3-Keto-22-epi-28-nor-cathasterone, a brassinosteroid-related metabolite from Cystoseira myrica

Article abstract of DOI:10.1016/j.steroids.2009.06.008

Bioassay-guided purification of an ethanolic extract of Cystoseira myrica against HEPG-2 (liver) and HCT116 (colon) human cancer cell lines led to the isolation of 3-keto-22-epi-28-nor-cathasterone, 1 and cholest-4-ene-3,6-dione, 2. This finding allowed u

Full text of DOI:10.1016/j.steroids.2009.06.008

Steroids 74 (2009) 927–930
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Steroids
journal homepage: www.elsevier.com/locate/steroids
3-Keto-22-epi-28-nor-cathasterone, a brassinosteroid-related
metabolite from Cystoseira myrica
Abdel-Hamid A. Hamdya, Elsayed A. Aboutablb, Somayah Sameera, Ahmed A. Husseinc,∗
,
Ana R. Díaz-Marrerod, José Dariasd, Mercedes Cuetod,∗∗
a Chemistry of Natural and Microbial Products Department, NRC, Cairo, Egypt
b Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Chemistry of Medicinal Plants Department, NRC, Cairo, Egypt
d Instituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico F. Sánchez, 3, Apdo. 195, 38206 La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2009
Received in revised form 23 June 2009
Accepted 24 June 2009
Available online 2 July 2009
Bioassay-guidedpurificationofanethanolicextractof CystoseiramyricaagainstHEPG-2(liver)andHCT116
(colon) human cancer cell lines led to the isolation of 3-keto-22-epi-28-nor-cathasterone, 1 and cholest-
4-ene-3,6-dione, 2. This finding allowed us to report for the first time that a brassinosteroid-related
metabolite occurs in seaweed. These compounds showed activity in the range of 12.38–1.16 ␮M with
selective activity of compound 2 to liver cancer cell lines.
© 2009 Elsevier Inc. All rights reserved.
Keywords:
Cystoseira
C27-brassinosteroid-related
Cytotoxicity
1. Introduction
the cytotoxic activity of its ethanolic extract against HEPG-2 and
HCT116, IC50 16.10 and 6.96 ␮g/mL, respectively. Bioassay-guided
Despite that plants and animals have diverged more than 1 bil-
lion years ago, they both use polyoxygenated steroidal molecules
as hormones. Brassinosteroids (BRs), the most recently discovered
class of plant-specific steroid hormones, essential for growth and
development, are polyoxygenated plant steroids with structural
similarity to ecdysteroids, growth-regulating steroid hormones
found in vertebrates and insects [1]. More than 50 naturally occur-
ring BRs have been identified in a wide range of plant species being
fractionation of the extract led to the isolation of the novel com-
pound 1 and the known 2 [4].
2. Experimental
2.1. General procedures
Optical rotations were measured on a Perkin-Elmer model 343
Plus polarimeter using a Na lamp at 25 ◦C. IR spectra were obtained
on a Fourier Transform Infrared Spectrometer (JASCO FT/IR–6100
Japan). Analysis was performed using KBr method. 1H NMR and
13C NMR, HSQC, HMBC, COSY and NOESY spectra were measured
employing a Bruker AMX 500 instrument operating at 500 MHz
for 1H NMR and at 125 MHz for 13C NMR. Two-dimensional NMR
spectra were obtained with the standard Bruker software. EIMS and
HREIMS data were taken on a Micromass Autospec spectrometer.
Merck Si gel (70–230 mesh and 230–400 mesh, for gravity flow
and flash chromatography, respectively) was used for column chro-
matography. Merck 5554 Kieselgel 60 F254 sheets were used for TLC
analysis.
C28-BRs (i.e., casthasterone (CS), brassinolide (BL)) and C27-BRs (i.e.,
28-norcasthasterone) the most abundant and widely occurring [2]
(Fig. 1). Other brassinosteroids are distributed in a limited number
of plant species but two of them, epicasthasterone and (24S)-24-
ethylbrassinone, have been found in a freshwater green alga [3]
(Fig. 1).
Isolation of compound 1 from Cystoseira myrica (Fig. 2), allowed
us to report for the first time that a brassinosteroid-related metabo-
lite occurs in seaweed. The brown alga C. myrica was selected
for chemical study among several algal species of the Red Sea by
2.2. Biological material
∗
Corresponding author. Tel.: +20 2 33371499; fax: +20 2 33077931.
Corresponding author. Tel.: +34 922 252 144; fax: +34 922 260 135.
E-mail addresses: hussein-ahmed@lycos.com (A.A. Hussein),
∗∗
C. myrica was collected from the region of Fayed, Egypt, in April
mcueto@ipna.csic.es (M. Cueto).
2006 and was identified by Dr. Adel Fahmi, Faculty of Science, Ein
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.06.008

