Synthesis of a new cytotoxic cephalostatin/ritterazine analogue from hecogenin and 22-epi-hippuristanol

Article abstract of DOI:10.1016/j.bmc.2009.11.018

A new cephalostatin/ritterazine analogue was prepared from the commercially available hecogenin acetate and the natural cytotoxic steroid 22-epi-hippuristanol. The method involved the reductive dimerization of enaminoketones (condensation of α-aminoketones) and condensation between an enaminoketone and an α-hydroxyketone. The new analogue showed higher cytotoxic activity than the cytotoxic 22-epi-hippuristanol against MDA-MB-231, A-549 and HT-29 cultured tumor cell lines.

Full text of DOI:10.1016/j.bmc.2009.11.018

Bioorganic & Medicinal Chemistry 18 (2010) 58–63
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a new cytotoxic cephalostatin/ritterazine analogue from
hecogenin and 22-epi-hippuristanol
*
*
Javier Jesús Poza, Jaime Rodríguez , Carlos Jiménez
Departamento de Química Fundamental, Facultade de Ciencias, Universidade da Coruña, Campus da Zapateira, 15071 A Coruña, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2009
Revised 3 November 2009
Accepted 7 November 2009
Available online 12 November 2009
A new cephalostatin/ritterazine analogue was prepared from the commercially available hecogenin ace-
tate and the natural cytotoxic steroid 22-epi-hippuristanol. The method involved the reductive dimeriza-
tion of enaminoketones (condensation of
and an -hydroxyketone. The new analogue showed higher cytotoxic activity than the cytotoxic 22-epi-
hippuristanol against MDA-MB-231, A-549 and HT-29 cultured tumor cell lines.
Ó 2009 Elsevier Ltd. All rights reserved.
a-aminoketones) and condensation between an enaminoketone
a
Keywords:
Cephalostatin/ritterazine analogue
22-epi-Hippuristanol
Cytotoxic activity
Tumor cell lines
1. Introduction
worms. This low accessibility, along with their exceptional cyto-
toxic activity, has led to interest in the synthesis of these com-
pounds and their analogues as potential antitumor agents. Many
different routes for the construction of these pyrazine-based di-
meric steroids have been reported and reviewed.9 The synthesis
of new analogues and the evaluation of their cytotoxic activity
could provide new insights into the structure–activity relation-
ships and will help to identify the structural fragment(s) responsi-
ble for the high biological activity of these heterodimers.
The role of natural products in drug discovery has enjoyed a
renaissance in recent years.1 After a decade in the wilderness, nat-
ural products have made a comeback, especially in oncology.2
Three new drugs, ixabepilone (IxempraÒ), trabectedin (ET-743,
YondelisÒ), and temsirolimus (CCI-779, ToriselÒ), derived from nat-
ural products were very recently approved by the FDA for the
treatment of different cancers.3
The cephalostatins—along with the structurally related rittera-
zines—form a unique class of trisdecacyclic compounds that con-
sist of two steroidal units linked through a pyrazine ring.4 The
first cephalostatins were isolated by Pettit et al.5 from the Indian
Ocean tube worm Cephalodiscus gilchristi. Cephalostatin 1 (1)
exhibits extraordinarily high cytostatic activity against a broad
spectrum of cancer cell lines and proved to be one of the most
powerful cell growth inhibitors ever tested in the NCI. Compound
1 is considerably more active in vitro than paclitaxel and has an
unprecedented mechanism of action.6 More than 18 other cepha-
lostatins were characterized and they showed the same unique
cytotoxicity profile in the NCI-60 cell line panel.7 Closely related
ritterazines were isolated by Fusetani and co-workers from the
Japanese marine tunicate Ritterella tokioka8 and they showed a
similar pattern of cytotoxic activity.
