Synthesis of a new family of four deoxycholic acid derived chiral stationary phases and their evaluation in the HPLC resolution of racemic compounds

Article abstract of DOI:10.1016/S0957-4166(99)00337-7

Four new chiral selectors obtained by suitable derivatization of the hydroxyl groups of the deoxycholic acid with two identical (homoderivatized) or different (heteroderivatized) arylisocyanates have been prepared and linked covalently to silica gel to obtain new chiral stationary phases (CSPs) for the HPLC separation of enantiomers. The CSPs containing two identical substituents are able to enantiodiscriminate different classes of racemic compounds, or the same racemates to a different extent, a property which depends on the different electronic character of the arylcarbamate moieties. The heteroderivatized CSPs retain the character of the two homoderivatized phases: however, the relative position of the two different arylcarbamate moieties on the deoxycholic backbone strongly affects the enantiodiscrimination capability of these two CSPs.

Full text of DOI:10.1016/S0957-4166(99)00337-7

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3353–3364
Synthesis of a new family of four deoxycholic acid derived chiral
stationary phases and their evaluation in the HPLC resolution of
racemic compounds
A. Iuliano,a P. Salvadori a and G. Félix b,∗
aCentro CNR Macromolecole Stereordinate ed Otticamente Attive, Dipartimento di Chimica e Chimica Industriale,
via Risorgimento 35, 56126 Pisa, Italy
bLaboratoire d’Analyse Chimique par Reconnaissance Moléculaire, ENSCPB, Avenue Pey-Berland (BP 108), 33402 Talence,
France
Received 5 July 1999; accepted 9 August 1999
Abstract
Four new chiral selectors obtained by suitable derivatization of the hydroxyl groups of the deoxycholic acid with
two identical (homoderivatized) or different (heteroderivatized) arylisocyanates have been prepared and linked
covalently to silica gel to obtain new chiral stationary phases (CSPs) for the HPLC separation of enantiomers. The
CSPs containing two identical substituents are able to enantiodiscriminate different classes of racemic compounds,
or the same racemates to a different extent, a property which depends on the different electronic character of
the arylcarbamate moieties. The heteroderivatized CSPs retain the character of the two homoderivatized phases:
however, the relative position of the two different arylcarbamate moieties on the deoxycholic backbone strongly
affects the enantiodiscrimination capability of these two CSPs. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
One of the most simple, accurate and reliable methods for measuring the enantiomeric purity of
organic compounds is HPLC on a chiral stationary phase (CSP), which allows the direct separation of
enantiomers.1 For this reason the last 15 years have witnessed the development and use of various CSPs
based on cyclodextrines,2 cellulose derivatives,3 helical polymers,4 proteins5 and low molecular weight
optically active compounds.6 These last CSPs are very attractive not only for their effectiveness, but also
because each molecule linked to the silica gel is considered to act independently (they are also named
independent7 CSPs), so that the factors governing the enantiodiscrimination process can be determined
and, on this basis, the development of new, more efficient CSPs can be designed. Indeed, this has allowed
∗
Corresponding author.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00337-7

3354
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
the efficiency of some independent CSPs to be improved and new selectors, showing wider applicability,
to be found.6a
It is now generally accepted that one interaction which plays an important role in the enantiodiscri-
mination process exhibited by the independent CSPs is the π–π one,7 taking place between electro-
nically complementary aromatic moieties present on the selector and the select and, therefore, a CSP
showing wide applicability could be obtained by synthesizing a ‘biselector system’, i.e. a chiral molecule
possessing two aromatic rings having different electronic properties, not interacting between themselves,
so that they could be able to establish, independently, π–π enantioselective interactions with a wide
range of racemic solutes. Of course, the biselector system must possess other functional groups as well,
giving rise to further stereodependent interactions with the enantiomers of the analytes in order to have a
full separation.7
Bile acids look very interesting candidates to act as scaffolds on which to attach the above mentioned
aromatic rings. They possess a unique structure, endowed with hydroxyl groups having different reac-
tivities, providing the opportunity for the attachment of molecular units: therefore new chiral molecules
with properties determined by the nature and the arrangement of these units can be obtained. Indeed, bile
acids have been used for preparing molecular tweezers,8 supramolecular receptors,9 chiral auxiliaries for
asymmetric reactions,10 scaffolds for combinatorial chemistry11 and chiral stationary phases for HPLC.12
We addressed our attention to the use of deoxycholic acid 1, one of the most common and readily
available bile acids, for designing a series of chiral selectors for chromatography, in which structural
and/or electronic perturbations could be introduced easily. Compound 1 possesses a carboxylic function,
which can be exploited for linking to silica gel, and two hydroxyl groups, placed 6–6.5 Å apart8 on a
rigid backbone with different reactivities, owing to their different steric environments: by derivatizing the
two hydroxyl moieties with two different aromatic groups, these would be separated sufficiently to act
independently and the compound should behave as a biselector system (Fig. 1).
Figure 1.
We decided to derivatize the hydroxyl groups of 1 as arylcarbamates, given that this kind of derivatiza-
tion allows aromatic rings having different electronic properties to be introduced and, at the same time,
to have NH carbamate moieties able to afford hydrogen bonding interactions with the racemic selectants.
3,5-Dimethylphenylisocyanate was chosen as the reagent possessing an electron rich aromatic ring and
3,5-dichlorophenylisocyanate was the derivatizing agent characterized by an electron-poor aromatic
moiety. We report herein the synthesis of a new family of four CSPs derived from deoxycholic acid
and the preliminary results obtained in the separation of racemic compounds.
2. Results and discussion
2.1. Synthesis of the CSPs
Our synthetic route to the homoderivatized and heteroderivatized CSPs is summarized in Scheme 1.