928
A.-H.A. Hamdy et al. / Steroids 74 (2009) 927–930
Table 1
NMR data of compound 1 [500 MHz, ı ppm, (J) Hz, CDCl3].
#
ıH
ıC
38.1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.60 m; 2.08 m
2.33 m; 2.40 m
–
2.30 m; 2.57 m
2.58 m
37.4
211.3
36.9
57.5
208.9
46.6
37.9
53.5
41.2
21.7
39.4
43.3
56.2
24.1
27.2
53.1
12.0
12.5
42.2
12.4
73.9
27.5
36.0
28.1
22.4
22.9
–
1.98 dd (12.9; 12.9); 2.36 m
1.83 m
1.32 m
–
1.44 m; 1.65 m
␣: 1.24 m; ␤: 2.04 m
–
1.26 m
1.11 m; 1.56 m
1.36 m; 1.72 m
1.17 m
0.70 s
0.94 s
1.70 m
Fig. 1. Selected examples of C27-, C28- and C29-BRs.
0.92 d (6.6)
3.60 bd (9.8)
1.18 m; 1.23 m
1.15 m; 1.40 m
1.24 m
Shams University. The alga was washed with water, dried in the
shade, and ground to a fine powder with a blender.
0.87 d (6.6)
0.89 d (6.6)
2.3. Extraction and isolation
2.5 kg of air-dried C. myrica was soaked in ethanol 95% at
room temperature for 3 days, the process was repeated three
times. The combined ethanol extracts were filtered and evapo-
rated under reduced pressure to yield 115.0 g total extract. The total
crude extract was applied to a flash silica column (15 cm × 25 cm)
and eluted with a solvent gradient of increasing polarity from
hexane to ethyl acetate. Fractions of 250 ml were collected and
those exhibiting similar TLC profiles were combined. Fraction C-
9 (522.0 mg, IC50 = 0.8, 4.36 ␮g/mL) was eluted through silica gel
using hexane–EtOAc (75:25) from which sub-fraction 9-1 (60.0 mg)
was purified by prep-TLC, solvent system CHCl3–MeOH (99:1),
to yield compound 1 (37.0 mg). Fraction C-6 (2.12 g, IC50 = 10.7,
13.6 ␮g/mL) was chromatographed on silica gel and eluted with
hexane–EtOAc mixtures (3%, 5%, 10%, 20%, 50% and EtOAc 100%).
Sub-fraction 6-3 (165.0 mg) was eluted with hexane–EtOAc (9:1),
and purified on prep-TLC (CHCl3–hexane–MeOH (77:22:1)) to yield
compound 2 (15.0 mg).
2.3.1. Compound 1
White powder; [␣]20 −30 (c 0.33, CH2Cl2); IR (KBr) ꢀmax 3545,
3406, 2925, 2857, 1709, 1631, 1462, 1385, 1271, 1242, 1163, 1116,
1078, 1027, 798, 648, 611, 464 cm−1; 1H NMR and 13C NMR in CDCl3,
see Table 1; EIMS 70 eV m/z (rel. int.): 416 [M]+ (1), 398 [M−H2O]+
(11), 316 [M−C6H12O]+ (100), 287 [M−C8H17 O]+ (46), 83 (66);
HREIMS m/z 416.3306 (calcd. for C27H44O3, 416.3290), 398.3190
(calcd. for C27H42O2, 398.3185), 316.2388 (calcd. for C21H32O2,
316.2402), 287.2027 (calcd. for C19H27O2, 287.2011).
2.4. (R)- and (S)-MPA ester derivatives 1a and 1b
A solution of compound 1 (4.1 mg, 9.8 × 10−3 mmol) in 1.0 mL
of CH2Cl2 was treated with N,Nꢀ-dicyclohexylcarbodiimide
(9.9 mg, 4.8 × 10−2 mmol), 4-dimethylaminopyridine (5.3 mg,
4.3 × 10−2 mmol) and (R)-␣-methoxy-␣-phenylacetic acid (9.9 mg,
6.0 × 10−2 mmol) and stirred at room temperature for 5 h. After
filtration, the reaction mixture was purified by silica gel chro-
matography (hexane–EtOAc (1:1)) to give the (R)-MPA ester
derivative 1a (2.6 mg, 4.7 × 10−3 mmol, 48.3% yield). The same
experimental procedure was followed to obtain the (S)-MPA ester
derivative 1b (2.4 mg, 4.4 × 10−3 mmol, 44.9% yield).
2.5. Cytotoxicity
The method used was previously described [5]. Human cancer
cells were seeded into 96-multiwell plates at 104 cells per well.
After 24 h, the cells were washed and maintained with different
concentrations of isolate for 48 h, at 37 ◦C and 5% CO2. Triplicate
wells were prepared for each individual dose. Cytotoxicity was
determined using the colorimetric SRB assay and scored as percent-
age reduction of treated culture versus untreated control culture.
Fig. 2. Occurring 22-hydroxylated BRs-related compounds in C. myrica and Ara-
bidopsis seedlings.

