Facile synthesis of 5β-cholane-sym-triazine conjugates starting from metformin and bile acid methyl esters: Liquid and solid state NMR characterization and single crystal structure of lithocholyl triazine

Article abstract of DOI:10.1016/j.molstruc.2009.08.008

Four bile acid-triazine conjugates: N^2′,N^2′-dimethyl-6′-(3α-hydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (lithocholyl triazine, 4a), N^2′,N^2′-dimethyl-6′-(3α,7α-dihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (chenodeoxycholyl triazine, 4b), N^2′,N^2′-dimethyl-6′6′-(3α,12α-dihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (deoxycholyl triazine) (4c), and N^2′,N^2′-dimethyl-6′-(3α,7α,12α-trihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (cholyl triazine) (4d) have been prepared and characterized by liquid and solid state NMR. An improved synthetic method produced better yields and an easier purification procedure for 4d than reported in the literature. Single crystal structure of 4a is reported: empirical formula C₂₈H₄₇N₅O, monoclinic P2₁ space group with unit cell dimensions, a 18.7135(5) A?, b 7.4510(2) A?, c 19.3073(5) A?, β 95.7290(10)°, volume 2678.65(12) A?³.

Full text of DOI:10.1016/j.molstruc.2009.08.008

Journal of Molecular Structure 936 (2009) 270–276
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Facile synthesis of 5b-cholane-sym-triazine conjugates starting from metformin
and bile acid methyl esters: Liquid and solid state NMR characterization and
single crystal structure of lithocholyl triazine
*
Satu Ikonen , Salla Takala, Nonappa, Erkki Kolehmainen
Department of Chemistry, University of Jyväskylä, P.O. Box 35, FIN-40014 Jyväskylä, Finland
a r t i c l e i n f o
a b s t r a c t
0
0
2
2
0
0
0
0
Article history:
Four bile acid-triazine conjugates: N ,N -dimethyl-6 -(3
a
-hydroxy-5b-24-norcholyl)-1 ,3 ,5 -triazine-
0
0
Received 2 July 2009
Accepted 6 August 2009
Available online 12 August 2009
0
0
2
2
0
0
0
0
2
,4 -diamine (lithocholyl triazine, 4a), N ,N -dimethyl-6 -(3
triazine-2 ,4 -diamine (chenodeoxycholyl triazine, 4b), N ,N -dimethyl-6 6 -(3
a
,7
a
-dihydroxy-5b-24-norcholyl)-1 ,3 ,5 -
0
0
0
0
2
2
0 0
a
,12
a
-dihydroxy-5b-24-
0
0
0
0
0
0
0
2
2
0
norcholyl)-1 ,3 ,5 -triazine-2 ,4 -diamine (deoxycholyl triazine) (4c), and N ,N -dimethyl-6 -(3
a
,7a
,
0
0
0
0
0
12a-trihydroxy-5b-24-norcholyl)-1 ,3 ,5 -triazine-2 ,4 -diamine (cholyl triazine) (4d) have been prepared
Keywords:
Bile acid
Triazine
Synthesis
X-ray crystallography
NMR
and characterized by liquid and solid state NMR. An improved synthetic method produced better yields
and an easier purification procedure for 4d than reported in the literature. Single crystal structure of 4a is
reported: empirical formula C28
8.7135(5) Å, b 7.4510(2) Å, c 19.3073(5) Å, b 95.7290(10)°, volume 2678.65(12) Å .
Ó 2009 Elsevier B.V. All rights reserved.
47 5 1
H N O, monoclinic P2 space group with unit cell dimensions, a
3
1
1
. Introduction
The most abundant naturally occurring bile acids in higher ver-
presence of sodium methylate, method previously described for
the preparation of other triazines [5]. First two molecules (4a and
4d, see Scheme 1) and their characterization were published by us
already in 2006 in form of a poster [6]. Marin et al. [7] also published
the synthesis of 4d and two other triazines. In order to conjugate bile
acids with dimethylbiguanide they used 2-ethoxy-1-ethoxycar-
bonyl-1,2-dihydroquinoline (EEDQ) as a coupling agent and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) for neutralization of HCl of
metformin, which resulted in a complex mixture of products. They
also evaluated the cytostatic activity of these compounds in vitro
and antitumor effect in vivo, respectively. They found ursodeoxy-
cholic acid derived triazine to possess moderate non-specific toxic-
ity similar to that of cisplatin and when administered to tumor-
bearing mice, this compound prolonged the survival of these ani-
mals, while treatment with cisplatin failed to do so [7]. Inspired by
these results we finished the synthesis of other two triazines (4b
and 4c, see Scheme 1) with improved yields.
We have also thoroughly studied the structures of the prepared
triazines in solution by NMR spectroscopy and mass spectrometry
and in solid state by CP/MAS NMR spectroscopy; a technique
which nowadays has a significant contribution to the understand-
ing of the structures of polymorphs and solvates of pharmaceutical
significance [8]. The study of the polymorphic nature of pharma-
ceutical solids is particularly important since the different poly-
morphic forms may have different physical or chemical
properties along with the differences in pharmaceutical activity.
Studies on the polymorphism of several bile acid derivatives have
been reported from our laboratory [9]. Recently we also studied
tebrates are derivatives of cholanic acid, cyclopentanoperhydroph-
enanthrene ring-containing steroid consisting of 24 carbon atoms.
Bile acids form as end products of cholesterol metabolism in the li-
ver and their formation include several hydroxylations of sterol
nucleus followed by a loss of an isopropyl group form the side
chain to give primary bile acids which are further modified by
the bacteria in the intestines to give secondary bile acids. Bile acids
are natural substrates of enterohepatic circulation. Their main role
is the digestion of lipids and lipid soluble vitamins [1]. Bile acids
and their salts as well their derivatives are found to have numerous
applications in biology, medicine and physiology as well as in
supramolecular chemistry and nanoscience [2]. Several research
groups have recently reviewed the supramolecular and pharma-
ceutical use of bile acids and their derivatives [3] as well as the uti-
lization of bile acid transportation system for the improved drug
delivery [4].
It is known that N-substituted 2,4-diamino-6alkyl-sym-triazines
are formed via condensation of biguanides with acid derivatives
such as acid chlorides, esters, anhydrides, and imino esters [5]. Here-
in we report thesynthesis and structural characterization of four bile
acid derived triazines (see Scheme 1) utilizing the condensation
reaction of bile acid methyl ester with dimethylbiguanide in the
*
Corresponding author. Tel.: +358 14 260 2654; fax: +358 14 260 2501.
