Oxyphytosterols as active ingredients in wheat bran suppress human colon cancer cell growth: Identification, chemical synthesis, and biological evaluation

Article abstract of DOI:10.1021/jf506361r

Consumption of whole grains has been reported to be associated with a lower risk of colorectal cancer. Recent studies illustrated that phytochemicals in wheat bran (WB) may protect against colorectal cancer. There is a growing interest in the phytosterol contents of foods as either intrinsic or added components due to their beneficial health effects. However, little is known whether phytosterols in WB contribute the observed chemopreventative activity of the grain. In the present study, we directly purified and identified four oxyphytosterols 1-4 from sterol-enriched fraction of WB, and also successfully synthesized five sterol oxides 5-8 and 13. Using these nine compounds as references, we outlined a comprehensive profile of steroids in WB using tandem liquid chromatography mass spectrometry with electrospray ionization (LC-ESI/MSⁿ, n = 2-3) techniques for the first time. Among them, three sterol oxides 13, 14, and 18 are novel compounds, and 14 compounds 3, 4, 6-11, 13, 14, 16, and 18-20 were reported in WB for the first time. Our results on the inhibitory effects of available sterol oxides 1-8 and 13 against the growth of human colon cancer cells HCT-116 and HT-29 showed that compounds 2-8 exerted significant antiproliferative effects, with oxysterol 8 being the most active one in both cells. We further demonstrated that four most active sterol oxides 5-8 could induce cell death through the apoptosis pathway. Our results showed that phytosterols, particularly oxyphytosterols, in WB possess significant antiproliferative properties, and thereby may greatly contribute the observed chemoprevention of the whole grain wheat.

Full text of DOI:10.1021/jf506361r

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Article
Oxyphytosterols as Active Ingredients in Wheat
Bran Suppress Human Colon Cancer Cell Growth:
Identification, Chemical Synthesis and Biological Evaluation
Yingdong Zhu, Dominique Soroka, and Shengmin Sang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506361r • Publication Date (Web): 06 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human Colon Cancer Cell
Growth: Identification, Chemical Synthesis and Biological Evaluation
Yingdong Zhu, Dominique Soroka, and Shengmin Sang*
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Center for Excellence in PostꢀHarvest Technologies, North Carolina Agricultural and Technical
State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081,
United States
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Running title: Oxyphytosterols in Wheat Bran
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Corresponding authors:
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Shengmin Sang, Ph.D.
North Carolina Agricultural and Technical State University
North Carolina Research Campus
Tel: 704ꢀ250ꢀ5710; Fax: 704ꢀ250ꢀ5709
Email: ssang@ncat.edu or shengminsang@yahoo.com
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ABSTRACT
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Consumption of whole grains has been reported to be associated with a lower risk of colorectal
cancer. Recent studies illustrated that phytochemicals in wheat bran (WB) may protect against
colorectal cancer. There is a growing interest in the phytosterol contents of foods as either
intrinsic or added components due to their beneficial health effects. However, little is known
whether phytosterols in WB contribute the observed chemopreventative activity of the grain. In
the present study, we directly purified and identified four oxyphytosterols 1ꢀ4 from sterolꢀ
enriched fraction of WB, and also successfully synthesized five sterol oxides 5ꢀ8 and 13. Using
these nine compounds as references, we outlined a comprehensive profile of steroids in WB
using tandem liquid chromatography mass spectrometry with electrospray ionization (LCꢀ
ESI/MSn, n = 2ꢀ3) techniques for the first time. Among them, three sterol oxides 13, 14 and 18
are novel compounds, and fourteen compounds 3, 4, 6ꢀ11, 13, 14, 16 and 18ꢀ20 were reported in
WB for the first time. Our results on the inhibitory effects of available sterol oxides 1ꢀ8 and 13
against the growth of human colon cancer cells HCTꢀ116 and HTꢀ29 showed that compounds 2ꢀ
8 exerted significant antiproliferative effects, with oxysterol 8 being the most active one in both
cells. We further demonstrated that four most active sterol oxides 5ꢀ8 could induce cell death
through the apoptosis pathway. Our results showed that phytosterols, particularly
oxyphytosterols, in WB possess significant antiproliferative properties, and thereby may greatly
contribute the observed chemoprevention of the whole grain wheat.
KEYWORDS: Apoptosis, cytotoxicity, chemical synthesis, sterol oxide, wheat bran, colon
cancer
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INTRODUCTION
Epidemiological studies have shown that intake of wholeꢀgrains or high fiber, particularly
fiber from cereal grains such as wheat and rye, is associated with a lower risk of several lifestyleꢀ
related diseases such as type 2 diabetes,1, 2 cardiovascular diseases,2, 3 and some types of
cancers.4, 5 A metaꢀanalysis of six prospective studies of wholeꢀgrains and colorectal cancer
discovered a statistically significant 17% reduction in colorectal cancer risk for every threeꢀ
servingsꢀperꢀday increment of wholeꢀgrain intake.6 In 2011, the World Cancer Research
Fund/American Institute for Cancer Research upgraded that there is “convincing” evidence that
consumption of dietary fiber protects against colorectal cancer.7 Besides fiber, whole grains are
rich sources of phytochemicals, minerals, vitamins and other active substances with possible
health effects.8 Recently, Reddy et al. found that the lipid fraction of wheat bran (WB) strongly
inhibited colon tumor incidence, multiplicity, and volume in the F344 rat model.9 Our group
further demonstrated that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7%
in the Apcmin/+ mouse model.10 More recently, we investigated the antiꢀcancer ingredients in WB
oil using a bioassayꢀguided strategy, and identified alkylresorcinols (ARs) as major active
components in WB for colon cancer chemoprevention.11 In addition, we also found that
sphingolipids have a weak activity against the growth of human colon cancer cells.12 However,
the role of phytochemicals in WB oil accountable for the observed bioactivity of the whole grain
is not fully understood yet. Whether many other phytochemicals including phytosterols may
protect against chronic disease remains to be determined.
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Phytosterols are plant components that have a chemical structure similar to cholesterol except
for the addition of an extra methyl or ethyl group. In plants, more than 200 different types of
phytosterols have been reported with the most abundant species being βꢀsitosterol, campesterol
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and stigmasterol.13 Besides their cholesterolꢀlowering effects,14 other beneficial physiologic
properties of plant sterols include antiꢀcancer,15 antiꢀatherosclerotic16 and antiꢀinflammatory
effects.17 It is known that wheat is a rich source of diverse sterols, either in their free, conjugated,
or oxidized forms. Earlier study led to the identification of four oxyphytosterols including 5,6ꢀ
epoxysitosterols and 7ꢀhydroxysitosterols from wheat flour.18 Iwatsuki et al. isolated and
identified five free sterols and seven sterol ferulates from WB extract.19 Recently, Prinsen et al.
reported sterols, sterol glycosides, sterol esters, sterol ferulates, steroid hydrocarbons and steroid
ketones in WB by using gas chromatographyꢀmass spectrometry techniques.20 However, a
complete chemical profile and the antiꢀcancer activities of phytosterols in WB are still unclear.
In the present study, we purified four oxyphytosterols 1ꢀ4 from WB extract and synthesized four
sterol oxides 5ꢀ8, two ferulates 13 and 13a, and two azaꢀsterols 7a and 8a. Using liquid
chromatography mass spectrometry (LC/MS), we established the chemical profile of
phytosterols in WB. Furthermore, the structureꢀactivity relationship (SAR) of sterol oxides
against the growth of human colon cancer cells was discussed based on both purified and
synthesized compounds 1ꢀ8 and 13.
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MATERIALS AND METHODS
Materials. Cholesterol, βꢀsitosterol, stigmasterol and pregnenolone were purchased from
Sigma (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA). Anhydrous reactions were
carried out in ovenꢀdried glassware under a nitrogen atmosphere unless otherwise noted.
Analytical (250 ꢀm thickness, 2ꢀ25 ꢀm particle size) and preparative thin layer chromatography
(TLC) plates (1000 ꢀm thickness, 2ꢀ25 ꢀm particle size) were purchased from Sigma (St. Louis,
MO) and Sorbent Technologies (Atlanta, GA), respectively. Sephadex LHꢀ20 was purchased
from Sigma (St. Louis, MO). Medium pressure column chromatography was carried out over
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reverse phase silica gel (60 Å, Sigma, St. Louis, MO), equipped with LabAlliance Series I pump
and Spectra/chrom fraction collector CFꢀ2. LC/MSꢀgrade solvents and other reagents were
obtained from Thermo Fisher Scientific (Waltham, MA). HCTꢀ116 and HTꢀ29 human colon
cancer cells were obtained from American Type Tissue Culture (Manassas, VA). McCoy’s 5A
medium was purchased from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum
(FBS) and penicillin/streptomycin were purchased from Gemini BioꢀProducts (West Sacramento,
CA). MTT (3ꢀ(4,5ꢀdimethylthiaxolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) was procured from
CalbiochemꢀNovabiochem (San Diego, CA). Apoptag Plus Peroxydase In Situ Apoptosis
Detection Kit was purchased from Millipore (Billerica, MA).
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Preparation of Sterolꢀenriched Fraction from WB. Eight fractions (F1ꢀF8) were obtained
from WB oil, as described in our previous study.11 Briefly, wheat bran, provided by ConAgra
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Food Ingredients Company, was extracted by 95% ethanol at 40 C for 24 h to give an ethanol
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extract. After evaporation, the ethanol residue was then suspended in water and partitioned with
ethyl acetate (EA). After removal of EA in vacuo, the WB oil was obtained. The WB oil was
subjected to column chromatography (CC) over a normal phase silica gel eluted with a mixture
of hexane and EA, affording F1ꢀF8, which are stored at ꢀ80 oC till analysis. Phytosterols could be
detected in both F7 and F8. In order to avoid the analytical interference from ARs in F7, we
enriched sterols from F8. Specifically, part of F8 (6.0 g) was subjected to reverse phase (RP) C18
silica gel medium pressure CC (30 × 360 mm, 60 Å) eluted with 50% methanol, methanol and
methanol containing 0.2% formic acid (FA). Sphingolipids remained in acidic methanol eluate.
The neutral methanol fraction was further loaded on Sephadex LHꢀ20 CC eluted with 50%
ethanol and ethanol. The ethanol eluates were evaporated in vacuo, giving the sterolꢀenriched
fraction (2.0 g).