A.-H.A. Hamdy et al. / Steroids 74 (2009) 927–930
929
Fig. 4. Selected 1H NMR chemical shifts of 1, 10 and 11.
Fig. 3. Selected NOEs of compound 1.
agreement with the chemical shift of C-20 (ı 42.6) [12] and with
those reported for H3-21 and H-22, for the synthetic steroids 10
[13] and 11 [14] whose stereochemistries are 20S, 22R and 20R, 22R,
respectively (Fig. 4). Thus, the absolute configuration of 1 can be
established as 5S, 8S, 9S, 10R, 13S, 14S, 17R, 20S and 22R.
The IC50 values on the cell growth were obtained from the drug
dose–response curves using the software Origin 6.1.
3. Results and discussion
Investigation on the natural occurrence of 22-hydroxylated
steroids in wild-type Arabidopsis seedlings allowed the identifi-
cation, for the first time in the Plant Kingdom, of the C27-BRs 3–6
(Fig. 2). Both 3 and 6 partially rescued the det2 mutant phenotype
in the dark which supports the presence of an early C-22 oxida-
tion pathway [6]. Although little is known about the biosynthetic
pathways of C27-BRs such as 28-norcastasterone (Fig. 1), the find-
ing of compounds 3–6 supports the idea that C27-sterols are direct
precursors of the corresponding C27-BRs, and suggests that 3–6
3-Keto-22-epi-28-nor-cathasterone 1 is a C27-polyoxygenated
steroid with the following features: (a) like natural brassinosteroids
it possesses a common 5␣-cholestane skeleton; (b) it contains only
one hydroxyl group in the side chain like compounds 3–7 [6], and
cathasterone 8 [7], both identified in cultured cells of Catharan-
thus roseus; (c) A/B rings of 1 bear a 3,6-diketo functionality, which
is uncommon in C27-steroids. At present, only a few compounds,
in addition to 1 and 2, have been reported with that functional-
ity [8]. Among BRs only one compound bearing a 3,6-diketo group,
3-dehydroteasterone 9, has been identified [9].
derive from cholesterol via a biosynthetic pathway similar to C28
-
BRs biosynthesis [15]. In fact, cholesterol is the best substrate for
cytochrome P450, CYP90B1, that catalyses C-22 hydroxylation and,
according to kinetic analysis, the substrate specificity of CYP90B1
indicated that sterols with a C-5–C-6 double bond are preferred
substrates when compared with stanols, which have no double
bond at that position [16].
Compound 1 was active against human liver (HEPG-2) and colon
(HCT116) cancer cells with an IC50 2.96 ␮M and IC50 12.38 ␮M,
respectively. Compound 2 was found to be significantly cytotoxic
to both cell lines, with an IC50 of 5.63 and 1.16 ␮M, respectively. It
exhibited a particularly high cytotoxicity to liver cancer cells, where
the cell number of HEPG-2 fell below 10% that of the control at
3.79 ␮M (IC10).
Compound 1 was isolated as a white powder. NMR data coupled
with a molecular ion at m/z 416 (HREIMS) suggested a molecular
formula of C27H44O3, indicating six degrees of unsaturation. The 13
C
NMR data and a DEPT NMR experiment (Table 1) were consistent
for a C-27 sterol having two ketone and one hydroxyl groups. Com-
parison of 1H and 13C chemical shifts of 1 with those reported for
steroids possessing a diketo-3,6 nucleus such as stigmastane-3,6-
dieneone [10] indicated that 1 must be a 3,6-diketocholestane. 2D
NMR experiments, HSQC, HMBC, and COSY, allowed us to confirm
the position of the ketone groups at C-3 and C-6. The presence of
a hydroxyl group at C-22 was established by COSY correlations and
by the HMBC correlation of H3-21 (ıH 0.92) with the carbon that
bears the hydroxyl group (ıC 73.9, C-22).
The relative stereochemistry of 1 was assigned on the basis of a
2D NOESY experiment. The C-5/C-10; C-8/C-9 and C-13/C-14 trans
fused ring junctions were established by NOE correlations of H-
8 with H3-18 and H3-19. NOEs observed between H3-18 and H-20
indicateda␤ dispositionforthesidechain(Fig. 3). Theabsolutecon-
figuration of C-22 was established by derivatization with (R)- and
(S)-␣-methoxy-␣-phenylacetic acids (MPA). NMR analysis [11] of
the ꢁı values for the two MPA esters 1a and 1b gave clear evidence
to assign the absolute stereochemistry at C-22 as R (Table 2). The
observed NOEs between H-22 and H2-16 and between H3-21 and
H-12␤ indicated C-21 must be in ␣ disposition, therefore, consider-
ing that the absolute stereochemistry of C-20 has been established
as R, the absolute stereochemistry of C-20 must be S. This is in good
Acknowledgments
This work was supported by the Academy of Science and Tech-
nology (Egypt) and Ministerio de Educación y Ciencia (Spain)
(BIO2007-61745, SAF2006-03004). A.R.D.-M. acknowledges finan-
cial support from Programa Juan de la Cierva (Ministerio de
EducaciónyCienciaofSpain). Mr. E. PalmerandMr. D. Lilesprovided
technical support. The Government of Egypt granted permission for
the collection of the samples.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2009.06.008.
Table 2
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