E-mail address: satu.ikonen@jyu.fi (S. Ikonen).
0
022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2009.08.008

S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
271
7'
8'
pulse for proton and carbons were found to be 4.0 and 5
l
s at
power levels of ꢀ5.0 and ꢀ4.0 dB, respectively. The experiments
were conducted at contact time of 2 ms. A total of 10,000 scans
were recorded with 4 s recycle delay for each sample. All FIDs were
processed by exponential apodization function with line broaden-
ing of 20–40 Hz prior to FT. The N{ H}CP/MAS NMR experiments
were carried out for all samples at a 10 kHz spinning rate under
N
2'
1
'
3
'
N
N
2
1
22
6'
4'
15
1
1
R
N
NH2
1
8
20
2
3
5'
Hartmann–Hahn condition. The
were found to be 4.2 and 5
p/2pulses for proton and nitrogen
s at power levels of –4.6 and ꢀ0.8 dB,
l
12
17
13 16
19
respectively. The optimized contact time of 2 ms was used for effi-
cient polarization transfer with a 4 s recycle delay to acquire the
CP/MAS spectra. A total of 70,000 scans were acquired obtain the
11
1
4
15
1
4
9
2
3
10
5
8
7
1
3
15
CP/MAS spectra. The C and N CPMAS chemical shifts were ref-
erenced with those of glycine standard measured before the each
sample of 4a–4d.
6
2
HO
R
H
Molecular masses of the synthesis products were confirmed by
using Micromass LCT ESI-TOF mass spectrometer in positive ion
mode. Samples were prepared in HPLC grade solvents and diluted
1
2
4
4
4
4
a
b
c
d
: R =R =H
to concentrations ꢁ0.1–1.0
lg/L. Under these conditions all the
1
2
: R =H, R =OH
compounds yielded abundant molecular ion peaks by coordinating
1
2
+
+
: R =OH, R =H
hydrogen, sodium, and/or potassium ions ([M+H] , [M+Na] and/or
+
1
2
[M+K] , respectively).
: R =R =OH
2.2. X-ray crystallography
Scheme 1. Prepared molecules and the numbering of the carbon and nitrogen
atoms.
Crystals of 4a suitable for single crystal X-ray crystallography
were grown in ethyl acetate/hexane (1:1) solution. Data was col-
lected at 123(2) K on a Bruker-Nonius KappaCCD diffractometer
the polymorphic nature of naturally occurring bile acids [10].
Molecular structure and crystal packing of 4a was determined uti-
lizing single crystal X-ray crystallography.
a
with graphite monochromated Cu-K radiation (k = 1.54184).
COLLECT [15] data collection software was utilized and data was
processed with DENZO-SMN [16]. The reflections were corrected
for Lorenz polarization effects but absorption correction was not
used. Structure was solved by direct methods (SHELXS-96 [17])
and refined anisotropically by full matrix least squares on F values
2
. Experimental
2.1. Spectroscopy
(
SHELXL-97 [17]). The hydrogen atoms, except N–H and O–H, were
1H and 13C NMR experiments were run with a Bruker Avance
calculated to their idealized positions and those attached to N or O
were located from electron density map. Hydrogen atoms were re-
fined only isotropically with isotropic temperature factors (1.2 or
1.5 times the parent atom factor). PLATON [18]-program was used
in validation of the structure. Figures were drawn using Ortep-3 for
Windows [19] and Mercury [20]. Crystallographic data (excluding
structure factors) have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publication number
CCDC-738340 for 4a. Copies of the data can be obtained free of
charge on application to CCDC, 12 Union Road, Cambridge CB2
1EZ UK.
DRX 500 NMR spectrometer equipped with a 5 mm diameter broad
1
band inverse probehead working at 500.13 MHz for H, at
1
3
15
1
{
25.76 MHz for C, and at 50.68 MHz for N, respectively. The
1
13
H}- C NMR spectrum was measured in standard way using com-
posite pulse decoupling (waltz-16). Spectra were measured in
CDCl
3
and CD
3
OD at 303 K and 1H and C chemical shifts refer-
13
1
enced to the solvent signals (d = 7.26 for H and d = 77.0 ppm for
1
3
) and d = 3.10 for 1H and d = 49.0 ppm
OD)). The nitrogen chemical shifts were
measured by PFG H, N HMBC using 50 ms evolution delay for
C from int. TMS (CDCl
3
1
3
for C from int. TMS (CD
3
1
15
1
5
couplings and N NMR spectra were referenced to an external
nitromethane in a capillary inserted coaxially inside the NMR-tube
d = 0.0 ppm). The assignment of the individual H, C and N sig-
2.3. Synthesis and purification
1
13
15
(
1
13
nals was carried out utilizing the 2D pulse sequences PFG H,
C
All bile acids were purchased from commercial sources and
were used as such in synthetic procedures. All the used solvents
were of an analytical grade and water was deionized before use.
The methyl esters of bile acids were prepared according to known
sulphuric acid catalyzed method [21]. Silica 0.043–0.060 Å was
used in column chromatographic purifications.
HMQC [11] and PFG 1H, C HMBC [12] and PFG 1H, N HMBC
13
15
[
13] and by referring them with those reported in the literature
for similar type of compounds [14].
For CP/MAS NMR spectroscopy the compounds were crystal-
lized from various solvents: 4a from methanol and p-xylene, 4b
from p-xylene and DMF–CH
3
CN, 4c from p-xylene and EtOAc–
OH, respectively. The
C{ H}CP/MAS and N{ H}CP/MAS NMR spectra were recorded
on a Bruker AV 400 spectrometer equipped with a 4 mm standard
bore CP/MAS probehead whose channel was tuned to
0
0
2
2
0
0
0
0
CH CN and CH CN and 4d from CH
3
3
3
2.3.1. N ,N -dimethyl-6 -(3a-hydroxy-5b-24-norcholyl)-1 ,3 ,5 -
1
3
1
15
1
0
0
triazine-2 ,4 -diamine (lithocholyl triazine, 4a)
Sodium (0.14 g; 6.0 mmol) was placed in a round bottom
100 mL flask, fitted with a reflux condenser and a dropping funnel,
X
1
3
15
1
00.62 MHz for C and 40.55 MHz for N, respectively. The other
2
and portion (20 mL) of methanol added at once under N atmo-
1
channel was tuned to 400.13 MHz for broad band H decoupling.