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Purification of Oxyphytosterols 1ꢀ4 from Sterolꢀenriched Fraction and Their NMR
Data. Repeated purification of the sterolꢀenriched fraction (1.0 g) by RP C18 silica gel medium
pressure CC eluted with methanol, giving stigmastꢀ4ꢀenꢀ6βꢀolꢀ3ꢀone (1) (4 mg), stigmastꢀ4,22ꢀ
dienꢀ6βꢀolꢀ3ꢀone (2) (6 mg), stigmastꢀ5ꢀenꢀ3βꢀolꢀ7ꢀone (3) (8 mg), and stigmastꢀ5,22ꢀdienꢀ3βꢀolꢀ
7ꢀone (4) (5 mg).
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Stigmastꢀ4ꢀenꢀ6βꢀolꢀ3ꢀone (1) as a white solid: H NMR (700 MHz, Pyꢀd5) δ 1.64 (1H, m,
Hꢀ1a), 1.92 (1H, m, Hꢀ1b), 2.45 (1H, m, Hꢀ2a), 2.60 (1H, m, Hꢀ2b), 6.07 (1H, s, Hꢀ4), 4.57 (1H,
m, Hꢀ6), 1.28 (1H, m, Hꢀ7a), 2.21 (1H, m, Hꢀ7b), 2.30 (1H, m, Hꢀ8), 0.91 (1H, m, Hꢀ9), 1.48
(2H, m, Hꢀ11), 1.17 (1H, m, Hꢀ12a), 2.04 (1H, m, Hꢀ12b), 1.02 (1H, m, Hꢀ14), 1.16 (1H, m, Hꢀ
15a), 1.65 (1H, m, Hꢀ15b), 1.27 (1H, m, Hꢀ16a), 1.88 (1H, m, Hꢀ16b), 1.17 (1H, m, Hꢀ17), 0.74
(3H, s, Hꢀ18), 1.57 (3H, s, Hꢀ19), 1.43 (1H, m, Hꢀ20), 1.02 (3H, d, J = 6.6 Hz, Hꢀ21), 1.14 (1H,
m, Hꢀ22a), 1.43 (1H, m, Hꢀ22b), 1.28 (2H, m, Hꢀ23), 1.02 (1H, m, Hꢀ24), 1.71 (1H, m, Hꢀ25),
0.89 (3H, d, J = 6.5 Hz, Hꢀ26), 0.91 (3H, d, J = 6.5 Hz, Hꢀ27), 1.32 (2H, m, Hꢀ28), and 0.92 (3H,
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t, J = 6.6 Hz, Hꢀ29); C NMR (175 MHz, Pyꢀd5) δ 37.9 (t, Cꢀ1), 35.4 (t, Cꢀ2), 200.1 (s, Cꢀ3),
126.3 (d, Cꢀ4), 170.3 (s, Cꢀ5), 70.3 (d, Cꢀ6), 40.1 (t, Cꢀ7), 30.7 (d, Cꢀ8), 54.5 (d, Cꢀ9), 38.9 (s, Cꢀ
10), 21.7 (t, Cꢀ11), 40.4 (t, Cꢀ12), 43.2 (s, Cꢀ13), 56.7 (d, Cꢀ14), 25.0 (t, Cꢀ15), 29.0 (t, Cꢀ16),
56.6 (d, Cꢀ17), 12.5 (q, Cꢀ18), 20.0 (q, Cꢀ19), 36.8 (d, Cꢀ20), 19.4 (q, Cꢀ21), 34.6 (t, Cꢀ22), 26.9
(t, Cꢀ23), 46.5 (d, Cꢀ24), 30.0 (d, Cꢀ25), 19.6 (q, Cꢀ26), 20.4 (q, Cꢀ27), 23.9 (t, Cꢀ28), and 12.6
(q, Cꢀ29); positive ESIMS, m/z 429 [M + H]+
.
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Stigmastꢀ4,22ꢀdienꢀ6βꢀolꢀ3ꢀone (2) as a white solid: 1H NMR (700 MHz, Pyꢀd5) δ 6.07 (1H,
s, Hꢀ6), 5.24 (1H, dd, J = 15.2, 8.8 Hz, Hꢀ22), 5.09 (1H, dd, J = 15.2, 8.8 Hz, Hꢀ23), 4.56 (1H, m,
Hꢀ6), 2.61 (1H, m), 2.46 (1H, m), 2.28ꢀ2.18 (2H, m), 2.09 (1H, m), 2.01 (1H, m), 1.93 (1H, m),
1.78 (1H, m), 1.68ꢀ1.41 (5H, m), 1.37ꢀ1.0 (10H, m), 1.57 (3H, s, Hꢀ19), 1.12 (3H, d, J = 6.6 Hz,
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Hꢀ21), 0.94 (3H, t, J = 6.5 Hz, Hꢀ29), 0.92 (3H, d, J = 6.5 Hz, Hꢀ27), 0.89 (3H, d, J = 6.5 Hz, Hꢀ
13
26), and 0.76 (3H, s, Hꢀ18); C NMR (175 MHz, Pyꢀd5) δ 200.1 (s, Cꢀ3), 170.3 (s, Cꢀ5),139.2
(d, Cꢀ22), 130.0 (d, Cꢀ23), 126.3 (d, Cꢀ4), 73.0 (d, Cꢀ6), 56.7, 56.5, 54.6, 51.9, 43.0, 41.2, 40.2,
40.1, 38.8, 37.9, 35.1, 32.6, 30.7, 29.7, 26.1, 25.0, 21.9, 21.7, 21.6, 20.0, 19.6, 13.0 (q, Cꢀ29),
and 12.7 (q, Cꢀ18); positive ESIMS, m/z 427 [M + H]+
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7ꢀKetoꢀβꢀsitosterol (3) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.64 (1H, s,
Hꢀ6), 3.56 (1H, m, Hꢀ3), 2.48ꢀ2.45 (1H, m), 2.40ꢀ2.32 (2H, m), 2.29ꢀ2.25 (1H, m), 2.06ꢀ2.03 (1H,
m), 1.98ꢀ1.95 (1H, m), 1.90ꢀ1.88 (2H, m), 1.69ꢀ1.46 (6H, m), 1.37ꢀ1.00 (13H, m), 1.21 (3H, s,
Hꢀ19), 0.93 (3H, d, J = 6.5 Hz, Hꢀ21), 0.85 (3H, t, J = 7.3 Hz, Hꢀ29), 0.83 (3H, d, J = 6.8 Hz, Hꢀ
27), 0.81 (3H, d, J = 6.8 Hz, Hꢀ26), and 0.70 (3H, s, Hꢀ18); 13C NMR (150 MHz,
CD3OD+CDCl3) δ 204.5 (s, Cꢀ7), 168.7 (s, Cꢀ5), 126.2 (d, Cꢀ6), 70.9 (d, Cꢀ3), 55.8 (d, Cꢀ17),
51.1 (d, Cꢀ14), 51.1 (d, Cꢀ9), 46.9, 46.4, 44.1, 42.5, 39.7, 39.4, 37.3, 37.1, 34.9, 31.6, 30.1, 29.4,
27.2, 27.0, 23.9, 22.1, 20.2 (q, Cꢀ27), 19.5 (q, Cꢀ26), 19.4 (q, Cꢀ21), 17.7 (q, Cꢀ19), 12.4 (q, Cꢀ
29), and 12.3 (q, Cꢀ18); positive ESIMS, m/z 429 [M + H]+
.
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7ꢀKetoꢀstigmasterol (4) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.63 (1H, s,
Hꢀ6), 5.15 (1H, dd, J = 15.1, 8.7 Hz, Hꢀ23), 5.01 (1H, dd, J = 15.1, 8.7 Hz, Hꢀ22), 3.56 (1H, m,
Hꢀ3), 2.48ꢀ2.45 (1H, m), 2.40ꢀ2.34 (1H, m), 2.31ꢀ2.24 (2H, m), 2.07ꢀ1.99 (2H, m), 1.97ꢀ1.94
(1H, m), 1.90ꢀ1.87 (1H, m), 1.75ꢀ1.71 (1H, m), 1.62ꢀ1.38 (7H, m), 1.34ꢀ1.10 (7H, m), 1.20 (3H,
s, Hꢀ19), 1.01 (3H, d, J = 6.7 Hz, Hꢀ21), 0.83 (3H, d, J = 6.4 Hz, Hꢀ27), 0.79 (3H, t, J = 7.3 Hz,
Hꢀ29), 0.78 (3H, d, J = 6.4 Hz, Hꢀ26), and 0.70 (3H, s, Hꢀ18); 13C NMR (150 MHz,
CD3OD+CDCl3) δ 204.4 (s, Cꢀ7), 168.5 (s, Cꢀ5), 139.0 (d, Cꢀ22), 130.3 (d, Cꢀ23), 126.1 (d, Cꢀ6),
70.6 (d, Cꢀ3), 55.6 (d, Cꢀ17), 52.2, 51.0, 50.9, 46.2, 43.8, 42.3, 41.1, 39.4, 39.3, 37.2, 32.7, 31.4,
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29.8, 27.1, 26.1, 21.9, 21.8 (q, Cꢀ27), 21.4 (q, Cꢀ26), 19.4 (q, Cꢀ21), 17.7 (q, Cꢀ19), 12.6 (q, Cꢀ
29), and 12.6 (q, Cꢀ18); positive ESIMS, m/z 427 [M + H]+
.
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General Procedure A for the Synthesis of Sterol Acetates. A mixture of sterols (1.0 eq),
acetic anhydride (Ac2O) (10.0 eq) and pyridine (Py) (50.0 eq) was incubated at 37 oC overnight.
The solvent was removed in vacuo. The residue was reconstituted in ethyl acetate (EA), washed
with water, dried over Na2SO4, and filtered. The filtration was evaporated off, and the residue
was subjected to a short pad of Celite, eluting with a mixture of hexane/EA (20:1), to give sterol
acetates.
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General Procedure B for the Synthesis of 7ꢀKetosterol Acetates. Chromium trioxide
(CrO3) (15.0 eq) was suspended in dry dichloromethane (DCM) and stirred for 30 min at ꢀ20 oC.
3,5ꢀDimethylpyrazole (15.0 eq) was added in one portion and the reaction mixture was stirred for
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30 min at ꢀ20 C. Sterol acetates (1.0 eq) were added and the mixture was stirred at ꢀ20 C and
allowed to warm up to room temperature (rt) overnight. EA was then added and the brown
suspension was filtered through Celite. The filtration was concentrated, and the residue was
purified by CC or preꢀTLC to give 7ꢀketosterol acetates.