Some time ago we reported the isolation of novel polyoxygenat-
ed steroids from the Indonesian gorgonian Isis hippuris. The main
component of the steroid mixture was the known compound 22-
epi-hippuristanol (2), which has a spiroketal ring in the side chain
and showed significant cytotoxic activities against several cultured
cell tumor lines.10 As a continuation of our studies on cytotoxic ste-
roids form marine organisms,11 and due to the structural similari-
ties between 1 and the monomer unit of the aforementioned
pyrazine-based bis-steroids, we report here the synthesis of a
new unsymmetrical bis-steroidal pyrazine analogue, compound
4, from the commercially available hecogenin acetate (3) and
22-epi-hippuristanol (2) (Chart 1).
2. Results and discussion
2.1. Chemistry
The availability of the cephalostatins and ritterazines from their
natural sources is extremely limited. For example, only 139 mg of
cephalostatin 1 (1) could be isolated from 166 kg of ‘crude’ marine
In the synthesis of the bis-steroidal pyrazine 4—a cephalostatin
analogue—we employed the symmetrical and non-symmetrical
routes developed by Winterfeldt.12 The natural cytotoxic spiroketal
22-epi-hippuristanol (2) was first transformed into its enaminoke-
* Corresponding authors. Tel.: +34 981 167000; fax: +34 981 167065.
E-mail addresses: jaimer@udc.es (J. Rodríguez), carlosjg@udc.es (C. Jiménez).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.018

J. J. Poza et al. / Bioorg. Med. Chem. 18 (2010) 58–63
59
Chart 1.
tone derivative 7 and this was then coupled to enaminoketone 11
(symmetrical route) or the -hydroxyketone 12 (non-symmetrical
route) obtained from the commercially available low cost starting
enaminoketone 7 in the presence of ammonium acetate at elevated
temperature using the unsymmetrical route17 afforded 4 in 16%
yield after purification by HPLC (Scheme 3).
a
material hecogenin acetate (3).
The preparation of enaminoketone 7 began with the oxidation
of the natural product 2 with PDC in DMF to give the diketone 5
in almost quantitative yield (96%).13 The structure of 5 was con-
firmed from its (+)-HRESIMS and its NMR spectra. Thus, the ab-
2.2. Biological evaluations
The cytotoxic activity of the bis-steroid pyrazine analogues 4
and 13 were evaluated in vitro against MDA-MB-231, A-549 and
sence of signals corresponding to H3b (4.32 ppm) and H11
a
HT-29 tumor cells. The results, expressed as GI50 values in lM,
(4.05 ppm) of 2 in the 1H NMR spectrum of 5 and the presence of
carbon signals at 211.4 and 210.4 ppm, corresponding to two car-
bonyl groups, in the 13C NMR spectrum confirmed this transforma-
tion. The chemo-, regio- and stereoselective bromination of 5 with
are reported in Table 1 and show an increase in biological activity
for 4 in comparison to 2. Curiously, the other new synthetic deriv-
atives of 22-epi-hippuristanol, compounds 5–7, were inactive to
these tumor cells.
PTAP afforded the 2
a
-bromo-3,11-dione 6 in 68% yield. The bro-
Compound 4 shares a similar structural framework to rittera-
zines B, F–I, Y and cephalostatin 7, differing in the presence of an
additional methyl group at C24, the absence of D14,15 in the left-
hand part of the molecule, and the oxidation positions. Although
compound 4 shows molecular dissymmetry, which was considered
to be a prerequisite for tumor-inhibition, its moderate cytotoxic
mination of 5 was confirmed by the chemical shift of H2b at 4.78
(1H, dd, J = 13.2 and 6.4 Hz) in the 1H NMR spectrum of 6 along
with its (+)-HRESIMS. Finally, treatment of 2a-bromo-3,11-dione
6 with sodium azide in DMF and a catalytic amount of NaI, afforded
the enaminoketone 7 in 97% yield, with the structure confirmed by
its spectroscopic data (Scheme 1).
activity (at lM level) in relation to the former ritterazines (at nM
The 2a
-bromo-3,11-dione 10 was synthesized from hecogenin
level) indicates that the appropriate oxidation positions are essen-
acetate (3) by saponification of the C-3 acetate and subsequent
PDC oxidation and bromination. Treatment of the bromo ketone
10 with sodium azide afforded the enaminoketone 11,12 whereas
tial for exceptional potency.
