Tripodal bile acid architectures based on a triarylphosphine oxide core obtained by copper-catalysed [1,3]-dipolar cycloaddition: Synthesis and preliminary aggregation studies

Article abstract of DOI:10.1002/ejoc.201301443

We report the synthesis and aggregation behaviour of new water-soluble, bile acid derived tripodal architectures based on a core derived from triphenylphosphine oxide. We employed the well-established copper-catalysed [1,3]-dipolar cycloaddition (CuAAC) for the construction of these tripodal molecules. The aggregation behaviour of these molecules in aqueous media was studied by different analytical methods such as dye solubilisation, dynamic light scattering, NMR and AFM. These molecular architectures also offer an additional advantage in aiding understanding of the influence of the nature of the bile acid backbone and of the configuration at the steroid C-3 position in these architectures; to the best of our knowledge this has not been reported in the literature. The unique gelation properties of the α-derivatives were explained through molecular modelling studies and the mechanical behaviour of these gels was studied by rheology experiments. A series of new tripodal architectures were synthesised through coupling of bile acids (cholic and deoxycholic) to a triarylphosphine oxide core through click reactions, and their aggregation was studied by different analytical techniques. The influence of configuration on the aggregation processes of these tripodal architectures was highlighted. Copyright

Full text of DOI:10.1002/ejoc.201301443

FULL PAPER
DOI: 10.1002/ejoc.201301443
Tripodal Bile Acid Architectures Based on a Triarylphosphine Oxide Core
Obtained by Copper-Catalysed [1,3]-Dipolar Cycloaddition: Synthesis and
Preliminary Aggregation Studies
Bala N. S. Thota,[a] Aramballi J. Savyasachi,[a] Nikolay Lukashev,[b] Irina Beletskaya,*[b] and
Uday Maitra*[a]
Keywords: Tripodal molecules / Aggregation / Amphiphiles / Gels / Click chemistry / Bile acids
We report the synthesis and aggregation behaviour of new
water-soluble, bile acid derived tripodal architectures based
on a core derived from triphenylphosphine oxide. We em-
ployed the well-established copper-catalysed [1,3]-dipolar
cycloaddition (CuAAC) for the construction of these tripodal
molecules. The aggregation behaviour of these molecules in
aqueous media was studied by different analytical methods
such as dye solubilisation, dynamic light scattering, NMR
and AFM. These molecular architectures also offer an ad-
ditional advantage in aiding understanding of the influence
of the nature of the bile acid backbone and of the configura-
tion at the steroid C-3 position in these architectures; to the
best of our knowledge this has not been reported in the lit-
erature. The unique gelation properties of the α-derivatives
were explained through molecular modelling studies and the
mechanical behaviour of these gels was studied by rheology
experiments.
have a pseudo-phase binding region but no binding site.
The amphiphilicity of these bio-surfactants can be modu-
lated by attaching hydrophilic/polar groups to the steroidal
backbone[4] or by designing bile acid derived oligomers[5]
capable of exhibiting better self-assembly through possible
cooperative effects in the molecular architectures. Several
groups have investigated the aggregation behaviour of bile
acid derivatives containing hydrophilic residues attached to
the side chain or to the concave polar face of the steroidal
nucleus. In particular, hydrophilic polyethylene oxide chains
have been used for the construction of three-armed conju-
gates of bile acids.[4a,4b] Zhao et al. described a series of tris
(cationic, anionic, nonionic) derivatives of cholic acid.[4e]
Although there are many reports on the synthesis of bile-
acid-derived oligomeric systems in the literature, most of
them have been exploited for supramolecular purposes such
as adaptive systems,[6] light harvesting,[7] foldamers,[8] mo-
lecular umbrellas[9] etc., whereas their amphiphilic and sur-
factant behaviour has not been adequately studied. Zhu
et al. synthesised a cholic-acid-derived dimer (“Gemini” an-
alogue) with an aliphatic diacid spacer attached to the bile
acid through an ester linkage at the C-3 position of the
steroid.[5b] Tato and co-workers recently reported a dimeric
system of deoxycholic acid units joined through an EDTA
linker as a gemini surfactant analogue of deoxycholic and
cholic acids.[5c]
Introduction
Bile acid derived structures have become increasingly im-
portant in different fields of chemistry over recent years,
having found applications in pharmacology, supramolec-
ular chemistry and nanoscience.[1] They exhibit facial am-
phiphilic properties due to their possession of a convex
hydrophobic side and a concave hydrophilic “cavity” bear-
ing one to three hydroxy groups. The bile salts, with nega-
tively charged head groups, are important physiological sur-
factants that play a significant role in the body, serving as
natural solubilisers in the digestion of fats and metabolism
of cholesterol.[2] At lower concentrations bile salts form
small primary aggregates, in which the convex hydrophobic
surfaces of the monomers tend to be arranged in such a
way as to form a hydrophobic binding site in the interior.
An increase in the concentration of bile salts leads to ag-
glomeration of primary aggregates into larger “secondary”
aggregates, which have been shown to involve relatively hy-
drophilic binding sites.[3] This feature sets these systems
apart from common host–guest systems, in which only one
type of binding cavity is available, and from micelles, which
[a] Department of Organic Chemistry, Indian Institute of Science,
Bangalore 560012, India
http://orgchem.iisc.ernet.in/faculty/um/
[b] Department of Chemistry, Moscow State Lomonosov
University,
After the introduction of the “click reaction” by the re-
search groups of Sharpless[10] and Meldal[11] a decade ago,
copper-catalysed [1,3]-dipolar cycloaddition (CuAAC) has
become one of the most widely used methodologies for the
Vorobievy Gory d. 1 str. 3, Moscow 119992, Russia
E-mail: beletska@org.chem.msu.ru
http://www.chem.msu.su/eng/people/beletsk.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301443.
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Tripodal Bile Acid Architectures
construction of complex organic molecules and supra- showed such behaviour under similar conditions. Although
molecular architectures. The applications of the click reac- the aggregation properties of bile acid oligomers have been
tion have been reviewed in detail in the literature.[12] The reported previously, such a crucial influence of configura-
1,2,3-triazole moiety is not just a linker, but also a good tion at C-3 on their aggregation properties has not, to the
pharmacophore, and so it has been successfully used for the best of our knowledge, been demonstrated previously.
synthesis of biologically active molecules derived from bile
acids,[13a,13b] and we have recently used it for syntheses of
peptide/bile acid conjugates.[13c] Click reactions have also
been employed for syntheses of new tripodal amphiphilic
ligands (so-called “molecular pockets”)[14] and different
pincer and macrocyclic compounds (cholaphanes).[15,16]
Here we disclose the synthesis of a series of trimeric bile
Scheme 1. Design of molecular architectures.
