Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28

Article abstract of DOI:10.1016/j.bioorg.2020.104137

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

Full text of DOI:10.1016/j.bioorg.2020.104137

Journal Pre-proofs
Discovery of new benzhydrol biscarbonate esters as potent and selective
apoptosis inducers of human melanomas bearing the activated ERK pathway:
SAR studies on an ERK MAPK signaling modulator, ACA-28
Ryosuke Satoh, Naoya Hamada, Ami Yamada, Yuki Kanda, Fumihiro
Ishikawa, Teruaki Takasaki, Genzoh Tanabe, Reiko Sugiura
PII:
S0045-2068(20)31434-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104137
YBIOO 104137
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 November 2019
5 June 2020
22 July 2020
Please cite this article as: R. Satoh, N. Hamada, A. Yamada, Y. Kanda, F. Ishikawa, T. Takasaki, G. Tanabe, R.
Sugiura, Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human
melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104137
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Discovery of new benzhydrol biscarbonate esters as potent and
selective apoptosis inducers of human melanomas bearing the
activated ERK pathway: SAR studies on an ERK MAPK
signaling modulator, ACA-28
Ryosuke Satoha, Naoya Hamadaa, Ami Yamadab, Yuki Kandaa, Fumihiro Ishikawab,
Teruaki Takasakia, Genzoh Tanabeb,* and Reiko Sugiuraa,*
a Laboratory of Molecular Pharmacogenomics and b Laboratory of Organic Chemistry,
Faculty of Pharmacy, Kindai University, Kowakae 3-4-1, Higashiosaka City, Osaka,
577-8502, Japan.
Email addresses:
satohr@phar.kindai.ac.jp (R Satoh)
1933420001u@kindai.ac.jp (N Hamada)
1511610091j@kindai.ac.jp (A Yamada)
1745110004k@kindai.ac.jp (Y Kanda)
ishikawa@phar.kindai.ac.jp (F Ishikawa)
takasaki@phar.kindai.ac.jp (T Takasaki)
g-tanabe@phar.kindai.ac.jp (G Tanabe)
sugiurar@phar.kindai.ac.jp (R Sugiura)
*Corresponding author. Laboratory of Molecular Pharmacogenomics, Faculty of
Pharmacy, Kindai University, Kowakae 3-4-1, Higashiosaka City, Osaka 577-8502,
Japan.
Email address: sugiurar@phar.kindai.ac.jp (R Sugiura)
*Corresponding author. Laboratory of Organic Chemistry, Faculty of Pharmacy, Kindai
University, Kowakae 3-4-1, Higashiosaka City, Osaka 577-8502, Japan.
Email address: g-tanabe@phar.kindai.ac.jp (G Tanabe)
Color should be used for Graphical abstract and Table 1.
1

Discovery of new benzhydrol biscarbonate esters as potent and
selective apoptosis inducers of human melanomas bearing the
activated ERK pathway: SAR studies on an ERK MAPK
signaling modulator, ACA-28
Ryosuke Satoha, Naoya Hamadaa, Ami Yamadab, Yuki Kandaa, Fumihiro Ishikawab,
Teruaki Takasakia, Genzoh Tanabeb,* and Reiko Sugiuraa,*
a Laboratory of Molecular Pharmacogenomics and b Laboratory of Organic Chemistry,
Faculty of Pharmacy, Kindai University, Kowakae 3-4-1, Higashiosaka City, Osaka,
577-8502, Japan.
Highlight
・ ACA-28 (2a), an ERK-dependent apoptosis inducer preferentially kills melanoma
cells.
・Fifteen analogs of 2a were synthesized and analyzed for the anti-melanoma activity.
・This is the first SAR study to enhance selectivity and potency of 2a.
・2b and 2c have much higher selectivities and potencies to kill melanoma cells than 2a.
・The size of alkyl group at C-O-carbonate unit was crucial for cancer selectivity.
2

ABSTRACT
The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills
human melanoma cell lines by inducing ERK-dependent apoptosis has generated
significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here,
we successfully developed candidates (2b, 2c) both of which induce more potent and
selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the
structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic
prospect of these candidates against ERK-dependent cancers represented by melanoma.
Graphical abstract
An ERK signaling modulator [ACA-28 (2a)] preferentially kills melanoma cells by inducing ERK-dependent apoptosis. The first SAR
study revealed that new candidates (2b, 2c) induce more potent and selective apoptosis towards melanoma cells than did 2a.
O
Less selective
More potent
More selective
O
OR
2j: R = C6H13
O
IC50 (NHEM) = 8.7 μM
IC50 (SK-MEL-28) = 4.9 μM
2b: R = C2H5, 2c: R = C3H7
H3CO
O
IC50 (NHEM) = 10.0 ~ 10.1 μM
IC50 (SK-MEL-28) = 2.8 μM
SR = 1.8
ACA-28 (2a): R = CH3
modification
modification
SR = 3.6
IC50 (NHEM) = 10.1 μM
IC50 (SK-MEL-28) = 5.3 μM
2k: R = C8H17, 2l: R = C10H21
IC50 (NHEM) = >30 μM
IC50 (SK-MEL-28) = 7.5 ~ >30 μM
SR = 1.9
IC50 (NHEM)
IC50 (SK-MEL-28)
NHEM: human epidermal melanocytes
SK-MEL-28: human melanoma cells
SR = N/A
Selective Ratio (SR) =
Keywords:
ERK MAPK; Anti-melanoma activity; Apoptosis; ACA-28; SAR Study; Benzhydrol
derivatives
Abbreviations
ERK
ACA
SAR
Extracellular signal-regulated kinase
1'-Acetoxychavicol acetate
Structure-activity-relationship
MAPK Mitogen-activated protein kinase
RAS
Rat sarcoma
MEK
Mitogen-activated protein/extracellular signal-regulated kinase kinase
BRAF B-Raf proto-oncogene, serine/threonine kinase
3

NRAS Neuroblastoma ras viral oncogene homolog
HER2 Human epidermal growth factor receptor 2
ErbB2 V-erb-b2 erythroblastic leukemia viral oncogene homolog 2
NHEM Normal human epidermal melanocytes
SR
Selective ratio
IC50
PTSA
50% inhibitory concentration
Polar total surface area
DMEM Dulbecco’s Modified Eagle Medium
FBS Fetal bovine serum
4

1. Introduction
The RAS/RAF/MEK/ERK pathway plays a key role in regulating cell
proliferation, differentiation, and growth by transducing extracellular signals [1]. This
pathway is often activated in various tumours. Hyperactivation of the
RAS/RAF/MEK/ERK MAPK pathway has been reported in human cancer as a result of
the abnormal activation of receptor tyrosine kinases or gain-of-function mutations,
mainly in the RAS or RAF genes [2]. In fact, activation of ERK leads cells to acquire
many of the hallmarks of cancer, including uncontrolled proliferation and resistance to
apoptosis. Therefore, targeting and inhibiting the ERK signaling pathway is seen as an
attractive option to overcome the malignant phenotypes.
In particular, constitutive activation of RAF/MEK/ERK signaling is prevalent
in human melanomas, largely due to activating mutations in BRAF or NRAS genes,
which cause hyperactivation of ERK [2-4]. Melanoma represents the most aggressive and
the deadliest form of skin cancer, and the prognosis of patients with metastatic melanoma
is grim, with a 5-year survival rate between 5–19%. The poor prognosis is partly due to
its resistance to chemotherapies, such as Dacarbazine. Recently, the use of compounds
targeting components of the ERK signaling pathway, including two RAF inhibitors
[Vemurafenib (Zelboraf®)] and [dabrafenib (Tafinlar®)], and a MEK inhibitor
[Trametinib (Mekinist®)] has led to substantial improvements in clinical outcomes in the
treatment of melanoma. However, response rates are highly variable, and the efficacy of
these drugs is primarily limited by the development of drug resistance. Both intrinsic and
acquired resistance to RAF and MEK inhibitors are frequently associated with the
persistence of ERK signaling in the presence of this spectrum of drugs. Therefore, the
development of innovative approaches that exert selective cytotoxicity for cancer cells is
highly desirable.
Recently, we have established a cell-based yeast phenotypic screen [5-7] for
identifying compounds that modulate MAPK signaling by utilizing the phenotype of
calcineurin knockout cells and its reversal by MAPK modulation [8]. Our chemical
screening system successfully identified a benzhydrol-type analog [ACA-28 (2a)] of 1'-
acetoxychavicol acetate [ACA (1)], a natural seed with various biological properties
including antitumor and anti-inflammatory activities [9,10]. Notably, [ACA-28 (2a)]
turned out to serve as an agent to preferentially kill melanoma cells with hyperactive ERK,
including SK-MEL-2 and SK-MEL-28 [11]. Importantly, 2a specifically induced
5

