7b (76.8 mg, 78%, pale brown oil): IR (neat) 2924, 2860,
1624, 1601, 1493, 1454, 1431, 1290, 1240, 1095, 1051 cm-1; 1H-
NMR (400 MHz, CDCl3) δ 1.60-1.79 (m, 4H), 2.32 (s, 1.5H),
2.33 (s, 1.5H), 2.34 (s, 1.5H), 2.42 (s, 1.5H), 2.46 (dd, J = 8.3 Hz,
6.9 Hz, 1H), 2.59 (brdd, J = 8.3 Hz, 6.0 Hz, 1H), 2.70 (brdd, J =
8.3 Hz, 6.9 Hz, 1H), 2.71 (brdd, J = 8.3 Hz, 6.9 Hz, 1H), 4.46 (s,
1H), 4.56 (s, 1H), 5.03 (s, 1H), 5.04 (s, 1H), 6.85 (brd, J = 7.8 Hz,
1H), 6.91 (m, 1H), 7.04 (brd, J = 7.4 Hz, 1H), 7.10-7.22 (m, 5H),
7.29 (m, 3H), 7.42 (brs, 1H); 13C-NMR (100 MHz, CDCl3) δ 12.6,
14.2, 21.40, 21.43, 27.0, 27.4, 29.4, 29.6, 29.9, 30.1, 34.2, 37.5,
54.4, 55.9, 68.9 (2C), 111.48, 111.52, 120.9 (2C), 124.3, 124.8,
124.92, 124.96, 126.9, 127.0 (3C), 127.2, 127.3, 127.80, 127.82,
128.0, 128.12, 128.15, 128.5, 129.8 (2C), 129.97, 130.01, 130.7,
131.1, 134.35, 134.38, 137.7, 137.8, 139.47, 139.52, 140.0, 140.5,
156.2 (2C), 165.3, 168.3; HRMS-ESI (m/z): [M+H+] calcd for
C27H31ClNOS, 452.1809; found, 452.1808.
7g (8.3 mg, 88%): IR (neat) 3030, 2927, 2856, 1624, 1612,
1
1510, 1442, 1240, 1176, 1034, 825 cm-1; H-NMR (400 MHz,
CDCl3) δ 1.59-1.77 (m, 4H), 2.34 (s, 3H), 2.35 (s, 1.5H), 2.45 (s,
1.5H), 2.45-2.49 (m, 1H), 2.55-2.64 (m, 3H), 4.49 (s, 1H), 4.58 (s,
1H), 5.15 (s, 2H), 6.88-6.92 (m, 2H), 7.02-7.31 (m, 8H), 7.40
(brdd, J = 7.4 Hz, 1.4 Hz, 1H), 7.56 (brd, J = 6.8 Hz, 1H); 13C-
NMR (100 MHz, CDCl3) δ 12.7, 14.3, 21.41, 21.45, 26.7, 27.0,
30.9, 31.3, 34.1, 34.6, 34.7, 37.3, 54.4, 55.7, 67.1 (2C), 114.66,
114.70, 124.3, 124.9, 126.9 (4C), 127.2, 127.4, 128.0, 128.17,
128.22, 128.6, 128.7 (2C), 128.8 (2C), 129.3 (6C), 132.5 (2C),
134.6 (2C), 134.9 (2C), 137.8, 137.9, 139.6, 140.4, 156.6, 156.7,
164.8, 170.3; HRMS-ESI (m/z): [M+H+] calcd for C27H31ClNOS,
452.1809; found, 452.1809.
7c (85.4 mg, 72%, yellow oil): IR (neat) 2925, 2860, 1624,
1
1493, 1454, 1240, 1090, 1014, 810 cm-1; H-NMR (400 MHz,
CDCl3) δ 1.62-1.78 (m, 4H), 2.32 (s, 1.5H), 2.33 (s, 1.5), 2.34 (s,
1.5H), 2.41 (s, 1.5H), 2.44 (dd, J = 8.3 Hz, 6.4 Hz, 1H), 2.57
(brdd, J = 7.4 Hz, 6.4 Hz, 1H), 2.68 (brdd, J = 7.4 Hz, 6.9 Hz,
1H), 2.70 (brdd, J = 8.3 Hz, 6.9 Hz, 1H), 4.46 (s, 1H), 4.56 (s,
1H), 5.02 (s, 1H), 5.03 (s, 1H), 6.86 (brd, J = 8.3 Hz, 1H), 6.90
(m, 1H), 7.04 (m, 1H), 7.11-7.23 (m, 5H), 7.34 (s, 4H); 13C-NMR
(100 MHz, CDCl3) δ 12.6, 14.1, 21.38, 21.41, 27.0, 27.3, 29.4,
29.6, 29.87, 30.0, 34.2, 37.5, 54.4, 55.9, 68.92, 68.94, 111.43,
111.46, 120.8 (2C), 124.3, 124.8, 126.9, 127.0, 127.16, 127.24,
128.0, 128.1 (2C), 128.3 (2C), 128.4 (2C), 128.5, 128.59 (2C),
128.61 (2C), 129.89, 129.95, 130.6, 131.0, 133.37, 133.40, 135.8,
135.9, 137.7, 137.8, 140.0, 140.5, 156.16, 156.18, 165.1, 168.1;
HRMS-ESI (m/z): [M+H+] calcd for C27H31ClNOS, 452.1809;
found, 452.1808.
7h (64.6 mg, 65%, yellow oil): IR (neat) 3030, 2925, 2860,
1624, 1512, 1460, 1431, 1302, 1238, 1176, 1095, 1039, 827 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.59-1.77 (m, 4H), 2.34 (s, 3H),
2.35 (s, 1.5H), 2.45 (s, 1.5H), 2.45-2.48 (m, 1H), 2.54-2.63 (m,
3H), 4.48 (s, 1H), 4.58 (s, 1H), 5.01 (s, 2H), 6.85-6.89 (m, 2H),
7.04-7.19 (m, 5H), 7.20 (td, J = 7.4 Hz, 1.8 Hz, 1H), 7.30 (brs,
3H), 7.44 (brs, 1H); 13C-NMR (100 MHz, CDCl3) δ 12.7, 14.2,
21.40, 21.43, 26.7, 27.0, 30.9, 31.2, 34.1, 34.5, 34.7, 37.3, 54.4,
55.8, 69.1 (2C), 114.58, 114.62, 124.3, 124.8, 125.3 (2C), 127.2,
127.30 (2C), 127.34, 127.9 (2C), 128.0, 128.1, 128.2, 128.6,
129.3 (6C), 129.7 (2C), 134.4 (2C), 134.6, 134.9, 137.78, 137.83,
139.2, 139.3, 139.7, 140.4, 156.5, 156.6, 163.4, 168.3; HRMS-
ESI (m/z): [M+H+] calcd for C27H31ClNOS, 452.1809; found,
452.1809.
7d (5.9 mg, 84%, colorless oil): IR (neat) 3033, 2925, 2860,
1626, 1610, 1489, 1446, 1261, 1157, 1059, 1047, 1036, 877 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.59-1.79 (m, 4H), 2.34 (s, 3H),
2.35 (s, 1.5H), 2.45 (s, 1.5H), 2.47 (dd, J = 7.8 Hz, 7.4 Hz, 1H),
2.58-2.67 ( m, 3H), 4.49 (s, 1H), 4.59 (s, 1H), 5.16 (s, 1H), 5.16
(s, 1H), 6.78-6.85 (m, 3H), 7.02-7.32 (m, 6H), 7.40 (brdd, J = 7.4
Hz, 2.3 Hz, 1H), 7.57 (brdd, J = 7.4 Hz, 2.3 Hz, 1H); 13C-NMR
(100 MHz, CDCl3) δ 12.7, 14.3, 21.4, 21.5, 26.8, 27.1, 30.7, 30.9,
34.1, 35.5, 35.7, 37.5, 54.5, 56.0, 67.0 (2C), 111.8, 112.0, 115.10,
115.12, 121.3 (2C), 124.3, 124.8, 126.9 (2C), 127.2, 127.3, 128.0,
128.18, 128.21, 128.6, 128.78, 128.80, 128.9 (2C), 129.3 (3C),
129.4, 132.5 (2C), 134.8, 134.9, 137.8, 137.9, 140.0, 140.5, 143.8,
144.1, 158.6 (2C), 165.0, 168.1; HRMS-ESI (m/z): [M+H+] calcd
for C27H31ClNOS, 452.1809; found, 452.1809.
