Isolation, biological significance, synthesis, and cytotoxic evaluation of new natural parathiosteroids A-C and analogues from the soft coral Paragorgia sp

Article abstract of DOI:10.1021/jo801198u

(Chemical Equation Presented) Three unusual new steroid thioesters, parathiosteroids A-C (1a-3a), were isolated from the 2-propanol extract of the soft coral Paragorgia sp. collected in Madagascar. Their structures, determined by detailed spectroscopic analysis, were confirmed by synthesis and represent the first isolation of natural steroids bearing a C22 thioester in their side chain. These compounds displayed cytotoxicity against a panel of three human tumor cell lines at the micromolar level. The preparation of several analogues revealed structure/activity relationships in this type of steroids, for example, that the XCH₂CH₂NHCOCH₃ moiety (X = S, O, NH) in the side chain is essential for the antiproliferative activity, and a low degree of oxidation in the A-ring results in higher bioactivity. These natural products could be biosynthetic intermediates in the steroid side chain degradation pathway involving activation with CoA and β-oxidations.

Full text of DOI:10.1021/jo801198u

Isolation, Biological Significance, Synthesis, and Cytotoxic
Evaluation of New Natural Parathiosteroids A-C and Analogues
from the Soft Coral Paragorgia sp.
Javier Jesu´s Poza,† Rogelio Ferna´ndez,‡ Fernando Reyes,*,‡ Jaime Rodr´ıguez,*,† and
Carlos Jime´nez*,†
Departamento de Qu´ımica Fundamental, Facultade de Ciencias, Campus da Zapateira, UniVersidade da
Corun˜a, 15071 A Corun˜a, Spain, and Medicinal Chemistry Department, PharmaMar S.A.U., Pol. Ind. La
Mina Norte, AVda. de los Reyes 1, 28770 Colmenar Viejo, Madrid, Spain
jfreyes@pharmamar.com; jaimer@udc.es; carlosjg@udc.es
ReceiVed June 6, 2008
Three unusual new steroid thioesters, parathiosteroids A-C (1a-3a), were isolated from the 2-propanol
extract of the soft coral Paragorgia sp. collected in Madagascar. Their structures, determined by detailed
spectroscopic analysis, were confirmed by synthesis and represent the first isolation of natural steroids
bearing a C22 thioester in their side chain. These compounds displayed cytotoxicity against a panel of
three human tumor cell lines at the micromolar level. The preparation of several analogues revealed
structure/activity relationships in this type of steroids, for example, that the XCH2CH2NHCOCH3 moiety
(X ) S, O, NH) in the side chain is essential for the antiproliferative activity, and a low degree of
oxidation in the A-ring results in higher bioactivity. These natural products could be biosynthetic
intermediates in the steroid side chain degradation pathway involving activation with CoA and ꢀ-oxidations.
Introduction
bacteria, the aliphatic side chain at C-17 is removed in a process
similar to ꢀ-oxidation involving progressively shorter carboxylic
acids.4 A recent study of the aerobic degradation of the bile
acid cholate in a Pseudomonas sp. strain allowed a metabolic
pathway for cholate to be proposed that involves A-ring
oxidation, activation with CoA, and ꢀ-oxidation of the acyl side
chain. In that study, it was possible to isolate and identify by
NMR 7R,12R-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate
(DHOPDC) as an intermediate of the ꢀ-oxidation. The activation
of DHOPDC with CoA (see Figure 1) was detected by LC-
MS/MS, but the intermediate was not isolated.5 The discovery
of natural products related to these degradation intermediates
would be relevant in supporting the activation with CoA and
A great deal of work has been carried out on the bacterial
catabolism of steroids as a result of the possibility of producing
bioactive steroids from nature.1 Detailed knowledge of steroid
catabolism is essential to engineer strains for the biotransfor-
mation of sterols. While the enzymatic reactions involved in
A-ring oxidation have been studied in great detail since 1960,2
the degradation of steroid side chains has been developed mainly
on a phenomenological level by extracting and identifying
degradation intermediates from whole steroid-transforming
cultures.3 For this reason, most of the metabolic pathways
proposed for the side-chain cleavage of steroids have been
deduced from microbial degradation. Thus, in some species of
(4) (a) Murohisa, T.; Iida, M. J. Ferment. Bioeng. 1993, 75, 13–17. (b) Geize,
R. V.; Yam, K.; Heuser, T.; Wilbrink, M. H.; Hara, H.; Anderton, M. C.; Sim,
E.; Dijkhuizen, L.; Davies, J. E.; Mohn, W. W.; Eltis, L. D. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 1947–1952.
(5) Birkenmaier, A.; Holert, J.; Henrike, E.; Moeller, H.; Friemel, A.;
Schoenenberger, R.; Suter, M. J.-F.; Klebensberger, J.; Philipp, B. J. Bacteriol.
2007, 189, 7165–7173.
† Universidade da Corun˜a.
‡ PharmaMar S.A.U.
(1) Fernandes, P.; Cruz, A.; Angelova, B.; Pinheiro, H. M.; Cabral, J. M. S.
Enzyme Microb. Technol. 2003, 32, 688–705.
(2) Talalay, P. Annu. ReV. Biochem. 1965, 34, 347–380.
(3) Kieslich, K. J. Basic Microbiol. 1985, 25, 461–474.
7978 J. Org. Chem. 2008, 73, 7978–7984
10.1021/jo801198u CCC: $40.75 2008 American Chemical Society
Published on Web 09/17/2008

New Natural Parathiosteroids from Paragorgia sp.