acid conjugates assembled on a convenient triarylphosphine
oxide scaffold through 1,2,3-triazole linker moieties ob-
tained by click reactions between alkynes and azides. The
synthesised derivatives (Scheme 1) have three important
structural features: a) they are conjugated to taurine to en-
hance their aqueous solubility, as commonly found in physi-
Results and Discussion
The synthesis of the target molecules 17–20 is outlined
ological systems, b) two different bile acids, cholic acid and in Schemes 2 and 3. It starts from the methyl esters of de-
deoxycholic acid, with variable number of hydroxy groups oxycholic and cholic acids, which in turn were obtained
on the concave face (three and two, respectively) were cho- through acid-catalysed esterification of the corresponding
sen, thus offering a simple way to tune the hydrophilicity acids in dry MeOH. The 3α-mesyloxy derivatives 5 and 6
and, in turn, the aggregation properties of the derivatives, were synthesised by treatment of the methyl esters with
and c) the triazole systems are each connected to the 3-posi- mesyl chloride in the presence of triethylamine in dichloro-
tion of the steroid ring in either α- or β-fashion, which of- methane. Nucleophilic substitution of compounds 5 and 6
fers an advantage for understanding of the influence of con- with NaN3 in DMF then resulted in the 3β-azidoderivatives
figuration on aggregation in such architectures. In fact, the 7 and 8, respectively, in good yields. On the other hand, 3α-
aggregation studies suggested a strong influence of the con- azido derivatives 3 and 4 were obtained from the corre-
figuration at the C-3 position in the steroid, which was sponding esters of cholic/deoxycholic acid by means of the
manifested through the thermoreversible formation of hy- Mitsunobu reaction, followed by substitution of the mesyl-
drogels by the 3α derivatives in phosphate buffer at physio- ates with NaN3 by a previously described procedure[17] with
logical pH. On the other hand, none of the 3β derivatives some minor experimental changes as shown in Scheme 2.
Scheme 2. Synthesis of 3-azido derivatives of bile acid methyl esters.
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Scheme 3. Synthesis of trimer-taurine conjugates.
All the azido bile ester derivatives 3, 4, 7 and 8 were ine) in DMF at 90 °C, followed by treatment with Dowex-
hydrolysed to the corresponding azido acid derivatives 9, Na in methanol. All the new compounds were characterised
10, 11 and 12, respectively (Scheme 3) by heating in 5% by a combination of spectroscopic methods (see the Exp.
KOH (aq)/dioxane mixtures. These derivatives were coupled Sect.).
to the central phosphine oxide core (compound 21) under
click conditions with CuSO4 and sodium ascorbate in DMF
at 60 °C to afford the trimeric molecular architectures 13–
16 in good yields. The sodium salts of the trimer-taurine
Aggregation Studies
conjugates 17–20 were synthesised from the corresponding
All the taurine conjugates obtained were soluble in water
acid derivatives by coupling with taurine in the presence of to varying degrees, depending on the hydrophilicity of the
EEDQ[18] (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinol- bile acid backbone and the configuration of the steroid at
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Tripodal Bile Acid Architectures
C-3. Interestingly, the 3β derivatives 19 and 20 were found sult either of a demicellisation process or of faster dynamics
to be appreciably more soluble in water than the 3α ana- of the aggregates at higher temperatures. DLS experiments
logues 17 and 18. As had been expected, all compounds 17– were performed to aid understanding of the influence of
20 exhibited aggregation in aqueous solutions. We em- these factors in the improvement of resolution of NMR
ployed a well-established dye-solubilisation method for spectra at higher temperature.
study of their aggregation.[19] The hydrophobic dye
orange OT, practically insoluble in aqueous media, was
stirred in aqueous solutions of different concentrations of
17–20 (for details see the Exp. Sect.). With increasing con-
centration of the derivatives, the amount of dye solubilised
in the aqueous media increased. This suggests that these
derivatives were able to form aggregates containing hydro-
phobic domains for the encapsulation of the dye in the
aqueous medium. A plot of the absorbance of the dye (pro-
portional to the amount solubilised) against the concentra-
tion of the derivatives showed that all the molecules start
aggregating at micromolar concentrations (Figure 1). In the
concentration range from 0 to 250 μm there was initially a
rapid increase in the amount of the dye extracted, followed
by a much slower (and linear) increase. The first derivative
Figure 2. NMR spectra of compound 20 in different solvents and
at different temperatures. (a) 0.5 mm in D2O at 25 °C, (b) 0.5 mm
plot (change in absorbance per trimer vs. concentration of
trimer) clearly showed that above 100 μm the amount of the
dye extracted per constant amount of surfactant molecules
remained almost constant. This indicates the formation of
structurally stable aggregates above this concentration for
all the compounds under study.
in D2O at 80 °C, and (c) 1 mm in [D6]DMSO at 25 °C.
Dynamic Light Scattering (DLS) Studies
DLS studies provide information on the sizes of aggre-
gates and dynamics of these systems at different tempera-
tures. All the samples showed multimodal distributions in
the sizes of their aggregates at room temperature. DLS mea-
surements were also carried out at 70 °C to check whether
the improvement in the resolution of NMR spectra is due
to dissociation of aggregates or to any other structural
changes in the sample. These studies revealed the presence
of aggregates even at higher temperature (70 °C), which
suggested that the improved resolution in NMR spectra at
higher temperature is due to faster dynamics of the aggre-
gates. Surprisingly, the multimodal size distributions for
cholic acid aggregates became monodisperse [Figure 3;
sometimes small proportions (Ͻ4%) of smaller particles of
1–2 nm in size were observed], whereas in the case of deoxy-
Figure 1. Orange OT extraction data for all the compounds (17, 18,
19 and 20); a) absorbance of dye vs. concentration of trimer after
half dilution of the extracted samples with MeOH, and b) first de-
rivatives of the data.
The aggregation of these molecules at micromolar con-
centrations was also supported by NMR spectra (Figure 2)
of these compounds in D2O (0.5 mm) and DMSO (1 mm).
The aggregation is usually manifested in the NMR spectra
by loss of resolution of some or all resonances above the
critical aggregation concentration (CAC). Such changes
were indeed observed for all of the taurine conjugates 17–
20 at 0.5 mm and at 25 °C, confirming their aggregation at
micromolar concentrations, which is in agreement with the
dye extraction experiments. Variable-temperature NMR ex-
periments were performed to aid understanding of the in-
fluence of temperature on the aggregation process. These
studies showed an increase in the resolution of the NMR
spectra with temperature, but no well-resolved splitting was
achieved for these compounds, especially in the aromatic
Figure 3. DLS data for cholic acid derivatives: a) compound 18,
and b) compound 20. Red: room temperature. Green: 70 °C. Blue:
region. The improvement in the resolution might be the re- 24 h after heating of the sample (at room temperature).
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cholic derivatives the size distributions were found to re- ture of the 3β derivative a star-like geometry is seen, with
main multimodal even at 70 °C, though becoming narrower the phosphine oxide scaffold in the centre of the molecule
with heating. Interestingly, on lowering the temperature and the bile acid units extending outwards (Figure 4, b),
back to the ambient, the monodispersities of the aggregates to make the molecule more hydrophilic and less prone to
of cholic acid derivatives 18 and 20 (particles size of 50 nm aggregation (and gelation). The shape of the α derivatives,
to 70 nm) persisted for at least 24 h at room temperature.
possessing distinct hydrophilic and hydrophobic sides, on
the other hand, makes such molecules more prone to aggre-
gation and gelation.