3. Conclusions
treatment with K2CO3, acetone and water14 gave the
a-hydroxyk-
In summary, a new cephalostatin/ritterazine analogue was syn-
thesized from the natural cytotoxic steroid 22-epi-hippuristanol
and the commercially available hecogenin acetate using the sym-
metrical and unsymmetrical routes designed by Winterfeldt.
Although the new analogue 4 showed a higher cytotoxic activity
etone 12 (Scheme 2).
Reductive dimerization of the enaminoketones 7 and 11 with
palladium on charcoal/hydrogen (5%) afforded the C2-unsymmetri-
cal diketone 4 in 22% yield along with the corresponding autocon-
densation products, compounds 1315 and 1416 (Scheme 3).
Compounds 4 and 13 were purified by normal phase HPLC and
their structural characterization was supported by (+)-HRESIMS
and by 1D and 2D NMR experiments. For example, the absence
of proton signals corresponding to H1 of the enaminoketones 7
and 11 in the 1H NMR spectrum of 4, along with the quaternary
carbon signals at 148.5 and 148.3 ppm in the 13C NMR spectrum,
indicated the formation of the non-symmetrical bis-steroidal
(at the lM level) than its natural precursor 2, it was far less cyto-
toxic than the natural cephalostatin/ritterazine.
4. Experimental
Melting points (mp) were determined on electrothermal digital
melting point apparatus and are uncorrected. Nuclear magnetic
resonance spectra (proton and carbon) were recorded on Bruker
pyrazine. Condensation between the a-hydroxyketone 12 and the
Scheme 1. Reagents and conditions: (a) PDC, DMF, 96%; (b) PTAP, THF, 0 °C, 68%; (c) NaI, NaN3, DMF, 50 °C, 97%.

60
J. J. Poza et al. / Bioorg. Med. Chem. 18 (2010) 58–63
Scheme 2. Reagents and conditions: (a) KOH, MeOH, 99%; (b) PDC, DMF, 99%; (c) PTAP, THF, 0 °C, 63%; (d) NaI, NaN3, DMF, 50 °C, 92%; (e) K2CO3, Ac2O:H2O (1:1), 45 °C, 80%.
Scheme 3. Reagents and conditions: (a) H2, EtOAc, MeOH, HOAc, Pd/C, 22%; (b) NH4OAc, MeOH, CH2Cl2, 50 °C, 16%.
AC200 F, 300 or 500 Advance spectrometers at the University of A
Coruña, using CDCl3 and CD3OD as the solvents and internal stan-
dards. Multiplicities of 13C signals were obtained by DEPT. Med-
ium-pressure chromatographic separations were carried out on
Silica Gel 60 (230–400 mesh). FT-IR spectra were recorded on a
Bruker VECTOR22 spectrophotometer. LREIMS and LRFABMS were

J. J. Poza et al. / Bioorg. Med. Chem. 18 (2010) 58–63
61
Table 1
at 50 °C. The reaction was quenched by the addition of water
(50 ml) and the mixture was extracted with ether. The combined
extracts were washed with brine and dried over MgSO4. The crude
product was subjected to chromatography (silica gel, hexanes/
ethyl acetate, 7:3) to give enaminoketone 7 (65 mg, 97%) as a white
semi-solid. 1H NMR (300 MHz, CDCl3) dH: 6.57 (H1, 1H, br); 4.53
The in vitro activities (GI50 values in
dimer 13
lM) of 22-epi-hippuristanol 2, analogue 4, and
Compound
MDA-MB-231
A-549
HT-29
2
4
13
5.6
2.0
2.6
3.2
1.6
1.7
4.5
1.6
2.1
(H16a, 1H, m); 1.36 (H18, 3H, s); 1.31 (H27, 3H, s); 1.26 (H21,
3H, s); 1.12 (H19, 3H, s); 1.04 (H26, 3H, s); 0.99 (H28, 3H, d,
J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) dC: 210.2 (C11, s); 195.3
(C3, s); 137.8 (C2, s); 127.5 (C1, d); 118.6 (C22, s); 84.5 (C25, s);
81.3 (C20, s); 79.1 (C16, d); 63.2 (C17, d); 61.4 (C9, d); 57.1 (C12,
t); 54.7 (C14, d); 45.8 (C5, t); 45.2 (C13, s); 44.5 (C23, t); 40.4
(C24, d); 39.5 (C4, t); 35.7 (C8, d); 35.5 (C10, s); 32.1 (C7, t); 31.0
(C15, t); 29.0 (C26, q); 28.2 (C6, t); 25.9 (C18, q); 23.0 (C27, q);
17.7 (C21, q); 14.7 (C28, q); 11.4 (C19, q). LREIMS (70 eV, m/z%):
471 (M+, 19); 148 (100). (+)-LRFABMS m/z (%): 472 ([M+H]+, 13);
109 (100). (+)-HRESIMS: m/z 472.3063 [M+H]+ (calcd for
C27H42NO5, 472.3057).
