Synthesis of Bioactive Diarylheptanoids from Alpinia officinarum and Their Mechanism of Action for Anticancer Properties in Breast Cancer Cells

Article abstract of DOI:10.1021/acs.jnatprod.0c01012

An efficient synthesis of the Alpinia officinarum-derived diarylheptanoids, viz., enantiomers of a β-hydroxyketone (1) and an α,β-unsaturated ketone (2) was developed starting from commercially available eugenol. Among these, compound 2 showed a superior

Full text of DOI:10.1021/acs.jnatprod.0c01012

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Article
Synthesis of Bioactive Diarylheptanoids from Alpinia officinarum
and Their Mechanism of Action for Anticancer Properties in Breast
Cancer Cells
Sunita Gamre,# Mrityunjay Tyagi,# Sucheta Chatterjee, Birija S. Patro, Subrata Chattopadhyay,
and Dibakar Goswami*
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ABSTRACT: An efficient synthesis of the Alpinia officinarum-derived diarylheptanoids, viz., enantiomers of a β-hydroxyketone (1)
and an α,β-unsaturated ketone (2) was developed starting from commercially available eugenol. Among these, compound 2 showed
a superior antiproliferative effect against human breast adenocarcinoma MCF-7 cells. Besides reducing clonogenic cell survival,
compound 2 dose-dependently increased the sub G1 cell population and arrested the G2-phase of the cell cycle, as revealed by flow
cytometry. Mechanistically, compound 2 acts as an intracellular pro-oxidant by generating copious amounts of reactive oxygen
species. Compound 2 also induced both loss of mitochondrial membrane potential (MMP) as well as lysosomal membrane
permeabilization (LMP) in the MCF-7 cells. The impaired mitochondrial and lysosomal functions due to reactive oxygen species
(ROS)-generation by compound 2 may contribute to its apoptotic property.
anticancer properties in vitro,5b−e as well as antiviral
ne of the most challenging targets in modern science is
activities.5i−l
Oto find a cure for cancer, a disease with a high mortality
Breast cancer is the most common cancer and also the
leading cause of cancer mortality in women worldwide.6a
Although early diagnosis of breast cancer has reduced the
death rates in the long term,6b there is certainly a need for
better therapeutics. In India, the highest mortality rate is
associated with breast cancer patients among women.6c The
MeOH extract of A. officinarum has shown antiproliferative
activity against MCF-7 cells, in a dose- and time-dependent
manner.6d More recently, the EtOH extract of A. galangal was
reported to also induce apoptotic killing of MCF-7 cells, albeit
with significantly reduced cytotoxicity.6e Diarylheptanoids
rate even in developed countries. Among different avenues,
surgery and radiation therapy have enhanced overall survival in
patients with localized tumors. However, in the case of cancer
metastasis where the therapy needs to reach every part of the
body, chemotherapy remains the focus of attention.1 Although
combinatorial synthesis coupled with molecular docking
studies have led to many synthetic drugs for chemotherapy,
natural products still hold a stake of more than 60% of all the
FDA approved chemotherapeutic drugs.2 Secondary metabo-
lites from plants not only led to new cytotoxic chemicals but
also have given important clues toward structural refinements
for developing better chemotherapeutic drugs.3 The natural
diarylheptanoids isolated from the Zingiber, Curcuma, Alpinia,
Alnus, and Myrica genera have gained increasing attention due
to their numerous physiological activities,4a including anti-
cancer, anti-HIV, and anti-inflammatory properties.4 The
diarylheptanoids 1 and 2 (Figure 1), extracted from the
roots of Alpinia officinarum Hance,5a have shown considerable
Received: September 16, 2020
Published: February 15, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Chemical structures of the diarylheptanoids 1 and 2.
Scheme 1. Retrosynthesis of (R)- and (S)-1 and 2
a
Scheme 2. Resolution of Homoallylic Alcohol 7
a
(i) TBSCl/Im/DMAP/CH2Cl2/25 °C/3 h; (ii) BH3·Me2S/THF/0 °C, then 25 °C/4 h, then H2O2/NaOH/25 °C/ 2 h; (iii) PCC/CH2Cl2/25
°C/3 h; (iv) allyl bromide/Zn/aqueous THF/0 °C, then 25 °C/3 h; (v) amano lipase PS/DIP ether/vinyl acetate/25 °C/18 h.
possessing a catechol moiety, isolated from the EtOH extract
of A. officinarum, showed cytotoxicity against the human breast
adenocarcinoma MCF-7 cell line with IC50 values of 97.2 μM
and 20.4 μM.6f Compound (R)-1, cytotoxic against neuro-
blastoma (IMR32) cells,5h in contrast, was found to have no
antiproliferative activity against the MCF-7 cell line.6g Overall,
there are limited bioevaluations of pure compounds from
Alpinia against human breast cancer cells, while the results with
the plant extracts are substantially different. Mechanistic
studies on the anticancer action of the diarylheptanoids from
Alpinia are lacking. During our search for dietary small
molecules with attractive antiproliferative activities, encourag-
ing results were found with a few natural diarylnonanoids,
especially the malabaricones that could induce apoptotic death
of several cancer cells without showing animal toxicity.7 Hence,
in continuation of our endeavor toward exploring dietary small
molecules for strong antiproliferative activities, the relative
antiproliferative efficacies of compounds 1 and 2 against the
MCF-7 cells were evaluated and the mode of action of the best
candidate was assessed.
Chirality plays an important role in development of
anticancer drugs.8a−d From this perspective, compound 1 is a
suitable choice for a detailed structure−activity relationship
study and to determine whether the carbinol stereogenic
center in 1 plays any role in its anticancer activity against the
MCF-7 cell line. Evaluation of their pharmacological activities
required appreciable amounts of the compounds. However,
isolation of these compounds from Alpinia sp. is a laborious
task because of the coexistence of many similar compounds in
the plant extract.9a Also, (S)-1 is unavailable in the plant. The
only successful synthesis of ( )-1 was reported by Shioiri et al.
starting from 3-arylpropionic acid using direct C-acylation with
diethyl phosphorocyanidate as the key step.9b Compound 2
was prepared in a low yield by dehydration of 1 under acidic
conditions. The synthesis is not promising because of usage of
toxic and corrosive diethyl phosphorocyanidate.9c No direct
synthesis of either enantiomerically pure 1 or the conjugated
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ketone 2 has been reported to date. All these prompted
development of a practical and efficient synthesis of both (R)-1
and (S)-1, along with the conjugated ketone 2. Previously, we
have successfully synthesized several bioactive molecules by
employing chemo-enzymatic reactions.10a−f In the present
synthesis of (R)- and (S)-1 also, a biocatalytic reaction as the
key step was adopted. The synthesized compounds were used
for the biological evaluation.
RESULTS AND DISCUSSION
■
Chemistry. The retrosynthetic protocol (Scheme 1) was
developed focusing on the use of readily available, low-cost
starting materials as well as inexpensive reagents along with
operationally simple steps. For the synthesis of 1, it was
planned to open the ring of oxirane ring 1a regioselectively.
The oxirane 1a, in turn, may be obtained from the resolved
enantiomers of 1b, accessible from commercially available
eugenol (3). For the synthesis of 2, a late-step cross metathesis
between 2a and 2b was envisioned. Whereas 2b can be
prepared from 3-phenylpropanol, fragment 2a can be prepared
from eugenol (3).
Figure 2. Configurations of MTPA-esters derived from (R)-7 and
(S)-7.
with meta-chloroperbenzoic acid (mCPBA) (Scheme 3).
Regioselective ring opening of the epoxide with BnMgBr
yielded (R)-10 as a mixture of C-3 epimers. PCC oxidation
gave the ketone (R)-11. Attempts at desilylation with Bu4NF
gave a complex mixture of products which could not be
separated by column chromatography. Desilylation with HF-
pyridine afforded the target compound (R)-1 in 9% overall
yield starting from 3. Using a similar set of reactions, (S)-1 was
synthesized from (S)-7, which, in turn, was prepared by LiAlH4
reduction of acetate (S)-8. The overall yield of (S)-1 starting
from 3 was 6%. The attractive features of the synthesis are (i)
simple reaction conditions, (ii) use of inexpensive reagents and
starting materials, and (iii) its convergent nature wherein the
enantiomeric products of the enzyme-mediated resolution
were both utilized.