AFM Study of the Aggregates
Gels are viscoelastic materials, so they can store and dis-
sipate energy; this is characterised by storage modulus (GЈ)
and loss modulus (GЈЈ), respectively, by dynamic rheol-
ogy.[20] Two types of rheological experiments were per-
formed on these gels: 1) frequency sweep at a constant
stress of 1.0 Pa, and 2) stress sweep at a constant frequency
of 1.0 Hz. The three important mechanical parameters that
can be obtained from these experiments are elasticity (GЈ),
fragility (σ*) and stiffness (GЈ/GЈЈ). For a soft solid such as
a gel the storage modulus is greater than the loss modulus
and the GЈ/GЈЈ ratio defines the stiffness of the gel.
The aggregates of cholate derivatives 18 and 20 were also
characterised by atomic force microscopy (AFM). The
AFM images confirmed the presence of aggregates of
sizes Ͼ100 nm in both the samples and it was also observed
that in the case of the 3α derivative (compound 18) these
aggregates clustered as shown in the Supporting Infor-
mation. In the case of the 3β derivative 20 more isolated
aggregates were observed than in the case of compound 18.
Influence of Stereochemistry on Aggregation (Selective
Hydrogelation by the 3α Derivatives)
Dynamic frequency sweep experiments were carried out
with the gels of the two compounds 17 and 18 at 10% w/v
in phosphate buffer (0.1 m) at pH 7.5 and GЈ and GЈЈ were
measured at 1 Hz. Stress sweep experiments were also car-
ried out on the same gels at a constant frequency of 1 Hz
to measure yield stress (σ*). The data from these experi-
ments are shown in Figure 5 and tabulated in Table 1. From
the experimental data it can be observed that compound 18
showed higher GЈ values than compound 17, which indi-
cates more elastic behaviour for the cholate-derived gel than
for the deoxycholate gel. At the same time the deoxy-
cholate-derived gel showed more stiffness and higher yield
stress than its cholic counterpart, which could be because
of the greater hydrophobicity of the deoxy steroidal nucleus
leading to stronger aggregation.
Although no significant differences in the aggregation
properties of the 3α/3β derivatives could be observed from
the dye solubilisation studies in pure water, a strong in-
fluence of the C-3 configuration on their aggregation was
found in studies in 0.1 m phosphate buffer (pH 7.5). The 3α
cholic acid derivative 18, for example, formed a very strong,
transparent gel at 10% w/v in this medium. The 3β deriva-
tive 20 did not form a gel at similar concentrations and
ionic strengths. In view of these findings, we subsequently
investigated the behaviour of deoxycholate derivatives 17
and 19 under similar conditions. Interestingly, even in the
case of the deoxy derivatives the observations were similar
to those with the cholate trimers, with only the 3α derivative
17 showing gelation at similar concentrations. These results
demonstrate that the gelation is a unique property of the
3α derivatives, emphasising the importance of configuration
at the C-3 position of the steroid. The agglomeration of
aggregates observed in AFM images also supports the con-
cept of stronger aggregation in the case of compound 18.
Molecular modelling experiments (Spartan’08) were per-
formed on both compounds 18 and 20. In the optimised
geometry for the 3α derivative, the phosphine oxide group
was on one side of the molecule and all the hydrophilic
sulfonate groups were on the other, with the three steroidal
arms creating a cavity (Figure 4, a). In the optimised struc-
Figure 5. Rheological experimental data for gels of compound 17
and 18.
Table 1. Rheological data for the hydrogels of 17 and 18. The GЈ
and GЈЈ values were obtained from frequency sweep experiments
performed at a fixed stress of 1 Pa. The values are at f = 1 Hz. The
yield stress values were obtained from stress sweep experiments at
a fixed frequency of 1.0 Hz.
GЈ [Pa]
GЈ/GЈЈ
σ* [Pa]
17
18
9920
20700
13
5
1600
1500
Figure 4. Energy-minimised structures of (a) compound 18 and,
(b) compound 20.
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Tripodal Bile Acid Architectures
from each sample (1 mL) was diluted twofold with MeOH and ab-
sorption spectra for these samples were recorded.
Conclusions
In conclusion, we have synthesised new trimeric mo-
lecular architectures, each based on a central phosphine ox-
ide core to which the bile acids were attached through click
chemistry, and obtained a series of tripodal amphiphiles in
good yields. The aggregation of these molecules was studied
by dye-solubilisation experiments, and all the systems ex-
hibited aggregation at micromolar concentrations. These re-
sults were further supported by NMR experiments, which
showed aggregation at micromolar concentrations for these
molecules at room temperature and improved resolution in
the NMR spectra on heating. These aggregates were char-
acterised by DLS and AFM methods. Variable-temperature
DLS experiments suggested that the improvements in the
resolution of NMR spectra for these compounds were poss-
ibly due to improved dynamics, and not because of dissoci-
ation of the aggregates. These DLS experiments also re-
vealed that the cholic acid derivatives formed monodisperse
aggregates at elevated temperatures, and that these persisted
on annealing. Only the α derivatives, of both deoxycholic
and cholic acid derivatives, were found to be capable of ge-
lation, which emphasises the importance of stereochemistry
for defining the mode of aggregation. To the best of our
knowledge, this is the first instance in which stereochemis-
try has been demonstrated to have an influence on bile acid
derived supramolecular architectures. Energy-minimised
structures from molecular modelling studies suggested bet-
ter solubilisation for 3β derivatives than for 3α derivatives,
which could be a probable reason for the observed gelation
of the α derivatives.
Dynamic Light Scattering Studies: All the DLS measurements were
carried out with a Malvern Nano ZS instrument at 633 nm and a
4 mW He-Ne laser at a back-scattered angle of 173°. The samples
were prepared by dissolving the required amount of the compound
(1 mm) in milliQ water and filtered through 0.45 μm nylon mem-
brane before the measurements.
Rheology: Dynamic rheological measurements were carried out
with the gels and a AR 1000 rheometer (TA instruments) with a
plate-plate (the rotor was serrated) geometry (20 mm diameter,
400 μm gap). Each gel was introduced onto the stage (at 25 °C) as
a hot solution, which was allowed to form the gel. The geometry
gap was set to 500 μm after 5 min and the excess sample was
trimmed. The experimental gap of 400 μm was then set and the
gels were stabilised in the geometry gap for 30 min before the ex-
periment was started. The solvent evaporation was kept minimised
by use of a metallic cover as a solvent trap.
Atomic Force Microscopy: Each AFM sample was prepared by
drop-casting an aqueous solution (10 μL) of the compound (0.1%
w/v) on a freshly washed silicon wafer and allowing it to air-dry
for 5 h. It was then dried under vacuum for another 4–5 h and
AFM images were taken in the tapping mode with a Veeco Dimen-
sion 3100 SPM instrument.
