Synthesis of steroidal quinones and hydroquinones from bile acids by Barton radical decarboxylation and benzoquinone addition. Studies on their cytotoxic and antifungal activities

Article abstract of DOI:10.1016/j.steroids.2011.09.012

Twelve new hydroquinones and quinones (4a-c to 7a-c) derived from free or peracetylated bile acids were prepared by a Barton decarboxylation reaction, with subsequent trapping of the resulting free radical by benzoquinone. All new compounds were completel

Full text of DOI:10.1016/j.steroids.2011.09.012

Steroids 77 (2012) 45–51
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of steroidal quinones and hydroquinones from bile acids by Barton
radical decarboxylation and benzoquinone addition. Studies on their cytotoxic
and antifungal activities
Gastón E. Siless a, María E. Knott b, Marcos G. Derita c, Susana A. Zacchino c, Lydia Puricelli b,
Jorge A. Palermo a,
⇑
a UMYMFOR – Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2 (1428) Buenos Aires, Argentina
b Research Area, ‘‘Angel H. Roffo’’ Institute of Oncology, University of Buenos Aires, Av. San Martín 5481 (C1417DTB), Buenos Aires, Argentina
c Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (2000)-Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2011
Received in revised form 5 September 2011
Accepted 28 September 2011
Available online 6 October 2011
Twelve new hydroquinones and quinones (4a–c to 7a–c) derived from free or peracetylated bile acids
were prepared by a Barton decarboxylation reaction, with subsequent trapping of the resulting free rad-
ical by benzoquinone. All new compounds were completely characterized by 2D NMR techniques and
screened for antifungal and cytotoxic activity. One of the new hydroquinones (7b) showed promising
results against the human pancreatic ductal carcinoma cell line PANC1, with similar cytotoxic activity
as the commercial chemotherapy drug doxorubicin.
Keywords:
Bile acids
Ó 2011 Elsevier Inc. All rights reserved.
Barton decarboxylation
Quinones
Benzoquinone
Cytotoxic activity
1. Introduction
products, and to evaluate their biological activities. A careful revi-
sion of the literature on marine natural products revealed several
Marine natural products are well known for their novel struc-
tures and biological activities [1]. However, many of these fascinat-
ing compounds suffer a severe drawback in terms of availability. In
many cases, these are minor metabolites produced by slow-
growing and delicate marine invertebrates such as sponges or soft
corals. Large scale collections of these invertebrates are generally
not possible for practical and ecological reasons. Aquaculture of
these phyla of marine invertebrates, although much progress has
been made, in most cases is still not a reliable alternative for the
production of bioactive chemicals. All these factors contribute to
a lack of available substances for in vivo bioassays, and generally
stop the development of new drugs derived from marine organ-
isms at the discovery stage. As a result, only a few marine-derived
drugs have reached the market.
privileged structures, which are generally present in bioactive
compounds. One of such structures is a quinone or hydroquinone
moiety attached to a terpenoid skeleton [2]. There are several
examples of this kind of compounds in the literature, but the best
known are avarol and avarone,
a sesquiterpenoid-derived
quinone–hydroquinone pair originally isolated from the sponge
Dysidea avara, in which the privileged structure is attached to a dri-
mane skeleton [3]. Avarol and avarone display powerful antitumor
activity [4–6], a fact that has triggered the development of several
synthetic strategies and SAR studies for these compounds [7–9].
On the other hand, there are many examples of natural quinones
and hydroquinones that display antifungal activity, making the
incorporation of these structural fragments a good choice in the
preparation of new antifungals [10,11].
We have recently started a project on synthetic modifications of
abundant and easily accessible natural starting materials such as
bile acids and plant terpenoids. One of the aims of this project is
to incorporate to these starting natural substances, structural frag-
ments which are frequently found in bioactive marine natural
In one of the most successful avarol syntheses, the quinone
fragment was incorporated by means of a Barton decarboxylation
reaction on an adequate carboxylic acid, with subsequent trapping
of the resulting free radical by benzoquinone [12]. The Barton
decarboxylation reaction of O-acyl esters formed from carboxylic
acids and N-hydroxy-2-thiopyridone is a versatile tool that can
be used for functional group conversions and the formation of car-
bon-carbon bonds [13]. The N–O bond of Barton esters can be
cleaved either in thermal or photolytic conditions to generate a
⇑
Corresponding author. Tel.: +54 11 4576 3346; fax: +54 11 4576 3385.
E-mail address: palermo@qo.fcen.uba.ar (J.A. Palermo).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.09.012

46
G.E. Siless et al. / Steroids 77 (2012) 45–51
carboxyl radical and a pyridine-2-thyil radical. The carboxyl radical
then undergoes CO2 loss to generate an alkyl radical which can be
conveniently used to produce a variety of products. If the gener-
ated radical reacts with a conveniently substituted alkene, quinone
or aromatic group, a new carbon-carbon bond will be formed. In
the steroid field, the Barton reaction has been previously used for
the preparation of a brominated derivative of a cholanic acid which
was finally used for the synthesis of a one-carbon homolog of the
starting compound [14].
on an FT-IR Nicolet Magna 550 instrument. TLC was carried out
on Merck Sílicagel 60 F254 plates. TLC plates were sprayed with
2% vainillin in concentrated H2SO4. Merck Silicagel (230–
400 mesh) was used for column chromatography. Solid phase
extraction was performed with reversed-phase silica gel SPE car-
tridges (Thermo Scientific).
2.2. Chemistry
In the present work, the same strategy of reference [12] was ap-
plied to a series of readily available bile acids. In this way, twelve
new quinone or hydroquinone derivatives of free or peracetylated
cholic, lithocholic and chenodeoxycholic acids were synthesized.
The peracetylated bile acid derivatives were of particular interest
since peracetylated bile acids, probably of microbiological origin,
have been isolated from several marine invertebrates, where they
are believed to be involved in the chemical defense of these organ-
isms [15,16]. As such, the peracetylated quinones can be consid-
ered as hybrids of avarone and acetylated bile acids. All new
compounds were completely characterized by 2D NMR techniques
and screened for antifungal and cytotoxic activity. One of the new
hydroquinones showed promising results against human pancre-
atic carcinoma cell line PANC1 (see Fig. 1).
