Preparation and characterization of some keto-bile acid azines

Article abstract of DOI:10.1016/j.steroids.2007.06.005

New acyclic dimers of ketocholanic acids with hydrazine were obtained. Crystal structure was determined for the 3,7-dihydroxy-12-ketocholanic acid azine. Some distinctive ¹H NMR signals are assigned for the entire set of azines.

Full text of DOI:10.1016/j.steroids.2007.06.005

steroids 7 2 ( 2 0 0 7 ) 756–764
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Preparation and characterization of some
keto-bile acid azines
Valerio Bertolasia, Olga Bortolinib, Giancarlo Fantina,
Marco Fogagnoloa,∗, Daniela Perronec
a
`
`
Dipartimento di Chimica, Universita di Ferrara, Via Borsari 46, 44100 Ferrara, Italy
b
c
Dipartimento di Chimica, Universita della Calabria, Via Bucci 12 C, 87036 Rende CS, Italy
`
Dipartimento di Biologia ed Evoluzione, Universita di Ferrara, C.so Ercole I d’Este, 32, 44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
New acyclic dimers of ketocholanic acids with hydrazine were obtained. Crystal structure
was determined for the 3,7-dihydroxy-12-ketocholanic acid azine. Some distinctive 1H NMR
signals are assigned for the entire set of azines.
Received 26 February 2007
Received in revised form
22 June 2007
© 2007 Elsevier Inc. All rights reserved.
Accepted 26 June 2007
Published on line 6 July 2007
Keywords:
Steroids
Dimeric bile acids
Homo- and heteroazines
Synthesis
1.
Introduction
A–ring A connection are the most abundant, whereas ring
C–ring C connections are no so frequent [4,6]. For connection
Bile acids are versatile building blocks that have become
increasingly important in a number of fields, such as pharma-
cology, biomimetic and supramolecular chemistry [1]. A vast
amount of interesting medical applications based on these
derivatives have been reported as well [2]. The synthesis of
specific steroidal dimers and oligomers has become of inter-
est because of their possible application as catalysts for some
types of reactions and as pharmacologically active deriva-
tives, in particular in fungal infections [3]. A pointed out by
Li and Dias [4], initially the synthesis of dimeric derivatives
with various inter-steroid linkage occurred as by-product for-
mation. Only in recent years these compounds were produced
by design [5]. Of the dimeric bile acids those derived by ring
and numbering see Fig. 1.
In 1996 we reported the preparation of a ring C–ring C
connected 12-azine, starting from dehydrocholic acid (3,7,12-
triketo-5␤-cholan-24-oic acid), upon reduction and treatment
with NH2NH2 [7]. In the present paper we have modified and
extended our synthetic strategy to the preparation of new
acyclic homo and hetero dimers with various ring connec-
tions utilizing the bile acids 3␣-hydroxy-7-keto-5␤-cholan-
24-oic acid 1, 3␣,12␣-dihydroxy-7-keto-5␤-cholan-24-oic acid
2, 3␣-hydroxy-12-keto-5␤-cholan-24-oic acid 3 and 3␣,7␣-
dihydroxy-12-keto-5␤-cholan-24-oic acid 4 as building blocks.
The X-ray structure of the homo ring C–ring C connected azine,
derived from bile acid 4, is also reported and discussed.
∗
Corresponding author. Fax: +39 0532 240709.
E-mail address: fgr@unife.it (M. Fogagnolo).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.06.005

steroids 7 2 ( 2 0 0 7 ) 756–764
757
2.1.2. 3˛,12˛-Dihydroxy-7-keto-5ˇ-cholan-24-oic acid 2
The compound was prepared according to a literature proce-
dure [9]. The product was purified by column chromatography
on silica gel (ethyl acetate/ acetic acid 50:1): m.p. 195–198 ◦C;
1H NMR (CD3OD): ı 0.77 (s, 3H, H-18); 1.06 (d, J = 6.2 Hz, 3H, H-
21); 1.26 (s, 3H, H-19); 2.60 (dd, J = 12 Hz, 1H, H-8␤); 3.02 (dd,
J = 13; 6.6 Hz, 1H, H-6␤); 3.58 (br, 1H, H-3␤); 4.04 (s, 1H, H-12␤).
