Latifolicinin A from a Fermented Soymilk Product and the Structure-Activity Relationship of Synthetic Analogues as Inhibitors of Breast Cancer Cell Growth

Article abstract of DOI:10.1021/acs.jafc.5b04028

The functional components in soymilk may vary depending upon the fermentation process. A fermented soymilk product (FSP) obtained by incubation with the microorganisms of intestinal microflora was found to reduce the risk of breast cancer. Guided by the inhibitory activities against breast cancer cells, two cytotoxic compounds, daidzein and (S)-latifolicinin A, were isolated from the FSP by repetitive extraction and chromatography. Latifolicinin A is the n-butyl ester of β-(4-hydroxyphenyl)lactic acid (HPLA). A series of the ester and amide derivatives of (S)-HPLA and l-tyrosine were synthesized for evaluation of their cytotoxic activities. In comparison, (S)-HPLA derivatives exhibited equal or superior inhibitory activities to their l-tyrosine counterparts, and (S)-HPLA amides showed better cytotoxic activities than their corresponding esters. In particular, (S)-HPLA farnesyl amide was active to triple-negative MDA-MB-231 breast cancer cells (IC₅₀ = 27 μM) and 10-fold less toxic to Detroit-551 normal cells.

Full text of DOI:10.1021/acs.jafc.5b04028

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Article
Latifolicinin A from Fermented Soymilk Product
and the Structure–Activity Relationship of Synthetic
Analogs as Inhibitors of Breast Cancer Cell Growth
Yi-Yu Ke, Chen Hsuan Tsai, Hui-Ming Yu, Yu-Chen Jao, Jim-Min Fang, and Chi-Huey Wong
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04028 • Publication Date (Web): 24 Oct 2015
Downloaded from http://pubs.acs.org on October 29, 2015
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Table of Contents Graphic
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Latifolicinin A from Fermented Soymilk Product and the Structure–Activity Relationship of
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Synthetic Analogs as Inhibitors of Breast Cancer Cell Growth
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Yi-Yu Ke,† Chen-Hsuan Tsai,§ Hui-Ming Yu,† Yu-Chen Jao,† Jim-Min Fang,*,†,§ and Chi-Huey
Wong*,†
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† The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang,
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Taipei, 115, Taiwan.
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§ Department of Chemistry, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei,
106, Taiwan.
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Table of Contents categories: (1) Bioactive Constituents and Functions; (2) Food and
Beverage Chemistry/Biochemistry; (3) Chemical Aspects of Biotechnology/Molecular
Biology.
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ABSTRACT
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The functional components in soymilk may vary depending on the fermentation process.
A fermented soymilk product (FSP) obtained by incubation with the microorganisms of
intestinal microflora was found to reduce the risk of breast cancer. Guided by the inhibitory
activities against breast cancer cells, two cytotoxic compounds, daidzein and (S)-latifolicinin
A, were isolated from FSP by repetitive extraction and chromatography. Latifolicinin A is the
n-butyl ester of β-(4-hydroxyphenyl)lactic acid (HPLA). A series of the ester and amide
derivatives of (S)-HPLA and L-tyrosine were synthesized for evaluation of their cytotoxic
activities. In comparison, (S)-HPLA derivatives exhibited equal or superior inhibitory
activities to their L-tyrosine counterparts, and (S)-HPLA amides showed better cytotoxic
activities than their corresponding esters. In particular, (S)-HPLA farnesyl amide was active to
the triple-negative MDA-MB-231 breast cancer cell (IC50 = 27 μM) and 10-fold less toxic to
Detroit-551 normal cell.
Keywords:
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Soymilk; Fermentation; Breast cancer; Latifolicinin; Organic synthesis; Cytotoxicity.
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INTRODUCTION
Breast cancer is a heterogeneous disease that often occurs in the pre- and post-menopause
period of women. In addition to human epidermal growth factor receptor 2 (HER2), the
hormone-sensitive breast cancer cells also contain estrogen receptor (ER) and progesterone
receptor (PR) as the biomarkers.1–3 When ER is stimulated by estrogen, a series of signals are
triggered to promote the growth of ER-positive breast tumor. The ER antagonist, e.g.
tamoxifen,4, 5 is used for treatment of the hormone-sensitive breast cancer. MCF-7 is a
triple-positive breast cancer cell line because it is ER- and PR-positive with overexpression of
HER 2.2 On the other hand, MDA-MB-231 is coined as a triple-negative breast cancer (TNBC)
cell line because it has low expression of HER2 and no expression of ER or PR.2, 6 Prognosis
and treatment of TNBCs are difficult since most drugs only target one of the three
receptors.7–9 The combinatorial therapies are more effective to TNBCs, but may also raise the
risk of side effects.