7i (56.0 mg, 77%, yellow oil): IR (neat) 3032, 2927, 2858,
1
1626, 1510, 1493, 1462, 1240, 1176, 1092, 1014, 812 cm-1; H-
NMR (400 MHz, CDCl3) δ 1.58-1.74 (m, 4H), 2.33 (s, 3H), 2.34
(s, 1.5H), 2.45 (s, 1.5H), 2.45-2.48 (m, 1H), 2.54-2.61 (m, 3H),
4.49 (s, 1H), 4.58 (s, 1H), 5.00 (s, 2H), 6.84-6.88 (m, 2H), 7.03-
7.19 (m, 5H), 7.21 (td, J = 7.8 Hz, 0.9 Hz, 1H), 7.35 (s, 4H); 13C-
NMR (100 MHz, CDCl3) δ 12.7, 14.3, 21.42, 21.45, 26.7, 27.0,
31.0, 31.3, 34.1, 34.5, 34.7, 37.4, 54.4, 55.8, 69.2 (2C), 114.61
(2C), 114.64 (2C), 124.3, 124.9, 127.2, 127.4, 128.0, 128.16,
128.20, 128.6, 128.66 (4C), 128.69 (4C), 129.3 (4C), 133.6 (2C),

134.6, 134.9, 135.67, 135.69, 137.8, 137.9, 139.7, 140.4, 156.6,
156.7, Signals of carbons (SC=N) were weak; [M+H+] calcd for
C27H31ClNOS, 452.1809; found, 452.1808.
0.58H); 13C-NMR (100 MHz, CDCl3) δ 19.2, 21.3, 21.4, 26.51,
26.53, 27.1, 27.4, 27.48, 27.51, 29.6, 29.7, 29.9, 46.6, 46.9, 62.3,
62.5, 67.0, 81.2, 83.3, 91.3, 92.7, 111.6, 121.0, 124.0, 126.9,
127.0, 127.2, 127.6, 127.7, 128.4, 128.8, 129.0, 129.1, 129.2,
129.3, 129.6, 129.7, 130.0, 130.39, 130.43, 132.3, 132.4, 132.7,
132.95, 133.01, 135.0, 135.1, 135.5, 135.6, 139.0, 156.1, 171.4,
173.4, 174.1, 176.3, 193.0, 197.8; HRMS-ESI (m/z): [M+Na+]
calcd for C47H50ClNNaO5Si, 794.3039; found, 794.3036.
7j (36.0 mg, 64%): IR (neat) 3066, 3035, 2925, 2860, 1626,
1493, 1452, 1381, 1344, 1289, 1240, 1112, 1090, 1045, 1026,
856 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.61-1.78 (m, 4H), 2.33
(s, 1.5H), 2.34 (s, 1.5H), 2.36 (s, 1.5H), 2.40 (m, 1.5H), 2.45
(brdd, J = 7.8 Hz, 7.4 Hz, 1H), 2.61-2.74 (m, 3H), 4.49 (s, 1H),
4.57 (s, 1H), 5.06 (s, 1H), 5.08 (s, 1H), 6.87-6.92 (m, 2H), 7.05
(brd, J = 7.4 Hz, 1H), 7.11-7.20 (m, 4H), 7.21 (td, J = 7.4 Hz,
1.8Hz, 1H), 7.28-7.45 (m, 5H); 13C-NMR (100 MHz, CDCl3) δ
12.9, 14.3, 21.41, 21.45, 27.1, 27.5, 29.3, 29.6, 29.9, 30.0, 34.3,
37.3, 54.3, 55.4, 69.8 (2C), 111.55, 111.61, 120.7 (2C), 124.4,
125.0, 126.9, 127.0 (4C), 127.1 (2C), 127.3, 127.5, 127.69,
127.72, 128.1, 128.2, 128.3, 128.49 (4C), 128.54, 128.7, 129.88,
129.92, 130.7, 130.9, 137.38, 137.44, 137.8, 138.0, 156.5 (2C),
Signals of carbons (SC=N) were weak; HRMS-EI (m/z): [M+]
calcd for C27H31NOS, 417.2126; found, 417.2107.
12b (9.9 mg, 53%, tautomeric mixture: 1.33:1 in CDCl3): IR
(neat) 3074, 2931, 2856, 1743, 1668, 1647, 1601, 1495, 1452,
1429, 1240, 1113, 1039 cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.94
(s, 5.1H), 0.95 (s, 3.9H), 1.57-1.87 (m, 4H), 2.30 (s, 1.7H), 2.32
(s, 1.3H), 2.67-2.74 (m, 2H), 2.91-3.12 (m, 2H), 3.98-4.11 (m,
2H), 4.48-4.56 (m, 3H), 5.02 (brs, 0.9H), 5.03 (brs, 1.1H), 6.83-
6.86 (m, 1H), 6.87-6.92 (m, 1H), 6.95-7.00 (m, 2H), 7.10-7.43
(m, 14H), 7.62-7.69 (m, 4H), 10.4 (brm, 0.43H), 11.3 (brm,
0.57H);13C-NMR (100 MHz, CDCl3) δ 19.2, 21.35, 21.38, 26.51,
26.54, 27.1, 27.4, 27.48, 27.52, 29.7, 29.8, 46.6, 46.9, 62.3, 62.5,
69.0, 81.3, 83.3, 91.3, 92.7, 111.6, 121.0, 124.0, 124.98, 125.02,
127.0, 127.1, 127.66, 127.69, 127.9, 129.06, 129.13, 129.6, 129.7,
129.8, 129.9, 130.1, 130.45, 130.50, 132.69, 132.74, 132.98,
133.03, 134.33, 134.35, 135.0, 135.1, 135.55, 135.64, 139.0,
139.5, 156.1, 171.5, 173.4, 174.1, 176.3, 193.1, 197.9; HRMS-
ESI (m/z): [M+Na+] calcd for C47H50ClNNaO5Si, 794.3039;
found, 794.3035.
7k (61.7 mg, 82%): IR (neat) 3026, 2925, 2864, 1624, 1493,
1
1454, 1419, 1327, 1240, 1165, 1126, 1066, 1018, 825 cm-1; H-
NMR (400 MHz, CDCl3) δ 1.62-1.81 (m, 4H), 2.32 (s, 1.5H),
2.34 (m, 3H), 2.42 (s, 1.5H), 2.47 (brdd, J = 8.3 Hz, 6.9 Hz, 1H),
2.62 (m, 1H), 2.71 (brdd, J = 7.4 Hz, 6.9 Hz, 1H), 2.73 (brdd, J
= 7.4 Hz, 6.9 Hz, 1H), 4.48 (s, 1H), 4.58 (s, 1H), 5.12 (s, 1H),
5.13 (s, 1H), 6.86 (brd, J = 8.3 Hz, 1H), 6.92 (m, 1H), 7.05 (brd,
J = 6.9 Hz, 1H), 7.10-7.23 (m, 5H), 7.54 (d, J = 8.0 Hz, 1H),
7.64 (d, J = 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 12.7,
14.2, 21.40, 21.43, 27.1, 27.4, 29.4, 29.6, 29.9, 30.0, 34.2, 37.4,
54.4, 55.7, 68.90, 68.93, 111.44, 111.46, 121.0 (2C), 124.1 (m,
2C), 124.3, 124.9, 125.5 (m, 2C), 126.9 (4C), 127.0 (2C), 127.1
(2C), 127.3, 127.4, 128.0, 128.19, 128.22, 128.6, 129.9 (m, 2C),
130.0, 130.1, 130.7, 131.0 137.85, 137.91, 139.9, 140.4, 141.5
(2C), 156.08, 156.11, Signals of carbons (SC=N) were weak; 19F-
NMR (376Hz, CDCl3) δ -62.42, -62.36; HRMS-EI (m/z): [M+]
calcd for C28H30F3NOS, 485.2000; found, 485.2002.