Characterisation for Compounds. General Procedure for the Synthe-
sis of Methyl 3α-Mesyloxy-12α-hydroxy-5β-cholan-24-oate (5) and
Methyl 3α-Mesyloxy-7α,12α-dihydroxy-5β-cholan-24-oate (6): Tri-
ethylamine (1.0 mL, 7.2 mmol) and mesyl chloride (7.2 mmol) were
added at 0 °C under argon to a solution of compound 1 or com-
pound 2 (4.75 mmol) in dichloromethane (20 mL). The reaction
mixture was brought to room temperature and left stirring at this
temperature for 2.5 h. Completion of the reaction was checked by
TLC, and the reaction mixture was then diluted with CHCl3
(50 mL), washed with dil. HCl, saturated aq. NaHCO3 and water
and dried with anhyd. Na2SO4. The filtered solution was concen-
trated and dried under vacuum to afford the crude product, which
was purified by silica gel column chromatography.
Experimental Section
General: All reactions were carried out in oven-dried glassware.
TLC was performed on pre-coated glass plates (0.25 mm silica gel
with fluorescent UV254) purchased from Aldrich. After elution the
plates were developed by the Libermann–Burchard reagent. Com-
mercial grade solvents were distilled prior to use in column
chromatography. Silica gel (100–200 mesh) columns were run under
gravity. 1H and 13C NMR was recorded with a Bruker 400 MHz
spectrometer in deuterated solvents as indicated, with TMS or re-
sidual solvent signals as the internal standards. Infrared spectra
were recorded with a JASCO-70 FT-IR spectrophotometer. Mass
spectra were obtained with Micromass ESI-TOF equipment. Melt-
ing points were recorded with a SRS Digimelt instrument. The
rheological experiments were performed with a Stress-Controlled
AR1000 (TA instruments) rheometer. Molecular modelling studies
were performed by use of the AM1 semi-empirical method in Spar-
tan’08 software.
Methyl 3α-Mesyloxy-12α-hydroxy-5β-cholan-24-oate (5): The pure
product (2.05 g, 86%) was obtained from compound 1 (2.0 g). Elu-
ent for column chromatography: EtOAc/PE 25%, m.p. 110–114 °C.
[α]2D2 = +21.5 (c = 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
4.66 (m, 1 H), 3.99 (s, 1 H), 3.66 (s, 3 H), 2.99 (s, 3 H), 2.41–1.03
(steroidal CH2 and CH), 0.97 (d, J = 6.4 Hz, 3 H), 0.92 (s, 3 H),
0.68 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 174.84, 82.83,
73.19, 51.70, 48.32, 47.52, 46.63, 42.24, 39.05, 36.05, 35.18, 34.98,
34.06, 33.74, 33.43, 31.19, 31.01, 28.75, 27.85, 27.55, 26.93, 26.06,
23.70, 23.06, 17.48, 12.88 ppm. IR (KBr): ν = 3623, 2944, 2872,
˜
1734, 1448, 1328, 1171 cm–1. HRMS: calcd. for C26H44O6S + Na
507.2756; found 507.2755. C26H44O6S (484.69): calcd. C 64.43, H
9.15, S 6.62; found C 63.28, H 8.58, S 6.92.
Methyl 3α-Mesyloxy-7α,12α-dihydroxy-5β-cholan-24-oate (6): The
pure product (1.97 g, 84%) was obtained from compound 2 (2.0 g).
Eluent for column chromatography: EtOAc/PE 40%, m.p. 95–
1
Dye Solubilisation: Dye solubilisation experiments were carried out
with compounds 17–20. Aqueous stock solutions of the com-
pounds with the highest concentration necessary for the experiment
were prepared. Samples of different concentrations of compounds
were prepared by dilution of each stock solution with water. A
known amount of the lipophilic dye (orange OT, 5 mg) was added
to each sample and the heterogeneous mixture was stirred at room
temperature for 20 h. The samples were then filtered through nylon
membranes (0.45 μm) to remove the excess solid dye. An aliquot
99 °C. [α]2D2 = + 34.6 (c 1, CHCl3). H NMR (400 MHz, CDCl3):
δ = 4.52 (m, 1 H), 3.99 (s, 1 H), 3.87 (s, 1 H), 3.67 (s, 3 H), 2.99
(s, 3 H), 2.4–1.1 (steroidal CH2 and CH), 0.98 (d, J = 6.4 Hz, 3 H),
0.91 (s, 3 H), 0.69 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 174.91, 82.92, 72.99, 68.26, 51.66, 47.31, 46.64, 41.98, 41.54,
39.58, 39.01, 36.18, 35.30, 34.92, 34.60, 34.29, 31.16, 30.96, 28.36,
28.03, 27.56, 26.69, 23.25, 22.42, 17.45, 12.64 ppm. IR (KBr): ν =
˜
3552, 2945, 2872, 1732, 1346, 1171 cm–1. HRMS: calcd. for
C26H44O7S + Na 523.2705; found 523.2702.
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General Procedure for the Synthesis of Methyl 3β-Azido-12α- Methyl 3α-Azido-12α-hydroxy-5β-cholan-24-oate (3): The pure
hydroxy-5β-cholan-24-oate (7) and Methyl 3β-Azido-7α,12α-di- product (1.5 g, 71%) was obtained from compound 1 (2.0 g). Elu-
hydroxy-5β-cholan-24-oate (8): Sodium azide (10 mmol) was added
under inert atmosphere to a solution of compound 5 or compound
6 (4.0 mmol) in dry DMF (30 mL) in a 100 mL round-bottomed
flask, and the mixture was left stirring at 85 °C for 8 h. Progress of
the reaction was monitored by TLC. After the completion of the
reaction, DMF was distilled off under reduced pressure, and the
reaction mixture was dissolved in CHCl3 (60 mL). This organic
solution was washed with water, dried with anhyd. Na2SO4, fil-
tered, concentrated and dried under high vacuum to afford the
crude product. This was purified by column chromatography on
silica gel to afford the pure product as a white solid.
ent for column chromatography: EtOAc/PE 15%, m.p. 108–114 °C.
1H NMR (400 MHz, CDCl3): δ = 3.98 (br. s, 1 H), 3.67 (s, 3 H),
3.33 (m, 1 H), 2.41–1.01 (steroidal CH2 and CH), 0.97 (d, J =
6.4 Hz, 3 H), 0.92 (s, 3 H), 0.68 (s, 3 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 174.82, 73.15, 61.36, 51.62, 48.26, 47.43, 46.59, 42.46,
36.09, 35.50, 35.17, 34.29, 33.77, 32.55, 31.15, 30.98, 28.79, 27.52,
27.13, 26.79, 26.13, 23.70, 23.35, 17.42, 12.84 ppm. IR (KBr): ν =
˜
3522, 2941, 2087, 1720 cm–1. HRMS: calcd. for C25H41N3O3 + Na
454.3046; found 454.3048. C25H41N3O3 (431.62): calcd. C 69.57, H
9.57, N 9.74; found C 70.40, H 9.22, N 8.52.
Methyl 3α-Azido-7α,12α-dihydroxy-5β-cholan-24-oate (4): The pure
product (1.7 g, 64%) was obtained from compound 2 (2.3 g). Elu-
ent for column chromatography: EtOAc/PE 25%, m.p. 108–110 °C.