Soy products traditionally taken by Asian people as dietary food and nutrient supplements
have also become popular in western world. Moderate consumption of soy foods appears to
be safe and may reduce the risk of breast cancer. The isoflavone constituents in soy foods,10
such as daidzein, 1,11 and genistein10, have chemical structures similar to estrogens. Due to
the structural similarity, isoflavones may function as estrogen surrogate or as ER
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antagonist.11–13 It is still disputable whether isoflavones have beneficial or detrimental effects
associated with breast cancers.14
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Soymilk is an aqueous extract of soybean. The functional components in soymilk may
vary depending on the fermentation process.15 Soymilk can be processed with
microorganisms such as Lactobacillus bacteria to release healthful metabolites.16 In a
previous study,17 a fermented soymilk product (FSP) obtained by incubation with the
microorganisms of intestinal microflora was found to induce apoptosis of MCF-7 breast
cancer cells. We report herein the isolation of daidzein, 1,12 and latifolicinin A, 4b,18, 19
(Figure 1) possessing cytotoxic activity from FSP. A variety of latifolicinin analogs were also
synthesized for evaluation of their cytotoxic activities.
Materials and Methods
General. All the reagents were commercially available (Sigma-Aldrich Co., St. Louis,
MI) (Acros Organics N.V., Geel, Belgium), and used without further purification unless
indicated otherwise. All solvents (Merck Millipore, Darmstadt, Germany) were anhydrous
grade unless indicated otherwise. Reactions were magnetically stirred and monitored by
thin-layer chromatography on silica gel (Merck Millipore, Darmstadt, Germany). Preparative
thin-layer chromatography was performed using 20 cm × 20 cm glass plate of 2 mm SiO2
thickness (Sigma-Aldrich Co., St. Louis, MI). Flash chromatography was performed on silica
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gel (40–63 μm particle size) (Merck Millipore, Darmstadt, Germany) and LiChroprep RP-18
(40–63 μm particle size) (Merck Millipore, Darmstadt, Germany). High-performance liquid
chromatography (HPLC) (Agilent Technologies, Santa Clara, CA) was performed using a
4-channel programmable pump and a UV/VIS detector for monitoring at 254 nm wavelength.
Melting points were recorded on a melting point apparatus (Yanaco New Science Inc., Kyoto,
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Japan). H and C NMR spectra were recorded on 400 or 600 MHz spectrometers (Bruker
Corp., Billerica, MA). Chemical shifts are given in δ values relative to tetramethylsilane
(TMS); coupling constants J are given in Hz. Internal standards were CHCl3 (δH = 7.24),
CDCl3 (δC = 77.0 for the central line of triplet), CH3OD (δH = 3.31), and CD3OD (δC = 49.0).
The splitting patterns are reported as s (singlet), d (doublet), q (quartet), m (multiplet), br
(broad) and dd (doublet of doublets). Optical rotations were recorded on a digital polarimeter
(JASCO International Co. Ltd., Tokyo, Japan). Electrospray ionization high-resolution mass
spectra (ESI–HRMS) were recorded on a Daltonics BioTOF III high-resolution mass
spectrometer (Bruker Corp., Billerica, MA).
Fermented soymilk product. A concentrated solution of fermented soymilk product
(FSP) was prepared and provided by Microbio Co., Ltd., Taipei, Taiwan).20 In brief, soymilk
was fermented by a co-cultural symbiotic system of Lactobacillus paracasei, Lactobacillus
bulgaricus and Saccharomyces cerevisiae. The FSP consists of a mixture of soybean extracts
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and the secondary metabolites of these microorganisms. The FSP was subjected to a
sterilization process to ensure no contamination with any food-borne pathogens.
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Extraction and isolation of active compounds. The above-prepared concentrated FSP
(0.5 L) was lyophilized to give 47.2 g of powder sample, which was mixed with water (1 L)
and extracted with n-hexane (1 L) for three times to give the hexane layer (Y-H, 16.3 mg) and
water layer (Y-W1). The Y-W1 layer was extracted with ethyl acetate (1 L) to give the EtOAc
layer (Y-EA, 486.5 mg) and water layer (Y-W2). The Y-W2 layer was further extracted with
n-butanol (1 L) to give the butanol layer (Y-B, 3.6 g) and water layer (Y-W3, 42.0 g). Finally,
the Y-B fraction (3.6 g) was dissolved in CHCl3 and extracted with CHCl3–water (1:1, v/v) for
5 times to give daidzein, 1 (75.5 mg), water layer (Y-BW, 1.2 g), and CHCl3 layer (Y-BC, 1.4
g). The Y-BC fraction was divided into 14 portions and respectively subjected to preparative
thin-layer chromatography using n-hexane–EtOAc–water (10:5:1) as the mobile phase to give
latifolicinin A, 4b (0.94 mg).
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Cell culture. MDA-MB-231, MCF-7 and Detroit-551 cell lines were obtained from the
American Type Culture Collection (Rockville, MD). These cells were grown followed by
ATCC propagation protocol. In brief, MDA-MB-231 cells were cultured in Leibovitz's L-15
medium containing 10% fetal bovine serum (FBS). MCF-7 cells were cultured in Eagle's
minimum essential medium (EMEM) containing 10% FBS and 0.01 mg/mL bovine insulin.
