New propanoyloxy derivatives of 5β-cholan-24-oic acid as drug absorption modifiers

Article abstract of DOI:10.1016/j.steroids.2013.02.001

A series of final twelve propanoyloxy derivatives of 5β-cholan-24-oic acid (O-propanoyl derivatives of cholic acid) as potential drug absorption modifiers (skin penetration enhancers, intestinal absorption promoters) was generated by multistep synthesis. Structure confirmation of all generated compounds was accomplished by ¹H NMR, ¹³C NMR, IR and MS spectroscopy methods. All the prepared compounds were analyzed using RP-TLC, and their lipophilicity (R_M) was determined. The hydrophobicity (log P), solubility (log S), polar surface area (PSA) and molar volume (MV) of the studied compounds were also calculated. All the target compounds were tested for their in vitro transdermal penetration effect and as potential intestinal absorption enhancers. The cytotoxicity of all the evaluated compounds was evaluated against normal human skin fibroblast cells. Their anti-proliferative activity was also assessed against human cancer cell lines: T-lymphoblastic leukemia cell line and breast adenocarcinoma cell line. One compound showed selective cytotoxicity against human skin fibroblast cells and another compound possessed the highest cytotoxicity against all the tested cell lines. Only one compound expressed anti-proliferative effect on leukemia cancer cells without affecting the growth of normal cells, which should be promising in potential development of new drugs. Most of the target compounds showed minimal anti-proliferative activity (IC₅₀ > 37 μM), indicating they would have moderate cytotoxicity when administered as chemical absorption modifiers. The relationships between the lipophilicity/polarity and the chemical structure of the studied compounds as well as the relationships between their chemical structure and enhancement effect are discussed in this article.

Full text of DOI:10.1016/j.steroids.2013.02.001

Steroids 78 (2013) 435–453
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New propanoyloxy derivatives of 5b-cholan-24-oic acid as drug
absorption modifiers
b
c
d
a
a,e
Lenka Coufalová a, , Lech Mrózek , Lucie Rárová , Lukáš Placek , Radka Opatrilová , Jirí Dohnal
,
⇑
ˇ
ˇ
ˇ
f
g
g
g
a,e,
⇑
ˇ
Katarína Král’ová , Oldrich Paleta , Vladimír Král , Pavel Drašar , Josef Jampílek
a Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic
b BorsodChem MCHZ, s.r.o., Chemicka 1/2039, 709 03 Ostrava-Marianske Hory, Czech Republic
c Centre of the Region Hana for Biotechnological and Agricultural Research, Department of Growth Regulators, Faculty of Science, Palacky University, Slechtitelu 11,
783 71 Olomouc, Czech Republic
d Pragolab s.r.o., Nad Krocinkou 55/285, 190 00 Prague 9, Czech Republic
e Research Institute for Pharmacy and Biochemistry, Lidicka 1879/48, 602 00 Brno, Czech Republic
f Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
g Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of final twelve propanoyloxy derivatives of 5b-cholan-24-oic acid (O-propanoyl derivatives of
cholic acid) as potential drug absorption modifiers (skin penetration enhancers, intestinal absorption pro-
moters) was generated by multistep synthesis. Structure confirmation of all generated compounds was
accomplished by 1H NMR, 13C NMR, IR and MS spectroscopy methods. All the prepared compounds were
analyzed using RP-TLC, and their lipophilicity (RM) was determined. The hydrophobicity (log P), solubility
(log S), polar surface area (PSA) and molar volume (MV) of the studied compounds were also calculated.
All the target compounds were tested for their in vitro transdermal penetration effect and as potential
intestinal absorption enhancers. The cytotoxicity of all the evaluated compounds was evaluated against
normal human skin fibroblast cells. Their anti-proliferative activity was also assessed against human can-
cer cell lines: T-lymphoblastic leukemia cell line and breast adenocarcinoma cell line. One compound
showed selective cytotoxicity against human skin fibroblast cells and another compound possessed the
highest cytotoxicity against all the tested cell lines. Only one compound expressed anti-proliferative
effect on leukemia cancer cells without affecting the growth of normal cells, which should be promising
in potential development of new drugs. Most of the target compounds showed minimal anti-proliferative
Received 17 September 2012
Received in revised form 18 January 2013
Accepted 9 February 2013
Available online 19 February 2013
Keywords:
Cholic acid derivatives
Lipophilicity
Solubility
Transdermal penetration enhancers
Intestinal absorption promoters
In vitro cytotoxicity
activity (IC50 > 37 lM), indicating they would have moderate cytotoxicity when administered as chemical
absorption modifiers. The relationships between the lipophilicity/polarity and the chemical structure of
the studied compounds as well as the relationships between their chemical structure and enhancement
effect are discussed in this article.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
during discovery and development, since insufficient solubility
may compromise other property assays, mask additional undesir-
Development in the field of pharmaceutical administration has
resulted in the discovery of highly sophisticated drug delivery sys-
tems that allow for the maintenance of a constant drug level in an
organism. Contrary to these revolution biopharmaceutical results,
over the last ten years, the number of poorly soluble drugs has
steadily increased. Literature states that about 60% of all drugs
coming directly from synthesis are nowadays poorly soluble. Com-
pounds with insufficient solubility carry a higher risk of failure
able properties, influence both pharmacokinetic and pharmacody-
namic properties of the compound and finally may affect the
developability of the compound. Poor solubility in water correlates
with poor bioavailability. If there is no way to improve drug solu-
bility, it will not be able to be absorbed from the gastrointestinal
tract into the bloodstream and reach the site of action [1,2].
Modification/optimization of unfavorable physico-chemical
properties of these drugs is possible through increasing their water
solubility or improving permeability. Generally, these strategies are
based on a few fundamental concepts: change of ionizability, lipo-
philicity, polarity or change of hydrogen bond donors or acceptors.
Pre-formulation/formulation can be another and mostly successful
strategy for improving aqueous solubility and/or permeability and
⇑
Corresponding authors. Tel.: +420 72125 6271 (J. Jampílek), +420 54156 2923
(L. Coufalová).
E-mail addresses: coufaloval@vfu.cz (L. Coufalová), josef.jampilek@gmail.com (J.
Jampílek).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.02.001

436
L. Coufalová et al. / Steroids 78 (2013) 435–453
subsequently bioavailability. For example, selection of a suitable
salt, particle size reduction (till nano size) connected with an in-
crease of the surface area, change of polymorphic forms, selection
of appropriate excipients to function as solubilizers/transporters
(surfactants or pharmaceutical complexing agents, permeability
enhancers) can be used for the oral dosage form [3]. One of possibil-
ities to modify/optimize drug unfavorable physico-chemical prop-
erties is to use absorption modifiers, e.g. bile acid derivatives [4–8].
Cholic acid is one of the most important human bile acids. Bile
acid derivatives/analogs are an important class of compounds with
a range of pharmacological activities. Nontoxic bile acid/salt deriv-
atives (as amphiphilic compounds) are used widely in drug formu-
lations as excipients (intestinal absorption enhancers, promoters)
and can influence gastrointestinal solubility, absorption and chem-
ical/enzymatic stability of drugs [8–17]. Cholic acid derivatives
were studied also as transdermal penetration enhancers [8,18–
26]. The reason for their effect may be their specific features in sol-
vation and self-assembly [27–35].
Transdermal therapeutic systems are an excellent alternative to
conventional pharmaceutical administration forms. However, the
application of transdermal drug delivery faces the problem of
insufficient or no penetration of active pharmaceutical substances
through the skin, as the outermost layer of skin, namely the stra-
tum corneum (SC), forms a strong barrier for most of exogenous
substances including drugs [6,26]. Transdermal penetration
enhancers are special pharmaceutical excipients that interact with
skin components, to increase absorption of drugs to blood circula-
tion after topical application. Numerous compounds of different
chemical structures were evaluated as penetration enhancers and
several possible mechanisms of action of enhancers have been
hypothesized, but exact mechanisms have not been elucidated
[26]. Transdermal chemical penetration enhancers are compounds
which can partition into and interact with the SC constituents
when incorporated into a transdermal formulation, thereby reduc-
ing the resistance of the skin to drug diffusion [26].
The multistep synthesis of a series of long chain propanoyloxy
derivatives of 5b-cholan-24-oic acid prepared as new potential
transdermal chemical penetration enhancers and/or as intestinal
drug absorption modifiers is described herein. This study is based
on recently published results of the enhancement effect of cholic
acid derivatives O-substituted by mono-, di- and tri-acyl C4, C10
and C16 linear saturated chains [8]. The design of all new long-
chain substituents of hydroxyl moieties of cholic acid is based on
isosteric replacement of oxygen for carbon with the aim to increase
the polar surface area and thus influence lipophilicity, polarity and
interactions with components in the SC as well as in the phospho-
lipid membrane.
Various acyl chain lengths (diethyleneglycol, decanoyl-diethyl-
eneglycol, bis(decanoyl)-glycerol, tocopherol) as well as different
degrees (mono-, di- and tri-substituted derivatives) and positions
(3 and/or 7 and/or 12 positions) of substitution of the compounds
discussed in this paper impart specific solvation and surface
features influencing structural modifications of biological
membranes. The traditional lipophilicity parameter, log P, is a
well-known physico-chemical descriptor widely used in QSAR
analysis. In some experimental studies of penetration enhance-
ment, the lipophilicity (non-polarity) of enhancers was measured
and the corresponding relationship between enhancer lipophilicity
and penetration enhancement potency was investigated [8,26,36–
38]. Therefore both the experimentally determined lipophilicity
(logarithm of the retention factor, RM values) and calculated lipo-
philicity (log P) as well as other molecular descriptors of all the fi-
nal compounds were investigated in this article.
Primary in vitro screening of transdermal penetration effect of
all the final synthesized compounds was performed using a Franz
cell [26,39], and intestinal absorption enhancement effect was
evaluated using PAMPA (parallel artificial membrane permeability
assays) experiments [40–42]. All the discussed compounds were
evaluated for their anti-proliferative activity against the T-lympho-
blastic leukemia cell line and the breast adenocarcinoma cell line
as well as for their cytotoxicity against normal human skin fibro-
blast cells. The relationships between the lipophilicity/solubility
and the chemical structure of the studied compounds as well as
the relationships between their chemical structure and activity
(enhancement effect) (SAR) are discussed in this article.
2. Result and discussion
2.1. Chemistry
Various synthetic pathways leading to preparation of acyl chlo-
rides 4, 8, 13 and 16 are described in Schemes 1–4. The hydroxyl
moieties of benzyl 3a,7a,12a-trihydroxy-5b-cholanoate (benzyl
cholate, 17) were subsequently acylated by those acyl chlorides
[8,43].
The reaction of mono benzyl alcohol 1 with succinic acid anhy-
dride provided benzylated acid 2 [44] and subsequent hydrogenol-
ysis of the benzyl ether group in the presence of palladium catalyst
yielded hydroxy acid 3. The reaction of benzylated acid 2 with oxa-
lyl chloride gave acyl chloride 4 that was used for acylation of ben-
zyl cholate (17) and preparation of O-substituted cholic acid
derivatives, see Scheme 1.
The free hydroxyl group of monobenzyl alcohol 1 was acylated
with decanoyl chloride providing benzyl derivative 5. Hydrogenol-
ysis of this compound afforded hydroxy derivative 6, which by
reaction with succinic acid anhydride led to 7. This acid was trea-
ted with oxalyl chloride to give acyl chloride 8, see Scheme 2.
Acylation of benzylated glycerol 9 [45] with decanoyl chloride
gave ester 10. Cleavage of the benzyl group in 10 led to alcohol
11, which by treatment with succinic acid anhydride gave 12.
The latter by reaction with oxalyl chloride afforded acid chloride
13, see Scheme 3.
(+/ꢀ)-
a
-Tocopherol hemisuccinate 15 was prepared by a reac-
-tocopherol (14) with succinic acid anhydride [46]
tion of (+/ꢀ)-
a
and then converted to acyl chloride 16 [47] by the standard proce-
dure with oxalyl chloride, see Scheme 4.
The reaction of benzyl 3a,7a,12a-trihydroxy-5b-cholan-24-
oate (benzyl cholate, 17) [43] with acyl chlorides in the presence
O
O
b
a
O
OH
O
O
OH
Cl
HO
O
BnO
OH
BnO
O
3
O
1
2
O
c
O
O
BnO
O
O
4
Scheme 1. Synthesis of 3-[2-(2-hydroxyethoxy)ethoxycarbonyl]propanoic acid (3) and 3-[2-(2-benzyloxyethoxy)ethoxycarbonyl]propanoyl chloride (4): (a) succinic acid
anhydride, DMAP, CH2Cl2, 22 °C; (b) H2, Pd/C, MeOH, 22 °C; (c) (COCl)2, toluene, 22 °C.