12c (50.4 mg, 53%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3070, 2929, 2858, 1741, 1668, 1647, 1601, 1495, 1452,
1429, 1240, 1113, 1039, 810 cm-1; 1H-NMR (400 MHz, CDCl3) δ
0.94 (s, 5.2H), 0.95 (s, 3.8H), 1.59-1.84 (m, 4H), 2.31 (s, 1.7H),
2.33 (s, 1.3H), 2.63-2.74 (m, 2H), 2.91-3.10 (m, 2H), 3.98-4.14
(m, 2H), 4.48-4.53 (m, 3H), 5.01 (brs, 0.84H), 5.02 (brs, 1.2H),
6.83-6.92 (m, 2H), 6.95-7.01 (m, 2H), 7.10-7.26 (m, 4H), 7.29-
7.43 (m, 10H), 7.63-7.70 (m, 4H), 10.4 (brm, 0.42H), 11.3 (brm,
0.58H);13C-NMR (100 MHz, CDCl3) δ 19.2, 21.35, 21.38, 26.51,
26.53, 27.1, 27.3, 27.45, 27.50, 29.7, 29.8, 46.6, 46.9, 62.3, 62.5,
69.0, 81.3, 83.3, 91.3, 92.8, 111.6, 120.9, 124.0, 127.1, 127.6,
127.7, 128.4, 128.6, 128.7, 129.07, 129.15, 129.6, 129.7, 129.98,
130.00, 130.41, 130.44, 132.7, 132.7, 132.97, 133.01, 133.5,
135.0, 135.1, 135.5, 135.6, 135.8, 139.0, 156.2, 171.5, 173.4,
174.0, 176.3, 193.1, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H50ClNNaO5Si, 794.3039; found, 794.3040.
7l (68.4 mg, 91%): IR (neat) 3014, 2925, 2860, 1624, 1608,
1
1522, 1493, 1452, 1346, 1240, 1111, 1055, 854 cm-1; H-NMR
(400 MHz, CDCl3) δ 1.64-1.80 (m, 4H), 2.32 (s, 1.5H), 2.34 (m,
3H), 2.45 (s, 1.5H), 2.47 (brdd, J = 7.8 Hz, 6.9 Hz, 1H), 2.63 (m,
1H), 2.73 (brdd, J = 7.8 Hz, 6.9 Hz, 1H), 2.74 (brdd, J = 7.4 Hz,
6.9 Hz, 1H), 4.48 (s, 1H), 4.57 (s, 1H), 5.16 (s, 1H), 5.17 (s,
1H),6.83 (m, 1H), 6.94 (m, 1H), 7.05 (m, 1H), 7.10-7.23 (m, 5H),
7.59 (m, 2H), 8.24 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 12.7,
14.3, 21.38, 21.43, 27.0, 27.3, 29.4, 29.6, 29.9, 30.0, 34.2, 37.4,
54.4, 55.7, 68.49, 68.53, 111.4 (2C), 121.3 (2C), 123.76 (2C),
123.79 (2C), 124.3, 124.9, 127.0, 127.1, 127.2 (3C), 127.3 (2C),
127.4, 128.0, 128.18, 128.20, 128.6, 130.1, 130.2, 130.7, 131.0,
137.8, 137.9, 140.3 (2C), 144.8, 144.9, 147.4 (2C), 155.8 (2C),
Signals of carbons (SC=N) were weak; HRMS-EI (m/z): [M+]
calcd for C27H30N2O3S, 462.1977; found, 462.1973.
12d (5.5 mg, 87%, tautomeric mixture: 1.27:1 in CDCl3): IR
(neat) 3070, 2931, 2858, 1745, 1670, 1649, 1603, 1444, 1155,
1134, 1115, 1061, 1041 cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.93
(s, 5.0H), 0.94 (s, 4.0H), 1.57-1.84 (m, 4H), 2.31 (s, 1.7H), 2.33
(s, 1.3H), 2.55-2.65 (m, 2H), 2.90-3.13 (m, 2H), 3.98-4.12 (m,
2H), 4.48-4.56 (m, 3H), 5.13 (br, 2H), 6.73-6.81 (m, 3H), 6.97-
7.02 (m, 2H), 7.11-7.30 (m, 6H), 7.33-7.42 (m, 7H), 7.54-7.70
(m, 4H), 10.4 (brm, 0.44H), 11.3 (brm, 0.56H); 13C-NMR (100
MHz, CDCl3) δ 19.2, 21.37, 21.40, 26.5, 26.6, 27.12, 27.14, 27.2,
27.5, 29.7, 31.10, 31.15, 35.3, 46.6, 46.9, 62.3, 62.5, 66.9, 81.3,
83.3, 91.4, 92.8, 112.0, 115.1, 121.27, 121.30, 124.0, 126.9,
127.68, 127.71, 128.8, 128.9, 129.1, 129.2, 129.3, 129.4, 129.66,
129.74, 132.5, 132.68, 132.71, 133.0, 133.1, 134.8, 135.0, 135.1,
135.56, 135.65, 139.0, 143.47, 143.51, 158.6, 171.5, 173.3, 174.0,
176.3, 193.2, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H50ClNNaO5Si, 794.3039; found, 794.3052.
4.2.5. Representative procedure for transformation of 7 to 12
To a mixture of 7a and MS4A, a solution of 8 (15.0 mg, 33.2
µmol) in CH2Cl2 (205 µL) was added. The reaction mixture was
stirred for 30 h and filtered through Celite. The filtrate was
concentrated in vacuo. Further purification was carried out by
silica gel column chromatography (eluent: hexane/ethyl acetate =
4) to give 12a (11.1 mg, 65%, tautomeric mixture: 1.38:1 in
CDCl3) as a colorless oil. IR (neat) 3068, 2929, 2858, 1741, 1670,
1647, 1601, 1495, 1452, 1238, 1130, 1113, 1039 cm-1; 1H-NMR
(400 MHz, CDCl3) δ 0.94 (s, 5.2H), 0.96 (s, 3.8H), 1.60-1.89 (m,
4H), 2.30 (s, 1.7H), 2.32 (s, 1.3H), 2.68-2.78 (m, 2H), 2.93-3.11
(m, 2H), 3.98-4.11 (m, 2H), 4.47-4.53 (m, 3H), 5.14 (brs, 2H),
6.88-7.00 (m, 4H), 7.11-7.30 (m, 6H), 7.33-7.43 (m, 7H), 7.54
(m, 1H), 7.62-7.69 (m, 4H), 10.4 (brm, 0.42H), 11.3 (brm,
12e (5.9 mg, 50%, tautomeric mixture: 1.27:1 in CDCl3): IR
(neat) 3070, 3047, 2929, 2858, 1741, 1670, 1649, 1601, 1429,
1259, 1155, 1132, 1113, 1041 cm-1; 1H-NMR (400 MHz, CDCl3)
δ 0.94 (s, 5.0H), 0.96 (s, 4.0H), 1.56-1.83 (m, 4H), 2.31 (s, 1.7H),
2.34 (s, 1.3H), 2.54-2.64 (m, 2H), 2.88-3.12 (m, 2H), 3.97-4.12
(m, 2H), 4.48-4.56 (m, 3H), 5.00 (br, 2H), 6.74-6.80 (m, 3H),
6.97-7.03 (m, 2H), 7.12-7.31 (m, 6H), 7.34-7.44 (m, 6H), 7.62-

7.70 (m, 4H), 10.4 (brm, 0.44H), 11.3 (brm, 0.56H); 13C-NMR
(100 MHz, CDCl3) δ 19.2, 21.38, 21.41, 26.5, 26.6, 27.11, 27.14,
27.2, 27.5, 29.7, 31.1, 31.2, 35.3, 46.6, 46.9, 62.3, 62.5, 69.0,
81.3, 83.3, 91.4, 92.8, 112.0, 112.1, 115.02, 115.04, 121.3, 124.0,
125.3, 125.4, 127.4, 127.69, 127.72, 128.0, 129.1, 129.2, 129.4,
129.66, 129.74, 129.8, 132.68, 132.71, 133.0, 133.1, 134.4, 135.0,
135.1, 135.57, 135.65, 139.1, 139.2, 143.49, 143.53, 158.6, 171.5,
173.3, 173.9, 176.3, 193.2, 197.9; HRMS-ESI (m/z): [M+Na+]
calcd for C47H50ClNNaO5Si, 794.3039; found, 794.3055.