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General Procedure C for the Synthesis of 7ꢀKetosterols. LiOH (4.0 eq) was added at rt to
a stirred solution of 7ꢀketosterol acetates (1.0 eq) in THF/MeOH/H2O (3:1:1). The resulting
mixture was stirred at rt for 2 h. The organic solvents were evaporated and the residue was
diluted with water, and extracted with EA. The organic layer was washed with water and brine,
dried over Na2SO4 and concentrated in vacuo. The residue was purified by preꢀTLC to give 7ꢀ
ketosterols.
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General Procedure D for the Synthesis of 7βꢀHydroxysterols. A suspension of 7ꢀ
ketosterols (1.0 eq) and cerium chloride heptahydrate (4.0 eq) in methanol was stirred at 0 oC for
10 min. Sodium borohydride (2.5 eq) was added to the suspension and the mixture was stirred at
rt for 4 h–18 h. The reaction solution was partitioned between water and EA. The organic layer
was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by preꢀTLC to give 7βꢀhydrolxysterols.
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General Procedure E for the Synthesis of 3ꢀOꢀ(4ꢀAcetoxyferuloyl)ꢀoxysterols. Transꢀ4ꢀ
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acetoxy ferulic acid (1.1 eq) and 7ꢀoxysterols (1.0 eq) were dissolved in dry DCM at 0 C. DCC
(3.0 eq) and DMAP (2.5 eq) were added, and the mixture was stirred at rt for 6 h. The solid
formed was filtered off, and the solution was washed with water, 10% HOAc and again with
water. The organic extracts were dried over Na2SO4 and concentrated in vacuo. The residue was
purified by preꢀTLC to give 3ꢀOꢀ(4ꢀacetoxyferuloyl)ꢀoxysterols.
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General Procedure F for the Synthesis of Oxysterol Ferulates. 3ꢀOꢀ(4ꢀAcetoxyferuloyl)ꢀ
oxysterols (1.0 eq) was dissolved in a mixture of DCM/MeOH (2:1, 3 mL). K2CO3 (0.2 eq) was
added, and the mixture was stirred at rt for 5 h. The reaction was quenched by adding a saturated
aqueous NH4Cl solution. The organic layers were separated, washed with water and brine, dried
over Na2SO4 and concentrated under reduced pressure. The residue was purified by preꢀTLC
(H/E = 3:1) to give oxysterol ferulates.
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Synthesis of Sterol Oxides 3ꢀ8.
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βꢁSitosterol acetate (22). Procedure A was followed by using βꢀsitosterol (414 mg, 1.0
mmol), Ac2O (0.94 mL, 10.0 mmol) and Py (4.07 mL, 50.0 mmol), giving the title compound
(456 mg, yield: 100%) as a white solid: positive ESIMS, m/z 457 [M + H]+.
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197
198
199
200
201
202
Stigmasterol acetate (23). Procedure A was followed by using stigmasterol (412 mg, 1.0
mmol), Ac2O (0.94 mL, 10.0 mmol) and Py (4.07 mL, 50.0 mmol), giving the title compound
1
(454 mg, yield: 100%) as a white solid: H NMR (600 MHz, CDCl3) δ 5.39 (1H, d, J = 4.7 Hz,
Hꢀ6), 5.16 (1H, dd, J = 15.1, 8.6 Hz, Hꢀ23), 5.03 (1H, dd, J = 15.1, 8.6 Hz, Hꢀ22), 4.62 (1H, m,
Hꢀ3), 2.35ꢀ2.31 (2H, m), 2.10ꢀ1.95 (4H, m), 2.05 (3H, s, OAcꢀ3), 1.90ꢀ1.85 (2H, m), 1.76ꢀ1.68
(1H, m), 1.62ꢀ1.39 (10H, m), 1.32ꢀ1.12 (5H, m), 1.11ꢀ0.92 (4H, m), 1.14 (3H, s, Hꢀ19), 1.13 (3H,
d, J = 6.7 Hz, Hꢀ21), 0.86 (3H, d, J = 6.4 Hz, Hꢀ27), 0.82 (3H, t, J = 7.3 Hz, Hꢀ29), 0.81 (3H, d,
J = 6.4 Hz, Hꢀ26), and 0.71 (3H, s, Hꢀ18); positive ESIMS, m/z 455 [M + H]+
.
203
204
205
206
207
208
209
Cholesterol acetate (24). Procedure A was followed by using cholesterol (386 mg, 1.0
mmol), Ac2O (0.94 mL, 10.0 mmol) and Py (4.07 mL, 50.0 mmol), giving the title compound
1
(428 mg, yield: 100%) as a white solid: H NMR (600 MHz, CDCl3) δ 5.36 (1H, d, J = 5.0 Hz,
Hꢀ6), 4.59 (1H, m, Hꢀ3), 2.34ꢀ2.28 (2H, m), 2.04ꢀ1.93 (2H, m), 2.03 (3H, s, OAcꢀ3), 1.88ꢀ1.78
(3H, m), 1.65ꢀ1.40 (8H, m), 1.38ꢀ1.20 (4H, m), 1.18ꢀ0.90 (9H, m), 1.02 (3H, s, Hꢀ19), 0.90 (3H,
d, J = 6.5 Hz, Hꢀ21), 0.86 (3H, d, J = 6.6 Hz, Hꢀ27), 0.85 (3H, d, J = 6.6 Hz, Hꢀ26), and 0.66
(3H, s, Hꢀ18); positive ESIMS, m/z 429 [M + H]+
.
210
211
212
213
7ꢁKetositosterol acetate (25). Procedure B was followed by using βꢀsitosterol acetate (25)
(200 mg, 0.44 mmol), CrO3 (658 mg, 6.58 mmol) and dimethylpyrazole (632 mg, 6.58 mmol) in
dry DCM (20 mL). The resulting residue was purified by preꢀTLC (H/E = 8:1) to give the title
compound (138 mg, yield: 67%) as a white solid: positive ESIMS, m/z 471 [M + H]+
.
214
215
216
7ꢁKetostigmasterol acetate (26). Procedure B was followed by using stigmasterol acetate (26)
(454 mg, 1.0 mmol), CrO3 (1.5 g, 15.0 mmol) and dimethylpyrazole (1.44 g, 15.0 mmol) in dry
DCM (50 mL). The resulting residue was purified by CC (H/E = 15:1, 10:1 and 8:1) to give the
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title compound (269 mg, yield: 57%) as a white solid: H NMR (600 MHz, CDCl3) δ 5.68 (1H,
Hꢀ6), 5.16 (1H, dd, J = 15.1, 8.6 Hz, Hꢀ23), 5.01 (1H, dd, J = 15.1, 8.6 Hz, Hꢀ22), 4.70 (1H, m,
Hꢀ3), 2.55ꢀ2.51 (1H, m), 2.47ꢀ2.42 (1H, m), 2.39ꢀ2.33 (1H, m), 2.24ꢀ2.20 (1H, m), 2.04ꢀ1.93
(4H, m), 2.03 (3H, s, OAcꢀ3), 1.79ꢀ1.48 (7H, m), 1.41ꢀ1.10 (8H, m), 1.20 (3H, s, Hꢀ19), 1.01
(3H, d, J = 6.7 Hz, Hꢀ21), 0.83 (3H, d, J = 6.3 Hz, Hꢀ27), 0.79 (3H, t, J = 7.3 Hz, Hꢀ29), 0.78
(3H, d, J = 6.3 Hz, Hꢀ26), and 0.68 (3H, s, Hꢀ18); positive ESIMS, m/z 469 [M + H]+
.
223
224
225
226
227
228
229
230
7ꢁKetocholesterol acetate (27). Procedure B was followed by using cholesterol acetate (27)
(428 mg, 1.0 mmol), CrO3 (1.5 g, 15.0 mmol) and dimethylpyrazole (1.44 g, 15.0 mmol) in dry
DCM (50 mL). The resulting residue was purified by CC (H/E = 15:1, 10:1, and 8:1) to give the
title compound (260 mg, yield: 60%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.69 (1H, s,
Hꢀ6), 4.70 (1H, m, Hꢀ3), 2.55ꢀ2.51 (1H, m), 2.48ꢀ2.35 (2H, m), 2.26ꢀ2.18 (1H, m), 2.07ꢀ1.82 (4H,
m), 2.06 (3H, s, OAcꢀ3), 1.71ꢀ1.47 (5H, m), 1.40ꢀ0.99 (13H, m), 1.20 (3H, s, Hꢀ19), 0.91 (3H, d,
J = 6.5 Hz, Hꢀ21), 0.86 (3H, d, J = 6.6 Hz, Hꢀ27), 0.85 (3H, d, J = 6.6 Hz, Hꢀ26), and 0.67 (3H, s,
Hꢀ18); positive ESIMS, m/z 443 [M + H]+
.
231
232
233
7ꢁKetositosterol (3). Procedure C was followed by using 7ꢀketoꢀβꢀsitosterol acetate (100 mg,
0.21 mmol) and LiOH (20 mg, 0.84 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting
residue was purified by preꢀTLC (H/E = 2:1) to give the title compound (3) (90 mg, yield: 100%).
234
235
236
237
7ꢁKetostigmasterol (4). Procedure C was followed by using 7ꢀketoꢀstigmasterol acetate (181
mg, 0.39 mmol) and LiOH (37 mg, 1.55 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting
residue was purified by preꢀTLC (C/M = 20:1) to give the title compound (4) (166 mg, yield:
100%).