1
TABLE 1. NMR Data for Parathiosteroid A (1a) in CDCl3, B (2a) in CD3OD, and C (3a) in CDCl3/CD3OD at 500 MHz for H and 125 MHz
for 13C
parathiosteroid A (1a)
δH, mult, J in Hz
parathiosteroid B (2a)
δH, mult, J in Hz
parathiosteroid C (3a)
δH, mult, J in Hz
C
δC, mult
δC, mult
δC, mult
1
2
3
4
155.7, CH
127.5, CH
186.3, C
7.05, d, 10.1
6.23, dd, 10.1, 1.9
159.2, CH
125.8, CH
200.7, C
7.31, d, 10.1
5.85, dd, 10.1, 1.0
125.9, CH
112.4, CH
154.3, C
7.09, d, 8.5
6.58, dd, 8.5, 2.6
123.8, CH
6.07, d, 1.9
39.7, CH2
2.44, dd, 17.8, 14.2; 2.19,
ddd, 17.8, 4.0, 1.0
1.96, m
114.8, CH
6.51, d, 2.6
2.78, m
5
6
169.0, C
32.7, CH2
43.6, CH
26.6, CH2
137.5, C
29.4, CH2
2.47, td, 13.3, 5.1; 2.36,
ddd, 13.3, 3.7, 2.7
1.95, m; 1.04, m
1.64, m
1.48, m
7
8
9
10
11
33.5, CH2
35.4, CH
52.1, CH
43.5, C
30.4, CH2
35.0, CH
49.4, CH
38.3, C
1.77, m; 1.04, m
1.56, m
1.07, m
28.6, CH2
38.8, CH
43.6, CH
131.3, C
26.5, CH2
1.87, m; 1.31, m
1.35, m
2.18, m
1.07, m
22.7, CH2
1.71, m
20.2, CH2
1.86, ddd, 13.5, 7.0, 3.5
1.54, m
2.27, m; 1.47, m
12
39.1, CH2
1.97, m; 1.28, m
38.9, CH2
2.04, ddd, 13.0, 3.5, 3.5; 1.34,
ddd, 13.0, 13.0, 4.0
39.6, CH2
2.07, dd, 8.8, 2.5; 1.49, m
13
14
15
16
17
18
19
20
21
22
24
25
27
28
NH
42.9, C
42.2, C
42.9, C
54.7, CH
24.4, CH2
27.1, CH2
52.4, CH
12.2, CH3
18.6, CH3
51.7, CH
17.7, CH3
203.8, C
28.1, CH2
39.8, CH2
170.2, C
23.2, CH3
1.04, m
55.1, CH
23.2, CH2
26.3, CH2
52.2, CH
10.6, CH3
11.1, CH3
51.1, CH
16.2, CH3
202.4, C
26.9, CH2
38.2, CH2
171.4, C
20.5, CH3
1.17, m
54.7, CH
23.7, CH2
27.0, CH2
52.8, CH
11.6, CH3
1.28, m
1.61, m; 1.18, m
1.74, m; 1.37, m
1.68, m
0.76, s
1.25, s
1.64, m; 1.17, m
1.75, m; 1.41, m
1.68, dd, 9.8, 8.8
0.78, s
1.07, s
2.65, dq, 10.5, 6.8
1.23, d, 6.8
1.72, m; 1.20, m
1.77, m; 1.40, m
1.74, m
0.75, s
2.63, dq, 10.1, 6.8
1.20, d, 6.8
51.7, CH
17.1, CH3
203.6, C
27.5, CH2
38.9, CH2
171.9, C
2.66, dq, 10.2, 6.8
1.24, d, 6.8
2.99, ddd, 6.5, 5.6, 0.9
3.42, ddd, 6.5, 5.7, 2.7
2.99, td, 6.7, 1.5
3.32a
2.99, ddd, 7.1, 6.9, 0.8
3.32a
1.96, s
6.01, brs
1.93, s
21.6, CH3
1.94, s
a Under the solvent signal.
the presumptive ꢀ-oxidation pathway for degradation of the side
chain of steroid compounds.
containing both a thioester and an acetamide, an element of
structural novelty without precedent in marine natural products.
Their structures were established by spectroscopic methods
(mainly 1D and 2D NMR) and confirmed by synthesis. Because
of the cytotoxic activities displayed by the natural steroids,
several analogues were prepared in order to deduce some
structure-activity relationships.
Results and Discussion
Samples of Paragorgia sp., collected by bottom trawling near
Madagascar, were extracted with 2-propanol. Bioassay-guided
fractionation of this organic extract, employing RP-18 silica gel
chromatography and reversed phase semipreparative HPLC,
yielded the new parathiosteroids A-C (1a-3a), which were
isolated as the main components responsible for the cytotoxic
activity.
FIGURE 1. Structure of DHOPDC-CoA.
During the course of our screening program aimed at the
identification of new bioactive steroids from marine organisms,6
we focused our attention on the gorgonian Paragorgia sp.
because of the cytotoxicity found in its 2-propanol extracts.
Previous studies carried out on the chemistry of species of the
genus Paragorgia are restricted to two accounts of the isolation
of xeniolides from extracts of Paragorgia arborea.7 Bioassay-
guided fractionation of the organic material yielded three novel
cytotoxic steroid derivatives named parathiosteroids A-C
(1a-3a). The structures of the new metabolites incorporate an
A-ring with different degrees of unsaturation and a side chain
The (+)-HRFABMS found for the [M + H]+ pseudomo-
lecular ion at m/z 444.2572 and the presence of 26 signals in
the 13C NMR spectrum allowed us to deduce the molecular
formula of C26H37NO3S for parathiosteroid A (1a). The 1H NMR
spectrum displayed signals for four methyl groups at δH 0.76
(s), 1.25 (s), and 1.20 (d, J ) 6.8 Hz), attributable to the presence
of a steroidal framework in the molecule, and at 1.96 (s)
characteristic of an acetyl group. Signals in the 13C NMR
spectrum for a conjugated carbonyl group at δC 186.3 (C3),
together with those of three sp2 methines at δC 155.7 (C1), 127.5
(C2), and 123.8 (C4) and one sp2 quaternary carbon at δC 169.0
(C5), along with correlations observed in the HMBC spectrum,
were consistent with the presence of a cross-conjugated ketone
(6) (a) Rodr´ıguez, J.; Nun˜ez, L.; Peixinho, S.; Jime´nez, C. Tetrahedron Lett.