2.2.1. Preparation of thiohydroxamic esters (2a–c)
In a flask protected from light with aluminum foil at 0 °C, per-
acetylated acid 1a (26 mg, 0.062 mmol) and 2-mercaptopyridine
N-oxide (39.6 mg, 0.311 mmol) in 1 mL of dry dichloromethane
were added. A solution of DCC (17 mg, 0.082 mmol) in 1 mL of
CH2Cl2 was then added and the mixture was stirred for 2 h at room
temperature. The solvent was removed by evaporation and the
obtained residue was purified by solid phase extraction on re-
versed-phase silicagel, eluting with methanol: water (7:3) to re-
move excess 2-mercaptopyridine N-oxide and then acetone to
recover the Barton ester. The acetone fraction was concentrated
carefully, with protection from light and water bath temperature
below 30 °C, to afford 28 mg of thiohydroxamate ester 2a (yield
87%). 1H NMR (200 MHz, CDCl3): 7.70(1H, dd, J = 8.4, 1.5 Hz, H-
600), 7.56(1H, dd, J = 7.0, 0.9 Hz, H-300), 7.21(1H, br t, J = 8.4 Hz, H-
500), 6.64(1H, td, J = 7.0, 1.5 Hz, H-400), 4.88(1H, br s, H-7), 4.59(1H,
m, H-3), 2.71(2H, m,H-23), 2.06(3H, s, Ac), 2.03 (3H, s, Ac),
0.99(3H, d, J = 5.5 Hz, H-21), 0.93(3H, s, H-19), 0.67(3H, s, H-18).
Using the same procedure described above for compound 2a,
reaction of 163 mg of peracetylated acid 1b in 4 mL of dichloro-
methane afforded 197.5 mg of thiohydroxamic ester 2b (yield
2. Experimental
2.1. General
Bile acids were obtained from commercial sources and used as
such or recrystallized prior to use when necessary. Peracetylated
compounds were obtained by standard procedures, using Ac2O/
DMAP/Py. Solvents were distilled for chromatography; CH2Cl2
was distilled from phosphorous pentoxide. NMR spectra were re-
corded on Bruker AC-200 (200.13 MHz) and Bruker Avance II
(500.13 MHz) spectrometers, using the signals of residual non-
deuterated solvents as internal reference. All 2D NMR experiments
(COSY, DEPT-HSQC, HMBC, and NOESY) were performed using
standard pulse sequences. HRMS were acquired on a Bruker micrO-
TOF-Q II spectrometer. UV spectra were obtained on a Hewlett
Packard 8453 spectrophotometer and IR spectra were obtained
1
96%). H NMR (200 MHz, CDCl3): 7.69(1H, d, J = 8.4 Hz, H-600),
7.57(1H, d, J = 7.1 Hz, H-300), 7.21(1H, t, J = 8,4 Hz, H-500), 6.63(1H,
t, J = 7.1 Hz, H-400), 4.72(1H, m, H-3), 2.70(2H, m, H-23), 2.03 (3H,
s, H-10), 0.98(3H, d, J = 6,0 Hz, H-21), 0.93(3H, s, H-19), 0.66(3H, s,
H-18).
Using the same procedure described above for compound 2a,
reaction of 114 mg of peracetylated acid 1c in 2 mL of dichloro-
methane afforded 115.6 mg of thiohydroxamic ester 2c (yield
84%). 1H NMR (200 MHz, CDCl3): 7.69(1H, dd, J = 8.4, 1.8 Hz, H-
600), 7.56(1H, dd, J = 6.8, 1.5 Hz, H-300), 7.21(1H, ddd, J = 8,4 Hz,
6.8, 1.5 Hz, H-500), 6.64(1H, ddd, J = 6.8, 6.8, 1.6 Hz, H-400),
Fig. 1. Synthesis of quinones and hydroquinones derived from bile acids.

G.E. Siless et al. / Steroids 77 (2012) 45–51
47
5.11(1H, br s, H-11), 4.91(1H, br s, H-7), 4.58(1H, m, H-3), 2.70(2H,
m, H-23), 2.15(3H, s, Ac-11), 2.09(3H, s, Ac-3), 2.04(3H, s, Ac-7),
0.92(6H, br s, H21, H-19), 0.75(3H, s, H-18).
J = 11.7, 4.8 Hz, H-23a), 2.66(1H, td, J = 11.7, 4.4 Hz, H-23b),
2.02(3H, s, Ac), 1.03 (3H, d, J = 6.2 Hz, H-21), 0.93(3H, s, H-19),
0.63(3H, s, H-18).
Using the same procedure described above, reaction of 19.3 mg
of thiohydroxamic ester 2c resulted in 16.7 mg of quinone 3c (yield
2.2.2. Decarboxylation and quinone addition – preparation of
quinones (3a–c)
1
79%). H NMR (CDCl3, 200 MHz): 8.31(1H, d, J = 4.7 Hz, H-600),
7.58(1H, dd, J = 7.8, 7.7 Hz, H-400), 7.35(1H, d, J = 7.8 Hz, H-300),
7.05(1H, dd, J = 7.7, 4.7 Hz, H-500), 6.81(2H, br s, H-50, H-60),
5.08(1H, br s, H-11), 4.91(1H, br s, H-7), 4.57(1H, m, H-3),
2.12(3H, s, Ac-11), 2.09(3H, s, Ac-3), 2.05(3H, s, Ac-7), 0.93(3H, d,
J = 5.5 Hz, H-21), 0.91(3H, s, H-19), 0.71(3H, s, H-18).