Detroit-551 were cultured in EMEM containing 10% FBS. MDA-MB-231 cells were
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incubated at 37 C with 100% air atmosphere without CO2, whereas MCF-7 and Detroit-551
cells were incubated at 37 C in 95% air and 5% CO2 atmosphere. The medium was changed
twice a week, and cells were split at about 80% confluence. Second to tenth passage cells
were used in experiments.
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Determination of cell growth inhibition. Cells were seeded at a density of 3  103
(MDA-MB-231), 8  103 (MCF-7) or 2  103 (Detroit-551) cells per well in 96-well plate for
24 h. Then, the FSP extract or test compound were dosed into the well in triplicate for 72 h
incubation. Finally, cell viability was measured using the CellTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay reagent (Promega Corporation, Madison, WI)
according to the manufacturer’s instructions. The absorbance was measured using
SpectraMax M5 (Molecular Devices, Sunnyvale, CA) for formazan product at a wavelength
of 490 nm with a reference wavelength of 650 nm. The absorbance of each well was corrected
with reference to the blank. Percent inhibition of cell survival is expressed as:
[1 – (absorbance of treated cells / absorbance of control cells)]  100%.
Inhibitor IC50 values, i.e. the concentrations of the compound required for 50% cell
viability were determined from dose–response curves by plotting the percent inhibition of cell
viability versus inhibitor concentrations using Prism 5 (GraphPad Software, Inc., San Diego,
CA).
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Synthesis of (S)-3-(4-hydroxyphenyl)lactic acid (HPLA), (S)-3, (R)-enantiomer and
racemic mixture. A solution of (4-hydroxyphenyl)pyruvic acid, 2, (3.0 g, 16.7 mmol) and
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Et3N (2.3 mL, 16.7 mmol) in N,N-dimethylformamide (DMF, 100 mL) was stirred at –40 C
for 10 min. A solution of (+)-diisopinocampheylchloroborane21, 22 ((+)-Ipc2BCl) (16.0 g, 50.0
mmol) in THF (50 mL) was injected over a period of 30 min. The mixture was stirred and
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warmed from –40 C to room temperature over a period of 12 h. The mixture was quenched
with saturated NaHCO3(aq), and concentrated under reduced pressure. The residue was
acidified with 1 M HCl(aq) to pH 3, and extracted with EtOAc (50 mL × 3). The organic layer
was washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure.
The residue was purified by chromatography on a silica gel column (5 cm inner diameter (i.d.)
× 30 cm length) with elution of MeOH/CH2Cl2 [1:9 v/v (300 mL) to 1:4 (500 mL)] to afford
the reduction product HPLA, 3,21, 23 (2.16 g, 71%) predominating in the (S)-enantiomer
(>91% ee as inferred from the HPLC analysis of the related (S)-Mosher ester24 of (S)-5b).
1
C9H10O4; [α]20 = –10.6 (c 1.2, MeOH); TLC (MeOH/CH2Cl2, 1:4) Rf = 0.4; H NMR
D
(CD3OD, 400 MHz) δ 7.07 (2 H, d, J = 8.5 Hz), 6.69 (2 H, d, J = 8.5 Hz), 4.26 (1 H, dd, J =
13
7.8, 4.5 Hz), 2.99 (1 H, dd, J = 13.9, 4.4 Hz), 2.81 (1 H, dd, J = 14.0, 7.9 Hz); C NMR
(CD3OD, 150 MHz) δ 177.6, 157.1, 131.7 (2 ×), 129.7, 116.1 (2 ×), 73.2, 40.9; ESI–HRMS
calcd. for C9H11O4: 183.0652, found: m/z 182.0648 [M + H]+.
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By a similar procedure, reduction of (4-hydroxyphenyl)pyruvic acid, 2, with (–)-Ipc2BCl
gave (R)-3 (>90% ee as inferred from the HPLC analysis of the (S)-Mosher ester of (R)-5b).
The physical and spectral properties of (R)-3 were the same as that described for (S)-3 except
for the optical rotation [α]20D = +10.6 (c 1.2, MeOH). The racemic sample (±)-3 was prepared
by catalytic hydrogenation of a solution of oxoacid 2 in EtOAc (4 atm of H2, Pd/C, room
temperature, 24 h).
General procedure for the synthesis of (S)-HPLA esters 4a–4i. Method A. A mixture of
HPLA, (S)-3, (26 mg, 0.14 mmol)) and a drop of concentrated H2SO4 in n-butanol (4 mL) was
heated at 80 oC for 2 h. The mixture was extracted with NaHCO3 (aq) and EtOAc. The organic
layer was washed with brine, dried over MgSO4, filtered and concentrated under reduced
pressure. The residue was purified by chromatography on a silica gel column (1 cm i.d. × 8
cm) with elution of EtOAc/n-hexane (3:7 v/v, 50 mL) to afford the n-butyl ester, latifolicinin
A,18 (S)-4b, (28 mg, 82%).