A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
3355
Scheme 1. Reagents and conditions: (a) tributylamine, dioxane; (b) ethyl chloroformate, 10°C; (c) allylamine, 10°C to rt; (d)
ArNCO (2.5 equiv.) toluene, reflux; (e) ArNCO (1.2 equiv.) toluene–THF, DMAP, rt; (f) Ar1NCO (1.2 equiv.), toluene, reflux;
(g) 3-mercaptopropyltrimethoxysilane (5 equiv.), ArBN CHCl3, reflux; (h) silica gel, toluene, reflux
Deoxycholic acid was converted to the corresponding allylamide by the mixed anhydride method,13
in 88% yield. This derivatization allows a terminal double bond to be introduced, which is well known
to be a suitable moiety for linking chiral selectors to silica gel.14 The amide bond thus introduced also
acts as a protecting group for the carboxylic function in the following reactions: the amide function is
hardly hydrolyzable and therefore very resistant to possible acidic or basic chromatographic conditions.
Furthermore, given that it is well known that the best results of enantioseparation are achieved when the
chiral system is remote from the silica gel,7a the conversion of the carboxylic function to the N-allylamide
moiety, followed by reaction of the terminal double bond with mercaptopropyltrimethoxysilane, allows
the chiral auxiliary to be connected to the silica gel by means of a long tether (11 units from silica gel to
cholestanic skeleton), which is a sufficient distance of the selector from the supporting material.
The heteroderivatized selectors were obtained exploiting the different reactivity of the 3- and 12-

3356
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
hydroxyl groups: in fact, by reacting 2 with an isocyanate at room temperature in the presence of a
catalytic amount of DMAP, only the hydroxyl group at the position 3 is converted to the corresponding
carbamate, whereas the reaction of the 12-hydroxyl moiety takes place only on warming the reaction
mixture to reflux in toluene. This allows the two different heteroderivatized selectors to be obtained
simply by changing the order of introduction of the arylisocyanates. As shown in Scheme 1, the
homoderivatized selectors were obtained simply by reacting 2 with the arylisocyanate at reflux in toluene.
The derivatization reaction of the 12-OH group for preparing both the homo- and the heteroderivatized
selectors afforded the corresponding carbamates only in moderate yield (∼60%), even if an excess of
arylisocyanate was employed. This occurs because a by-product, originating from the attack of the
isocyanate on the amide nitrogen of the 3-arylcarbamoyl derivative, is formed to a significant extent
during the reaction: however, this did not constitute a serious drawback, because the by-product was
easily removed by flash chromatography.†
In order to prepare the CSPs, selectors 4a–d were reacted with a fivefold excess of mercaptopropyltri-
methoxysilane in the presence of AIBN for 20 h. Under these experimental conditions 4a–d were fully
converted to the corresponding trimethoxysilyl derivatives, which were separated from the excess of
mercaptopropyltrimethoxysilane simply by washing with pentane. The grafting to silica gel was carried
out in toluene at reflux for 24 h and the CSPs A–D, after being thoroughly washed and dried, were packed
into stainless steel columns of 25 or 15 cm (internal diameter 4.6 mm).
2.2. Use of CSPs A–D in the resolution of some racemic compounds
Compounds 6–13 (Fig. 2), being representative examples of different classes of analytes, were used to
test the enantiodiscriminating properties of CSPs A–D.
Figure 2.
As the data reported in Table 1 show, CSP B separates the π-donor racemic compounds 6–8 better than
CSP A does, whereas it does not enantiodiscriminate π-acceptor analytes 9 and 10, these being separated
by CSP A. As far as the retention times are concerned, it is of note that π-donor racemic compounds 6–8
are strongly retained on CSP B, whereas lower k0 values are obtained on CSP A for the same analytes.
†
The formation of the by-product could be avoided using the N-methyl-N0allylamide of the deoxycholic acid: work is in
progress on this topic.

A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
3357
Table 1
Chromatographic resolutiona of compounds 6–13 on CSPs A–D
On the contrary, π-acceptor racemates 9 and 10 are better retained on CSP A than on CSP B. Given that
the only difference between these two phases is the electronic nature of the aryl moieties, linked to the
deoxycholic skeleton by means of identical carbamate functions, the different retention times observed
for the same compound on the two CSPs are certainly attributable to different π–π interactions, taking
place between the aromatic ring of the selectant and those of the selectors. These results demonstrate the
complementary characteristics of the deoxycholic based selectors, bearing the 3,5-dichlorophenyl and
3,5-dimethylphenyl moieties in the separation of racemic compounds possessing an aromatic π-donor or
π-acceptor group.
The heteroderivatized CSP C, bearing a 3,5-dichlorophenylcarbamate moiety at position 12 and a
3,5-dimethylphenylcarbamate group at position 3, shows a low efficiency both in the separation of
6–8 with respect to CSP B and in the enantiodiscrimination of compound 9 with respect to CSP A;
furthermore compound 10 is not resolved at all. The retention times for π-donor racemic compounds
6–8 are higher than those observed for the same analytes on CSP A and lower than those obtained on
CSP B; likewise, the π-acceptor racemates 9 and 10 are better retained on CSP C than on CSP B, but
less well retained than on CSP A. These data suggest that the two different aryl moieties of CSP C are
still able to exhibit π–π interactions with the aromatic complementary groups present on the substrates,
although, as the k0 values indicate, to a less extent than the homoderivatized phases. However, whilst
the role played by the 3,5-dichlorophenylcarbamate moiety at position 12 in the enantiodiscrimination
process of CSP C is preserved (even if the separations are poorer than CSP B, which possesses two 3,5-
dichlorophenylcarbamate groups), the effect of the 3,5-dimethylphenylcarbamate moiety at position 3 is
almost completely lost.
Interestingly, CSP D, the mixed phase bearing a 3,5-dichlorophenylcarbamate moiety at position 3
and a 3,5-dimethylphenylcarbamate group at position 12, shows, as expected, the same characteristics
of CSP C, as far as the retention times are concerned, the k0 values being similar to those obtained
with CSP C, but behaves quite differently with respect to CSP C as far as the enantiodiscrimination
process is concerned. This phase is able to separate both π-donor and π-acceptor analytes: its enan-
tiodiscriminating capability towards π-acceptor compounds 9 and 10 remains almost the same as CSP
A, notwithstanding that it possesses only one 3,5-dimethylphenylcarbamate moiety, whereas it shows a
slight decrease towards π-donor analytes 6–8 with respect to CSP B. This means that the chiral auxiliary
of CSP D acts as a biselector system, being able to enantiodiscriminate racemic compounds possessing
both π-donor and π-acceptor aromatic rings.
As far as the separation of the benzodiazepine enantiomers 11–13 is concerned, the data reported