Thiohydroxamate ester 2a (27.8 mg, 0.047 mmol) and p-benzo-
quinone (76.3 mg, 0.706 mmol) were dissolved in 4 mL of dry
dichloromethane under nitrogen atmosphere at 0 °C. The reaction
mixture was subsequently irradiated using a single 300 W tung-
sten lamp, while maintaining the temperature at 0 °C in an ice
bath. Solvent evaporation followed by solid-phase extraction of
the residue on reversed-phase using methanol: water (1:1) and
then acetone as eluants, afforded a crude product which could be
used as such or further purified by column chromatography on sil-
ica using ciclohexane: ethyl acetate (8:2), yielding 25.5 mg (83%) of
pure quinone 3a. 1H NMR (200 MHz, CDCl3): 8.31(1H, br d,
J = 4.9 Hz, H-600), 7.57(1H, td, J = 7.9, 1.8 Hz, H-400), 7.34(1H, br d,
J = 7.9 Hz, H-300), 7.05(1H, dd, J = 7.9, 5.1 Hz, H-500), 6.82(2H, br s,
H-50, H-60), 4.87(1H, br s, H-7), 4.59(1H, m, H-3), 2.84(1H, td,
J = 11.9, 5.3 Hz, H-23a), 2.67(1H, td, J = 11.3, 5.1 Hz, H-23b),
2.05(3H, s, Ac), 2.03(3H, s, Ac), 1.04(3H, d, J = 6.6 Hz, H-21),
0.93(3H, s, H-19), 0.63(3H, s, H-18).
2.2.3. Reduction to steroidal peracetylated hydroquinones (4a–c)
2.2.3.1. 23-Hydroquinoyl-24-nor-5b-cholane-3a,7a-diyl diacetate
(4a). A solution of quinone 3a (42.8 mg, 0.066 mmol) in 2 mL of
DME was added to a suspension of an excess of activated Raney
Nickel in 2 mL of DME and refluxed for 30 min while stirring. The
reaction mixture was then filtered over a small pad of celite and
the solvent was evaporated to obtain a crude residue which could
be used as such or further purified by column chromatography on
silica using ciclohexane: ethyl acetate (7:3), yielding 44.8 mg (95%)
of pure hydroquinone 4a. 1H NMR (500 MHz, CDCl3): 6.63(1H, d,
J = 8.3 Hz, H-60), 6.61(1H, d, J = 3.0 Hz, H-30), 6.54(1H, dd, J = 8.3,
3.0 Hz, H-50), 4.88(1H, br q, J = 3 Hz, H-7), 4.59(1H, m, H-3),
4.48(1H, s, OH), 4.4(1H, s, OH), 2.62(1H, ddd, J = 13.9, 11.6,
5.2 Hz, H-23a), 2.41(1H, ddd, J = 13.9, 11.3, 5.7 Hz, H-23b),
2.06(3H, s, Ac), 2.04(3H, s, Ac), 1.04(3H, d, J = 6.6 Hz, H-23),
Using the same procedure described above for compound 3a,
reaction of 83.1 mg of thiohydroxamic ester 2b resulted in
53.8 mg of quinone 3b (yield 61%). 1H NMR (200 MHz, CDCl3):
8.32(1H, br d, J = 4.4 Hz, H-600), 7.57(1H, td, J = 7.7, 2.2 Hz, H-400),
7.34(1H, br d, J = 8.0 Hz, H-300), 7.04(1H, ddd, J = 7.3, 5.1, 1.1 Hz,
H-500), 6.81(2H, br s, H50, H-60), 4.72(1H, br s, H-3), 2.85H, td,
0.93(3H, s, H-19), 0.66(3H, s, H-18). 13C NMR (125 MHz, CDCl3):
+
see Table 1. ESI-MS m/z [M+Na]+ 563.3355 (calc. for C33H48NaO6
,
Table 1
13C NMR (125 MHz) data for compounds 4a–c, 5a–c, 6a–c, 7a–b.
C
4aa
4ba
4ca
5aa
5ba
35.3
5ca
34.9
6aa
35.2
6ba
35.5
6cb
35.4
7ab
35.9
7bb
35.1
1
34.8
34.8
34.9
35.0
2
3
4
5
6
7
8
9
26.7
74.0
34.5
40.8
31.3
71.2
37.8
33.9
34.0
20.6
39.4
42.7
50.3
23.5
28.0
55.8
11.5
22.6
35.6
18.6
35.9
26.5
147.1
130.3
116.6
149.1
113.1
115.9
170.6
21.4
170.4
21.5
32.4
74.6
27.1
42.1
28.5
26.5
35.2
40.6
32.1
21.0
40.4
42.9
56.7
24.4
27.2
56.2
12.3
23.5
36.2
18.9
36.1
26.8
147.5
130.7
116.9
149.5
113.3
116.1
170.9
21.7
27.1
74.3
34.8
41.1
31.4
71.0
37.9
29.1
34.5
25.8
75.3
45.3
43.5
23.0
27.3
47.6
12.4
22.7
35.2
18.2
36.1
26.8
147.5
130.6
116.9
149.8
113.3
116.1
170.8
21.8
170.7
21.7
170.9
21.6
26.9
74.3
34.9
41.1
31.5
71.4
38.0
34.2
34.8
20.8
39.6
42.9
50.4
23.7
28.2
55.7
11.9
22.8
35.7
18.7
34.2
25.7
187.7
150.4
132.4
187.9
136.4
136.9
170.8
21.6
170.6
21.8
32.5
74.6
26.8
42.1
28.5
26.5
36
40.6
34.8
21.1
40.3
43
56.7
24.4
27.2
56.1
12.2
23.5
35.9
18.7
34.4
25.9
187.7
150.7
132.5
188.1
136.5
137
27.1
74.3
34.8
41.1
31.5
70.9
38.0
29.1
34.5
25.8
75.6
45.3
43.6
23.0
27.4
47.4
22.8
12.4
35.2
18.0
34.2
25.7
187.7
138.2
132.7
188.0
136.5
137.0
170.7
21.8
170.6
21.6
170.8
21.7
30.8
72.2
40.1
41.6
34.8
68.7
39.6
33.0
35.5
20.7
39.8
42.9
50.6
23.9
28.4
55.7
11.9
22.9
35.9
18.7
34.4
25.9
187.7
150.6
132.5
188.1
136.4
137.0
30.8
72.1
36.7
42.3
28.4
26.6
36.0
40.6
34.8
21.0
40.4
43.0
56.7
24.4
27.4
56.0
12.2
23.6
35.9
18.7
34.4
25.9
187.7
132.5
188.1
150.7
136.4
137.1
30.7
72.2
40.0
41.6
34.9
68.5
39.8
26.9
34.7
27.7
73.1
46.7
42.2
23.3
25.7
47.2
12.8
22.7
35.7
17.8
34.4
26.0
182.2
150.5
132.5
182.4
136.5
137.0
36.6
72.9
40.5
43.2
31.4
69.1
40.6
34.1
36.2
21.8
41.1
43.7
51.5
24.6
29.3
57.5
12.2
23.4
37.1
19.3
37.5
27.9
149.1
131.9
117.5
151.0
113.7
116.6
30.4
69.9
36.3
40.1
26.9
26.1
35.4
41.5
34.2
20.4
39.7
42.3
56.1
23.8
27.8
55.6
11.9
23.3
35.2
18.6
35.9
26.3
147.4
129.7
116.2
149.6
112.6
115.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
10
20
30
40
50
60
3-Ac
170.9
21.7
7-Ac
12-Ac
a
In CDCl3.