2.1.3. 3˛-Hydroxy-12-keto-5ˇ-cholan-24-oic acid 3
To
a r.t. stirred solution of methyl deoxycholate (2.5 g,
6.1 mmol) and triethylamine (1.28 mL, 9.2 mmol) in CH2Cl2
(40 mL) was added drop by drop a solution of pivaloyl chloride
(1.13 mL, 9.2 mmol) in CH2Cl2 (20 mL). After 24 h the reaction
Fig. 1 – Bile acid steroid skeleton.
mixture was treated with aqueous solution of NaHCO3 (30 mL).
The organic phase was separated, dried on anhydrous Na2SO4,
concentrated and the expected derivative (6), purified by col-
umn chromatography (SiO2, cyclohexane/ ethyl ether 75/25),
was obtained as sirup in 73% yield (2.2 g). 1H NMR (CDCl3): ı
0.71 (s, 3H, 18-H3); 0.94 (s, 3H, 19-H3); 1.0 (d, J = 6.1 Hz, 3H, 21-
H3), 1.2 (s, 9H, COOt-Bu); 3.67 (s, 3H, COOMe); 4.0 (s, 1H, H-12␤);
4.70 (br, 1H, H-3␤).
A stirred solution of pivaloyl derivative (2 g, 4.1 mmol) in
acetone at 0 ◦C (40 mL) was treated with the Jones’ reagent
until a slight permanent orange color was obtained. After
10 min at r.t. the reaction mixture was quenched with 2-
propanol (0.5 mL), filtered over celite, concentrated, treated
with an aqueous solution of NaHCO3 and extracted with ethyl
acetate. After removal of the solvent in a vacuum, the residue
was dissolved in dioxane (20 mL), treated with aqueous NaOH
15% (10 mL) and refluxed for 6 h. Most of the organic solvent
was evaporated, the residue was cooled in an ice-bath and
acidified with HCl 30%. The expected product 3 was obtained
as white solid (1.3 g, yield 82%); m.p. 165–167 ◦C; 1H NMR
(CD3OD): ı 0.8 (d, J = 6.2 Hz, 3H, H-21); 1.10 (s, 3H, H-18); 1.12
(s, 3H, H-19); 2.62 (dd, J = 12.2 Hz, 1H, H-11␤); 3.59 (br, 1H, H-3␤).
2.
Experimental
Melting points are uncorrected and were determined on a 510
Bu¨ cki Meltig Point instrument. TLC were performed on pre-
coated Silica Gel plates (thickness 0.25 mm, Merck) and Silica
Gel (Fluka, Kiesegel 60, 70–230 mesh) was used for prepara-
tive column chromatography. The 1H and 13C NMR spectra
were recorded in CDCl3 or CD3OD solutions, in 5 mm tubes,
at room temperature, with a Varian Gemini 300 spectrometer
operating at 300 MHz (1H) and 75 MHz (13C), with TMS as exter-
nal standard. Mass spectra were run on MICROMASS ZMD2000
instrument operating in electrospray ionization. X-ray struc-
ture were collected at room temperature, 295 K, on a Nonius
Kappa CCD diffractometer with graphite monochromated Mo
˚
K␣ radiation (ꢀ = 0.7107 A).
2.1.
Preparation of bile acids 1–4
2.1.1. 3˛-Hydroxy-7-keto-5ˇ-cholan-24-oic acid 1
The compound was prepared according to a literature pro-
cedure [8]. M.p. 208–211 ◦C; 1H NMR (CD3OD): ı 0.73 (s, 3H,
H-18); 0.98 (d, J = 6.2 Hz, 3H, H-21); 1.28 (s, 3H, H-19); 2.60 (dd,
J = 10.9 Hz, 1H, H-8␤); 3.03 (dd, J = 12.1; 4.8 Hz, 1H, H-6␤); 3.58 (br,
1H, H-3␤).
2.1.4. 3˛,7˛-Dihydroxy-12-keto-5ˇ-cholan-24-oic acid 4
The compound was prepared from methyl 3␣,7␣-diacethoxy-
12-hydroxycholanate via oxidation with the Jones’ reagent

758
steroids 7 2 ( 2 0 0 7 ) 756–764
according to a literature procedure [10] followed by saponifica-
tion as described for compound 3. The expected product was
obtained as white solid (1.1 g, 87%); m.p. 219–221 ◦C; 1H NMR
(CD3OD): ı 0.90 (d, J = 6.3 Hz, 3H, H-21); 1.10 (s, 3H, H-18); 1.13
(s, 3H, H-19); 2.62 (dd, J = 13.3, 1H, H-11␤); 3.42 (br, 1H, H-3␤);
3.94 (brs, 1H, H-7␤).