3358
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
in Table 1 show that this new family of selectors exhibits quite good enantioselectivity towards these
pharmaceuticals, the α values ranging from 1.11 to 1.77: furthermore all the CSPs show the same trend
in the enantioseparation of these racemates, the best separation being achieved always in the case of
compound 12.
Comparing the enantiodiscrimination capability of CSP A and CSP B towards the benzodiazepines,
we can observe that both the CSPs work rather well with this kind of racemic compound, although
CSP B shows higher enantioselectivity. This means that both 3,5-dichlorophenylcarbamate and 3,5-
dimethylphenylcarbamate linked to the deoxycholic scaffold constitute chiral selectors suitable for the
enantioseparation of the benzodiazepines, even if to a different extent. Interestingly, CSP B, which
affords better separations than CSP A, also shows higher retention times than CSP A, suggesting that
the derivatization with the 3,5-dichlorophenylisocyanate is more suitable to afford stronger and more
enantioselective interactions between the chiral selector and the benzodiazepine selectants.
The retention times of the benzodiazepines with the two heteroderivatized CSPs are intermediate
between those obtained for CSP A and those observed for CSP B: therefore CSP C and CSP D behave,
as far as the retention is concerned, toward the benzodiazepines just as toward the racemates 6–10.
As far as the enantiodiscriminating capability is concerned, CSP C shows lower α values for all the
benzodiazepines than CSP A and CSP B, whereas the enantioseparations obtained with CSP D are the
most efficient. Given that CSP C and CSP D are different only in the relative position of the two aryl
groups, the different behaviour exhibited in the separation of the benzodiazepine enantiomers is certainly
attributable only to the different location of the two aryl moieties in the deoxycholic skeleton. So, when
the 3,5-dimethylphenylcarbamate moiety is located at position 12 and 3,5-dichlorophenylcarbamate at
position 3, as in CSP D, the action of the two aryl groups on the enantioseparation process is reinforced,
whereas, when their position is exchanged, as in CSP C, they cannot work in a synergetic way, giving
rise to worse separations.
In conclusion, starting from deoxycholic acid, a new family of efficient chiral selectors, contai-
ning two identical or different aromatic substituents, has been synthesized and used for the HPLC
separation of racemic compounds. The results obtained in the enantioseparation of selected racemic
compounds have allowed us to establish the complementary character of the selectors containing two 3,5-
dimethylphenylcarbamate or two 3,5-dichlorophenylcarbamate moieties toward the enantioseparation of
π-donor and π-acceptor analytes.
Deoxycholic acid has been shown to be suitable for the preparation of heteroderivatized selectors,
which have been obtained by derivatizing the two hydroxyl groups with two different arylisocyanates.
The two heteroderivatized selectors have been found to be non-equivalent in the enantioseparation of
racemic compounds, their effectiveness depending on the relative position of the two different carbamate
moieties on the deoxycholic backbone. Only when the two derivatizing groups take ‘matched’15 posi-
tions, as in CSP D, does the chiral auxiliary behave as a biselector system, being able to separate both π-
donor and π-acceptor analytes. On the contrary, if the two different arylcarbamates take ‘mismatched’15
positions, as in CSP C, poorer separations are obtained.
3. Experimental
3.1. General
1H and 13C NMR spectra were recorded in CDCl3 on a Varian Gemini-200 or a Bruker 200 MHz
NMR spectrometer, using TMS as external standard. The following abbreviations are used: s=singlet,