[α]2D3 = +46.2 (c = 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
3.99 (s, 1 H), 3.86 (s, 1 H), 3.67 (s, 3 H), 3.15 (m, 1 H), 2.4–1.1
(steroidal CH2 and CH), 0.98 (d, J = 6.4 Hz, 3 H), 0.91 (s, 3 H),
0.69 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 174.81, 72.97,
68.21, 61.25, 51.50, 47.15, 46.48, 41.84, 41.75, 39.33, 35.40, 35.30,
35.23, 34.71, 34.49, 30.98, 30.75, 28.13, 27.45, 26.75, 26.49, 23.14,
Methyl 3β-Azido-12α-hydroxy-5β-cholan-24-oate (7): The pure
product (1.36 g, 86%) was obtained from compound 5 (1.72 g).
Eluent for column chromatography: EtOAc/PE 15–20%, m.p. 126–
129 °C. [α]2D4 = 32.7 (c 1, CHCl3). H NMR (400 MHz, CDCl3): δ
1
= 3.99 (br. s, 1 H), 3.95 (br. s, 1 H), 3.66 (s, 3 H), 2.41–1.04 (steroi-
dal CH2 and CH), 0.97 (d, J = 6.4 Hz, 3 H), 0.94 (s, 3 H), 0.69 (s,
3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 174.82, 73.34, 58.86,
51.67, 48.49, 47.58, 46.67, 37.42, 35.97, 35.18, 34.63, 33.41, 31.19,
31.02, 30.66, 30.25, 28.91, 27.56, 26.58, 26.10, 24.73, 23.70, 23.67,
22.52, 17.26, 12.43 ppm. IR (KBr):
ν = 3449, 2940, 2102,
˜
17.51, 12.89 ppm. IR (KBr): ν = 2937, 2088, 1732, 1451, 1275,
˜
1748 cm–1. HRMS: calcd. for C25H41N3O4 + Na 470.2995; found
470.2718.
1177 cm–1. HRMS: calcd. for C25H41N3O3 + Na 454.3046; found
454.3044. C25H41N3O3 (431.62): calcd. C 69.57, H 9.57, N 9.74;
found C 69.33, H 9.27, N 9.89.
General Procedure for the Synthesis of 3α-Azido-12α-hydroxy-5β-
cholanic Acid (9), 3β-Azido-12α-hydroxy-5β-cholanic Acid (11), 3α-
Azido-7α,12α-dihydroxy-5β-cholanic Acid (10) and 3β-Azido-
7α,12α-dihydroxy-5β-cholanic Acid (12): The methyl ester of the
corresponding acid (3.2 mmol) was dissolved in 1,4-dioxane
(30 mL), aq. KOH (5% w/v, 30 mL) was added, and the mixture
was stirred at 80 °C for 3 h. After completion of the reaction, the
mixture was concentration to half the volume under reduced pres-
sure and the aqueous layer was acidified with dil. HCl to a pH of
5–6. Precipitation of the product was observed during the acidifica-
tion and the compound was extracted with EtOAc (3ϫ30 mL).
The combined organic layer was washed with water and dried with
anhyd. Na2SO4. It was filtered, concentrated and dried to afford
the crude product, which was purified by column chromatography
on silica gel.
Methyl 3β-Azido-7α,12α-dihydroxy-5β-cholan-24-oate (8): The pure
product (1.7 g, 79%) was obtained from compound 6 (2.25 g). Elu-
ent for column chromatography: EtOAc/PE 30–35%, m.p. 160–
164 °C. [α]2D3 = + 23.4 (c = 1, CHCl3). 1H NMR (400 MHz, CDCl3):
δ = 3.99 (s, 1 H), 3.91 (s, 1 H), 3.87 (s, 1 H), 3.67 (s, 3 H), 2.55–
1.12 (steroidal CH2 and CH), 0.98 (d, J = 6.4 Hz, 3 H), 0.93 (s, 3
H), 0.70 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 174.86,
73.09, 68.54, 58.84, 51.69, 47.39, 46.69, 42.09, 39.57, 36.88, 35.29,
35.20, 34.19, 33.15, 31.19, 30.97, 30.61, 28.61, 27.59, 26.33, 24.68,
23.31, 23.02, 17.47, 12.67 ppm. IR (KBr): ν = 3236, 2943, 2919,
˜
2098, 1743, 1729, 1455, 1438 cm–1. HRMS: calcd. for C25H41N3O4
+ Na 470.2995; found 470.2993.
General Procedure for the Synthesis of Methyl 3α-Azido-12α-
hydroxy-5β-cholan-24-oate (3) and Methyl 3α-Azido-7α,12α-di-
hydroxy-5β-cholan-24-oate (4): Triphenylphosphine (14 mmol) was
added to a solution of compound 1/compound 2 (5 mmol) in dry
THF (50 mL). MsOH (12 mmol) and DIAD (14 mmol) were added
to this solution, followed by the addition of DMAP (12 mmol).
The temperature was raised to 40 °C, and the mixture was stirred
for 8 h. At the end of the reaction, the reaction mixture was filtered
through a sintered funnel containing celite, and the filtrate was con-
centrated under reduced pressure and dried. This reaction mixture
was dissolved in CHCl3, washed with dil. HCl, saturated aq.
NaHCO3 solution and water, dried with anhyd. Na2SO4, concen-
trated under reduced pressure and purified by column chromatog-
raphy on a silica column to afford the methyl 3β-mesyloxydeoxy-
cholate or the methyl β-mesyloxycholate with a small amount of
an impurity. These compounds were used directly in the next step.
Sodium azide (10 mmol) was added under an inert atmosphere to
a solution of the methyl 3β-mesyloxydeoxycholate or the methyl
3β-mesyloxycholate in dry DMF (20 mL) and the mixture was left
stirring at 80 °C for 8 h. Progress of the reaction was monitored by
TLC. After the completion of the reaction, the solvent was removed
under reduced pressure. The reaction mixture was dissolved in
CHCl3 (120 mL), washed with water and dried with anhyd.
Na2SO4. This organic phase was filtered, concentrated and purified
by column chromatography on silica gel.
3α-Azido-12α-hydroxy-5β-cholanic Acid (9): The pure acid (1.37 g,
89%) was obtained from compound 3 (1.6 g). Eluent for column
chromatography: EtOH/CHCl3 5%, m.p. 85–88 °C. [α]2D2 = +55.8
1
(c = 1, MeOH). H NMR (400 MHz, [D6]DMSO): δ = 11.90 (br.
s, 1 H), 4.18 (d, J = 4 Hz, 1 H), 3.78 (br. s, 1 H), 3.427 (m, 1 H),
2.26–0.96 (steroidal CH2 and CH), 0.91 (d, J = 6.4 Hz, 3 H), 0.87
(s, 3 H), 0.60 (s, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
174.88, 71.03, 60.37, 47.36, 46.17, 45.98, 41.53, 35.48, 34.87, 33.71,
32.77, 31.89, 30.77, 30.72, 28.47, 27.08, 26.58, 26.08, 25.85, 23.40,
22.88, 16.90, 12.39 ppm. IR (KBr): ν = 3481, 2940, 2866, 2091,
˜
1709 cm–1. HRMS: calcd. for C24H39N3O3 + Na 440.2889; found
440.2887.