apoptosis via selective stimulation of the ERK phosphorylation in melanoma cells and
HER2/ErbB2 overproducing cells wherein ERK signaling is hyperactivated [11]. The
unique feature of ACA-28 to induce "ERK-dependent apoptosis" represents a conceptual
breakthrough, which contrasts with the current strategy to develop ERK signaling
inhibitors for cancer therapy. To the best of our knowledge, no studies focusing on the
structure-activity-relationship (SAR) aspect of the ERK-dependent apoptosis inducers
have been studied to date. Here, we report, as the first SAR studies of 2a, the
pharmacological evaluation of 2a analogs on their abilities to selectively inhibit cell
growth of melanoma cells by inducing ERK-dependent apoptosis.
Although 2a preferentially inhibited the growth of melanoma cells, it also
affects the cell viability of normal human epidermal melanocytes (NHEM), particularly
at concentrations higher than 10 M (Fig. 1) [11]. This prompted us to improve the
potency and selectivity of 2a towards cancer cells and thus spare normal cells. For this
purpose, it is imperative to analyze the structural basis of ACA-28, which provides
selective ERK-dependent apoptosis. In order to evaluate the selectivity of the compound,
the ratio of IC50 values of a compound in terms of growth inhibition against melanoma
and NHEM cells was calculated (hereafter referred to as “selective ratio (SR)”). By
focusing on two carbonate areas at the position of C4 and C in the structure of 2a, fifteen
analogs (2b–2n, 3a, and 3n) were synthesized (Fig. 1). As the results of their biological
evaluation, seven derivatives (2b–2e, 2g, 2i, and 3a) were shown to inherit the unique
biological activity of 2a to preferentially inhibit the growth of melanoma cells, but with
enhanced potency and selectivity than did 2a. Importantly, 2b and 2c induced cancer-
specific ERK-dependent apoptosis with much higher potencies than did 2a. These
findings suggest 2b and 2c as new lead compounds to combat ERK-dependent cancers
such as melanomas.
2. Results and discussion
First, fifteen analogs (2b–2n, 3a, and 3n) were synthesized following procedure depicted
in Scheme 1. Commercially available 4-hydroxybenzophenone (4) was treated with
methyl chloroformate and propionyl chloride to give the corresponding esters [5a (R =
OCH3), 5b (R = C2H5)] in good yields. Subsequently, NaBH4 reduction of 5a and 5b were
conducted under the pH-controlled conditions, because the yield of a target (6a) was
reduced by hydrolysis of the base labile phenolic ester function when 5a was treated under
6

the usual NaBH4 reduction conditions. As a result, the benzhydrol intermediates (6a, 6b)
were obtained in 80%-91% yields. Finally, acylation of the secondary hydroxyl of 6a and
6b with appropriate acylating reagent afforded targets (2b–2n, 3a) with different acyl
moieties at the two oxygens. The other candidate (3n) having two propionyl moieties was
synthesized in good yield by NaBH4 reduction of 4, followed by acylation of the resultant
diol (7) with propionyl chloride. All the synthetic compounds were characterized by the
1
combination of H and 13C NMR spectroscopy, infra-red spectroscopy, and high-
resolution mass spectrometry.
To evaluate and compare the potency and selectivity of the synthesized ACA-
28 analog compounds (2b–2n, 3a, and 3n), IC50 values, and selective ratios (SRs) of each
compound in SK-MEL-28 cells and NHEM cells were calculated (Table 1). Although the
effects on cell growth inhibition of 2a against SK-MEL-28 cells have previously been
reported,11 its activities were re-evaluated in comparison with newly synthesized
candidates under the same experimental conditions. As shown in Fig. 2, 2a killed
approximately 60% of SK-MEL-28 cells at 6 M concentration (IC50 = 5.3 M), whereas
about 80% of NHEM cells maintained viability (IC50 = 10.1 M), indicating that 2a
exhibited selectivity for the growth inhibition of melanoma cells (SR = 1.9). Also, the
preferential growth inhibition of SK-MEL-28 cells over the NHEM cells was observed
when cells were treated with 6 to 15 M concentrations of 2a.
Analysis of the IC50 values for the series of C4-methyl carbonate-type analogs
(2b–2n) revealed that three compounds (2b, 2c, and 2e), which have C2 or C3 alkyl
moiety as a component of the carbonate unit at the C carbon, exhibited stronger
inhibition of SK-MEL-28 cells than did 2a at 6 M concentration (average IC50 = 2.8–
3.2 M). In contrast, the NHEM cells treated with 2b, 2c, and 2e, maintained viability
at a similar degree as that with 2a (IC50: 2a 10.1 M 2b 10.0 M, 2c 10.1 M, 2e 10.9
M), indicating that 2b, 2c, and 2e achieved higher selectivity against melanoma cells
(SR >3.0). Especially, the potencies of 2b and 2c to inhibit melanoma cell growth were
outstanding with IC50 values of less than 3 M (IC50: 2b 2.8 M, 2c 2.8 M).
Approximately 60% of melanoma cells were killed upon 3 M treatment of 2b and 2c,
whereas NHEM cells exhibited a markedly high survival rate of up to 90% (IC50: 2b 10.0
M, 2c 10.1 M) (SR = 3.6). Interestingly, despite 2f having a propargyl group, which is
constructed by three carbons, 2f exhibited a low potency and a poor selectivity for the
inhibition of melanoma cells (IC50 = 5.5 M and the SR = 2.2). Although substitution
7

with the C4 or C6 alkyl group maintained moderate anti-melanoma activities of 2g, 2h,
2i, and 2j (IC50: 2g 3.8 M, 2h 4.2 M, 2i 4.3 M, 2j 4.9 M), their potencies were
inferior to those of 2b-2e. Further elongation by the introduction of n-octyl (C8) and n-
decyl (C10) groups led to a significant reduction in anti-melanoma activity as evidenced
by the high IC50 values of 2k (7.5 M) and 2l (>30 M) on melanoma cell growth. Thus,
it was noteworthy that the introduction of C2 or C3 alkyl groups to the carbonate moiety
at the C carbon, by enhancing its hydrophobic property, contributes to preferentially
improve the activity of this series of compounds against melanoma cells while
maintaining the viability of NHEM cells. The size of alkyl groups (C2 or C3) and/or their
hydrophobic property of alkyl groups were seemed to involve in the onset of good cell
selectivity. The boiled egg analysis [12] using calculated physicochemical properties,
such as lipophilicity (logP) and polar total surface area (PTSA), of the thirteen candidates
(2a-2m) by using the SwissADME server [13] suggested that nine (2a-2i) compounds
were blood-brain-barrier permeable and the remaining four (2j-2m) were absorbable in
the gastrointestinal tract (Fig. S1, S2). This would suggest that a delicate balance between
WlogP (from 3.38 to 4.16) and PTSA (71.1) values was important for achieving both
excellent potency and selectivity towards SK-MEL-28 cells (Table S1). Additionally,
although WlogP (4.16) and PTSA (71.1) values of 2c and 2d were the same with each
other, a significant difference was detected with respect to their SR (2c/2d = 3.6/2.9). The
bulkiness of the substituent was suggested to contribute to the intensity of the activity and
selectivity.
Next, three isosteres (2n, 3a, and 3n) of 2a were tested. The results showed the
following potencies: 3a showed substantially similar anti-melanoma activity (IC50 = 4.1
M) and a cell-selectivity (SR = 2.1) compared with those of 2a, thus indicating that
bioisosteric replacement of the oxygen atom of the carbonate unit at the C4 carbon of 2a
with methylene unit has little effect on the potency and cell-selectivity.
In contrast, a similar modification regarding the carbonate unit at the C carbon
of 2a resulted in a significant reduction in the cell growth inhibition ability of 2n towards
SK-MEL-28 (IC50 = 13.3 M) and NHEM cells (IC50 = 29.0 M). Compound (3n) also
showed similar cytotoxic properties to that of 2n.
Although the potencies of 2n and 3n against melanoma cells were weakened as compared
with 2a by replacing the oxygen atom of carbonate unit at the C carbon with methylene
unit, the SR was maintained (≒ 2). We have previously shown that 2a induced selective
8