Approximately 100 mg of dried and finely powdered samples were
sphere. When the reaction had ceased, another portion (20 mL)
of methanol was added after which dimethylbiguanide hydrocho-
ride (0.83 g; 5.0 mmol) in methanol was added dropwise. The
resulting reaction mixture was stirred for 30 min at ambient
temperature. A solution of methyl lithocholate (1.95 g; 5.0 mmol)
packed in the ZrO
2
rotor closed with Kel-F cap and spun at 10 kHz
rate. The C{ H}CP/MAS NMR was carried out for all samples un-
der Hartmann–Hahn conditions with TPPM decoupling. The /2
1
3
1
p

2
72
S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
47 5 2
ESI-TOF MS (MeOH/HCOOH 0.1%) m/z: C28H N O
. Found: [M+H]+
486.44 ([4c+H] requires 486.38). Anal. calcd. for C28H N O : C
47 5 2
in methanol was added to the reaction mixture through the drop-
ping funnel. Reaction mixture was then heated to reflux and reflux-
+
ing continued for 48 h under N
and precipitated product was filtered, washed with methanol and
2
. Mixture was then cooled to ꢀ5 °C
69.24; H 9.75; N 14.42. Found: C 69.23; H 9.69; N 14.26.
0
0
dried in vacuo to give 0.71 g of 4a as white powder (yield 29%).
2
2
0
2
1
4
.3.2.3. N ,N -dimethyl-6 -(3a a,12a-trihydroxy-5b-24-norcholyl)-
,7
1
500 MHz) d ppm: 1H NMR (CDCl
H NMR (CDCl
3
3
500 MHz) d
0
0
0
0
0
,3 ,5 -triazine-2 ,4 -diamine (cholyl triazine) (4d). Yield 0.785 g of
d as white powder (yield 39%). H NMR (CD
0
ppm: 5.04 (br. s, 2H, 4NH
2
), 3.66 (m, 1H, 3CH), (br. s, 6H, 7 /
1
3
OD 500 MHz) d
0
8
CH
3
), 2.58–2.34 (2H, ddqAB, dH
A
= 2.55 ppm and dH
, –OH), 0.98 (d,
), 0.66 (s, 3H, 18CH ).
3
126 MHz) d ppm: 179.2 (C6 ), 166.9 (C2 ), 165.8
B
= 2.37 ppm,
ppm: 3.95 (m, 1H, 12CH), 3.78 (m, 1H, 7CH) 3.78 (m, 1H, 3CH),
J
ab = ꢀ14.0 Hz), 1.87–0.98 (steroidal –CH and –CH
2
0
0
3
.11 (br. s, 6H, 7 /8 CH
dH
CH
3 A
), 2.56–2.32 (ddqAB, dH = 2.35 ppm and
3
H, J = 6.4 Hz, 21CH ), 0.93 (s, 3H, 19CH
3
3
3
B
= 2.53 ppm, Jab = ꢀ13.3 Hz), 2.30–0.93 (steroidal –CH and –
1
3
0
0
C NMR (CDCl
2
), 1.06 (d, 3H, J = 6.0 Hz, 21CH
3
) 0.91 (s, 3H, 19CH
3
) 0.70 (s,
0
(
(
C4 ), 72.0 (C3), 56.7 (C14), 56.4 (C17), 42.9 (C13), 42.3 (C5), 40.6
13
0
3
H, 18CH
3
). C NMR (CDCl
C2 ), 165.7 (C4 ), 73.1 (C12), 72.1 (C3), 68.6 (C7), 47.3 (C17), 46.7
3
126 MHz) d ppm: 179.2 (C6 ), 166.8
0
0
C9), 40.4 (C12), 36.7 (C4), 36.2 (C7 /8 ), 36.0 (C20), 35.9 (C23),
0
0
(
3
2
5.8 (C8), 35.5 (C1), 34.7 (C10), 33.8 (C22), 30.8 (C2), 28.4 (C16),
7.4 (C6), 26.6 (C7), 24.4 (C15), 23.5 (C19), 21.0 (C11), 18.7
0
0
(
3
(
C13), 42.0 (C14), 41.7 (C5), 39.9 (C4), 39.8 (C8), 36.3 (C7 /8 ),
5.8 (C23), 35.7 (C20), 34.8 (C6), 34.9 (C10), 35.5 (C1), 33.8
C22), 30.7 (C2), 28.4 (C11), 27.7 (C16), 26.8 (C9), 23.4 (C15),
(
[
(
47 5
C21), 12.2 (C18). ESI-TOF MS (MeCN) m/z: C28H N O. Found:
+
+
+
M+H] 470.35 ([4a+H] requires 470.73) [M+Na] 492.33
2
C
5
5
1
2.7 (C19), 17.8 (C21), 12.8 (C18). ESI-TOF MS (MeCN) m/z:
[4a+Na]+ requires 492.71). Anal. calcd. for C28
H
47
N
5
O
1
ꢂ½CH OH:
+
+
3
28
47 5 3
H N O . Found: [M+H] 502.40 ([4d+H] requires 502.73),
C 70.47; H 10.17; N 14.42. Found: C 70.83; H 10.00; N 14.24.
24.39 (([4d+Na] requires 524.71), 540.37 ([4d+K]+ requires
+
40.82). Anal. calcd. for C28 O: C 65.85; H 9.47; N
H
47
N
5
O
3
ꢂ½H
2
2
2
.3.2. General method for preparation of other triazines
0 0
3.71. Found: C 66.20; H 9.40; N 13.72.
2
2
0
0
0
0
.3.2.1. N ,N -dimethyl-6 -(3a,7a-dihydroxy-5b-24-norcholyl)-1 ,3 ,5 -
0
0
triazine-2 ,4 -diamine (chenodeoxycholyl triazine, 4b). Sodium
0.184 g; 8.0 mmol) was placed in a round bottom 250 mL flask, fitted
with a reflux condenser and a dropping funnel, and portion (15 mL) of
methanoladdedatonce underN atmosphere. When thereaction had
3
. Results and discussion
(
3.1. Synthesis
2
ceased, another portion (15 mL) of methanol was added after which
dimethylbiguanide (1.325 g; 8.0 mmol) in 30 mL of methanol was
added dropwise. Resulting reaction mixture was stirred for 30 min.