For the synthesis of the enantiomers of 1 (Scheme 2), the
phenolic group of eugenol (3) was silylated with tert-
butyldimethylslilyl chloride (TBSCl)/imidazole (Im)/4,4′-
dimethylaminopyridine (DMAP), and the olefinic function of
the resultant compound 4 was subjected to hydroboration-
oxidation to afford the alcohol 5. Its oxidation with pyridinium
chlorochromate (PCC) to the aldehyde 6 followed by Luche’s
allylation11 gave the homoallylic alcohol ( )-7. For its
resolution, the Amano lipase (from Pseudomonas fluorescens)-
catalyzed transesterification ( )-7 with vinyl acetate in
diisopropyl ether (DIP) was attempted. This afforded the
(R)-alcohol 7 and (S)-acetate 8 in 85 and >99% ee,
respectively, after 45% conversion (cf. HPLC). A second
transesterification (12% conversion, cf. HPLC) of the resolved
(R)-7 under the same conditions improved its ee to >96%. The
enantiomeric purities of (R)-7 and (S)-8 were assessed by
chiral-phase HPLC analyses of the purified products using a
chiral-phase OJ-H column and isocratic elution (flow rate 1
mL/min, monitored by absorbance at 220 nm) with
isopropanol−n-hexane 1:99. The absolute configurations of
the products were assigned following Kazlauskas’ empirical rule
that is widely accepted to define for lipase-catalyzed acylation
of secondary carbinols.12a In addition, the % ee values of the
enantiomeric alcohols (R)-7 and (S)-7 were determined from
the relative intensities of the methoxy resonances of the
corresponding α-methoxytrifluoromethyl phenyl acetates
(MTPA), prepared using (R)-MTPA chloride.12b The absolute
configurations of both (R)-7 and (S)-7 could also be
For the synthesis of 2 (Scheme 4), 3-phenylpropan-1-ol
(12) was oxidized with PCC to aldehyde 13. Treatment of 13
with vinylmagnesium bromide gave the racemic alcohol 14,
which was oxidized with PCC to get the conjugated ketone 15.
The aldehyde 6, prepared from eugenol (Scheme 2), was
subjected to a Wittig olefination using methyltriphenylphos-
phonium iodide to afford the olefin 16. The olefin 16 was
desilylated using Bu4NF, and the resultant product subjected
to Hoveyda−Grubbs II catalyzed cross-metathesis with 15 to
afford the target compound 2 in 30% overall yield starting from
the commercially available compound 12. The (E) geometry of
1
2 was confirmed from the J-value (16.0 Hz) of the H NMR
olefinic doublet at δ 6.11. Compounds (R)-1, (S)-1, and 2
1
were unequivocally characterized using H NMR, 13C NMR,
and elemental analysis.9b,13 The purity of the compounds was
determined using HPLC, and >95% pure compounds were
used for the biological assays.
1
ascertained from the H NMR spectra after calculating the
Biological Study. Compound 2 Reduces Viability and
Proliferation of MCF-7 Cells. Initially, the effects of the
alpinoids [(R)-1, (S)-1, and 2] on metabolic viability of MCF-
7 cells were assessed by the MTT assay. The results (Figure 3)
showed that all the test compounds reduced the cell viability
concentration-dependently. Similar activities were observed for
(R)-1 and (S)-1 but were much reduced compared with that of
2. The IC50 values of the enantiomers of 1 were >50 μM, while
that (IC50 = 3.9 0.8 μM) of compound 2 was significantly
lower. This result showed that compound 1 was largely
inactive compared to compound 2. Under the same
experimental conditions, the IC50 value of the positive control,
paclitaxel was 2.1 0.3 μM. Compound 2 was also cytotoxic
to cells of the human osteosarcoma (U2-OS) cell line with an
ΔδRS values of the benzylic as well as homobenzylic protons,
according to the Mosher and modified Mosher methods
(Supporting Information). Figure 2 depicts the two
diaseteromers obtained from (R)-7 and (S)-7, respectively,
1
and the shielding effects that contribute to the H NMR
spectra of both acetates.12c
After each reaction, the insoluble enzyme was recovered by
filtration, the filtrate concentrated in vacuum, and the residue
subjected to silica gel column chromatography to obtain the
individual products. The recovered lipase was reused at least
three times without any significant loss of enzyme activity.
Next, (R)-7 was silylated (TBSCl/Im/DMAP) to give 9, and
its olefinic function converted to the corresponding epoxide
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a
Scheme 3. Synthesis of (R)-1 and (S)-1 .
a
(i) TBDMSCl/Im/CH2Cl2/3 h; (ii) mCPBA/CH2Cl/0 °C, then 25 °C/3 h, then BnMgBr/THF/16 h; (iii) Dess−Martin periodinane/CH2Cl2/0
°C/2 h; (iv) HF−Py/CH3CN−Py/0 °C−25 °C/5 h; (v) LAH/Et2O/0 °C/2 h.
a
Scheme 4. Synthesis of 2
a
(i) PCC/CH2Cl2; (ii) vinylmagnesium bromide/THF/−78 °C; (iii) MePh3PI/n-BuLi/−78 °C/THF; (iv) (a) Bu4NF /THF/0 °C; (b) 15/
Hoveyda−Grubbs II/toluene/reflux.
IC50 value of 10.7 1.4 μM but did not show significant toxic
effects to human normal intestinal (INT-407) cells up to a
concentration of 50 μM. The vehicle (0.1% DMSO) was
nontoxic to all the used cell lines. We also performed MTT
assays using respective compounds without cells, but the test
compounds did not show any significant change in absorbance
at 570 nm, indicating that the test compounds did not react
with MTT at the given experimental conditions, excluding the
chance of false positives. The results with compound (R)-1
corroborates a previously reported IC50 value with the same
cell line.6g Comparison of the MTT data with a previous
report6f with the (R)-1 and 2 analogues bearing a catechol
moiety suggested that introduction of hydroxy groups in the
phenyl ring does not alter the cytotoxicity appreciably. The
results further suggested that the presence of the stereogenic
carbinol center in compound 1 may contribute to its cytotoxic
properties to MCF-7 cells. Additionally, 2 showed impressive
cytotoxicity (IC50 0.1 μM) to the human neuroblastoma IMR-
32 cell line.5h It is generally believed that the presence of a 4-
hydroxy-3-methoxyphenyl moiety improves the cytotoxic
properties of diarylheptanoids.14a However, the MTT assay
results with compounds 1 and 2 indicated that the substituted
phenyl ring may not be a critical structural determinant for the
cytotoxic activities against MCF-7 cells. The superior cytotoxic
effect of 2 compared to the enantiomers of 1 may be attributed
to the presence of an α,β-unsaturated carbonyl moiety in 2, as
reported earlier.14b Previously, it has been suggested that the
α,β-unsaturated carbonyl compounds-mediated toxicity is
attributable to formation of Michael-type adducts with the
nucleophilic sulfhydryl groups of protein thiols.14c,d This may
explain why compound 2 showed better cytotoxicity than the
enantiomers of 1. Since compound 2 was found to be the most
cytotoxic, it was chosen for further studies to establish its
molecular mechanism of action in MCF-7 cells.
Next, the clonogenic assay was carried out to assess the
effect of compound 2 on MCF-7 cells. Compared to the
vehicle treated cells, compound 2 inhibited colony formation
in a concentration dependent manner on the 10th day,
reflecting loss of cell proliferation (Figure 4). Previously, it was
reported that the A. officinarum extract significantly decreased
proliferation of MCF-7 cells in a dose dependent manner. Our
results indicated that the efficacy of crude extracts against
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cells.6d This variation may be due to the presence of other
molecules in the crude extract of A. officinarum.
Compound 2 Induces Oxidative Stress in MCF-7 Cells.