2.2.6. Heteroazine (connection 12-12) 12
Thirty-seven percent yield, column chromatography (SiO2,
ethyl acetate/cyclohexane/acetic acid 30:20:1), m.p. 195–202 ◦C
with dec 1H NMR (CD3OD): ı 0.99–1.07 (m, 18H); 3.4 (br, 1H, H-
3␤); 3.51 (br, 1H, H-3␤); 3.71 (dd, J = 12 3.5 Hz, 1H, H-11␣); 3.80
(dd, J = 12.2 3.5 Hz, 1H, H-11␣), 3.88 (brs, 1H, H-7␤). Selected 13
C
NMR resonances (CD3OD): ı 178.33 (24-C); 175.31 (12-C); 175.83
2.2.
General procedure for the synthesis of the azines
(12-C).
2.6 mmol of the appropriate ketocholanic acid 1–4 (or 1.3 mmol
of each one of the two different bile acids for the preparation
of heteroazines) were dissolved in 5 mL of glacial acetic acid,
under gentile heating. Hydrazine hydrate (13 mmols, 80%) was
slowly added to the mixture. The reaction was left under stir-
ring at room temperature for 24 h, poured in a flask containing
30 g of water and ice crushed. The resulting crude white solid
was filtered, washed with distilled water and dried in air. The
pure azines were obtained by recrystallization or by column
chromatography.
2.2.7. Heteroazine (connection 7-12) 13
Forty-five percent yield, column chromatography (SiO2, ethyl
acetate/cyclohexane/acetic acid 40:10:1), m.p. 228–235 ◦C dec.;
1H NMR (CD3OD): ı 0.74 (s, 3H, H-18); 1.0 (m, 9H, H-18, H-19,
H-21); 1.08 (d, J = 6.3 Hz, 3H, H-21); 1.15 (s, 3H, H-19); 2.85 (d,
J = 12.7 Hz, 1H, H-6␣); 2.92 (dd, J = 12.3; 3.5 Hz, 1H, H-11␣); 3.39
(br, 1H, H-3␤); 3.55 (br, 1H, H-3␤); 3.88 (brs, 1H, H-7␤). Selected
13C NMR resonances (CD3OD): ı 178.54; 178.46 (24-C); 170.53
(12-C); 165.99 (7-C). ESI-MS: [M+H]+ m/z 793.7.
2.2.8. Heteroazine (connection 7-12) 14
2.2.1. Homoazine (connection 7-7) 7
Forty-seven percent yield, column chromatography (SiO2,
ethyl acetate/cyclohexane/acetic acid 30:20:1), m.p. 205–210 ◦C
dec. 1H NMR (CD3OD): ı 0.75 (s, 3H); 0.97–1.07 (m, 12H); 1.1
(s, 3H); 2.79 (d, J = 10.1 Hz, 1H, H-6␣ or 11␣); 2.82 (dd, J = 9.6;
2.6 Hz, 1H, H-11␣ or 6␣); 2.52 (br, 2H, H-3␤); 4,01 (brs, 1H, H-
12␤). Selected 13C NMR resonances (CD3OD): ı 178.44 (24-C);
170.53 (12-C); 165.38 (7-C).
Ninety-one percent yield, crystallized from ethanol, m.p.
163–170 ◦C with dec. 1H NMR (CDCl3): ı 0.77 (s, 6H, H-18); 1.01 (d,
J = 6.3 Hz, 6H, H-21); 1.14 (s, 6H, H-19); 3.04 (d, J = 12.6 Hz, 2H, H-
6␣); 3.50 (br, 2H, H-3␤). Selected 13C NMR resonances (CD3OD):
ı 178.20 (24-C); 167.89 (7-C).
2.2.2. Homoazine (connection 7-7) 8
Ninety-three percent yield, crystallized from ethanol, m.p.
223–230 ◦C with dec; 1H NMR (CD3OD): ı 0.79 (s, 6H, H-18);
1.05 (d, J = 6.6 Hz, 6H, H-21); 1.13 (s, 6H, H-19); 2.98 (dd, J = 12.6;
1.4 Hz, 2H, H-6␣); 3.50 (br, 2H, H-3␤); 4.02 (brs, 2H, H-12␤).
Selected 13C NMR resonances (CD3OD): ı 178.32 (24-C); 166.91
(7-C).