cell growth inhibition against multiple melanoma cell lines, including SK-MEL-28, SK-
MEL-2, and MeWo, all of which have the highly activated ERK MAPK signaling as
compared with the normal melanocyte. To investigate if the newly synthesized fifteen
analogs (2b–2n, 3a, and 3n) of 2a also exhibited selectivity against melanomas, we
calculated the IC50 values and SRs of the fifteen analogs against SK-MEL-2 and MeWo
cells in comparison with NHEM cells. The results are substantially similar to those
obtained with SK-MEL-28 cells and further corroborated the conclusion that 2b and 2c
have superior anti-melanoma activity with a good cell-selectivity (Fig. S3 and Table S2).
As aforementioned, four C4-carbonate analogs 2b and 2c exhibited prominent
cytotoxicity for melanoma cells (IC50 <3 M) with a high SR (=3.6). We, therefore,
selected 2b and 2c to further analyze their ability to induce ERK-dependent apoptosis in
comparison with those of the prototype compound ACA-28 (2a), and a less potent and
non-selective derivative (2l). As shown in Figure 3A (upper panel), the immunoblotting
analysis showed that 2b and 2c significantly stimulated ERK and ELK1 (a transcription
factor as an established ERK substrate) phosphorylation in SK-MEL-28 cells, but not in
NHEM cells. The levels of ERK and ELK1 phosphorylation upon 2b and 2c treatment in
SK-MEL-28 cells seemed higher than those upon 2a. In contrast, the activation of ERK
by a less-potent and non-selective compound 2l remained unchanged. In order to
quantitatively evaluate the ability of these compounds to induce ERK and ELK1
phosphorylation, the relative phosphorylation levels in SK-MEL-28 cells were quantified
(Fig. 3A lower panel). The effect of 2b on ERK activation was far exceeded 150% (up to
180%), as compared with 2a, which induced approximately 150% activation when
phosphorylation levels upon DMSO treatment were set as 100%. In particular, 2c
achieved approximately 210% ERK activation. More importantly, the ERK activation
induced by 2b and 2c in NHEM cells was almost undetectable, again confirming the
highly selective nature of 2b and 2c to induce melanoma-specific ERK activation.
Moreover, significant cleavage of the Caspase-3 concomitant with ERK
activation was observed in SK-MEL-28 cells treated with 2a, 2b, or 2c, but not with
DMSO or 2l, indicating that 2b or 2c, similar to 2a induced ERK-dependent apoptosis.
The induction of apoptosis was stronger in cells treated with 2b or 2c as compared with
2a, further proving that 2b and 2c are more potent inducers of ERK-dependent apoptosis
in SK-MEL-28 cells than 2a. In contrast, the cleaved Caspase-3 was barely detectable in
NHEM cells treated with all the compounds examined, thus confirming that 2b and 2c,
9

similarly to 2a, induced cancer-specific apoptosis. Importantly, MEK inhibitor U0126
attenuated the cleavage of Caspase-3 by 2b and 2c, suggesting these compounds induce
apoptosis through the activation of ERK signaling (Fig 3B). We also performed cell-
based assays of apoptotic cells to investigate the effects of the ACA derivatives (2a, 2b,
and 2c) and Cisplatin (positive control) as an apoptosis inducer (Fig. S4). As a result,
both of the two cell-based assays, (Caspase-Glo® 3/7 Assay System: Fig. S4, upper panel,
and RealTime Glo Annexin V Apoptosis Assay: Fig. S4, lower panel), reproduced the
results of the caspase cleavage assays confirming the impact of ACA derivatives (2a, 2b,
and 2c) to induce apoptosis, which is significantly stronger than that by Cisplatin (at 10
μM). Altogether, 2b and 2c were more specific and potent inducers of ERK-dependent
apoptosis than 2a.
Finally, we also performed the in vitro kinase assay experiments to investigate
the effects of the ACA derivatives (2b and 2l, a good and a poor ERK signaling stimulator,
respectively), on the ERK kinase activity using ELK1, a transcription factor as an
established ERK substrate (Fig. 4). As a result, we obtained the data showing the effect
of 2b, but not 2l, to stimulate ERK kinase activity in vitro, which correlated well with the
ERK phosphorylation levels in melanoma cells. Thus, although the mechanism and
targets of ACA derivatives remain unknown, these results confirmed that ACA
derivatives modulate ERK phosphorylation levels, which leads to biochemical ERK
kinase activity. Regarding the experiments to investigate the binding ability of the
compounds on the components of the ERK signaling pathway, including ERK MAPK,
will be important issues for future study.
3. Conclusion
In summary, with the aim of developing new therapeutic agents for targeting
the ERK signaling pathway, here we performed the first SAR study of ACA-28 (2a) to
develop its ability to selectively inhibit cancer cell growth by inducing apoptosis with
ERK hyperactivation, by focusing on two methyl carbonate moieties at C4- and C-
oxygens of 2a. The substitution of the methyl group of the carbonate unit at the C-
oxygen with the other alkyl groups longer than eight carbons (2k and 2l) was not
preferable for the anti-melanoma activity. Compounds (2a–2j) bearing the alkyl groups
with the carbons-length less than six were favorable for the onset of the potent activity.
The optimal substitution pattern for the activity was obtained when the alkyl group was
10

two or three carbons (2b–2e) in length, indicating that 2b–2e were potent apoptosis
inducers in melanoma. Particularly, the ethylated and n-propylated analogs (2b and 2c)
showed good activity (IC50 = 2.8 M) in this series of candidates. It was noteworthy that
2b and 2c exhibited higher selectivity ratios between SK-MEL-28 and NHEM cells than
2a. Regarding the bioisosteric replacement of oxygen atoms at both carbonate units of 2a
with methylene moiety, the bioisostere (3a) was revealed to show potent activity, with
the same degree as those of 2b. On the other hand, regarding the bioisosteric replacements
of oxygen atoms at C-carbonate units of 2a and 3a with methylene moiety, the
bioisosteres (2n and 3n) significantly decreased the activity against SK-MEL-28.
As aforementioned, this first SAR study on ACA-28 (2a) is a significant step
forward for the development of new anti-cancer measures against ERK-dependent
cancers. However, critical questions remain, including the identification of far more
potent and selective inducers of ERK-dependent apoptosis than 2b and 2c, as well as
clarification of the mechanisms underlying ACA-28-induced cancer cell killing to
establish therapeutic measures to combat incurable diseases associated with deteriorated
ERK signaling. Performing further SAR studies, and creating appropriate probes to find
out what are the potential molecular targets of ACA-28 (2a) are currently underway in
our laboratory.
4. Experimental
4.1. General experimental procedures
Mps were determined on a hot-stage melting point apparatus and are uncorrected. IR
spectra were measured on an FT-IR spectrophotometer. NMR spectra were recorded on
an FT-NMR spectrometer (1H, 400, 500, or 800 MHz; 13C, 125, or 200 MHz). Chemical
shifts () and coupling constants (J) are given in ppm and Hz, respectively.
Tetramethylsilane (TMS) was used as an internal standard for 1H NMR measurements in
CDCl3, whereas 13C NMR measurements utilized the solvent signal (77.0 ppm) of CDCl3
for this purpose. For the measurement of 1H and 13C NMR spectra in CD3OD, the solvent
signals 33.1 ppm and 49.0 ppm were used, respectively, as internal standards. Low-
resolution and high-resolution mass spectra were recorded on an orbitrap mass
spectrometer (ESI). All the organic extracts were dried over anhydrous Na2SO4 prior to
evaporation.
11

4.1.1. Acylation of 4-hydroxybenzophenone (4)
4.1.1.1. Benzophenone-4-yl methyl carbonate (5a)
Methyl chloroformate (1.6 mL, 20.7 mmol) was added to a solution 4-
hydroxybenzophenone (4, 1.98 g, 10 mmol) in dry pyridine (20 mL) at 0 °C. After being
stirred at 0 °C for 1 h, the reaction mixture was poured into with ice-water (100 mL) and
the resulting mixture was extracted with ethyl acetate (50 mL x 1, 30 mL x 2). The extract
was successively washed with 10% ice-cooled sulfuric acid, aqueous sodium hydrogen
carbonate and brine and condensed in vacuo to give a colorless solid (2.57 g), which on
recrystallization from a mixture of n-hexane and ethyl acetate, gave the title compound
(5a, 2.42 g, 95%) as colorless needles, mp. 95–96 °C, lit. [14] mp. 94–94 °C. IR (KBr):
1762, 1651 cm-1. 1H NMR (500 MHz, CDCl3) δ: 3.94 (3H, s, OCH3), 7.31 (2H, dm, J =
8.9, arom.), 7.49 (2H, tm, J = 7.4, arom.), 7.60 (1H, tm, J = 7.4, arom.), 7.80 (2H, dm, J
= 7.4, arom.), 7.87 (2H, dm, J = 8.9, arom.).13C NMR (125 MHz, CDCl3) δ: 55.6 (OCH3),
120.9/128.3/129.9/131.7/132.5 (d, arom.), 135.3/137.4/153.6/154.1 (s, arom. and
CH3OCO2C6H4), 195.4 (C6H4COC6H5). HRMS (ESI) m/z: [M+Na]+ Calcd for
C15H12O4Na 279.0628; Found 279.0631.
4.1.1.2. Benzophenone-4-yl propionate (5b)
According to the method used for the preparation of 5a, 4-hydroxybenzophenone (4, 300
mg, 1.52 mmol) was treated with propionyl chloride (264 mL, 3.02 mmol) in dry pyridine
(3 mL) at 0 °C for 30 min. Work-up gave a colorless oil (395 mg), which on column
chromatography (CHCl3) gave the title compound (5b, 369 mg, 96%) as a colorless
viscous oil. IR (neat): 1767, 1659 cm-1. 1H NMR (500 MHz, CDCl3) δ: 1.29 (3H, t, J =
7.6, CH2CH3), 2.64 (2H, q, J = 7.6, CH2CH3), 7.22 (2H, dm, J = 8.9, arom.), 7.49 (2H,
tm, J = 7.4, arom.), 7.60 (1H, tm, J = 7.4, arom.), 7.80 (2H, dm, J = 7.4, arom.), 7.86 (2H,
13
dm, J = 8.9, arom.). C NMR (125 MHz, CDCl3) δ: 9.0 (CH2CH3), 27.8 (CH2CH3),
121.5/128.3/129.9/131.7/132.4 (d, arom.), 134.9/137.5/154.0 (s, arom.), 172.5
(C2H5CO2C6H4), 195.6 (C6H4COC6H5) HRMS (ESI) m/z: [M+Na]+ Calcd for
C16H14O3Na 277.0835; Found 277.0837.
4.1.2. NaBH4 reduction of 4-acyloxybenzophenone (5a and 5b)
4.1.2.1 Benzhydrol-4-yl methyl carbonate (6a)
To a mixture of benzophenone-4-yl methyl carbonate (5a, 1.09 g, 4.26 mmol),
12