Redox imbalance or ROS (reactive oxygen species) generation
in cancer cells can lead to cell death. DCFH-DA dye is
frequently used to quantify the generation of ROS or oxidative
stress. Compound 2 did not induce ROS generation in the
cells up to 3 h, but when incubated for 6 h, a significant
increase in oxidative stress (measured via DCF fluorescence)
was observed (Figure 6). In this study, H2O2 was used as the
positive control for the assay. The study demonstrated that
compound 2 acts as a pro-oxidant in MCF-7 cells, perturbs cell
signaling mechanisms via ROS-generation, and eventually
induces apoptosis in MCF-7 cells. Curcumin, a representative
diarylheptanoid with antiproliferative activities, has also been
reported to rapidly increase the ROS level in H-ras-
Transformed MCF10A human breast epithelial cells and also
in human breast cancer T-78 cells, but only at a relatively
higher concentration.15a,b It is worth mentioning that ROS
generation at such a low concentration of any diarylheptanoids
is unprecedented. The low IC50 value of compound 2
compared to ∼30 μM of curcumin15c also corroborates this
observation. A similar mechanism for compound 2 has not
been reported so far.
Figure 3. MCF-7 cells (5000 cells per well in 96 well plate) were
treated with vehicle (control) or compounds (R)-1, (S)-1, 2, and
paclitaxel for 72 h and the cell viability was assessed by MTT assay.
The survival results are expressed in percentage considering that of
the untreated control cells as 100. All the experiments were repeated
three times with similar results. All determinations were made in three
replicates, and the values are means SEM. *p < 0.01 and **p <
0.001 (one-way ANOVA) compared to the vehicle control.
Compound 2 Induces Loss of Mitochondrial Membrane
Potential (MMP) in MCF-7 Cells. Loss of mitochondrial
membrane potential (MMP, ΔΨM) is another hallmark of
apoptotic initiator signal, and oxidative stress is known to
impair the mitochondrial activity in cancer cells. Hence, we
assessed the dose dependent effect of compound 2 on MMP
loss using the voltage-sensitive fluorescent cationic dye, JC-1.16
JC-1 exhibits potential-dependent accumulation in mitochon-
dria giving a red fluorescence. In this study, N-(3-
chlorophenyl)carbonohydrazonoyl dicyanide (CCCP), a
known chemical inhibitor of oxidative phosphorylation, was
used as the positive control. The MMP loss was quantified in
terms of a decrease in red fluorescence of the JC-1 dye.
Treatment with compound 2 decreased the red fluorescence of
the cells in a concentration dependent manner (Figure 7).
These results indicated that compound 2 can elicit
mitochondrial dysfunction in MCF-7 cells.
MCF-7 breast cancer cells may be attributable to the presence
of compound 2 as one of the active principles.
Sub-G1 Assay and Cell Cycle Quantification. To check if
the cell death induced by compound 2 is apoptotic, the sub-G1
population of the untreated and treated cells were quantified.
The hypodiploid DNA content (indicating sub-G1 population)
was analyzed as an apoptosis marker by flow cytometry. The
apoptotic nuclei appear as broad hypodiploid DNA peaks
before the G1-phase. Compound 2 increased the apoptotic
cells (Figure 5A) in a concentration-dependent manner (16.3%
at 5 μM and 27.5% at 10 μM), compared to 5.4% in the vehicle
treated cells.
Many established anticancer agents act by inducing DNA
damage followed by cell cycle arrest, which later culminates
into induction of apoptosis. Hence, the effects of compound 2
on cell cycle progression of MCF-7 cells were assessed by flow
cytometry. As shown in Figure 5B, treatment with compound 2
led to arrest of cells in the G2-phase of the cell cycle.
Quantified data of the above experiments are shown in Figure
5C. Together, the results suggested that the compound 2-
mediated cell cycle arrest may be attributable to cell death.
Previously, it has been reported that the MeOH extract of A.
officinarum induces S-phase cell cycle progression of MCF-7
Compound 2 Also Induces Lysosomal Membrane
Permeabilization (LMP). LMP has also emerged as an inducer
of apoptosis. It is associated with lysosomal membrane rupture
leading to leakage of intralysosomal content into the
cytoplasm.17a This can be detected using a lysosomotropic
dyelike acridine orange (AO). Here, LMP was examined from
Figure 4. (A) MCF-7 cells (1000 cells per well) were grown in a 6-well plate and treated with compound 2 (0−5 μM) for 10 days, stained with
0.5% crystal violet, and the colonies of viable cells were photographed and counted. (B) The surviving fraction was calculated considering that of
untreated cells as 1 after correcting the plate efficiency. All the experiments were repeated three times with similar results. All determinations were
made in three replicates and values in the graph are means SEM. *p < 0.05 and **p < 0.01 compared to vehicle control (one-way ANOVA).
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Figure 5. (A) Sub-G1 cell population analyses. MCF-7 cells (1 × 105 cells/well in a 6-well plate) were incubated with different concentrations of
compound 2 for 48 h, and the sub-G1 populations analyzed by flow cytometry. (B) Cell cycle analysis. MCF-7 cells were incubated with vehicle or
compound 2 (5 and 10 μM) for 24 h, followed by staining with 0.1% sodium citrate solution containing Triton-X-100, propidium iodide, and
RNase. Cells were acquired by a Partec flow cytometer and analyzed by the Flowjo software. (C) Quantification of the cell cycle assay is shown. All
the experiments were repeated three times in three replicates, and the values are means SEM. *p < 0.05 and **p < 0.01 compared to vehicle
(one-way ANOVA). Representative histograms are shown.
Figure 6. Involvement of intracellular ROS in compound 2-induced cytotoxicity to the MCF-7 cells. (A) ROS generation by compound 2. MCF-7
cells (1 × 105 cells/well in 6-well plate) were treated with different concentrations of compound 2 (5 and 10 μM) for different periods. The ROS
levels were measured from the DCFDA fluorescence, analyzed by flow cytometry, considering that of vehicle control cells as 1. (B) Quantification
of the ROS levels measured by the DCFDA assay (6 h). For the positive control, cells were also treated with H2O2 (100 μM). All the experiments
were repeated three times in three replicates, and the values are shown as means SEM. *p < 0.05 and **p < 0.01 compared to control (one-way
ANOVA).
the increase in the green fluorescence of AO in control
compared to compound 2-treated cells. Similar to ROS
generation kinetics, compound 2 dose-dependently induced
significant LMP in the cells at 6 h, but not up to 3 h, post-
treatment (Figure 8). The results suggested that compound 2
can cause LMP in MCF-7 cells. It is well established that
apoptotic cell death is characterized by partial lysosomal
membrane leakage, whereas massive rupture followed by rapid
leakage of intralysosomal contents into the cytosol lead to
necrosis.17b The results indicated that the elevated ROS level is
the principal factor leading to LMP and finally to apoptosis.
In conclusion, dietary phenolics especially the alpinoids
show promise as anticancer agents but were not adequately
investigated in human breast cancer cells. Given their low
natural abundances and isolation difficulties in appreciable
amounts for physiological studies, an efficient synthesis of two
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Figure 7. Loss of mitochondrial membrane potential (MMP) in the MCF-7 cells by compound 2. (A, B) The cells (1 × 105 cells/well, 6 well plate)
were incubated with vehicle (control) and compound 2 (5 and 10 μM) for 6 h, stained with JC-1 for 30 min, and the MMP loss was quantified by
flow cytometry from the decrease in red fluorescence. CCCP (10 μM) was used as the positive control for the assay. The mean fluorescence
intensity (MFI) was quantified, and the fold change with respect to (wrt) to the vehicle was plotted. All the experiments were repeated five times
with three replicates, and the values are means SEM. *p < 0.05 compared to control (one-way ANOVA). Representative histograms are shown
for part A, while part B is the quantified data.