Method B. A mixture of (S)-3 (50 mg, 0.27 mmol), K2CO3 (38 mg, 0.27 mmol) and
farnesyl bromide (81 μL, 0.30 mmol) in DMF (5 mL) was stirred at room temperature for 8 h.
The mixture was concentrated under reduced pressure. The residue was added to H2O (2 mL),
and extracted with EtOAc (5 mL × 3). The organic phase was washed with brine, dried over
MgSO4, filtered and concentrated under reduce pressure. The residue was purified by
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chromatography on a silica gel column (1 cm i.d. × 8 cm) with elution of EtOAc/n-hexane
(v/v 3:7, 50 mL) to afford the farnesyl ester (S)-4h (44 mg, 42%).
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(S)-3-(4-Hydroxyphenyl)lactic acid farnesyl ester (4h): C24H34O4; syrup; [α]28
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= –34.8 (c 2.2, CH2Cl2); IR νmax (neat) 3403, 2965, 2924, 2855, 1732, 1615, 1516, 1446, 1377,
1217, 1107, 1085, 931, 829 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.01 (2 H, d, J = 8.4 Hz),
6.67 (2 H, dd, J = 5.6, 2.8 Hz), 5.35–5.31 (1 H, m), 5.09–5.05 (2 H, m), 4.67 (3 H, d, J = 7.2
Hz), 4.39 (1 H, s), 3.03 (1 H, dd, J = 14.2, 4.4 Hz), 2.87 (2 H, dd, J = 13.2, 6.6 Hz), 2.20–2.00
(6 H, m), 1.98–1.94 (2 H, t, J = 8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.3, 154.7, 143.5,
135.6, 131.4, 130.6 (2 ×), 127.8, 124.2, 123.4, 117.4, 115.3 (2 ×), 71.4, 62.5, 39.6, 39.5, 39.4,
26.7, 26.1, 25.7, 17.7, 16.5, 16.0; ESI–HRMS calcd for C24H35O4: 385.2379, found: m/z
385.2391 [M + H]+.
General procedure for the synthesis of (S)-HPLA amides 10b, 10d, 10f, 10h and 10i.
A mixture of (S)-3 (317 mg, 1.74 mmol) and acetic anhydride (0.95 mL, 10 mmol) in pyridine
(5 mL) was stirred at room temperature for 3 h. The mixture was concentrated under reduced
pressure. The residue was added to 1 M HCl(aq) (5 mL), and extracted with EtOAc (15 mL ×
3). The organic phase was washed with brine, dried over MgSO4, filtered and concentrated
under reduced pressure to afford a diacetylation product (S)-7a (413 mg, 90%).
The above-prepared diacetate (S)-7a (413 mg, 1.57 mmol) was treated with NaHCO3
(252 mg, 3 mmol) in MeOH–H2O solution (5 mL, 1:1 v/v) at room temperature for 8 h. The
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residue was added to 1 M HCl(aq) to pH 3, and then extracted with EtOAc (15 mL × 4). The
organic phase was washed with brine, dried over MgSO4, filtered and concentrated under
reduced pressure to afford monoacetate (S)-7 (309 mg, 88%).
A mixture of (S)-7 (32 mg, 0.14 mmol), diisopropylethylamine (DIPEA) (0.05 mL, 0.15
mmol) and isopropyl chloroformate (i-PrOCOCl) (0.15 mL, 0.15 mmol) in DMF (2 mL) was
stirred for 0.5 h at 0 oC. Dodecylamine (0.03 mL, 0.14 mmol) was added, and the mixture was
stirred for 1 h. The mixture was concentrated under reduced pressure. The residue was
purified by chromatography on a silica gel column (1 cm i.d. × 8 cm length) with elution of
EtOAc/n-hexane [1:9 v/v (30 mL) to 2:1 (50 mL)] to afford compound (S)-9f (32 mg), which
was treated with 1 M NaOH (0.5 mL) in MeOH (2 mL) for 20 min at room temperature. The
mixture was concentrated under reduced pressure. The residue was neutralized with 1 M
HCl(aq), and extracted with EtOAc (5 mL × 3). The combined organic layers were washed with
brine, dried over MgSO4, filtered and concentrated under reduced pressure to afford amide
(S)-10f (25 mg, 50% overall yield from (S)-7).