Solution of methyl chenodeoxycholate (1.626 g; 4.0 mmol) in 10 mL
of methanol was added to the reaction mixture through the dropping
funnel. Reaction mixture was then heated to reflux and refluxing con-
The synthetic route to bile acid triazines is outlined in the
scheme 2. The preparation of bile acid triazines started by conver-
sion of each bile acid 1a–d to their methyl esters 2a–d utilizing
Fischer esterification reaction which is widely used in preparation
of simple bile acid alkyl esters [21]. The formation of the bile acid
substituted triazine ring was condensation reaction of dimethyl-
biguanide with bile acid ester in the presence of sodium methylate.
Two equivalents of both dimethyl biguanide hydrochloride and
base against the ester were used and the reaction mixtures were
refluxed from 19 to 48 h. While, according to Marin et al. [7] the
EEDQ/DCU-coupling method produced a complex mixture of prod-
ucts thus requiring two successive column chromatographic puri-
fications, here the main impurity in the crude product was bile acid
ester and only one column chromatographic purification was suffi-
cient enough to give pure products 4b–d. In the case of 4a, the
product was precipitated out from the reaction mixture at ꢀ5 °C
and it was purified simply by washing the precipitate with meth-
anol. Yields of 4a–d varied from 29% to 39%. Better yields were thus
obtained using this method compared to the previously reported
method where for example the yield of 4d was 21%.
tinued for 19 h under N
tion and residue dissolved in 120 mL of CHCl
2
. Mixture was then concentrated by evapora-
, solution washed with
and evap-
3
5
ꢃ 40 mL of H
2
O and 2 ꢃ 40 mL of brine, dried over MgSO
4
orated to dryness. Crude product was purified by column chromatog-
raphy using EtOAc:MeOH (9:1) as an eluent. Fractions containing the
product werecombined, evaporatedanddried invacuo togive 0.727 g
of 4b as a white powder (yield 37%).
1
H NMR (CDCl
3
500 MHz) d ppm: 5.10 (br. s, 2H, 4NH
2
), 3.83 (m,
), 2.58–2.34
= 2.36 ppm, Jab = 13.6 Hz),
2
, –OH), 0.98 (d, 3H, J = 6.5 Hz,
0
0
1
(
H, 7CH), 3.446 (m, 1H, 3CH) 3.12 (br. s, 6H, 7 /8 CH
2H, ddqAB, dH = 2.56 ppm and dH
.24–0.94 (steroidal –CH and –CH
1CH ), 0.90 (s, 3H, 19CH ), 0.65 (s, 3H, 18CH
26 MHz) d ppm: 178.9 (C6 ), 166.6 (C2 ), 165.6 (C4 ), 72.0 (C3),
8.5 (C7), 56.1 (C17), 50.5 (C14), 42.7 (C13), 41.6 (C5), 39.9 (C4),
3
A
B
2
2
1
6
3
13
3
3
3
). C NMR (CDCl
3
0
0
0
0
0
9.7 (C12), 39.5 (C8), 39.5 (C22), 36.1 (C7 /8 ), 35.7 (C23), 35.6
(
(
C20), 35.4 (C1), 35.1 (C10), 34.6 (C6), 32.9 (C9), 30.7 (C2), 28.2
C16), 23.7 (C15), 22.8 (C19), 20.6 (C11), 18.5 (C21), 11.8 (C18).
3.2. NMR spectroscopy
ESI-TOF MS (MeOH/HCOOH 0.1%), m/z:
[
(
C
28
H
47
N
5
O
2
.
Found:
M+H] 486.41 ([4b+H] requires 486.38) [M+Na] 508.39
[4b+Na]+ requires 508.36). Anal. calcd. for C28
: C 69.24;
+
+
+
13C and 15N NMR chemical shifts and selected H NMR chemical
1
H
47
N
5
O
2
shifts for 4a–d in CDCl
1–3. Our assignment of the C NMR chemical shifts for 4d in
CD OD is in agreement with that made by Marin et al. [7].
Very different types of nitrogen atoms are present in these com-
3 3
and/or CD OD are presented in the Tables
1
3
H 9.75; N 14.42. Found: C 69.12; H 9.67; N 14.30.
3
0
0
2
2
0
0
0
0
2
.3.2.2. N ,N -dimethyl-6 -(3
a
,12
a-dihydroxy-5b-24-norcholyl)-1 ,3 ,5 -
0
0
0
0
0
triazine-2 ,4 -diamine (deoxycholyl triazine) (4c). Yield 0.653 g of white
powder (34%). H NMR (CDCl
pounds, the endocyclic s-triazine N-atoms (N(1 ), N(3 ) and N(5 )),
the aniline-like N-atom (N(4 )) and tertiary non-aromatic N-atom
(N(2 )). When the samples were measured in CDCl
(C4 ) is more shielded compared to the carbon at-
(C2 ), but when the sample 4d was measured in
OD the order of their chemical shifts was opposite, illustrating
the importance of the solvent effect. The signal for protons of C(7 /
8 ) is very broad in solution at 303 K which probably results from
dynamics of the methyl groups bound to nitrogen. This disables
the observation of the N chemical shift for N(2 ). When the sam-
ples of 4b and 4c in CDCl and 4d in CD OD were heated to 333 or
1
0
3
500 MHz) d ppm: 5.09 (br. s, 2H,
0
0
4
8
NH
CH
2
), 3.99 (br. t, 1H, 12CH), 3.59 (m, 1H, 3CH), 3.11 (br. s, 6H, 7 /
3
, the carbon at-
0
0
3
), 2.60–2.36 (ddqAB, dH
A
= 2.57 ppm and dH
B
= 2.38 ppm,
tached to –NH
tached to N(CH
CD
2
0
J
3
ab = ꢀ13.7 Hz), 1.91–0.93 (steroidal –CH and –CH
2
, ꢀOH), 1.02 (d,
3
)
2
1
3
H, J = 6.0 Hz, 21CH
3
), 0.90 (s, 3H, 19CH
3
), 0.67(s, 3H, 18CH
3
).