In CD3OD.
b

48
G.E. Siless et al. / Steroids 77 (2012) 45–51
563.3349). IR (film, cmꢀ1): 3420.8, 2940.4, 2881.2, 1742.6, 1722.9,
1518.8, 1459.6, 1393.8, 1249.0, 1202.9, 1038.4, 755.4. aD (CHCl3,
cmꢀ1): 2933.8, 2868.0, 1749.2, 1670.2, 1459.6, 1367.5, 1249.0,
1038.4, 906.8, 748.8. UV (CHCl3, cmꢀ1):
254 = 2071.4, 324 = 658.6.
e
e
c = 0.29) = +4.83°. UV (CHCl3, 1/Mcm):
e241 = 1209,
e293 = 2260.
2.2.4.3.
23-(1,4-Benzoquinoyl)-24-nor-5b-cholane-3a,7a,12a-triyl
2.2.3.2. 23-Hydroquinoyl-24-nor-5b-cholane-3
a
-yl acetate
triacetate (5c). Using the same procedure described above, reaction
of 25.9 mg of hydroquinone 4c resulted in 24.8 mg of quinone 5c
(Yield 96%). 1H NMR (CDCl3, 500 MHz): 6.75(1H, d, J = 10.1 Hz, H-
60), 6.72(1H, dd, J = 10.1, 2.4 Hz, H-50), 6.54(1H, d, J = 2.4 Hz, H-30),
5.10(1H, dd, J = 2.85, 2.8 Hz, H-12), 4.91(1H, dd, J = 2.74, 5.4 Hz,
H-7), 4.57(1H, s, H-3), 2.48(1H, dddd, J = 15.1, 11.5, 5.9, 1.4 Hz,
H-23a), 2.26(1H, dddd, J = 15.1, 11.2, 5.1, 1.3 Hz, H23-b), 2.15(3H,
s, Ac-12), 2.05(3H, s, Ac-7), 2.09(3H, s, Ac-3), 0.92(3H, s, H-18),
0.89(3H, d, J = 6.6 Hz, H-21), 0.74(3H, s, H-19). 13C NMR
(125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 619.3264 (calc.
(4b). Using the same procedure described above for compound
3a, reaction of 8.7 mg of quinone 3b resulted in 5.0 mg of hydro-
quinone 4b (yield 70%). 1H NMR (500 MHz, CDCl3): 6.63(1H, d,
J = 8.3 Hz, H-60), 6.61(1H, d, J = 3.0 Hz, H-30), 6.54(1H, dd, J = 8.3,
3.0 Hz, H-50), 4.72(1H, m, H-3), 4.48(1H, s, OH), 4.4(1H, s, OH),
2.62(1H, ddd, J = 13.9, 11.6, 5.2 Hz, H-23a), 2.41(1H, ddd, J = 13.9,
11.3, 5.7 Hz, H-23b), 2.06(3H, s, Ac), 2.04(3H, s, Ac), 1.04(3H, d,
J = 6.6 Hz, H-23), 0.93(3H, s, H-19), 0.66(3H, s, H-18). 13C NMR
(125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 505.3285 (calc.
for C31H46NaO4, 505.3294). IR (film, cmꢀ1): 3394.5, 2940.4, 2868.0,
1709.7, 1510.0, 1466.2, 1393.0, 1281.9, 1189.0, 1031.8, 775.1. aD
for
35H52NO8, 614.3687). IR (film, cmꢀ1): 2953.6, 2874.6, 1729,
1663.6, 1472.8d, 1380.6, 1262.2, 1031.8, 755.4. aD (CHCl3,
C
35H48NaO8, 619.3241), [M+NH4]+ 614.3708 (calc. for
C
(CHCl3, c = 0.45) = 14.66°. UV (CHCl3, cmꢀ1):
e250 = 276.3, e288 =
330.
c = 0.62) = +51.1°.UV (CHCl3, 1/Mcm): e301 = 504, e320 = 498, e260 =
2648.
2.2.3.3. 23-Hydroquinoyl-24-nor-5b-cholane-3a,7a,12a-triyl triace-
tate (4c). Using the abovementioned procedure, reaction of
2.2.5. Cleavage of acetates – preparation of quinones (6a–c)
2.2.5.1. 23-(1,4-Benzoquinoyl)-3 ,7 -dihydroxy-24-nor-5b-cholane
37.3 mg of quinone 3c afforded 25.9 mg of hydroquinone 4c (Yield
a
a
1
82%). H NMR (CDCl3, 500 MHz): 6.61(1H, d, J = 2.95 Hz, H-30),
(6a). To a solution of quinone 5a (16.3 mg, 0.0303 mmol) in 1 mL
of dry THF at 0 °C, LiAlH4 (200 mg, excess) was added, and the reac-
tion mixture was strirred for 30 min in an ice/brine bath. Methanol,
water and 1 M aqueous hydrochloric acid were sequentially added
in order to destroy the excess of LiAlH4, and the mixture was then
extracted twice with EtOAc. The combined organic phases were
washed with NaHCO3 (ss) and water, and finally evaporated at re-
duced pressure. The residue was redissolved in 4 mL of ethyl ether
and treated with an excess of MnO2. The reaction mixture was stir-
red at 25 °C for 30 min, filtered through a small pad of celite and
the solvent was removed by evaporation at reduced pressure.