recorded on a Thermo MAT-95XP spectrometer, while (+)–(ꢀ)
HRESIMS were measured on Applied Biosystems QSTAR Elite.
4.1. 22-epi-Hippuristan-3,11-dione (5)
22-epi-Hippuristanol (2) (0.1 g, 0.22 mmol) was treated with
PDC (0.2 g, 0.5 mmol) in anhydrous DMF (2 ml) in the presence of
activated molecular sieves and stirred at room temperature for
6 h. Insoluble materials were filtered off through a Celite pad and
the filtrate was concentrated in vacuo. The residue was purified by
column chromatography (silica gel, hexanes/ethyl acetate, 8:2) to
afford the 3,11-dione 5 (95 mg, 96%) as a white semi-solid: mp
220 °C, lit. 229.5–232 °C;18 IR (neat) mmax 3434, 2924, 1701, 1050,
4.4. Hecogenin-3-one (9)
Hecogenin (1.0 g, 2.3 mmol) was treated with PDC (2.0 g,
5.0 mmol) in anhydrous DMF (20 ml) in a similar way to 2. The
product was purified by column chromatography (silica gel, hex-
anes/ethyl acetate, 8:2) to give the 3,12-dione 9 (0.99 g, 99%) as a
;
1031, 1009, 970, 923, 874 cmꢀ1 1H NMR (300 MHz, CDCl3) dH:
4.48 (H16 , 1H, m); 1.30 (H18, 3H, s); 1.27 (H27, 3H, s); 1.21 (H21,
a
3H, s); 1.07 (H19, 3H, s); 0.97 (H26, 3H, s); 0.94 (H28, 3H, d,
J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) dC: 211.4 (C11, s); 210.4 (C3,
s); 118.5 (C22, s); 84.3 (C25, s); 81.8 (C20, s); 79.0 (C16, d); 64.0
(C17, d); 63.3 (C9, d); 58.1 (C12, t); 55.7 (C14, d); 46.9 (C5, d); 45.8
(C13, s); 44.3 (C23, t); 40.9 (C24, d); 39.6 (C4, t); 37.9 (C1, t); 37.1
(C2, t); 35.7 (C8, d); 35.2 (C10, s); 32.3 (C7, t); 31.5 (C15, t); 29.0
(C26, q); 28.2 (C6, t); 26.0 (C18, q); 22.9 (C27, q); 17.5 (C21, q);
14.0 (C28, q); 11.0 (C19, q). LREIMS (70 eV, m/z%): 458 (M+, 4); 315
(100). (+)-LRFABMS m/z (%): 459 ([M+H]+, 37); 154 (100). (+)-HRE-
SIMS: m/z 459.3104 [M+H]+ (calcd for C28H43O5, 459.3105).