6.98-7.08 (m, 4H), 7.12-7.15 (m, 1H), 7.20-7.26 (m, 1H), 7.34-
7.42 (m, 10H), 7.63-7.70 (m, 4H), 10.4 (brm, 0.42H), 11.3 (brm,
0.58H);13C-NMR (100 MHz, CDCl3) δ 19.2, 21.36, 21.39, 26.51,
26.54, 27.07, 27.12, 27.2, 27.5, 31.4, 31.5, 34.4, 46.6, 46.9, 62.2,
62.5, 69.2, 81.3, 83.3, 91.3, 92.8, 114.7, 124.0, 127.59, 127.62,
127.66, 127.69, 128.67, 128.69, 129.07, 129.15, 129.25, 129.29,
129.6, 129.7, 132.6, 132.7, 132.9, 133.0, 133.6, 134.26, 134.32,
135.0, 135.1, 135.5, 135.6, 139.0, 156.7, 171.5, 173.3, 174.0,
176.2, 193.2, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H50ClNNaO5Si, 794.3039; found, 794.3045.
12f (15.6 mg, 67%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3051, 2929, 2856, 1741, 1668, 1647, 1601, 1493, 1448,
12j (24.9 mg, 59%, tautomeric mixture: 1.33:1 in CDCl3): IR
(neat) 3032, 2929, 2858, 1743, 1670, 1647, 1601, 1498, 1452,
1
1429, 1408, 1259, 1132, 1113, 1041 cm-1; H-NMR (400 MHz,
1
CDCl3) δ 0.94 (s, 5.3H), 0.96 (s, 3.7H), 1.57-1.83 (m, 4H), 2.31
(s, 1.8H), 2.34 (s, 1.2H), 2.54-2.63 (m, 2H), 2.90-3.12 (m, 2H),
3.98-4.15 (m, 2H), 4.47-4.56 (m, 3H), 4.99 (brs, 0.82H), 5.00
(brs, 1.2H), 6.74-6.79 (m, 3H), 6.98-7.03 (m, 2H), 7.1-7.26 (m,
3H), 7.34-7.43 (m, 10H), 7.63-7.7 (m, 4H), 10.4 (brm, 0.41H),
11.3 (brm, 0.59H); 13C-NMR (100 MHz, CDCl3) δ 19.2, 21.38,
21.40, 26.5, 26.6, 27.1, 27.2, 27.5, 31.1, 31.2, 35.3, 46.6, 46.9,
62.3, 62.5, 69.0, 81.3, 83.3, 91.4, 92.8, 112.0, 112.1, 114.99,
115.03, 121.2, 124.0, 127.68, 127.71, 128.7, 128.8, 129.1, 129.2,
129.4, 129.66, 129.74, 132.66, 132.69, 133.0, 133.1, 133.6, 135.0,
135.1, 135.56, 135.64, 139.1, 143.46, 143.51, 158.6, 171.5, 173.3,
173.9, 176.3, 193.2, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H50ClNNaO5Si, 794.3039; found, 794.3033.
1406, 1342, 1240, 1113, 1039, 924, 823 cm-1; H-NMR (400
MHz, CDCl3) δ 0.94 (s, 5.1H), 0.96 (s, 3.9H), 1.57-1.85 (m, 4H),
2.30 (s, 1.7H), 2.32 (s, 1.3H), 2.66-2.75 (m, 2H), 2.91-3.11 (m,
2H), 3.98-4.11 (m, 2H), 4.46-4.53 (m, 3H), 5.06 (br, 2H), 6.87-
6.91 (m, 2H), 6.94-6.99 (m, 2H), 7.10-7.31 (m, 5H), 7.32-7.44
(m, 10H), 7.63-7.70 (m, 4H), 10.4 (brm, 0.43H), 11.3 (brm,
0.57H); 13C-NMR (100 MHz, CDCl3) δ 19.2, 21.37, 21.39, 26.52,
26.55, 27.1, 27.3, 27.5, 29.7, 29.8, 46.6, 46.9, 62.3, 62.5, 69.8,
81.3, 83.3, 91.3, 92.7, 111.6, 120.7, 124.1, 127.05, 127.08,
127.67, 127.70, 127.8, 128.5, 129.06, 129.13, 129.6, 129.7, 129.9,
130.0, 130.4, 130.5, 132.70, 132.74, 132.98, 133.03, 135.0, 135.1,
135.56, 135.65, 137.4, 139.0, 156.5, 171.5, 173.5, 174.1, 176.3,
193.1, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H51NO5SiNa, 760.3429; found, 760.3429.
12g (58.3 mg, 66%, tautomeric mixture: 1.33:1 in CDCl3): IR
(neat) 3070, 2929, 2856, 1741, 1668, 1647, 1601, 1510, 1442,
12k (38.0 mg, 61%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 2931, 2860, 1741, 1670, 1649, 1601, 1493, 1454, 1429,
1
1429, 1238, 1130, 1113, 1038, 823 cm-1; H-NMR (400 MHz,
1
CDCl3) δ 0.94 (s, 5.1H), 0.96 (s, 3.9H), 1.57-1.81 (m, 4H), 2.32
(s, 1.7H), 2.34 (s, 1.3H), 2.52-2.61 (m, 2H), 2.88-3.14 (m, 2H),
3.98-4.12 (m, 2H), 4.47-4.56 (m, 3H), 5.12 (br, 2H), 6.87-6.91
(m, 2H), 6.97-7.15 (m, 5H), 7.19-7.30 (m, 4H), 7.33-7.44 (m,
6H), 7.53-7.57 (m, 1H), 7.63-7.70 (m, 4H), 10.4 (brm, 0.43H),
11.3 (brm, 0.57H); 13C-NMR (100 MHz, CDCl3) δ 19.2, 21.3,
21.4, 26.5, 26.5, 27.0, 27.1, 27.5, 31.38, 31.44, 34.4, 34.7, 46.6,
46.9, 62.2, 62.4, 67.1, 81.3, 83.3, 91.3, 92.8, 114.7, 124.0, 126.9,
127.58, 127.65, 127.7, 128.2, 128.5, 128.7 128.8, 129.06, 129.13,
129.3, 129.6, 129.7, 132.4, 132.6, 132.7, 132.9, 133.0, 134.27,
134.32, 134.8, 135.0, 135.1, 135.5, 135.6, 139.0, 156.7, 171.5,
173.3, 174.0, 176.2, 193.1, 197.9; HRMS-ESI (m/z): [M+Na+]
calcd for C47H50ClNNaO5Si, 794.3039; found, 794.3046.