tetrahydrofuran (24 mL) and acetate buffer (pH = 5, 4 mL) was added NaBH4 (177 mg,
4.65 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h and then stirred at room
temperature for another 30 min. The reaction mixture was poured into with ice-water (50
mL), and the resulting mixture was extracted with ethyl acetate (50 mL x 1, 30 mL x 2).
The extract was washed with brine and condensed in vacuo to give a colorless oil (1.06
g), which on column chromatography (CHCl3) gave the titled compound (6a, 1.00 g,
91%) as a colorless oil. IR (neat): 3421, 1767 cm-1. 1H NMR (500 MHz, CDCl3) δ: 2.34
(1H, d, J = 3.1, OH), 3.89 (3H, s, OCH3), 5.83 (1H, d, J = 3.1, CHOH), 7.13 (2H, dm, J
= 8.7, arom.), 7.25–7.41 (7H, m, arom.). 13C NMR (125 MHz, CDCl3) δ: 55.4 (OCH3),
75.6 (CHOH), 121.0/126.5/127.65/127.73/128.6 (d, arom.), 141.6/143.4/150.3 (s, arom.),
154.2 (CH3OCO2). HRMS (ESI) m/z: [M+Na]+ Calcd for C15H14O4Na 281.0784; Found
281.0788.
4.1.2.2. Benzhydrol-4-yl propionate (6b)
According to the method used for the preparation of 6a, benzophenone-4-yl propionate
(5b, 350 mg, 1.38 mmol) was treated with NaBH4 (52 mg, 1.38mmol) in a mixture of
tetrahydrofuran (8 mL) and acetate buffer (pH = 5, 2 mL) at 0 °C for 2 h. Work-up gave
a colorless oil (430 mg), which on column chromatography (CHCl3→CHCl3/CH3OH,
20:1) gave the titled compound (6b, 282 mg, 80%) as a colorless oil. IR (neat): 3449,
1759 cm-1. 1H NMR (500 MHz, CDCl3) δ: 1.25 (3H, t, J = 7.6, COCH2CH3), 2.31 (1H,
d, J = 3.0, OH), 2.57 (2H, q, J = 7.6, COCH2CH3), 5.83 (1H, d, J = 3.0, CHOH), 7.40
(2H, dm, J = 8.6, arom.), 7.27 (1H, m, arom.), 7.31–7.41 (6H, m, arom.). 13C NMR (125
MHz, CDCl3) δ: 9.0 (COCH2CH3), 27.7 (COCH2CH3), 75.7 (CHOH),
121.5/126.5/127.6/127.7/128.5 (d, arom.), 141.2/143.5/150.0 (s, arom.), 173.0
(C2H5CO2). HRMS (ESI) m/z: [M+Na]+ Calcd for C16H16O3Na 279.0992; Found
279.0995.
4.1.3. Acylation of compound (6a)
4.1.3.1. Ethyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2b)
Ethyl chloroformate (200 L, 2.1 mmol) was added to a solution of 6a (271 mg, 1.05
mmol) in dry pyridine (2 mL) at 0 °C. After being stirred at 0 °C for 1.5 h, the reaction
mixture was poured into ice-water (20 mL), and the resulting mixture was extracted with
ethyl acetate (10 mL x 3). The extract was successively washed with 10% ice-cooled
13

sulfuric acid, aqueous sodium bicarbonate, and brine and condensed in vacuo to give a
colorless solid (320 mg), which on column chromatography (n-hexane–AcOEt, 10/1)
gave the title compound (2b, 298 mg, 86%) as colorless needles (from n-hexane), mp.
1
69–70 °C. IR (KBr): 1774, 1751 cm-1. H NMR (500 MHz, CDCl3) δ: 1.31 (3H, t, J =
7.0, OCH2CH3), 3.89 (3H, s, OCH3), 4.02 (2H, q, J = 7.0, OCH2CH3), 6.70 (1H, s,
CHOCO2), 7.16 (2H, dm, J = 8.7, arom.), 7.27–7.41 (7H, m, arom.). 13C NMR (125 MHz,
CDCl3) δ: 14.2 (OCH2CH3), 55.4 (OCH3), 64.3 (OCH2CH3), 79.9 (CHOCO2),
121.1/126.9/128.2/128.3/128.6 (d, arom.), 137.6/139.3/150.7 (s, arom.), 154.1
(CH3OCO2C6H4), 154.4 (CHOCO2C2H5). HRMS (ESI) m/z: [M+Na]+ Calcd for
C18H18O6Na 353.0996; Found 353.0996.
4.1.3.2. 4-(Methoxycarbonyloxy)benzhydryl n-propyl carbonate (2c)
According to the method used for the preparation of 2b, 6a (208 mg, 0.81 mmol) was
treated with n-propyl chloroformate (280 L, 2.48 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (282 mg), which on column chromatography
(CHCl3) gave the title compound (2c, 239 mg, 86%) as a colorless viscous oil. IR (neat):
1
1767, 1748 cm-1. H NMR (500 MHz, CDCl3) δ: 0.95 (3H, t, J = 7.4, OCH2CH2CH3),
1.69 (2H, qt, J = 7.4, 6.7, OCH2CH2CH3), 3.90 (3H, s, OCH3), 4.10 (2H, t, J = 6.7,
OCH2CH2CH3), 6.70 (1H, s, CHOCO2), 7.15 (2H, dm, J = 8.5, arom.), 7.28–7.37 (5H,
m, arom.), 7.39 (2H, dm, J = 8.5, arom.). 13C NMR (125 MHz, CDCl3) δ: 10.1
(OCH2CH2CH3), 22.0 (OCH2CH2CH3), 55.4 (OCH3), 69.9 (OCH2CH2CH3), 79.9
(CHOCO2), 121.1/126.9/128.2/128.3/128.6 (d, arom.), 137.6/139.3/150.7 (s, arom.),
n
154.1 (CH3OCO2C6H4O), 154.6 (CHOCO2 C3H7). HRMS (ESI) m/z: [M+Na]+ Calcd for
C19H20O6Na 367.1152; Found 367.1147.
4.1.3.3. Isopropyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2d)
According to the method used for the preparation of 2b, 6a (207 mg, 0.80 mmol) was
treated with isopropyl chloroformate (290 L, 2.53 mmol) in dry pyridine (2 mL) at 0 °C
for 2 h. Work-up gave a colorless oil (280 mg), which on column chromatography
(CHCl3) gave the title compound (2d, 229 mg, 83%) as a colorless viscous oil. IR (neat):
1
1767, 1744 cm-1. H NMR (500 MHz, CDCl3) δ: 1.286/1.294 [each 3H, d, J = 6.2,
OCH(CH3)2], 3.89 (3H, OCH3), 4.88 [1H, sept, J = 6.2, 6.2, OCH(CH3)2], 6.69 (1H, s,
CHOCO2), 7.15 (2H, dm, J = 8.7, arom.), 7.27–7.36 (5H, m, arom.), 7.39 (2H, dm, J =
14