Figure 8. Loss of lysosomal membrane permeabilization (LMP) of the MCF-7 cells by compound 2. The cells (1× 105 cells/well in 6 well plate)
were incubated with compound 2 (5 and 10 μM) for different periods, stained with AO (10 μM), and analyzed by flow cytometry. The lysosomal
membrane integrity was quantified from the percentage of cells showing increased green florescence of AO (6 h). The mean fluorescence intensity
(MFI) in the green channel was quantified, and the fold change wrt to the vehicle was plotted. All the experiments were repeated five times in three
replicates, and the values are shown as means SEM. *p < 0.05 compared to control (one-way ANOVA).
alpinoids, viz., the enantiomers of a β-hydroxyketone
(compound 1) and a α,β-conjugated ketone (compound 2)
diarylheptanoids, was developed for the first time. In contrast
to compound 1, the superior anticancer activity of compound
2 against the MCF-7 cells revealed a key role of the α,β-
conjugated carbonyl moiety, but not the carbinol functionality
and its absolute configuration in the designated property of the
diarylheptanoids. The results showed that cytotoxic action of
compound 2 is through oxidative stress/ROS generation. This,
in turn, leads to MMP loss, LMP, and induced apoptosis, as is
observed with several natural and synthetic chemotherapeutic
molecules. Given the encouraging biological findings of the
present investigation with compound 2 and its availability in
appreciable amounts via the efficient synthesis, compound 2
appears to be a potential natural anticancer agent against
human breast cancers and warrants detailed investigations in
preclinical tumor models.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All chemicals (AR grade),
except those indicated below, were purchased from Sigma-Aldrich
(Mumbai, India) and used without further purification. Dulbecco’s
modified Eagle’s Medium (DMEM) and antibiotic antimycotic
solution were purchased from HiMedia Laboratories (Mumbai,
India). Fetal bovine serum (FBS) was procured from Gibco Life
Technologies (Mumbai, India). All solvents (AR grade) used for
synthesis were purchased from SRL (Mumbai, India). The IR spectra
(KBr pellets) were recorded with a Bruker Tensor II spectropho-
tometer. [α]D values are given in deg cm2 g−1. The 1H and 13C NMR
spectra were recorded with a Varian 500 MHz NMR spectrometer,
and the NMR spectra were processed using the Bruker TOPSPIN
software or MestReNova-14.0 software.
1-tert-Butyldimethylsilyloxy-2-methoxy-4-allybenzene (4).18a To
a stirred and cooled (0 °C) solution of 3 (5.00 g, 30.47 mmol),
imidazole (3.11 g, 45.67 mmol), and DMAP (catalytic) in CH2Cl2
(30 mL), TBSCl (5.51 g, 36.54 mmol) was added dropwise, and the
mixture was stirred for another 3 h at room temperature. After the
reaction was complete (cf., TLC), it was poured into ice cold water
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(30 mL), the organic layer separated, and the aqueous portion
extracted with CHCl3 (3 × 20 mL). The combined organic extracts
were washed with water (2 × 20 mL) and brine (1 × 10 mL) and
dried. Removal of solvent in vacuo followed by purification of the
residue by column chromatography (silica gel, 0−5% EtOAc−n-
27.36 mmol) in diisopropyl ether (10 mL) was agitated on an orbital
shaker at 110 rpm for 18 h. The reaction mixture was filtered and the
solution concentrated in vacuo. The crude product was subjected to
column chromatography (silica gel, 0−10% EtOAc−n-hexane) to
afford alcohol (R)-7 and acetate (S)-8. For resolution of (R)-7, the
same conditions were used.
1
hexane) afforded pure 4 (7.81 g, 92%) as a colorless oil. H NMR
(500 MHz, CDCl3) δ 0.15 (6H, s), 1.00 (9H, s), 3.32 (2H, d, J = 6.5
Hz), 3.79 (3H, s), 5.04−5.09 (2H, m), 5.93−6.01 (1H, m), 6.63−
6.64 (1H, m), 6.67−6.69 (1H, m), 6.77 (1H, d, J = 8 Hz). 13C NMR
(125 MHz, CDCl3) δ −4.7, 18.4, 25.7, 39.9, 55.5, 112.7, 115.4, 120.7,
133.4, 137.8, 143.3, 150.8. Anal. Found: C, 69.37; H, 9.42. Calcd for
C19H26O2Si: C, 69.01; H, 9.41%.
(R)-1-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)hex-5-en-
3-ol [(R)-7].18d Yield: 2.13 g (46%); colorless oil; [α]2D2 +11.6 (c 1.8 in
CHCl3); ee = 98% (determined by HPLC; column AD-H, eluent,
isopropanol−n-hexane 5:95; flow rate, 0.5 mL/min). H NMR (500
1
MHz, CDCl3) δ 0.14 (6H, s), 0.99 (9H, s), 1.65 (1H, br s), 1.73−
1.78 (2H, m), 2.16−2.20 (1H, m), 2.30−2.33 (1H, m), 2.59−2.64
(1H, m), 2.70−2.74 (1H, m), 3.66−3.68 (1H, m), 3.78 (3H, s),
5.12−5.15 (2H, m), 5.78−5.84 (1H, m), 6.64 (1H, dd, J = 6.5 and 1.5
Hz), 6.68 (1H, d, J = 1.5 Hz), 6.75 (1H, d, J = 8.0 Hz). 13C NMR
(125 MHz, CDCl3) δ −4.6, 18.4, 25.7, 31.8, 38.6, 42.0, 55.5, 70.0,
112.5, 118.3, 120.4, 120.7, 134.7, 135.4, 143.0, 150.7. Anal. Found: C,
68.04; H, 9.55. Calcd for C19H32O3Si: C, 67.81; H, 9.58%.
(S)-1-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)hex-5-en-
3-yl acetate [(S)-8]. Yield: 2.22 g (43%); colorless oil; [α]2D2 −3.7 (c
1.0 in CHCl3); ee = 97% (determined by HPLC; column AD-H,
eluent, isopropanol−n-hexane 5:95; flow rate, 0.5 mL/min). 1H NMR
(500 MHz, CDCl3) δ 0.13 (6H, s), 0.98 (9H, s), 1.82−1.86 (2H, m),
2.04 (3H, s), 2.31−2.33 (2H, m), 2.50−2.53 (1H, m), 2.55−2.61
(1H, m), 3.78 (3H, s), 4.91−4.98 (1H, m), 5.05−5.09 (2H, m),
5.71−5.77 (1H, m), 6.60 (1H, dd, J = 8.0 and 1.5 Hz), 6.64 (1H, d, J
= 1.5 Hz), 6.74 (1H, d, J = 8.0 Hz). 13C NMR (125 MHz, CDCl3) δ
−4.7, 18.4, 21.2, 25.7, 31.4, 35.4, 38.7, 55.5, 72.9, 112.4, 117.8, 120.3,
120.7, 133.5, 134.9, 143.1, 150.7, 170.8. Anal. Found: C, 66.52; H,
9.18. Calcd for C21H34O4Si: C, 66.62; H, 9.05%.
3-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)propan-1-ol
(5).18b To a stirred and cooled (0 °C) solution of 4 (7.7 g, 27.69
mmol) in THF (50 mL), BH3-DMS (95%, 1.65 mL, 16.59 mmol) was
added dropwise. The mixture was brought to room temperature, and
stirred for an additional 4 h, treated with EtOH (25 mL) followed by
aqueous 3 N NaOH (10 mL). The solution was cooled to 0 °C, H2O2
(10 mL, 30% solution) was added slowly, and the reaction mixture
was stirred at room temperature for another 2 h. The organic layer
was separated, and the aqueous portion was extracted with EtOAc (3
× 50 mL). The combined organic extracts were washed with water (2
× 50 mL) and brine (1 × 50 mL) and dried. Removal of solvent in
vacuo followed by purification of the residue by column
chromatography (silica gel, 0−15% EtOAc−n-hexane) afforded pure
1
5 (5.98 g, 73%) as a yellow oil. H NMR (500 MHz, CDCl3) δ 0.15
(6H, s), 1.00 (9H, s), 1.61 (1H, br s), 1.85−1.91 (2H, m), 2.65 (2H,
t, J = 7.5 Hz), 3.68 (2H, t, J = 6.5 Hz), 3.79 (3H, s), 6.64 (1H, dd, J =
8.0 and 1.5 Hz), 6.69 (1H, d, J = 2.0 Hz), 6.76 (1H, d, J = 8.0 Hz).
13C NMR (125 MHz, CDCl3) δ −4.7, 18.4, 25.7, 31.8, 34.3, 55.5,
62.4, 112.6, 120.5, 120.7, 135.2, 143.1, 150.8. Anal. Found: C, 64.83;
H, 9.38. Calcd for C16H28O3Si: C, 64.82; H, 9.52%.