2.2.9. Heteroazine (connection 7-12) 15
Fifty-seven percent yield, column chromatography (SiO2, ethyl
acetate/cyclohexane/acetic acid 20:30:1), mp. 176–180 ◦C with
dec. 1H NMR (CD3OD): ı 0.76 (s, 3H, H-18); 0.96–1.06 (m, 15H);
2.81 (m, 2H, H-6␣, H-11␣); 3.54 (br, 2H, H-3␤). Selected 13C NMR
resonances (CD3OD): ı 178.30 (24-C); 178.35 (24-C); 170.60 (12-
C); 165.76 (7-C).
2.2.3. Heteroazine (connection 7-7) 9
Forty-one percent yield, column chromatography (SiO2, ethyl
acetate/cyclohexane/acetic acid 30:20:1); m.p. 228–233 ◦C dec.
1H NMR (CD3OD): ı 0.76 (s, 3H, H-18); 0.78 (s, 3H, H-18); 1.0 (d,
J = 6.4 Hz, 3H, H-21); 1.06 (d, J = 6.3 Hz, 3H, H-21); 1.12 (s, 3H, H-
19); 1.14 (s, 3H, H-19); 3,02 (d, J = 12.8 Hz, 2H, H-6␣); 3.50 (br,
2H, H-3␤); 4.03 (brs, 1H, H-12␤). Selected 13C NMR resonances
(CD3OD): ı 178.48 (24-C); 178.41 (24-C); 167.73 (7-C); 167.41
(7-C).
2.2.10. Heteroazine (connection 7-12) 16
Thirty-eight percent yield, column chromatography (SiO2,
ethyl acetate/cyclohexane/acetic acid 25:25:1), m.p. 201–206 ◦C
with dec. 1H NMR (CD3OD):ı 0.78 (s, 3H, H-18); 0.97–1.1 (m, 12H);
1.15 (s, 3H, H-19); 2.88 (dd, J = 13; 1.5 Hz, 1H, H-6␣ or 11␣); 2.94
(dd, J = 13.5; 3.6 Hz, 1H, H-11␣ or 6␣); 3.39 (br, 1H, H-3␤); 3.55 (br,
1H, H-3␤); 3.87 (brs, 1H, H-7␤); 4.03 (brs, 1H, H-12␤). Selected 13
NMR resonances (CD3OD): ı 178.50 (24-C); 170.48 (12-C); 165.63
C
(7-C).
2.2.4. Homoazine (connection 12-12) 10
Eighty-six percent yield, column chromatography (SiO2, ethyl
acetate/cyclohexane/acetic acid 20:30:1), m.p. 135–140 ◦C dec.
1H NMR (CD3OD): ı 1.02 (m, 12H); 1.06 (s, 6H); 3.56 (br, 2H,
H-3␤); 3.69 (dd, J = 12; 3,5 Hz, 2H, H-11␣). Selected 13C NMR res-
onances (CD3OD): ı 178,21 (24-C); 175,43 (12-C). ESI-MS: [M+H]+
m/z 777.6.
3.
Results and discussion
3.1.
Chemistry
The keto-bile acid precursors 1 and 2 were easily prepared
from the corresponding commercial available chenodeoxy-
cholic and cholic acids by selective oxidation of the hydroxyl
group at position C(7) with N-bromosuccinimide [8,9]. The keto
derivative 3 was prepared in good yield from methyl deoxy-
cholate 5 following an unprecedented reported procedure,
consisting in the selective protection of the hydroxyl group
at C(3) with pivaloyl chloride, followed by Jones’ oxidation and
saponification, according to Scheme 1.
2.2.5. Homoazine (connection 12-12) 11
Ninety-six percent yield, crystallized from methanol,
m.p. 240–245 ◦C dec. 1H NMR (CD3OD):
ı 0.97–1.03 (m,
18H); 3.34 (br, 2H, H-3␤); 3.63 (dd, J = 9.2; 3,6 Hz, 2H, H-
11␣); 3.83 (brs, 2H, H-7␤). Selected 13C NMR resonances
(CD3OD): ı 178.34 (24-C); 174.35 (12-C). ESI-MS: [M+H]+ m/z
809.6.

steroids 7 2 ( 2 0 0 7 ) 756–764
759
Scheme 1
The keto-bile acid 4 was prepared from methyl 3␣,7␣-
interest was isolated.
diacethoxy-12-hydroxycholanate via oxidation with the Jones’
reagent, followed by saponification [10].
AcOH
1 + 2
equimolar amounts
+ NH2–NH2 −→ 9 +
7 + 8
by product homoazines
(2)
ꢀꢁꢂꢃ
ꢀꢁꢂꢃ
r.t.,24 h
With the four precursors in our hands we have under-
taken the synthesis of the different azines shown in Fig. 2.