1325, 1242, 1165, 1124, 1113, 1066, 1039, 823 cm-1; H-NMR
(400 MHz, CDCl3) δ 0.94 (s, 5.2H), 0.95 (s, 3.8H), 1.57-1.86 (m,
4H), 2.30 (s, 1.3H), 2.32 (s, 1.7H), 2.67-2.78 (m, 2H), 2.92-3.12
(m, 2H), 3.98-4.11 (m, 2H), 4.48-4.58 (m, 3H), 5.12 (br, 2H),
6.83-6.86 (m, 1H), 6.88-6.93 (m, 1H), 6.96-7.09 (m, 2H), 7.11-
7.25 (m, 4H), 7.33-7.43 (m, 6H), 7.51-7.55 (m, 2H), 7.60-7.70
(m, 6H), 10.4 (brm, 0.42H), 11.3 (brm, 0.58H); 13C-NMR (100
MHz, CDCl3) δ 19.2, 21.3, 21.4, 26.50, 26.53, 27.1, 27.4, 27.48,
27.54, 29.7, 29.8, 46.6, 46.9, 62.3, 62.5, 68.90, 68.93, 81.3, 83.3,
91.3, 92.8, 111.49, 111.52, 121.1, 124.0, 124.1 (m), 125.5 (m),
126.9, 126.98, 127.03, 127.2, 127.62, 127.66, 127.70, 128.6,
129.1, 129.2, 129.6 (m), 129.6, 129.7, 130.05, 130.08, 130.12,
130.39, 130.43, 132.6, 132.7, 132.96, 133.02, 135.0, 135.1,
135.53, 135.55, 135.64, 139.0, 141.4, 156.1, 171.5, 173.4, 174.0,
176.3, 193.1, 197.9; 19F-NMR (376Hz, CDCl3) δ -62.4; HRMS-
ESI (m/z): [M+Na+] calcd for C48H50F3NO5SiNa, 828.3303;
found, 828.3303.
12h (51.2 mg, 73%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3032, 2929, 2856, 1741, 1668, 1647, 1601, 1510, 1429,
1238, 1132, 1113, 1039, 823 cm-1; 1H-NMR (400 MHz, CDCl3) δ
0.94 (s, 5.2H), 0.96 (s, 3.8H), 1.57-1.81 (m, 4H), 2.32 (s, 1.7H),
2.34 (s, 1.3H), 2.51-2.60 (m, 2H), 2.88-3.14 (m, 2H), 3.98-4.12
(m, 2H), 4.47-4.56 (m, 3H), 4.98 (brs, 2H), 6.84-6.88 (m, 2H),
6.98-7.09 (m, 4H), 7.12-7.15 (m, 1H), 7.20-7.25 (m, 1H), 7.28-
7.31 (m, 3H), 7.33-7.44 (m, 7H), 7.63-7.69 (m, 4H), 10.4 (brm,
0.42H), 11.3 (brm, 0.58H); 13C-NMR (100 MHz, CDCl3) δ 19.2,
21.36, 21.39, 26.52, 26.55, 27.07, 27.13, 27.2, 27.5, 31.4, 31.5,
34.4, 46.6, 46.9, 62.2, 62.5, 69.1, 81.3, 83.3, 91.4, 92.8, 114.7,
124.0, 125.28, 127.34, 127.59, 127.62, 127.67, 127.70, 128.0,
128.6, 129.1, 129.2, 129.28, 129.31, 129.6, 129.7, 129.8, 132.6,
132.7, 132.9, 133.0, 134.3, 134.39, 134.42, 135.0, 135.1, 135.5,
135.6, 139.0, 139.2, 156.7, 171.5, 173.3, 174.0, 176.3, 193.2,
197.9; HRMS-ESI (m/z): [M+Na+] calcd for C47H50ClNNaO5Si,
794.3039; found, 794.3043.
12l (41.9 mg, 55%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 2934, 2855, 1740, 1668, 1647, 1601, 1521, 1494, 1428,
1
1346, 1300, 1241, 1134, 1112, 1089, 1041, 824 cm-1; H-NMR
(400 MHz, CDCl3) δ 0.93 (s, 5.3H), 0.95 (s, 3.7H), 1.60-1.87 (m,
4H), 2.31 (s, 1.8H), 2.33 (s, 1.2H), 2.66-2.79 (m, 2H), 2.92-3.14
(m, 2H), 4.00-4.14 (m, 2H), 4.50-4.60 (m, 3H), 5.157 (br, 0.8H),
5.163 (br, 1.2H), 6.81-6.84 (m, 1H), 6.89-6.94 (m, 1H), 6.97-7.10
(m, 2H), 7.12-7.25 (m, 4H), 7.32-7.43 (m, 6H), 7.57-7.69 (m,
6H), 8.22-8.26 (m, 2H), 10.4 (brm, 0.41H), 11.3 (brm, 0.59H);
13C-NMR (100 MHz, CDCl3) δ 19.2, 21.35, 21.37, 26.49, 26.52,
27.1, 27.4, 27.5, 27.6, 29.7, 29.8, 29.9, 46.6, 46.9, 62.2, 62.5,
68.48, 68.53, 81.3, 83.3, 91.3, 92.8, 111.4, 111.5, 121.3, 123.8,
124.0, 127.19, 127.22, 127.3, 127.59, 127.65, 127.7, 129.1, 129.2,
129.59, 129.63, 129.7, 130.12, 130.15, 130.38, 130.42, 132.6,
132.7, 132.9, 133.0, 135.0, 135.1, 135.50, 135.53, 135.6, 139.0,
144.7, 147.39, 147.42, 155.78, 155.80, 171.5, 173.3, 173.9, 176.3,
193.1, 197.9; HRMS-ESI (m/z): [M+Na+] calcd for
C47H50N2O7SiNa, 805.3280; found, 805.3280.
12i (49.4 mg, 80%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3030, 2929, 2858, 1741, 1668, 1645, 1601, 1510, 1238,
1132, 1113, 1039, 1014, 822 cm-1; 1H-NMR (400 MHz, CDCl3) δ
0.94 (s, 5.2H), 0.96 (s, 3.8H), 1.57-1.81 (m, 4H), 2.32 (s, 1.7H),
2.34 (s, 1.3H), 2.51-2.59 (m, 2H), 2.88-3.13 (m, 2H), 3.98-4.15
(m, 2H), 4.48-4.56 (m, 3H), 4.98 (br, 2H), 6.83-6.87 (m, 2H),
4.2.6. Representative procedure for transformation of 12 to 6

1
To a solution of 12a (10.0 mg, 12.9 µmol) in DMF (80 µL), a
solution of TASF (4.3 mg, 15.5 µmol) in DMF (11.9 µL) was
added at 0 °C. The mixture was stirred for 5 h, then a solution of
phosphate buffer (pH 7.0) and ethyl acetate were added. The
organic layer was separated, and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were dried over
anhydrous magnesium sulfate and concentrated in vacuo (DMF
was removed by azeotropic evaporation with toluene). Further
purification was carried out by preparative thin layer
chromatography (eluent: hexane/ethyl acetate = 1/4) and high-
performance liquid chromatography (column: SenshuPak,
PEGASIL Silica SP100, 10×250 mm, eluent: hexane/ethyl
acetate = 1/2) to give 6a (4.2 mg, 61%) as a white amorphous
solid (tautomeric mixture: 1.38:1 in CDCl3). IR (neat) 3444, 2930,
1736, 1645, 1600, 1494, 1442, 1407, 1343, 1238, 1033 cm-1; 1H
NMR (400 MHz, CDCl3) δ 1.57-1.70 (m, 2H), 1.78-1.88 (m,
2H), 1.98 (brt, J = 6.9 Hz, 0.58H), 2.05 (brt, J = 6.9 Hz, 0.42H),
2.34 (br, 3H), 2.75 (brt, J = 7.4 Hz, 2H), 2.91-3.13 (m, 2H), 3.92-
3.98 (m, 2H), 4.48 (br, 1.2H), 4.49 (br, 0.8H), 4.51 (brt, J = 4.1
Hz, 0.42H), 4.53 (brt, J = 4.1 Hz, 0.58H), 5.14 (br, 2H), 6.89-
6.94 (m, 2H), 6.96-7.00 (m, 2H), 7.12-7.31 (m, 6H), 7.36-7.39
(m, 1H), 7.52-7.56 (m, 1H), 10.3 (br, 0.42H), 11.2 (br, 0.58H);
13C NMR (100 MHz, CDCl3) δ 21.4, 27.3, 27.4, 27.7, 29.7, 29.8,
29.9, 46.9, 47.2, 61.7, 61.8, 67.12, 67.14, 80.6, 82.3, 90.3, 91.8,
111.7, 121.0, 124.25, 124.34, 126.96, 127.03, 127.2, 127.9,
128.0, 128.5, 128.9, 129.1, 129.2, 129.3, 129.4, 130.1, 130.3,
130.4, 132.46, 132.51, 134.5, 134.6, 135.0, 139.1, 156.2, 171.1,
174.1, 174.6, 175.9, 193.2, 197.5; HRMS-ESI (m/z): [M+Na+]
calcd for C31H32ClNNaO5, 556.1861; found, 556.1860.