3α-Azido-7α,12α-dihydroxy-5β-cholanic Acid (10): The pure prod-
uct (0.96 g, 83%) was obtained from compound 7 (1.2 g). Eluent
for column chromatography: EtOH/CHCl3 8–10%, m.p. 172–
176 °C. [α]2D2 = +42.9 (c = 1, MeOH). 1H NMR (400 MHz, [D6]-
DMSO): δ = 11.9 (br. s, 1 H), 4.13 (br. s, 2 H), 3.78 (s, 1 H), 3.62
(s, 1 H), 3.23 (m, 1 H), 2.39 (q, J = 12.8 Hz, 1 H), 2.22–1.1 (steroi-
dal CH2 and CH), 0.93 (d, J = 6.4 Hz, 3 H), 0.84 (s, 3 H), 0.59 (s,
3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 174.92, 70.94,
66.08, 60.75, 46.07, 45.73, 41.48, 41.35, 35.02, 34.97, 34.46, 34.28,
30.80, 30.78, 28.40, 27.26, 27.19, 26.28, 26.09, 22.70, 22.43, 16.93,
1412
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1406–1415

Tripodal Bile Acid Architectures
12.28 ppm. IR (KBr): ν = 3473, 2938, 2869, 2092, 1709 cm–1.
HRMS: calcd. for C24H39N3O4 + Na 456.2838; found 456.2835.
(br. s, 3 H), 8.29 (s, 3 H), 8.09 (d, J = 6.4 Hz, 6 H), 7.78 (dd, J =
8.4, 11.2 Hz, 6 H), 5.42 (s, 6 H), 4.49 (m, 3 H), 4.16 (br. s, 3 H),
3.77 (s, 3 H), 2.31–0.99 (steroidal CH2 and CH), 0.92–0.90 (18 H,
merged d and s), 0.610 (s, 9 H) ppm. 13C NMR (100 MHz, [D6]-
DMSO): δ = 174.92, 164.69, 141.11, 136.55 (d, J = 100 Hz), 132.83,
132.09 (d, J = 10 Hz), 129.55 (d, J = 11 Hz), 123.13, 79.16, 71.17,
59.99, 58.40, 47.32, 46.26, 46.04, 41.89, 35.54, 35.18, 34.88, 33.85,
33.18, 32.85, 30.79, 28.53, 27.57, 27.09, 26.56, 25.78, 23.41, 22.91,
˜
3β-Azido-12α-hydroxy-5β-cholanic Acid (11): The pure product
(1.32 g, 93%) was obtained from compound 4 (1.48 g). Eluent for
column chromatography: EtOH/CHCl3 5%, m.p. 85–88 °C. [α]2D3
=
+30.5 (c = 1, MeOH). 1H NMR (400 MHz, [D6]DMSO): δ = 11.89
(br. s, 1 H), 4.14 (s, 1 H), 4.07 (br. s, 1 H), 3.78 (s, 1 H), 2.25–0.97
(steroidal CH2 and CH), 0.91 (d, J = 6.4 Hz, 3 H), 0.88 (s, 3 H),
0.60 (s, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 174.88,
79.15, 71.04, 58.24, 47.42, 46.18, 46.01, 37.09, 35.37, 34.89, 34.11,
32.35, 30.80, 30.72, 30.45, 29.62, 28.65, 27.09, 26.12, 25.75, 24.02,
16.92, 12.37 ppm. IR (KBr): ν = 3676, 3323, 2868, 1644, 1566,
˜
1366, 1336 cm–1. HRMS: calcd. for C102H138N9O16P
1799.9970; found 1799.9842.
+ Na
23.38, 16.90, 12.40 ppm. IR (KBr): ν = 2940, 2871, 2102, 1709,
˜
Compound 15 (Trimer, β-DC–COOH): The pure product (970 mg,
84%) was obtained from compound 21 (280 mg). Eluent for col-
umn chromatography: EtOH/CHCl3 10–15%, m.p. 206–209 °C.
1449 cm–1. HRMS: calcd. for C24H39N3O3 + Na 440.2889; found
440.2889.
[α]2D3 = +18.5 (c = 1, MeOH). H NMR (400 MHz, [D6]DMSO): δ
1
3β-Azido-7α,12α-dihydroxy-5β-cholanic Acid (12): The product
(1.24 g, 88%) was obtained from compound 8 (1.46 g). Eluent for
column chromatography: EtOH/CHCl3 10%, m.p. 212–216 °C.
= 11.94 (br. s, 3 H), 8.31 (s, 3 H), 8.09 (d, J = 8 Hz, 6 H), 7.78 (dd,
J = 3.2, 8 Hz, 6 H), 5.42 (s, 6 H), 4.73 (s, 3 H), 4.20 (s, 3 H), 3.80
(s, 3 H), 2.37–0.92 (steroidal CH2 and CH), 0.92 (d, J = 6.4 Hz, 9
H), 0.81 (s, 9 H), 0.61 (s, 9 H) ppm. 13C NMR (100 MHz, [D6]-
DMSO): δ = 174.92, 164.67, 141.14, 136.52 (d, J = 100 Hz), 132.83,
132.05 (d, J = 10 Hz), 129.51 (d, J = 12 Hz), 124.13, 79.13, 71.05,
58.44, 55.98, 47.44, 46.19, 46.01, 36.86, 35.37, 34.90, 33.84, 32.65,
30.83, 30.74, 30.38, 29.16, 28.63, 27.10, 26.00, 25.60, 24.17, 23.38,
1
[α]2D5 = +24 (c = 1, MeOH). H NMR (400 MHz, [D6]DMSO): δ =
11.89 (br. s, 1 H), 4.13 (d, J = 3.6 Hz, 1 H), 4.11 (d, J = 3.6 Hz, 1
H), 3.99 (s, 1 H), 3.78 (d, J = 2.8 Hz, 1 H), 3.62 (s, 1 H), 3.30 (s,
1 H), 2.26–1.13 (steroidal CH2 and CH), 0.92 (d, J = 6.4 Hz, 3 H),
0.85 (s, 3 H), 0.59 (s, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO):
δ = 175.40, 71.37, 66.56, 58.74, 46.48, 46.17, 41.76, 37.41, 35.42,
35.10, 34.47, 32.95, 31.22, 31.17, 30.91, 28.99, 27.64, 26.11, 24.49,
16.91, 12.39 ppm. IR (KBr): ν = 3682, 3328, 2942, 1720, 1568,
˜
1403, 1281 cm–1. HRMS: calcd. for C102H138N9O16P
1799.9970; found 1799.9858.
+ Na
23.34, 23.14, 17.33, 12.71 ppm. IR (KBr): ν = 3319, 2925, 2872,
˜
2098, 1715, 1450 cm–1. HRMS: calcd. for C24H39N3O4 + Na
456.2838; found 456.2835.
Compound 14 (Trimer, α-C–COOH): The pure product (0.83 g,
79%) was obtained from compound 21 (250 mg). Eluent for col-
Compound 21: DBU (3.6 mL, 24.4 mmol) and propargyl bromide
(2.2 mL, 24.4 mmol) were added to a solution of tris(p-carb-
oxyphenyl)phosphine oxide[21] (2.5 g, 6.1 mmol) in DMF (25 mL),
and the mixture was left stirring at 60 °C for 12 h. Progress of the
reaction was monitored by TLC, and at the end of the reaction the
solvent was removed under reduced pressure. The reaction mixture
was dried, dissolved in CHCl3 (100 mL), washed with dil. HCl,
saturated NaHCO3 solution and water and then dried with anhyd.