The term homo refers to the azines obtained starting from the
same keto-bile acids, while the term hetero is used for azines
derived from two different bile acid precursors.
The synthetic procedure is general, simple and straightfor-
ward. The appropriate ketocholanic acid 1–4 was dissolved in
glacial acetic acid and the hydrazine hydrate was slowly added
to the mixture. The reaction mixture was left under stirring at
room temperature for 24 h, followed by the usual work-up, Eq.
(1).
3.2.
NMR spectra
As pointed out in several publications the 1H NMR spectra of
bile acids are typified in two major zones, e.g. the methylene-
methine envelope centred in the 0.5–2.5 ppm region and the
portion of the spectra between 3 and 5 ppm [11]. Oxo-hydroxy
bile acids exhibited additional and specific signals in the range
2.5–3.6 ppm, likely due to the presence of the carbonyl groups
on selected adjacent protons [12]. A similar deshielding effect
induced by the C N groups of the azine may be found in the 1H
NMR spectra of compounds 7–16, on the protons closest to this
group [6]. Table 1 reports the related resonances multiplicities
and coupling constants.
AcOH
1 − 4 + NH2–NH2 −→ Azine
(1)
r.t.,24 h
For the preparation of heteroazines equimolar amounts of
two different bile acid building blocks are used. Heteroazine
9, for example, is obtained by reacting a 1:1 mixture of bile
acids 1 and 2 with hydrazine hydrate in AcOH, Eq. (2), 12 was
prepared from a mixture of 3 and 4, 13 by reacting 1 and 4, 14
from a mixture of 2 and 3, 15 from 1 and 3 and 16 was pre-
pared from a mixture of 2 and 4. The obvious consequence is
that the homoazines 7, 8, 10, 11, derived from an unique keto-
cholanic acid precursor, are obtained in nearly quantitative
yield (86–96%), Eq. (1), whereas for heterozines 9, 12, 13–16,
derived from two different ketocholanic acid precursors, the
yield is lower (37–57%), since the concomitant formation of
the related homoazines are observed (TLC) as by-products, but
not quantified, Eq. (2). After work-up, only the heteroazine of
For the azines 10–12, C(12)-C(12) connected, the specific
resonances have to be assigned to one of the two protons at
C(11), either equatorial (␣) or axial (␤). The multiplicity of the
signal, a doublet of doublet, is characterized by two J values:
the first one in the range of 9.2–12 Hz (geminal coupling) and
the second one of about 3.5 Hz, attributable to a vicinal cou-
pling to the axial proton at C(9) position. This considerations
unequivocally attribute the specific resonances to the equato-
rial C(11) protons. Similar arguments may be used to assign to
the equatorial C(11) protons the specific resonances, collected
in Table 1, for compounds 13–16. On the other hand, the dis-
tinctive resonances found for the azines 7–9 and 13–16 are
assigned to the equatorial C(6) protons. Further support to this
attribution may be found in independent experiments carried

760
steroids 7 2 ( 2 0 0 7 ) 756–764
Fig. 2 – Structure of the homo- and heteroazines of this study.
out on cholanoic acid 2 and on the corresponding azine 8. As
shown in Scheme 2 and described in Section 2, bile acid 2 is
characterized, among others, by two NMR resonances at ı 2.60
and 3.02, respectively. Treatment of this compound with basic
deuterated water leads to the disappearance of the signal at
3.02 ppm, attributable to axial C(6)-hydrogen [12]. Exchange of
the conformationally forced hydrogen at carbon C(8), in fact, is
prevented. The deuterated azine 8bis, prepared starting from
the deuterated cholanoic acid 2bis, showing the absence of
the resonance at 2.98 ppm, clearly assigned this resonance to
C(6)-hydrogens.
3.3.
X-ray structure determination
For the azine 11 the formation of crystals has permit-
ted the X-ray structure of this compound. X-ray diffraction
data for compound 11 were collected at room tempera-
ture, 295 K, on a Nonius Kappa CCD diffractometer with

steroids 7 2 ( 2 0 0 7 ) 756–764
761
Table 1 – 1H NMR chemical shifts, multiplicities and coupling constants of ␣-protons adjacent to the C N groups of 7–16
Diazine
7-7 connection
12-12 connection
7-12 connection
7
8
9
10
11
12
13
14
15
16
Proton
ı (ppm)
Multip.