A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
3359
d=doublet, dd=double doublet, t=triplet, m=multiplet, br=broad. TLC analyses were performed on
silica gel 60 Macherey–Nagel sheets; flash chromatographic separations were carried out on adequate
dimension columns using silica gel 60 (230–400 mesh). HPLC analyses were performed on a JASCO PU-
980 intelligent HPLC pump equipped with a JASCO UV-975 detector. Optical rotations were measured
with a JASCO DIP-360 digital polarimeter. Melting points were taken using a Kofler Reichert–Jung
apparatus and are uncorrected. The IR spectra were recorded on a Perkin–Elmer 1710 spectrophotometer.
Toluene, THF and dioxan were refluxed over sodium benzophenone and distilled before use. Allylamine
and tributylamine were distilled over CaH2. Unless otherwise specified the reagents were used without
any purification.
Standard procedures were used for preparing racemic amides, the DNB derivative of the valinol and
the anilide of naproxen.16
3.2. N-Allyl-deoxycholan-24-amide 2
To a solution of 6 g (0.0153 mol) of deoxycholic acid in 150 ml of dry dioxan, dry tri-n-butylamine
(3.64 ml, 0.0153 mol) was added and the solution was kept at 10°C. Ethylchloroformate (1.47 ml, 0.0153
mol) in dry dioxan (5 ml) was added dropwise (10 min) and the reaction mixture stirred for 10 min at the
same temperature. Dry allylamine (2.8 ml, 0.038 mol) in dry dioxan (5 ml) was added dropwise at 10°C
and the mixture was stirred for 30 min at 10°C, then at room temperature for 2.5 h.
The reaction mixture was poured in water (400 ml) and the product was extracted with ethyl
acetate (4×100 ml). The collected organic extracts were washed with 10% hydrochloric acid, water,
a saturated solution of NaHCO3 and water, in that order, then dried over Na2SO4. The solvent was
evaporated under reduced pressure and the crude product was purified by column chromatography (SiO2,
dichloromethane:ethanol 88:12) affording 5.7 g (0.0134 mol, 88% yield) of the amide. Mp 89–90°C.
[α]21 +39.7 (c 1.02, CH2Cl2). H NMR (200 MHz, CDCl3, δ): 5.75 (m, 1H, CH_); 5.48 (t, 1H, NH);
1
D
5.08 (m, 2H, _CH2); 3.89 (br t, 1H, H12); 3.81 (tt, 2H, CH2-NH); 3.53 (m, 1H, H3); 2.10–0.75 (m, 28H,
steroidal CH and CH2 and OH); 0.92 (d, 3H, CH320); 0.82 (s, 3H, CH3); 0.60 (s, 3H, CH3). 13C NMR
(200 MHz, CDCl3, δ): 173.53 (C_O), 134.44 (C_CH2), 116.07 (C_CH2), 73.04 (C12), 71.60 (C3),
48.09, 46.98, 46.48, 42.09, 41.83, 36.38, 36.00, 35.26, 34.11, 33.58, 33.36, 31.69, 30.41, 28.56, 27.49,
27.15, 26.13, 23.66, 29.08, 17.38, 12.67. IR (KBr): 3380, 3310, 2925, 2861, 1645, 1549, 1017. Anal.
calcd for C27H45NO3: C, 75.13; H, 10.51; N, 3.24. Found: C, 75.17; H, 10.53; N, 3.25.
3.3. 3-Arylcarbamoyl-N-allyl-deoxycholan-24-amide 3c,d: general procedure
To a solution of 3.7 mmol of 1 in dry THF (50 ml) 4.5 mmol of arylisocyanate and 25 mg (0.2
mmol) of DMAP were added and the mixture was stirred at room temperature for 15 h. The THF was
evaporated under reduced pressure and the crude product was purified by column chromatography (SiO2,
dichloromethane:acetone 90:10).
3.3.1. 3-(3,5-Dimethylphenyl)carbamoyl-N-allyl-deoxycholan-24-amide 3c
25
1
Yield 1.7 g, 78%. Mp 120–122°C. [α] =61.7 (c=1.02, CH2Cl2). H NMR (200 MHz, CDCl3, δ):
D
7.04 (s, 2H, aromatics); 6.65 (s, 2H, aromatic and NH carbamate); 5.75 (m, 1H, CH_); 5.62 (t, 1H, NH);
5.08 (m, 2H, _CH2); 4.58 (m, 1H, H3); 3.89 (br t, 1H, H12); 3.81 (t, 2H, CH2-NH); 2.10–0.75 (m,
27H, steroidal CH and CH2 and OH); 0.92 (d, 3H, CH320); 0.82 (s, 3H, CH3); 0.60 (s, 3H, CH3). 13C
NMR (200 MHz, CDCl3, δ): 173.56 (amide C_O), 153.45 (carbamate C_O), 138.42 (aromatic), 138.03
(aromatic) 134.30 (C_CH2), 124.74 (aromatic), 116.42 (aromatic), 116.09 (C_CH2), 74.90 (C3), 73.10