C
3
0
0
0
0
NMR (CDCl
3
126 MHz) d ppm: 178.9 (C6 ), 166.6 (C2 ), 165.6 (C4 ),
0
7
3
3
2
6.7 (C12), 71.8 (C3), 48.3 (C14), 47.5 (C17), 46.6 (C13), 42.1 (C5),
0
0
6.5 (C4), 36.1 (C8), 36.1 (C7 /8 ), 35.8 (C23), 35.4 (C20), 35.3(C1),
4.1 (C10), 33.7 (C9), 33.6 (C22), 30.6 (C2), 28.6 (C11), 27.5 (C16),
7.2 (C6), 26.2 (C7), 23.7 (C15), 23.1 (C19), 17.6 (C21), 12.8 (C18).
1
5
0
3
3

S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
273
O
O
1
R
OH
1
R
OMe
MeOH, H SO , reflux, 48 h
2
4
2
HO
R
H
2
HO
R
H
1
2
1
1
1
1
a
b
c
d
: R =R =H
1
2
: R =H, R =OH
-
2a d
1
2
: R =OH, R =H
1
2
: R =R =OH
NH NH
i) Na, MeOH, rt, 0.5 h
ii) 2a-d, MeOH, reflux, 19-48 h
-
4a d
N
NH NH2 *HCl
Scheme 2. Preparation of the bile acid triazines.
Table 1
can be detected directly without the magnetization transfer from
1
3C NMR chemical shifts of 4a–c in CDCl
4a 4b 4c
35.5 35.4 35.3
3
and 4d in CDCl
3
and CD
3
OD at 303 K.
0
0
the proton, and thus the chemical shifts also for N(3 ) and N(2 )
1
5
C
4d (CDCl
3
)
4d (CD
3
OD)
are observed in solid state for 4a. Table 3 lists the N NMR chem-
3 3
ical shifts for 4a–c in in CDCl , for 4d in CD OD and for 4a also in
solid state.
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
C1)
C2)
C3)
C4)
C5)
C6)
C7)
C8)
35.5
30.7
72.1
39.9
41.7
34.8
68.6
39.8
26.8
34.9
28.4
73.1
46.7
42.0
23.4
27.7
47.3
12.8
22.7
35.7
17.8
33.8
35.8
179.2
166.8
165.7
36.3
36.6
31.4
73.1
40.7
43.4
30.8
72.0
36.7
42.3
27.4
26.6
35.8
40.6
34.7
21.0
40.4
42.9
56.7
24.4
28.4
56.4
12.2
23.5
36.0
18.7
33.8
35.9
179.2
166.9
165.8
36.2
30.7
72.0
39.9
41.6
34.6
68.5
39.5
32.9
35.1
20.6
39.7
42.7
50.5
23.7
28.2
56.1
11.8
22.8
35.6
18.5
39.5
35.7
178.9
166.6
165.6
36.1
30.6
71.8
36.5
42.1
27.2
26.2
36.1
33.7
34.1
28.6
76.7
46.6
48.3
23.7
27.5
47.5
12.8
23.1
35.4
17.6
33.6
35.8
178.9
166.6
165.6
36.1
For CP/MAS experiments, the compounds were crystallized
from various solvents and solvent-mixtures including methanol,
p-xylene, acetonitrile, ethyl acetate, and dimethylformamide
*
35.3
1
3
(
DMF)-acetonitrile. C CP/MAS spectra of 4a crystallized both
69.2
41.2
28.1
*
from methanol and from p-xylene were of good quality with sharp
peaks. The steroidal methylene and methine regions are generally
crowded and not well resolved compared to the other regions. Be-
cause of the varying number of hydroxyl groups and the triazine
C9)
*
C10)
C11)
C12)
C13)
C14)
C15)
C16)
C17)
C18)
C19)
C20)
C21)
C22)
C23)
35.3
29.8
74.2
47.7
43.2
24.4
28.9
48.5
13.2
23.3
37.2
18.0
35.3
36.7
180.0
166.8
168.0
36.6
1
3
ring, we divide the C CP/MAS spectra resonances to heteroaro-
matic region (160–180 ppm), hydroxyl region (60–80 ppm), and
aliphatic region containing tertiary, methylene and methine car-
bons (20–50 ppm) and methyl carbons (10–20 ppm). Table 4 col-
1
3
lects some selected C NMR chemical shifts of 4a–4d in solid state.
1
3
Fig. 1 presents selected spectra from the C CP/MAS experi-
ments. Crystallization of 4a from both methanol and p-xylene re-
13
sulted in highly crystalline material from which the C CP/MAS
15
spectra with sharp peaks could be obtained. Also the N CP/MAS
1
5
0
spectrum was of high quality enabling the assignment of the
N
C6 )
0
13
C2 )
chemical shifts in solid state. However, C CP/MAS spectra of 4a
from different solvents were similar.
0
C4 )
0
0
*
C7 /8 )
Crystallization of 4b from p-xylene afforded as well crystalline
material which seems to be a mixture of at least two polymorphic
*
Peaks overlapped.
forms. In the contrary, crystals obtained from DFM-CH
3
CN show
1
3
0
0
3
37 K, respectively, the signal for C(7 /8 )–H sharpened signifi-
only one form which C CP/MAS spectrum show sharp signals.
1
3
0
cantly and the coupling from the methyl protons to N(2 ) could
be detected. Only in the case of 4a in CDCl at 333 K still no cou-
3
pling was detected. However, in the CP/MAS NMR N nucleus
The C CP/MAS spectrum displayed doublet resonance for most
of the carbons which probably arises from the existence of two
non-equivalent molecules in the asymmetric unit in its crystal lat-
15
Table 2
Selected 1H chemical shifts of 4a–c in CDCl
3
3
and 4d in CD OD at 303 K.