1258, 1157, 1089, 1035 cm-1; H-NMR (400 MHz, CDCl3) δ
1.52-1.69 (m, 2H), 1.75-1.84 (m, 2H), 1.99 (brt, J = 6.4 Hz,
0.58H), 2.05 (brt, J = 6.4 Hz, 0.42H), 2.35 (brs, 3H), 2.64 (brt, J
= 7.8 Hz, 2H), 2.92-3.12 (m, 2H), 3.91-4.02 (m, 2H), 4.51 (br,
1.2H), 4.52 (br, 0.8H), 4.52 (brt, J = 4.1 Hz, 0.42H), 4.55 (brt, J
= 4.1 Hz, 0.58H), 5.14 (s, 2H), 6.78-6.83 (m, 3H), 6.98-7.03 (m,
2H), 7.13-7.32 (m, 5H), 7.38-7.41 (m, 1H), 7.54-7.57 (m, 1H),
10.3 (br, 0.42H), 11.3 (br, 0.58H); 13C-NMR (100 MHz, CDCl3)
δ 21.4, 26.9, 27.0, 27.3, 27.6, 31.0, 35.2, 35.3, 46.9, 47.2, 61.6,
61.8, 70.0, 80.7, 82.5, 90.5, 91.9, 112.0, 112.1, 115.1, 115.2,
121.3, 124.2, 124.3, 126.9, 127.8, 128.0, 128.8, 128.9, 129.17,
129.19, 129.3, 129.4, 129.5, 132.6, 134.5, 134.6, 134.8, 139.2,
143.37, 143.41, 158.6, 171.2, 173.9, 174.4, 175.8, 193.3, 197.5;
HRMS-ESI (m/z): [M+Na+] calcd for C31H32ClNNaO5, 556.1861;
found, 556.1865.
6e (3.6 mg, 61%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3444, 2930, 1736, 1645, 1599, 1506, 1441, 1409, 1344,
1
1257, 1150, 1096, 1035 cm-1; H-NMR (400 MHz, CDCl3) δ
1.52-1.68 (m, 2H), 1.75-1.84 (m, 2H), 2.00 (brt, J = 6.4 Hz,
0.58H), 2.05 (brt, J = 6.9 Hz, 0.42H), 2.35 (s, 3H), 2.63 (t, J =
7.4 Hz, 2H), 2.93-3.12 (m, 2H), 4.51 (br, 1.2H), 4.52 (m, 1.2H),
4.55 (brt, J = 3.7 Hz, 0.58H), 5.01 (s, 2H), 6.77-6.81 (m, 3H),
6.98-7.04 (m, 2H), 7.13-7.26 (m, 3H), 7.30 (brs, 3H), 7.43 (brs,
1H), 10.3 (br, 0.42H), 11.2 (br, 0.58H); 13C-NMR (100 MHz,
CDCl3) δ 21.4, 26.9, 27.0, 27.2, 27.6, 31.0, 35.2, 46.9, 47.2, 61.6,
61.7, 69.0, 80.7, 82.5, 90.5, 91.9, 111.95, 112.03, 115.05, 115.11,
121.3, 124.2, 124.3, 125.4, 127.4, 127.8, 127.95, 128.01, 129.16,
129.19, 129.35, 129.39, 129.43, 129.44, 129.8, 134.4, 134.6,
139.1, 139.2, 143.36, 143.41, 158.5, 171.2, 173.8, 174.4, 175.8,
193.2, 197.5; HRMS-ESI (m/z): [M+Na+] calcd for
C31H32ClNNaO5, 556.1861; found, 556.1864.
6b (3.3mg, 58%, tautomeric mixture: 1.27:1 in CDCl3): IR
(neat) 3445, 2930, 1738, 1646, 1600, 1495, 1453, 1408, 1344,
1
1239, 1113, 1085, 1048 cm-1; H-NMR (400 MHz, CDCl3) δ
6f (4.3 mg, 72%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3445, 2932, 1736, 1645, 1600, 1494, 1446, 1408, 1343,
1258, 1156, 1087, 1034, 1016, 812 cm-1; H-NMR (400 MHz,
1.53-1.71 (m, 2H), 1.76-1.86 (m, 2H), 1.97 (dd, J = 7.8 Hz, 6.0
Hz, 0.56H), 2.03 (dd, J = 7.4 Hz, 6.0 Hz, 0.44H), 2.339 (s, 1.3H),
2.342 (s, 1.7H), 2.73 (brt, J = 7.4 Hz, 2H), 2.91-3.14 (m, 2H),
3.90-4.00 (m, 2H), 4.49 (br, 1.1H), 4.50 (br, 0.9H), 4.51 (brt, J =
4.1 Hz, 0.44H), 4.54 (brt, J = 4.1 Hz, 0.56H), 5.03 (brs, 2H), 6.86
(brd, J = 7.8 Hz, 1H), 6.89-6.93 (m, 1H), 6.96-7.01 (m, 2H),
7.12-7.19 (m, 3H), 7.22-7.31 (m, 4H), 7.42 (brm, 1H), 10.3 (br,
0.44H), 11.2 (br, 0.56H); 13C-NMR (100 MHz, CDCl3) δ 21.4,
27.27, 27.32, 27.4, 27.7, 29.76, 29.81, 46.9, 47.2, 61.7, 61.8, 69.0,
80.6, 82.3, 90.3, 91.8, 111.6, 121.0, 124.26, 124.34, 125.1, 127.0,
127.2, 127.9, 128.0, 129.1, 129.2, 129.3, 129.4, 129.86, 129.90,
130.1, 130.39, 130.4, 134.4, 134.5, 134.6, 139.1, 139.5, 156.2,
171.1, 174.0, 174.6, 175.8, 193.2, 197.5; HRMS-ESI (m/z):
[M+Na+] calcd for C31H32ClNNaO5, 556.1861; found, 556.1855.