1997, 38, 1833–1836. (b) Gonza´lez, N.; Barral, M. A.; Rodr´ıguez, J.; Jime´nez,
C. Tetrahedron 2001, 57, 3487–3497.
(7) (a) D’Ambrosio, M.; Guerriero, A.; Pietra, F. Z. Naturforsch. 1984, 39,
1180–1183. (b) Stonik, V. A.; Makar’eva, T. N.; Dmitrenok, A. S. Khim. Prir.
Soedin. 1990, 125–126.
J. Org. Chem. Vol. 73, No. 20, 2008 7979

Poza et al.
TABLE 2. In Vitro Antitumor Activities (GI50 in µM) of
Parathiosteroids A-C and Analogues
in the A-ring. The NMR data for this part of the molecule (see
Table 1) are in agreement with those observed for other steroids
containing the same A-ring in the tetracyclic framework.8 A
signal observed at δC 203.8 was assigned to a thioester carbonyl
group that was placed at C22 by cross-peaks observed with H17,
H20, and H21 in the HMBC experiment. Finally, COSY
correlations from H25 to H24 and the NH signal at δH 6.01, as
well as the key HMBC correlations H24/C22, H25/C27, NH/
C25, NH/C27, and H28/C27, established the side chain structure
of parathiosteroid A as depicted in 1a. Correlations observed
in the NOESY spectrum [Me19 (δ 1.25) with H6ax (δ 2.47)
and H8 (δ 1.64); Me18 (δ 0.76) with H12eq (δ 1.97), H8, and
H20 (δ 2.63); Me21 (δ 1.20) with H12 eq (δ 1.97); H12ax (δ
1.28) with H9 (δ 1.07) and H14 (δ 1.04)] are in agreement with
the usual configuration of natural steroids (8ꢀ,9R,10ꢀ,13ꢀ,
14R,17ꢀ,20S*).
Parathiosteroid B (2a) was isolated as an amorphous white
solid of molecular formula C26H39NO3S, according to its (+)-
HRFABMS and 13C NMR spectra. Comparison of the NMR
data of this compound with those of 1a revealed the absence
of one of the double bonds present in parathiosteroid A as
the only noticeable difference between the two compounds.
Indeed, signals for just one conjugated double bond were
observed in the spectra of 2a (δC 159.2, 125.8/δH 7.31, 5.85).
This unsaturation was placed between C1/C2 on the basis of
correlations observed in the HMBC spectrum. The downfield
shift of C3 by 14.4 ppm with respect to 1a, as a consequence
of the disappearance of the ∆4 double bond present in
parathiasteroid A, corroborated the structure proposed for 2a
(see Table 1).
The third compound in the series, parathiosteroid C (3a), gave
a [M + H]+ peak at m/z 430.2420, which is consistent with a
molecular formula of C25H35NO3S. The most evident differences
found between the 1H NMR spectra of 3a and 1a are the
disappearance of the singlet methyl group at C19 and the
downfield shift of the A-ring protons in 3a. These findings,
together with the changes observed in the proton-proton
coupling constants of H1, H2, and H4 with respect to 1a, were
in agreement with the aromatization of the A-ring of the steroidal
nucleus. The presence of six sp2 carbon signals (one of them
oxygenated) in the 13C NMR and DEPT-135 spectra (see Table
1) and correlations observed in the HMBC spectrum cor-
roborated this proposal. Examples of other marine natural
products with an aromatic A-ring are represented by geodisterol,
a polyoxygenated sterol isolated from the tropical marine sponge
Geodia sp.,9 and two 19-norpregnane derivatives obtained from
samples of soft corals of the genus Capnella.10
compound
MDA-MB-231
A-549
HT-29
1a
2a
3a
10a
22a
1b
47
13
88
19
33
15
13
38
14
31
6.5
20
6.9
14
10
15
15
2b
10
3b
10b
22b
1c
4.6
62
70
4.6
53
90
3.0
37
38
10c
39
79
72
29), lung (A-549), and breast (MDA-MB-231), with GI50 values
in the micromolar range (see Table 2). In particular, parathios-
teroid B (2a) showed a selective cytotoxicity against HT-29
with a GI50 of 6.5 µM.
In order to confirm the proposed structures and deduce some
structure-activity relationships, the natural products and new
analogues with different oxidation degrees in the A-ring and
different heteroatoms in the side chain were synthesized from
the commercially available 20-(hydroxymethyl)-pregnan-1,4-
dien-3-one (4) and (+)-estrone (12). The synthesis of parathios-
teroid A (1a) began with the oxidation of 20-(hydroxymethyl)-
pregnan-1,4-dien-3-one (4). Direct conversion of the primary
hydroxy group at C22 to the acid was precluded by steric
hindrance. Thus, compound 4 had to be oxidized in two steps
to the corresponding carboxylic acid, first to the aldehyde 5
with PDC in DMF and then to the acid 6 with TEMPO, NaClO2,
and NaClO.11 Thioesterification of the carboxylic acid of 6 with
N-acetylcysteamine (NAC) using p-toluenesulfonyl chloride
(TsCl) and N-methylimidazole, according to the method devel-
oped by Tanabe,12 yielded 1a in moderate yield (65%), again
due to steric hindrance at the C22 carbonyl group (Scheme 1).
Treatment of the acid 6 with N-acetylethanolamine or N-(2-
aminoethyl)acetamide under the same conditions as before gave
analogues 1b and 1c, respectively.