L. Coufalová et al. / Steroids 78 (2013) 435–453
437
a
O
O
O
c
b
HO
OBn
C9H19COO
O
OBn
d
C9H19COO
OH
1
5
6
O
c
O
OH
O
Cl
C9H19COO
O
C9H19COO
O
O
O
7
8
Scheme 2. Synthesis of 3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]propanoic acid (7) and 3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]propanoyl chloride (8): (a)
C9H19COCl, pyridine, DMAP, CH2Cl2, 22 °C; (b) H2, Pd/C, AcOEt, 40 °C; (c) succinic acid anhydride, DMAP, CH2Cl2, 22 °C; (d) (COCl)2, toluene, DMF, 22 °C.
HO
HO
C9H19OCO
C9H19OCO
C9H19OCO
C9H19OCO
OBn
OBn
OH
Cl
c
a
b
10
11
9
O
O
O
OH
c
C9H19OCO
C9H19OCO
C9H19OCO
C9H19OCO
d
O
O
O
13
12
Scheme 3. Synthesis of 3-[2,3-bis(decanoyloxy)propyloxycarbonyl]propanoic acid (12) and 3-[2,3-bis(decanoyloxy)propyloxycarbonyl]propanoyl chloride (13): (a)
C9H19COCl, pyridine, DMAP, CH2Cl2, 22 °C; (b) H2, Pd/C, 22 °C; (c) succinic acid anhydride, DMAP, CH2Cl2, 22 °C; (d) (COCl)2, toluene, 22 °C.
O
OH
O
a
b
OH
O
O
O
3
3
14
15
O
O
b
Cl
O
O
3
16
Scheme 4. Synthesis of (+/ꢀ)-
a-tocopherol hemisuccinate (15) and 3-{[2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl]oxycarbonyl}propanoyl chloride (16):
(a) succinic acid anhydride, DMAP, CH2Cl2, 22 °C; (b) (COCl)2, toluene, 22 °C.
of 4-dimethylaminopyridine (DMAP) under mild conditions fol-
their potency of penetration enhancement was investigated [26].
In the current investigation we examined both the calculated lipo-
philicity (log P) and the experimental lipophilicity RM data [48] as
well as calculated solubility (log S), polar surface area (PSA) and
molar volume (MV) of all compounds to determine if these factors
play a role in their penetration enhancement potency.
lowed by hydrogenolysis of protecting groups provided
3a-
mono(acyloxy) derivatives 19, 26, 32 and 38, see Scheme 5.
The preparation of 7
and 40 included the protection of 3
ter 17 by the benzyloxycarbonyl group, resulting in compound 20
[8] and subsequent acylation of both free 7 ,12 -dihydroxy moie-
a,12a
-bis(acyloxy) derivatives 22, 28, 34
a-hydroxy group of benzyl es-
a
a
However, the algorithms used in calculations of descriptors,
especially log P and log S, in principle do not take into account
the aspects of configuration and conformation. The positions of
substituents are implemented in the calculation protocol, however,
the prediction depends on the number of similar compounds in the
database. Hence, having in mind the importance of permeability
and solubility (polarity) for biological activity [49], this study com-
pares calculated log P and log S values with the related experimen-
tal parameter, the RM (linear physico-chemical descriptor in TLC)
values of the final derivatives, as a measure of their lipophilicity.
The RM values were determined by reverse phase thin layer chro-
matography (RP-TLC). RM represents the logarithmic ratio of dis-
tances between solute spot and solvent front on the one hand
and the migration distance of the solute on the other hand [48].
Solubility was estimated using the software that is applied by
many industrial companies, ACD/Solubility DB. This program cal-
culates aqueous solubility values at any pH under the standard
conditions (and zero ionic strength). The accuracy of calculations
for simple structures is usually better than 0.2–0.5 logarithmic
units. So, it is not derived from log P and takes into account not
only the pH (solubility as a function of pH) but compares the frag-
mental estimations [50] with experimental material from ca. 6000
compounds databased. Nevertheless, it is important to note that all
ties. Finally, the benzyloxycarbonyl group was selectively cleaved
by hydrogenolysis on palladium catalyst with simultaneous depro-
tection of the carboxy group. This method, including the protection
of 3
was developed for the selective preparation of 7
noyloxy)-3 -hydroxy-5b-cholan-24-oic acid [8]. Hydrogenolysis
a-hydroxy group in ester 17 by the benzyloxycarbonyl moiety,
a
,12 -bis(deca-
a
a
of the benzylether group in diethyleneglycol series was success-
fully performed only in hydrogen atmosphere on palladium cata-
lyst, see Scheme 5.
3a,7a,12a-Tris(acyloxy) derivatives 24, 30, 36 and 42 were pre-
pared by a reaction of benzyl cholate 17 with acyl chlorides in the
presence of benzyltriethylammonium chloride (BTEAC) as a phase
transfer catalyst with subsequent hydrogenolysis of the protecting
group, see Scheme 5.
2.2. Lipophilicity and solubility of the prepared compounds
The well-known physico-chemical descriptors such as lipophil-
icity, solubility, polar surface area and molar volume are largely
employed during quantitative structure–activity relationship anal-
ysis. In a number of studies examining penetration enhancement,
the relationship between lipophilicity and other descriptors and

438
L. Coufalová et al. / Steroids 78 (2013) 435–453
OH
RCOO
RCOO
C2H4COOBn
d
C2H4COOBn
b
C2H4COOH
BnOCO
BnOCO
HO
OH
OCOR
OCOR
H
H
H
20
21,27,33,39
22,28,34,40
c
OH
OH
OH
C2H4COOBn
a
C2H4COOBn
b
C2H4COOH
HO
ROCO
ROCO
OH
OH
OH
H
H
H
17
18,25,31,37
19,26,32,38
RCOO
C2H4COOH
RCOO
C2H4COOBn
d
b
ROCO
OCOR
ROCO
OCOR
H
H
24,30,36,42
23,29,35,41
18,21,23
19,22,24
R= Bn(OCH2CH2)2OCOCH2CH2
R= H(OCH2CH2)2OCOCH2CH2
25,26,27,28,29,30 R= C9H19CO(OCH2CH2)2OCOCH2CH2
31,32,33,34,35,36 R= C9H19OCOCH2CH(C9H19OCO)CH2OCOCH2CH2
37,38,39,40,41,42 R= tocopherylOCOCH2CH2
Scheme 5. Synthesis of target mono-, bis- and tris(acyl) derivatives (a) RCOCl, DMAP, CH2Cl2, 22 °C; (b) HCOONH4, Pd/C or H2, Pd/C, 40 °C; (c) BnOCOCl, pyridine, 0 °C; (d)
RCOCl, BTEAC, CaH2, toluene, 112 °C.
log P and log S values calculated using various programmes should
be understood as approximate. The results are shown in Table 1
and illustrated in Fig. 1.
anomalies were observed. Within Group 1 solubility decreased
with the decreasing number of substituents, i.e. 3 (acid) > 24
(tris) > 22 (bis) > 19 (mono), while within Groups 2–4 bis(substi-
tuted) derivatives 28, 34 and 40 unexpectedly expressed much
lower polarity/solubility than the mono(substituted) (Group 2
and Group 3) or tris(substituted) (Group 4) compounds. This trend
is also reflected in poor correlation of polarity/solubility (log S
data) with the experimentally determined lipophilicity (RM), see
Fig. 1.
All these differences could be probably explained by different
solvation and other types of non-covalent interactions (especially
van der Waals interactions, hydrogen bonds, dipole–dipole interac-
tions) of hydroxyl moieties in the C(3), C(7) and C(12) positions of the
steroidal skeleton as well as free hydroxyl moieties in aliphatic
chains in the case of diethyleneglycols (Group 1). These interac-
tions are extremely important for the significantly different enhan-
cer effect of these bis(substituted) derivatives, see below. All these
unexpected differences influence enhancement effect. It is not pos-
sible to explain these specific properties/behavior of bis(substi-
tuted) derivatives using PSA that logically increases the number
of polar atoms in the molecule.
The compounds under investigation could be divided into four
groups based on the substituents on the steroidal scaffold: Group
1 includes diethyleneglycols 19, 22 and 24 and starting acid 3;
Group 2 contains decanoyl-diethyleneglycols 26, 28 and 30 and
starting acid 7; Group 3 is composed of bis(decanoyl)-glycerols
32, 34 and 36 and starting acid 12; Group 4 includes tocopherol
substituted compounds 38, 40 and 42 and starting acid 15.
The calculated log P data and the determined RM values corre-
spond to the expected trend in lipophilicity, increasing within
the series of the evaluated compounds as follows: Group 1 (dieth-
yleneglycols) < Group
2 (decanoyl-diethyleneglycols) < Group 3
[bis(decanoyl)-glycerols] < Group 4 (tocopherol derivatives). This
dependence is approximately linear. As expected, compound 42
possessed the highest lipophilicity. Contrary to all expectations
and calculated log P and log S data, compound 24 showed the low-
est lipophilicity (RM). According to ACD, the lowest lipophilicity
was computed for compound 3 (log P = ꢀ0.42), and the lowest
polarity/solubility was computed for compound 40 (log
S = ꢀ11.0). Noteworthy dissimilarity between Group 1 and other
groups can be found. Starting acid 3 in Group 1 possessed higher
lipophilicity than substituted 5b-cholan-24-oic acid derivatives
19, 22 and 24 within this group, and it can be stated that within
this series lipophilicity decreased with substitution, i.e. that lipo-
philicity increased subsequently: 24 (trisubstituted) < 22 (disubsti-
tuted) < 19 (monosubstituted) < 3 (starting acid). In general, it can
be stated for Groups 2–4 that the starting acid showed the lowest
lipophilicity (according to the RM values) together with the highest
solubility (according to the log S values), i.e. that lipophilicity in-
creased subsequently: starting acid < mono(substituted) com-
pound < bis(substituted) compound < tris(substituted) compound.
As expected, the highest solubility/polarity (log S) was shown
by Group 1, and polarity decreased with the chain length:
Group 1 > Group 2 > Group 3 > Group 4, nevertheless interesting
Generally, it could be concluded that the prediction power of
used experimental RM or calculated values for extrapolation of
transport modifications may be a good tool for searching potential
absorption modifiers.
2.3. In vitro screening of transdermal penetration-enhancing effect
The penetration enhancement effect of the prepared com-
pounds was evaluated using theophylline as a model penetrant
and propylene glycol/water 1:1 (v/v) as a donor vehicle by means
of a Franz cell [26,39]. Theophylline was used as a model drug of
medium polarity (log P = ꢀ0.06) [51,52], as it has been extensively
studied in transdermal penetration experiments [53,54]. Most of
the studies involved the use of propylene glycol (PG) or its mixture
with water or ethanol as a donor vehicle. Previous studies have