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7βꢁHydroxysitosterol (5). Procedure D was followed by using 7ꢀketoꢀβꢀstigmasterol (3) (30
mg, 0.07 mmol), CeCl3·7H2O (104 mg, 0.28 mmol) and NaBH4 (7 mg, 0.18 mmol) in MeOH (3
mL). The mixture was stirred at rt for 18 h. The residue was purified by preꢀTLC (C/M = 20:1)
1
to give the title compound (5) (17 mg, yield: 57%) as a white solid: H NMR (600 MHz,
CD3OD+CDCl3) δ 5.24 (1H, s, Hꢀ6), 3.72 (1H, d, J = 8.0 Hz, Hꢀ7), 3.41 (1H, m, Hꢀ3), 2.26ꢀ2.18
(2H, m), 2.04ꢀ2.02 (1H, m), 1.87ꢀ1.78 (4H, m), 1.70ꢀ1.66 (1H, m), 1.55ꢀ1.33 (6H, m), 1.32ꢀ0.99
(13H, m), 1.06 (3H, s, Hꢀ19), 0.94 (3H, d, J = 6.5 Hz, Hꢀ21), 0.86 (3H, t, J = 7.4 Hz, Hꢀ29), 0.84
(3H, d, J = 6.8 Hz, Hꢀ27), 0.83 (3H, d, J = 6.8 Hz, Hꢀ26), and 0.72 (3H, s, Hꢀ18); 13C NMR (150
MHz, CD3OD+CDCl3) δ 144.0 (s, Cꢀ5), 127.4 (d, Cꢀ6), 73.7 (d, Cꢀ7), 72.1 (d, Cꢀ3), 57.7 (d, Cꢀ
17), 56.8 (d, Cꢀ14), 50.0, 47.2, 44.0, 42.5, 41.2, 41.0, 38.2, 37.5, 37.4, 35.1, 32.2, 30.3, 29.6, 27.3,
27.1, 24.1, 22.2, 20.2 (q, Cꢀ27), 19.5 (q, Cꢀ26), 19.4 (q, Cꢀ21), 19.3 (q, Cꢀ19), 12.3 (q, Cꢀ29),
and 12.3 (q, Cꢀ18); positive ESIMS, m/z 431 [M + H]+
.
250
251
252
253
254
255
256
257
258
259
260
7βꢁHydroxystigmasterol (6). Procedure D was followed by using 7ꢀketoꢀstigmasterol (4) (100
mg, 0.23 mmol), CeCl3·7H2O (350 mg, 0.94 mmol) and NaBH4 (22 mg, 0.58 mmol) in MeOH (5
mL). The mixture was stirred at rt for 6 h. The residue was purified by preꢀTLC (C/M = 20:1) to
1
give the title compound (6) (50 mg, yield: 50%) as a white solid: H NMR (600 MHz,
CD3OD+CDCl3) δ 5.24 (1H, s, Hꢀ6), 5.16 (1H, dd, J = 15.1, 8.8 Hz, Hꢀ23), 5.03 (1H, dd, J =
15.1, 8.8 Hz, Hꢀ22), 3.71 (1H, d, J = 8.2 Hz, Hꢀ7), 3.42 (1H, m, Hꢀ3), 2.29ꢀ2.20 (2H, m), 2.06ꢀ
1.99 (2H, m), 1.87ꢀ1.69 (4H, m), 1.58ꢀ1.36 (7H, m), 1.31ꢀ1.11 (9H, m), 1.06 (3H, s, Hꢀ19), 1.03
(3H, d, J = 6.7 Hz, Hꢀ21), 0.85 (3H, d, J = 6.3 Hz, Hꢀ27), 0.81 (3H, t, J = 7.1 Hz, Hꢀ29), 0.80
(3H, d, J = 6.3 Hz, Hꢀ26), and 0.73 (3H, s, Hꢀ18); 13C NMR (150 MHz, CD3OD+CDCl3) δ 144.0
(s, Cꢀ5), 139.7 (d, Cꢀ22), 130.5 (d, Cꢀ23), 127.3 (d, Cꢀ6), 73.7 (d, Cꢀ7), 72.0 (d, Cꢀ3), 57.7 (d, Cꢀ
17), 56.7, 52.7, 50.0, 43.8, 42.4, 41.8, 41.1, 40.8, 38.2, 37.5, 33.1, 32.2, 30.3, 27.3, 26.5, 22.2,
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21.9 (q, Cꢀ27), 21.5 (q, Cꢀ26), 19.5 (q, Cꢀ21), 19.4 (q, Cꢀ19), 12.7 (q, Cꢀ29), and 12.6 (q, Cꢀ18);
positive ESIMS, m/z 429 [M + H]+
.
263
264
265
266
267
268
269
270
271
272
273
7ꢁKetocholesterol (7). Procedure C was followed by using 7ꢀketoꢀcholesterol acetate (260
mg, 0.61 mmol) and LiOH (59 mg, 2.44 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting
residue was purified by preꢀTLC (CHCl3/MeOH = 15:1) to give the title compound (7) (223 mg,
yield: 91%) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.63 (1H, s, Hꢀ6), 3.55 (1H,
m, Hꢀ3), 2.48ꢀ2.45 (1H, m), 2.41ꢀ2.35 (2H, m), 2.30ꢀ2.26 (1H, m), 2.06ꢀ2.04 (1H, m), 1.98ꢀ1.96
(1H, m), 1.90ꢀ1.87 (2H, m), 1.63ꢀ1.46 (5H, m), 1.39ꢀ0.99 (13H, m), 1.22 (3H, s, Hꢀ19), 0.94 (3H,
d, J = 6.4 Hz, Hꢀ21), 0.87 (3H, d, J = 6.6 Hz, Hꢀ27), 0.86 (3H, d, J = 6.6 Hz, Hꢀ26), and 0.71
13
(3H, s, Hꢀ18); C NMR (150 MHz, CD3OD+CDCl3) δ 204.5 (s, Cꢀ7), 168.8 (s, Cꢀ5), 126.3 (d,
Cꢀ6), 71.0 (d, Cꢀ3), 56.1 (d, Cꢀ17), 51.3 (d, Cꢀ14), 51.2 (d, Cꢀ9), 46.5, 44.2, 42.6, 40.6, 39.9, 39.5,
37.5, 37.3, 36.9, 31.7, 29.5, 29.0, 27.3, 24.9, 23.2 (q, Cꢀ27), 23.0 (q, Cꢀ26), 22.2, 19.4 (q, Cꢀ21),
17.1 (q, Cꢀ19), and 12.4 (q, Cꢀ18); positive ESIMS, m/z 401 [M + H]+.
274
275
276
277
278
279
280
281
282
7βꢁHydroxycholesterol (8). Procedure D was followed by using 7ꢀketoꢀcholesterol (7) (100
mg, 0.26 mmol), CeCl3·7H2O (388 mg, 1.04 mmol) and NaBH4 (25 mg, 0.65 mmol) in MeOH (8
mL). The mixture was stirred at rt for 4 h. The residue was purified by preꢀTLC (C/M = 15:1) to
1
give the title compound (8) (58 mg, yield: 58%) as a white solid: H NMR (600 MHz,
CD3OD+CDCl3) δ 5.24 (1H, s, Hꢀ6), 3.72 (1H, d, J = 8.2 Hz, Hꢀ7), 3.42 (1H, m, Hꢀ3), 2.27ꢀ2.18
(2H, m), 2.05ꢀ2.02 (1H, m), 1.89ꢀ1.78 (4H, m), 1.56ꢀ1.25 (10H, m), 1.18ꢀ0.99 (9H, m), 1.06 (3H,
s, Hꢀ19), 0.93 (3H, d, J = 6.5 Hz, Hꢀ21), 0.87 (3H, d, J = 6.6 Hz, Hꢀ27), 0.86 (3H, d, J = 6.6 Hz,
Hꢀ26), and 0.71 (3H, s, Hꢀ18); 13C NMR (150 MHz, CD3OD+CDCl3) δ 143.9 (s, Cꢀ5), 127.3 (d,
Cꢀ6), 73.6 (d, Cꢀ7), 72.0 (d, Cꢀ3), 57.6 (d, Cꢀ17), 56.9 (d, Cꢀ14), 49.9, 43.9, 42.4, 41.1, 40.9, 40.6,
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38.2, 37.4, 37.3, 37.0, 32.2, 29.6, 29.0, 27.2, 24.9, 23.2 (q, Cꢀ27), 23.0 (q, Cꢀ26), 22.2, 19.5 (q,
Cꢀ21), 19.4 (q, Cꢀ19), and 12.4 (q, Cꢀ18); positive ESIMS, m/z 403 [M + H]+
.
285
Synthesis of Sterol Ferulates 13 and 13a.
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3ꢁOꢁ(4ꢁAcetoxyferuloyl)ꢁ7βꢁhydroxycholesterol (28). Transꢀ4ꢀacetoxy ferulic acid was
prepared followed procedure A by using transꢀferulic acid (582 mg, 3.0 mmol), Ac2O (2.8 mL,
o
30.0 mmol) and Py (12 mL, 150 mmol). The mixture was incubated at 37 C overnight. The
organic solvent was removed in vacuo to give transꢀ4ꢀacetoxy ferulic acid (700 mg, yield: 100%)
as a white solid: 1H NMR (600 MHz, CDCl3) δ 7.73 (1H, d, J = 15.9 Hz, Hꢀ1), 7.14 (1H, d, J =
8.1 Hz, Hꢀ6'), 7.12 (1H, s), 7.06 (1H, d, J = 8.1 Hz, Hꢀ6'), 6.39 (1H, d, J = 15.9 Hz, Hꢀ2), 3.87
(3H, s, OCH3ꢀ3'), and 2.32 (3H, s, OAcꢀ4'); positive ESIMS, m/z 237 [M + H]+
.
293
294
295
296
297
298
299
300
301
Procedure E was followed by using transꢀ4ꢀacetoxy ferulic acid (20 mg, 0.08 mmol), 7βꢀ
hydroxycholesterol (31 mg, 0.08 mmol), DCC (50 mg, 0.24 mmol) and DMAP (24 mg, 0.2
mmol) in dry DCM (1 mL). The resulting residue was purified by preꢀTLC (H/E = 4:1) to give
the title compound (21 mg, yield: 42%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.34 (1H,
s, Hꢀ6), 4.75 (1H, m, Hꢀ3), 3.86 (1H, d, J = 8.3 Hz, Hꢀ7), 2.48ꢀ2.32 (2H, m), 2.15ꢀ0.99 (24H, m),
1.06 (3H, s, Hꢀ19), 0.93 (3H, d, J = 6.5 Hz, Hꢀ21), 0.87 (3H, d, J = 6.5 Hz, Hꢀ27), 0.86 (3H, d, J
= 6.5 Hz, Hꢀ26), 0.70 (3H, s, Hꢀ18), and 4ꢀacetyl ferulate [7.62 (1H, d, J = 15.9 Hz, Hꢀ7'), 7.10
(1H, d, J = 8.1 Hz, Hꢀ6'), 7.09 (1H, s, Hꢀ2'), 7.04 (1H, d, J = 8.1 Hz, Hꢀ5'), 6.35 (1H, d, J = 15.9
Hz, Hꢀ8'), 3.85 (3H, s, OMeꢀ3'), and 2.31 (3H, s, OAcꢀ4')]; positive ESIMS, m/z 621 [M + H]+
.