3-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)propanal
(6).18c To a stirred mixture of 5 (5.90 g, 19.92 mmol) and NaOAc (10
mol %) in CH2Cl2 (30 mL) was added PCC (6.43 g, 29.85 mmol) in
lots. After stirring for 3 h (cf., TLC), the reaction mixture was diluted
with Et2O (30 mL), and the supernatant passed through a pad of
silica gel. Removal of solvent in vacuo followed by column
chromatography of the residue (silica gel, 0−10% EtOAc−n-hexane)
furnished pure 6 (5.27 g, 90%) as a colorless oil. 1H NMR (500 MHz,
CDCl3) δ 0.15 (6H, s), 0.99 (9H, s), 2.74−2.77 (2H, m), 2.87−2.91
(2H, m), 3.79 (3H, s), 6.63 (1H, dd, J = 8.0 and 1.5 Hz), 6.68 (1H, d,
J = 2.0 Hz), 6.76 (1H, d, J = 8.0 Hz), 9.82 (1H, s). 13C NMR (125
MHz, CDCl3) δ −4.7, 18.4, 25.7, 27.9, 45.5, 55.5, 112.5, 120.3, 120.9,
133.7, 143.5, 150.9, 201.8. Anal. Found: C, 65.37; H, 8.97. Calcd for
C16H26O3Si: C, 65.26; H, 8.90%.
(R)-6-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-3-tert-
butyldimethylsilyloxyhex-1-ene [(R)-9]. Following the same proce-
dure described before, alcohol (R)-7 (2.00 g, 5.94 mmol) was silylated
using imidazole, DMAP, and TBSCl to afford pure (R)-9 (2.49 g,
93%) as a colorless oil. [α]2D3 +2.0 (c 1.2 in CHCl3). H NMR (500
1
MHz, CDCl3) δ 0.06 (6H, d, J = 2.5 Hz), 0.14 (6H, s), 0.91 (9H, s),
0.99 (9H, s), 1.70−1.77 (2H, m), 2.26−2.27 (2H, m), 2.49−2.55
(1H, m), 2.60−2.67 (1H, m), 3.73−3.75 (1H, m), 3.79 (3H, s),
5.03−5.07 (2H, m), 5.78−5.87 (1H, m), 6.61 (1H, dd, J = 8.0 and 1.5
Hz), 6.66 (1H, d, J = 2.0 Hz, 1H), 6.75 (1H, d, J = 8.0 Hz). 13C NMR
(125 MHz, CDCl3) δ −4.7, −4.5, −4.3, 18.2, 18.4, 25.8, 25.9, 31.5,
38.7, 41.9, 55.5, 71.5, 112.5, 116.8, 120.4, 120.7, 135.2, 136.0, 142.9,
150.7. Anal. Found: C, 66.39; H, 10.31. Calcd for C25H46O3Si2: C,
66.61; H, 10.29%.
(3R/S,5R)-7-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-5-
tert-butyldimethylsilyloxy-1-phenylheptan-3-ol [(R)-10]. To a
stirred and cooled (0 °C) solution of alkene (R)-9 (2.25 g, 4.99
mmol) in CH2Cl2(20 mL), a solution of m-CPBA (2.23 g, 9.98 mmol,
77%) in CH2Cl2 (10 mL) was added dropwise. After stirring the
mixture for 3 h at room temperature, it was poured into ice-cold water
(50 mL), the organic layer was separated, and the aqueous portion
was extracted with CHCl3 (3 × 20 mL). The combined organic
extracts were washed with water (2 × 20 mL), brine (1 × 15 mL), and
dried. Removal of solvent in vacuo afforded the epoxide (1.98 g),
which was used for the next step without further purification.
To a stirred solution of the Grignard reagent prepared from BnBr
(1.81 g, 10.60 mmol) and Mg turnings (309 mg, 12.72 mmol) in
THF (20 mL), a solution of the above epoxide (1.98 g, 4.24 mmol) in
THF (20 mL) was added dropwise. After stirring the mixture
overnight until completion of the reaction (cf., TLC), the mixture was
treated with aqueous saturated NH4Cl, the organic layer separated,
and the aqueous portion extracted with Et2O (2 × 20 mL). The
combined organic extracts were washed with water (2 × 20 mL),
brine (1 × 15 mL), and dried. The residue was purified by column
chromatography (silica gel, 0−10% Et2O−n-hexane) to afford the C-3
epimeric mixture of (R)-10 (1.87 g, 67% in two steps) as a colorless
(3R/S)-1-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)hex-5-
en-3-ol (7).18d To a stirred and cooled (0 °C) solution of the
aldehyde 6 (5.10 g, 17.34 mmol) and allyl bromide (2.99 mL, 34.64
mmol) in moist THF (30 mL) was added Zn dust (2.83 g, 43.28
mmol) followed by a few drops of aqueous saturated NH4Cl. After
stirring for 3 h (cf., TLC), the reaction was quenched with aqueous
saturated NH4Cl solution (10 mL) and the mixture was filtered
through a pad of silica gel. The organic layer was separated, and the
aqueous portion was extracted with EtOAc (3 × 30 mL). The
combined organic extracts were washed with water (2 × 30 mL) and
brine (1 × 20 mL) and dried. Removal of solvent in vacuo followed
by purification of the residue by column chromatography (silica gel,
0−10% EtOAc−n-hexane) afforded ( )-7 (4.72 g, 81%) as a colorless
1
oil. H NMR (500 MHz, CDCl3) δ 0.14 (6H, s), 0.99 (9H, s), 1.61
(1H, brs merged with H2O), 1.74−1.79 (2H, m), 2.12−2.21 (1H, m),
2.29−2.35 (1H, m), 2.59−2.65 (1H, m), 2.70−2.76 (1H, m), 3.64−
3.68 (1H, m), 3.79 (3H, s), 5.12−5.16 (2H, m), 5.78−5.86 (1H, m),
6.64 (1H, dd, J = 8.0 and 1.5 Hz), 6.69 (1H, d, J = 1.5 Hz), 6.75 (1H,
d, J = 8.0 Hz). 13C NMR (125 MHz, CDCl3) δ −4.7, 18.4, 25.7, 31.7,
38.6, 42.0, 55.5, 70.0, 112.6, 118.2, 120.4, 120.7, 134.7, 135.4, 143.1,
150.7. Anal. Found: C, 67.75; H, 9.48. Calcd for C19H32O3Si: C,
67.81; H, 9.58%.
oil. [α]2D2 −18.5 (c 1.0 in CHCl3). H NMR (500 MHz, CDCl3) δ
1
0.08−0.10 (6H, three s), 0.16 (6H, s), 0.92 and 0.94 (9H, two s), 1.01
(9H, s), 1.66−1.73 (3H, m), 1.81−1.85 (2H, m), 1.88−1.91 (1H, m),
2.54 (2H, t, J = 7.5 Hz), 2.70−2.72 (1H, m), 2.80−2.82 (1H, m),
Lipase-Catalyzed Transesterification of ( )-7. A mixture of ( )-7
(4.60 g, 13.68 mmol), Amano lipase (1.0 g), and vinyl acetate (2.35 g,
359
https://dx.doi.org/10.1021/acs.jnatprod.0c01012
J. Nat. Prod. 2021, 84, 352−363

Journal of Natural Products
pubs.acs.org/jnp
Article
3.17 and 3.42 (1H, two broad s), 3.79 (3H, s), 4.02−4.04 (2H, two
m), 6.61 (1H, dd, J = 8.0 and 2.0 Hz), 6.66 (1H, d, J = 1.5 Hz), 6.77
(1H, d, J = 8.0 Hz). 7.18−7.26 (3H, m), 7.27−7.31 (2H, m). 13C
NMR (125 MHz, CDCl3) δ −4.6, −4.0, 17.9, 18.4, 25.7, 25.8, 30.7,
31.7, 31.8, 31.9, 37.9, 39.5, 39.6, 39.8, 41.4, 42.9, 55.5, 67.7, 70.2,
71.2, 72.6, 112.4, 120.3, 120.7, 125.7, 128.1, 128.3, 128.4, 128.5,
130.7, 135.2, 135.4, 142.2, 142.3, 143.1, 150.7, 150.8. Anal. Found: C,
68.51; H, 10.10. Calcd for C32H54O4Si2: C, 68.76; H, 9.74%.