H
4a
4b
4c
4d
C(3)–H
C(7)–H
3.62 (m, 1H)
–
–
0.66 (s, 3H)
0.93 (s, 3H)
0.98 (d, 3H, J = 6.4 Hz)
3.45 (m, 1H)
3.83 (t, 1H)
–
0.65 (s, 3H)
0.90 (s, 3H)
0.98 (d, 3H, J = 6.5 Hz)
3.59 (m, 1H)
–
3.99 (br. t, 1H)
0.67 (s, 3H)
0.90 (s, 3H)
1.02 (d, 3H, J = 6.0 Hz)
3.36 (m, 1H)
3.78 (m, 1H)
3.95 (m, 1H)
0.70 (s, 3H)
0.91 (s, 3H)
1.06 (d, 3H, J = 6.0 Hz)
C(12)–H
C(18)–H
C(19)–H
C(21)–H
C(23)–H
2.34–2.58 (ddqAB, dH
and dH
A
= 2.55 ppm
2.34–2.58 (ddqAB, dH
and dH
A
= 2.56 ppm
2.36–2.60 (ddqAB, dH
and dH
A
= 2.57 ppm
2.32–2.56 (ddqAB, dH
A
= 2.35 ppm
B
= 2.37 ppm, Jab = ꢀ14.0 Hz)
B
= 2.36 ppm, Jab = ꢀ13.6 Hz)
B
= 2.38 ppm, Jab = ꢀ13.7 Hz)
and dH
3.11 (br. s, 2H)
Not obs.
B
= 2.53 ppm, Jab = ꢀ13.3 Hz)
0
0
C(7 /8 )–H
3.12 (br. s, 2H)
5.04 (br. s, 2H)
3.12 (br. s, 2H)
5.10 (br. s, 2H)
3.11 (br. s, 2H)
5.09 (br. s, 2H)
0
N(4 )–H

2
74
S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
Table 3
1
5N chemical shifts (from external CH
3
NO
2
) of 4a–c in CDCl
3
at 303 K/333 K, 4d in CD
3
OD at 337 K and 4a in solid state at 296 K.
N
4a
4a
4b
4c
4d
0
a
a
b
a
b
c
c
N(1 )
ꢀ168.8
ꢀ173.7
ꢀ309.5
ꢀ206.8
ꢀ299.0
ꢀ187.2
ꢀ169.3 /ꢀ168.1
ꢀ169.7 /ꢀ168.3
ꢀ171.8
ꢀ305.4
0
a
a
*
a
b
b
a
b
N(2 )
Not obs.
Not obs.
Not obs. /ꢀ305.7
Not obs. /ꢀ305.7
0
a
a
Not obs.c
N(3 )
ꢀ198.4 /Not obs.
ꢀ198.7
0
a
*
a
b
b
a
b
b
c
N(4 )
ꢀ304.0
ꢀ303.8 /ꢀ303.6
ꢀ303.6 /ꢀ304.0
Not obs.
0
a
a
a
c
N(5 )
ꢀ178.9
ꢀ177.0 /ꢀ174.8
ꢀ177.3 /ꢀ175.1
ꢀ181.3
a
b
c
3
3
3
03 K.
33 K.
37 K.
*
Shifts may be interchanged.
Table 4
Selected 13C NMR chemical shifts of 4a–d from CP/MAS NMR experiments at 303 K.
C
4a (MeOH)
4a (p-xylene)
3
4b (DMF–CH CN)
4c (CH
3
CN)
4d (MeOH)
(
(
(
(
(
(
(
(
(
C3)
C7)
70.1
–
–
70.5
–
–
73.1/70.1
66.7/65.0
–
12.4/11.8
22.8
71.4
–
–b
b
–
–
b
C12)
C18)
C19)
C21)
73.2
12.9
20.5
15.8
179.1
15.0
23.9
18.9
176.4
165.2
165.2
15.5
24.4
19.3
177.0
165.6
165.6
11.6
23.5
17.4
177.1
165.9c
163.8c
18.1/17.8
178.5
0
C6 )
0
a
a
a
a
c
c
c
c
C2 )
167.3
164.5
167.1
164.1
0
C4 )
a
b
c
Peaks overlap.
Could not be assigned.
Could be interchanged.
tice. We have found similar behaviour with the naturally occurring
bile acids: the crystals containing two molecules per asymmetric
unit always have the doublet resonance patterns in their 13C CP/
MAS spectra. This has been explained to be due to the differences
in the side chain as well as in the hydrogen bonding modes of these
non-equivalent molecules [10]. In the case of crystals of 4b from
f
3
DMF–CH CN largest differences in the chemical shift values be-
e
tween the two peaks in the doublet resonances are found in the hy-
droxyl region which supports the existence of two non-equivalent
molecules with different hydrogen bonding systems. However,
more experiments are needed in order to prove this hypothesis.
Unfortunately the quality of the crystals obtained from 4b was
not suitable for structural analysis by single crystal X-ray
diffraction.
d
c
3
Crystallization of 4c from EtOAc–CH CN afforded again highly
crystalline material, but despite of the careful drying of the sub-
stance there are extra peaks in CP/MAS spectrum resulting from
the residual EtOAc present in the sample. When the same sample
3
was crystallized from pure CH CN it resulted in crystalline mate-
rial, though the quality of the spectrum was not as high but most
of the peaks were still well resolved. This spectrum differs
slightly from the previous one especially in the hydroxyl region.
This could be explained by different hydrogen bonding mode in
these crystals.
b
In the case of 4d crystallization of the sample turned out to be
demanding. Finally, crystalline material was obtained by crystalli-
zation of 4d from methanol, however, its crystallinity was low
compared to the other samples and as a result the resolution of
1
3
a
the peaks in the C CP/MAS spectrum was poor. Most of the char-
acteristic peaks could still be assigned from this spectrum. Instead
of the expected three peaks, there are four peaks at the hydroxyl
region. This cannot be explained by residual solvent, since the res-
onance for methanol should be more shielded. Thus the probable
explanation is that also in the case of 4d there exists more than
one polymorphic form in this solid.
2
00
150
100
50
0
ppm (f1)
These preliminary studies show that 4a–4c form highly crystal-
line material upon recrystallization from various solvents and at
least in the case of 4b and 4c there can be found more than one
Fig. 1. 13C CP/MAS spectra of (a) 4a from p-xylene, (b) 4b from p-xylene, (c) 4b from
DMF-acetonitrile, (d) 4c from ethyl acetate-acetonitrile, (e) 4c from acetonitrile, and
(
f) 4d from methanol measured at 100 MHz at 303 K.

S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
275
Table 5
crystallographically independent molecules in an asymmetric unit.
In the Fig. 2 the molecular structure of molecule A is shown.
Comparison of themoleculeAand moleculeB showsthatthey are
similar on their steroidal skeleton and the differences are found in
the side chain. Conformation of the bile acid side chain can be de-
scribed using the value of the C17–C20–C22–C23 dihedral angle or
more comprehensively by examining all of the four dihedral angles
in the side chain using letters t (trans), g (gauche) or i (intermediate)
Crystallographic data and parameters for 4a.