302
303
304
3ꢁOꢁ(4ꢁAcetoxyferuloyl)ꢁ7ꢁketocholesterol (29). Procedure E was followed by using transꢀ4ꢀ
acetoxy ferulic acid (18 mg, 0.075 mmol), 7ꢀketoꢀcholesterol (20 mg, 0.05 mmol), DCC (31 mg,
0.15 mmol) and DMAP (16 mg, 0.13 mmol) in dry DCM (1 mL). The resulting residue was
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purified by preꢀTLC (H/E = 4:1) to give the title compound (18 mg, yield: 58%) as a white solid:
1H NMR (600 MHz, CDCl3) δ 5.72 (1H, s, Hꢀ6), 4.85 (1H, m, Hꢀ3), 2.64ꢀ2.61 (1H, m), 2.56ꢀ
2.51 (1H, m), 2.42ꢀ2.38 (1H, m), 2.26ꢀ2.21 (1H, m), 2.08ꢀ1.98 (2H, m), 1.93ꢀ1.87 (1H, m), 1.77ꢀ
1.74 (1H, m), 1.58ꢀ1.48 (5H, m), 1.37ꢀ0.99 (13H, m), 1.20 (3H, s, Hꢀ19), 0.92 (3H, d, J = 6.5 Hz,
Hꢀ21), 0.85 (3H, d, J = 6.5 Hz, Hꢀ27), 0.84 (3H, d, J = 6.5 Hz, Hꢀ26), 0.68 (3H, s, Hꢀ18), and 4ꢀ
acetyl ferulate [7.63 (1H, d, J = 15.9 Hz, Hꢀ7'), 7.10 (1H, d, J = 8.2 Hz, Hꢀ6'), 7.09 (1H, s, Hꢀ2'),
7.04 (1H, d, J = 8.2 Hz, Hꢀ5'), 7.35 (1H, d, J = 15.9 Hz, Hꢀ8'), 3.84 (3H, s, OMeꢀ3'), and 2.31
(3H, s, OAcꢀ4')]; positive ESIMS, m/z 619 [M + H]+
.
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
7ꢁKetocholesterol ferulate (13). Procedure F was followed by using 3ꢀOꢀ(4ꢀ
acetoxyferuloyl)ꢀ7ꢀketocholesterol (15 mg, 0.024 mmol) and K2CO3 (0.52 mg, 0.0049 mmol) in
DCM/MeOH (2:1, 3 mL). The residue was purified by preꢀTLC (H/E = 3:1) to give the title
1
compound 13 (10 mg, yield: 71%) as a white solid: H NMR (600 MHz, CDCl3) δ 5.72 (1H, s,
Hꢀ6), 4.84 (1H, m, Hꢀ3), 2.65ꢀ2.51 (2H, m), 2.44ꢀ2.37 (1H, m), 2.28ꢀ2.20 (1H, m), 2.10ꢀ1.85 (4H,
m), 1.80ꢀ1.48 (3H, m), 1.40ꢀ1.20 (8H, m), 1.18ꢀ0.99 (6H, m), 1.23 (3H, s, Hꢀ19), 0.91 (3H, d, J
= 6.5 Hz, Hꢀ21), 0.86 (3H, d, J = 6.5 Hz, Hꢀ27), 0.85 (3H, d, J = 6.5 Hz, Hꢀ26), 0.68 (3H, s, Hꢀ
18), and ferulate moiety [7.60 (1H, d, J = 15.8 Hz, Hꢀ7'), 7.06 (1H, d, J = 8.2 Hz, Hꢀ6'), 7.02 (1H,
s, Hꢀ2'), 6.90 (1H, d, J = 8.2 Hz, Hꢀ5'), 6.26 (1H, d, J = 15.8 Hz, Hꢀ8'), and 3.92 (3H, s, OMeꢀ3')];
13C NMR (150 MHz, CDCl3) δ 202.0 (s, Cꢀ7), 164.0 (s, Cꢀ5), 126.7 (d, Cꢀ6), 72.1 (d, Cꢀ3), 54.8,
50.0, 49.8, 45.4, 43.1, 39.5, 38.7, 38.4, 37.9, 36.2, 36.1, 35.7, 28.5, 28.0, 27.5, 26.3, 23.8, 22.8 (q,
Cꢀ27), 22.6 (q, Cꢀ26), 21.2, 18.9 (q, Cꢀ21), 17.3 (q, Cꢀ19), 12.0 (q, Cꢀ18), and ferulate moiety
[166.4 (s, C=O, Cꢀ9'), 148.0 (s, Cꢀ4'), 146.8 (s, Cꢀ3'), 145.1 (d, Cꢀ7'), 126.9 (s, Cꢀ1'), 123.1 (d, Cꢀ
6'), 115.4 (d, Cꢀ8'), 114.7 (d, Cꢀ5'), 109.3 (d, Cꢀ2'), and 55.9 (q, OMeꢀ3')]; positive ESIMS, m/z
577 [M + H]+
.
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7βꢁHydroxycholesterol ferulate (13a). Procedure F was followed by using 3ꢀOꢀ(4ꢀ
acetoxyferuloyl)ꢀ7βꢀhydroxycholesterol (21 mg, 0.034 mmol) and K2CO3 (1.0 mg, 0.0068 mmol)
in DCM/MeOH (2:1, 3 mL). The residue was purified by preꢀTLC (H/E = 3:1) to give the title
compound 13a (16 mg, yield: 80%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.34 (1H, s,
Hꢀ6), 4.76 (1H, m, Hꢀ3), 3.87 (1H, d, J = 7.8 Hz, Hꢀ7), 2.48ꢀ2.37 (2H, m), 2.05ꢀ1.79 (5H, m),
1.70ꢀ1.22 (10H, m), 1.18ꢀ0.99 (9H, m), 1.09 (3H, s, Hꢀ19), 0.93 (3H, d, J = 6.5 Hz, Hꢀ21), 0.87
(3H, d, J = 6.5 Hz, Hꢀ27), 0.86 (3H, d, J = 6.5 Hz, Hꢀ26), 0.70 (3H, s, Hꢀ18), and ferulate moiety
[7.60 (1H, d, J = 15.9 Hz, Hꢀ7'), 7.06 (1H, dd, J = 8.2, 1.6 Hz, Hꢀ6'), 7.03 (1H, d, J = 1.6 Hz, Hꢀ
2'), 6.91 (1H, d, J = 8.2 Hz, Hꢀ5'), 6.27 (1H, d, J = 15.9 Hz, Hꢀ8'), and 3.92 (3H, s, OMeꢀ3')]; 13C
NMR (150 MHz, CDCl3) δ 142.5 (s, Cꢀ5), 126.3 (d, Cꢀ6), 73.4 (d, Cꢀ7), 73.3 (d, Cꢀ3), 55.8 (d),
55.4 (d), 48.2, 42.9, 40.8, 39.6, 39.5, 37.8, 36.7, 36.6, 36.2, 35.7, 28.5, 28.0, 27.9, 26.4, 23.8,
22.8 (q, Cꢀ27), 22.6 (q, Cꢀ26), 21.0, 19.2 (q, Cꢀ19), 18.8 (q, Cꢀ19), 11.8 (q, Cꢀ18), and ferulate
moiety [166.6 (s, C=O, Cꢀ9'), 147.9 (s, Cꢀ4'), 146.7 (s, Cꢀ3'), 144.7 (d, Cꢀ7'), 127.0 (s, Cꢀ1'),
123.1 (d, Cꢀ6'), 115.9 (d, Cꢀ8'), 114.7 (d, Cꢀ5'), 109.3 (d, Cꢀ2'), and 55.9 (q, OMeꢀ3')]; positive
ESIMS, m/z 579 [M + H]+
.
343
Synthesis of Azaꢀoxysterols 7a and 8a.
344
345
346
347
348
349
Pregnenolone acetate (30). Procedure A was followed by using pregnenolone (1.58 g, 10.0
mmol) and Ac2O (4.7 mL, 50.0 mmol) in Py (20 mL), giving the title compound (3.58 g, yield:
1
100%) as a white solid: H NMR (600 MHz, CDCl3) δ 5.35 (1H, m, Hꢀ6), 4.32 (1H, m, Hꢀ3),
2.58ꢀ2.52 (2H, m), 2.39ꢀ2.31 (1H, m), 2.23ꢀ2.12 (1H, m), 2.12 (3H, s, Hꢀ21), 2.04 (3H, s, OAcꢀ
3), 2.0ꢀ1.2 (16H, m), 1.37 (3H, s, Hꢀ19), and 0.64 (3H, s, Hꢀ18); positive ESIMS, m/z 359 [M +
H]+
.
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356
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20ꢁAzaꢁcholesterol acetate (31). Pregnenolone acetate (358 mg, 1.0 mmol) was dissolved in
10 mL methanol, and isopentylamine (522 mg, 6.0 mmol) was added. pH value of the solution
was adjusted to around 6 by adding HOAc (1.0 mL). Then 10 mL of THF and NaBH3CN (82 mg,
1.1 mmol) in methanol (2 mL) were added, the reaction mixture was stirred under reflux
overnight. After evaporation in vacuo, the resulted residue was resuspended in 10 mL water, and
the pH was adjusted to 8 by 10% Na2CO3 solution. The solution was extracted with DCM (10
mL × 3), washed with water (10 mL) and brine (10 mL), dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by CC (H/E = 5:1; H/E/TEA = 5:1:0.1 and
3:1:0.1) to give the title compound (446 mg, yield: 85%) as a colorless gum: 1H NMR (600 MHz,
CDCl3) δ 5.37 (1H, m, Hꢀ6), 4.60 (1H, m, Hꢀ3), 2.90ꢀ2.75 (1H, m, Hꢀ20), 2.38ꢀ2.28 (2H, m, Hꢀ
23), 2.05 (3H, s, OAcꢀ3), 2.02ꢀ1.92 (1H, m), 1.90ꢀ1.80 (2H, m), 1.75ꢀ1.41 (12H, m), 1.40ꢀ1.10
(8H, m), 1.01 (3H, s, Hꢀ19), 0.98ꢀ0.84 (9H, m, Hꢀ21, Hꢀ26 and Hꢀ27), and 0.73 (3H, s, Hꢀ18);
positive ESIMS, m/z 430 [M + H]+
.