L. Coufalová et al. / Steroids 78 (2013) 435–453
439
Table 1
Comparison of determined Rm with calculated lipophilicity (log P) and solubility (log S) values; calculated values of polar surface area (PSA [Å2]) and molar volume (MV [cm3]).
(Log P, log S and MV were calculated using ACD ver. 12, PSA was calculated using CambrigeSoftware ChemBio3D, ver. 12.).
indicated that PG by itself (or a PG/water co-solvent system) does
not interfere with membranes, but rather exhibits a synergistic ef-
fect in combination with other penetration enhancers [55–57].
Porcine ear skin was selected for initial evaluation of enhancement
effect of prepared compounds as this tissue is a suitable in vitro
model of human skin [58,59]. Porcine skin has been shown to be
histologically and biochemically similar to human skin, therefore
full-thickness pig ear skin has been used in numerous
percutaneous absorption studies [60]. Nevertheless, for testing of
hydrophobic penetrants, dermatomed skin has been recommended
[61]. The skin penetration experiments were performed using sta-
tic Franz diffusion cells [39].
As it could be expected, the presented transdermal penetration
enhancement results of the target compounds and the data ob-
tained using PAMPA method (see below) are different (see Table 2,
where the enhancement ratios (ERs) are presented). The principal
difference is probably due to different properties of the transport
‘‘membrane’’; the PAMPA diaphragm is an artificial model of cell

440
L. Coufalová et al. / Steroids 78 (2013) 435–453
40.0
34.0
28.0
22.0
16.0
10.0
4.0
R = 0.9522
S
P
R = 0.7883
-2.0
-8.0
-14.0
-0.9
-0.4
0.1
0.6
1.1
1.6
2.1
2.6
RM
Fig. 1. Comparison of log P (squares, y-axis, positive values) and/or log S (triangles, y-axis, negative values) values (lipo/hydrophilicity properties) computed using two
programs (ACD/LogP DB, ACD/LogS DB) with the determined RM values.
lipid bilayer ‘‘simulating’’ intestine wall, whereas the ear porcine
skin is a much more complex naturally constructed barrier.
The effect of target 5b-cholan-24-oic acid derivatives on pene-
tration of theophylline through the porcine skin is presented in
Fig. 2 (dotted columns). Control experiments were run with only
theophylline in the donor vehicle in the absence of any enhancer.
The used system PG/water ensured entire solubility of theophyl-
line, which is crucial for evaluation of penetration of theophylline
through membranes. This solvent system also provides stable
emulsion or microsuspension of the investigated enhancer
[8,26,36–38,51–57]. A tested enhancer cannot be completely dis-
solved, similarly as in topical formulations, because by penetration
of enhancer to the skin the dynamic balance (dissolved/undis-
solved part) is continuously changing. The investigated enhancer
should be understood as a pharmaceutical excipient influencing
only drug transport across the skin barrier [5,6].
[bis(decanoyl)-glycerols] and Group 4 (tocopherols) is low in com-
parison with Group 1 and Group 2. This observation confirms the
fact that highly lipophilic branched long-chains or chains contain-
ing cycles (aromatic or saturated ring) are not preferred substitu-
ents of transdermal chemical penetration enhancers [26].
From the results presented in Table 2 and Fig. 2 (dotted col-
umns) it is evident that transdermal penetration-enhancement
effect is strongly dependent on balanced lipo/hydrophilic proper-
ties (lipophilicity/solubility-polarity) of individual discussed
enhancers due to their optimal interaction with skin components.
The dependences of the transdermal penetration-enhancement
effect (ER) on the lipophilicity expressed by experimentally
determined RM values or by log P as well as on the molar volume
(MV [cm3]) for the studied steroid-like compounds are shown in
Figs. 3A–C.
From the above mentioned Figs. 3A–C, where starting acids (3,
The highest enhancement ratios (ERs) were obtained with 19
[mono(diethyleneglycol)] and 24 [tris(diethyleneglycol)] deriva-
tives with ERs of 2.63 and 2.50, respectively, see Table 2, Fig. 2
(dotted columns).
The results of one-way analysis of the variance (ANOVA) test
complemented by the Bonferroni’s multicomparison test are pre-
sented in Table 2, where differences were considered significant
at P = 0.05. Whereas no significant differences between transder-
mal ER of some tested acids were found, namely between 7 and
15, considerable differences between determined ER were
estimated for studied steroid-like compounds. Due to structural
difference the starting acids cannot be compared with derivatives
of 5b-cholan-24-oic acid.
7, 12, 15) are not shown, it is evident that for 3
a-mono(substi-
tuted) and 3 ,7 ,12 -tris(substituted) compounds the transder-
a
a
a
mal ER enhancement effect linearly decreases with the increasing
lipophilicity or with the increasing molar volume. After the elimi-
nation of compound 32 (that showed lower ER than expected) the
correlation coefficient for linear regression including the rest seven
compounds was ꢀ0.967 for RM, ꢀ0.970 for log P and ꢀ0.923 for
MV. On the other hand, no definite dependence of ER of 7a,12a-
bis(substituted) compounds on the above mentioned descriptors
was found. The compounds 22, 34 and 40 showed significantly
lower enhancement effect than mono(substituted) and tris(substi-
tuted) derivatives, but it is problematic to explain the reason of the
too high effectiveness of compound 28.
Generally, it can be postulated that the highest enhancement ef-
fect within the individual series was shown by mono(substituted)
and tris(substituted) derivatives, whereas bis(substituted)
derivatives possessed the lowest enhancing effect, i.e. penetra-
tion-enhancement effect decreased as follows: mono(substi-
tuted) > tris(substituted) > bis(substituted) > starting acid, except
Group 3, where starting acid 12 showed higher ER value than
bis(substituted) derivative 34. While the most active compounds
were from Group 1, it seems that decanoyl-diethyleneglycol
(Group 2, compounds 26, 28, 30) is the most advantageous substi-
tuent, because this series expressed the highest well-balanced
enhancement ratios without any evident dependence of enhance-
ment effect on the position of the substituent in the steroid
skeleton. The penetration-enhancement effect of both Group 3
In our previous paper [8] we found that the dependence of ER
enhancing effect on log S was a mirror image to the dependence
of transdermal ER on lipophilicity. This was also confirmed in pres-
ent study (figure not shown), only with lower correlation coeffi-
cients. The definite dependence of transdermal ER on PSA (see
Fig. 3D) cannot be determined, however from Table 1 it can be
concluded that PSA value approx. 160 Å2 [compound 19,
mono(diethyleneglycol)] is the most advantageous for the highest
enhancement effect within the discussed types of substituents,
nevertheless within tris(substituted) derivatives secondary maxi-
mum can be found [PSA about 380 Å2 for compound 24,
tris(diethyleneglycol)].
The above described molecular descriptors characterize proper-
ties of the compounds, nevertheless a mechanism of the higher

L. Coufalová et al. / Steroids 78 (2013) 435–453
441
Table 2
The effect of target 5b-cholan-24-oic acid derivatives on the
penetration of theophylline through the artificial polyvinylidene
fluoride (PVDF) PAMPA membrane is presented in Fig. 2 (hatched
columns). Control experiments were run with only theophylline
in the donor vehicle in the absence of any enhancer. The used sol-
vents ensure solubility of theophylline as a model penetrating
compound, which is important for facilitation of penetration
through a membrane. This system also supports the stability of
the microsuspension of the tested enhancer. The same as for the
skin, the ratio between theophylline penetration with and without
an enhancer was determined for the selected concentration based
on the previous experience [36–38]. Only if some of evaluated po-
tential enhancers expressed significant enhancer effect, the influ-
ence of the enhancer concentration on enhancement effect would
be investigated.
Penetration in Franz cells is different from that in PAMPA tests,
because the real skin is used as a barrier in Franz cells. Another
parameter that may influence the measurements is solubility (sol-
vation) supramolecular superassembly properties of cholic acid
derivatives (donors), but explanation of this parameter is not the
aim of this study. The difference between both experiments can
also be seen in the geometrical arrangement of the experiment:
in the PAMPA method a donor is below the diaphragm, whereas
in the Franz cell the donor is above it. The greatest differences were
noticeable in the results of the starting acids and especially
bis(diethyleneglycol) derivative 22 and bis[bis(decanoyl)-glycerol]
derivative 34.
Enhancement ratios (ERs) of the prepared target compounds and in vitro anti-
proliferative activity/cytotoxicity of tested compounds on normal and cancer cell
lines. IC50 (lmol/l) assessed by Calcein AM assay of surviving cells (T-lymphoblastic
leukemia CEM, breast adenocarcinoma MCF7, human foreskin fibroblasts BJ). ERs data
for skin are expressed as mean SD (n = 5 experiments), ERs data for PAMPA are
expressed as mean SD (n = 4 experiments) and anti-proliferative activity of the
compounds are expressed as mean SD (n = 3 experiments). The means followed by
different letters are significantly different at P = 0.05. The starting acids were
compared separately from the final steroid-like compounds.
Comp. ERs
Skin
Cell lines IC50 [lmol/l]
PAMPA
CEM
MCF7
BJ
3
1.88 0.11y
2.63 0.47e
2.27 0.19x
2.45 0.20bc
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
>37
19
22
24
7
1.54 0.25ab 2.66 0.05cde
2.50 0.19e
1.47 0.26x
2.41 0.07e
2.75 0.24e
3.03 0.10y
2.72 0.09de
26
28
30
12
32
34
36
15
38
40
42
2.28 0.14de 2.86 0.03e
2.36 0.24de 2.68 0.15cde
1.60 0.18xy 2.43 0.19x
2.01 0.20cd 2.23 0.08a
26.6 2.9a >37
>37
>37
>37
>37
>37
>37
>37
>37
>37
18.5 1.4a
>37
>37
>37
>37
1.20 0.08a
2.67 0.17cde
1.99 0.41cd 2.53 0.10bcd >37
1.50 0.20x
2.26 0.12de 2.23 0.22a
1.61 0.20b 2.52 0.18bcd >37
1.85 0.25bc 2.36 0.10ab
>37
2.33 0.09x
26.9 7.7a 35.8 3.6 31.8 3.8b
>37
>37
>37
>37
>37
>37
>37
enhancement effect of
3a-mono(substituted) and 3a,7a,12a-
The highest enhancement ratios (ERs) were obtained for com-
pounds 28 [bis(decanoyl-diethyleneglycol)], 24 [tris(diethylene-
glycol)] and 26 [mono(decanoyl-diethyleneglycol)]: 2.86, 2.75
and 2.72, respectively. 3-[2-(2-Decanoyloxyethoxy)ethoxycar-
bonyl]propanoic acid (7) expressed the highest intestinal absorp-
tion-promoting effect (ER = 3.03). According to the above
presented data, see Table 2, Fig. 2 (hatched columns), it can be con-
cluded that most discussed compounds showed noteworthy intes-
tinal absorption-enhancing effect. It can be stated that trends of
intestinal permeability enhancement effect in this study are con-
trary to those of transdermal enhancement effect, as also discussed
in [8]. While transdermal enhancement effect was the highest for
mono(substituted) and tris(substituted) derivatives and the lowest
for bis(substituted) compounds, in PAMPA experiments bis(substi-
tuted) compounds showed high activities within individual series,
see Table 2, Fig. 2 (hatched columns). This fact can be probably
connected with the above discussed specific non-covalent interac-
tions of hydroxyl moieties, i.e. specific lipo/hydrophilic interactions
and properties (see Section 2.2).
tris(substituted) derivatives would be also explained by substitu-
tion of C(3) position of bile acid by an aliphatic chain, which can
simulate 3-O-cholesterol-esters that belong among major skin
components and thus, due to structural similarity, the discussed
compounds can easier intercalate between skin components in
the SC and disrupt ceramide–ceramide, ceramide–cholesterol or
cholesterol–cholesterol bonds and, by doing this, form a ‘‘channel’’
in the SC. Conversely, the free hydroxyl moiety in C(3) position of
bile acid (7a,12a-bis(substituted) derivatives) can interact with
skin components, which can result in more impermeable SC
[26,62].
2.4. In vitro screening of intestinal absorption-enhancing effect
(PAMPA experiments)
Parallel artificial membrane permeability assays (PAMPA) have
become a very useful and quite cheap tool for predicting intestinal
drug permeability and are well-suited as a ranking tool for the
assessment of compounds with passive intestinal transport mech-
anisms. An absorption study of binary mixtures or final formula-
tions is also possible on PAMPA plates [40–42].
The results of the one-way analysis of the variance (ANOVA)
test complemented by the Bonferroni’s multicomparison test are
presented in Table 2 where differences were considered significant
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
control
3
19
22
24
7
26
28
30
12
32
34
36
15
38
40
42
Compounds
skin
PAMPA
Fig. 2. Enhancement ratios (ERs) through porcine ear skin and PAMPA of the prepared target compounds. Control experiments used theophylline in the donor vehicle without
any enhancer. ERs data for skin were calculated from 5 experiments, ERs data for PAMPA were calculated from 4 experiments.