8.7 arom.). 13C NMR (125 MHz, CDCl3) δ: 21.8 [OCH(CH3)2], 55.4 (OCH3), 72.4
[OCH(CH3)2], 79.7 (CHOCO2), 121.1/126.9/128.2/128.3/128.6 (d, arom.),
iso
137.7/139.5/150.7 (s), 154.0/154.1 (CH3OCO2C6H4 and CHOCO2 C3H7). HRMS (ESI)
m/z: [M+Na]+ Calcd for C19H20O6Na 367.1152; Found 367.1148.
4.1.3.4. Allyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2e)
According to the method used for the preparation of 2b, 6a (200 mg, 0.78 mmol) was
treated with allyl chloroformate (270 L, 2.56 mmol) in dry pyridine (2 mL) at 0 °C for
2 h. Work-up gave a colorless oil (278 mg), which on column chromatography (CHCl3)
gave the title compound (2e, 225 mg, 85%) as a colorless waxy solid, mp. 54–55 °C. IR
(KBr): 1767, 1751 cm-1. 1H NMR (500 MHz, CDCl3) δ: 3.89 (3H, s, OCH3), 4.63 (2H,
ddd, J = 5.9, 1.4, 1.1, CH2CH=CH2), 5.26 (1H, ddt, J = 10.5, 1.4, 1.1, CH2CH=CHH),
5.35 (1H, ddt, J = 17.2, 1.4, 1.4 CH2CH=CHH), 5.92 (1H, ddt, J = 17.2, 10.5, 5.9
CH2CH=CH2), 6.70 (1H, s, CHOCO2), 7.16 (2H, dm, J = 8.7, arom.), 7.28–7.36 (5H, m,
arom.), 7.39 (2H, dm, J = 8.7, arom.). 13C NMR (125 MHz, CDCl3) δ: 55.4 (OCH3), 68.7
(CH2CH=CH2), 80.1 (CHOCO2), 119.1 (CH2CH=CH2), 121.1 (CH2CH=CH2),
126.9/128.2/128.3/128.6/131.4 (d, arom.), 137.4/139.2/150.8 (s, arom.), (s), 154.1
(CH3OCO2C6H4), 154.3 (CHOCO2CH2CH=CH2). HRMS (ESI) m/z: [M+Na]+ Calcd for
C19H18O6Na 365.0996; Found 365.0996.
4.1.3.5. 4-(Methoxycarbonyloxy)benzhydryl propagyl carbonate (2f)
According to the method used for the preparation of 2b, 6a (160 mg, 0.62 mmol) was
treated with propagyl chloroformate (180 L, 1.88 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (263 mg), which on column chromatography (n-
hexane–AcOEt, 5/1) gave the title compound (2f, 197 mg, 93%) as a colorless
1
microcrystalline solid, mp. 59–60 °C. IR (KBr): 3291, 2129, 1761 cm-1. H NMR (500
MHz, CDCl3) δ: 2.53 (1H, t, J = 2.6, CH2C≡CH), 3.89 (3H, s, OCH3), 4.73 (2H, d, J =
2.6, CH2C≡CH), 6.71 (1H, s, CHOCO2), 7.16 (2H, dm, J = 8.9, arom.), 7.28–7.36 (5H,
m, arom.), 7.39 (2H, dm, J = 8.9, arom.). 13C NMR (125 MHz, CDCl3) δ: 55.4 (OCH3),
55.5 (CH2C≡CH), 75.9 (CH2C≡CH), 76.7 (CH2C≡CH), 80.6 (CHOCO2),
121.1/126.9/128.29/128.8.34/128.6 (d, arom.), 137.1/138.9/150.8 (s, arom.), (s),
153.8/154.0 (CH3OCO2C6H4 and CHOCO2CH2C≡CH). HRMS (ESI) m/z: [M+Na]+
Calcd for C19H16O6Na 363.0839; Found 363.0827.
15

4.1.3.6. n-Butyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2g)
According to the method used for the preparation of 2b, 6a (200 mg, 0.78 mmol) was
treated with n-butyl chloroformate (330 L, 2.54 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (272 mg), which on column chromatography
(CHCl3) gave the title compound (2g, 252 mg, 91%) as a colorless viscous oil, IR (neat):
1763, 1745 cm-1. 1H NMR (500 MHz, CDCl3) δ: 0.92 (3H, t, J = 7.4, OCH2CH2CH2CH3),
1.34–1.43 (2H, m, OCH2CH2CH2CH3), 1.61–1.68 (2H, m, OCH2CH2CH2CH3), 3.89 (3H,
s, OCH3), 4.14 (2H, t, J = 6.7, OCH2CH2CH2CH3), 6.69 (1H, s, CHOCO2), 7.15 (2H, dm,
J = 8.7, arom.), 7.27–7.36 (5H, m, arom.), 7.38 (2H, dm, J = 8.7, arom.). 13C NMR (125
MHz, CDCl3) δ: 13.6 (OCH2CH2CH2CH3), 18.9 (OCH2CH2CH2CH3), 30.6
(OCH2CH2CH2CH3), 55.4 (OCH3), 68.2 (OCH2CH2CH2CH3), 79.9 (CHOCO2),
121.0/126.9/128.2/128.3/128.6 (d, arom.), 137.6/139.4/150.8 (s, arom.), 154.1
n
(CH3OCO2C6H4), 154.6 (CHOCO2 C4H9). HRMS (ESI) m/z: [M+Na]+ Calcd for
C20H22O6Na 381.1309; Found 381.1307.
4.1.3.7. tert-Butyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2h)
A mixture of 6a (100 mg, 0.39 mmol), di-tert-butyl carbonate (170 mg, 0.78 mmol), 4-
dimethylaminopyridine (14.2 mg, 0.12 mmol) and dry pyridine (2 mL) was stirred at 0 °C
for 3 h. The reaction mixture was poured into ice-water (20 mL) and the resulting mixture
was extracted with ethyl acetate (10 mL x 3). The extract was successively washed with
10% ice-cooled sulfuric acid, aqueous sodium bicarbonate and brine and condensed in
vacuo to give a colorless oil (156 mg), which on column chromatography (n-hexane–
AcOEt, 10/1) gave the title compound (2h, 122 mg, 89%) as a colorless microcrystalline
1
solid, mp. 87–88 °C. IR (neat): 1767, 1743 cm-1. H NMR (500 MHz, CDCl3) δ: 1.47
[9H, s, OC(CH3)3], 3.88 (3H, s, OCH3), 6.64 (1H, s, CHOCO2), 7.15 (2H, dm, J = 8.6,
13
arom.), 7.27–7.36 (5H, m, arom.), 7.38 (2H, dm, J = 8.6, arom.). C NMR (125 MHz,
CDCl3) δ: 27.7 [OC(CH3)3], 55.4 (OCH3), 79.1 (CHOCO2), 82.6 [OC(CH3)3],
121.0/126.9/128.0/128.2/128.6 (d, arom.), 137.9/139.6/150.6 (s), 152.8/154.1
(CH3OCO2C6H4 and CHOCO2tertC4H9) HRMS (ESI) m/z: [M+Na]+ Calcd for
C20H22O6Na 381.1309; Found 367.1297.
4.1.3.8. Isobutyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2i)
16

According to the method used for the preparation of 2b, 6a (200 mg, 0.78 mmol) was
treated with isobutyl chloroformate (330 L, 2.51 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (275 mg), which on column chromatography
(CHCl3) gave the title compound (2i, 236 mg, 85%) as a colorless viscous oil, IR (neat):
1767, 1748 cm-1. 1H NMR (800 MHz, CDCl3) δ: 0.94 [6H, d, J = 6.7, OCH2CH(CH3)2],
1.98 (1H, nonet, J = 6.7, OCH2CH(CH3)2], 3.90 (3H, s, OCH3), 3.93 (2H, d, J = 6.7,
OCH2CH(CH3)2], 6.70 (1H, s, CHOCO2), 7.16 (2H, dm, J = 8.7, arom.), 7.29–7.37 (5H,
m, arom.), 7.39 (2H, dm, J = 8.7, arom.). 13C NMR (200 MHz, CDCl3) δ: 18.9
[OCH2CH(CH3)2], 27.8 [OCH2CH(CH3)2], 55.4 (OCH3), 77.4 [OCH2CH(CH3)2], 79.9
(CHOCO2), 121.1/126.9/128.2/128.3/128.6 (d, arom.), 137.6/139.4/150.8 (s), 154.1
iso
(CH3OCO2C6H4), 154.7 (CHOCO2 C4H9. HRMS (ESI) m/z: [M+Na]+ Calcd for
C20H22O6Na 381.1309; Found 367.1309.
4.1.3.9. n-Hexyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2j)
According to the method used for the preparation of 2b, 6a (202 mg, 0.78 mmol) was
treated with n-hexyl chloroformate (407 L, 2.50 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (317 mg), which on column chromatography
(CHCl3) gave the title compound (2j, 268 mg, 89%) as a colorless viscous oil. The oil
turned into a waxy solid on keeping in the freezer, mp. 38–39 °C. IR (neat): 1767, 1748
cm-1. 1H NMR (500 MHz, CDCl3) δ: 0.88 [3H, t, J = 7.0, OCH2CH2(CH2)3CH3], 1.24–
1.39 [6H, m, OCH2CH2(CH2)3CH3], 1.62–1.70 [2H, m, OCH2CH2(CH2)3CH3], 3.89 (3H,
s, OCH3), 4.13 [2H, t, J = 6.8, OCH2CH2(CH2)3CH3], 6.69 (1H, s, CHOCO2), 7.15 (2H,
dm, J = 8.7, arom.), 7.27–7.37 (5H, m, arom.), 7.39 (2H, dm, J = 8.7, arom.). 13C NMR
(125 MHz, CDCl3) δ: 13.9 [OCH2(CH2)4CH3], 22.5/25.3/28.6/31.4 [OCH2(CH2)4CH3],
55.4 (OCH3), 68.5 [OCH2(CH2)4CH3], 79.9 (CHOCO2), 121.1/126.9/128.2/128.3/128.6
(d, arom.), 137.6/139.4/150.8 (s, arom.), 154.1 (CH3OCO2C6H4), 154.6
n
(CHOCO2 C6H13). HRMS (ESI) m/z: [M+Na]+ Calcd for C22H26O6Na 409.1622; Found
409.1617.
4.1.3.10. 4-(Methoxycarbonyloxy)benzhydryl n-octyl carbonate (2k)
According to the method used for the preparation of 2b, 6a (202 mg, 0.78 mmol) was
treated with n-octyl chloroformate (500 L, 2.54 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (329 mg), which on column chromatography
17