Na2SO4. It was filtered, concentrated and dried to afford the crude
product (2.9 g), which was purified by column chromatography on
silica gel with EtOAc/CHCl3 10–20% as eluent to provide the pure
product (2.8 g, 88%), m.p. 179–182 °C. 1H NMR (CDCl3,
400 MHz): δ = 8.18 (dd, J = 2 Hz, 8 Hz, 6 H), 7.75 (dd, J = 8 Hz,
12 Hz, 6 H), 4.95 (s, 6 H), 2.53 (s, 3 H) ppm. 13C NMR (CDCl3,
100 MHz): δ = 164.82, 136.61 (J = 100 Hz), 133.21, 132.23 (J =
umn chromatography: EtOH/CHCl3 15%, m.p. 210–214 °C. [α]2D2
=
+29.1 (c = 1, MeOH). 1H NMR (400 MHz, [D6]DMSO): δ = 11.93
(br. s, 3 H), 8.16 (s, 3 H), 8.12 (dd, J = 2.4, 8.4 Hz, 6 H), 7.79 (dd,
J = 2.8, 8.4 Hz, 6 H), 5.44 (s, 6 H), 4.33 (m, 3 H), 4.14–4.11 (m, 6
H), 3.82 (s, 3 H), 3.65 (s, 3 H), 2.80 (q, J = 12.8 Hz, 3 H), 2.28–
1.05 (steroidal CH2 and CH), 0.95 (d, J = 6.4 Hz, 9 H), 0.92 (s, 9
H), 0.62 (s, 9 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
174.88, 164.70, 141.03, 136.52 (d, J = 101 Hz), 132.80, 132.07 (d,
J = 9 Hz), 129.52 (d, J = 12 Hz), 122.92, 71.02, 66.06, 60.39, 58.40,
46.12, 45.78, 41.72, 41.38, 36.86, 35.29, 34.96, 34.41, 30.8, 30.77,
28.43, 27.17, 26.2, 22.69, 22.46, 16.94, 12.27 ppm. IR (KBr): ν =
˜
3450, 2939, 2870, 1720, 1618, 1364, 1271, 1097 cm–1. HRMS: calcd.
for C102H138O19N9P + Na 1846.9738; found 1846.9665.
Compound 16 (Trimer, β-C–COOH): The product (1.07 g, 82%)
was obtained from compound 21 (300 mg). Eluent for column
10 Hz), 130.04 (J = 12 Hz), 77.30, 75.60, 53.10 ppm. IR (KBr): ν
˜
= 3290, 2126, 1722 cm–1. HRMS [M + Na]: calcd. for C30H21O7P
+ Na 547.0923; found 547.0925.
chromatography: EtOH/CHCl3 20%, m.p. 209–212 °C. [α]2D3
=
+16.5 (c = 1, MeOH). 1H NMR (400 MHz, [D6]DMSO): δ = 11.90
(br. s, 3 H), 8.29 (s, 3 H), 8.09 (d, J = 6 Hz, 6 H), 7.78 (dd, J =
3.2, 8.4 Hz, 6 H), 5.42 (s, 6 H), 4.64 (s, 3 H), 4.21 (s, 3 H), 4.15 (s,
3 H), 3.80 (s, 3 H), 3.65 (s, 3 H), 2.95 (dt, J = 4.8, 14 Hz, 3 H),
2.24–1.16 (steroidal CH2 and CH), 0.93 (d, J = 6 Hz, 9 H), 0.78 (s,
9 H), 0.60 (s, 9 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
174.91, 164.70, 141.08, 136.52 (d, J = 100 Hz), 132.86, 132.07 (d,
J = 10 Hz), 129.52 (d, J = 12 Hz), 124.12, 79.14, 70.97, 66.15,
58.46, 56.06, 46.10, 45.77, 41.34, 39.52, 36.80, 34.99, 34.42, 33.97,
32.05, 30.81, 30.47, 28.58, 27.21, 26.13, 24.25, 22.89, 22.73, 16.94,
General Procedure for Click Reactions between Phosphine Oxide
Core Trispropargyl Ester 21 and Azido Bile Acids: An azido-acid
compound (3 mmol) was added to a solution of compound 21
(0.8 mmol) in distilled DMF (15 mL), followed by the addition of
an aq. solution of CuSO4 (0.1 m, 0.04 mmol) and sodium ascorbate
(0.2 m, 0.2 mmol). The reaction mixture was left stirring at 60 °C
for 5 h. Completion of the reaction was monitored by TLC, DMF
was removed under reduced pressure, and the reaction mixture was
dissolved in EtOH/CHCl3 (50%), adsorbed over silica gel and puri-
fied by column chromatography to afford the pure product. Be-
cause AcOH (0.2%) was used in the eluent during the purification,
after column purification the compound was precipitated three or
four times from EtOAc.
12.29 ppm. IR (KBr): ν = 3437, 2938, 2869, 1720, 1625, 1457, 1271,
˜
1098 cm–1. HRMS calcd. for C102H138O19N9P + Na 1846.9738;
found 1846.9735.
General Procedure for the Synthesis of Trimer–Taurine Conjugates
17–20: EEDQ (2.5 mmol) was added to a solution of the trimer tris
acid (13–16, 0.5 mmol) in dry DMF (20 mL), and the mixture was
heated to 60 °C. Taurine (3 mmol) and triethylamine (3 mmol) were
Compound 13 (Trimer, α-DC–COOH): The product (1.57 g, 82%)
was obtained from compound 21 (412 mg). Eluent for column
chromatography: EtOH/CHCl3 10–15%, m.p. 227–230 °C. [α]2D3
=
+28.3 (c = 1, MeOH). 1H NMR (400 MHz, [D6]DMSO): δ = 11.96 added to this reaction mixture after 5 min and left stirring at 90 °C
Eur. J. Org. Chem. 2014, 1406–1415
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1413

I. Beletskaya, U. Maitra et al.
FULL PAPER
for 4 h. The progress of the reaction was monitored by TLC. After
complete consumption of starting material, the solvent was re-
moved under reduced pressure and the reaction mixture was dried.
It was then dissolved in ethanol (10 mL), filtered to remove excess
taurine, adsorbed over silica gel and purified by column
chromatography to afford the pure product as a white solid (0.2%
acetic acid was added to the eluent during the column purification).
Sodium salts of these conjugates were prepared by stirring with
Dowex-Na+ in methanol. Each mixture was filtered to remove
Dowex and the filtrate was concentrated, dried and characterised.