442
L. Coufalová et al. / Steroids 78 (2013) 435–453
B
2.8
19
A
2.8
2.6
19
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
24
30
24
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
38
-0.97033
26
26
30
-0.96691
28
38
28
36
42
32
42
32
36
40
22
22
40
34
34
0.0
5.0
10.0
15.0
20.0
log P
25.0
30.0
35.0
40.0
-0.9
-0.4
0.1
0.6
1.1
1.6
2.1
2.6
RM
C
D
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
19
19
24
26
24
30
-0.92317
30
28
26
38
38
28
32
36
36
32
42
22
42
40
22
40
34
34
100
150
200
250
PSA [Å2]
300
350
400
400
600
800
1000
1200
1400
1600
1800
MV [cm3]
Fig. 3. The dependences of transdermal penetration-enhancement effect ER on the lipophilicity RM (Fig. 3A), log P (Fig. 3B), molar volume (MV [cm3]) (Fig. 3C) and polar
surface area (PSA [Å2]) (Fig. 3D) of the studied steroid-like target compounds.
at P = 0.05. Whereas no significant differences between intestinal
ER of some tested acids were found, namely between 3, 12 and
15, considerable differences between determined ER were esti-
mated for studied steroid-like compounds. As mentioned above,
the starting acids cannot be compared with derivatives of
5b-cholan-24-oic acid due to structural difference.
were ꢀ0.973 (RM), ꢀ0.970 (log P) and ꢀ0.920 (MV). The mono-
substituted compounds 19, 32 and 38 showed significantly lower
enhancement effect than bis(substituted) and tris(substituted)
compounds. Considerably higher efficiency was shown by com-
pound 26. However, from four ER values that are available for
mono-substituted compounds it is not possible to determine
clearly whether in this case the dependence would also be bi-
phasic.
The definite dependence of ER on PSA (see Fig. 4D) the same as
the definite dependence of transdermal penetration-enhancement
effect ER on PSA (Fig. 3D) cannot be determined; however from Ta-
ble 1 it can be concluded that the optimum of PSA value was ap-
prox. 234 Å2 [compound 28, bis(decanoyl-diethyleneglycol)].
As all the decanoyl-diethyleneglycol (Group 2) and diethylene-
glycol derivatives (Group 1) expressed balanced transdermal and
intestinal permeability enhancement effect, contrary to the rest
of the discussed compounds, it can be suggested that these com-
pounds as amphiphilic compounds showed high hydrotropic effect,
and they can reduce the resistance of membranes to drug diffusion
[63,64].
The higher intestinal absorption enhancement effect of the dis-
cussed compounds compared with the transdermal penetration-
enhancement effect, especially low transdermal enhancement ef-
fect of bis(substituted) derivatives, can be also explained by a
‘‘cut-off’’ effect [8,65,66]. This ‘‘cut-off’’ effect can be observed for
n-alkyl substituted series of amphiphilic compounds. The inversion
of effect is connected with the structural diversity of the skin and
the PAMPA membrane. The hydrophobic parts of surfactants inter-
act with lipidic parts of biological membranes. However, the water
solubility of surfactants with longer alkyl chains is limited, and too
large values of surfactant partition coefficient do not enable the
Based on the results, decanoyl-diethyleneglycol (Group 2) seems
to be the most advantageous substituent of hydroxyl moieties of
cholic acid. The intestinal absorption-enhancing effect depending
on substituents decreased subsequently: Group 2 (decanoyl-dieth-
yleneglycols) > Group 1 (diethyleneglycols) > Group 3 [bis(deca-
noyl)-glycerols] > Group 4 (tocopherols). While all compounds
within Group 2 showed effect, within Group 3 and Group 4 only
di- and tri-substituted derivatives can be considered to have effect,
i.e. compounds with high lipophilicity and low polarity within indi-
vidual series. Starting acids 12 and 15 were more active than
mono(substituted) derivatives 32 and 38. A totally opposite trend
can be observed within Group 1. Tris(substituted) derivative 24
showed the highest intestinal absorption-enhancing effect, but this
compound possessed the highest hydrophilicity as well as polarity/
solubility. Similarly to the results obtained for ER-skin penetration
enhancing effect, also intestinal penetration enhancing effect signif-
icantly depended on the lipophilicity and the molar volume of tested
steroid-like compounds (Fig. 4A–C).
From the mentioned above Figs. 4A–C, where starting acids (3,
7, 12, 15) are not shown, it is evident that for di- and tri-substi-
tuted compounds the dependence of intestinal ER enhancement ef-
fect on the lipophilicity or molar volume shows bilinear course.
The highest ER penetration enhancing effect was observed for com-
pound 28 (RM = ꢀ0.15, log P = 12.85, MV = 949 cm3), and the corre-
lation coefficients evaluated for descending part of the dependence

L. Coufalová et al. / Steroids 78 (2013) 435–453
443
penetration of such molecules through hydrophilic (aqueous) re-
gions of biological membranes. Consequently, the final concentra-
tion of long-chain surfactants in the membrane will be lower than
that of surfactants with shorter alkyl chains, which results in the
loss of effect. It is suggested that the lateral expansion of the
phospholipid bilayer of biological membranes caused by the inter-
calation of long-chain amphiphilic molecules between phospho-
lipid molecules and the mismatch between their hydrocarbon
chains lengths result in the creation of free volume in the bilayer
hydrophobic region [66]. According to the free volume theory the
extent of membrane disturbance due to surfactant incorporation
depends on the size of free volume created under its alkyl chain,
which can be then filled up with chains of neighboring lipids, as
well as on the partition coefficient of the surfactants [65,67].
Therefore the most effective disturbance of the membrane and
thus the highest inhibitory effect will be exhibited by surfactants
with middle alkyl chain length ensuring not only sufficiently high
free volume under the alkyl chain but also a high concentration of
the surfactant in the membrane due to the suitable value of the
surfactant partition coefficient [68].
[tris(decanoyl-diethyleneglycol)] showed selective cytotoxicity
against human skin fibroblast cells. Based on these observations
both compounds cannot be used as penetration modifiers. But
compound 28 expressed cytotoxicity effect only against leukemia
cells without affecting the growth of normal BJ cells, which should
be promising in potential development of new anti-proliferative
drugs.
Other discussed tested compounds demonstrated poor anti-
proliferative effect (or insignificant cytotoxicity effect) against all
the cell lines, with IC50 values greater than 37 lmol/l, see Table 2.
These results suggest that the poor anti-proliferative activity of tar-
get compounds will lead to limited cytotoxicity if they are used
in vivo as absorption modifiers.
In the cytotoxicity test against BJ, the IC50 value of compound 30
(18.5
pound 15 (31.8
other hand, no significant difference was found between IC50 val-
ues against CEM cells of compound 28 (26.6 mol/l) and com-
l
mol/l) significantly differed from the IC50 value of com-
lmol/l) at the probability level P = 0.05. On the
l
pound 15 (26.9 lmol/l) (see Table 2).
3. Conclusions
2.5. In vitro cytotoxicity/anti-proliferative assays
In this paper a series of twelve final propanoyloxy derivatives of
5b-cholan-24-oic acid with diethyleneglycol, decanoyl-diethylene-
glycol, bis(decanoyl)-glycerol and tocopherol chains were prepared
by multistep synthesis as new potential transdermal penetration
enhancers and intestinal drug absorption modifiers. Experimental
(relative) lipophilicity RM, calculated from RP-TLC measurements
was compared with predicted log P and log S values. The deter-
mined RM values as well as the calculated log P and log S values
as well as polar surface areas and molar volumes of the target com-
pounds were compared with the membrane permeability influence
To evaluate the cytotoxic properties of the tested compounds,
cells of different histopathological origin were used: a T-lympho-
blastic leukemia cell line (CEM), a breast adenocarcinoma cell line
(MCF7), and, as controls, normal human skin fibroblast cells (BJ).
Treatment with compounds 28 [bis(decanoyl-diethyleneglycol)]
and 15 (3-{[2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chro-
man-6-yl]oxycarbonyl}propanoic acid) resulted in dose-dependent
inhibition of cancer cells viability. Compound 15 showed the high-
est cytotoxicity against all the tested cell lines and compound 30
B
A
3.0
28
3.0
2.8
2.6
2.4
2.2
2.0
28
24
-0.97023
2.8
24
34
26
-0.97277
26
22
19
30
22
30
36
34
2.6
2.4
2.2
2.0
40
40
36
42
42
19
38
32
38
32
0.0
5.0
10.0
15.0
20.0
log
25.0
30.0
35.0
40.0
-0.9
-0.4
0.1
0.6
1.1
1.6
2.1
2.6
RM
P
C
D
3.0
2.8
2.6
2.4
2.2
2.0
3.0
2.8
2.6
2.4
2.2
2.0
28
28
22
24
26
30
26
24
-0.92012
34
22
30
34
40
36
36
40
19
42
19
42
38
32
38
32
400
600
800
1000
1200
1400
1600
1800
100
150
200
250
PSA [Å2]
300
350
400
MV [cm3]
Fig. 4. The dependences of intestinal absorption enhancement effect ER on the lipophilicity RM (Fig. 4A), log P (Fig. 4B), molar volume (MV [cm3]) (Fig. 4C) and polar surface
area (PSA [Å2]) (Fig. 4D) of the studied steroid-like target compounds.