(S)-6-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-3-tert-
butyldimethylsilyloxy-hex-1-ene [(S)-9]. Using the same procedure
as described before, (S)-7 (1.2 g, 3.57 mmol) was silylated using
imidazole, DMAP, and TBSCl to afford pure (S)-9 (1.46 g, 91%) as a
colorless oil. [α]2D2 −2.1 (c 1.1 in CHCl3). 1H NMR (500 MHz,
CDCl3) δ 0.06 (6H, d, J = 2.5 Hz), 0.14 (6H, s), 0.91 (9H, s), 0.99
(9H, s), 1.70−1.77 (2H, m), 2.26−2.27 (2H, m), 2.49−2.55 (1H, m),
2.60−2.66 (1H, m), 3.73−3.75 (1H, m), 3.79 (3H, s), 5.03−5.07
(2H, m), 5.79−5.86 (1H, m), 6.61 (1H, d, J = 8.0 Hz), 6.66 (1H, s),
6.75 (1H, d, J = 8.0 Hz). 13C NMR (125 MHz, CDCl3) δ −4.7, −4.5,
−4.3, 18.1, 18.4, 25.7, 25.9, 31.4, 38.7, 41.8, 55.5, 71.5, 112.5, 116.8,
120.4, 120.6, 135.2, 136.0, 150.7. Anal. Found: C, 66.53, H, 10.28.
Calcd for C25H46O3Si2: C, 66.61; H, 10.29%.
(R)-7-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-5-tert-
butyldimethylsilyloxy-1-phenyl-3-oxoheptane [(R)-11]. To a cooled
(0 °C) and stirred solution of (R)-10 (1.6 g, 2.86 mmol) in CH2Cl2
(10 mL), Dess−Martin periodinane (1.82 g, 4.29 mmol) was added
portionwise. After completion of the reaction (2 h, cf., TLC)), the
mixture was diluted with CHCl3 (10 mL) and treated successively
with aqueous saturated NaHCO3 (5 mL) and aqueous saturated
Na2S2O3 (35 mL). The mixture was stirred for 30 min, the organic
layer separated, and the aqueous portion extracted with CHCl3 (2 ×
10 mL). The combined organic extracts were dried, concentrated in
vacuo, and subjected to column chromatography (silica gel, 0−5%
EtOAc−n-hexane) to obtain (R)-11 (1.27 g, 80%) as a colorless oil.
(3S/R,5S)-7-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-5-
tert-butyldimethylsilyloxy-1-phenylheptan-3-ol [(S)-10]. Using the
same procedure as described before, alkene (S)-7 (1.40 g, 3.10 mmol)
was converted to the epoxide and treated with BnMgBr to afford the
C-3 epimeric mixture of (S)-10 (1.09 g, 63% over two steps) as a
colorless oil. [α]2D2 +17.0 (c 1.0 in CHCl3). H NMR (500 MHz,
1
CDCl3) δ 0.07−0.09 (6H, four s), 0.15 (6H, s), 0.91 and 0.92 (9H,
two s), 1.00 (9H, s), 1.64−1.74 (3H, m), 1.78−1.90 (3H, m), 2.21
(1H, br s), 2.52 (t, J = 8.5 Hz) and 2.55−2.63 (m) (2H), 2.64−2.74
(1H, m), 2.75−2.85 (1H, m), 3.79 (3H, s), 3.90−3.97 and 3.99−4.06
(2H, two m), 6.59 (1H, d, J = 8.0 Hz), 6.64 (1H, s), 6.76 (1H, d, J =
7.5 Hz), 7.16−7.24 (3H, m), 7.25−7.31 (2H, m). 13C NMR (125
MHz, CDCl3) δ −4.7, −4.6, −4.0, 17.9, 18.4, 25.7, 25.8, 30.7, 31.8,
31.9, 38.0, 39.5, 39.6, 39.9, 41.4, 42.9, 55.5, 67.8, 70.2, 71.2, 72.6,
112.5, 120.4, 120.7, 125.7, 128.3, 128.4, 128.5, 135.2, 135.4, 142.3,
143.2, 150.8. Anal. Found: C, 68.47, H, 9.76. Calcd for C32H54O4Si2:
C, 68.76; H, 9.74%.
[α]2D3 −18.4 (c 1.6 in CHCl3). H NMR (500 MHz, CDCl3) δ 0.01
1
(3H, s), 0.06 (3H, s), 0.14 (6H, s), 0.88 (9H, s), 0.99 (9H, s), 1.70−
1.79 (2H, m), 2.46−2.51 (1H, m), 2.54−2.58 (2H, m), 2.61−2.66
(1H, m), 2.74−2.77 (2H, m), 2.86−2.90 (2H, m), 3.79 (3H, s),
4.19−4.23 (1H, m), 6.59 (1H, dd, J = 8.0 and 1.5 Hz), 6.64 (1H, d, J
= 1.5 Hz), 6.75 (1H, d, J = 8.0 Hz), 7.16−7.20 (3H, m), 7.26−7.29
(2H, m). 13C NMR (125 MHz, CDCl3) δ −4.7, 18.0, 18.4, 25.8, 25.9,
30.0, 31.0, 39.5, 46.1, 50.2, 55.5, 68.7, 112.5, 120.3, 120.7, 126.0,
128.3, 128.5, 135.5, 141.0, 150.7, 185.6. Anal. Found: C, 69.11; H,
9.31. Calcd for C32H52O4Si2: C, 69.01; H, 9.41%.
(S)-7-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)-5-tert-
butyldimethylsilyloxy-1-phenyl-3-oxoheptane [(S)-11]. Using the
same procedure as described before, (S)-10 (1.0 g, 1.79 mmol) was
oxidized using Dess−Martin periodinane to afford ketone (S)-11 (807
(R)-1-(4′-Hydroxy-3′-methoxyphenyl)-7-phenyl-5-oxoheptan-3-
ol [(R)-1]. To a cooled (0 °C) solution of (R)-9 (1.2 g, 2.15 mmol) in
10 mL of MeCN−pyridine (20:1 v/v), 0.16 mL of HF−pyridine
solution (70% HF) was added. The reaction mixture was gradually
warmed to room temperature and stirred for another 5 h (cf., TLC).
The reaction mixture was again cooled to 0 °C, and 2.5 mL of
saturated aqueous NaHCO3 was added slowly followed by 1.5 mL of
water. The organic layer was separated, and the aqueous portion was
extracted with Et2O (2 × 10 mL). The combined organic extracts
were washed with water (2 × 10 mL) and brine (1 × 10 mL) and
dried. Removal of solvent in vacuo followed by purification of the
residue by column chromatography (silica gel, 0−20% EtOAc−n-
hexane) afforded pure (R)-1 (0.551 g, 78%) as a colorless oil. [α]D23
−12.2 (c 1.2 in CHCl3) [literature18e [α]D −12.1 (CHCI3)]. 1H
NMR (500 MHz, CDCl3) δ 1.58−1.67 (2H, m merged with br s),
1.75−1.79 (1H, m), 2.53−2.63 (3H, m), 2.69−2.77 (3H, m), 2.88−
2.92 (2H, m), 3.88 (3H, s), 4.02−4.05 (1H, m), 5.47 (1H, s), 6.68
(1H, dd, J = 8.0 and 2.0 Hz), 6.70 (1H, d, J = 2.0 Hz), 6.83 (1H, d, J
= 8.0 Hz), 7.16−7.21 (3H, m), 7.26−7.30 (2H, m). 13C NMR (125
MHz, CDCl3) δ 29.5, 31.4, 38.3, 45.0, 49.3, 55.9, 66.9, 111.1, 114.3,
120.9, 126.2, 128.2, 128.5, 133.7, 140.6, 143.7, 146.4, 211.1. Anal.
Found: C, 73.44; H, 7.43. Calcd for C20H24O4: C, 73.15; H, 7.37%.