Parathiosteroid B (2a) was synthesized from the Birch
reduction product of 4. Treatment of 4 with seven equivalents
of lithium in liquid ammonia and anhydrous THF at -78 °C
for 3 h, using NH4Cl as the proton donor, gave diol 7 in
quantitative yield.13 The carbon chemical shift of the C19
methyl group at 11.8 ppm in the 13C NMR spectrum of 7
was consistent with 5R steroid models.14 Oxidation of the
two hydroxyl groups in 7 to the corresponding carbonyl
carbons present in 8 was achieved with PDC in DMF. The
synthesis of a 3-keto-∆1 steroid intermediate by bromination
at C2 followed by dehydrohalogenation15 of 8 was compli-
cated by the fact that the treatment of 8 with phenyltrim-
ethylammonium perbromide (PTAP) gave epimerization at
C20. For that reason, the aldehyde group in 8 was oxidized
to the corresponding carboxylic acid to give 9, which was
(11) Zhao, M. M.; Li, J.; Mano, E.; Song, Z. J.; Tschaen, D. M. Org. Synth.
2005, 81, 195–199.
Compounds 1a-3a displayed cytotoxic properties against a
panel of three human tumor cell lines, including colon (HT-
(12) Wakasugi, K.; Iida, A.; Misaki, T.; Nishii, Y.; Tanabe, Y. AdV. Synth.
Catal. 2003, 345, 1209–1214.
(13) Rabinowitz, M. H.; Djerassi, C. J. Am. Chem. Soc. 1992, 114, 304–
317.
(14) The carbon chemical shift of the C19 methyl group for 5ꢀ steroid models
resonates around 24 ppm. Goad, L. J.; Akihisa, T. Analysis of Sterols; Chapman
& Hall: London, 1997.
(15) (a) Ferraboschi, P.; Colombo, D.; Prestileo, P. Tetrahedron: Assymetry
2003, 14, 2781–2785.
(8) (a) Lievens, S. C.; Hope, H.; Molinski, T. F. J. Nat. Prod. 2004, 67,
2130–2132. (b) Mellado, G. G.; Zub´ıa, E.; Ortega, M. J.; Lo´pez-Gonza´lez, P. J.
J. Nat. Prod. 2005, 68, 1111–1115.
(9) Wang, G.-Y.-S.; Crews, P. Tetrahedron Lett. 1996, 37, 8145–8146.
(10) Blackman, A. J.; Heaton, A.; Skelton, B. W.; White, A. H. Aust. J. Chem.
1985, 38, 565–573.
7980 J. Org. Chem. Vol. 73, No. 20, 2008

New Natural Parathiosteroids from Paragorgia sp.
SCHEME 1. Synthesis of Parathiosteroid A and Analogues
SCHEME 2. Synthesis of Parathiosteroid B and Analogues
then submitted to thioesterification with N-acetylcysteamine
to afford analogue 10a. Analogues 10b and 10c were also
obtained from 9 when it was treated with N-acetylethanola-
mine or N-(2-aminoethyl)acetamide, respectively, under the
same conditions used for thioesterification. The chemo-,
regio-, and stereoselective bromination of 10a and 10b at
C2 with PTAP yielded 11a and 11b, respectively, without
epimerization. Dehydrobromination of both compounds using
LiBr and Li2CO3 in refluxing dimethylformamide gave
parathiosteroid B (2a) and its analogue 2b, respectively
(Scheme 2).
activity, compound 17 was obtained from 16 by removal of the
silyl protecting group under the same conditions.
Additional analogues were obtained from the partial Birch
reduction of 4. Thus, treatment of 4 with 1.5 equiv of lithium
in liquid ammonia and anhydrous THF at -78 °C for 20 min,
using NH4Cl as the proton donor, gave alcohol 19 in 85% yield.
Oxidation of alcohol 19 to aldehyde 20 and then to acid 21,
followed by thioesterification and esterification gave analogues
22a and 22b (Scheme 4).
Spectroscopic data for all synthetic products are identical to
those of the natural products. Cytotoxic activity of all analogues
was evaluated in vitro against HT-29, A-549, and MDA-MB-
231 tumor cells. The results, expressed as GI50 values in µM,
are reported in Table 2.
Parathiosteroid C (3a) was synthesized using (+)-estrone (12)
as starting material, which was transformed to the desired
homoallylic alcohol 13 following the same synthetic strategy
used by Nemoto et al. in the preparation of an analogue of OSW-
First, we evaluated the influence of different oxidation degrees
at the A-ring keeping the same side chain. Among those steroids
bearing a thioester at C22 (natural steroids (1a-3a) and
analogues 10a and 22a), parathioesteroid B (2a) with a ∆1
double bond and analogue 10a, without any C-C double bond
on the A-ring, were the most active and selective against HT-
29 cells (GI50 of 6.5 and 6.9 µM, respectively). This fact
suggested that a lower oxidation level on the A-ring results in
higher cytotoxic activity. The same behavior was found in those
steroids containing an oxygen atom instead of a sulfur atom at
C23 position (analogues 1b-3b, 10b, and 22b), where the
analogue 10b, without any C-C double bond on the A-ring,
was the most potent within this group and also the most active
of all the compounds tested. Although analogues 1c and 10c,
bearing a NH group at C23, were active, they displayed lower
potency than their corresponding counterparts bearing a C22-
1.16 Subsequent stereoselective hydrogenation of the hindered
16
∆
double bond of 1317 gave alcohol 14 in 80% yield.
Installation of the side chain was completed by oxidation of
alcohol 14 to aldehyde 15 and then to acid 16, followed by
thioesterification and esterification to give 18a and 18b by the
same methodology as before. Finally, removal of tert-butyl-
diphenylsilyl (TBDMS) ether using TBAF and acetic acid (pH
5) in dry THF18 gave the desired natural product 3a and its
analogue 3b, respectively (Scheme 3). In order to evaluate the
significance of the presence of the side chain on the biological
(16) Matsuya, Y.; Masuda, S.; Ohsawa, N.; Adam, S.; Eustache, J.; Sukenaga,
Y.; Kamoshita, K.; Nemoto, H. Eur. J. Org. Chem. 2005, 803–808.