444
L. Coufalová et al. / Steroids 78 (2013) 435–453
studied by the PAMPA method and transdermal absorption in vitro.
The ability of the final compounds to enhance the penetration of
theophylline through porcine skin was examined using a Franz cell,
and the intestinal drug absorption enhancing effect was evaluated
by means of PAMPA experiments. All these derivatives, based on
isosteric replacement of oxygen for carbon in the alkyl chain, ex-
pressed much higher enhancement effect than the primary C4,
CH2Cl2 (200 ml), and DMAP (9.95 g, 81.4 mmol) was added. The
solution was stirred at ambient temperature for 1 h. Then AcOH
(1 ml) was added and the mixture was evaporated under reduced
pressure. The crude product was purified by gradient Fc (toluene/
AcOEt/AcOH 150:50:2 to 25:75:1). This provided an oily liquid.
Yield: 10 g (51%). 1H NMR (250 MHz, CDCl3), d: 2.64 (s, 4H, COCH2-
CH2CO), 3.60–3.72 (m, 6H, PhCH2OCH2CH2OCH2CH2), 4.22–4.30
(m, 2H, CH2CH2OCO), 4.57 (s, 2H, PhCH2O), 7.30–7.37 (m, 5H, Ph),
10.53 (s, 1H, COOH). 13C NMR (62.5 MHz, CDCl3), d: 29.0, 29.0,
63.9, 69.1, 69.4, 70.6, 73.3, 127.7, 127.9, 128.4, 138.1, 172.2 (COOR),
177.3 (COOH).
C10 and C16 linear acyloxy derivatives of 5b-cholic acid. The highest
transdermal penetration-enhancement effect in this study was
exhibited by compounds 19 [mono(diethyleneglycol)] and 24
[tris(diethyleneglycol)], while the highest intestinal absorption
enhancement effect was expressed by compounds 28 [bis(deca-
noyl-diethyleneglycol)], 24 [tris(diethyleneglycol)] and 26
[mono(decanoyl-diethyleneglycol)]. Generally, it can be postulated
that the highest transdermal enhancement effect within the indi-
vidual series was shown by mono-substituted derivatives, whereas
bis(substituted) derivatives demonstrated the highest intestinal
permeability enhancement effect. Decanoyl-diethyleneglycol and/
or diethyleneglycol are the most preferred substituents of cholic
acid hydroxyl moieties. All the compounds were additionally eval-
uated their cytotoxicity against two human cancer cell lines and
against normal human skin fibroblasts. Compound 28 showed
anti-proliferative effect on cancer cells without affecting the
growth of normal cells, which suggests that this compound would
have low cytotoxic side-effects when administered as an enhancer/
excipient. Ten other target compounds exhibited limited cytotoxic-
ity, so they could be used as absorption modifiers. The obtained
data provided important parameters for correlations between sol-
ubility/lipophilicity and enhancement effect, and noteworthy
structure–activity relationships were found for subsequent struc-
ture optimization and rationalization of the design of novel poten-
tial cholic acid-type enhancers.
4.1.2. 3-[2-(2-Hydroxyethoxy)ethoxycarbonyl]propanoic acid (3)
To the solution of acid 2 (2.56 g, 8.64 mmol) in MeOH (100 ml)
10% Pd/C (0.27 g) was added, and the mixture was stirred under
hydrogen for 1 h. Then the mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by gradient Fc (toluene/AcOEt/AcOH 150:50:2 to
25:75:1). This provided an oily liquid. Yield: 1.2 g (67%). 1H NMR
(250 MHz, CDCl3), d: 2.62–2.69 (m, 4H, COCH2CH2CO), 3.55–3.68
(m, 2H, CH2OH), 3.65–3.78 (m, 4H, CH2OCH2), 4.24–4.32 (m, 2H,
CH2OCO), 6.37 (s, 2H, OH). 13C NMR (62.5 MHz, CDCl3), d: 29.1,
29.3, 61.7, 63.7, 69.1, 72.3, 172.3 (COOR), 176.5 (COOH). IR
(cmꢀ1):
m(CH) 2930, m(C@O) 1725, m(CO) 1162, 1126, 1061. Anal.
Calc. for C8H14O6 (206.19): 46.60% C, 6.84% H; found: 45.33% C,
7.19% H. HR-MS: for C8H15O6 [M + H]+ calculated: 207.0869 m/z;
found: 207.08655.
4.1.3. 3-[2-(2-Benzyloxyethoxy)ethoxycarbonyl]propanoyl chloride
(4)
To the solution of acid 2 (5.33 g, 17.99 mmol) in toluene
(7.5 ml) oxalyl chloride (7.5 ml, 88.63 mmol) was added at ambi-
ent temperature, after 30 min DMF (2 drops) was added, and then
the mixture was stirred for 5 min. After that the mixture was evap-
orated under reduced pressure. Crude acyl chloride 4 (6.0 g) was
used in the subsequent acylation reaction without further
purification.
4. Experimental
4.1. Chemistry
All reagents were purchased from Sigma–Aldrich (Schnelldorf,
Germany) and Merck (Darmstadt, Germany). Kieselgel 60, 0.063–
0.200 mm (Merck, Darmstadt, Germany) was used for flash chro-
matography (Fc). Thin layer chromatography (TLC) experiments
were performed on aluminium foil-backed silica gel 40 F254 plates
(Merck, Darmstadt, Germany). Detection was performed by spray-
ing with a solution of 20 g of Ce(SO4)2 in 200 ml 10% H2SO4 and
subsequent heating. The melting points were determined on a Boe-
tius apparatus (Nagema, Germany) and are uncorrected. Infrared
(IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for
Nicolet™ 6700 FT-IR Spectrometer (Thermo Scientific, USA). The
spectra were obtained by accumulation of 256 scans with 2 cmꢀ1
resolution in the 4000–600 cmꢀ1 region. Parameter ‘‘zero-filling’’
was 0. Happ-Gensel apodisation function was used. Elemental
analyses were performed, using a Vario EL III Universal CHNOS Ele-
mental Analyzer (Elementar Analysensysteme, Germany). All 1H-
and 13C NMR spectra were recorded on a Bruker Avance-250 FT-
NMR spectrometer (250 MHz for 1H-NMR and 62.9 MHz for 13C
NMR, Bruker Comp., Karlsruhe, Germany). Chemicals shifts are re-
ported in ppm (d) using internal Si(CH3)4 as the reference, with dif-
fuse, easily exchangeable signals being omitted. Mass spectra were
measured using a LTQ Orbitrap Hybrid Mass Spectrometer (Ther-
mo Electron Corporation, USA) with direct injection into an APCI
source (400 °C) in the negative mode.
4.1.4. 2-(2-Benzyloxyethoxy)ethyl decanoate (5)
The solution of decanoyl chloride (9.5 ml, 45.78 mmol) in CH2-
Cl2 (20 ml) was slowly added dropwise at 0 °C (ice bath) under ar-
gon to the solution of 2-(2-benzyloxyethoxy)ethanol [44] (1,
8.01 g, 40.82 mmol), pyridine (3.6 ml, 44.6 mmol) and DMAP
(0.50 g, 4.09 mmol) in CH2Cl2 (30 ml). Then the mixture was stirred
and warmed to ambient temperature. After 2 h it was poured into
aqueous AcOH (6%), and the aqueous layer was washed with CH2-
Cl2. The combined organic layers were washed with water, brine,
dried with anhydrous MgSO4 and evaporated under reduced pres-
sure. The crude product was purified by gradient Fc (CH2Cl2/MeOH/
AcOH 900:7.5:3 to 150:5:1). This provided an oily liquid. Yield:
14.0 g (97%). 1H NMR (250 MHz, CDCl3), d: 0.88 (m, 3H, CH3),
1.18–1.40 (m, 12H, 6 ꢁ CH2), 1.62 (m, 2H, CH2CH2CH2COO), 2.32
(m, 2H, CH2COO), 3.60–3.74 (m, 6H, 3 ꢁ CH2OCH2), 4.20–4.27 (m,
2H, CH2OCO), 4.57 (s, 2H, PhCH2OCH2), 7.25–7.37 (m, 5H, Ph). 13C
NMR (62.5 MHz, CDCl3), d: 14.2, 22.8, 25.0, 29.3, 29.4, 29.5, 31.9,
34.3, 63.5, 69.4, 69.5, 70.8, 73.4, 127.8, 127.9, 128.5, 138.3, 174.0
(COOR).
4.1.5. 2-(2-Hydroxyethoxy)ethyl decanoate (6)
Decanoate 5 (14.04 g, 40.06 mmol) was dissolved in AcOEt
(300 ml), 10% Pd/C (1.33 g) was added, and the mixture was stirred
under hydrogen at 40 °C for 24 h. Then additional 10% Pd/C was
added (1.01 g), and the mixture was stirred under hydrogen at
40 °C for 24 h. Then the mixture was filtered through celite and
evaporated under reduced pressure. The crude product was puri-
fied by gradient Fc (toluene/AcOEt/AcOH 90:15:1 to 50:50:1). This
4.1.1. 3-[2-(2-Benzyloxyethoxy)ethoxycarbonyl]propanoic acid (2)
2-(2-Benzyloxyethoxy)ethanol [44] (1, 12.96 g, 66 mmol) and
succinic acid anhydride (8.12 g, 81.1 mmol) were dissolved in