N-Dodecyl (S)-3-(4-hydroxyphenyl)lactamide, 10f: C21H35NO3; white solid; mp
93.4–94.6 oC; [α]23D = –4.6 (c 1.6, MeOH); IR νmax (neat) 3301, 2922, 2851, 1744, 1658, 1369,
1221, 1194, 911, 760 cm–1; 1H NMR (CD3OD, 400 MHz) δ 7.66 (1 H, t, J = 5.5 Hz), 7.05 (2
H, d, J = 8.5 Hz), 6.68 (2 H, d, J = 8.5 Hz), 4.16 (1 H, dd, J = 7.2, 4.1 Hz), 3.23–3.05 (2 H, m),
2.96 (1 H, dd, J = 14.0, 4.1 Hz), 2.75 (1 H, dd, J = 14.0, 7.3 Hz), 1.41 (2 H, quint), 1.36–1.16
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(18 H, m), 0.90 (3 H, t, J = 6.7 Hz); 13C NMR (CD3OD, 100 MHz) δ 176.4, 157.0, 131.7 (2 ×),
129.6, 115.9 (2 ×), 74.1, 41.0, 40.1, 33.1, 30.8, 30.7, 30.6, 30.5, 30.4, 27.9, 23.7, 14.4;
ESI–HRMS calcd for C21H36NO3: 350.2690, found: m/z 350.2687 [M + H]+.
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Synthesis
of
-tyrosine
L
ester:
N-dodecyl
(S)-2-amino-3-(4-hydroxyphenyl)propanoate, 12f. A mixture of compound (S)-11 (100 mg,
0.35 mmol), Cs2CO3 (60 mg, 0.19 mmol) and 1-iodododecane (0.01 mL, 0.39 mmol) in DMF
(5 mL) was stirred for 8 h at room temperature. The mixture was concentrated under reduced
pressure. The residue was purified by chromatography on a silica gel column (1 cm i.d. × 10
cm length) with elution of EtOAc/n-hexane [1:9 v/v (40 mL) to 1:1 (60 mL)] to afford the
ester product (130 mg). The ester product was treated with 2 M HCl in EtOAc for 1 h at room
temperature to remove the Boc protecting group, and then concentrated under reduced
pressure to afford (S)-12f as the hydrochloride salt (107 mg, 83% overall yield). C21H36NO3Cl;
o
white solid; mp 129.2–131.8 C; [α]23 = +20.7 (c = 1.0, MeOH); IR νmax (neat) 3297, 2958,
D
1
2921, 2856, 2623, 1740, 1618, 1517, 1356, 1234, 849, 735; H NMR (CD3OD, 400 MHz) δ
7.07 (2 H, d, J = 8.4 Hz), 6.78 (2 H, d, J = 8.4 Hz), 4.22–4.15 (3 H, m), 3.12 (1 H, d, J = 6.8
Hz), 1.63–1.58 (2 H, m), 1.63–1.58 (2 H, m), 1.34–1.25 (18 H, m), 0.90 (3 H, t, J = 6.8 Hz);
13C NMR (100 MHz, CD3OD) δ 170.8, 158.9, 132.0 (2 ×), 126.2, 117.4 (2 ×), 68.1, 55.9, 37.3,
33.6, 31.3, 31.2, 31.1, 31.0, 30.8, 27.4, 24.2, 14.9; ESI–HRMS of (S)-12f (as the HCl salt)
calcd for C21H36NO3: 350.2695 [M – Cl]+, found: m/z 350.2691.
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General procedure for the synthesis of
-tyrosine esters: N-dodecyl
L
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(S)-2-amino-3-(4-hydroxyphenyl)propylamide, 13f. A mixture of compound (S)-11 (281 mg,
1.00 mmol), DIPEA (0.19 mL, 1.10 mmol) and i-PrOCOCl (1.10 mL, 0.56 mmol) in DMF
(10 mL) was stirred for 0.5 h at 0 oC. After addition of n-dodecylamine (0.25 mL, 1.10 mmol),
the mixture was stirred for 1 h, and concentrated under reduced pressure. The residue was
purified by chromatography on a silica gel column (1.5 cm i.d. × 15 cm length) by elution
with EtOAc/n-hexane [1:9 v/v (50 mL) to 1:1 (100 mL)] to afford an amide product (332 mg).
The amide product was treated with 2 M HCl in EtOAc for 1 h at room temperature to remove
the Boc protecting group, and then concentrated under reduced pressure to afford (S)-13f as
the hydrochloride salt (300 mg, 74% overall yield). C21H37N2O2Cl; white solid; mp
o
129.2–131.8 C; [α]23 = +34.1 (c 1.0, MeOH); IR νmax (neat) 3343, 2920, 2851, 1661 ,1613,
D
1
1551, 1517 ,1467, 1452, 1273, 1205, 837; H NMR (CD3OD, 400 MHz) δ 7.01 (2 H, d, J =
8.8 Hz), 6.70 (2 H, d, J = 8.4 Hz), 3.46 (1 H, t, J = 7.0 Hz), 3.19–3.12 (1 H, m), 3.09–3.02 (1
H, m), 2.86 (1 H, dd, J = 13.4, 7.0 Hz), 2.74 (1 H, dd, J = 13.6, 6.8 Hz), 1.43–1.35 (2 H, m),
1.35–1.15 (18 H, m), 0.90 (3 H, t, J = 6.8 Hz); 13C NMR (100 MHz, CD3OD) δ 170.0, 158.7,
132.1 (2 ×), 126.6, 117.3 (2 ×), 56.6, 41.1, 38.5, 33.6, 31.2 (2 ×), 31.1 (2 ×), 31.0, 30.9, 30.6,
28.4, 24.2, 14.9; ESI–HRMS of (S)-13f (as the HCl salt) calcd for C21H37N2O2: 349.2855
[M – Cl]+, found: m/z 349.2847.