(17) Katona, B. W.; Rath, N. P.; Anant, S.; Stenson, W. F.; Covey, D. F. J.
Org. Chem. 2007, 72, 9298–9307.
(18) Konda, Y.; Takahashi, Y.; Arima, S.; Sato, N.; Takeda, K.; Dobashi,
K.; Baba, M.; Harigaya, Y. Tetrahedron 2001, 57, 4311–4321.
J. Org. Chem. Vol. 73, No. 20, 2008 7981

Poza et al.
SCHEME 3. Synthesis of Parathiosteroid C and Analogues
SCHEME 4. Synthesis of Analogues with ∆4
thioester or ester group, suggesting that the substitution of a
sulfur or oxygen atom at C23 by a NH group results in a
decrease of cytotoxic activity. Compounds 6, 9, 17, and 21 were
inactive, demonstrating that the C23/C28 moiety is required for
biological activity.
in the preparation of coenzyme A esters at C22 as tools for
biosynthetic studies into steroid side chain cleavage.19
Experimental Section
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 incubated for 72 h in the presence or
absence of test compounds (at 10 concentrations ranging from 40
to 0.01 µg/mL). For quantitative estimation of cytotoxicity, the
colorimetric sulforhodamine B (SRB) method was used essentially
performed as described previously.20 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. Sulforhodamine B was then
extracted in 10 mM trizma base solution, and the absorbance was
measured at 490 nm. Results are expressed as GI50, the concentra-
tion that causes 50% inhibition in cell growth after correction for
cell count at the start of the experiment (NCI algorithm).
The isolation of 1a-3a is remarkable not only because they
are the first natural steroids isolated that bear a C22 thioester
but also because they could be biosynthetic intermediates for
the degradation pathway of the steroid side chain through
activation with CoA and ꢀ-oxidation. In such a case, this is the
first report of the isolation of a degradation intermediate bearing
a fragment of a steroid-CoA thioester.
In summary, we have isolated and characterized three new
steroid thioesters, parathiosteroids A-C (1a-3a) from the
2-propanol extract of the soft coral Paragorgia sp., collected
in Madagascar. The structures of these compounds were
determined on the basis of detailed spectroscopic analysis and
confirmed by synthesis. The compounds also displayed signifi-
cant cytotoxic activity. The preparation of different analogues
revealed several structure/activity relationships in this type of
steroids, for example, that the XCH2CH2NHCOCH3 moiety (X
) S, O, NH) in the side chain is essential for the antiproliferative
activity and a low degree of oxidation on A-ring results in higher
bioactivity. Additionally, a degradation pathway of the steroid
side chain through activation with CoA and ꢀ-oxidation could
be involved in the biosynthesis of these metabolites. Finally,
the synthetic strategy developed here could also be very useful
Collection, Extraction and Isolation. Paragorgia sp. was
collected by bottom trawling near Madagascar Island at a depth of
(19) Kurosawa, T.; Fujiwara, M.; Nakano, H.; Sato, M.; Yoshimura, T.;
Murai, T. Steroids 2001, 66, 499–504.
(20) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica,
D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst.
1990, 82, 1107–1112.
7982 J. Org. Chem. Vol. 73, No. 20, 2008

New Natural Parathiosteroids from Paragorgia sp.
723/790 m. The material was identified by Dr. Pablo Lo´pez from
the University of Sevilla (Spain). A voucher specimen is deposited
at PharmaMar (ORMA 001992). The frozen organism (90 g) was
triturated and then extracted with 2-propanol (3 × 200 mL). The
combined extracts were concentrated to yield a crude of 1.07 g.
This material was subjected to VLC on Lichroprep RP-18 with a
stepped gradient from H2O to MeOH. Compounds 1a (35 mg), 2a
(2.0 mg), and 3a (5.6 mg) were isolated from fractions eluting with
MeOH by semipreparative HPLC (SymmetryPrep C-18, 7.8 mm
× 150 mm, gradient H2O/CH3CN from 50% to 100% CH3CN, UV
detection).
32.8 (t); 27.2 (t); 24.4 (t); 22.8 (t); 18.6 (q); 16.9 (C19, q); 12.2
(C18, q); LREIMS (70 eV, m/z %): 342 (M+, 4); 84 (100); (+)-
LRFABMS m /z (%): 343 ([M + H]+, 15); 137 (100); (+)-
HRESIMS: m/z 343.2283 [M + H]+ (calcd for C22H31O3, 343.2267).
Synthetic Parathiosteroid A (1a). TsCl (36 mg, 0.18 mmol) in
dry CH3CN (3 mL) was added to a solution of (20S)-3-oxopregna-
1,4-diene-20-carboxylic acid (6) (50 mg, 0.15 mmol) and N-
methylimidazole (35 µL, 0.44 mmol) in dry CH3CN (3 mL) under
an argon atmosphere, and the mixture was stirred under reflux (85
°C) for 1 h. N-Acetylcysteamine (16 µL, 0.14 mmol) was added at
85 °C, and the mixture was stirred at the same temperature for
24 h. Water was added, and the resulting mixture was extracted
with EtOAc (2 × 15 mL). The organic phase was washed with
water, brine, dried (MgSO4), and concentrated. The crude obtained
was subjected to column chromatography (silica gel, n-hexanes/
EtOAc 4:1), and the product was purified by HPLC (Sharlau C18,
flow rate 1 mL/min, retention time 34 min, CH3CN/H2O, 7:3) to
give 1a (32 mg, 65%) as a white amorphous solid. [R]25D +6.8 (c
0.2, MeOH); LREIMS (70 eV, m/z %): 443 (M+, 8); 82 (100);
(+)-LRFABMS, m/z (%): 466 ([M + Na]+, 8); 444 ([M + H]+,
38); 121 (100); (+)-HRESIMS: m/z 444.2550 [M + H]+ (calcd
for C26H38NO3S, 444.2567), 466.2373 [M + Na]+ (calcd for
C26H37NO3SNa, 466.2386).