The crude product was purified by column chromatography on sil-
ica using a ciclohexane:ethyl acetate gradient to give 4.5 mg of qui-
none 6a (Yield 40%). 1H NMR (500 MHz, CDCl3): 6.76(1H, d,
J = 10.0 Hz, H-60), 6.71(1H, dd, J = 10.0, 2.5 Hz, H-30), 6.56(1H, dt,
J = 2.4, 1.3 Hz, H-30), 3.86(1H, br s, H-7), 3.47(1H, m, H-3),
2.49(1H, dddd, J = 15.1, 11.3, 4.5, 1.4 Hz, H-23a), 2.30(1H, dddd,
J = 15.1, 11.1, 5.1, 1.3 Hz, H-23b), 0.91(3H, s, H-19), 1.00(3H, d,
J = 6.9 Hz, H-19), 0.67(3H, s, H-18). 13C NMR (125 MHz, CDCl3):
see Table 1. ESI-MS m/z [M+Na]+ 477.2960 (calc. for C29H42NaO4,
477.2981). IR (film, cmꢀ1): 3394.5, 2940.4, 2868.0, 1663.6,
1492.5, 1084.5, 906.8, 768.6. aD (CHCl3, c = 0.84) = +5.59° UV
6.63(1H, d, J = 8,5 Hz, H-60), 6.54(1H, dd, J = 8.5, 2.95 Hz, H-50),
5.11(1H, br dd, J = 2.6, 2.8 Hz, H-12), 4.91(1H, br dd, J = 2.9,
5.7 Hz, H-7), 4.58(1H, m, H-3), 2.62(1H, ddd, J = 13.5, 11.9, 4.8 Hz,
H-23a), 2.39(1H, ddd, J = 13.5, 11.2, 5.3 Hz, H-23b), 2.15(3H, s,
Ac-12), 2.08(3H, s, Ac-3), 2.05(3H, s, Ac-7), 0.93(3H, d, J = 4.6 Hz,
H-21), 0.92(3H, s, H-19), 0.72(3H, s, H-18). 13C NMR (125 MHz,
CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 621.3419 (calc. for
C
35H50NaO8, 621.3398), [M+NH4]+ 616.3860 (calc. for C35H54NO8,
616.3844). IR (film, cmꢀ1): 3401.1, 2947.0, 2861.4, 1742.6 br,
1643.9d, 1466.2d, 1387.2d, 1255.6 br, 1038.4, 768.6. aD (MeOH,
c = 0.28) = +45.4°. UV (MeOH, 1/Mcm): e229 = 961, e296 = 888.
2.2.4. Oxidation to peracetilated steroidal quinones (5a-c)
2.2.4.1. 23-(1,4-benzoquinoyl)-24-nor-5b-cholane-3 -diyl diace-
a7a
tate (5a). A solution of hydroquinone 4a (44.3 mg, 0.082 mmol)
in 5 mL of ethyl ether was treated with an excess of MnO2 and
strirred at 25 °C for 30 min. The reaction mixture was then filtered
over a small pad of celite yielding a crude product after solvent re-
moval. Purification by column chromatography on silica using
ciclohexane:ethyl acetate (9:1) afforded 40 mg (91%) of pure qui-
none 5a. 1H NMR (500 MHz, CDCl3): 6.76 (1H, d, J = 10.1 Hz,
H-60), 6.71(1H, dd, J = 10.1, 2.4 Hz, H-50), 6.55(1H, br d, J = 2.4 Hz,
H30), 4.88(1H, br s, H-7), 4.59(1H, m, H-3), 2.49(1H, ddd, J = 15.0,
12.0, 3.8 Hz, H-23a), 2.29(1H, ddd, J = 15.0, 11.8, 4.9, H-23b),
2.06(3H, s, Ac), 2.04(3H, s, Ac), 1.00(3H, d, J = 6.6 Hz, H-21),
(CHCl3, 1/Mcm):
e
248 = 2890,
e256 = 2960,
e294 = 916.3.
2.2.5.2. 23-(1,4-Benzoquinoyl)-3a-hydroxy-24-nor-5b-cholane
(6b). Using the same procedure described above for compound 6a,
reaction of.11.8 mg of quinone 5b resulted in 8.4 mg of quinone
6b (yield 78%). 1H NMR (500 MHz, CDCl3): 6.75(1H, d, J = 10.2 Hz,
H-60), 6.71(1H, dd, J = 10.1, 2.5, H-50), 6.56(1H, br s, J = 2.5 Hz, H-
30), 3.63(1H, m, H-3), 2.49(1H, dddd, J = 15.1, 11.2, 4.8, 1.4 Hz,
H-23a), 2.30(1H, dddd, J = 15.1, 11.2, 5.2. 1.1 Hz, H-23b), 0.92(3H,
s, H-19), 0.99(3H, d, J = 6.6 Hz, H-21), 0.65(3H, s, H-18). 13C NMR
(125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 461.3035 (calc.
for C29H42NaO3, 461.3026) [M+NH4]+ 456.3475 (calc. for C29H46NO3,
456.3472). aD (CHCl3, c = 0.43) = +12.1°. IR (film, cmꢀ1): 3355.0,
2947.0, 2874.6, 1663.6, 1466.2w, 1301.6d, 1038.4, 762.0 m. UV
0.94(3H, s, H-19), 0.64(3H, s, H-18). 13C NMR (125 MHz, CDCl3):
see Table 1. ESI-MS m/z [M+Na]+ 561.3206 (calc. for C33H46NaO6
,
+
561.3187), [M+NH4]+ 556.3656 (calc. for C33H50NO6+, 556.3633).
IR (film, cmꢀ1): 2947.0, 2881.2, 1736.0, 1663.6, 1380.6, 1262.2,
1025.2, 906.8, 768.6. aD (CHCl3, c = 1.24) = +7.58°. UV (CHCl3,
1/Mcm):
e254 = 3096.8,
e288 = 535.2.
2.2.4.2.