(CHCl3) gave the title compound (2k, 272 mg, 84%) as a colorless viscous oil, IR (neat):
1767, 1748 cm-1. 1H NMR (500 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.2,
OCH2CH2(CH2)5CH3], 1.22–1.38 (10H, m, OCH2CH2(CH2)5CH3], 1.62–1.70 (2H, m,
OCH2CH2(CH2)5CH3], 3.90 (3H, s, OCH3), 4.13 (2H, t, J = 6.7, OCH2CH2(CH2)5CH3],
6.70 (1H, s CHOCO2), 7.15 (2H, dm, J = 8.7, arom.), 7.27–7.37 (5H, m, arom.), 7.39 (2H,
dm, J = 8.7, arom.). 13C NMR (125 MHz, CDCl3) δ: 14.1 [OCH2(CH2)6CH3],
22.6/25.6/28.6/29.10/29.13/31.7
[OCH2(CH2)6CH3],
55.4
(OCH3),
68.5
[OCH2(CH2)6CH3], 79.8 (CHOCO2), 121.1/126.9/128.2/128.3/128.6 (d, arom.),
n
137.6/139.3/150.7 (s, arom.), 154.0 (CH3OCO2C6H4), 154.6 (CHOCO2 C8H17). HRMS
(ESI) m/z: [M+Na]+ Calcd for C24H30O6Na 437.1935; Found 437.1933.
4.1.3.11. n-Decyl 4-(methoxycarbonyloxy)benzhydryl carbonate (2l)
According to the method used for the preparation of 2b, 6a (200 mg, 0.78 mmol) was
treated with n-decyl chloroformate (575 L, 2.50 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (350 mg), which on column chromatography
(CHCl3) gave the title compound (2l, 308 mg, 90%) as a colorless viscous oil, IR (neat):
1763, 1748 cm-1. 1H NMR (800 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.2,
OCH2CH2(CH2)7CH3], 1.21–1.36 (14H, m, OCH2CH2(CH2)7CH3], 1.63–1.69 (2H, m,
OCH2CH2(CH2)7CH3]), 3.89 (3H, s, OCH3), 4.13 (2H, t, J = 6.7, OCH2CH2(CH2)7CH3],
6.69 (1H, s, CHOCO2), 7.15 (2H, dm, J = 8.7, arom.), 7.27–7.37 (5H, m, arom.), 7.38
13
(2H, dm, J = 8.7, arom.). C NMR (200 MHz, CDCl3) δ: 14.1 [OCH2(CH2)8CH3],
22.7/25.6/28.6/29.2/29.3/29.46/29.48/31.9 [OCH2(CH2)8CH3], 55.4 (OCH3), 68.5
[OCH2(CH2)8CH3], 79.9 (CHOCO2), 121.1/126.9/128.2/128.3/128.6 (d, arom.),
n
137.6/139.4/150.8 (s, arom.), 154.1 (CH3OCO2C6H4), 154.6 (CHOCO2 C10H21). HRMS
(ESI) m/z: [M+Na]+ Calcd for C26H34O6Na 465.2248; Found 465.2249.
4.1.3.12. 4-(Methoxycarbonyloxy)benzhydryl phenyl carbonate (2m)
According to the method used for the preparation of 2b, 6a (200 mg, 0.78 mmol) was
treated with phenyl chloroformate (316 L, 2.50 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless viscous oil (312 mg), which on column chromatography
(CHCl3) gave the title compound (2m, 244 mg, 83%) as a colorless microcrystalline solid,
mp. 86–87 °C. IR (neat): 1763 cm-1. 1H NMR (800 MHz, CDCl3) δ: 3.90 (3H, s, OCH3),
6.79 (1H, s, CHOCO2), 7.16 (2H, dm, J = 7.3, arom.), 7.19 (2H, dm, J = 8.7, arom.), 7.23
18

(1H, tm, J = 7.3, arom.), 7.33 (1H, tm, J = 7.3, arom.), 7.35–7.39 (4H, m, arom.), 7.41
13
(2H, dm, J = 7.3), 7.44 (2H, dm, J = 8.7, arom.). C NMR (200 MHz, CDCl3) δ: 55.4
(OCH3), 80.9 (CHOCO2), 121.0/121.2/126.1/127.0/128.4/128.5/128.7/129.4 (d, arom.),
137.1/138.9/150.9/151.1 (s, arom.), 152.9/154.1 (CH3OCO2C6H4 and CHOCO2C6H5).
HRMS (ESI) m/z: [M+Na]+ Calcd for C22H18O6Na 401.0996; Found 401.0996.
4.1.3.13. 4-(Methoxycarbonyloxy)benzhydryl propionate (2n)
Propionyl chloride (203 L, 2.32 mmol) was added to a solution of 6a (200 mg, 0.78
mmol) in dry pyridine (2 mL) at 0 °C. After being stirred at 0 °C for 1 h, the reaction
mixture was poured into ice-water (30 mL), and the resulting mixture was extracted with
ethyl acetate (10 mL x 3). The extract was successively washed with 10% ice-cooled
sulfuric acid, aqueous sodium bicarbonate, and brine and condensed in vacuo to give a
colorless oil (380 mg), which on column chromatography (CHCl3) gave the title
compound (2n, 219 mg, 90%) as a colorless viscous oil. IR (neat): 1767, 1740 cm-1. 1H
NMR (500 MHz, CDCl3) δ: 1.17 (3H, t, J = 7.6, COCH2CH3), 2.45 (3H, t, J = 7.6,
COCH2CH3), 3.89 (3H, s, OCH3), 6.89 (1H, s, CHOCO), 7.14 (2H, dm, J = 8.6, arom.),
13
7.27–7.39 (7H, m, arom.). C NMR (125 MHz, CDCl3) δ: 9.02 (COCH2CH3), 27.8
(COCH2CH3), 55.4 (OCH3), 75.9 (CHOCO), 121.0/127.0/128.0/128.3/128.5 (d, arom.),
138.2/139.9/150.5 (s, arom.), 154.1 (CH3OCO2C6H4), 172.3 (CHOCOC2H5). HRMS
(ESI) m/z: [M+Na]+ Calcd for C18H18O5Na 337.1046; Found 337.1047.
4.1.3.14. Methyl 4-propionyloxybenzhydryl carbonate (3a)
According to the method used for the preparation of 2b, 6b (240 mg, 0.94 mmol) was
treated with methyl chloroformate (180 L, 2.32 mmol) in dry pyridine (2 mL) at 0 °C
for 1 h. Work-up gave a colorless oil (302 mg), which on column chromatography
(CHCl3) gave the title compound (3a, 246 mg, 84%) as a colorless viscous oil. IR (neat):
1
1755 (absorptions due to two carbonyl stretching overlap) cm-1. H NMR (500 MHz,
CDCl3) δ: 1.24 (3H, t, J = 7.6, COCH2CH3), 2.56 (2H, t, J = 7.6, COCH2CH3), 3.78 (3H,
s, OCH3), 6.70 (1H, s, CHOCO2), 7.06 (2H, dm, J = 8.6, arom.), 7.27–7.39 (7H, m, arom.).
13C NMR (125 MHz, CDCl3) δ: 8.98 (COCH2CH3), 27.7 (COCH2CH3), 54.9 (OCH3),
80.2 (CHOCO2), 121.6/126.8/128.2(2C)/128.6 (d, arom.), 137.0/139.3/150.4 (s, arom.),
155.0 (CHOCOCH3), 172.8 (C2H5CO2C6H4). HRMS (ESI) m/z: [M+Na]+ Calcd for
C18H18O5Na 337.1046; Found 337.1051.
19