white semi-solid. 1H NMR (300 MHz, CDCl3) dH: 4.34 (H16
a, 1H,
m); 3.49 (H26b, 1H, ddd, J = 10.9, 4.4, 2.0 Hz); 3.34 (H26a, 1H, t,
J = 10.9 Hz); 2.54 (H17, 1H, dd, J = 9.0, 8.0 Hz); 2.48 (H11b, 1H,
dd, J = 10.5, 13.5 Hz); 2.38 (H8, 1H, br s); 2.43–2.32 (H2, 2H, m);
2.17 (H11a, 1H, dd, J = 10.5, 5.0 Hz); 2.20–1.90 (H4, 2H, m); 1.25
(H18, 3H, s); 1.09 (H21, 3H, d, J = 7.0 Hz); 1.06 (H19, 3H, s); 0.79
(H27, 3H, d, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) dC: 212.8 (C12,
s); 210.7 (C3, s); 109.2 (C22, s); 79.1 (C16, d); 66.8 (C26, t); 55.5;
55.1 (C13, s); 54.8; 53.5; 46.2; 44.4; 42.2; 37.8; 37.7; 36.2 (C10,
s); 34.2; 31.5; 31.4; 31.2; 31.1; 30.2; 28.5; 28.7; 17.1 (C27, q);
16.0 (C21, q); 13.2 (C19, q); 11.2 (C18, q). LREIMS (70 eV, m/z%):
428 (M+, 33); 139 (100). (+)-LRFABMS m/z (%): 429 ([M+H]+, 57);
133 (100). (+)-HRESIMS: m/z 429.2998 [M+H]+ (calcd for
C27H41O4, 429.2999).
4.2. 2a-Bromo-22-epi-hippuristan-3,11-dione (6)
A cold (0 °C) solution of trimethyl(phenyl)ammonium perbro-
mide (80 mg, 0.2 mmol) in dry THF (2 ml) was added dropwise over
a period of 3 h to a solutionof the 3,11-dione5 (95 mg, 0.21 mmol) in
dry THF (2 ml). After a further 45 min, the reaction was quenched by
the addition of saturated aqueous sodium hydrogen carbonate
(5 ml). The product was extracted with EtOAc and the organic layer
was washed with brine and dried over MgSO4. The crude product
was subjected to chromatography (silica gel, hexanes/ethyl acetate,
4.5. 2a-Bromohecogenin-3-one (10)
A solution of 3,12-dione 9 (6.0 g, 14.0 mmol) in dry THF (20 ml)
was reacted with trimethyl(phenyl)ammonium perbromide (5.0 g,
12.5 mmol) in dry THF (20 ml) in a similar way to 5. The product
was purified by column chromatography (silica gel, hexanes/ethyl
9:1) to give 2
talline product: mp 115–121 °C; IR (neat) mmax 3494, 2925, 1702,
1025, 970, 923, 871 cmꢀ1 1H NMR (300 MHz, CDCl3) dH: 4.78
(H2b,1H, dd, J = 13.2, 6.4 Hz); 4.48 (H16 , 1H, m); 1.30 (H18, 3H,
a-bromo-3,11-diketone 6 (76 mg, 68%) as a white, crys-
acetate, 9:1) to give 2
white semi-solid. 1H NMR (300 MHz, CDCl3) dH: 4.70 (H2b, 1H, dd,
J = 13.2, 6.4 Hz); 4.32 (H16 , 1H, m); 3.46 (H26b, 1H, m); 3.32
a-bromo-3,12-diketone (10) (4.5 g, 63%) as a
;
a
a
s); 1.28 (H27, 3H, s); 1.24 (H21, 3H, s); 1.07 (H19, 3H, s); 0.98
(H26, 3H, s); 0.94 (H28, 3H, d, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3)
dC: 211.4 (C11, s); 210.4 (C3, s); 118.5 (C22, s); 84.3 (C25, s); 81.8
(C20, s); 79.0 (C16, d); 64.0 (C17, d); 63.3 (C9, d); 57.1 (C12, t);
56.1 (C2, d); 54.7 (C14, d); 45.7 (C5, d); 45.3 (C13, s); 44.8 (C23, t);
40.6 (C24, d); 39.9 (C4, t); 37.4 (C1, t); 35.8 (C8, d); 35.6 (C10, s);
32.6 (C7, t); 31.8 (C15, t); 28.7 (C26, q); 28.2 (C6, t); 25.9 (C18, q);