(3ꢀ,5r,20S)-20-Methylpregnane-3,21-diol (7). A three-necked
flask was fitted with a dropping funnel, a coldfinger condenser filled
with liquid N2 and an inlet tube connected to an ammonia source,
with the gas dried using KOH. Argon was swept through the system
for 10 min and then ammonia (40 mL) was trapped in the flask.
Lithium wire (7 equiv) was cut into small pieces and added. After
stirring for 1 h, (20S)-20-hydroxymethylpregna-1,4-dien-3-one (4)
(1 g, 1.5 mmol) in THF (20 mL) was added dropwise, and the
mixture was stirred at -78 °C over 3 h. The cooling bath was
removed, and the mixture was allowed to warm to -40 °C for 20
min. The flask was dipped into a cooling bath and anhydrous NH4Cl
was added during 2 h (note: take care! vigorous reaction). The
mixture turned white and pasty. Most of the ammonia was removed
with a stream of argon. The residue was diluted with ether, washed
with brine and dried. Evaporation left a white solid that was
subjected to column chromatography (silica gel, n-hexanes/EtOAc
4:1) to afford (20S)-20-methylpregnane-3,21-diol (7) (0.99 g, 99%)
as a white amorphous solid. [R]25D +6.6 (c 0.1, CH2Cl2); 1H NMR
(300 MHz, CDCl3) δ (ppm): 3.75-3.33 (H22, H22′, 2H, m); 3.39
- 3.33 (H3R, 1H, m); 1.26 (H19, 3H, s); 1.04 (H21, 3H, d, J )
6.8 Hz); 0.68 (H18, 3H, s); 13C NMR (75 MHz, CDCl3) δ (ppm):
70.9 (C3, d); 67.6 (C22, t); 55.8 (C14, d); 53.9 (C17, d); 52.1 (d);
44.4 (d); 42.2 (s); 39.4 (s); 38.3 (t); 37.7 (d); 36.5 (t); 35.1 (t);
35.0 (d); 31.6 (t); 31.1 (t); 28.2 (t); 27.3 (t); 23.8 (t); 20.8 (C11, t);
16.2 (C21, q); 11.8 (C19, q); 11.7 (C18, q); LREIMS (70 eV, m/z
%): 334 (M+, 78); 84 (100); (-)-HRESIMS: m/z 333.2894 [M -
H]+ (calcd for C22H37O2, 333.2788).
Parathiosteroid A (1a). Amorphous white solid. [R]25 +5.9
D
(c 0.2, MeOH); IR (KBr) νmax 3291, 3080, 2939, 1657, 1621, 1599,
1550, 1438, 1403, 1371, 1291, 1240, 1204, 1009, 964, 930, 886
1
cm-1; H (500 MHz) and 13C NMR (125 MHz) see Table 1; (+)-
FABMS m/z 444 [M + H]+; (+)-HRFABMS m/z 444.2572 [M +
H]+ (calcd for C26H38NO3S, 444.2567).
Parathiosteroid B (2a). Amorphous white solid. [R]25 +66.2
D
(c 0.2, MeOH); IR (KBr) νmax 3301, 3080, 2939, 1684, 1546, 1442,
1373, 1274, 1262, 1237, 1199, 1176, 1161, 1107, 966, 935 cm-1
;
1H (500 MHz) and 13C NMR (125 MHz) see Table 1; (+)-FABMS
m/z 446 [M + H]+; (+)-HRFABMS m/z 446.2710 [M + H]+ (calcd
for C26H40NO3S, 446.2723).
Parathiosteroid C (3). Amorphous white solid. [R]25 +11.2
D
(c 0.2, MeOH); IR (KBr) νmax 3384, 3148, 2947, 2875, 1679, 1646,
1605, 1572, 1532, 1496, 1452, 1428, 1383, 1359, 1293, 1252, 968
1
cm-1; H (500 MHz) and 13C NMR (125 MHz) see Table 1; (+)-
FABMS m/z 430 [M + H]+; (+)-HRFABMS m/z 430.2420 [M +
H]+ (calcd for C25H36NO3S, 430.2410).
(20S)-3-Oxopregna-1,4-diene-20-carboxaldehyde (5). A mix-
ture of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (4) (1.0 g,
3.0 mmol) with PDC (4.0 g, 10.0 mmol) in anhydrous DMF (50
mL) in the presence of activated molecular sieves was stirred at
room temperature for 5 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,
n-hexanes/EtOAc 4:1) affording (20S)-3-oxopregna-1,4-diene-20-
carboxaldehyde (5) (0.99 g, 99%) as a white amorphous solid. [R]25
D
+13.8 (c 1.4, CH2Cl2). 1H NMR (300 MHz, CDCl3) δ (ppm): 9.55
(H22, 1H, d, J ) 3.0 Hz); 7.04 (H1, 1H, d, J ) 10.1 Hz); 6.23
(H2, 1H, dd, J ) 10.1, 1.9 Hz); 6.07 (H4, 1H, d, J ) 1.7 Hz); 1.23
(H19, 3H, s); 1.12 (H21, 3H, d, J ) 6.8 Hz); 0.78 (H18, 3H, s);
13C NMR (75 MHz, CDCl3) δ (ppm): 204.3 (C22, d); 186.0 (C3,
s); 168.7 (C5, s); 155.4 (C1, d); 127.0 (C2, d); 123.4 (C4, d); 54.3
(C14, d); 51.8 (C17, d); 50.4 (d); 48.9 (d); 43.1 (s); 42.8 (s); 38.6
(d); 35.0 (t); 33.1 (t); 32.3 (t); 26.4 (t); 24.2 (t); 22.3 (t); 18.2 (q);
12.9 (C19, q); 12.0 (C18, q); LREIMS (70 eV, m/z %): 326 (M+,
6); 122 (100); (+)-LRFABMS m /z (%): 327 ([M + H]+, 72); 121
(100).