4.1.4. 4-Hydroxybenzhydrol (7)
4-Hydroxybenzophenone (4, 1.98 g, 10 mmol) was treated with NaBH4 (1.51 g, 40 mmol)
in a mixture of THF (30 mL) and H2O (10 ml). While the mixture was heated under reflux
for 9 h, the color of the mixture was gradually changed from pale yellow to colorless. The
reaction mixture was poured into with ice-cooled water (100 mL), and the reaction was
quenched by the addition of NH4Cl. The resulting mixture was extracted with ethyl
acetate (100 mL x 1, 50 mL x 2). The extract was washed with brine and condensed in
vacuo to give the practically pure title compound (7, 1.99 g, quant.) as a colorless
microcrystalline solid, mp. 169–170 °C, lit. [15] mp. 159–162 °C. 1H NMR (400 MHz,
CDCl3) δ: 2.14 (1H, d, J = 2.8, OH), 4.77 (1H, s OH), 5.80 (1H, d, J = 2.8, CHOH), 7.20–
7.40 (9H, m, arom.).
4.1.5. 4-Hydroxybenzhydrol dipropionate (3n)
According to the method used for the preparation of 2n, 7 (200 mg, 1.0 mmol) was treated
with propionyl chloride (262 L, 3.0 mmol) in dry pyridine (2 mL) at 0 °C for 2 h. Work-
up gave a colorless oil (319 mg), which on column chromatography (CHCl3) gave the
title compound (3n, 284 mg, 91%) as a colorless viscous oil. IR (neat): 1759, 1743 cm-1.
1H NMR (500 MHz, CDCl3) δ: 1.17 (3H, t, J = 7.6, CHOCOCH2CH3), 1.25 (3H, t, J =
7.6, C6H4OCOCH2CH3), 2.44 (2H, t, J = 7.6, CHOCOCH2CH3), 2.57 (2H, t, J = 7.6,
C6H4OCOCH2CH3), 6.89 (1H, s, CHOCO2), 7.05 (2H, dm, J = 8.6, arom.), 7.26–7.36
13
(7H, m, arom.). C NMR (125 MHz, CDCl3) δ: 9.03 (CHOCOCH2CH3), 27.7/27.8
(CHOCOCH2CH3), 76.3 (CHOCO2), 121.5/127.0/127.9/128.2/128.5 (d, arom.),
137.8/140.1/150.3 (s, arom.), 172.8/173.3 (COC2H5). HRMS (ESI) m/z: [M+Na]+ Calcd
for C19H20O4Na 335.1254; Found 335.1256.
4.2. Cell culture
SK-MEL-28 cells were obtained from the JCRB Cell Bank of National Institutes of
Biomedical Innovation, Health and Nutrition (Japan). NHEM cells were purchased from
KURABO Bio-Medical department (Japan). SK-MEL-28 and NHEM cells were fed with
the culture medium of Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai tesque,
Japan) supplemented with 10% fetal bovine serum (FBS) (Gibco® Thermo Fisher
Scientific, USA) and that of DermaLife® M Comp kit (KURABO, Japan), respectively.
20

The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C.
4.3. WST-8 assay
WST-8 assay was performed as previously described [11].
4.4. Protein expression and Western blot analysis
Protein expression and Western blot analysis were performed as previously described
[11]. The following primary antibodies were used anti-α-tubulin (Sigma-Aldrich, USA),
anti-caspase-3 (full length and cleaved Caspase-3), anti-phospho-p44/42 MAPK
Thr202/Tyr204 (phospho-ERK1/2), anti-p44/42 MAPK (total ERK1/2), anti-phospho-
ELK1 Ser383 (phospho-ELK1) and anti-ELK1 (total ELK1) (Cell Signaling Technology,
USA). “Relative quantification (%)” was calculated by dividing the phosphorylated
protein (phospho-ERK1/2 or phospho-ELK1) intensity by the total protein (total ERK1/2
or total ELK1) intensity.
4.5. Cell-based apoptosis assay
Apoptosis-inducing activity of the compounds was detected using the Caspase-Glo® 3/7
Assay System (Promega, USA) and the RealTime Glo Annexin V Apoptosis Assay
(Promega, USA) according to the manufacturer’s instructions. 2 × 104 cells/well were
plated in a 96-well white microplate and incubated at 37°C for 24 hours. After the
incubation, 2× compounds diluted by the medium were added into each well at a final
concentration of 1×. After the additional 48 hours incubation, Caspase-Glo® 3/7 Reagent
or 2× Detection Reagent was added into each well, and the plate was incubated for 30
min or 180 min, respectively. After the incubation, the luminescence was measured using
a CentroXS3 LB960 plate reader (Berthold, Germany).
4.6. Immunoprecipitation
HeLa cells that have been co-transfected with pFLAG-CMV2-ERK1 and pFLAG-
CMV2-ERK2 were lysed in TNE buffer (20 mM Tris– HCl (pH 7.5), 150 mM NaCl, 2
mM EDTA, 1% NP-40, 50 mM sodium fluoride) with a mixture of protease inhibitors (1
mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, and 10
μg/ml leupeptin) and a mixture of phosphatase inhibitors (5 mM sodium fluoride, 5 mM
Na3PO4, 10 mM Na2H2P2O7, 10 mM Na2MoO4). The lysates were centrifuged and FLAG-
21

fused proteins in the supernatants were immunoprecipitated using an EZview™ Red
ANTI-FLAG® M2 Affinity Gel (Merck Millipore, USA).
4.7. In vitro kinase assay
In vitro kinase reactions consisted of immunoprecipitated proteins (FLAG-ERK1 and
FLAG-ERK2), 5 μg/mL recombinant GST-His fused human ELK1 protein (Sino
Biological, China), 1× kinase buffer (Cell Signaling Technology, USA) and 20 μM ATP
(Nacalai tesque, Japan). Mixtures were incubated at 37°C for 30 min then the reactions
were stopped by adding 3× SDS-PAGE loading buffer.
Author contributions
GT and R Sugiura designed this project. R Satoh, NH, AY, FI, TT, GT carried out
experiments. R Satoh, NH, AY, YK, FI, TT, GT and R Sugiura analyzed the data. R Satoh,
GT and R Sugiura wrote the manuscript. All authors reviewed the manuscript.
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgments
We would like to thank Dr. William Figoni for critical reading of the manuscript.
Formatting of funding sources
This study was supported by the MEXT-Supported Program for the Strategic Research
Foundation at Private Universities, 2014–2018 [S1411037 (R Sugiura)], a Grant-in-Aid
for scientific research from the Japan Society for the Promotion of Science [JSPS
(KAKENHI), 19H03376 (R Sugiura), 17K08377 (GT), 20K06494 (R Satoh)], a grant
from the Hoansha Foundation, 2017–2020 (GT) and by the Antiaging Project for Private
Universities (R Sugiura and GT).
References
[1] M. Qi and E. A. Elion, MAP kinase pathways, J. Cell Sci. 118 (2005) 3569-3572.
[2] Y. Pylayeva-Gupta, E. Grabocka and D. Bar-Sagi, RAS oncogenes: weaving a
tumorigenic web, Nat. Rev. Cancer 11 (2011) 761-774.
22

[3] P. M. Pollock and P. S. Meltzer, A genome-based strategy uncovers frequent BRAF
mutations in melanoma, Cancer Cell 2 (2002) 5-7.
[4] A. Gorden, I. Osman, W. Gai, D. He, W. Huang, A. Davidson, A. N. Houghton, K.
Busam and D. Polsky, Analysis of BRAF and N-RAS mutations in metastatic melanoma
tissues, Cancer Res. 63 (2003) 3955-3957.
[5] R. Sugiura, T. Toda, H. Shuntoh, M. Yanagida and T. Kuno, pmp1+, a suppressor of
calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast, EMBO
J. 17 (1998) 140-148.
[6] R. Sugiura, A. Kita, Y. Shimizu, H. Shuntoh, S. O. Sio and T. Kuno, Feedback
regulation of MAPK signalling by an RNA-binding protein, Nature 424 (2003) 961-965.
[7] R. Satoh, Y. Matsumura, A. Tanaka, M. Takada, Y. Ito, K. Hagihara, M. Inari, A.
Kita, A. Fukao, T. Fujiwara, S. Hirai, T. Tani and R. Sugiura, Spatial regulation of the
KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export
system in Schizosaccharomyces pombe, Mol. Microbiol. 104 (2017) 428-448.
[8] R. Sugiura, A. Kita, N. Tsutsui, O. Muraoka, K. Hagihara, N. Umeda, T. Kunoh, H.
Takada and D. Hirose, Acremomannolipin A, the potential calcium signal modulator with
a characteristic glycolipid structure from the filamentous fungus Acremonium strictum,
Bioorg. Med. Chem. Lett. 22 (2012) 6735-6739.
[9] M. Ohnishi, T, Tanaka, H. Makita, T. Kawamori, H. Mori, K. Satoh, A. Hara, A.
Murakami, H. Ohigashi and K. Koshimizu, Chemopreventive effect of a xanthine oxidase
inhibitor, 1'-acetoxychavicol acetate, on rat oral carcinogenesis, Jpn. J. Cancer Res. 87
(1996) 349-356.
[10] Y. Nakamura, A. Murakami, Y. Ohto, K. Torikai, T. Tanaka and H. Ohigashi,
Suppression of tumor promoter-induced oxidative stress and inflammatory responses in
mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate, Cancer Res.
58 (1998) 4832-4839.
[11] R. Satoh, K. Hagihara, K. Matsuura, Y. Manse, A. Kita, T. Kunoh, T. Masuko, M.
Moriyama, H. Moriyama, G. Tanabe, O. Muraoka and R. Sugiura, Identification of ACA-
28, a 1'-acetoxychavicol acetate analogue compound, as a novel modulator of ERK
MAPK signaling, which preferentially kills human melanoma cells, Genes Cells 22
(2017) 608-618.
[12] A. Daina and V. Zoete, A BOILED-Egg To Predict Gastrointestinal Absorption and
Brain Penetration of Small Molecules, ChemMedChem 11 (2016) 1117-1121.
23

[13] A. Daina, O. Michielin and V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules,
Sci. Rep. 7 (2017) doi:10.1038/srep42717.
[14] E. Fischer, Carbomethoxy Derivatives of Phenolcarboxylic Acids and Their
Utilization for Syntheses. III, Ber. Deutsch Chem. Ges. 42 (1909) 1015–1022.
[15] D. Lie and W. Peter, Chemistry of photogenerated -phenyl-substituted o-, m-, and
p-quinone methides from phenol derivatives in aqueous solution, Can. J. Chem. 86 (2008)
105–118.
24