23-(1,4-Benzoquinoyl)-24-nor-5b-cholane-3
a
-yl
acetate
(5b). Using the same procedure described above, reaction of
34.6 mg of hydroquinone 4b resulted in 31.4 mg of quinone 5b
(Yield 86%).1H NMR (500 MHz, CDCl3): 6.75(1H, d, J = 10.1 Hz, H-
60), 6.71(1H, dd, J = 10.1, 2.6 Hz, H-50), 6.55(1H, m, H-30), 4.72(1H,
m, H-3), 2.49(1H, dddd, J = 15.1, 11.3, 4.7, 1.4 Hz, H-23a),
2.29(1H, dddd, J = 15.41, 11.1, 5.2, 1.2 Hz, H-23b), 2.03 (3H, s,
Ac). 0.93(3H, s, H-19), 0.99(3H, d, J = 6.6 Hz, H-21), 0.65(3H, s, H-
18). 13C NMR (125 MHz, CDCl3): See Table 1. ESI-MS m/z [M+Na]+
503.3127 (calc. for C31H44NaO4, 503.3137), [M+H]+ 481.3324 (calc.
for C31H45O4, 481.3318). aD (CHCl3, c = 1.28) = +28.63°. IR (film,
(CHCl3, 1/Mcm):
e258 = 2982,
e
289 = 1185,
e324 = 548.
2.2.5.3. 23-(1,4-Benzoquinoyl)-3
a
,7 ,12 -trihydroxy-24-nor-5b-cho-
a
a
lane (6c). Using the same procedure described above for com-
pound 6a, reaction of 25.0 mg of quinone 5c resulted in 5.7 mg of
quinone 6c (yield 29%) after purification by preparative TLC. 1H
NMR (500 MHz, CD3OD): 6.56(1H, dt, J = 2.4, 1.4 Hz, H-30),
6.75(1H, d, J = 10.1 Hz, H-60), 6.71(1H, dd, J = 10.1, 2.5 Hz, H-50),

G.E. Siless et al. / Steroids 77 (2012) 45–51
49
4.00(1H, br t, J = 2.6 Hz, H-12), 3.86(1H, br dd, J = 2.7, 5.7 Hz, H-7),
2.3. Cytotoxicity evaluation
3.46(1H, m, H-3), 2.50(1H, dddd, J = 15.2, 11.7, 4.8, 1.5 Hz, H-23a),
2.32(1H, dddd, J = 15.2, 10.9, 5.4, 1.0 Hz, H-23b), 1.06(3H, d,
J = 6.5 Hz, H-21), 0.90(3H, s, H-19), 0.70(3H, s, H-18). 13C NMR
(125 MHz, CD3OD): see Table 1. ESI-MS m/z [M+Na]+ 493.2925
(calc. for C29H42NaO5, 493.2930). IR (film, cmꢀ1): 3369.5, 2944.2,
2863.5, 1727.1, 1661.1, 1631.8, 1463.1, 1389.8, 1272.5, 1206.5,
The effect of the different compounds on cell growth was as-
sayed on log phase unsynchronized monolayers of two different
cell lines: LM3 (murine mammary adenocarcinoma) and PANC1
(human ductal pancreatic carcinoma) [17]. LM3 cells were cultured
at 37 °C in plastic flasks in MEM medium (Gibco; InvitrogenCorp,
1081.9. UV (MeOH, 1/Mcm):
332 = 395.
e
209 = 5500,
e
253 = 1190,
e
294 = 857,
Carlsbad, Calif) supplemented with 10% FCS, 2 mM
and 80 g/ml Gentamicin. PANC1 cells were cultured at 37 °C in
RPMI 1640 (Gibco; InvitrogenCorp, Carlsbad, Calif) supplemented
with 10% FCS, 2 mM -glutamine and 80 g/ml Gentamicin. Both
L-glutamine
e
l
L
l
2.2.6. Reduction to steroidal hydroquinones (7a–c)
2.2.6.1. 23-Hydroquinoyl-3 ,7 -dihydroxy-24-nor-5b-cholane
cells lines were cultured in a humidified air atmosphere with 5%
CO2. Serial passages were made by treatment of confluent mono-
layers with 0.25% trypsin and 0.02% EDTA in Ca+2 and Mg+2-free
PBS. Cells were periodically determined to be mycoplasma free
by the Hoechst’s method.
a
a
(7a). Using the same procedure described previously for the prep-
aration of compound 4a, reaction of quinone 6a (8.6 mg,
0.019 mmol) in 1 mL of CH2Cl2 resulted, after preparative TLC chro-
matography, in 2.2 mg of hydroquinone 7a (yield 25%). 1H NMR
(500 MHz, CD3OD): 6.56(1H, d, J = 8.5 Hz, H-60), 6.51 (1H, d,
J = 3.2 Hz, H-30), 6.42(1H, dd, J = 8.5, 3.1 Hz, H-50), 3.80(1H, br s,
H-7), 3.37(1H, m, H-3), 2.60(1H, d, J = 13.4 Hz, H-23), 2.40(1H, d,
J = 13.4 Hz, H-500), 2.27(1H, ddd, J = 13, 12, 11 Hz, H-4a), 0.93(3H,
s, H-19), 1.04(3H, d, J = 6.6 Hz, H-21), 0.68(3H, s,H-18). 13C NMR
(125 MHz, CD3OD): see Table 1. ESI-MS m/z [M+Na]+ 479.3135
(calc. for C29H44NaO4+, 479.3132). IR (film, cmꢀ1): 3374.8, 2933.8,
2868.0, 1729(d), 1459.6, 1216.1, 1077.9, 755.4. aD (MeOH,
The bioassays were carried on as follows: 3 ꢁ 103 LM3 cells/
well or 4 ꢁ 103 PANC1 cells/well in complete medium were seeded
in 96 multiwell plates. After 24 h, cells received serial doses of the
compounds (0.01–100 lM) or vehicle (DMSO) in medium plus 2%
SFB for 3 days. As control, the cytotoxic activity of doxorubicin
was also tested. Medium with freshly added compounds was chan-
ged every two days. Viability was assessed by reduction of the tet-
razolium salt (MTS) to the formazan product in viable cells (Cell
Titer 96 TM, Promega Corp) as calculated by the 492/620 nm
absorbance ratio. IC (Inhibitory Concentration)50 was defined as
the concentration of the compound required for 50% cell growth
inhibition.
c = 0.2) = +1.5°. UV (MeOH, 1/Mcm):
295 = 920.
e226 = 1003, e252 = 746,
e
2.2.6.2. 23-Hydroquinoyl-3a-hydroxy-24-nor-5b-cholane (7b). Using
the same procedure, reaction of 8.4 mg of quinone 6b resulted in
5.8 mg of hydroquinone 7b (yield 69%). 1H NMR (500 MHz, CDCl3):
6.43(1H, br d, J = 2.95 Hz,H-30), 6.53(1H, d, J = 8.5 Hz, H-60),
6.35(1H, dd, J = 8.5, 2.95 Hz, H-50), 3.36(1H, m, H-3), 2.48(1H, m,
H-23a), 2.29(1H, ddd, J = 13.4, 10.9, 6.2 Hz, H-23b), 0.87(3H, s, H-
19), 0.96(3H, d, J = 6.5 Hz, H-21), 0.60(3H, s, H-18), 8.48(1H, s,
OH-40), 8.41(1H, s, OH-10), 4.45(1H, OH-3). 13C NMR (125 MHz,
CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 463.3189 (calc. for
2.4. Antifungal activity evaluation
2.4.1. Microorganisms and media
For the antifungal evaluation, standardized strains from the
American Type Culture Collection (ATCC), Rockville, MD, USA Can-
dida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32264
were used.