J (Hz)
6a
3.04
d
6a
2.98
dd
12.6
1.4
6a
3.02
d
11b
3.69
dd
12.0
3.5
11b
3.63
dd
9.2
3.6
11␤
3.71
dd
12.0
3.5
6␤
2.85
d
6␤c
2.79
d
6.11
2.81
m
6␤c
2.88
dd
13.0
1.5
12.6
12.8
12.7
10.1
–
J (Hz)
Proton
ı (ppm)
Multip.
J (Hz)
11␣
3.80
dd
12.2
4.4
11␤
2.92
dd
12.3
3.5
11␤c
2.82
dd
9.6
2.6
11␤c
2.94
dd
13.5
3.6
J (Hz)
For C(6) and C(11) positions ␣-proton is equivalent to equatorial proton while ␤-proton is equivalent to axial proton.
a
Assigned to both equatorial protons at carbons C(6) of the first and second cholanic framework forming the azine.
Assigned to both equatorial protons at carbons C(11) of the first and second cholanic framework forming the azine.
The assignment along the column could be reversed.
b
c
formed using PARST [15] and PLATON [16] systems of
programs.
˚
graphite monochromated Mo K␣ radiation (ꢀ = 0.7107 A). The
structure was solved by direct methods (SIR97) [13] and
refined (SHELXL-97) [14] by full matrix least squares with
anisoptropic non-H atoms and hydrogen atoms included on
calculated positions riding on their carrier atoms, except
for some OH hydrogens which were refined isotropically.
The hydrogen positions of the water molecules could
not be determined. All other calculations have been per-
3.3.1. Crystal data
11,
C
48H76N2O8·2H2O; monoclinic, space group P21,
◦
˚
a = 12.9669(2), b = 18.6740(3), c = 12.2846(3) A, ˇ = 118.8423(7) ,
V = 2404.69(8) A , Z = 2, Dc = 1.167 g cm−3
.
Intensity data
3
˚
collected with ꢁ ≤ 27.5◦; 10,508 independent reflections mea-
Scheme 2

762
steroids 7 2 ( 2 0 0 7 ) 756–764
Fig. 3 – ORTEP view and atom numbering of compound 11 displaying the thermal ellipsoids at 30% probability.
sured; 8681 observed [I > 2ꢂ(I)]. Final R index = 0.0653 (observed
reflections), Rw (all reflections) = 0.1924 and S = 1.057.
Complete crystallographic data (excluding structural fac-
tors) have been deposited with the Cambridge Crystallo-
graphic Data Centre and allocated the deposition number
CCDC 637989. These data can be obtained, free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html or on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 1223
336033 or e-mail: deposit@ccdc.cam.ac.uk].
Fig. 5 – The hydrogen bond system involved in the
formation of the hydrophilic channels.
ORTEP [17] view of compound 11 is given in Fig. 3. The
molecular fragment in Fig. 4 shows the configurations and the
conformation determined by the azino linkage. The cholic acid
moieties, A and B, forming the molecular dimer, exhibit two
different carboxylic side-chain conformations: full extended,
t t t t, for A and folded t g t g for B, Table 2 [18].
Fig. 4 – Molecular fragment of compound 11 showing the E
configuration of C12A N1 and C12B N2 double bonds, and
the trans conformation around the N1–N2 single bond.

steroids 7 2 ( 2 0 0 7 ) 756–764
763
Table 2 – Selected torsion angles (◦) for compound 11
C13A-C17A-C20A-C22A: ␺
C17A-C20A-C22A-C23A: ␺
C20A-C22A-C23A-C24A: ␺
179.2(3)
−176.8(4)
174.5(4)
t
t
t
t
C13B-C17B-C20B-C22B: ␺
C17B-C20B-C22B-C23B: ␺
C20B-C22B-C23B-C24B: ␺
−179.1(3)
64.7(4)
155.5(4)
95.8(5)
t
g
t
1
2
3
1
2
3
C22A-C23A-C24A-O28A: ␺
160.5(5)
C22B-C23B-C24B-O28B: ␺
g
4
4
C12A-N1-N2-C12B
C11A-C12A-N1-N2
C13A-C12A-N1-N2
−137.1(3)
3.2(4)
170.8(3)
t
C11B-C12B-N2-N1
C13B-C12B-N2-N1
3.2(4)
170.9(3)
E
E
Carboxylic side-chain conformations; configurations and conformation around the azino linkage.
Table 3 – Hydrogen bond parameters (A, ◦) for compound 11
˚
D–H. . .A
Symm. Op.