(20S)-3-Oxopregna-1,4-diene-20-carboxylic Acid (6). (20S)-
3-Oxopregna-1,4-diene-20-carboxaldehyde (5) (0.5 g, 1.5 mmol)
was dissolved in a 1.3:1 CH3CN/pH 6.5 buffer solution mixture
(10 mL). TEMPO (0.13 g, 0.7 mmol), NaClO2 (0.3 g, 0.12 mmol),
and NaClO (2 mL) were added. The mixture was stirred at room
temperature for 24 h, and after that time an aqueous saturated
Na2SO3 solution (25 mL) was added. The pH was set to 3 by adding
aqueous 2 N HCl solution. The mixture was extracted with EtOAc
(3 × 30 mL) and washed with brine (30 mL). The solvent was
dried over anhydrous sodium sulfate and removed under vacuum.
The crude was purified by column chromatography (silica gel,
n-hexanes/EtOAc 7:3) affording the pure (20S)-3-oxopregna-1,4-
Compound 11a. A cold (0 °C) solution of phenyltrimethylam-
monium perbromide (PTAP) (25 mg, 0.65 mmol) in dry THF (3
mL) was added dropwise to a solution of 10a (30 mg, 0.7 mmol)
in dry THF (4 mL) over a period of 3 h. After a further 45 min,
the reaction was quenched by the addition of a saturated sodium
hydrogen carbonate aqueous solution (10 mL). The aqueous layer
was extracted with EtOAc, and the combined organic layers were
washed with water and dried over MgSO4. The crude product was
subjected to column chromatography (silica gel, n-hexanes/EtOAc
4:1), and the product was purified by HPLC (Sharlau C18, flow
rate 1 mL/min, retention time 74 min, CH3CN/H2O, 7:3) to give
diene-20-carboxylic acid (6) (0.37 g, 75%) as a white amorphous
11a (17 mg, 62%) as a white amorphous solid. [R]25 +190.0 (c
D
1
1
solid. [R]25 -18.8 (c 1, CH2Cl2). H NMR (300 MHz, CDCl3) δ
0.1, CH2Cl2); H NMR (300 MHz, CDCl3) δ (ppm): 5.78 (NH,
D
(ppm): 7.05 (H1, 1H, d, J ) 10.1 Hz); 6.24 (H2, 1H, dd, J ) 10.1,
1.9 Hz); 6.08 (H4, 1H, d, J ) 1.4 Hz); 1.24 (H21, 3H, d, J ) 6.6
Hz); 1.23 (H19, 3H, s); 0.78 (H18, 3H, s); 13C NMR (75 MHz,
CDCl3) δ (ppm): 186.5 (C3, s); 181.7 (C22, s); 169.3 (C5, s); 155.9
(C1, d); 127.5 (C2, d); 123.8 (C4, d); 55.0 (C14, d); 52.3 (C17, d);
52.2 (d); 43.5 (s); 42.7 (s); 42.3 (d); 39.3 (t); 35.5 (d); 33.5 (t);
1H, br s.); 4.73 (H2ꢀ, 1H, dd, J ) 13.1, 6.3 Hz); 3.43 (H25, 2H,
m); 2.99 (H24, 2H, m); 2.65 (H4a, 1H, m); 2.42 (H1a, 1H, m);
1.96 (H28, 3H, s); 1.21 (H21, 3H, d, J ) 6.8 Hz); 1.09 (H19, 3H,
s); 0.71 (H18, 3H, s); 13C NMR (75 MHz, CDCl3) δ (ppm): 204.2
(C22, s); 201.1 (C3, s); 170.2 (C27, s); 55.6 (C14, d); 54.4 (C17,
d); 53.5 (C20, d); 52.6 (C9, d); 51.8 (C2, d); 51.6 (C12, t); 47.4
J. Org. Chem. Vol. 73, No. 20, 2008 7983

Poza et al.
(C5, d); 43.9 (C13, s); 42.9 (C4, t); 40.0 (C1, t); 39.4 (C25, t);
39.0 (C10, s); 34.9 (C8, d); 31.4 (t); 28.3 (C24, t); 28.1 (t); 27.3
(t); 24.3 (t); 23.3 (C28, q); 21.4 (C11, t); 17.8 (C21, q); 12.3 (C19,
q); 12.1 (C18, q); LREIMS (70 eV, m/z %): 525 (M+, 5); 117 (100);
(+)-LRFABMS m /z (%): 526 ([M + H]+, 6); 154 (100); (+)-
HRESIMS: m/z 526.1992 [M + H]+ (calcd for C26H41NO3SBr,
526.1985).
3H, s); 0.19 (Si(CH3)2, 6H, s); 13C NMR (75 MHz, CDCl3) δ (ppm):
153.6 (C3, s); 138.3 (C5, s); 133.8 (C10, s); 126.5 (C1, d); 120.3
(C4, d); 117.5 (C2, d); 68.4 (C22, t); 55.6 (C14, d); 53.0 (C17, d);
44.2 (C13, s); 43.3 (C20, d); 40.2 (C12, t); 39.2 (d); 39.2 (d); 30.1
(t); 28.2 (t); 28.1 (t); 27.1 (t); 26.1 (SiC(CH3)3, q); 24.4 (C11, t);
18.5 (SiC(CH3)3, s); 17.1 (C21, q); 12.5 (C18, q); -4.4 (Si(CH3)2,
q); LREIMS (70 eV, m/z %): 428 (M+, 57); 371 (100); (+)-
LRFABMS m /z (%): 429 ([M + H]+, 68); 428 (100); (+)-
HRESIMS: m/z 429.3169 [M + H]+ (calcd for C27H45O2Si,
429.3183).