1
CDCl3) δ 1.53-1.68 (m, 2H), 1.74-1.84 (m, 2H), 2.00 (brt, J = 6.4
Hz, 0.59H), 2.05 (brt, J = 6.9 Hz, 0.41H), 2.35 (s, 3H), 2.63 (brt,
J = 7.4 Hz, 2H), 2.93-3.12 (m, 2H), 3.91-3.99 (m, 2H), 4.51 (br,
1.2H), 4.53 (m, 1.2H), 4.55 (brt, J = 4.1 Hz, 0.59H), 5.00 (brs,
2H), 6.76-6.80 (m, 3H), 6.99-7.04 (m, 2H), 7.14-7.21 (m, 2H),
7.24-7.29 (m, 1H), 7.35 (s, 4H), 10.3 (br, 0.41H), 11.3 (br,
0.59H); 13C-NMR (100 MHz, CDCl3) δ 21.4, 26.9, 27.0, 27.2,
27.6, 30.9, 35.20, 35.24, 46.9, 47.2, 61.6, 61.7, 69.1, 80.7, 82.5,
90.5, 91.9, 112.0, 112.1, 115.07, 115.13, 121.2, 124.2, 124.3,
127.8, 128.0, 128.7, 128.8, 129.17, 129.20, 129.37, 129.40, 133.7,
134.5, 134.6, 135.6, 139.2, 143.36, 143.41, 158.6, 171.2, 173.9,
174.4, 175.8, 193.3, 197.5; HRMS-ESI (m/z): [M+Na+] calcd for
C31H32ClNNaO5, 556.1861; found, 556.1867.
6c (4.4 mg, 80%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3392, 2930, 1738, 1644, 1600, 1494, 1452, 1409, 1343,
1239, 1086, 1049, 1015, 810 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.56-1.70 (m, 2H), 1.74-1.84 (m, 2H), 1.95-2.03 (m, 1H), 2.35
(br, 3H), 2.71 (brt, J = 7.4 Hz, 2H), 2.91-3.11 (m, 2H), 3.90-4.01
(m, 2H), 4.48 (br, 1.2H), 4.49 (br, 0.8H), 4.50 (brt, J = 4.6 Hz,
0.41H), 4.54 (brt, J = 4.1 Hz, 0.59H), 5.02 (s, 2H), 6.86 (brd, J =
8.3 Hz, 1H), 6.90 (brt, J = 7.4 Hz, 1H), 6.96-7.02 (m, 2H), 7.13-
7.19 (m, 3H), 7.23-7.28 (m, 1H), 7.34 (brs, 4H), 10.3 (br, 0.41H),
11.2 (br, 0.59H); 13C-NMR (100 MHz, CDCl3) δ 21.4, 27.2, 27.3,
27.3, 27.6, 29.61, 29.64, 29.7, 29.8, 46.8, 47.2, 61.6, 61.8, 69.1,
80.6, 82.4, 90.4, 91.9, 111.6, 121.0, 124.2, 124.3, 127.2, 127.9,
128.0, 128.5, 128.7, 129.16, 129.18, 129.36, 129.39, 130.0, 130.3,
130.4, 133.6, 134.5, 134.6, 135.8, 139.2, 156.3, 171.2, 174.0,
174.5, 175.9, 193.2, 197.5; HRMS-ESI (m/z): [M+Na+] calcd for
C31H32ClNNaO5, 556.1861; found, 556.1865.
6g (4.5 mg, 59%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3391, 2924, 1736, 1644, 1598, 1509, 1442, 1408, 1343,
1237, 1177, 1085, 1033, 825 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.53-1.68 (m, 2H), 1.72-1.82 (m, 2H), 1.97-2.08 (m, 1H), 2.36
(brs, 3H), 2.60 (brt, J = 7.4 Hz, 2H), 2.92-3.13 (m, 2H), 3.91-
4.03 (m, 2H), 4.51 (br, 1.2H), 4.52 (br, 1.2H), 4.55 (brt, J = 4.1
Hz, 0.59H), 5.13 (s, 2H), 6.88-6.92 (m, 2H), 6.98-7.04 (m, 2H),
7.07-7.11 (m, 2H), 7.13-7.18 (m, 1H), 7.23-7.31 (m, 3H), 7.37-
7.41 (m, 1H), 7.54-7.58 (m, 1H), 10.3 (br, 0.42H), 11.3 (br,
0.58H); 13C-NMR (100 MHz, CDCl3) δ 21.4, 26.9, 27.0, 27.3,
27.6, 31.3, 34.4, 46.9, 47.2, 61.6, 61.8, 67.1, 80.7, 82.5, 90.4,
91.9, 114.7, 124.2, 124.3, 126.9, 127.8, 127.9, 128.7, 128.9,
129.15, 129.18, 129.3, 129.4, 132.5, 134.19, 134.22, 134.5, 134.6,
134.8, 139.1, 156.7, 171.2, 173.9, 174.5, 175.8, 193.2, 197.5;
HRMS-ESI (m/z): [M+Na+] calcd for C31H32ClNNaO5, 556.1861;
found, 556.1865.
6d (3.7 mg, 67%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3390, 2937, 1736, 1645, 1600, 1506, 1443, 1407, 1344,

6h (5.1 mg, 64%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3441, 2930, 1736, 1645, 1600, 1510, 1437, 1408, 1344,
1237, 1084, 1032, 826 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.52-
1.68 (m, 2H), 1.72-1.82 (m, 2H), 2.00-2.10 (m, 1H), 2.36 (brs,
3H), 2.59 (brt, J = 7.4 Hz, 2H), 2.91-3.13 (m, 2H), 3.94-4.00 (m,
2H), 4.51 (br, 1.2H), 4.52 (br, 1.2H), 4.55 (brt, J = 4.1 Hz,
0.59H), 5.00 (s, 2H), 6.85-6.89 (m, 2H), 6.98-7.04 (m, 2H), 7.06-
7.11 (m, 2H), 7.13-7.18 (m, 1H), 7.24-7.28 (m, 1H), 7.28-7.31
(m, 3H), 7.43 (br, 1H), 10.3 (br, 0.41H), 11.3 (br, 0.59H); 13C-
NMR (100 MHz, CDCl3) δ 21.4, 26.9, 27.0, 27.3, 27.6, 31.3,
34.32, 34.35, 46.9, 47.2, 61.6, 61.7, 69.1, 80.7, 82.5, 90.4, 91.9,
114.7, 124.2, 124.3, 125.3, 127.4, 127.8, 127.9, 128.0, 129.1,
129.2, 129.3, 129.4, 129.8, 134.2, 134.3, 134.4, 134.5, 134.6,
139.16, 139.20, 156.7, 171.2, 173.9, 174.4, 175.8, 193.2, 197.5;
HRMS-ESI (m/z): [M+Na+] calcd for C31H32ClNNaO5, 556.1861;
found, 556.1866.
HRMS-ESI (m/z): [M+Na+] calcd for C32H32F3NO5Na, 590.2125;
found, 590.2125.
6l (22.7 mg, 82%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3406, 2933, 2864, 1736, 1645, 1599, 1520, 1452, 1410,
1346, 1240, 1111, 1049, 825 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.57-1.70 (m, 2H), 1.78-1.87 (m, 2H), 2.07 (brt, J = 6.0 Hz, 1H),
2.35 (brs, 3H), 2.74 (brt, J = 7.4 Hz, 2H), 2.95-3.15 (m, 2H),
3.93-4.02 (m, 2H), 4.52 (br, 1.2H), 4.54 (br, 1.2H), 4.55 (brt, J =
4.1 Hz, 0.59H), 5.16 (s, 2H), 6.84 (brd, J = 8.7 Hz, 1H), 6.93 (brt,
J = 7.4 Hz, 1H), 6.97-7.03 (m, 2H), 7.13-7.20 (m, 3H), 7.23-7.28
(m, 1H), 7.58-7.62 (m, 2H), 8.23-8.28 (m, 2H), 10.3 (br, 0.41H),
11.2 (br, 0.59H); 13C-NMR (100 MHz, CDCl3) δ 21.4, 27.27,
27.32, 27.4, 27.7, 29.7, 29.8, 46.8, 47.2, 61.5, 61.6, 68.5, 68.6,
80.8, 82.6, 90.5, 91.9, 111.5, 121.3, 123.9, 124.16, 124.24, 127.2,
127.3, 127.4, 127.8, 127.9, 129.1, 129.2, 129.3, 129.4, 130.1,
130.3, 130.4, 134.4, 134.6, 139.2, 144.7, 147.5, 155.8, 171.2,
173.8, 174.4, 175.9, 193.2, 197.6; HRMS-ESI (m/z): [M+Na+]
calcd for C31H32N2O7Na, 567.2102; found, 567.2102.