3360
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
(C12), 48.16, 47.10, 46.42, 41.64, 35.67, 35.07, 34.85, 34.03, 33.57, 33.21, 32.43, 31.54, 28.56, 27.44,
26.88, 26.73, 26.00. 23.56, 22.94, 21.27, 17.30, 12.61. IR (KBr): 3335, 3077, 2936, 2861, 1713, 1653,
1616, 1540, 1448, 1373, 1330, 1270, 1222, 1081, 1017. Anal. calcd for C36H54N2O4: C, 74.70; H, 9.40;
N, 4.84. Found: C, 74.75; H, 9.42; N, 4.85.
3.3.2. 3-(3,5-Dichlorophenyl)carbamoyl-N-allyl-deoxycholan-24-amide 3d
24
1
Yield 1.6 g, 70%. Mp 118–120°C. [α] =56.6 (c=1.12, CH2Cl2). H NMR (200 MHz, CDCl3 δ):
D
7.95 (br s, 1H, NH carbamate); 7.42 (d, 2H, aromatics); 6.92 (t, 1H, aromatic) 5.75 (m, 2H, CH_ and
NH amide); 5.08 (m, 2H, _CH2); 4.61 (m, 1H, H3) 3.89 (t, 1H, H12); 3.81 (t, 2H, CH2-NH); 2.10–0.75
(m, 27H, steroidal CH and CH2 and OH); 0.92 (d, 3H, CH320); 0.82 (s, 3H, CH3); 0.60 (s, 3H, CH3).
13C NMR (200 MHz, CDCl3 δ): 173.68 (amide C_O), 153.14 (carbamate C_O), 140.77 (aromatic),
135.07 (aromatic) 134.15 (C_CH2), 122.66 (aromatic), 116.66 (aromatic), 116.40 (C_CH2), 75.44
(C3), 73.31 (C12), 48.25, 47.41, 46.56, 42.00, 41.82, 35.33, 34.67, 34.15, 33.71, 33.45, 32.45, 31.72,
28.66, 27.66, 26.92, 26.53, 26.10, 23.66, 23.01, 17.32, 12.73. IR (KBr): 3284, 3077, 2935, 2870, 1715,
1655, 1590, 1535, 1443, 1405, 1372, 1307, 12407, 1214, 1110, 1061, 1023, 985, 920. Anal. calcd for
C34H48Cl2N2O4: C, 65.90; H, 7.81; Cl, 11.44; N, 4.52. Found: C, 65.94; H, 7.82; Cl, 11.47; N, 4.54.
3.3.3. 3,12-Diarylcarbamoyl-N-allyl-deoxycholan-24-amide 4a,b: general procedure
A mixture of 3 mmol of 1, dry toluene (25 ml) and arylisocyanate (10.2 mmol) was stirred under
reflux for 20 h. The toluene was removed under vacuum and the crude product was purified by column
chromatography (SiO2, dichloromethane:acetone 95:5).
3.3.4. 3,12-Bis-(3,12-dimethylphenyl)carbamoyl-N-allyl-deoxycholan-24-amide 4a
24
1
Yield 1.4 g, 64%. Mp 128–130°C. [α] =90.3 (c=0.975, CH2Cl2). H NMR (200 MHz, CDCl3, δ):
D
7.04 (s, 2H, aromatics); 6.89 (s, 2H, aromatics); 6.65 (br s, 1H, NH carbamate); 6.65 (s, 1H, aromatic);
6.60 (s, 1H, aromatic); 6.48 (br s, 1H, NH carbamate); 5.72 (m, 1H, CH_); 5.52 (br t, 1H, NH amide);
5.08 (m, 3H, _CH2 and H12); 4.58 (m, 1H, H3); 3.78 (t, 2H, CH2-NH); 2.20 (s, 6H CH3); 2.18
(s, 6H, CH3); 2.10–0.75 (m, 26H, steroidal CH and CH2); 0.82 (s, 3H, CH3); 0.79 (d, 3H, CH320);
0.68 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, δ): 173.27 (amide C_O), 153.10 (carbamate C_O),
138.52 (aromatic), 138.45 (aromatic), 138.04 (aromatic), 137.80 (aromatic), 134.26 (C_CH2), 124.74
(aromatic), 116.39 (aromatic), 116.14 (aromatic) 116.01 (C_CH2), 76.37 (C12), 75.00 (C3), 48.38,
47.64, 45.12, 41.75, 35.47, 34.31, 34.48, 34.26, 33.66, 33.45, 32.56, 31.44, 27.35, 26.80, 25.86, 25.79,
23.37, 22.83, 21.28, 17.59, 12.41. IR (KBr): 3342, 3075, 2936, 2861, 1715, 1656, 1634, 1608, 1554,
1544, 1447, 1378, 1327, 1268, 1223, 1191, 1159, 1081, 1012. Anal. calcd for C45H63N3O5: C, 74.45; H,
8.75; N, 5.79. Found: C, 74.49; H, 8.77; N, 5.80.
3.3.5. 3,12-Bis-(3,5-dichlorophenyl)carbamoyl-N-allyl-deoxycholan-24-amide 4b
25
1
Yield 1.44 g, 62%. Mp 148–151°C. [α] =85.0 (c=1, CH2Cl2). H NMR (200 MHz, CDCl3, δ):
D
7.42 (d, 2H, aromatics); 7.29 (d, 3H, aromatics and NH carbamate); 6.98 (t, 1H, aromatic); 6.96 (t, 1H,
aromatic); 6.83 (br s, 1H, NH carbamate); 5.68 (m, 2H, CH_ and NH amide); 5.05 (m, 3H, _CH2 and
H12); 4.54 (m, 1H, H3); 3.78 (t, 2H, CH2-NH); 2.30–0.75 (m, 26H, steroidal CH and CH2); 0.82 (s,
3H, CH3); 0.78 (d, 3H, CH320); 0.64 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, δ): 173.47 (amide
C_O), 152.80 (carbamate C_O), 152.71 (carbamate C_O), 140.49 (aromatic), 140.05 (aromatic),
135.26 (aromatic), 135.16 (aromatic), 133.97 (C_CH2), 122.98 (aromatic), 122.88 (aromatic), 117.01
(aromatic), 116.64 (aromatic) 116.45 (C_CH2), 77.37 (C12), 76.07 (C3), 49.51, 47.62, 45.19, 41.91,
41.79, 35.46, 34.66, 34.41, 34.32, 33.65, 33.56, 32.52, 31.51, 27.38, 26.64, 25.88, 25,66, 23.37, 22.81,