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N-Dodecyl (S)-2-dodecylamino-3-(4-hydroxyphenyl)propylamide, 14f. To a mixture
of compound (S)-13f (63 mg, 0.16 mmol) and 1-dodecanal (0.04 mL, 0.18 mmol) in EtOH (3
mL) a solution of NaBH3CN (12 mg, 0.18 mmol) in EtOH (0.6 mL) was added in three
portions at room temperature. The solution was adjusted to pH 4–6 by addition of HCl
solution (2 M in EtOH, 0.1 mL). The mixture was stirred for 20 min, neutralized with
saturated NaHCO3(aq) (1 mL), and extracted with Et2O (15 mL × 3). The combined organic
phase was dried over MgSO4, concentrated under reduced pressure, and purified by
chromatography on a silica gel column (1 cm i.d. × 10 cm length) with elution of
EtOAc/n-hexane [1:9 v/v (30 mL), 2:3 (30 mL), to 4:1 (50 mL)] to afford compound (S)-14f
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254
255
256
(43 mg, 52%). C33H60N2O2; yellow oil; [α]25 = +11.7 (c 2.0, MeOH); IR νmax (neat) 3305,
D
1
2958, 2925, 2856, 1650 , 1569, 1516 ,1467, 1254, 825; H NMR (CD3OD, 400 MHz) δ 7.37
(NH, t, J = 5.6 Hz), 7.00 (2 H, d, J = 8.4 Hz), 6.80 (2 H, d, J = 8.4 Hz), 3.30–3.16 (3 H, m),
3.08 (1 H, dd, J = 14.0, 4.0 Hz), 2.54 (1 H, dd, J = 14.0, 9.6 Hz), 2.38 (2 H, t, J = 7.0 Hz),
1.46 (2 H, J = 6.4 Hz), 1.26–1.15 (40 H, m), 0.85 (6 H, t, J = 7.0 Hz); 13C NMR (100 MHz,
CD3OD) δ 174.5, 155.6, 130.0, 129.8 (2 ×), 116.7 (2 ×), 64.3, 48.9, 39.1, 38.6, 33.6, 31.9 (2
×), 29.9 (2 ×), 29.63 (2 ×), 29.60 (2 ×), 29.5 (2 ×), 29.4 (2 ×), 29.3 (2 ×), 29.2 (2 ×), 27.1, 27.0,
22.7 (2 ×), 14.1 (2 ×); ESI–HRMS calcd for C33H61N2O2: 517.4733, found: m/z 517.4741 [M
+ H]+.
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RESULTS AND DISCUSSION
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Isolation of cytotoxic compounds from fermented soymilk product. The powder
sample of FPS was partitioned between n-hexane and water. The aqueous layer (Y-W1) was
extracted successively with ethyl acetate and n-butanol. The hexane-, EtOAc- and butanol
layers were found to possess modest inhibitory activity against the growth of MDA-MB-231
cancer cells with the IC50 values of 0.49, 0.48 and 0.43 mg/mL, respectively. The butanol
layer (Y-B) was further extracted with CHCl3–water, and the active compounds daidzein, 1,12
and (S)-latifolicinin A, 4b,18, 19 were isolated from the chloroform layer (Y-BC).
Identification of (S)-latifolicinin A as the bioactive enantiomer. Latifolicinin A is the
n-butyl ester of 2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, 3, which is also known as
β-(4-hydroxyphenyl)lactic acid (HPLA).23, 25 Latifolicinin A was firstly isolated from the fruit
of Cordia latifolia (Boraginaceae, forget-me-not family) along with its analogs, including the
ethyl ester 4a (latifolicinin B), the methyl ester (latifolicinin C) and latifolicinin D that is a
derivative of latifolicinin acid having an anisole moiety instead of the phenol group.18, 19
Latifolicinins A–D were found to have modest larvicidal activities against the
yellow-fever-transmitting mosquito, but no significant antibacterial activity.18, 19 HPLA and
latifolicinin C have been reported to possess antioxidant activity.26, 27 The HPLA fragment has
also been found as a moiety in the structures of aquatic peptides isolated from the
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cyanobacterium Microcystis aeruginosa.23 However, the absolute configuration of active
HPLA derivatives was not clearly defined in these studies.