Strains were grown on Sabouraud-chloramphenicol agar slants
for 48 h at 30 °C and maintained on slopes of Sabouraud-dextrose
agar (SDA, Oxoid) and sub-cultured every 15 days to prevent pleo-
morphic transformations. Inocula were obtained according to re-
ported procedures and adjusted to 1–5 ꢁ 103 cells with colony
forming units (CFU)/mL [18].
C
29H44NaO3, 463.3183). IR (film, cmꢀ1): 3374.8, 2933.8, 2861.4,
1466.2, 1038.4, 769. aD (MeOH, c = 0.22) = +19.1°. UV (MeOH,
1/Mcm): 223 = 1006, 295 = 934.
e
e
2.2.6.3. 23-Hydroquinoyl-3a,7a,12a-trihydroxy-24-nor-5b-cholane
(7c). Using the same procedure, reaction of 5.7 mg of quinone 6c
resulted in 4.3 mg of hydroquinone 7c (yield 75%). 1H NMR
(200 MHz, CD3OD): 6.50(1H, d, J = 2.6 Hz, H-30), 6.53(1H, d,
J = 9.0 Hz, H-60), 6.40(1H, dd, J = 8.7, 3.0 Hz, H-50), 3.89(1H, br s,
H-12), 3.28(1H, m, H-3), 2.51(1H, m, H-23a), 2.33(1H, m, H-23b),
0.98(3H, d, J = 6.0 Hz, H-21), 0.79(3H, s, 1H-19), 0.59(3H, s,
H-18).ESI-Q-TOF: 490.35412). ESI-MS m/z [M+Na]+ 495.3090 (calc.
2.4.2. Antifungal susceptibility tests
Minimum Inhibitory Concentration (MIC) of each compound
was determined by using broth microdilution techniques accord-
ing to the guidelines of the National Committee for Clinical Labora-
tory Standards (CLSI) for yeasts (M27-A3) [18]. MIC values were
determined in RPMI-1640 (Sigma, St Louis, Mo, USA) buffered to
pH 7.0 with MOPS. Microtiter trays were incubated at 35 °C in a
moist, dark chamber, and MICs were visually recorded at 48 h.
For the assay, stock solutions of pure compounds were two-fold
for
29H48NO5, 490.3533). IR (film, cmꢀ1): 3406.1, 2914.9, 2856.2,
1463.1, 1382.5, 1206.5, 1089.2. UV (MeOH, 1/Mcm): 211 = 2392,
227 = 1080, 293 = 770.
C
29H44NaO5, 495.3086), [M+NH4]+ 490.3541 (calc. for
C
e
e
e
diluted with RPMI from 256–0.98
lg/mL (final volume = 100
ll)
and a final DMSO concentration 61%. A volume of 100
l
l of inocu-
lum suspension was added to each well with the exception of the
sterility control where sterile water was added to the well instead.
Amphotericin B, fluconazole, and itraconazole (Sigma, St. Louis,
MO, USA), were used as positive controls.
2.2.7. Reduction of 4a–c to 7a–c
Using the same procedure described in Section 2.2.5 for the
reduction of quinones 5a–c, treatment of hydroquinones 4a–c with
LiAlH4 in THF produced the corresponding deacetylated hydroqui-
nones 7a–c (yields: 7a: 80%, 7b: 96%, 7c: 71%).
3. Results and discussion
2.2.8. Oxidation of 7a–c to 6a–c
Using the same procedure described in Section 2.2.4 for the oxi-
dation of hydroquinones 4a–c, oxidation of hydroquinones 7a–c
with MnO2 in Et2O gave the corresponding deacetylated quinones
6a–c (yields: 7a: 90%, 7b: 81%, 7c: 85%).
In the present work, Barton esters 2a–c were prepared from
peracetylated chenodeoxicholic, lithocholic and cholic acids
respectively, by reaction with 2-mercaptopyridine N-oxide and
DCC in dry CH2Cl2 at room temperature. The yields obtained with

50
G.E. Siless et al. / Steroids 77 (2012) 45–51
Table 2
IC50 values (
the free bile acids as starting materials were considerably lower.
The choice of acetate as protective group was decided on the basis
of its presence in many marine natural products and the above-
mentioned interest in the peracetylated bile acid derivatives as
‘‘natural product like’’ compounds. The general synthetic route
would provide sequential access to both free and peracetylated
quinones and hydroquinones. In order to obtain reasonable yields
of Barton esters, an excess of 2-mercaptopyridine N-oxide had to
be employed. A quick purification of the labile thiohydroxamate
esters, with minimal handling and efficient removal of the excess
of 2-mercaptopyridine N-oxide, was then mandatory. This could
be achieved by SPE on reversed phase cartridges. An initial elution
with MeOH: H2O (7:3) completely removed the excess of 2-
mercaptopyridine N-oxide, allowing the recovery of the thiohydr-
oxamate esters by a subsequent elution of the cartridge with
acetone. These esters were immediately used without further
purification.