A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
3361
17.61, 12.42. IR (KBr): 3438, 3075, 2936, 2861, 1715, 1649, 1592, 1533, 1447, 1410, 1250, 1212. Anal.
calcd for C41H51Cl4N3O5: C, 60.97; H, 6.36; Cl, 17.56; N, 5.20. Found: C, 61.00; H, 6.37; Cl, 17.59; N,
5.22.
3.4. 3-Arylcarbamoyl-12-arylcarbamoyl0 -N-allyl-deoxycholan-24-amide: general procedure
To a solution of 3 (2.2 mmol) in dry toluene (25 ml), the arylisocyanate (2.7 mmol) was added and
the solution was stirred at reflux for 24 h. The toluene was evaporated at reduced pressure and the crude
product was purified by flash chromatography (SiO2, dichloromethane:acetone 95:5).
3.4.1. 3-(3,5-Dimethylphenyl)carbamoyl-12-(3,5-dichlorophenyl)carbamoyl-N-allyl-deoxycholan-24-
amide 4c
25
1
Yield 1.1 g, 65%. Mp 130–132°C. [α] =86.1 (c=1.03, CH2Cl2). H NMR (200 MHz, CDCl3, δ):
D
7.42 (br s, 3H, aromatic and NH carbamate); 6.89 (br s, 3H, aromatics); 6.60 (s, 1H, aromatic); 6.55 (br
s, 1H, carbamate); 5.72 (m, 1H, CH); 5.52 (br t, 1H, NH amide); 5.08 (m, 3H, _CH2 and H12); 4.58 (m,
1H, H3); 3.78 (t, 2H, CH2-NH); 2.20 (s, 6H CH3); 2.10–0.75 (m, 26H, steroidal CH and CH2); 0.79 (d,
3H, CH320); 0.77 (s, 3H, CH3); 0.68 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, δ): 173.31 (amide
C_O), 153.33 (carbamate C_O), 152.34 (carbamate C_O), 140.63 (aromatic), 136.63 (aromatic),
137.64 (aromatic), 135.03 (aromatic), 134.13 (C_CH2), 124.93 (aromatic), 122.63 (aromatic), 117.03
(aromatic), 116.31 (aromatic), 116.4 (10C_CH2), 76.98 (C12), 75.34 (C3), 49.22, 47.77, 45.09, 41.67,
35.34, 34.95, 34.25, 33.96, 33.76, 33.67, 32.71, 31.48, 27.44, 27.06, 26.90, 25.97, 25.39, 23.32, 22.83,
21.38, 17.60, 12.47. IR (KBr): 3321, 3075, 2957, 2861, 1715, 1651, 1616, 1589, 1541, 1450, 1407, 1375,
1260, 1242, 1218, 1194, 1159, 1114, 1087. Anal. calcd for C43H57Cl2N3O5: C, 67.35; H, 7.49; Cl, 9.25;
N, 5.48. Found: C, 67.39; H, 7.51; Cl, 9.27; N, 5.51.
3.4.2. 3-(3,5-Dichlorophenyl)carbamoyl-12-(3,5-dimethylphenyl)carbamoyl-N-allyl-deoxycholan-24-
amide 4d
25
1
Yield 1.0 g, 60%. Mp 137–139°C. [α] =77.9 (c=0.78, CH2Cl2). H NMR (200 MHz, CDCl3):
D
7.22 (d, 2H, aromatics); 7.02 (s, 2H, aromatics); 6.92 (d, 1H, aromatic); 6.71 (br s, 1H, carbamate);
6.69 (br s, 1H, NH carbamate); 6.62 (s, 1H, NH carbamate); 5.72 (m, 1H, CH_); 5.49 (br s, 1H, NH
amide); 5.05 (m, 3H, _CH2 and H12); 4.55 (m, 1H, H3); 3.69 (t, 2H, CH2-NH); 2.22 (s, 6H CH3);
2.10–0.75 (m, 26H, steroidal CH and CH2); 0.84 (s, 3H, CH3); 0.71 (d, 3H, CH3); 0.68 (s, 3H, CH3).
13C NMR (200 MHz, CDCl3, δ): 173.31 (amide C_O), 153.33 (carbamate C_O), 152.34 (carbamate
C_O), 140.63 (aromatic), 136.63 (aromatic), 137.64 (aromatic), 135.03 (aromatic), 134.13 (C_CH2),
124.93 (aromatic), 122.63 (aromatic), 117.03 (aromatic), 116.31 (aromatic), 116.4 (10C_CH2), 76.98
(C12), 75.34 (C3), 49.60, 47.69, 45.22, 41.99, 41.80, 35.63, 34.96, 34.66, 34.44, 34.04, 33.54, 32.46,
31.56, 27.40, 26.85, 26.74, 26.01, 25.86, 23.47, 22.90, 21.32, 17.66, 12.46. IR (KBr): 3438, 3075, 2936,
2862, 1711, 1647, 1590, 1536, 1449, 1409, 1296, 1243, 1216, 1190. Anal. calcd for C43H57Cl2N3O5: C,
67.35; H, 7.49; Cl, 9.25; N, 5.48. Found: C, 67.40; H, 7.52; Cl, 9.27; N, 5.50.
3.5. 3,12-Diarylcarbamoyl-N-(3-trimethoxysilylpropylthio)propyl-deoxycholan-24-amide 5a–d: gene-
ral procedure
To a solution of 5, 1.4 mmol in dry CHCl3 (10 ml) freshly distilled 3-mercaptopropyltrimethoxysilane
(1.3 ml, 7 mmol) and AIBN (0.05 g, 0.3 mmol) were added and the mixture was stirred under reflux
for 20 h. The solvent was eliminated by evaporation under reduced pressure and the residual oil was