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We thus prepared (S)-HPLA (Figure 1B) by an enantioselective reduction of
3-(4-hydroxyphenyl)-2-oxopropanoic acid, 2, with (+)-diisopinocampheylchloroborane
((+)-Ipc2BCl) according to the previously reported method.21, 22, 27, 28 The product (S)-3b was
subjected to an acid-catalyzed esterification in n-butanol to give (S)-latifolicinin A, (S)-4b,
which was treated with iodomethane in the presence of K2CO3 for selective alkylation of the
phenol group to yield (S)-5b. The Mosher ester24 (S,S)-6b derived from (S)-5b and
(S)-MTPA-Cl was determined to have 91.2% diastereomeric excess (de) by the NMR and
HPLC analyses (Figure 2).29 Accordingly, the above-prepared HPLA, 3, and latifolicinin A,
4b, should have at least 91.2% enantiomeric excess (ee) predominating in the (S)-enantiomers
because the alkylation and ester formation under both basic and neutral conditions would
hardly change the structural configuration.28
By a similar procedure, reduction of α-oxoacid 2 with (–)-Ipc2BCl afforded (R)-HPLA,23,
30 which underwent esterification in n-butanol to give (R)-latifolicinin A in >90% ee. Our cell
viability assays indicated that (S)-latifolicinin A possessed inhibitory activities against
MDA-MB-231 and MCF-7 cancer cells with IC50 in sub-millimolar range (Table 1), whereas
the enantiomer (R)-4b was inactive (IC50 > 2 mM). We also found that (S)-5b having an
anisole moiety instead of the phenol group in (S)-4b did not show any significant inhibition
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against MDA-MB-231 or MCF-7 cells, indicating the phenol group was essential for the
cytotoxic activity.
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Synthesis of (S)-HPLA esters and amides. In order to explore more potent cytotoxic
agents, we synthesized a series of (S)-latifolicinin analogs 4a and 4c–4i (Figure 1B). Thus, the
above-prepared (S)-3b was treated with appropriate n-alkyl alcohols using concentrated
H2SO4 as the catalyst to give the desired alkyl esters 4a and 4c–4f (method A). In another
approach (method B), alkylation reactions of (S)-3b with geranyl chloride, farnesyl bromide
and 3-(5-bromopentoxy)-estra-1,3,5(10)-triene-17β-ol in the presence of a base (K2CO3 or
Cs2CO3) occurred selectively at the secondary hydroxyl group to afford esters 4g, 4h and 4i.
Furthermore, (S)-HPLA amides 10b, 10d, 10f, 10h and 10i bearing butyl, octyl, dodecyl,
farnesyl and (estradiol-3-yl)pentyl substituents, respectively, were prepared as shown in
Figure 3. The above-prepared (S)-3b was first reacted with acetic anhydride in pyridine to
give the diacetylation product (S)-7a, which was treated with NaHCO3, a weak base, to give
monoacetate (S)-7 by selective removal of the acetyl group on the phenyl moiety. Compound
(S)-7 was activated with isopropyl chloroformate to a mixed anhydride, and then treated with
appropriate amines (8b, 8d, 8f, 8h and 8i) to produce the corresponding amide derivatives.
Compounds 10b, 10d, 10f, 10h and 10i predominating in the (2S)-configuration were
obtained in 50–72% overall yields after saponification of the acetyl group.
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Synthesis of L-tyrosine esters and amides. As L-tyrosine bearing α-amino group is
structurally similar to (S)-HPLA that has α-hydroxyl group, the ester and amide derivatives of
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-tyrosine were also prepared (Figure 4) for comparison of their cytotoxic activities. The
alkylation reaction of N-Boc-tyrosine (S)-11 with dodecyl iodide was carried out, followed by
removal of the Boc protecting group in acidic conditions, to provide the tyrosine ester (S)-12f
in 83% yield. Alternatively, N-Boc-tyrosine was subjected to amidation reactions with
dodecylamine and phytylamine31, respectively, to give the amide derivatives (S)-13f and
(S)-13j after removal of the Boc group. Compound (S)-13f was further treated with dodecanal
in the presence of NaBH3CN to give the reductive amination product (S)-14f.
L
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Measurement of cell viability and determination of IC50 values. Table 1 shows the
cytotoxic activities of the above-prepared ester and amide derivatives of (S)-HPLA and
-tyrosine. As the alkyl chains elongated from 2 to 12 carbons, the (S)-HPLA esters 4a–4f
L
appeared to possess increasing cytotoxicity against MDA-MB-231 and MCF-7 breast cancer
cells (entries 1–6). The cytotoxic activities of (S)-HPLA amides 10b, 10d and 10f also
increased as the lengths of their alkyl substituents increased (entries 10–12). In a similar trend,
the farnesyl ester (S)-4h was about 5-fold more potent than the geranyl ester (S)-4g against
both MDA-MB-231 and MCF-7 cancer cells (entries 7 and 8). In comparison, the (S)-amide
derivatives 10d, 10f and 10h showed somewhat better cytotoxic activities than their
corresponding (S)-esters 4d, 4f and 4h. The -tyrosine dodecyl amide (13f) was about 11-fold
L
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more potent than the -tyrosine dodecyl ester (12f) against both breast cancer cells (entries 15
L
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and 16). However, the cytotoxic activities of the double-alkylated compound (S)-14f greatly
decreased by >30 fold when another dodecyl substituent was introduced to the N2-position of
-tyrosine (cf. entries 16 and 18). The (S)-HPLA derivatives 4f and 10f appeared to have the
L
cytotoxic activities superior or equal to their -tyrosine counterparts 12f and 13f (cf. entries 6
L
vs. 15, and 12 vs. 16, respectively).