4
a
Empirical formula
Molecular weight
Crystal system
Space group
a (Å)
C
28 47 5
H N O
469.71
Monoclinic
P2
18.7135(5)
7.4510(2)
19.3073(5)
90
95.7290(10)
90
2678.65(12)
4
1
b (Å)
c (Å)
[
22]. Here we use only three dihedral angles because of the shorten-
a
(°)
b (°)
(°)
ing of the side chain with one carbon, when the ring is formed. In the
case of 4a both molecules A and B have the side chain conformation
ttt, but the dihedral angle C17A–C20A–C22A–C23A is 5.7° larger
thanthedihedralangleC17B–C20B–C22B–C23B. Alsootherdihedral
angles in the side chain differ to some extent. This causes a slightly
different twist of the side chain and triazine part in two independent
molecules. Although the differences are quite small and the struc-
c
3
Volume (Å )
Z
Densitycalc (g cm 3
ꢀ
)
1.165
123(2)
0.555
1032
T (K)
Abs. coefficient (mmꢀ1)
F(0 0 0)
1 1 1
ture resembles orthorhombic P2 2 2 space group, the structure
h range (°)
Index ranges
3.14–67.08
ꢀ22 ꢄ h ꢄ 20
could not be solved in this higher symmetry. Addsym in PLATON
[18], which uses the Le Page algorithm for missing symmetry, was
used to check the structure for missing symmetry, but no higher
crystallographic symmetry was found. Instead the program sug-
gested the existence of potential pseudosymmetry, i.e. slightly dis-
torted higher symmetry, in these crystals. Crystal packing and the
hydrogen bonding network of 4a are illustrated in the Fig. 3. Hydro-
gen bonding geometries are listed in the Table 6. Both molecules A
and B form similar hydrogen bonds. Three different types of classical
hydrogen bonds are found in these crystals.
ꢀ
8 ꢄ k ꢄ 8
ꢀ
22 ꢄ l ꢄ 22
Reflections collected
Internal consistency
10158
0.0861
4913/7/632
0.987
Data/restraints/parameters
Goodness-of-fit on P2
Final R indices [I > 2
R indices (all data)
r(I)]
R
1
= 0.0676
wR = 0.1748
= 0.0744
wR = 0.1856
2
R
1
2
Extinction coefficient
Largest diffraction peak and hole (e Åꢀ3)
0.0050(9)
0.290 and ꢀ0.354
4
. Conclusions
form i.e. polymorph or solvate. In order to explore the potential
polymorphic nature of these solids, it is, however, necessary to per-
form a more detailed study. Thus extensive crystallization from
several solvents followed by the study of the solids by CP/MAS
NMR spectroscopy, powder X-ray diffraction and when possible
also structure determination by single crystal X-ray crystallogra-
phy are needed.
Condensation reaction between bile acid esters and dimethyl-
biguanide in the presence of sodium methoxide is shown to be a
facile method in preparation of cholane–triazine conjugates. The
3.3. X-ray crystallography
Table 5 summarizes the crystal data and parameters for 4a. Se-
lected geometrical parameters are presented in the Table 6. Com-
Fig. 2. Ortep-3 plot of the molecular structure of 4a (molecule A) showing thermal
ellipsoids with 50% probability.
pound 4a crystallizes in monoclinic space group P2
1
with two
Table 6
Selected geometrical parameters of 4a.
X–Y
D(X–Y) (Å)
1.317(5)/
W–X–Y–Z
(WXYZ)/°
D–Hꢂ ꢂ ꢂA
N(4A)–HNA2ꢂ ꢂ ꢂN(3A)#1
d(Dꢂ ꢂ ꢂA) (Å)
2.977(5)
(DHA) (°)
0
0
C(6 A)–N(1 A),
C13A–C17A–C20A–C22A/
170.6(3)/
136(5)
0
0
0
C(6 B)–N(1 B)
1.313(5)
1.367(5)/
C13B–C17B–C20B–C22B
C17A–C20A–C22A–C23A/
172.6(3)
ꢀ163.8(3)/
0
N(1 A)–C(2 A),
N(4A)–HNA1ꢂ ꢂ ꢂO(1A)#2
O(1A)–H(1OA)ꢂ ꢂ ꢂN(5A)#3
N(4B)–HNB1ꢂ ꢂ ꢂO(1B)#4
N(4B)–HNB2ꢂ ꢂ ꢂN(3B)#5
2.884(4)
2.794(5)
2.875(4)
2.993(5)
164(6)
169(5)
162(5)
133(4)
0
0
0
N(1 B)–C(2 B)
1.366(5)
1.340(5)/
C17B–C20B–C22B–C23B
C20A–C22A–C23A–C6 A/
ꢀ158.1(3)
0
0
C(2 A)–N(3 A),
173.6(3)/
0
0
0
0
0
C(2 A)–N(3 A)
1.339(5)
1.338(5)/
C20B–C22B–C23B–C6 B
C22A–C23A–C6 A–N1 A/
175.2(3)
ꢀ92.8(4)/
0
0
N(3 A)–C(4 A),
0
0
0
0
0
0
N(3 B)–C(4 B)
1.342(5)
1.339(5)/
C22B–C23B–C6 B–N1 B
ꢀ94.0(4)
N(5 A)–C(6 A),
86.6(4)/
0
0
C22A–C23A–C6 A–N5 A
0
0
0
0
N(5 B)–C(6 B)
1.351(5)
1.340(5)/
86.2(4)
ꢀ178.4(4)/
0
0
0
0
C(2 A)–N(2 A),
N1 A–C2 A–N2 A–C7 A/
O(1B)–H(1OB)ꢂ ꢂ ꢂN(5B)#6
2.803(5)
163(4)
0
0
0
0
0
0
0
0
0
0
0
0
C(2 B)–N(2 B)
1.344(5)
1.336(5)/
N1 B–C2 B–N2 B–C7 B
ꢀ177.9(4)
C(4 A)–N(4 A),
N1 A–C2 A–N2 A–C8 A/
11.1(6)/
0
0
0
0
0
0
N(4 B)–N(4 B)
1.336(5)
N1 B–C2 B–N2 B–C8 B
12.0(6)
Symmetry transformations used to generate equivalent atoms: #1 ꢀx, y + 1/2, ꢀz + 2; #2 x ꢀ 1, y, z; #3 ꢀx + 1, y ꢀ 1/2, ꢀz + 2; #4 x + 1, y, z; #5 ꢀx + 2, y + 1/2, ꢀz + 1; #6
x + 1, y ꢀ 1/2, ꢀz + 1.