3362
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
dispersed in pentane (40 ml), giving a solid which was filtered and washed with pentane (5×30 ml). The
pure product 5a–d was obtained in quantitative yield.
3.5.1. 3,12-Bis-(3,5-dimethylphenyl)carbamoyl-N-(3-trimethoxysilylpropylthio)propyl-deoxycholan-24-
amide 5a
26
1
Yield 1.29 g. Mp 89–91°C. [α] =71.6 (c=1, CH2Cl2). H NMR (200 MHz, CDCl3): 7.04 (s, 2H,
D
aromatic); 6.89 (s, 2H, aromatic); 6.65 (br s, 1H, NH carbamate); 6.65 (s, 1H, aromatic); 6.60 (s, 1H,
aromatic); 6.48 (sl s, 1H, NH carbamate); 5.52 (br s, 1H, NH amide); 5.08 (br s, 1H, H12); 4.58 (m, 1H,
H3); 3.57 (s, 9H, OCH3); 3.33 (dd, 2H, CH2-CO); 2.55 (t, 4H, CH2-S); 2.20 (s, 6H CH3); 2.18 (s, 6H,
CH3); 2.10–0.75 (m, 32H, steroidal CH and CH2, chain CH2); 0.82 (s, 3H, CH3); 0.79 (d, 3H, CH320);
0.68 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, δ): 173.43 (amide C_O), 153.11 (carbamate C_O),
138.76 (aromatic), 138.64 (aromatic), 138.00 (aromatic), 137.82 (aromatic), 124.96 (aromatic), 116.31
(aromatic), 76.74 (C12), 75.12 (C3), 50.51, 50.46, 49.56, 47.66, 45.27, 41.61, 38.66, 35.64, 34.36, 34.74,
34.43, 34.07, 33.60, 32.59, 31.60, 29.41, 29.21, 27.44, 26.88, 26.03, 25.67, 23.49, 22.98, 21.36, 21.32,
17.66, 12.47. IR (KBr): 3331, 3075, 2936, 2861, 1725, 1704, 1650, 1613, 1549, 1447, 1378, 1271, 1223,
1191, 1159, 1079, 1015. Anal. calcd for C51H79N3O8SSi: C, 66.41; H, 8.63; N, 4.56; S, 3.48; Si, 3.04.
Found: C, 66.45; H, 8.65; N, 4.58; S, 3.45; Si, 3.05.
3.5.2. 3,12-Bis-(3,5-dichlorophenyl)carbamoyl-N-(3-trimethoxysilylpropylthio)propyl-deoxycholan-24-
amide 5b
Yield 1.4 g. Mp 110–112°C. [α]28=78.3 (c=0.95, CH2Cl2). 1H NMR (200 MHz, CDCl3): 7.42 (d, 2H,
D
aromatics); 7.29 (d, 3H, aromatics and NH carbamate); 6.98 (t, 1H, aromatic); 6.96 (t, 1H, aromatic);
6.78 (br s, 1H, NH carbamate); 5.60 (t, 1H, NH amide); 5.02 (br, 2H, H12); 4.54 (m, 1H, H3); 3.48 (s,
9H, OCH3); 3.26 (dd, 2H, CH2-NH); 2.48 (t, 4H, CH2-S); 2.30–0.75 (m, 32H, steroidal CH and CH2,
chain CH2); 0.82 (s, 3H, CH3); 0.78 (d, 3H, CH320); 0.64 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3,
δ): 173.64 (amide C_O), 152.76 (carbamate C_O), 152.63 (carbamate C_O), 140.33 (aromatic),
140.02 (aromatic), 135.28 (aromatic), 135.20 (aromatic), 123.13 (aromatic), 116.96 (aromatic), 116.68
(aromatic) 76.39 (C12), 76.00 (C3), 50.54, 49.65, 47.69, 45.28, 41.79, 36.76, 35.58, 34.99, 34.48, 34.02,
33.69, 33.47, 31.62, 29.47, 29.13, 27.51, 26.69, 25.91, 23.44, 22.95, 18.30, 17.65, 12.42. IR (KBr): 3342,
3085, 2936, 2861, 1734, 1709, 1653, 1634, 1589, 1538, 1449, 1437, 1410, 1378, 1306, 1247, 1215, 1191,
1162, 1114, 1071. Anal. calcd for C47H67Cl4N3O8SSi: C, 56.23; H, 6.73; Cl, 14.12; N, 4.19; S, 3.19; Si,
2.80. Found: C, 56.26; H, 6.74; Cl, 14.15; N, 4.20; S, 3.18; Si, 2.81.
3.5.3. 3-(3,5-Dimethylphenyl)carbamoyl-12-(3,5-dichlorophenyl)carbamoyl-N-(3-trimethoxysilyl-
propylthio)propyl-deoxycholan-24-amide 5c
25
1
Yield 1.34 g. Mp 107–110°C. [α] =60.0 (c=0.98, CH2Cl2). H NMR (200 MHz, CDCl3, δ): 7.42
D
(br s, 3H, aromatic and NH carbamate); 6.89 (br s, 3H, aromatic); 6.60 (s, 1H, aromatic); 6.55 (br s, 1H,
carbamate); 5.42 (br s, 1H, NH amide); 5.08 (br s, 1H, H12); 4.58 (m, 1H, H3); 3.57 (s, 9H, OCH3); 3.33
(dd, 2H, CH2-CO); 2.55 (t, 4H, CH2-S); 2.10–0.75 (m, 32H, steroidal CH and CH2, chain CH2); 0.79 (d,
3H, CH320); 0.77 (s, 3H, CH3); 0.68 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, δ): 173.65 (amide
C_O), 153.34 (carbamate C_O), 152.67 (carbamate C_O), 138.56 (aromatic), 137.61 (aromatic),
135.04 (aromatic), 124.89 (aromatic), 122.51 (aromatic), 116.96 (aromatic), 116.17 (aromatic), 76.99
(C12), 75.26 (C3), 50.47, 49.25, 47.57, 45.11, 41.81, 38.68, 35.38, 34.96, 34.29, 35.80, 33.67, 32.71,
31.53, 29.34, 29.09, 27.44, 27.02, 26.68, 25.93, 25.50, 23.34, 22.84, 21.38, 17.58, 12.44. IR (KBr): 3326,
3085, 2957, 2861, 1731, 1704, 1650, 1586, 1533, 1442, 1410, 1323, 1260, 1218, 1191, 1154, 1095, 1015.