363
364
365
366
367
368
369
370
371
372
7ꢁKetoꢁ20ꢁazaꢁcholesterol acetate (32). Procedure B was followed by using 20ꢀazaꢀ
cholesterol acetate (446 mg, 1.04 mmol), CrO3 (1.56 g, 15.6 mmol) and dimethylpyrazole (1.50
g, 15.6 mmol) in dry DCM (50 mL). The mixture was stirred at rt for 6 h. After addition of EA,
the mixture was filtered through a short pad of Celite eluted by a mixture of EA and TEA (30:1),
and the filtration was concentrated in vacuo. The resulted residue was purified by CC (H/E = 5:1;
H/E/TEA = 5:1:0.1 and 3:1:0.1) to give the title compound (314 mg, yield: 68%) as a yellow
gum: 1H NMR (600 MHz, CDCl3) δ 5.70 (1H, m, Hꢀ6), 4.71 (1H, m, Hꢀ3), 2.98ꢀ2.80 (1H, m, Hꢀ
20), 2.58ꢀ2.41 (2H, m, Hꢀ23), 2.30ꢀ2.10 (2H, m), 2.05 (3H, s, OAcꢀ3), 2.02ꢀ1.20 (19H, m), 1.18
(3H, s, Hꢀ19), 0.98ꢀ0.84 (9H, m, Hꢀ21, Hꢀ26 and Hꢀ27), and 0.75 (3H, s, Hꢀ18); positive ESIMS,
m/z 444 [M + H]+
.
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377
378
379
380
381
382
383
7ꢁKetoꢁ20ꢁazaꢁcholesterol (7a). Procedure C was followed by using 7ꢀketoꢀ20ꢀazaꢀ
cholesterol acetate (314 mg, 0.71 mmol) and LiOH (68 mg, 2.84 mmol) in THF/MeOH/H2O
(3:1:1, 5 mL). The mixture was stirred at rt for 1 h. After removal of the organic solvent, the
residue was partitioned between water (5 mL) and DCM (5 mL). DCM extracts were washed
with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by
CC (H/E/TEA = 5:1:0.5, 3:1:0.5, 2:1:0.5 and 1.5:1:0.5) to give the title compound (7a) (240 mg,
yield: 85%) as a white solid: 1H NMR (700 MHz, CDCl3) δ 5.69 (1H, s, Hꢀ6), 3.67 (1H, m, Hꢀ3),
2.97ꢀ2.86 (1H, m, Hꢀ20), 2.53ꢀ2.49 (2H, m, Hꢀ23), 2.41ꢀ2.38 (1H, m), 2.25ꢀ2.21 (1H, m), 1.96ꢀ
1.87 (2H, m), 1.85ꢀ1.77 (2H, m), 1.70ꢀ1.66 (2H, m), 1.64ꢀ1.58 (2H, m), 1.57ꢀ1.50 (1H, m), 1.45ꢀ
1.21 (10H, m), 1.18 (3H, s, Hꢀ19), 0.94 (3H, d, J = 6.5 Hz, Hꢀ21), 0.93 (6H, d, J = 6.5 Hz, Hꢀ
26/27), and 0.78 (3H, s, Hꢀ18); positive ESIMS, m/z 402 [M + H]+
.
384
385
386
387
388
389
390
391
392
393
394
395
7βꢁHydroxyꢁ20ꢁazaꢁcholesterol (8a). Procedure D was followed by using 7ꢀketoꢀ20ꢀazaꢀ
cholesterol (7a) (284 mg, 0.71 mmol), CeCl3·7H2O (1.59 g, 4.26 mmol) and NaBH4 (108 mg,
o
o
2.84 mmol) in MeOH (10 mL) at ꢀ10 C. The mixture was stirred at ꢀ10 C for 5 min and
quenched with water (1 mL). After removal of most of the organic solvent, the residue was
partitioned between water (10 mL) and DCM (10 mL). DCM extracts were washed, dried and
concentrated. The resulted residue was purified by CC (H/E/TEA = 5:1:0.1, 2:1:0.1, 2:1:0.5, and
1
1:1:0.5) to give the title compound (8a) (100 mg, yield: 35%) as a white solid: H NMR (700
MHz, CDCl3) δ 5.27 (1H, s, Hꢀ6), 3.83 (1H, d, J = 8.1 Hz, Hꢀ7), 3.54 (1H, m, Hꢀ3), 2.99 (1H, m,
Hꢀ20), 2.88 (1H, m, Hꢀ23a), 2.68 (1H, m, Hꢀ23b), 2.35ꢀ2.31 (1H, m), 2.28ꢀ2.22 (1H, m), 1.98ꢀ
1.83 (5H, m), 1.66ꢀ1.57 (4H, m), 1.53ꢀ1.46 (4H, m), 1.42ꢀ1.30 (4H, m), 1.25ꢀ1.16 (4H, m), 1.16
(3H, s, Hꢀ19), 0.94 (3H, d, J = 6.7 Hz, Hꢀ21), 0.93 (3H, d, J = 6.5 Hz, Hꢀ27), 0.92 (3H, d, J = 6.5
Hz, Hꢀ26), and 0.73 (3H, s, Hꢀ18); 13C NMR (175 MHz, CDCl3) δ 143.5 (s, Cꢀ5), 125.6 (d, Cꢀ6),
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398
73.3 (d, Cꢀ7), 71.3 (d, Cꢀ3), 63.2 (d, Cꢀ20), 55.2 (d, Cꢀ17), 48.1, 45.9, 43.5, 42.9, 41.7, 40.6, 37.7,
37.5, 37.0, 36.6, 31.5, 28.2, 26.7, 26.3, 22.6, 22.4 (q, Cꢀ26/27), 20.8 (q, Cꢀ21), 19.1 (q, Cꢀ19),
and 12.7 (q, Cꢀ18); positive ESIMS, m/z 404 [M + H]+
.
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
LC/MS Analysis. LC/MS analysis was carried out with a ThermoꢀFinnigan Spectra System
consisting of an Accela high speed MS pump, an Accela refrigerated autosampler, and an LCQ
Fleet ion trap mass detector (Thermo Electron, San Jose, CA, USA) incorporated with an
electrospray ionization (ESI) interface. A GeminiꢀNX C18 column (150 mm × 3.0 mm i.d., 5 ꢀm,
Phenomenex) was used to analyze authentic sterol oxides and the sterolꢀenriched fraction with a
flow rate of 0.3 mL/min. The binary mobile phase system consisted of water with 0.2% acetic
acid (AA) as phase A and methanol with 0.2% AA as phase B. The column was eluted by a
gradient progress (80 to 90% B from 0 to 10 min; 90 to 95% B from 10 to 20 min; 95ꢀ99% B
from 20 to 30 min; 99ꢀ100% B from 30 to 45 min; 100% B from 45 to 65 min, and then 80% B
from 65 to 70 min). The injection volume was 10 ꢀL for each sample. The column temperature
was maintained at 20 °C. To optimize the mass spectrometric parameters, each standard
dissolved in methanol (10 µM) was infused in the ESI source by a syringe pump (flow rate 10
µL/min) and analyzed in positive ion mode. The optimized parameters for analysis of
oxyphytosterols are: capillary voltage 16 V, spray voltage 4.6 kV, tube lens offset 70 V, capillary
o
temperature 260 C, and sheath gas (nitrogen) flow rate 25 (arbitrary units). The structural
information of compounds 1ꢀ21 were obtained by tandem mass spectrometry (MS/MS) through
collisionꢀinduced dissociation (CID) with a relative collision energy setting of 35%. Data
acquisition was performed with Xcalibur version 2.0 (Thermo Electron, San Jose, CA, USA).
1
417
418
Nuclear Magnetic Resonance (NMR). H, 13C NMR, and twoꢀdimensional (2ꢀD) NMR
spectra were recorded on a Bruker AVANCE 600 MHz or 700 MHz spectrometer (Bruker Inc.,
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421
Silberstreifen, Rheinstetten, Germany). Compounds were analyzed in CDCl3, CD3OD or
Pyridineꢀd5. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and br
(broad). The 13C NMR spectra are proton decoupled.
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
MTT Assay. Cell growth inhibition was determined by a 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) colorimetric assay.21 Human colon cancer cells HCTꢀ116
and HTꢀ29 were plated in 96ꢀwell microtiter plates with 5000 cells/well and allowed to attach for
24 h at 37 ºC. The test compounds in dimethyl sulfoxide (DMSO) were added to cell culture
medium to the desired final concentrations (final DMSO concentrations for control and
treatments were 0.1 %). After the cells were cultured for 24 h, the medium was aspirated and
cells were treated with 200 ꢀL fresh medium containing 2.41 mmol/L MTT. After incubation for
3 h at 37 ºC, the medium containing MTT was aspirated, 100 ꢀL of DMSO was added to
solubilize the formazan precipitate, and plates were shaken gently for an hour at room
temperature. Absorbance values were derived from the plate reading at 550 nm on a Biotek
microtiter plate reader (Winooski, VT). The reading reflected the number of viable cells and was
expressed as a percentage of viable cells in the control. Both HCTꢀ116 and HTꢀ29 cells were
cultured in McCoy’s 5A medium. All of the above media were supplemented with 10 % fetal
bovine serum, 1 % penicillin/streptomycin, and 1 % glutamine, and the cells were kept in a 37 ºC
incubator with 95% humidity and 5% CO2.
437
438
439
440
441
Apoptotic Assay. The Cell Death Detection ELISA (Enzymeꢀlinked immunoabsorbant assay)
Plus kit from Roche (Mannheim, Germany) was used. HCTꢀ116 and HTꢀ29 cells were seeded in
o
96ꢀwell plates at 3000 cells/well and incubated at 37 C in 5% CO2 incubator. After 24 hours,
fresh media supplemented with DMSO (control), 5, 6, 7, or 8 (5, 10, 20 or 30 ꢀM; final DMSO
concentration for control and treatments was 0.1%) were added to the wells. After 48 h, the
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444
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447
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461
462
463
464
microplate was centrifuged for 10 min at 1200 rpm, and the supernatant was removed. Then, 200
µL of the lysis buffer was added in each well and the microplate was incubated for 30 min at
room temperature. The plate was then centrifuged for 10 min at 1200 rpm and 20 µL of the
supernatant was transferred to streptavidinꢀcoated microwells. ELISA assay was performed
according to manufacturer’s instruction. Absorbance in each well was measured at 405 nm in
absorbance units (AU), and the enrichment factor (EF) in small nucleosomes was calculated with
the formula EF = AU(treated)/AU(DMSO). The experiment was repeated independently to confirm the
results.
Statistical Analysis. For simple comparisons between two groups, twoꢀtailed Student’s tꢁtest
was used. A pꢀvalue of less than 0.05 was considered statistically significant in all the tests.