22.5 (C27, q); 17.4 (C21, q); 14.2 (C28, q); 11.4 (C19, q). (+)-LRFABMS
m/z (%): 539 ([M+H]+, 5) (81Br)/537 ([M+H]+, 2) (79Br); 154 (100). (+)-
(H26a, 1H, t, J = 11.0 Hz); 2.23 (H11a, 1H, dd, J = 10.5, 5.0 Hz);
2.20–1.90 (H4, 2H, m); 1.22 (H18, 3H, s); 1.05 (H21, 3H, d,
J = 6.8 Hz); 1.05 (H19, 3H, s); 0.78 (H27, 3H, d, J = 6.5 Hz). 13C NMR
(75 MHz, CDCl3) dC: 211.8 (C12, s); 200.2 (C3, s); 109.2 (C22, s);
78.9 (C16, d); 66.8 (C26, t); 61.8; 55.2; 54.4; 53.5 (C2, d); 53.4
(C13, s); 50.8; 46.9; 43.5; 42.1; 39.2 (C10, s); 37.6; 33.7; 31.3;
31.0; 30.8; 30.1; 28.7; 27.9; 17.0 (C27, q); 15.9 (C21, q); 13.2 (C19,
q); 11.8 (C18, q). LREIMS (70 eV, m/z%): 508 (M+, 15) (81Br)/506
(M+, 8) (79Br); 139 (100). (+)-LRFABMS m/z (%): 509 ([M+H]+, 36)
HRESIMS: m/z 537.2212 [M+H]+
(
79Br) (calcd for C28H42O579Br,
( (
81Br)/507 ([M+H]+, 28) 79Br); 132 (100). (+)-HRESIMS: m/z
537.2210).
507.2114 [M+H]+ (79Br) (calcd for C27H40O479Br, 507.2104).
4.3. 2-Amino-22-epi-hippurist-1-en-3,11-dione (7)
-Bromo-3,11-diketone 6 (76 mg, 0.14 mmol) was dissolved
4.6. 2-Aminohecogenin-1-en-3-one (11)
2a
2a-Bromo-3,12-diketone 10 (4.0 g, 7.9 mmol) was dissolved in
in DMF (5 ml) and NaN3 (0.1 g, 8.0 mmol) and a catalytic amount
DMF (100 ml) and was reacted with NaN3 (5.2 g, 80 mmol) and a
of NaI was added. The suspension was stirred for 1 h under argon
catalytic amount of NaI in a similar way as 6. The product was puri-

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J. J. Poza et al. / Bioorg. Med. Chem. 18 (2010) 58–63
fied by column chromatography (silica gel, hexanes/ethyl acetate,
7:3) to give the enaminoketone 11 (3.2 g, 92%) as a yellow solid
which was crystallized from diethyl ether/ethyl acetate: mp 235–
240 °C; IR (neat) mmax 3447, 3349, 2929, 1703, 1667, 1633, 1038,
1.13 (H210, 3H, d, J = 6.5 Hz); 1.12 (H19, 3H, s); 1.07 (H190, 3H, s);
1.03 (H26, 3H, s); 0.99 (H28, 3H, d, J = 6.8 Hz); 0.84 (H270, 3H, d,
13
J = 6.4 Hz). C NMR (125 MHz, CDCl3) dC: 213.0 (C120, s); 204.5
(C11, s); 148.5/148.5/148.3/148.3 (C2, C20, C3, C30, s); 117.2 (C22,
s); 109.4 (C220, s); 84.5 (C25, s); 81.9 (C20, s); 79.8 (C16, d); 79.2
(C160, d); 66.9 (C260, t); 65.6; 63.6; 57.0; 55.7; 55.0; 54.8; 53.5;
45.3; 45.4; 42.5; 41.6; 41.0; 37.7; 37.1; 36.1; 35.3; 34.2; 33.8;
32.8; 32.2; 31.9; 31.4; 31.2; 30.6; 30.3; 30.1; 29.8; 29.7; 29.6;
29.4; 28.9; 28.1; 27.1; 26.4; 22.7 (C26, q); 19.8 (C21, q); 17.2
(C270, q); 16.0 (C210, q); 14.2 (C28, q); 13.1 (C19, q); 13.8 (C180,
q); 11.7 (C190, q). (+)-LRFABMS m/z (%): 879 ([M+H]+, 18); 133
(100). HREIMS (70 eV, m/z%): 878.5771 [M+] (calcd for
C55H78N2O7, 878.5804). (+)-HRESIMS: m/z 879.5888 [M+H]+ (calcd
for C55H79N2O7, 879.5882).