A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
3363
Anal. calcd for C49H73Cl2N3O8SSi: C, 61.10; H, 7.64; Cl, 7.36; N, 4.36; S, 3.33; Si, 2.92. Found: C,
61.16; H, 7.63; Cl, 7.34; N, 4.37; S, 3.32; Si, 2.90.
3.5.4. 3-(3,5-Dichlorophenyl)carbamoyl-12-(3,5-dimethylphenyl)carbamoyl-N-(3-trimethoxysilyl-
propylthio)propyl-deoxycholan-24-amide 5d
25
1
Yield 1.34 g. Mp 105–107°C. [α] =77.3 (c=0.85, CH2Cl2). H NMR (200 MHz, CDCl3): 7.21
D
(d, 2H, aromatic); 7.02 (s, 2H, aromatic); 6.92 (t, 1H, aromatic); 6.80 (br s, 1H, carbamate); 6.72 (br
s, 1H, carbamate); 6.62 (s, 1H, aromatic); 5.64 (br s, 1H, NH amide); 5.00 (br s, 1H, H12); 4.55
(m, 1H, H3); 3.50 (s, 9H, OCH3); 3.24 (dd, 2H, CH2-NH); 2.46 (t, 4H, CH2-S); 2.10–0.75 (m, 32H,
steroidal CH and CH2, chain CH2); 0.84 (s, 3H, CH3); 0.71 (d, 3H, CH3); 0.68 (s, 3H, CH3). 13C
NMR (200 MHz, CDCl3, δ): 173.46 (amide C_O), 153.24 (carbamate C_O), 152.51 (carbamate C_O),
140.56 (aromatic), 138.60 (aromatic), 137.24 (aromatic), 135.20 (aromatic), 124.96 (aromatic), 123.00
(aromatic), 116.57 (aromatic), 116.36 (aromatic), 76.73 (C12), 75.66 (C3), 49.54, 47.59, 45.23, 41.73,
38.64, 35.58, 34.94, 34.63, 34.39, 34.01, 33.54, 32.40, 31.34, 29.36, 29.16, 27.42, 26.81, 26.72, 26.01,
25.80, 23.45, 22.92, 22.84, 21.34, 17.69, 12.43. IR (KBr): 3331, 3085, 2936, 2861, 1720, 1651, 1586,
1538, 1453, 1410, 1373, 1245, 1218, 1191, 1079. Anal. calcd for C49H73Cl2N3O8SSi: C, 61.10; H, 7.64;
Cl, 7.36; N, 4.36; S, 3.33; Si, 2.92. Found: C, 61.14; H, 7.62; Cl, 7.38; N, 4.37; S, 3.34; Si, 2.93.
3.6. Preparation of the CSPs A–D: general procedure
A solution of the silane 5 (12 mmol) in 15 ml of dry toluene was added dropwise to 3.5 g of spherical
silica gel (200 Å, 5 µm), previously dried at 180°C at 0.1 mm Hg for 15 h, slurried in 15 ml of dry
toluene and the mixture was gently stirred at reflux for 24 h. The mixture, cooled at room temperature,
was filtered and washed with toluene (3×30 ml), dichloromethane (3×30 ml), methanol (3×30 ml), THF
(3×30 ml) and pentane (3×30 ml), then dried at 50°C at 0.1 mm Hg.
The amount of chiral selector linked to silica gel was determined by elemental analysis.
CSP A: C% 9.36; H% 1.44; N% 0.63 corresponding to 0.155 mmol/g and 0.442 µmol/m2.
CSP B: C% 10.11; H% 1.34; N% 0.68; Cl% 2.45 corresponding to 0.173 mmol/g and 0.494 µmol/m2.
CSP C: C% 8.62; H% 1.30; N% 0.61 corresponding to 0.148 mmol/g and 0.423 µmol/m2.
CSP D: C% 9.51; H% 1.38; N% 0.57 Cl% 1.16 corresponding to 0.154 mmol/g and 0.440 µmol/m2.
The derivatized silica gels were slurried in dichloromethane and packed into 25 cm (15 cm for CSP D)
stainless steel columns at 400 bar using acetone.
References
1. Satinder, A. In Chiral Separation: Applications and Technology; ACS: Washington, DC, 1997.
2. Chang, C.; Reid III, G. L.; Chen, S.; Chang, C. D.; Armstrong, D. W. Trends Anal. Chem. 1993, 12, 144.
3. Yashima, E.; Okamoto, Y. Bull. Chem. Soc. Jpn. 1995, 68, 3289.
4. Okamoto, Y. Chem.-Tech. 1987, 176.
5. Aubry, A. F.; Markoglou, N.; Descorps, V.; Felix, G.; Wainer, I. W. G. J. Chromatogr., A 1994, 695, 1.
6. (a) Welch, C. J. J. Chromatogr., A 1994, 666, 3. (b) Salvadori, P.; Rosini, C.; Pini, D.; Altemura, P.; Bertucci, C.; Uccello
Barretta, G. Chirality 1989, 1, 161. (c) Gasparrini, F.; Misiti, D.; Villani, C.; La Torre, F.; Sinibaldi, M. J. Chromatogr., A
1988, 457, 235. (d) Oi, S.; Ono, H.; Tanaka, H.; Masayuki, S.; Myano, S. J Chromatogr., A 1994, 679, 35.
7. (a) Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989, 89, 347. (b) Welch, C. J.; Pirkle, W. H. J. Liq. Chromatogr. 1992, 15,
1947.
8. D’Souza, L. D; Maitra, U. J. Org. Chem. 1996, 61, 9494.

3364
A. Iuliano et al. / Tetrahedron: Asymmetry 10 (1999) 3353–3364
9. (a) Li, Y.; Dias, J. R. Chem. Rev. 1997, 97, 283. (b) Wallimann, P.; Marti, T.; Fürer, A.; Diederich, F. Chem. Rev. 1997, 97,
1567. (c) Davis, A. P. Chem. Soc. Rev. 1993, 243. (d) Davis, A. P.; Gilmer, J. F.; Perry, J. J. Angew. Chem., Int. Ed. Engl.
1996, 35, 1312. (e) Davis, A. P.; Menzer, S.; Walsh, J. J.; Williams, D. J. J. Chem. Soc., Chem. Commun. 1996, 453.
10. (a) Maitra, U.; Mathivanan, P. Tetrahedron: Asymmetry 1994, 5, 1171. (b) Maitra, U.; Bag, B. G. J. Org. Chem. 1992, 57,
6979. (c) Maitra, U.; Mathivanan, P. J. Chem. Soc., Chem. Commun. 1993, 1469.
11. (a) Boyce, R.; Li, G.; Nestler, H. P.; Suenaga, T.; Still, W. C. J. Am. Chem. Soc. 1994, 116, 7955. (b) Cheng, Y.; Suenaga,
T.; Still, W. C. J. Am. Chem. Soc. 1996, 118, 1813.
12. Vaton-Chanvrier, L.; Peulon, V.; Combret, Y.; Combret, J. Chromatographia 1997, 46, 613.
13. Bellini, A. M.; Quaglio, M. P.; Guarneri, M.; Cavazzini, G. Eur. J. Med. Chem. 1983, 18, 185.
14. (a) Rosini, C.; Bertucci, C.; Pini, D.; Altemura, P.; Salvadori, P. Tetrahedron Lett. 1985, 28, 3361. (b) Lienne, M.;
Macaudière, P.; Caude, M.; Rosset, R.; Tambuté, A. Chirality 1989, 1, 45. (c) Gasparrini, F.; Misiti, D.; Villani, C. J.
Org. Chem. 1995, 60, 4314.
15. Masamune, S.; Choy, W.; Petersen, J.; Sita, L. R. Angew. Chem., Int. Ed. Engl. 1985, 24, 1.
16. Uccello-Barretta, G.; Iuliano, A.; Franchi, E.; Balzano, F.; Salvadori, P. J. Org. Chem. 1998, 63, 9197.