6i (3.7 mg, 70%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3452, 2929, 1736, 1644, 1599, 1510, 1440, 1408, 1344,
1238, 1091, 1033, 1015, 814 cm-1; 1H-NMR (400 MHz, CDCl3) δ
1.52-1.67 (m, 2H), 1.72-1.81 (m, 2H), 1.96-2.07 (m, 1H), 2.36
(brs, 3H), 2.59 (brt, J = 7.4 Hz, 2H), 2.92-3.13 (m, 2H), 3.93-
4.00 (m, 2H), 4.51 (br, 1.2H), 4.53 (br, 1.2H), 4.55 (brt, J = 4.1
Hz, 0.59H), 4.99 (s, 2H), 6.85-6.89 (m, 2H), 6.96-7.04 (m, 2H),
6.98-7.04 (m, 2H), 7.05-7.10 (m, 1H), 7.24-7.29 (m, 1H), 7.35 (s,
4H), 10.3 (br, 0.41H), 11.3 (br, 0.59H); 13C-NMR (100 MHz,
CDCl3) δ 21.4, 26.96, 27.02, 27.3, 27.6, 31.3, 34.4, 46.9, 47.2,
61.6, 61.8, 69.2, 80.7, 82.4, 90.5, 91.9, 114.7, 124.2, 124.3, 127.8,
128.0, 128.7, 129.17, 129.19, 129.3, 129.36, 129.39, 133.7,
134.18, 134.22, 134.5, 134.6, 135.6, 139.2, 156.8, 171.2, 173.9,
174.5, 175.8, 193.3, 197.5; HRMS-ESI (m/z): [M+Na+] calcd for
C31H32ClNNaO5, 556.1861; found, 556.1867.
4.2.7. Inhibitory activity for VHR
VHR activity was measured in a 96-well microtiter plate using
O-methylfluorescein phosphate (OMFP) (Sigma, St. Louis, MO)
as a substrate. Purified VHR (22 ng) was preincubated at 37 °C
for 30 min in the reaction buffer (50 mM succinate, 1 mM
EDTA, 150 mM NaCl, 1 mM dithiothreitol, pH 6.0, 0.001% NP-
40) in the presence or absence of
a test compound.
Dephosphorylation reaction was started by addition of OMFP (10
µM). The mixture was incubated at 37 °C for 10 min.
Fluorescence emission from the dephosphorylated product was
measured with
a
SpectraMax M5 (Molecular Devices,
Sunnyvale, CA) using a 485 nm excitation filter and 530 nm
emission filter. The IC50 value for each cell line was determined
from the sigmoid dose-response curve using the Origin8J
program.
6j (11.4 mg, 78%, tautomeric mixture: 1.38:1 in CDCl3): IR
(neat) 3423, 2935, 2864, 1738, 1645, 1599, 1498, 1450, 1406,
1344, 1238, 1113, 1084, 1030 cm-1; 1H-NMR (400 MHz, CDCl3)
δ 1.54-1.70 (m, 2H), 1.76-1.85 (m, 2H), 2.07 (brt, J = 6.9 Hz,
0.58H), 2.12 (brt, J = 6.9 Hz, 0.42H), 2.34 (brs, 3H), 2.72 (brt, J
= 7.4 Hz, 2H), 2.90-3.11 (m, 2H), 3.91-4.00 (m, 2H), 4.45 (br,
0.8H), 4.46 (br, 1.2H), 4.50 (brt, J = 4.1 Hz, 0.42H), 4.53 (brt, J
= 4.1 Hz, 0.58H), 5.06 (s, 2H), 6.87-6.92 (m, 2H), 6.94-7.00 (m,
2H), 7.12-7.19 (m, 3H), 7.21-7.25 (m, 1H), 7.27-7.32 (m, 1H),
7.34-7.43 (m, 4H), 10.3 (br, 0.42H), 11.2 (br, 0.58H); 13C-NMR
(100 MHz, CDCl3) δ 21.4, 27.2, 27.3, 27.6, 29.6, 29.7, 29.8, 46.8,
47.2, 61.6, 61.8, 69.8, 80.6, 82.4, 90.3, 91.8, 111.7, 120.8, 124.27,
124.34, 127.1, 127.2, 127.8, 127.9, 128.0, 128.5, 129.1, 129.2,
129.31, 129.35, 130.0, 130.36, 130.40, 134.5, 134.6, 137.3, 139.1,
156.5, 171.2, 174.1, 174.6, 175.9, 193.2, 197.5; HRMS-ESI
(m/z): [M+Na+] calcd for C31H33NO5Na, 522.2251; found,
522.2251.
4.2.8. Anti-proliferative activity for HeLa cells
Proliferation of HeLa cells in the presence of RE derivatives
was evaluated as follows. HeLa cells were plated at 3 x 103
cells/well in 96-well microtiter plates and treated with
compounds at the indicated concentrations. After 48 h treatment,
Alamar BlueTM (Invitrogen) was added to the wells, and the plate
was incubated for 3 h at 37 °C under 5 % CO2 in air.
Fluorescence was measured with a SpectraMax M5 (Molecular
Devices) using a 530 nm-excitation filter and a 590 nm-emission
filter. The IC50 value for each cell line was determined from the
sigmoid dose-response curve using the Origin8J program.
Acknowledgments
We thank the Collaboration Promotion Unit (RIKEN Global
Research Cluster) for HRMS analysis and helpful discussions.
This work was partially supported by Project Funding from
RIKEN and a Grant-in Aid for Scientific Research on Innovative
Area (Grant Number 24102534).
6k (17.4 mg, 72%, tautomeric mixture: 1.44:1 in CDCl3): IR
(neat) 3423, 2929, 2864, 1738, 1645, 1601, 1493, 1452, 1412,
1
1325, 1240, 1163, 1120, 1066, 1018, 823 cm-1; H-NMR (400
MHz, CDCl3) δ 1.56-1.70 (m, 2H), 1.76-1.86 (m, 2H), 2.05-2.13
(m, 1H), 2.34 (brs, 3H), 2.73 (brt, J = 7.4 Hz, 2H), 2.92-3.14 (m,
2H), 3.90-4.01 (m, 2H), 4.50 (br, 1.2H), 4.51 (br, 1.2H), 4.53 (brt,
J = 4.1 Hz, 0.59H), 4.53 (brt, J = 4.1 Hz, 0.58H), 5.12 (s, 2H),
6.86 (brd, J = 8.3 Hz, 1H), 6.92 (brt, J = 6.9 Hz, 1H), 6.96-7.02
(m, 2H), 7.12-7.20 (m, 3H), 7.22-7.27 (m, 1H), 7.54 (brd, J = 8.3
Hz, 2H), 7.64 (brd, J = 8.3 Hz, 2H), 10.3 (br, 0.41H), 11.2 (br,
0.59H); 13C-NMR (100 MHz, CDCl3) δ 21.4, 27.3, 27.4, 27.6,
29.6, 29.8, 46.8, 47.2, 61.6, 61.7, 69.0, 80.7, 82.5, 90.4, 91.9,
111.6, 121.1, 124.1 (m), 124.2, 124.3, 125.5 (m), 127.0, 127.1,
127.2, 127.8, 127.9, 129.1, 129.2, 129.3, 129.4, 129.6 (m), 130.1,
130.3, 130.4, 134.5, 134.6, 139.2, 141.4, 156.1, 171.2, 174.0,
174.5, 175.9, 193.2, 197.5; 19F-NMR (376Hz, CDCl3) δ -62.4;
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Supplementary Material
1H NMR, 13C NMR, and data from biochemical and biological
experiments.