1007, 954, 868 cmꢀ1
;
1H NMR (300 MHz, CDCl3) dH: 5.87 (H1, 1H,
br s); 4.34 (H16
a, 1H, m); 3.47 (H26b, 1H, m); 3.34 (H26a, 1H, t,
J = 10.8 Hz); 1.25 (H18, 3H, s); 1.08 (H19, 3H, s); 1.07 (H21, 3H,
d, J = 6.8 Hz); 0.79 (H27, 3H, d, J = 6.6 Hz). 13C NMR (75 MHz,
CDCl3) dC: 211.9 (C12, s); 194.7 (C3, s); 137.7 (C2, s); 123.5 (C1,
d); 108.8 (C22, s); 78.6 (C16, d); 66.4 (C26, t); 55.1 (C13, s); 54.8;
53.2; 51.8; 44.1; 41.8; 39.6 (C10, s); 38.0; 37.3; 34.0; 31.0; 30.6;
30.5; 29.7; 28.3; 26.6; 16.6 (C27, q); 15.6 (C21, q); 13.4 (C19, q);
12.8 (C18, q). LREIMS (70 eV, m/z%): 441 (M+, 19); 118 (100). (+)-
LRFABMS m/z (%): 442 ([M+H]+, 91); 133 (100). (+)-HRESIMS: m/z
442.2959 [M+H]+ (calcd for C27H40NO4, 442.2951).
Spectral data for 13: 1H NMR (500 MHz, CDCl3) dH: 4.40 (H16
a,
1H, m); 3.54 (H26b, 1H, m); 3.39 (H26a, J = 11.0 Hz); 1.29 (H18,
3H, s); 1.10 (H21, 3H, d, J = 6.3 Hz); 1.08 (H19, 3H, s); 0.84 (H27,
3H, d, J = 6.5 Hz). 13C NMR (125 MHz, CDCl3) dC: 213.3 (C12, s);
148.4/148.4 (C2, C3, s); 109.2 (C22, s); 78.8 (C16, d); 66.9 (C26,
t); 55.4; 54.9; 51.8; 49.7; 45.4; 43.1; 42.8; 37.6; 36.2; 35.3; 34.0;
31.5; 31.2; 30.5; 29.7; 28.8; 28.2; 17.5 (C27, q); 16.2 (C21, q);
13.3 (C18, q); 11.8 (C19, q). LREIMS (70 eV, m/z%): (+)-848 (M+,
14); 139 (100). LRFABMS m/z (%): 849 ([M+H]+, 9); 133 (100).
(+)-HRESIMS: m/z 849.5775 [M+H]+ (calcd for C54H77N2O6,
849.5776).
4.7. 2
a
-Hydroxyhecogenin-3-one (12)
A mixture of 2
a-bromo-3,12-diketone 10 (0.20 g, 0.39 mmol),
K2CO3 (1.0 g, 7.2 mmol), acetone (15 ml), and water (5 ml) was
heated at 45 °C for 15 h. The mixture was diluted with water and
then partly evaporated in vacuo. The residue was extracted with
CHCl3. The extract was dried over MgSO4 and evaporated to give
the 2
a-hydroxy-3,12-diketone (8) (0.14 g, 80%) as a white semi-so-
lid. 1H NMR (300 MHz, CDCl3) dH: 5.80 (OH, 1H, br s); 4.39 (H16
a,
1H, m); 4.28 (H2b, 1H, dd, J = 12.0, 7.0 Hz); 3.53 (H26b, 1H, m);
3.38 (H26a, 1H, t, J = 10.8 Hz); 1.28 (H18, 3H, s); 1.11 (H21, 3H,
d, J = 6.8 Hz); 1.10 (H19, 3H, s); 0.83 (H27, 3H, d, J = 6.8 Hz). 13C
NMR (75 MHz, CDCl3) dC: 212.4 (C12, s); 210.2 (C3, s); 109.3
(C22, s); 79.1 (C16, d); 72.5 (C2, d); 66.9 (C26, t); 55.2; 55.1;
54.6; 48.0; 47.7; 42.2; 42.3; 37.9; 37.5; 33.6; 31.4; 31.2; 31.1;
30.2; 29.7; 28.8; 28.2; 17.7 (C27, q); 16.0 (C21, q); 13.3 (C19, q);
12.6 (C18, q). LREIMS (70 eV, m/z%): 444 (M+, 23); 139 (100). (+)-
LRFABMS m/z (%): 445 ([M+H]+, 18); 154 (100). (+)-HRESIMS: m/z
445.2961 [M+H]+ (calcd for C27H41O5, 445.2948).