D–H
H. . .A
D. . .A
D–H. . .A
O25A-H25A. . .O29B
O29A-H29A. . .O27A
O28A-H28A. . .O2w
O25B-H25B. . .O2w
O29B-H29B. . .O27B
O28B-H28B. . .O1w
O1w-H. . .O25A
x, y, z
1.01(5)
0.81(3)
0.82a
1.90(5)
2.08(4)
1.81
2.07
1.97(4)
1.83
2.822(5)
2.812(4)
2.621(8)
2.589
2.860(5)
2.614(3)
2.699(5)
151(4)
150(4)
172
120
173(4)
160
−x, 1/2 + y, 1 − z
1 − x, y − 1/2, 1 − z
x − 1, y, z
0.82a
x − 1, y, z − 1
0.89(4)
0.82a
2 − x, 1/2 + y, 2 − z
1 − x, y − 1/2, 1 − z
a
Calculated hydrogens.
Both C12A N1 and C12B N2 double bonds display E con-
figurations, while the conformation around the N1–N2 single
bond can be classified trans owing to the C12A N1–N2 C12B
torsion angle value of −137.1(3)◦. The overall structure of
the dimer is stabilized by an intramolecular O25A-H. . .O29B
hydrogen bond.
X-ray crystallographic studies have revealed that cholic
acid frequently forms antiparallel layer structures consisting
of alternate stacking of lipophilic and hydrophilic layers and
that host cavities are formed in the lipophilic layers which
include a large variety of guest molecules [19–23]. Conversely,
in this dimeric cholic acid derivative, the hydrogen bonded
Fig. 6 – The unit cell of compound 11 as viewed down the crystallographic c-axis showing the channels containing water
molecules.

764
steroids 7 2 ( 2 0 0 7 ) 756–764
[8] Hofmann AF. The preparation of chenodeoxycholic acid and
its glycine and taurine conjugates. Acta Chem Scand
1963;17:173–86.
[9] Fieser LF, Rajagopalan S. Selective oxidation with
N-bromosuccinimide. I. Cholic acid. J Am Chem Soc
1949;71:3935–8.
[10] Fieser LF, Rajagopalan S. Oxidation of steroids. III. Selective
oxidations and acylations in the bile acid series. J Am Chem
Soc 1950;72:5530–6.
[11] Brane S, Kirk DN. The bile acids, chemistry, physiology and
metabolism, vol. 4. New York: Plenum; 1988.
[12] Bettarello L, Bortolini O, Fantin G, Guerrini A. Mixed
oxo-hydroxo bile acids as actual or potential impurities in
ursodeoxycholic acid preparation: a 1H and 13C NMR study. Il
Farmaco 2000;55:51–5.
[13] Altomare A, Burla MC, Camalli M, Cascarano MG,
Giacovazzo C, Guagliardi A, et al. SIR97: a new tool for crystal
structure determination and refinement. J Appl Crystallogr
1999;32:115–6.
[14] Sheldrick GM. Program for refinement of crystal structures,
SHELXL-97. Germany: University of Go¨ttingen; 1996.
[15] Nardelli M. PARST95—an update to PARST: a system of
Fortran routines for calculating molecular structure
parameters from the results of crystal structure analyses. J
Appl Crystallogr 1995;28:659.
[16] Spek AL. Single-crystal structure validation with the
program PLATON. J Appl Crystallogr 2003;36:7–13.
[17] Burnett MN, Johnson CK. ORTEP-III, Report ORNL-6895. Oak
Ridge National Laboratory, Oak Ridge, TN; 1996.
[18] Bertolasi V, Ferretti V, Pretto L, Fantin G, Fogagnolo M,
Bortolini O. Hydrogen-bonded aggregations of oxo-cholic
acids. Acta Crystallogr Sect B 2005;61:346–56.
multilayer arrangement of cholic acid host is absent. Instead
of lipophilic layers, the structure exhibits hydrophilic chan-
nels built up by the hydroxyl groups, except O25A-H, of a
cholic acid dimer and the carboxylic groups of other dimers,
forming with water molecules a three-dimensional array of
hydrogen bonds which stabilize the crystal lattice (Table 3;
Fig. 5). In the crystal packing the hydrophilic channels, where
the water molecules O1w and O2w are trapped, run along the
crystallographic c-axis, as shown in Fig. 6. The formation of
hydrophilic channels in crystal lattices of cholic acid (or cholic
acid derivatives) inclusion complexes is uncommon. Only few
examples of these crystals containing water and/or alcohol
molecules are reported, for instance: deoxycholic acid with
ethanol and water [24], cholic acid with water [25], cholic acid
with methanol or ethanol or 1-propanol [26].