Barton esters 2a–c were then subjected to photolytic decarbox-
ylation in the presence of benzoquinone as radical trap, by irradi-
ation with a 300 W tungsten lamp at 0° in CH2Cl2 under N2
atmosphere to yield the corresponding 2-thiopyridyl 1,2 quinone
adducts 3a–c [19]. The excess of benzoquinone was removed
quickly and efficiently by SPE on reversed-phase by elution with
MeOH:H2O (1:1), and then adducts 3a–c were recovered by subse-
quent elution of the SPE cartridge with acetone. As expected, 2-
thioprydil sulfides of the decarboxylated bile acids were the main
byproducts of this reaction, but in most cases the crude adducts
could be used as such without further purification by column chro-
matography. Reductive desulfurization of adducts 3a–c with Raney
Ni in CH2Cl2 yielded crude hydroquinones 4a–c in fairly good
yields. These products were oxidized to the corresponding qui-
nones 5a–c by MnO2 in ether.
l
M) for the in vitro screening against tumor cell lines.
Compound
PANC1 (pancreas)
LM3 (murine)
7a
7b
7c
>50
>50
12.1 7.5
>50
5.6 1.0
>50
Doxorubicin
Lithocholic acid
20.2 3.3
>100
0.48 0.1
>100
mammary adenocarcinoma) and PANC1 (human ductal pancreatic
carcinoma). LM3 is an aggressive murine tumor cell line with high
metastatic capacity to lung, originally developed at the Institute of
Oncology A.H. Roffo [17]. The well known in vivo behavior of this
cell line makes it a good model for further studies. On the other
hand, PANC1 is a pancreatic carcinoma cell line that is quite resis-
tant to chemotherapeutic agents. For comparison, doxorubicin was
used as positive standard. Of the twelve tested compounds, only
the free hydroquinones 7a–c displayed some cytotoxic activity.
The results are shown in Table 2, and indicate that only compound
7b (the free hydroquinone derived from lithocholic acid) showed
remarkable cytotoxic activity against both cell lines. PANC1 is a
very resistant tumor cell line as shown by the high IC50 value for
doxorubicin. Compound 7b was as active as doxorubicin against
this cell line. Interestingly the corresponding hydroquinones de-
rived from the other two bile acids (7a and 7c) also displayed cell
cytotoxic activity but with IC50 values larger than 50
lithocholic acid was also tested for comparison, and displayed
IC50 values larger than 100 M.
All the abovementioned compounds were also tested for anti-
fungal activity against C. albicans and C. neoformans. Unfortunately,
lM, while
l
in all cases the MIC50 values were higher than 400 lM showing
that these compounds are not good promissory antifungal com-
pounds for further research. Nevertheless, this lack of activity
against fungal cells indicates a selective toxicity of these steroidal
quinones and hydroquinones against tumor cell lines enhancing
their importance as cytotoxic entities.
Cleavage of the acetate groups was the most problematic step of
the syntheses. Initial attempts under basic conditions (4% KOH in
dioxane:methanol or K2CO3 in methanol:water) produced decom-
position of the compounds and led to complex mixtures. The en-
hanced acidity of H-23s, vicinal to the quinone group, may be
one of the causes of this unstability under basic conditions [20].
For this reason, acidic and neutral conditions were also tested.
Transesterification in the presence of KCN in methanol under re-
flux also produced complex mixtures [21], as well as the use of
(Bu3Sn)2O in a variety of solvents (MeOH, EtOH, and toluene) under
reflux [22,23].
Lewis acids were also tested: ZnCl2 in MeOH under reflux failed,
while the use of Sc(OTf)3 under the same conditions only produced
the deprotection of the C-3 hydroxyl in compound 5a, leaving the
C-7 acetate intact. The use of microwave heating with Sc(OTf)3 on
compound 5b produced 50% of 6b, while under the same condi-
tions, compound 5c only produced 20% of the C-3 deacetyl deriva-
tive, leaving C-7 and C-12 acetates intact.
All these unproductive attempts, led to the use of a strong
reducing agent such as LiAlH4 in THF for the deprotection of qui-
nones 5a–c. This reaction gave mixtures of the deacylated qui-
nones and hydroquinones which, for ease of purification were
oxidized with MnO2 to obtain the fully deacetylated quinones
6a-c, with a good yield for compound 6b (78% for both steps)
and relatively lower yields for compounds 6a and 6c. Finally, com-
pounds 6a–c were reduced to the corresponding fully deacylated
hydroquinones 7a–c with Raney Ni in DME, using basically the
same technique as for the preparation of compounds 3a–c.
In order to circumvent the low-yielding deprotection step, com-
pounds 7a–c could be prepared directly from 4a–c by reduction
with LiAlH4 in THF with good yields. Compounds 6a-c could then
be prepared from 7a–c by MnO2 oxidation.
4. Conclusion
In the present work, 12 new quinones and hydroquinones de-
rived from free or peracetylated bile acids were synthesized. All
the compounds were fully characterized by spectroscopical tech-
niques and studies of their cytotoxic and antifungal activities were
performed. One of the new compounds, 7b, showed good IC50 val-
ues against both tested tumor cell lines, especially against the qui-
mioresistant PANC1 cell line, where 7b was twice as active as
doxorubicin. In general, the yields were very good except for the
deacetylation step. However, the reduction of 5b, precursor of
the most bioactive compound 7b, with LiAlH4 proceeded with a
fairly good yield. On the other hand, direct reduction of acetylated
hydroquinones 4a–c with LiAlH4 gave better yields of compounds
7a–c, which could then be oxidized to 6a–c with MnO2. These
exciting preliminary results will lead to the preparation of a library
of derivatives of compound 7b, and to a more extensive and thor-
ough exploration of the scope and mechanism of action of this no-
vel substance.
Acknowledgements
We are indebted to Mrs. Alicia Rivelli for expert assistance with
cell culture techniques. Research at the University of Buenos Aires
was supported by Grants from CONICET (PIP No. 516-2009) and
UBA (X163 Programación 2008–2010). S.A.Z. thanks ANPCyT for a
research Grant (PICT 0608). G.E.S. and M.G.D. thank CONICET for
The effect on cell growth of compounds 4a–c, 5a–c, 6a–c and
7a–c, was tested against two different cell lines: LM3 (murine

G.E. Siless et al. / Steroids 77 (2012) 45–51
51
derived metabolite xestoquinone and its family. Biosci Biotechnol Biochem
2005;69:1749–52.
[11] Arif T, Bhosale JD, Kumar N, Mandal TK, Bendre RS, Lavekar GS, et al. Natural
doctoral and postdoctoral fellowships respectively. M.E.K. thanks
ANPCyT for a doctoral fellowship.
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