4.9. Biological activity
A-549 (ATCC CCL-185), lung carcinoma; HT-29 (ATCC HTB-38),
colorectal carcinoma, and MDA-MB-231 (ATCC HTB-26), breast
adenocarcinoma cell lines were obtained from the ATCC. Cell lines
were maintained in RPMI medium supplemented with 10% fetal
calf serum (FCS), 2 mM L-glutamine and 100 U/ml penicillin and
streptomycin, at 37 °C and 5% CO2. Triplicate cultures were incu-
bated for 72 h in the presence or absence of test compounds (at
ten concentrations ranging from 40 to 0.01 lg/ml). For quantitative
4.8. Compounds 4 and 13
estimation of cytotoxicity, the colorimetric sulforhodamine B (SRB)
method was used essentially performed as described previously.19
Briefly, cells were washed twice with PBS, fixed for 15 min in 1%
glutaraldehyde solution, rinsed twice in PBS, and stained in 0.4%
SRB solution for 30 min at room temperature. Cells were then
rinsed several times with 1% acetic acid solution and air-dried. Sul-
forhodamine B was then extracted in 10 mM trizma base solution
and the absorbance measured at 490 nm. Results are expressed as
GI50, the concentration that causes 50% inhibition in cell growth
after correction for cell count at the start of the experiment (NCI
algorithm).
4.8.1. Method A
A mixture of enaminoketone 7 (12 mg, 0.025 mmol) and enami-
noketone 12 (12 mg, 0.025 mmol) was dissolved in EtOAc (3 ml).
The solution was mixed with MeOH (0.1 ml) and two drops of ace-
tic acid. The catalyst [65 mg of Pd/C (5%)] was added and the com-
pound was hydrogenated at room temperature overnight. After the
reaction was complete the mixture was filtered and the filtrate was
washed with saturated aqueous sodium bicarbonate and saturated
aqueous sodium chloride, dried over MgSO4 and concentrated un-
der reduced pressure. The residue was purified by HPLC (Scharlau
C18, flow rate 1.5 ml/min, EtOAc/Hex, 1:1) to give 5 mg of 4 (reten-
tion time 68 min) as a white semi-solid in 22% yield and 3 mg of
compound 13.
Acknowledgments
This work was financially supported by Grants from the Minis-
try of Science and Innovation of Spain (CTQ2008-04024 and
AGL2009-12266-C02-02). We thank Ricardo F. Mendoça for his
helpful contribution to this project. We are grateful to Pharmamar
S.A. for the pharmacological assays. J.P. thanks the Xunta de Galicia
for a fellowship. We also thank to Servizos de Apoio a Investigación
from University of A Coruña (SAI-UDC) for the analytical support.
4.8.2. Method B
A
solution of enaminoketone
NH4OAc (9 mg) in moist MeOH (5 ml < 0.1% H2O) was heated un-
der reflux for 30 min. A solution of 2 -hydroxy-3,12-diketone 12
7 (10 mg, 0.021 mmol) and
a
(10 mg, 0.022 mmol) in CH2Cl2 (0.6 ml) was added and the result-
ing suspension was heated under reflux for 3 h. The mixture was
quenched with H2O, extracted with CH2Cl2 and the organic layer
was washed with brine and dried over MgSO4. The solvent was re-
moved and the residue was purified by HPLC as described above to
give 3 mg of 4 as a white semi-solid in 16% yield.
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