L. Coufalová et al. / Steroids 78 (2013) 435–453
445
provided an oily liquid. Yield: 7.9 g (76%). 1H NMR (250 MHz,
CDCl3), d: 0.88 (m, 3H, CH3), 1.20–1.40 (m, 12H, 6 ꢁ CH2), 1.62
(m, 2H, CH2CH2CH2COO), 2.33 (m, 2H, CH2COO), 3.57–3.63 (m,
2H, CH2OH), 3.67–3.78 (m, 4H, CH2OCH2), 4.20–4.28 (m, 2H, CH2-
OCO). 13C NMR (62.5 MHz, CDCl3), d: 14.2, 22.7, 25.0, 29.2, 29.3,
29.5, 31.9, 34.3, 61.8, 63.3, 69.3, 72.5, 173.9 (COOR).
24 h, filtered and evaporated under reduced pressure. The crude
product was purified by Fc (CH2Cl2/MeOH/AcOH 300:10:3). This
provided an oily liquid. Yield: 12.37 g (49%). 1H NMR (250 MHz,
CDCl3), d: 0.88 (m, 6H, 2 ꢁ CH3), 1.20–1.40 (m, 24H, 2ꢁ(CH2)6),
1.54–1.71 (m, 4H, 2 ꢁ CH2CH2COO), 2.20–2.40 (m, 5H, 2 ꢁ CH2-
COO, OH), 3.72 (d, 2H, CH2OH, J = 5.05 Hz), 4.22 (dd, 1H,
2J = 11.93 Hz, 3J = 5.77 Hz), 4.33 (dd, 1H, 2J = 11.93 Hz,
3J = 4.43 Hz), 5.02–5.15 (m, 1H). 13C NMR (62.5 MHz, CDCl3), d:
14.2, 22.8, 25.0, 25.1, 29.2, 29.2, 29.4, 29.5, 31.9, 34.2, 34.4, 61.6,
62.2, 72.3, 173.6 (COOR), 173.9 (COOR).
4.1.6. 3-[2-(2-Decanoyloxyethoxy)ethoxycarbonyl]propanoic acid (7)
Decanoate 6 (7.92 g, 30.42 mmol), succinic acid anhydride
(3.79 g, 37.9 mmol) and DMAP (4.50 g, 36.8 mmol) were dissolved
in CH2Cl2 (120 ml) and stirred at ambient temperature for 2 h.
After addition of concentrated AcOH (3 ml, 52.4 mmol) the mixture
was poured into water. The mixture was extracted with CH2Cl2.
The combined organic layers were washed with brine, dried with
anhydrous MgSO4 and evaporated under reduced pressure. The
crude product was purified by Fc (AcOEt/AcOH 100:1) and crystal-
lized from toluene/petroleum ether (6:1). This provided a white
crystalline compound. Yield: 9.3 g (84%). Mp. 36 °C. 1H NMR
(250 MHz, CDCl3), d: 0.88 (m, 3H, CH3), 1.2–1.4 (m, 12H,
6 ꢁ CH2), 1.62 (m, 2H, CH2CH2CH2COO), 2.33 (m, 2H, CH2COO),
2.60–2.75 (m, 4H, COCH2CH2CO), 3.66–3.74 (m, 4H, CH2OCH2),
4.20–4.30 (m, 4H, 2 ꢁ CH2OCO), 10.84 (s, 1H, COOH). 13C NMR
(62.5 MHz, CDCl3), d: 14.2, 22.7, 25.0, 28.9, 29.0, 29.2, 29.3, 29.5,
31.9, 34.3, 63.3, 63.9, 69.1, 69.2, 172.2 (COOR), 174.0 (COOR),
4.1.10. 3-[2,3-Bis(decanoyloxy)propyloxycarbonyl]propanoic acid (12)
Succinic acid anhydride (3.75 g, 37.37 mmol) was added to the
solution of compound 11 (12.37 g, 30.88 mmol) and DMAP (4.52 g,
36.99 mmol) in CH2Cl2 (120 ml), and the mixture was stirred for
2.5 h. Then AcOH (1 ml) was added, and the mixture was evapo-
rated under reduced pressure. The crude product was purified by
gradient Fc (CH2Cl2/AcOEt/AcOH 800:65:8.5 and 800:150:9). This
provided an oily liquid. Yield: 12.2 g (79%). 1H NMR (250 MHz,
CDCl3), d: 0.88 (m, 6H, 2 ꢁ CH3), 1.20–1.40 (m, 24H, 2ꢁ(CH2)6),
1.54–1.69 (m, 4H, 2 ꢁ CH2CH2COO), 2.26–2.37 (m, 4H, 2 ꢁ CH2-
COO), 2.60–2.72 (m, 4H, COCH2CH2CO), 4.00–4.38 (m, 4H, 2 ꢁ CH2-
OCO), 5.21–5.32 (m, 1H, CHOCO), 9.79 (s, COOH). 13C NMR
(62.5 MHz, CDCl3), d: 14.2, 22.8, 24.9, 25.0, 28.8, 28.9, 29.2, 29.2,
29.4, 29.5, 32.0, 34.2, 34.3, 62.14, 62.8, 68.9, 171.7 (COOR), 173.1
177.8 (COOH). IR (cmꢀ1):
1694, (CH) 1325, (CO) 1176, 1132. Anal. Calc. for C18H32O7
m(CH) 2953, 2915, 2848, m(C@O) 1724,
m
m
(COOR), 173.5 (COOR), 177.7 (COOH). IR (cmꢀ1):
2917, 2851, (C@O) 1730, 1697, (CH) 1386, (CO) 1172. Anal. Calc.
m(CH) 2953,
(360.44): 59.98% C, 8.95% H; found: 59.45% C, 9.15% H. HR-MS:
m
m
m
for
C
18H31O7 [MꢀH]ꢀ calculated: 359.2070 m/z; found:
for C27H48O8 (500.67): 64.77% C, 9.66% H; found: 66.08% C, 10.09%
H. HR-MS: for C27H47O8 [MꢀH]ꢀ calculated: 499.3271 m/z; found:
499.32578 m/z.
359.20435 m/z.
4.1.7. 3-[2-(2-Decanoyloxyethoxy)ethoxycarbonyl]propanoyl chloride
(8)
4.1.11. 3-[2,3-Bis(decanoyloxy)propyloxycarbonyl]propanoyl chloride
(13)
To the solution of acid 7 (6.07 g, 16.84 mmol) in toluene (25 ml)
oxalyl chloride (7.0 ml, 82.72 mmol) was added at ambient tem-
perature and after 15 min DMF (2 drops). Then the mixture was
stirred for 20 min and evaporated under reduced pressure. Crude
acyl chloride 8 (6.9 g) was used in the subsequent acylation reac-
tion without further purification.
To the solution of acid 12 (6.99 g, 13.96 mmol) in toluene
(12 ml) oxalyl chloride (6 ml, 70.90 mmol) was added at ambient
temperature, after 15 min DMF (2 drops) was added, and the mix-
ture was stirred for 20 min. Then the mixture was evaporated un-
der reduced pressure. Crude acyl chloride 13 (7.5 g) was used in
the subsequent acylation reaction without further purification.
4.1.8. 1,2-O,O0-Bisdecanoyl-3-O00-benzyl-rac-glycerol (10)
Decanoyl chloride (35 ml, 168.7 mmol) dissolved in CH2Cl2
(50 ml) was slowly (during 30 min) added dropwise to the cooled
(15 °C) solution of 1-O-benzyl-rac-glycerol [45] (9, 15.05 g,
82.59 mmol), DMAP (0.16 g, 1.31 mmol) and pyridine (15 ml,
185.8 mmol) in CH2Cl2 (100 ml). The mixture was stirred at ambi-
ent temperature for 2.5 h and then heated at 40 °C for 8.5 h with
elimination of air humidity. Then glacial AcOH (4 ml) was added
to the mixture. The water layer was separated and washed with
CH2Cl2. The organic layer was washed with water and brine, dried
with anhydrous MgSO4 and evaporated under reduced pressure.
The crude product was purified by gradient Fc (CH2Cl2/AcOEt
100:0 to 100:6). This provided an oily liquid. Yield: 35.2 g (87%).
1H NMR (250 MHz, CDCl3), d: 0.88 (m, 6H, 2 ꢁ CH3), 1.15–1.40
(m, 24H, 2ꢁ(CH2)6), 1.60 (m, 4H, 2 ꢁ CH2CH2COO), 2.23–2.37 (m,
4H, 2 ꢁ CH2COO), 3.59 (d, 2H, CH2OCH2Ph, J = 5.18 Hz), 4.19 (dd,
1H, 2J = 11.89 Hz, 3J = 6.42 Hz), 4.35 (dd, 1H, 2J = 11.89 Hz,
3J = 3.84 Hz), 4.54 (2H, PhCH2), 5.19–5.30 (m, 1H), 7.25–7.40 (m,
5H, Ph). 13C NMR (62.5 MHz, CDCl3), d: 14.3, 22.8, 25.0, 25.1,
29.2, 29.2, 29.4, 29.6, 32.0, 34.24, 34.5, 62.8, 68.4, 70.1, 73.4,
127.8, 127.9, 128.6, 137.8, 173.3 (COOR), 173.6 (COOR).
4.1.12. (+/ꢀ)-
Succinic acid anhydride (3.64 g; 36.38 mmol) was added to the
solution of -tocopherol (14, 12.62 g; 29.3 mmol) and DMAP
a-Tocopherol hemisuccinate (15)
a
(4.76 g, 38.96 mmol) in CH2Cl2 (200 ml), and the mixture was stir-
red at ambient temperature for 2 h [46]. Then the mixture was
evaporated under reduced pressure. The crude product was puri-
fied by Fc (CH2Cl2/MeOH/AcOH 30:1.5:0.3). This provided an oily li-
quid. Yield: 15.8 g (99%). 1H NMR (250 MHz, CDCl3), d: 0.81–0.90
(m, 12H, 4 ꢁ CH3), 0.95–1.65 (m, 25H), 1.60–1.90 (m, 2H), 1.96
(s, 3H, ArCH3), 2.00 (s, 3H, ArCH3), 2,08 (s, 3H, ArCH3), 2.58 (m,
2H), 2.75–2.97 (m, 4H, COCH2CH2CO). 13C NMR (62.5 MHz, CDCl3),
d: 11.9, 12.2, 13.0, 19.7, 19.8, 19.8, 19.8, 19.9, 19.7, 19.8, 19.8, 19.8,
19.9, 20.7, 21.2, 22.8, 22.9, 24.0, 24.6, 24.9, 28.1, 28.7, 29.1, 31.2,
32.9, 32.9, 37.4, 37.5, 37.6, 37.7, 39.5, 75.2, 117.6, 123.2, 125.0,
126.8, 140.6, 149.6, 170.9 (COOR), 178.2 (COOH). IR (cmꢀ1):
m(CH) 2952, 2922, 2866, m(C@O) 1745, 1692, m(CH) 1460, m(CO)
1152, 1105, 1087. Anal. Calc. for C33H54O5 (530.78): 74.67% C,
10.25% H; found: 74.52% C, 10.56% H.
4.1.13. 3-{[2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-
6-yl]oxycarbonyl} propanoyl chloride (16)
4.1.9. 1,2-O,O0-Bisdecanoyl-rac-glycerol (11)
To the solution of compound 15 (2.72 g, 5.13 mmol) in toluene
(4 ml) oxalyl chloride (2 ml, 23.64 mmol) was added, and the mix-
ture was stirred for 2 h [47]. Then the mixture was evaporated un-
der reduced pressure. The crude acyl chloride 16 (2.9 g) was used
in the subsequent acylation reaction without further purification.
The solution of compound 10 (30.76 g, 62.68 mmol) in AcOEt
(300 ml) was stirred with 10% Pd/C (3.07 g) under hydrogen at
ambient temperature for 22 h. Then a further portion of 10% Pd/C
(1 g) was added, and the mixture was stirred under hydrogen for