O
O
O
R
O
CH3

O
O
4
H3CO
O
H3C
O
2
ACA (1)
O
a : R = OCH3 (ACA-28)
b : R = OC2H5
k: R = OCH2(CH2)6CH3
l : R = OCH2(CH2)8CH3
m : R = OC6H5
c : R = OCH2CH2CH3
d : R = OCH(CH3)2
e : R = OCH2CH=CH2
f : R = OCH2C≡ CH
g: R = OCH2(CH2)2CH3
h: R = OC(CH)3
O
R
n : R = C2H5
O
H3CH2C
O
3
i: R = OCH2CH(CH3)2
j : R = OCH2(CH2)4CH3
a : R = OCH3
n : R = CH2CH3
Fig. 1. Chemical structures of ACA (1), ACA-28 (2a) and analogs (2b–2n, 3a, and 3n)
of 2a
O
O
ClCO2CH3 or C2H5COCl
O
pyridine, 0 °C
R
O
(95%-96%)
HO
5
4
a : R = OCH3, b : R = C2H5
NaBH4
THF/H2O, 50 °C
NaBH4, THF,
acetate buffer (pH 5.0), 0°C
(80%-91%)
(quant.)
OH
OH
O
R
O
6
HO
7
a : R = OCH3, b : R = C2H5
ClCO2R', Boc2O or C2H5COCl
pyridine, 0 °C
R' = (CH2)nCH3 (n = 0, 1, 2, 3, 5, 7, 9)
(CH2)nCH(CH3)2 (n = 0, 1),
CH2CH=CH2, CH2CH≡CH, C6H5
(83%-91%)
ClCO2CH3
pyridine, 0 °C
C2H5COCl
pyridine, 0 °C
(91%)
(84%)
2b-2n
3a
3n
Scheme 1. Syntheses of analogs (2b–2n, 3a, and 3n) of 2a
25

120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
2a
SR = 1.9
2b
SR = 3.6
2c
SR = 3.6
2d
SR = 2.9
NHEM
SK-MEL-28
(μM)
(μM)
(μM)
(μM)
30
0
3
6
10
IC50(NHEM)=10.1 μM
IC50(SK-MEL-28)=5.3 μM
15
30
0
3
6
10
IC50(NHEM)=10.0 μM
IC50(SK-MEL-28)=2.8 μM
15
30
0
3
6
10
IC50(NHEM)=10.1 μM
IC50(SK-MEL-28)=2.8 μM
15
30
0
3
6
10
IC50(NHEM)=8.4 μM
IC50(SK-MEL-28)=2.9 μM
15
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
2e
SR = 3.4
2f
SR = 2.2
2g
SR = 2.1
2h
SR = 1.3
(μM)
(μM)
(μM)
(μM)
30
0
3
6
10
IC50(NHEM)=10.9 μM
IC50(SK-MEL-28)=3.2 μM
15
30
0
3
6
10
IC50(NHEM)=12.3 μM
IC50(SK-MEL-28)=5.5 μM
15
30
0
3
6
10
IC50(NHEM)=8.1 μM
IC50(SK-MEL-28)=3.8 μM
15
30
0
3
6
10
IC50(NHEM)=5.5 μM
IC50(SK-MEL-28)=4.2 μM
15
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
2i
SR = 2.6
2j
SR = 1.8
2k
SR = N/A
2l
SR = N/A
0
3
6
10
IC50(NHEM)=11.0 μM
IC50(SK-MEL-28)=4.3 μM
15
30 (μM)
0
3
6
10
IC50(NHEM)=8.7 μM
IC50(SK-MEL-28)=4.9 μM
15
30 (μM)
0
3
6
10
IC50(NHEM)>30 μM
IC50(SK-MEL-28)=7.5 μM
15
30 (μM)
0
3
6
10
IC50(NHEM)>30 μM
IC50(SK-MEL-28)>30 μM
15
30 (μM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
2m
SR = 1.4
2n
SR = 2.2
3a
SR = 2.1
3n
SR = 1.9
(μM)
(μM)
(μM)
(μM)
30
0
3
6
10
IC50(NHEM)=13.9 μM
IC50(SK-MEL-28)=10.0 μM
15
30
0
3
6
10
IC50(NHEM)=29.0 μM
IC50(SK-MEL-28)=13.3 μM
15
30
0
3
6
10
IC50(NHEM)=8.7 μM
IC50(SK-MEL-28)=4.1 μM
15
30
0
3
6
10
IC50(NHEM)=21.7 μM
IC50(SK-MEL-28)=11.4 μM
15
Fig. 2. Cell growth inhibition induced by the compounds against melanoma cells (SK-
MEL-28) and normal melanocytes (NHEM). Viability of NHEM and SK-MEL-28 after
exposure to the indicated concentrations of the compounds for 48 h, expressed as the
relative percentage against that of DMSO treatment (0 μM). Columns, means; bars, SD;
SR, selective ratio; n = 7.
26

NHEM
SK-MEL-28
A
-
2a
2l
2b
2c
-
2a
2l
2b
2c
phospho-ERK1/2
total ERK1/2
phospho-ELK1
total ELK1
full length Caspase-3
cleaved Caspase-3
Tubulin
**
250
200
150
100
50
**
-
2a 2l 2b 2c
0
NHEM
SK-MEL-28
(n=5)
**
*
350
300
250
200
150
100
50
-
2a 2l 2b 2c
0
NHEM
-
SK-MEL-28
(n=5)
U0126
B
-
2b
2c
-
2b
2c
phospho-ERK1/2
total ERK1/2
full length Caspase-3
cleaved Caspase-3
Tubulin
27

Fig. 3. 2b and 2c are superior apoptosis inducers than 2a (ACA-28) in melanoma cells.
(A) Cleavage of Caspase-3 was more effectively induced after treatment with 2b or 2c as
compared with that with 2a in melanoma cells (upper panel). Relative quantification of
ERK1/2 MAPK phosphorylation and ELK1 phosphorylation in NHEM and SK-MEL-28
cells upon each compound treatment (lower panel). Phosphorylation levels
(phosphorylated protein intensity/total protein intensity) after treatment with DMSO (-)
were set as 100% in each cell. Columns, means; bars, SE; n = 5. **P < 0.01, *P < 0.05,
significantly different from 2a by Williams’ test. (B) MEK inhibitor U0126 attenuates
2b- and 2c-induced apoptosis. Simultaneous treatment of 10 μM U0126 reversed 2b- and
2c-induced phosphorylation of ERK1/2 and apoptosis in SK-MEL-28 cells.
FLAG-ERK1/2
FLAG-ERK1/2
-
-
+
-
-
+
-
2l 2b
-
2l
2b
-
2l 2b
250
200
150
100
50
phospho-ELK1
total ELK1
*
300
200
100
0
phospho-ERK1/2
total ERK1/2
0
(n=3)
(n=3)
Fig. 4. Immunoprecipitated FLAG-ERK1/2 derived from HeLa cells that were treated
with 2b phosphorylates the target protein ELK1 in vitro. Relative quantification of ELK1
phosphorylation (center panel). Phosphorylation levels (phosphorylated protein
intensity/total protein intensity) of no addition of FLAG-ERK1/2 were set as 100%.
Relative quantification of ERK1/2 phosphorylation (right panel). Phosphorylation levels
of using FLAG-ERK1/2 derived from DMSO treated HeLa cells were set as 100%.
Columns, means; bars, SE; n = 3. *P < 0.05, significantly different from 2a by Williams’
test.
28

Table 1. IC50 values of compounds
Compound
IC50 (NHEM)
10.1 μM
10.0 μM
10.1 μM
8.4 μM
IC50 (SK-MEL-28)
5.3 μM
Selective Ratio (SR)
2a (ACA-28)
1.9
3.6
3.6
2.9
3.4
2.2
2.1
1.3
2.6
1.8
N/A
N/A
1.4
2.2
2.1
1.9
2b
2c
2d
2e
2f
2.8 μM
2.8 μM
2.9 μM
10.9 μM
12.3 μM
8.1 μM
3.2 μM
5.5 μM
2g
2h
2i
3.8 μM
5.5 μM
4.2 μM
11.0 μM
8.7 μM
4.3 μM
2j
4.9 μM
2k
2l
>30 μM
>30 μM
13.9 μM
29.0 μM
8.7 μM
7.5 μM
>30 μM
10.0 μM
13.3 μM
4.1 μM
2m
2n
3a
3n
21.7 μM
11.4 μM
SR= 3.5~
IC50 (SK-MEL-28)= ~3.0 μM
IC50 (SK-MEL-28)= 3.1~4.0 μM
IC50 (SK-MEL-28)= 6.1~10.0 μM
IC50 (SK-MEL-28)= 10.1 μM~
SR= 2.5~3.4
SR= 1.5~1.9
SR= ~1.4
29