Compound 17. Acid 16 (20 mg, 0.02 mmol) was dissolved in
dry THF (2 mL), and then TBAF (100 µL) and acetic acid (25 µL)
were added to the solution, which was stirred overnight at room
temperature. The reaction was quenched by the addition of water
(2 mL). The mixture was extracted with EtOAc (3 × 10 mL) and
washed with brine (10 mL). The solvent was dried over anhydrous
sodium sulfate and removed under vacuum to give a residue that
was purified by column chromatography (silica gel, n-hexanes/
Synthetic Parathiosteriod B (2a). Dehydrobromination was
achieved by treatment of 2-bromo derivative 11a (30 mg, 0.06
mmol) with LiBr (35 mg, 0.36 mmol) and Li2CO3 (31 mg, 0.36
mmol) in DMF (6 mL) at reflux for 2 h. The reaction progress was
monitored by TLC (n-hexanes/EtOAc 7:3). After reaction comple-
tion, the mixture was poured into cool water (10 mL), and the
precipitate was recovered by filtration. The crude product was
subjected to column chromatography (silica gel, n-hexanes/EtOAc
4:1) and then purified by HPLC (Sharlau C18, flow rate 1 mL/
min, retention time 61 min, CH3CN/H2O, 7:3) to give 2a (17 mg,
54%) as a white amorphous solid. [R]25 +70.8 (c 0.2, MeOH);
D
1H NMR (500 MHz, CDCl3) δ (ppm): 7.31 (H1, 1H, d, J ) 10.2
Hz); 5.85 (H2, 1H, dd, J ) 10.2, 1.0 Hz); 3.44 (H25, 2H, m); 3.01
(H24, 2H, dt, J ) 6.7, 1.5 Hz); 2.65 (H20, 1H, dq, J ) 10.5, 6.8
Hz); 2.43 (H4b, 1H, dd, J ) 17.8, 14.2 Hz); 1.96 (H28, 3H, s);
1.22 (H21, 3H, d, J ) 6.8 Hz); 1.02 (H19, 3H, s); 0.73 (H18, 3H,
s);13C NMR (125 MHz, CDCl3) δ (ppm): 204.2 (C22, s); 200.2
(C3, s); 170.2 (C27, s); 158.3 (C1, d); 127.5 (C2, d); 55.6 (C14,
d); 52.6 (C17, d); 51.8 (C20, d); 49.8 (C9, d); 44.2 (C5, d); 43.0
(C13, s); 40.9 (C12, t); 40.0 (C4, t); 39.5 (C25, t); 38.9 (C10, s);
35.6 (C8, d); 31.2 (C7, t); 28.1 (C24, t); 27.6 (C16, t); 27.5 (C6, t);
24.2 (C15, t); 23.3 (C28, q); 21.2 (C11, t); 17.7 (C21, q); 13.0
(C19, q); 12.4 (C18, q); LREIMS (70 eV, m/z %): 445 (M+, 8);
119 (100); (+)-LRFABMS m /z (%): 446 ([M + H]+, 14); 154
(100); (+)-HRESIMS: m/z 446.2738 [M + H]+ (calcd for
C26H40NO3S, 446.2723).
Compound 14. Palladium on carbon (0.35 g, 5%), unsaturated
alcohol 13 (0.5 g, 1 mmol), and methanol (20 mL) were added to
a hydrogenation bottle. The mixture was hydrogenated at 70 psi
during 10 h, and then the solution was passed through Celite to
remove the palladium catalyst. After evaporation of the solvent
under reduced pressure, the residue was purified by column
chromatography (silica gel, n-hexanes/EtOAc 7:3) to afford 14 (0.4
g, 80%) as a white amorphous solid. [R]25D +62.3 (c 0.04, CH2Cl2);
1H NMR (300 MHz, CDCl3) δ (ppm): 7.11 (H1, 1H, d, J ) 8.3
Hz); 6.60 (H2, 1H, d, J ) 8.3, 2.6 Hz); 6.55 (H4, 1H, d, J ) 2.6
Hz); 3.67 (H22a, 1H, m); 3.40 (H22b, 1H, m); 2.80 (H6, 2H, m);
1.11 (H21, 3H, d, J ) 6.6 Hz); 0.99 (SiC(CH3)3, 9H, s); 0.73 (H18,
EtOAc 7:3) to afford 17 (8 mg, 45%) as a white amorphous solid.
1
[R]25 +92.1 (c 0.04, CHCl3); H NMR (500 MHz, CDCl3) δH:
D
7.19 (H1, 1H, d, J ) 8.5 Hz); 6.60 (H2, 1H, d, J ) 8.5, 2.6 Hz);
6.57 (H4, 1H, d, J ) 2.6 Hz); 2.87 (H6, 2H, m); 1.31 (H21, 3H, d,
J ) 7.9 Hz); 0.78 (H18, 3H, s). 13C NMR (125 MHz, CDCl3) δC:
179.2 (C22, s); 153.3 (C3, s); 138.4 (C5, s); 132.8 (C10, s); 126.5
(C1, d); 115.3 (C4, d); 112.6 (C2, d); 55.1 (C14, d); 52.7 (C17, d);
43.7 (C20, d); 42.2 (C13, s) 39.7 (d); 38.8 (t); 29.7 (d); 29,6 (t);
27.6 (t); 27.4 (t); 26.7 (t); 19.4 (t); 17.2 (C21, q); 12.2 (C18, q);
LREIMS (70 eV, m/z %): 328 (M+, 100); HREIMS: m/z 328.2029
[M]+ (calcd for C21H28O3, 328.2033).
Acknowledgment. This work was financially supported by
Grants from the Xunta de Galicia (PGIDIT05RMA10302PR)
and the Ministerio de Educacio´n y Ciencia (CQT2005-00793).
J.J.P. thanks the Xunta de Galicia for a grant. We also wish to
thank P. Lo´pez for assistance in the taxonomic identification
of the specimen and L. F. Garc´ıa-Ferna´ndez for performing the
cytotoxicity assays.
Supporting Information Available: Other experimental
procedures and 1H and 13C NMR spectra for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO801198U
7984 J. Org. Chem. Vol. 73, No. 20, 2008