446
L. Coufalová et al. / Steroids 78 (2013) 435–453
4.1.14. Benzyl 3
benzyloxyethoxy)ethoxycarbonyl]propanoyloxy}-7
5b-cholan-24-oate (18)
a
-{3-[2-(2-
135.6, 136.2, 154.8 (OCOO), 174.2 (COOR). HR-MS: for C39H51O7
a
,12a-dihydroxy-
[MꢀH]ꢀ calculated: 631.3713 m/z; found: 631.3726 m/z [8].
Acyl chloride 4 (4.58 g, 14.55 mmol) dissolved in CH2Cl2 (20 ml)
was slowly added dropwise to the solution of benzyl cholate (17,
6.00 g, 12.03 mmol) and DMAP (1.77 g, 14.49 mmol) in CH2Cl2
(40 ml) at 0 °C under argon. Then the mixture was stirred and
warmed to an ambient temperature. After 3 h it was poured into
aqueous AcOH (250 ml, 1.2%), and the aqueous layer was washed
with CH2Cl2. The combined organic layers were dried with anhy-
drous MgSO4 and evaporated under reduced pressure. The crude
product was purified by Fc (CH2Cl2/MeOH/AcOH 300:10:1). This
provided a colorless oil. Yield: 1.9 g (20%). 1H NMR (250 MHz,
CDCl3), d: 0.66 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.96 (d, 3H, CH3,
J = 5.71 Hz), 1.00–2.50 (m, 26H), 2.52–2.67 (m, 4H, COCH2CH2CO),
3.60–3.75 (m, 6H, 3 ꢁ CH2OCH2), 3.82 (m, 1H, C(7)H), 3.96 (m, 1H,
4.1.17. Benzyl 3a-benzyloxycarboxy-7a,12a-bis{3-[2-(2-
benzyloxyethoxy)ethoxycarbonyl] propanoyloxy}-5b-cholan-24-oate
(21)
The mixture of ester 20 (2.52 g, 3.98 mmol), BTEAC (0.36 g,
1.58 mmol) and CaH2 (0.67 g, 15.90 mmol) in toluene (40 ml)
was mixed with the solution of crude acyl chloride 4 (4.98 g,
15.83 mmol) in toluene (20 ml), and the resulting mixture was re-
fluxed under argon for 4 h. Then the reaction mixture was poured
into the mixture of aqueous AcOH (6%) and toluene. After shaking
the aqueous layer was washed with toluene. The combined organic
layers were washed with brine, dried with anhydrous MgSO4 and
evaporated under reduced pressure. The crude product was puri-
fied by Fc (toluene/Et2O/TEA 80:20:1 and toluene/AcOEt/TEA
60:30:1). This provided a colorless oil. Yield: 3.3 g (69%). 1H NMR
(250 MHz, CDCl3), d: 0.68 (s, 3H, CH3), 0.79 (d, 3H, CH3,
J = 5.71 Hz), 0.91 (s, 3H, CH3), 0.95–2.50 (m, 24H), 2.50–2.75 (m,
8H, 2 ꢁ COCH2CH2CO), 3.56–3.72 (m, 12H, 6 ꢁ CH2OCH2), 4.20–
4.30 (m, 4H, 2 ꢁ CH2OCO), 4.45 (m, 1H, C(3)H), 4.55 (s, 2H, PhCH2-
OCH2), 4.56 (s, 2H, PhCH2OCH2), 4.92 (m, 1H, C(7)H), 5.09 (2H,
PhCH2OCO), 5.10 (m, 1H, C(12)H), 5.14 (s, 2H, PhCH2OCOO),7.30–
7.40 (m, 20H, 4 ꢁ Ph). 13C NMR (62.5 MHz, CDCl3), d: 12.3, 17.5,
22.6, 22.9, 25.6, 26.8, 27.4, 28.9, 29.0, 29.1, 29.5, 29.6, 30.8, 31.3,
31.4, 34.4, 34.6, 34.7, 34.7, 37.9, 41.0, 43.4, 45.3, 47.4, 63.9, 64.0,
66.2, 69.2, 69.4, 69.5, 69.6, 70.6, 70.7, 71.1, 73.4, 75.7, 78.3,
127.7, 127.9, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 135.4,
136.2, 138.3, 154.7 (OCOO), 171.8 (COOR), 171.9 (COOR), 172.3
(COOR), 172.4 (COOR), 174.0 (COOR).
C(12)H), 4.20–4.30 (m, 2H, CH2OCO), 4.57 (s, 2H, PhCH2OCH2),
4.58 (m, 1H, C(3)H), 5.11 (2H, PhCH2OCO), 7.32–7.38 (m, 10H,
2 ꢁ Ph). 13C NMR (62.5 MHz, CDCl3), d: 12.6, 17.4, 22.6, 23.2,
26.7, 26.7, 27.5, 28.4, 29.2, 29.4, 29.6, 30.9, 31.4, 34.5, 34.8, 34.9,
35.2, 35.2, 39.6, 41.3, 42.0, 46.6, 47.3, 63.9, 66.2, 68.3, 69.2, 69.4,
70.7, 73.0, 73.4, 74.8, 127.7, 127.8, 128.3, 128.3, 128.5, 128.6,
136.2, 138.2, 171.9 (COOR), 172.5 (COOR), 174.2 (COOR).
4.1.15. 7a,12a-Dihydroxy-3a-{3-[2-(2-
hydroxyethoxy)ethoxycarbonyl]propanoyloxy}-5b-cholan-24-oic acid
(19)
Benzyl ester 18 (1.49 g, 1.92 mmol) was dissolved in MeOH
(60 ml), and 10% Pd/C (0.12 g) was added. The mixture was stirred
under hydrogen at 40 °C for 1.5 h. Then the suspension was filtered
through celite and evaporated under reduced pressure. The crude
product was purified by Fc (CH2Cl2/MeOH/AcOH 150:15:1). This
provided a colorless oil. Yield: 0.8 g (69%). 1H NMR (250 MHz,
CDCl3), d: 0.69 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.99 (d, 3H, CH3,
J = 5.56 Hz), 1.00–2.47 (m, 27H), 2.53–2.70 (m, 4H, COCH2CH2CO),
3.55–3.80 (m, 6H, 3 ꢁ CH2OCH2), 3.85 (m, 1H, C(7)H), 3.99 (m, 1H,
4.1.18. 3a-Hydroxy-7a,12a-bis{3-[2-(2-
hydroxyethoxy)ethoxycarbonyl]propanoyloxy}-5b-cholan-24-oic acid
(22)
The mixture of ester 21 (3.19 g, 2.68 mmol), MeOH (160 ml),
AcOEt (40 ml) and 10% Pd/C (0.62 g) was stirred under hydrogen
at 40 °C for 5.5 h. Then the mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by Fc (CH2Cl2/MeOH/AcOH 150:15:1). This provided a col-
orless oil. Yield: 1.6 g (75%). 1H NMR (250 MHz, CDCl3), d: 0.72 (s,
3H, CH3), 0.81 (d, 3H, CH3, J = 5.86 Hz), 0.89 (s, 3H, CH3), 0.95–
2.50 (m, 26H), 2.60–2.75 (m, 8H, 2 ꢁ COCH2CH2CO), 3.44 (m, 1H,
C
(12)H), 4.20–4.30 (m, 2H, CH2OCO), 4.58 (m, 1H, C(3)H), 4.85 (s,
OH). 13C NMR (62.5 MHz, CDCl3), d: 12.6, 17.3, 22.6, 23.2, 26.6,
26.7, 27.6, 28.3, 29.4, 29.7, 30.9, 31.1, 34.5, 34.8, 34.9, 35.2, 35.4,
39.6, 41.3, 41.9, 46.6, 47.0, 61.6, 63.8, 68.4, 69.1, 72.5, 73.1, 75.0,
171.9 (COOR), 172.5 (COOR), 178.5 (COOH). IR (cmꢀ1):
m(OH)
3441, 3260, m(CH) 2936, 2871, m(C@O) 1713, m(CH) 1382, m
(CO)
C
(3)H), 3.55–3.62 (m, 4H, 2 ꢁ CH2OH), 3.65–3.75 (m, 8H, 2 ꢁ CH2-
1163. Anal. Calc. for C32H52O10 (596.74): 64.41% C, 8.78% H; found:
62.78% C, 8.93% H. HR-MS: for C32H52O10 [MꢀH]ꢀ calculated:
595.3482 m/z; found: 595.3441 m/z.
OCH2), 4.00–4.45 (s, OH), 4.18–4.37 (m, 4H, 2 ꢁ CH2OCO), 4.94
(m, 1H, C(7)H), 5.10 (m, 1H, C(12)H). 13C NMR (62.5 MHz, CDCl3),
d: 12.2, 17.6, 22.6, 23.0, 25.4, 27.4, 28.8, 29.3, 29.4, 29.74, 29.8,
30.3, 30.7, 31.5, 34.4, 34.8, 35.1, 38.0, 39.1, 41.0, 43.4, 45.2, 47.1,
61.6, 61.7, 63.9, 63.9, 69.1, 71.6, 72.6, 72.7, 76.0, 171.7 (COOR),
4.1.16. Benzyl 3a-benzyloxycarboxy-7a,12a-dihydroxy-5b-cholan-24-
oate (20)
172.5 (COOR), 178.3 (COOH). IR (cmꢀ1):
2867, (C@O) 1724,
m(OH) 3412,
m(CH) 2927,
The solution of benzyl cholate 17 (5.03 g, 10.1 mmol) in dry pyr-
idine (75 ml) was cooled to 0 °C and mixed with BnOCOCl (4.37 g,
25.6 mmol) under argon. Then the mixture was stirred for 4 h, and
a further portion of BnOCOCl (4.38 g, 25.7 mmol) was added. After
20 h the solvents were evaporated under reduced pressure. The
residue was dissolved in toluene and washed with aqueous AcOH
(17%). The aqueous layer was washed with toluene. Combined or-
ganic extracts were washed with brine and evaporated under re-
duced pressure. The crude product was purified by Fc (CH2Cl2/
MeOH/AcOH 300:5:1). This provided a colorless oil. Yield: 3.0 g
(46%). 1H NMR (250 MHz, CDCl3), d: 0.65 (s, 3H, CH3), 0.89 (s, 3H,
CH3), 0.96 (d, 3H, CH3, J = 5.95 Hz), 1.00–2.50 (m, 27H), 3.83 (m,
1H, C(7)H), 3.95 (m, 1H, C(12)H), 4.45 (m, 1H, C(3)H), 5.10 (2H,
PhCH2), 5.12 (s, 2H, PhCH2), 7.30–7.40 (m, 10H, 2 ꢁ Ph). 13C NMR
(62.5 MHz, CDCl3), d: 12.6, 17.4, 22.5, 23.3, 26.7, 26.7, 27.5, 28.4,
31.0, 31.4, 34.4, 34.8, 34.9, 35.2, 35.2, 39.7, 41.3, 42.0, 46.6, 47.3,
66.2, 68.3, 69.3, 72.9, 78.6, 128.3, 128.3, 128.3, 128.5, 128.6,
m
m(CO) 1161, 1125, 1067. Anal. Calc. for
C
40H64O15 (784.93): 61.21% C, 8.22% H; found: 59.19% C, 8.49% H.
HR-MS: for C40H63O15 [MꢀH]ꢀ calculated: 783.4167 m/z; found:
783.4159 m/z.
4.1.19. Benzyl 3a,7a,12a-tris{3-[2-(2-
benzyloxyethoxy)ethoxycarbonyl]propanoyloxy}-5b-cholan-24-oate
(23)
The mixture of benzyl ester 17 (1.50 g, 3.01 mmol), BTEAC
(0.26 g, 1.14 mmol), CaH2 (0.77 g, 18.29 mmol) and toluene
(25 ml) was mixed with the solution of crude acyl chloride 4
(5.66 g, 17.99 mmol) in toluene (25 ml), and the mixture was re-
fluxed for 3 h. Then the reaction mixture was poured into the mix-
ture of aqueous AcOH (6%) and toluene. After shaking the aqueous
layer was washed with toluene. The combined organic layers were
washed with saturated aqueous NaHCO3 and brine, dried with
anhydrous MgSO4 and evaporated under reduced pressure. The