(S)-1-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)hex-5-en-
3-ol [(S)-(7)]. To a cooled (0 °C) and stirred solution of the acetate
(S)-8 (1.7 g, 4.49 mmol) in Et2O (25 mL) was added LiAlH4 (0.11 g,
3.0 mmol) in portions. After stirring for 2 h (cf., TLC), the mixture
was treated with aqueous saturated Na2SO4 to decompose excess
LiAlH4. The precipitate was filtered, washed with Et2O (25 mL), and
the filtrate concentrated to give a residue which on purification by
column chromatography (silica gel, 0−10% EtOAc−n-hexane)
afforded (S)-7 (1.25 g, 83%) as a colorless oil. [α]2D4 −10.8 (c 1.3
in CHCl3). 1H NMR (500 MHz, CDCl3) δ 0.15 (6H, s), 0.99 (9H, s),
1.67 (1H, br s), 1.74−1.79 (2H, m), 2.15−2.21 (1H, m), 2.30−2.35
(1H, m), 2.59−2.65 (1H, m), 2.70−2.77 (1H, m), 3.64−3.69 (1H,
m), 3.79 (3H, s), 5.13−5.16 (2H, m), 5.78−5.86 (1H, m), 6.65 (1H,
d, J = 7.5 Hz), 6.69 (1H, s), 6.76 (1H, d, J = 8.0 Hz). 13C NMR (125
MHz, CDCl3) δ −4.7, 18.4, 25.7, 31.7, 38.5, 42.0, 55.4, 70.0, 112.4,
118.3, 120.4, 120.7, 134.7, 135.4, 143.0, 150.7. Anal. Found: C, 67.64,
H, 9.57. Calcd for C19H32O3Si: C, 67.81; H, 9.58%.
mg, 81%) as a colorless oil. [α]2D3 +18.2 (c 1.0 in CHCl3). H NMR
1
(500 MHz, CDCl3) δ 0.01 (3H, s), 0.06 (3H, s), 0.15 (6H, s), 0.88
(9H, s), 0.99 (9H, s), 1.74−1.79 (2H, m), 2.46−2.52 (1H, m), 2.54−
2.59 (2H, m), 2.60−2.67 (1H, m), 2.73−2.78 (2H, m), 2.86−2.91
(2H, m), 3.79 (3H, s), 4.19−4.26 (1H, m), 6.59 (1H, dd, J = 8.0 and
2.0 Hz), 6.65 (1H, d, J = 1.5 Hz), 6.75 (1H, d, J = 8.0 Hz), 7.15−7.21
(3H, m), 7.25−7.30 (2H, m). 13C NMR (125 MHz, CDCl3) δ −4.7,
−4.6, 18.0, 18.4, 25.7, 25.9, 29.5, 31.1, 39.5, 46.1, 50.2, 55.5, 68.7,
112.5, 120.4, 120.7, 126.1, 128.3, 128.5, 135.5, 141.0, 143.1, 150.7,
208.7. Anal. Found: C, 69.40; H, 9.24. Calcd for C32H52O4Si2: C,
69.11; H, 9.41%.
(S)-1-(4′-Hydroxy-3′-methoxyphenyl)-7-phenyl-5-oxoheptan-3-
ol [(S)-1]. Using the same procedure as described before, (S)-11 (700
mg, 1.26 mmol) was desilylated to afford pure (S)-1 (310 mg, 75%)
as a colorless oil. [α]D23 +12.1 (c 1.0 in CHCl3). 1H NMR (500 MHz,
CDCl3) δ 1.62−1.70 (1H, m), 1.72−1.83 (1H, m), 2.53−2.65 (3H,
m), 2.69−2.78 (3H, m), 2.88−2.93 (2H, m), 3.13 (1H, br s), 3.86
(3H, s), 4.02−4.09 (1H, m), 5.65 (1H, s), 6.68 (1H, dd, J = 8.0 and
1.5 Hz), 6.71 (1H, d, J = 1.5 Hz), 6.83 (1H, d, J = 8.0 Hz), 7.14−7.22
(3H, m), 7.25−7.31 (2H, m). 13C NMR (125 MHz, CDCl3) δ 29.4,
31.3, 38.3, 44.9, 49.2, 55.8, 66.9, 111.1, 114.3, 120.9, 126.2, 128.2,
128.5, 133.6, 140.6, 143.7, 146.4, 211.0. Anal. Found: C, 73.22, H,
7.36. Calcd for C20H24O4: C, 73.15; H, 7.37%.
( )-5-Phenylpent-1-en-3-ol (14).18f To a mixture of 3-phenyl-
propanol 12 (0.200 g, 1.47 mmol) and NaOAc (10 mol %) in CH2Cl2
(20 mL) was added PCC (475 mg, 2.20 mmol) portionwise. After
stirring for 3 h (cf., TLC), the mixture was diluted with Et2O (10 mL)
and the supernatant passed through a pad of silica gel. Removal of
solvent in vacuo furnished 13 (179 mg), which was used for the next
step without further purification.
To a cooled (0 °C) and stirred solution of 13 (179 mg) in THF
(10 mL) was added vinylmagnesium bromide (2.0 mmol, 1.0 M in
THF). After stirring for 4 h at 0 °C (cf., TLC), the reaction was
quenched with aqueous 10% NH4Cl, the mixture filtered,
concentrated in vacuo, and the residue dissolved in Et2O (30 mL).
The organic layer was washed with water (2 × 20 mL) and brine (1 ×
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10 mL) and dried. Solvent removal in vacuo and column
penicillin, and 100 μg/mL streptomycin in a humidified incubator
under 5% CO2 at 37 °C. The stock solutions (50 mM) of (R)-1, (S)-
1, and 2 were prepared in DMSO and stored at −20 °C. The MCF-7
cells were seeded in 96-well plates at a density of 5 × 103 cells per well
in 100 μL of complete medium. After incubating overnight, cells were
treated with the test compounds (0−50 μM) for 72 h followed by
addition of MTT solution to a final concentration of 0.5 mg/mL.
After this, 100 μL of the solubilization solution (10% SDS in 0.01 N
HCl) was added into each well, the plate was allowed to stand
overnight in the incubator, and the absorbance at 570 nm was read. In
control experiments, compounds, without cells, were incubated with
MTT solution and no appreciable increase in the absorbance at 570
nm was noticed. The 50% inhibitory concentration (IC50) values were
calculated considering 100% survival in case where the cells were
treated with the vehicle control.
chromatography of the residue afforded ( )-14 (191 mg, 80% in
1
two steps). H NMR (500 MHz, CDCl3) δ 1.68 (1H, br s), 1.84−
1.89 (2H, m), 2.68−2.79 (2H, m), 4.12−4.16 (1H, m), 5.15 (1H, d, J
= 10.5 Hz), 5.25 (1H, d, J = 17.0 Hz), 5.88−5.95 (1H, m), 7.18−7.22
(3H, m), 7.26−7.31 (2H, m). 13C NMR (125 MHz, CDCl3) δ 31.6,
38.4, 72.4, 114.9, 125.8, 128.4, 140.9, 141.8. Anal. Found: C, 81.74;
H, 8.75. Calcd for C11H14O: C, 81.44; H, 8.70%.
5-Phenylpent-1-en-3-one (15).18g Using the same procedure as
described before, alcohol 14 (170 mg, 1.05 mmol) was oxidized using
PCC, followed by column chromatography to afford ketone 15 (144
1
mg, 86%). H NMR (500 MHz, CDCl3) δ 2.90−2.98 (4H, m), 5.84
(1H, d, J = 10.5 Hz), 6.22 (1H, d, J = 17.5 Hz), 6.33−6.39 (1H, m),
7.18−7.22 (3H, m), 7.28−7.31 (2H, m). 13C NMR (125 MHz,
CDCl3) δ 29.7, 41.1, 126.1, 128.2, 128.3, 128.4, 136.4, 141.0, 199.7.
Anal. Found: C, 82.33; H, 7.57. Calcd for C11H12O: C, 82.46; H,
7.55%.