ꢀ

2
76
S. Ikonen et al. / Journal of Molecular Structure 936 (2009) 270–276
Fig. 3. Crystal packing view along c-axis illustrating the hydrogen bonding network in crystals of 4a. Non-bonding hydrogens are omitted for clarity.
method reported in this study may be useful also in preparation of
some other nitrogen containing bile acid derivatives with potential
pharmaceutical use. Owing to the pharmaceutical potential of the
prepared cholane–triazine conjugates, also their polymorph and
solvate forming properties have been preliminary screened by
measuring their C and N CP/MAS NMR spectra, which are com-
pared with liquid state NMR chemical shifts. These studies arise
questions about the polymorphic or solvate forming nature of
these compounds and we hope we will be able to present more de-
tailed study of the polymorphic nature of these and similar com-
pounds which are currently being prepared. The information
gained from the solution NMR and solid state NMR here may serve
as reference material when dealing with some similar type of
compounds.
[3] (a) J. Tamminen, E. Kolehmainen, Molecules 6 (2001) 21;
(
(
(
b) E. Virtanen, E. Kolehmainen, Eur. J. Org. Chem. 16 (2004) 3385;
c) M. Calabresi, P. Andreozzi, C. La Mesa, Molecules 12 (2007) 1731;
d) A.P. Davis, Molecules 12 (2007) 2106;
(e) S. Bhat, U. Maitra, Molecules 12 (2007) 2181;
f) G.J. Jenkins, L.J. Hardie (Eds.), Bile Acids: Toxicology and Bioactivity, Royal
(
Society of Chemistry, Cambridge, 2008.
1
3
15
[
4] (a) W. Kramer, G. Wess, Eur. J. Clin. Invest. 26 (1996) 715;
(b) E. Sievänen, Molecules 12 (2007) 1859.
[
[
5] V.I. Kelarev, M.A. Silin, O.A. Borisova, Chem. Heterocycl. Compd. 39 (2003) 632.
6] S. Takala, S. Ikonen, E. Kolehmainen, Abstracts of XXVIII Finnish NMR
symposium, Kuopio, Finland, 2006, p. 13.
[7] M. Vallejo, M.A. Castro, M. Medarde, R.I.R. Macias, M.R. Romero, M.Y. El-Mir,
M.J. Monte, O. Briz, M.A. Serrano, J.J.G. Marin, Biochem. Pharmacol. 73 (2007)
1394.
[
8] (a) R.K. Harris, Analyst 131 (2006) 351;
(b) R.K. Harris, J. Pharm. Pharmacol. 59 (2007) 225;
(
(
c) M. Geppi, G. Mollica, S. Borsacchi, C.A. Veracini, Appl. Spectrosc. Rev. 43
2008) 202.
[
9] (a) A. Valkonen, E. Kolehmainen, M. Lahtinen, E. Sievänen, V. Noponen, M.
Tolonen, R. Kauppinen, Molecules 12 (2007) 2161;
Acknowledgements
(
b) A. Valkonen, M. Lahtinen, E. Kolehmainen, Steroids 73 (2008) 1228.
[
10] Nonappa, M. Lahtinen, S. Ikonen, E. Kolehmainen, R. Kauppinen, Cryst. Growth
Des. (2009), doi:10.1021/cg9005828.
11] A. Bax, M.F. Summers, J. Am. Chem. Soc. 108 (1986) 2093.
12] A. Bax, S. Subramanian, J. Magn. Reson. 67 (1986) 565.
13] A. Bax, R.H. Griffey, B.L. Hawkins, J. Magn. Reson. 55 (1983) 301.
The authors wish to thank Spec. Lab. Tech. Reijo Kauppinen for
his help in running the NMR spectra, and Lab. Tech. Elina
Hautakangas for elemental analysis. Financial support from the
Finnish Ministry of Education, National Graduate School of Organic
Chemistry and Chemical Biology (S. Ikonen) and from the Academy
of Finland (project no. 121805, Nonappa) is gratefully
acknowledged.
[
[
[
[14] (a) M. Amm, N. Platzer, J. Guilhem, J.P. Bouchet, J.P. Volland, Magn. Reson.
Chem. 36 (1998) 587;
(
b) J. Ray Dias, H. Gao, E. Kolehmainen, Spectrochim. Acta A. 56 (2000) 53.
[
15] COLLECT, Bruker AXS Inc., Madison, WI, 2004.
[16] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in
oscillation mode, in: C.W. Carter Jr., R.M. Sweet (Eds.), Methods in
Enzymology. Macromolecular Crystallography, Part A, vol. 276, Academic
Press, New York, 1997, pp. 307–326.
[
[
[
[
References
17] G.M. Sheldrick, Acta Crystallogr. A Found. Crystallogr. 64 (2008) 112.
18] A. Spek, J. Appl. Crystallogr. 36 (2003) 7.
19] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565.
20] C.F. Macrae, P.R. Edgington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor, M.
Towler, J. van de Streek, J. Appl. Crystallogr. 39 (2006) 453.
21] L.F. Fieser, S. Rajagopalan, J. Am. Chem. Soc. 72 (1950) 5530.
22] (a) E. Giglio, C. Quagliata, Acta Crystallogr. B Struct. Sci. 31 (1975) 743;
[
1] H. Danielsson, in: P.P. Nair, D. Kritchevsky (Eds.), The Bile Acids: Chemistry,
Physiology and Metabolism, vol. 2, Plenum Press, 3336 New York, 1973, pp. 1–
3
2.
[
2] (a) A. Enhsen, W. Kramer, G. Wess, Drug Discov. Today 3 (1998) 409;
[
[
(
(
(
b) S. Mukhopadhyay, U. Maitra, Curr. Sci. 87 (2004) 1666;
c) A. Hoffman, R.L. Hagey, Cell. Mol. Life Sci. 65 (2008) 2461;
d) Nonappa, U. Maitra, Org. Biomol. Chem. 6 (2008) 658.
(
b) K. Nakano, K. Sada, Y. Kurozumi, M. Miyata, Chemistry 7 (2001) 209.