RESULTS
Structural Elucidation of Oxyphytosterols 1ꢀ4. The chemical investigation on the sterolꢀ
enriched fraction yielded two unusual 3ꢀketophytosterols 1 and 2, and two 7ꢀketophytosterols 3
and 4 (Figure 1). Compound 1 had a molecular formula of C29H48O2 based on positive ESIꢀMS
at m/z 411 [M – H2O + H]+ and NMR data. One tertiary proton adjacent to oxygen atom, one
1
olefinic proton and six methyl groups in the H NMR spectrum of 1, and a ketone group, four
double bond signals and an oxygenated tertiary carbon in the 13C NMR spectrum of 1, suggested
1 is oxyphytosterol.22 Heteronuclear multiple bond correlation (HMBC) established a partial
structure of 4ꢀenꢀ6ꢀolꢀ3ꢀone in 1 (Figure 2). The fragment ion at m/z 253/393/429 [M – 2H2O –
C10H20 + H]+ in the MS3 spectra of 1 (Table 1), corresponding to the loss of two water molecules
followed by the cleavage of the side chain, further confirmed 1 as being stigmastꢀ4ꢀenꢀ6βꢀolꢀ3ꢀ
one, as previously described in extracts of wheat straw.23
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Compound 2 possessed a molecular formula of C29H46O2 based on positive ESIꢀMS at m/z
1
13
409 [M – H2O + H]+ and NMR data. Its H and C NMR data were very similar to those of 1
except for additional olefinic signals [δH 5.09 (1H, dd, J = 15.2, 8.8 Hz) and δH 5.24 (1H, dd, J =
15.2, 8.8 Hz); and δC 130.0 (d) and δC 139.2 (s)] in 2, indicating 2 is stigmastꢀ4,22ꢀdienꢀ6βꢀolꢀ3ꢀ
one, as previously described in extracts of wheat straw.23
470
471
472
473
474
475
476
477
Compound 3 had the same molecular formula of C29H48O2 to that of 1 based on positive ESIꢀ
1
13
MS at m/z 411 [M – H2O + H]+ and NMR data. The H and C NMR spectra of 3 were similar
to those of 1, suggesting 3 is also an oxyphytosterol. HMBC correlations established a partial
structure of 5ꢀenꢀ3ꢀolꢀ7ꢀone in 3 (Figure 2). The fragment ion at m/z 253/393/429 [M – 2H2O –
C10H20 + H]+ in the MS3 spectra of 3, corresponding to the loss of two water molecules followed
by the side chain, further confirmed 3 as being stigmastꢀ5ꢀenꢀ3βꢀolꢀ7ꢀone, namely 7ꢀ
ketositosterol, as previously reported from marine sponges.24 This is the first report of 7ꢀ
ketositosterol 3 in WB.
478
479
480
481
482
483
484
485
Compound 4 had a molecular formula of C29H46O2 based on positive ESIꢀMS at m/z 409 [M
– H2O + H]+ and NMR data. Its 1H and 13C NMR data were very similar to those of 3 except for
additional olefinic signals [δH 5.01 (1H, dd, J = 15.1, 8.7 Hz) and δH 5.15 (1H, dd, J = 15.1, 8.7
Hz); and δC 130.3 (d) and δC 139.0 (d)] in 4, indicating 4 is an oxidation product of stigmasterol.
Fragment ion at m/z 251/391/427 [M – 2H2O – C10H20 + H]+ in the MS3 spectra of 4 (Table 1),
following the same fragmentation patterns as 3, established 4 as being stigmastꢀ5,22ꢀdienꢀ3βꢀolꢀ
7ꢀone, namely 7ꢀketostigmasterol, as previously described elsewhere.25 This is the first report of
7ꢀketostigmasterol 4 in WB as well.
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Synthesis of Sterol Oxides 3ꢀ8, Sterol Ferulates 13 and 13a, and Azaꢀoxysterols 7a and
8a. Sterol oxides 3ꢀ8 were synthesized in house in quantities, following the method of McCarthy
with some modifications (Figure 3A).26 Briefly, acetylation of sterols yielded sterol acetates
22−24. The acetates were treated by the combination of chromium trioxide and dimethylpyrazole
490
491
492
493
494
495
496
497
498
499
500
501
to give 7ꢀketosterol acetates 25−27. After hydrolysis using lithium hydroxide, 7ꢀketosterols 3, 4
and 7 were obtained. Luche reduction of 7ꢀketosterols afforded 7βꢀhydroxysterols 5, 6 and 8.
Coupling reaction of 7ꢀketocholesterol 7 with 4ꢀacetoxy ferulic acid utilizing N,N′ꢀ
dicyclohexylcarbodiimide (DCC) and 4ꢀdimethylaminopyridine (DMAP) followed by
deacetylation yielded 7ꢀketocholesterol ferulate 13. Likewise, 7βꢀhydroxycholesterol ferulate
13a was obtained from 7βꢀhydroxycholesterol 8. The synthesis of azaꢀoxysterols 7a and 8a was
illustrated in Figure 3B. Reductive amination of pregnenolone acetate 30 with isopentylamine
catalyzed by NaBH3(CN) in MeOH/THF at pH 6 produced 20ꢀazaꢀcholesterol acetate 31.27
Oxidation of 31 by chromium trioxide followed by treatment with lithium hydroxide afforded 7ꢀ
ketoꢀ20ꢀazaꢀcholesterol 7a with a yield of 60%. Luche reduction of 7a gave 7βꢀhydroxylꢀ20ꢀazaꢀ
cholesterol 8a with a yield of 35%. This is the first report of synthesis for 7ꢀketoꢀ20ꢀazaꢀ
cholesterol 7a and 7βꢀhydroxyꢀ20ꢀazaꢀcholesterol 8a.
502
503
504
505
506
507
508
Characterization of Synthetic Sterol Oxides 5ꢀ8, Sterol Ferulates 13 and 13a, and Azaꢀ
oxysterols 7a and 8a. Compound 5 was reduced from 7ꢀketositosterol 3. Its molecular ion at m/z
431 [M + H]+ is two mass units higher than that of 3, indicating 5 is the reduction product of 3.
The appearance of one additional signal adjacent to oxygen atom [δH 3.72 (1H, d, J = 8.0 Hz)
and δC 73.7 (d)] in 5 confirmed the conversion of the carbonyl group in 3 into the hydroxyl group
in 5. The fragment ion at m/z 241/395/431 [M – 2H2O – C10H20 – CH2 + H]+ in the MS3 spectra
of 5 (Table 1), corresponding to the loss of two water molecules followed by the side chain and
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511
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one methyl group, further supported that 5 was stigmastꢀ5ꢀenꢀ3β,7βꢀdiol, namely 7βꢀ
hydroxysitosterol, as previously reported in wheat flour.18 Likewise, compound 6 was the
product of the reduction of 4 and subsequently established as being stigmastꢀ5,22ꢀdienꢀ3β,7βꢀ
diol, namely 7βꢀhydroxystigmasterol, as previously reported elsewhere.25
513
514
515
516
517
518
519
520
Compound 7, owning a molecular formula of C27H44O2 based on positive ESIꢀMS at m/z 383
1
13
[M – H2O + H]+ and NMR data, was synthesized from cholesterol. Its H and C NMR data
were very similar to those of 3, suggesting 7 is an oxidation product of cholesterol. The fragment
ion at m/z 253/365/401 [M – 2H2O – C8H16 + H]+ in the MS3 spectra of 7 (Table 1), in
agreement with the loss of two water molecules followed by the side chain, provided 7 as
cholestꢀ5ꢀenꢀ3βꢀolꢀ7ꢀone, namely 7ꢀketocholesterol, as previously described in marine sponges.24
Likewise, compound 8, as the reduction product of 7, was elucidated as being cholestꢀ5ꢀenꢀ
3β,7βꢀdiol, namely 7βꢀhydroxycholesterol, as previously reported in marine sponges.24
521
522
523
524
525
526
527
528
529
Compound 13, possessing a molecular formula of C37H52O5 based on its positive ESIꢀMS at
m/z 383 [M – C10H10O4 + H]+ and NMR data, was a ferulic acid conjugate of 7. HMBC
correlation between Hꢀ3 (δH 4.84) and the carbonyl group (Cꢀ9') of ferulate (δC 166.4)
established the linkage at Cꢀ3 between ferulate moiety and 7ꢀketocholesterol (Figure 2). The
fragment ion at m/z 383/577 [M – C10H10O4 + H]+ in the MS2 spectra of 13, corresponding to the
loss of a ferulic acid moiety, further confirmed 13 as being 7ꢀketocholesterol ferulate. Compound
13a was synthesized by the coupling reaction of ferulic acid with 8. Its NMR spectra were
similar to those of 13 except for an additional oxygenated signal [δH 3.87 (1H, d, J = 7.8 Hz) and
δC 73.4 (d)], coinciding 13a being 7βꢀhydroxycholesterol ferulate.
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531
532
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537
Compound 7a, having a molecular formula of C26H43NO2 based on its positive ESIꢀMS at
m/z 402 [M + H]+ and NMR data, was synthesized from pregnenolone through four steps. The 1H
NMR spectra of 7a was similar to that of 7ꢀketocholesterol 7. The major difference is the
appearance of two multiplets at δH 2.97ꢀ2.86 (1H, m, Hꢀ20) and δH 2.53ꢀ2.49 (2H, m, Hꢀ23) in
7a compared to those of 7, indicating 7a is 7ꢀketoꢀ20ꢀazaꢀcholesterol. Compound 8a was the
1
13
reductive product of 7a. The H and C NMR spectra of 8a showed an additional oxygenated
signal [δH 3.83 (1H, d, J = 8.1 Hz) and δC 73.3 (d)] compared to 7a, suggesting 8a is 7βꢀ
hydroxyꢀ20ꢀazaꢀcholesterol.
538
539
540
541
542
543
544
545
546
547
548
Fragmentation Patterns of Authentic Sterol Oxides. There are two major fragmentation
patterns of the authentic 7ꢀketosterols 3, 4 and 7 (Table 1) under ESI positive ionization mode,
as exemplified by compound 3 in Figure 4, pattern A): loss of two water molecules from parent
ion forms a stable resonance structure with the positive charge at Cꢀ7 position in MS2 spectra
(Figure 4A); and pattern B): cleavage of the side chain at the C17ꢀC20 bond in the fragments
obtained from pattern A via hydrogen shifts and inductive cleavages produces a typical daughter
ion in MS3 spectra (Figure 4B). These fragmentation patterns are consistent with the conclusions
that Jiang et al. reported previously.28 The observation from 7βꢀhydroxysterols 5, 6 and 8 led to a
characteristic fragmentation pattern C): the cleavage of the side chain followed by an additional
methyl group in the fragments obtained from pattern A gives characteristic tertiary daughter ions
in MS3 spectra (Table 1).