Clonogenic Survival Assay.19b This was carried out using a
procedure reported before. Briefly, 1 × 103 cells were seeded in a 6-
well plate overnight and treated with compound 2 (1 μM, 2.5 μM,
and 5 μM) for 10 days in a complete growth medium. The colonies
were fixed with MeOH, stained with 0.5% crystal violet in a 1:1
MeOH−water solution, the colonies counted, and the plates were
scanned. The percent survival was calculated considering that of
vehicle control as 100%.
4-(4′-tert-Butyldimethylsilyloxy-3′-methoxyphenyl)but-1-ene
(16).18g To a cooled (−40 °C) and stirred suspension of
methyltriphenylphosphonium iodide (4.04 g, 10.0 mmol) in THF
(30 mL) was added n-BuLi (6.79 mL, 10.19 mmol, 1.5 M in n-
hexane). After stirring the mixture for 0.5 h, aldehyde 6 (2.0 g, 6.79
mmol) in THF (20 mL) was added. The mixture was stirred at the
same temperature for 3 h and then at room temperature for 12 h,
poured into ice-cold water (25 mL), and extracted with EtOAc (3 ×
20 mL). The combined organic extracts were washed successively
with water (2 × 20 mL) and brine (1 × 10 mL) and dried. Solvent
removal in vacuo followed by column chromatography (silica gel, 0−
10% EtOAc−n-hexane) of the residue gave pure 16 (1.45 g, 73%). 1H
NMR (500 MHz, CDCl3) δ 0.13 (6H, s), 0.98 (9H, s), 2.31−2.36
(2H, m), 2.63 (2H, t, J = 8.0 Hz), 3.78 (3H, s), 4.94−5.04 (2H, m),
5.81−5.87 (1H, m), 6.20 (1H, d, J = 8.0 Hz), 6.67 (1H, s), 6.74 (1H,
d, J = 8.0 Hz). 13C NMR (125 MHz, CDCl3) δ −4.7, 18.4, 25.7, 35.1,
35.7, 55.5, 112.5, 114.8, 120.4, 120.6, 135.4, 138.2, 143.0, 150.6. Anal.
Found: C, 69.98; H, 9.61. Calcd for C17H28O2Si: C, 69.81; H, 9.65%.
1-Phenyl-7-(4′-hydroxy-3′-methoxyphenyl)hept-4E-en-3-one
(2).13 To a cooled (−78 °C) and stirred solution of 16 (200 mg, 0.68
mmol) in THF (10 mL) was injected Bu4NF (0.9 mL, 1.0 M in
THF). After stirring the mixture for 2 h, the mixture was poured into
ice-cold H2O (10 mL) and extracted with EtOAc (2 × 10 mL). The
organic extract was washed with water (2 × 10 mL) and brine (1 × 5
mL) and dried. Removal of solvent afforded the crude compound,
which was subjected to cross metathesis without further purification.
A solution of the above compound (98 mg) in toluene (5 mL) was
added to a mixture of conjugated ketone 15 (59 mg, 0.37 mmol) and
Hoveyda−Grubbs II catalyst (12.5 mg, 0.02 mmol) suspended in dry
and degassed toluene (5 mL). The reaction mixture was refluxed for
18 h and then filtered through a pad of silica gel. The filtrate was
concentrated in vacuo, and the residue chromatographed (silica gel,
Apoptosis and Cell Cycle Analyses. Briefly, the cells (1 × 105 /well
in a 6-well plate) were treated with compound 2 (5 μM and 10 μM)
for 48 h. The cells were collected by trypsinization, incubated with PI
(50 μg/mL) and RNase A (200 μg/mL) in 1 mL of hypotonic buffer
(0.1% sodium citrate plus 0.1% Triton X-100) for 30 min at 37 °C
and analyzed by flow cytometry.
Intracellular ROS Level Assay. The MCF-7 cells (1 × 105/well in
6-well plate) were treated with DCFH-DA (10 μM) for 30 min,
followed by washing and incubation with compound 2 (5 μM and 10
μM) or H2O2 (100 μM) for 6 h. After trypsinization followed by
washing with PBS, the cells were analyzed by flow cytometry. The
increased green fluorescence (λex. at 480 nm and λem. at 530 nm) of
oxidized DCFDA indicated the ROS levels.
Analysis of Mitochondrial Membrane Integrity. Briefly, the cells
(1 × 105 /well, 6 well plate) treated with compound 2 (5 μM and 10
μM) for 8 h were washed and stained with JC-1 dye (5 μg/mL) for 30
min, trypsinized, and examined by flow cytometry. N-(3-
chlorophenyl)carbonohydrazonoyl dicyanide (10 μM) was used as
the positive control for the assay. The MMP loss was quantified from
the decrease in red fluorescence of JC-1 dye.
Analysis of Lysosomal Membrane Permeablization (LMP).19c
The MCF7 cells (3 × 105/well) were incubated with compound 2
(0−10 μM), washed with phosphate buffered saline (PBS), and
incubated with acridine orange (10 μM) for 15 min at 37 °C. After
trypsinization followed by washing with cold PBS, the cells were
analyzed by flow cytometry. The lysosomal membrane integrity was
quantified from the percentage of cells showing reduced red
florescence of AO.
1
0−10% EtOAc−n-hexane) to give 2 (104 mg, 61%). H NMR (500
MHz, CDCl3) δ 2.47−2.52 (2H, m), 2.68−2.71 (2H, m), 2.83−2.91
(2H, m), 2.92−2.94 (2H, m), 3.87 (3H, s), 5.49 (1H, s), 6.11 (1H, d,
J = 16 Hz), 6.65−6.67 (2H, m), 6.80−6.85 (2H, m), 7.18−7.21 (3H,
m), 7.26−7.30 (2H, m). 13C NMR (125 MHz, CDCl3) δ 30.0, 34.1,
34.5, 41.7, 55.9, 110.8, 114.3, 120.9, 126.1, 128.3, 128.5, 130.6, 132.6,
141.2, 143.9, 146.4, 199.5. Anal. Found: C, 77.61; H, 7.39. Calcd for
C20H22O3: C, 77.39; H, 7.14%.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01012.
Cell Cultures. The MCF-7 cells were procured from the National
Centre for Cell Sciences, Pune, India, and the U2-OS cells were
procured from ATCC (American Type Culture Collection). Cells
were cultured with DMEM medium supplemented with 10% FBS, 2
mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
The cells were incubated at 37 °C in a humidified 5% CO2
atmosphere and were subcultured using 0.25% trypsin in
phosphate-buffered saline (PBS) every 3−4 days to maintain 50−
70% confluency.
1
Copies of H and 13C NMR spectra of all compounds
synthesized (PDF)
AUTHOR INFORMATION
■
Corresponding Author
MTT Assay.19a The antiproliferative activity of (R)-1, (S)-1, and 2
were determined using the MTT reduction assay. Briefly, the MCF-7
cells were grown in Dulbecco’s Modified Eagle Media (DMEM)
supplemented with 5% fetal bovine serum (FBS), 100 U/mL
Dibakar Goswami − Bio-Organic Division, Bhabha Atomic
Research Centre, Mumbai, India 400085; Homi Bhabha
National Institute, Mumbai, India 400094; orcid.org/
0000-0002-4608-767X; Email: dibakarg@barc.gov.in
361
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Article
M.; Yamamoto, R.; Yasukawa, K.; Kurokawa, M. J. Nat. Med. 2013,
67, 773−781.
Authors
Sunita Gamre − Bio-Organic Division, Bhabha Atomic
Research Centre, Mumbai, India 400085
Mrityunjay Tyagi − Bio-Organic Division, Bhabha Atomic
Research Centre, Mumbai, India 400085
Sucheta Chatterjee − Bio-Organic Division, Bhabha Atomic
Research Centre, Mumbai, India 400085
Birija S. Patro − Bio-Organic Division, Bhabha Atomic
Research Centre, Mumbai, India 400085; Homi Bhabha
National Institute, Mumbai, India 400094; orcid.org/
0000-0001-9161-5913
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Subrata Chattopadhyay − Bio-Organic Division, Bhabha
Atomic Research Centre, Mumbai, India 400085
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01012
Author Contributions
#S.G. and M.T. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the NMR facility at TIFR,
Mumbai, India, for recording NMR spectra.
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