12 Hz, 6 H), 5.48 (s, 6 H), 4.66 (br. s, 3 H), 3.98 (br. s, 3 H), 3.82
(br. s, 3 H), 3.59 (t, J = 7.2 Hz, 6 H), 3.04 (dt, J = 4.8, 14.4 Hz, 3
H), 2.97 (t, J = 7.2 Hz, 6 H), 2.37–1.08 (steroidal CH2 and CH),
1.04 (d, J = 6.4 Hz, 9 H), 0.88 (s, 9 H), 0.73 (s, 9 H) ppm. 13C
NMR (100 MHz, [D4]methanol): δ = 176.59, 166.47, 143.12, 136.86
(d, J = 100 Hz), 135.18, 133.35 (d, J = 10 Hz), 130.99 (d, J =
12 Hz), 125.43, 73.93, 68.90, 59.36, 58.73, 51.56, 48.04, 47.60,
43.02, 41.02, 38.53, 36.96, 36.62, 36.06, 35.06, 34.27, 33.65, 33.15,
31.80, 29.72, 28.71, 27.99, 25.71, 24.22, 23.48, 17.75, 13.03 ppm.
IR (KBr): ν = 3441, 2940, 1724, 1645 cm–1. HRMS: calcd. for
˜
C108H150O25N12Na3S3P + Na 2233.9320; found 2233.9265.
Compound 17 (Trimer, α-DC–CONHCH2CH2SO3Na): The prod-
uct (1.2 g, 78%) was obtained from compound 13 (1.3 g). Eluent
for column chromatography: MeOH/CHCl3 20–40%. [α]2D3 = +39.1
Supporting Information (see footnote on the first page of this arti-
cle): Copies of the 1H and 13C NMR spectra of compounds 13–20.
1
(c = 1, MeOH). H NMR (400 MHz, [D4]methanol): δ = 8.25 (s, 3
H), 8.19 (dd, J = 2.4, 8.4 Hz, 6 H), 7.79 (dd, J = 8.4, 12 Hz, 6 H),
5.47 (s, 6 H), 4.56 (m, 3 H), 3.98 (br. s, 3 H), 3.59 (t, J = 7.2 Hz,
6 H), 2.96 (t, J = 7.2 Hz, 6 H), 2.41–1.08 (steroidal CH2 and CH),
1.02–1.01 (18 H, merged doublet and singlet), 0.72 (s, 9 H) ppm.
13C NMR (100 MHz, [D4]methanol): δ = 176.54, 166.43, 143.37,
136.92 (d, J = 100 Hz), 135.96, 135.18, 133.37 (d, J = 10 Hz),
131.03 (d, J = 12 Hz), 124.08, 74.03, 62.67, 59.34, 51.54, 48.20,
47.7, 44.08, 37.37, 36.9, 36.80, 36.64, 35.46, 35.01, 34.90, 34.33,
Acknowledgments
The authors acknowledge the Russian foundation of basic research
(grant numbers 11-03-00265a and 08-03-91308INDa) and Dr. A. V.
Kazantsev for syntheses of some starting compounds. The IISc
Bangalore group thanks the Department of Science and Technol-
ogy (DST), New Delhi, for support of this collaborative research
33.12, 29.78, 28.92, 28.68, 28.07, 27.33, 24.88, 23.53, 17.75, (grant number INT/RFBR/P-03/9/9/08).
13.20 ppm. IR (KBr): ν = 3691, 3326, 2939, 2861, 1725, 1553 cm–1.
˜
HRMS: calcd. for C108H150N12O22Na3S3P + Na 2185.9473; found
2185.9449.
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Compound 19 (Trimer, β-DC–CONHCH2CH2SO3Na): The pure
product (948 mg, 84%) was obtained from compound 15 (1 g). Elu-
ent for column chromatography: MeOH/CHCl3 20–40%. [α]2D3
=
+20.3 (c = 1, MeOH). 1H NMR (400 MHz, [D4]methanol): δ =
8.19 (s, 3 H), 8.17 (d, J = 8 Hz, 6 H), 7.78 (dd, J = 8 Hz, 12 Hz, 6
H), 5.47 (s, 6 H), 4.73 (s, 3 H), 3.99 (s, 3 H), 3.59 (t, J = 6.8 Hz, 6
H), 2.96 (t, J = 6.8 Hz, 6 H), 2.49–1.06 (steroidal CH2 and CH),
1.03 (d, J = 10.4 Hz, 9 H), 0.89 (s, 9 H), 0.72 (s, 9 H) ppm. 13C
NMR (100 MHz, [D4]methanol): δ = 176.58, 166.46, 143.17, 136.86
(d, J = 103 Hz), 135.18, 133.34 (d, J = 10 Hz), 130.96 (d, J =
12 Hz), 125.44, 73.99, 59.35, 58.71, 51.50, 48.12, 47.67, 38.81,
37.22, 36.90, 36.74, 36.62, 35.49, 34.66, 34.26, 33.13, 31.77, 30.83,
29.96, 28.66, 27.49, 27.06, 25.63, 24.85, 24.05, 17.72, 13.21 ppm.
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IR (KBr): ν = 3683, 2938, 2869, 1725, 1623 cm–1. HRMS: calcd.
˜
for C108H150N12O22Na3S3P + Na 2185.9473; found 2185.9476.
Compound 18 (Trimer, α-C–CONHCH2CH2SO3Na): The pure
product (873 mg, 79%) was obtained from compound 14 (1.03 g).
Eluent for column chromatography: MeOH/CHCl3 30–40%. [α]2D3
1
= +26.5 (c = 1, MeOH). H NMR (400 MHz, [D4]methanol): δ =
8.18 (s, 3 H), 8.18 (dd, J = 2.4, 8.4 Hz, 6 H), 7.79 (dd, J = 8.4,
12.4 Hz, 6 H), 5.46 (s, 6 H), 4.42 (m, 3 H), 3.98 (br. s, 3 H), 3.81
(br. s, 3 H), 3.59 (t, J = 6.8 Hz, 6 H), 2.96 (t, J = 6.8 Hz, 6 H),
2.85 (q, J = 12 Hz, 3 H), 2.38–1.11 (steroidal CH2 and CH), 1.03
(d, J = 6.4 Hz, 9 H), 1.01 (s, 9 H), 0.73 (s, 9 H) ppm. 13C NMR
(100 MHz, [D4]methanol): δ = 176.57, 166.41, 143.35, 136.92 (d, J
= 100 Hz), 135.21, 133.38 (d, J = 10 Hz), 131.02 (d, J = 12 Hz),
123.86, 73.95, 68.81, 62.94, 59.33, 51.54, 48.13, 47.63, 43.66, 43.06,
41.07, 38.19, 36.98, 36.84, 36.64, 36.05, 35.56, 34.36, 33.16, 29.50,
28.99, 28.74, 28.06, 24.22, 23.04, 17.79, 13.04 ppm. IR (KBr): ν =
˜
3433, 2939, 1724, 1638 cm–1. HRMS: calcd. for C108H150O25N12-
Na3S3P + Na 2233.9320; found 2233.9307.
Compound 20 (Trimer, β-C–CONHCH2CH2SO3Na): The pure
product (860 mg, 73%) was obtained from compound 16 (1.06 g).
Eluent for column chromatography: MeOH/CHCl3 35–45%. [α]2D3
= +16 (c = 1, MeOH). 1H NMR (400 MHz, [D4]methanol): δ =
8.18 (dd, J = 2.4, 8.4 Hz, 6 H), 8.16 (s, 3 H), 7.78 (dd, J = 8.4,
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Received: September 22, 2013
Published Online: January 8, 2014
Eur. J. Org. Chem. 2014, 1406–1415
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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