The observation that water molecules are trapped within
the channels of crystals of compound 11 opens on the possibil-
ity to test azines in host–guest inclusion processes. Inclusion
of molecules within others is a well known phenomenon
observed both with achiral and chiral compounds, having
important application in the separation of isomers and enan-
tiomers [27]. In this frame we have carried out preliminary
tests using azines as hosts, experiments that give evidence
that derivative 8 is able to accommodate 2-methyl cyclohex-
anone within its channels, with a host to guest ratio of 1:1.
4.
Conclusions
[19] Nakano K, Sada K, Kurozumi Y, Miyata M. Importance of
packing coefficient of host cavities in the isomerization of
open host frameworks: guest-size-dependent isomerization
in cholic acid inclusion crystals with monosubstituted
benzenes. Chem Eur J 2001;7:209–20.
[20] Yoswathananont N, Sada K, Miyata M, Akita S, Nakano K.
Dependence of selective enclathration on types of cholic
acid crystals. Org Biomol Chem 2003;1:210–4.
[21] Nakano K, Mochizuki E, Yasui N, Morioka K, Yamauchi Y,
Kanehisa N, et al. Mechanism of selective and unselective
enclathration by a host compound possessing open, flexible
host frameworks. Eur J Org Chem 2003:2428–36.
[22] Bertolasi V, Bortolini O, Fogagnolo M, Fantin G, Pedrini P.
Enantioselective inclusion in bile acids: resolution of cyclic
ketones. Tetrahedron: Asymm 2001;12:1479–83.
[23] Bertolasi V, Bortolini O, Fantin G, Fogagnolo M, Pretto L.
Inclusion of cyclic carbonates by a cholic acid host: structure
and enantioselection. Tetrahedron: Asymm 2006;17:308–12.
[24] Candeloro De Sanctis S, Coiro VM, Giglio E, Pagliuca S, Pavel
NV, Quagliata C. The hexagonal phase of the 3:2:1 canal
complex between Deoxycholic acid, ethanol and water: an
inclusion compound with hydrophilic channels. Acta
Crystallogr Sect B 1978;34:1928–33.
Novel bile acid moieties containing the structures presented
in this paper have a great deal of potential in pharmacology,
biomimetic and supramolecular chemistry. In addition bile
acids are versatile building blocks for the design of frameworks
capable of ionic and molecular recognition. Preliminary results
on host–guest enclathration confirmed the capability of some
azines to include organic molecules. Work is in progress in this
field.
r e f e r e n c e s
[1] Virtanen E, Kolehmainen E. Use of bile acids in
pharmacological and supramolecular applications. Eur J Org
Chem 2004:3385–99, and references.
[2] Salunke DB, Hazra BG, Pore VS. Steroidal conjugates and
their pharmacological applications. Curr Med Chem
2006;13:813–47.
[3] Salunke DB, Hazra BG, Pore VS, Manoj KB, Pallavi BN,
Mukund VD. New steroidal dimers with antifungal and
antiproliferative activity. J Med Chem 2004;47:1591–4.
[4] Li Y, Dias JR. Dimeric and oligomeric steroids. Chem Rev
1997;97:283–304.
[5] Lotowski Z, Guzmanski D. Ne acyclic dimmers of cholic acid
with oxamide and hydrazide spacers. Monatsh Chem
2006;137:117–24.
[25] Shibakami M, Tamura M, Sekiya A. Topological and
nontopological rearrangement in crystal lattice of cholic
acid inclusion complex induced by guest exchange. J Am
Chem Soc 1995;117:4499–505.
[26] Jones EL, Nassimbeni LR. Crystal and molecular structures
of the inclusion compounds of cholic acid with methanol,
ethanol and 1-propanol. Acta Crystallogr Sect B
1990;46:399–405.
[6] Gao H, Dias JR. Cyclocholates with 12-oxo and 7,12-oxo
groups. Eur J Org Chem 1998:719–24.
[7] Bortolini O, Cova U, Fantin G, Medici A. A new
non-enzymatic route to chenodeoxycholic acid. Chem Lett
1996:335.
[27] Toda F, Bishpo R, editors. Separation and reactions in
organic supramolecular chemistry: perspectives in
supramolecular chemistry, vol. 8. Wiley; 2004. p. 123.