The (S)-HPLA derivatives 4i, 10f, 10h and 10i bearing geranyl, farnesyl and estradiol
moieties were found to be less toxic to Detroit-551 fibroblast cells derived from a skin biopsy
of a normal embryo (Table 1). Among them, (S)-HPLA farnesyl amide, 10h, showed 6–10
fold less toxic to Detroit-551 normal cell (IC50 = 261 μM) than MDA-MB-231 (IC50 = 27 μM)
and MCF-7 cells (IC50 = 43 μM) cancer cells.
It was interesting to note that the (S)-HPLA ester 4i and amide 10i, prepared by
incorporation of an estradiol moiety, also exhibited good inhibitory activities (IC50 ≈ 10–15
μM) against both ER-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells
(entries 9 and 14). L-Tyrosine phytyl amide (13j) also exhibited high activities (IC50 ≈ 5–9
μM, entry 17) against the two breast cancer cells, but it was equally toxic to Detroit-551 cell
(IC50 = 4.7 μM). However, one may not overlook the possible surfactant effect of the aliphatic
phytyl substituent on cell membranes, even though the mechanism of function is unclear.
Though (S)-HPLA esters (e.g. 4b, 4h and 4i) and (S)-HPLA amides (e.g. 10f, 10h and 10i)
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contain a structural scaffold similar to that of L-tyrosine, none of them at 400 μM showed
appreciable inhibition against tyrosine kinase in our preliminary test. Further studies are
needed to elucidate the real target protein(s) of (S)-HPLA esters and amides.
In summary, we have identified (S)-latifolicinin A, 4b, as a cytotoxic constituent in the
fermented soymilk product prepared by incubation with microorganisms of intestinal
microflora. (S)-Latifolicinin A possessed moderate inhibitory activities (IC50 ≈ 0.8 mM)
against triple-negative MDA-MB-231 and triple-positive MCF-7 breast cancer cells, whereas
its (R)-enantiomer and the anisole surrogate (S)-5b were inactive to the cancer cells. To
understand the structure–activity relationship, we have synthesized a series of (S)-HPLA
esters and amides. The cytotoxic activities of the (S)-HPLA derivatives enhanced as the
lengths of alkyl substituents increased. Among the examined compounds, (S)-HPLA farnesyl
amide, 10h, was active to the triple-negative MDA-MB-231 breast cancer cell (IC50 = 27 μM)
and 10-fold less toxic to Detroit-551 normal cell. The mechanism for the cytotoxic activity of
(S)-HPLA derivatives awaits further studies.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
http://pubs.acs.org. DOI:
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Flow chart for isolation of daidzein and (S)-latifolicinin A, additional synthetic procedure
and characterization of compounds, 1H and 13C NMR spectra, and HPLC diagram.
AUTHOR INFORMATION
Corresponding Author
*(J.-M.F) Phone: 8862-33661663. Fax: 8862-23637812. E-mail: jmfang@ntu.edu.tw.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Academia Sinica for financial support, Microbio Co., Ltd., Taipei, Taiwan for
providing the fermented soymilk product, and Dr. Ting-Jen Rachel Cheng (the Genomics
Research Center, Academia Sinica) for tyrosine kinase inhibition assay.
ABBREVIATIONS USED
Boc, tert-butoxycarbonyl; de, diastereomeric excess; ee, enantiomeric excess; EMEM, Eagle's
minimum essential medium; ER, estrogen receptor; FBS, fetal bovine serum; FSP, fermented
soymilk product; HER2, human epidermal growth factor receptor 2; HPLA,
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β-(4-hydroxyphenyl)lactic acid; Ipc2BCl, diisopinocampheylchloroborane; PR, progesterone
receptor; TNBC, triple-negative breast cancer.
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Figure Captions:
Figure 1. (A) Chemical structure of daidzein, 1. (B) Synthesis of (S)-HPLA esters 4a–4i.
Figure 2. Conversion of the synthesized sample of (S)-latifolicinin A, (S)-4b, to Mosher ester
for optical purity determination.
Figure 3. Synthesis of (S)-HPLA amides.
Figure 4. Synthesis of L-tyrosine ester and amides.
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