549
550
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Identification of Steroids 1ꢀ21 in WB by LCꢀESI/MSn Spectra. Oxyphytosterols 1ꢀ4 were
isolated from WB extract and identified by NMR experiments. Sterol oxides 5ꢀ8 and 13 in WB
were recognized by comparison of their retention times and tandem mass spectra with those of
the synthetic standards (Table 1). Compounds 9ꢀ12 and 14ꢀ20 were tentatively elucidated in WB
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based on the analysis of their tandem mass spectra, referring to fragmentation patterns observed
from known standards (Table 1 and Figure 4). Stigmasterol 21 was confirmed in WB by
comparison with its commercial authentic standard.
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As per the knowledge obtained above, compound 9 had two peak ions with loss of H2O at
m/z 397 [M – H2O + H]+ and 379 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and one
daughter ion with loss of the alkyl side chain at m/z 253 [M – 2H2O – C9H18 + H]+ in its MS3
spectra (pattern B) (Table 1), indicating 9 being campestꢀ5ꢀenꢀ3βꢀolꢀ7ꢀone, namely 7ꢀ
ketocampesterol.29 Compound 10, with two peak ions at m/z 399 [M – H2O + H]+ and 381 [M –
2H2O + H]+ in its MS2 spectra (pattern A), and one major daughter ion at m/z 241 [M – 2H2O –
C9H18 – CH2 + H]+ in its MS3 spectra (pattern C) (Table 1), was identified as campestꢀ5ꢀenꢀ
3β,7βꢀdiol, namely 7βꢀhydroxycampesterol.29 Similarly, compound 11, having two peak ions at
m/z 395 [M – H2O + H]+ and 377 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and one
tertiary ion at m/z 251 [M – 2H2O – C9H18 + H]+ in its MS3 spectra (pattern B), was proposed as
ergostꢀ4ꢀenꢀ3,6ꢀdione, as reported previously in wheat straw.30 Compound 12 owned two peak
ions at m/z 401 [M – H2O + H]+ and 383 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and
one daughter ion at m/z 243 [M – 2H2O – C9H18 – CH2 + H]+ in its MS3 spectra (pattern C), was
tentatively suggested as ergostaneꢀ3β,7βꢀdiol, as described previously elsewhere.31
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Compound 14, with a base peak at m/z 397 [M – C10H10O4 + H]+ in its MS2 spectra and a
major ion at m/z 379 [M – C10H10O4 – H2O + H]+ in its MS3 spectra, had similar fragmentation
patterns (loss of ferulic acid moiety) as that of analogue 13 (Table 1), indicating 14 is a sterol
ferulate. The molecular weight of 14 at m/z 591 [M + H]+ was 14 mass units higher than that of
13, suggesting that 14 is 7ꢀketocampesterol ferulate, which has never been reported before. In a
similar manner, compounds 15ꢀ17, with major peak ions at m/z 383 [M – C10H10O4 + H]+, 395
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[M – C10H10O4 + H]+ and 397 [M – C10H10O4 + H]+ in their respective MS2 spectra,
corresponding to loss of ferulic acid moiety, were identified as campesterol ferulate 15,
stigmasterol ferulate 16 and sitosterol ferulate 17, respectively. Of which, campesterol ferulate
15 and sitosterol ferulate 17 have been reported in WB previously,19 whereas stigmasterol
ferulate 16 was here described in WB for the first time. Peak ions at m/z 415 [M – 162 + H]+ and
397 [M – 180 + H]+ in MS2 spectra of 18 corresponded to the loss of a hexanose. Moreover, peak
ion at m/z 379 [M – 180 – H2O + H]+ in its MS3 spectra, similar to the fragmentation of 7ꢀ
ketocampesterol 9, tentatively suggested that 18 is 3ꢀOꢀglucopyranosylꢀ7ꢀketocampesterol,
which has never been reported before. Compound 19, bearing two peak ions at m/z 427 [M – 162
+ H]+ and 409 [M – 180 + H]+ in its MS2 spectra and one peak ion at m/z 391 [M – 180 – H2O +
H]+ in its MS3 spectra, was therefore proposed as 3ꢀOꢀglucopyranosylꢀ7ꢀketostigmasterol, as
reported previously in Rosa laevigata.32 Similarly, parent ion at m/z 591 [M + H]+ of 20 was 14
mass units higher than that of 18, indicating that 20 is 3ꢀOꢀglucopyranosylꢀ7ꢀketositosterol, as
described previously in Rosa laevigata.32
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Consequently, a total of twentyꢀone steroids 1ꢀ21 were identified and characterized from WB
extract, using NMR experiments, chemical synthesis approaches, and LCꢀESI/MSn techniques,
eventually leading to a comprehensive profile of steroids in WB, as shown in Figure 5. Among
them, 7ꢀketositosterol 3 and 7ꢀketostigmasterol 4 were found to be the major oxyphytosterols in
WB.
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In Vitro Cytotoxic Activity. Two human colon cancer cell lines HCTꢀ116 and HTꢀ29 were
treated with eight sterol oxides 1ꢀ8 and the nonꢀoxidized stigmasterol 21, with concentrations
ranging from 0 to 120 ꢀM. Seven sterol oxides 2ꢀ8 were found to be active against the growth of
both HCTꢀ116 and HTꢀ29 human colon cancer cells (Figures 6A and 6B). Nonꢀoxidized
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stigmasterol was inactive in both cells (IC50 >120
ꢀ
M, Figures 6A and 6B). Unusual 3ꢀ
ketophytosterols 1 and 2 were less active than 7ꢀketophytosterols 3, 4 and 7 in both cancer cell
lines, indicating an entity of 3ꢀolꢀ5ꢀenꢀ7ꢀone in the structure is more favorable for antiꢀcancer
activity than a 6ꢀolꢀ4ꢀenꢀ3ꢀone. In HCTꢀ116 cell lines, 7βꢀhydroxycholesterol 8 was found to be
the most active compound with an IC50 value of 13.20 ꢀM, followed by 7βꢀhydroxystigmasterol
6 (IC50: 15.73 ꢀM), 7βꢀHydroxylsitosterol 5 (IC50: 16.64 ꢀM), 7ꢀketocholesterol 7 (IC50: 24.78
ꢀM), 7ꢀketositosterol 3 (IC50: 32.66 ꢀM), and 7ꢀketostigmasterol 4 (IC50: 43.47 ꢀM) (Figure 6A).
The reduction products 5, 6 and 8 showed 2ꢀfold higher inhibitory effects than their respective 7ꢀ
keto precursors 3, 4 and 7 (Figure 6A), suggesting an entity of 5ꢀenꢀ3,7ꢀdiol in the structure
greatly increases the activity compared to an partial structure of 3ꢀolꢀ5ꢀenꢀ7ꢀone. 7βꢀ
Hydroxycholesterol 8, with eight carbons in the alkyl side chain, showed a better inhibitory
effect than 7βꢀhydroxysterols 5 and 6, both bearing ten carbons in alkyl side chains, indicating
that increasing the numbers of carbon in the alkyl side chain decreases the activity. In HTꢀ29 cell
lines, 7βꢀhydroxycholesterol 8 also showed the most potency with IC50 value of 10.09 ꢀM
(Figure 6B). Whereas, the rest of the sterol oxides 2ꢀ7 showed moderate inhibitory effects, with
IC50 values ranged from 29.57ꢀ60.46 ꢀM (Figure 6B). Notably, the two major oxyphytosterols 3
and 4 found in WB exerted great antiproliferative property in both cancer cell lines, indicating
oxyphytosterols may contribute to the observed chemopreventive effects of WB.
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We also measured the growth inhibitory effects of the ferulate conjugates of sterols, 7ꢀ
ketocholesterol ferulate 13 and 7βꢀhydroxycholesterol ferulate 13a. Our results indicated that
they were inactive in both cell lines (IC50s > 60 ꢀM), indicating a free hydroxyl group at Cꢀ3β in
both entities of 3ꢀolꢀ5ꢀenꢀ7ꢀone and 5ꢀenꢀ3,7ꢀdiol plays a crucial role in the observed activity.
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In order to increase the polarity of oxyphytosterols, we synthesized two azaꢀoxysterols 7a
and 8a by introducing an –NH group into Cꢀ20 in the side chain of oxysterols 7 and 8,
respectively. In HTꢀ29 cells, MTT assays showed that 7a exerted a similar activity (IC50: 35.33
ꢀM) to that of 7 (IC50: 36.23 ꢀM), and the activity of 8a was attenuated while still remaining
active (IC50: 54.26 ꢀM), compared to 8 (IC50: 10.09 ꢀM). In HCTꢀ116 cells, the activity of both
azaꢀoxysterols 7a and 8a were decreased (IC50 > 60 ꢀM for both) compared to oxysterols 7 and 8.
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Induction of Apoptosis in Colon Cancer Cells. Apoptosis is a mechanism often responsible
for the induction of cell death in response to internal or external stress. In order to clarify
whether oxyphytosterols exert antiproliferative properties via a pathway of apoptosis, we tested
the four most active sterol oxides 5ꢀ8 for apoptotic effects in human colon cancer cells HCTꢀ116
and HTꢀ29. The results are summarized in Figures 6C and 6D. In HCTꢀ116 cells, compounds 5ꢀ
7 showed significant apoptotic effects at the concentration of 30 ꢀM (Figure 6C), while
compound 8 exerted significant apoptotic effect at an even low concentration (20 ꢀM) compared
to DMSO control. In particular, the induction of apoptosis by 8 was significantly superior to
those of the rest 5ꢀ7 at the same concentration (20 ꢀM). In HTꢀ29 cells, compound 8 was also
found to be the most potent agent to induce apoptosis (Figure 6D). Compound 6 displayed a
significant apoptotic effect at the concentration of 30 ꢀM (Figure 6D). Compounds 5 and 7
showed more or less apoptotic effects at the concentration of 30 ꢀM.
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DISCUSSION
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In the present study, we outlined a comprehensive chemical profile of steroids in WB.
Notably, we observed that seventeen (1ꢀ14 and 18ꢀ20) out of twentyꢀone steroids (1ꢀ21) in WB
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