L. Coufalová et al. / Steroids 78 (2013) 435–453
447
crude product was purified by gradient Fc (hexane/Et2O/AcOH
25:25:1 to 0:50:1 and then Et2O/MeOH/AcOH 100:5:2). This pro-
vided a colorless oil. Yield: 2.8 g (69%). 1H NMR (250 MHz, CDCl3),
d: 0.69 (s, 3H, CH3), 0.80 (d, 3H, CH3, J = 5.69 Hz), 0.90 (s, 3H, CH3),
0.95–2.50 (m, 24H), 2.53–2.78 (m, 12H, 3 ꢁ COCH2CH2CO), 3.55–
3.75 (m, 18H, 9 ꢁ CH2OCH2), 4.20–4.30 (m, 6H, 3 ꢁ CH2OCO),
4.56 (s, 6H, 3 ꢁ PhCH2OCH2), 4.57 (m, 1H, C(3)H), 4.92 (m, 1H,
for 30 min. After that the suspension was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by gradient Fc (CH2Cl2/MeOH/AcOH 30:0.5:0.1 to
30:3:0.2). This provided an oily liquid. Yield: 0.4 g (68%). 1H NMR
(250 MHz, CDCl3), d: 0.70 (s, 3H, CH3), 0.88 (m, 3H, CH3), 0.91 (s,
3H, CH3), 1.00 (d, 3H, CH3, J = 5.93 Hz), 1.00–2.05 (m, 36H), 2.13–
2.50 (m, 6H), 2.53–2.70 (m, 4H, COCH2CH2CO), 3.66–3.73 (m, 4H,
CH2OCH2), 3.86 (m, 1H, C(7)H), 3.99 (m, 1H, C(12)H), 4.19–4.28 (m,
4H, 2 ꢁ OCH2CH2OCO), 4.59 (m, 1H, C(3)H). 13C NMR (62.5 MHz,
CDCl3), d: 12.7, 14.2, 17.5, 22.7, 22.8, 23.3, 25.1, 26.8, 26.9, 27.6,
28.5, 29.3, 29.3, 29.4, 29.6, 29.7, 30.9, 31.0, 32.0, 34.4, 34.5, 34.9,
35.0, 35.3, 35.4, 39.7, 41.4, 42.2, 46.7, 47.3, 63.4, 63.9, 68.4, 69.2,
69.3, 73.1, 74.9, 171.9 (COOR), 172.5 (COOR), 174.1 (COOR), 178.8
C
(7)H), 5.09 (2H, PhCH2OCO), 5.10 (m, 1H, C(12)H), 7.30–7.36 (m,
20H, 4 ꢁ Ph). 13C NMR (62.5 MHz, CDCl3), d: 12.3, 17.5, 22.6,
22.9, 25.7, 26.9, 27.3, 28.9, 29.0, 29.1, 29.2, 29.5, 29.6, 30.9,
31.3, 31.4, 34.4, 34.7, 34.8, 37.9, 41.0, 43.4, 45.3, 47.4, 63.9,
63.9, 66.2, 69.2, 69.5, 70.7, 71.2, 73.4, 74.5, 75.7, 127.7, 127.8,
128.3, 128.3, 128.5, 128.6, 136.2, 138.3, 171.7 (COOR), 171.8
(COOR), 171.9 (COOR), 172.2 (COOR), 172.3 (COOR), 172.4 (COOR),
173.9 (COOR).
(COOH). IR (cmꢀ1):
(CH) 1378, m(CO) 1162. Anal. Calc. for C42H70O11 (751.00): 67.17%
m(OH) 3487, m(CH) 2923, 2855, m(C@O) 1729,
m
C, 9.39% H; found: 66.50% C, 9.72% H. HR-MS: for C42H69O11 [MꢀH]ꢀ
4.1.20. 3
a,7a
,12a-Tris{3-[2-(2-hydroxyethoxy)ethoxycarbonyl]
calculated: 749.4840 m/z; found: 749.4801 m/z.
propanoyloxy}-5b-cholan-24-oic acid (24)
The solution of benzyl ester 23 (2.45 g, 1.84 mmol) in MeOH
(110 ml) and AcOEt (28 ml) was stirred with 10% Pd/C (0.43 g) under
hydrogen at 40 °C for 5.5 h. Then the mixture was filtered through
celite and evaporated under reduced pressure. The crude product
was purified by Fc (CH2Cl2/MeOH/AcOH 150:15:1). This provided
an oily liquid. Yield: 1.1 g (61%). 1H NMR (250 MHz, CDCl3), d: 0.71
(s, 3H, CH3), 0.80 (d, 3H, CH3, J = 5.41 Hz), 0.89 (s, 3H, CH3), 0.95–
2.45 (m, 24H), 2.55–2.80 (m, 12H, 3 ꢁ COCH2CH2CO), 3.53–3.62
(m, 6H, 3 ꢁ CH2OH), 3.64–3.74 (m, 12H, 6 ꢁ CH2OCH2), 4.0–4.3
(OH), 4.2–4.35 (m, 6H, 3 ꢁ CH2OCO), 4.55 (m, 1H, C(3)H), 4.91 (m,
1H, C(7)H), 5.10 (m, 1H, C(12)H). 13C NMR (62.5 MHz, CDCl3), d: 12.2,
17.5, 22.6, 22.9, 25.6, 26.8, 27.4, 28.9, 29.1, 29.2, 29.3, 29.6, 29.7,
30.7, 31.3, 34.4, 34.7, 34.7, 37.9, 40.9, 43.4, 45.2, 47.0, 61.6, 61.6,
61.7, 63.7, 63.7, 63.8, 69.1, 71.4, 72.6, 74.6, 75.8, 171.8 (COOR),
172.0 (COOR), 172.4 (COOR), 172.5 (COOR), 178.2 (COOH). IR
4.1.23. Benzyl 3a-benzyloxycarboxy-7a,12a-bis{3-[2-(2-
decanoyloxyethoxy)ethoxycarbonyl]propanoyloxy}-5b-cholan-24-
oate (27)
Ester 20 (2.09 g, 3.30 mmol), BTEAC (0.27 g, 1.19 mmol) and
CaH2 (0.53 g, 12.59 mmol) were dissolved in toluene (40 ml) and
mixed with the solution of crude acyl chloride
8 (4.84 g,
12.76 mmol) in toluene (20 ml). The mixture was refluxed under
argon for 3 h. The reaction mixture was poured into the mixture
of aqueous AcOH (3%) and toluene. After shaking the aqueous layer
was washed with toluene. The combined organic layers were
washed with brine, dried with anhydrous MgSO4 and evaporated
under reduced pressure. The crude product was purified by Fc (tol-
uene/AcOEt/TEA 80:20:1). This provided an oily liquid. Yield: 3.0 g
(68%). 1H NMR (250 MHz, CDCl3), d: 0.69 (s, 3H, CH3), 0.80 (d, 3H,
CH3, J = 5.78 Hz), 0.88 (m, 6H, 2 ꢁ CH3), 0.91 (s, 3H, CH3), 0.95–
2.20 (m, 50H), 2.20–2.50 (m, 6H), 2.50–2.80 (m, 8H, 2 ꢁ COCH2CH2-
CO), 3.60–3.72 (m, 8H, 2 ꢁ CH2OCH2), 4.16–4.29 (m, 8H, 4 ꢁ CH2-
OCO), 4.46 (m, 1H, C(3)H), 4.94 (m, 1H, C(7)H), 5.09 (2H, PhCH2O),
5.10 (m, 1H, C(12)H), 5.13 (s, 2H, PhCH2OCOO), 7.30–7.40 (m, 10H,
2 ꢁ Ph). 13C NMR (62.5 MHz, CDCl3), d: 12.3, 14.2, 17.5, 22.5,
22.7, 22.9, 25.0, 25.6, 26.8, 27.3, 28.9, 29.0, 29.1, 29.2, 29.4, 29.5,
30.8, 31.2, 31.4, 31.9, 34.3, 34.4, 34.6, 34.6, 34.7, 37.9, 41.0, 43.4,
45.2, 47.4, 63.3, 63.7, 63.8, 66.2, 69.1, 69.2, 69.3, 69.5, 71.1, 75.7,
78.3, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 135.4, 136.2, 154.6
(OCOO), 171.7 (COOR), 171.8 (COOR), 172.2 (COOR), 172.3 (COOR),
173.8 (COOR), 173.9 (COOR).
(cmꢀ1):
m(OH) 3445, m(CH) 2939, 2870, m(C@O) 1725, m(CH) 1381,
m
(CO) 1160, 1127. Anal. Calc. for C48H76O20 (973.11): 59.24% C,
7.87% H; found: 56.62% C, 8.03% H. HR-MS: for C48H75O20 [MꢀH]ꢀ
calculated: 971.4852 m/z; found: 971.4811 m/z.
4.1.21. Benzyl 3a-{3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]
propanoyloxy}-7
a
,12 -dihydroxy-5b-cholan-24-oate (25)
a
The solution of acyl chloride 8 (1.84 g, 4.85 mmol) in CH2Cl2
(20 ml) was slowly added to the solution of benzyl cholate (17,
2.01 g, 4.03 mmol) and DMAP (0.58 g, 4.75 mmol) in CH2Cl2
(30 ml) at 0 °C. The mixture was stirred at this temperature for
45 min. Then the mixture was warmed to an ambient temperature
and poured into aqueous AcOH (6%). The aqueous layer was washed
with CH2Cl2. The combined organic layers were washed with satu-
rated aqueous NaHCO3, dried with anhydrous MgSO4 and evapo-
rated under reduced pressure. The crude product was purified by
Fc (CH2Cl2/MeOH/AcOH 300:10:1). This provided an oily liquid.
Yield: 0.7 g (22%). 1H NMR (250 MHz, CDCl3), d: 0.67 (s, 3H, CH3),
0.88 (m, 3H, CH3), 0.90 (s, 3H, CH3), 0.98 (d, 3H, CH3, J = 5.87 Hz),
1.00–2.00 (m, 36H), 2.13–2.50 (m, 6H), 2.53–2.69 (m, 4H, COCH2-
CH2CO), 3.65–3.73 (m, 4H, CH2OCH2), 3.84 (m, 1H, C(7)H), 3.97 (m,
1H, C(12)H), 4.18–4.29 (m, 4H, 2 ꢁ OCH2CH2OCO), 4.59 (m, 1H,
4.1.24. 7
a
,12a-Bis{3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]
propanoyloxy}-3a-hydroxy-5b-cholan-24-oic acid (28)
The solution of HCOONH4 (0.54 g, 8.56 mmol) in MeOH (50 ml)
and 10% Pd/C (0.58 g) were added to the solution of ester 27
(2.90 g, 2.20 mmol) in AcOEt (50 ml). The mixture was stirred un-
der argon for 1.5 h. Then the mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by gradient Fc (CH2Cl2/MeOH/AcOH 100:5:1). This pro-
vided an oily liquid. Yield: 1.8 g (74%). 1H NMR (250 MHz, CDCl3),
d: 0.70 (s, 3H, CH3), 0.80 (d, 3H, CH3, J = 6.05 Hz), 0.85 (m, 6H,
2 ꢁ CH3), 0.88 (s, 3H, CH3), 0.95–2.15 (m, 51H), 2.15–2.45 (m,
6H), 2.55–2.75 (m, 8H, 2 ꢁ COCH2CH2CO), 3.43 (m, 1H, C(3)H),
3.60–3.73 (m, 8H, 2 ꢁ CH2OCH2), 4.14–4.28 (m, 8H, 4 ꢁ CH2OCO),
4.91 (m, 1H, C(7)H), 5.08 (m, 1H, C(12)H). 13C NMR (62.5 MHz,
CDCl3), d: 12.2, 14.2, 17.5, 22.5, 22.7, 22.9, 25.0, 25.4, 27.3, 28.8,
29.1, 29.2, 29.3, 29.5, 29.6, 30.4, 30.6, 30.8, 31.4, 31.9, 34.3, 34.4,
34.7, 35.1, 38.0, 39.0, 41.1, 43.3, 45.2, 47.2, 63.3, 63.8, 63.9, 69.0,
69.1, 69.2, 71.5, 71.6, 75.9, 171.7 (COOR), 171.7 (COOR), 172.3
(COOR), 172.4 (COOR), 174.0 (COOR), 179.0 (COOH). IR (cmꢀ1):
C
(3)H), 5.11 (2H, PhCH2), 7.32–7.38 (m, 5H, Ph). 13C NMR
(62.5 MHz, CDCl3), d: 12.7, 14.2, 17.5, 22.7, 22.8, 23.3, 25.0, 26.8,
26.9, 27.5, 28.6, 29.3, 29.3, 29.4, 29.5, 29.6, 31.0, 31.4, 32.0, 34.4,
34.5, 34.8, 35.0, 35.2, 35.3 39.7, 41.4, 42.2, 46.7, 47.4, 63.4, 63.8,
66.2, 68.3, 69.1, 69.3, 73.0, 74.9, 128.3, 128.4, 128.7, 136.3, 171.9,
172.4, 174.0, 174.1.
4.1.22. 3
a-{3-[2-(2-Decanoyloxyethoxy)ethoxycarbonyl]
propanoyloxy}-7
a
,12 -dihydroxy-5b-cholan-24-oic acid (26)
a
The solution of benzyl ester 25 (0.66 g, 0.79 mmol) in AcOEt
m(OH) 3445,
m(CH) 2924, 2854, m(C@O) 1728, m(CH) 1378, m(CO)
(15 ml) was stirred with 10% Pd/C (0.13 g) under hydrogen at 30 °C
1161. Anal. Calc. for C60H100O17 (1093.43): 65.91% C, 9.22% H;

448
L. Coufalová et al. / Steroids 78 (2013) 435–453
found: 64.13% C, 9.20% H. HR-MS: for C60H99O17 [MꢀH]ꢀ calcu-
4.1.28. 3
propanoyloxy}-7
a
-{3-[2,3-Bis(decanoyloxy)propyloxycarbonyl]
,12 -dihydroxy-5b-cholan-24-oic acid (32)
lated: 1091.6882 m/z; found: 1091.6843 m/z.
a
a
The solution of HCOONH4 (0.57 g, 9.04 mmol) in MeOH (20 ml)
and 10% Pd/C (0.26 g) were added to the solution of ester 31
(2.13 g, 2.17 mmol) in AcOEt (20 ml). The mixture was stirred under
argon for 1.5 h. Then the mixture was filtered through celite and
evaporated under reduced pressure. The crude product was purified
by Fc (CH2Cl2/MeOH/AcOH 300:5:1). This provided an oily liquid.
Yield: 1.3 g (68%). 1H NMR (250 MHz, CDCl3), d: 0.69 (s, 3H, CH3),
0.87 (m, 6H, 2 ꢁ CH3), 0.89 (s, 3H, CH3), 0.99 (d, 3H, CH3,
J = 5.91 Hz), 1.00–2.01 (m, 49H), 2.13–2.48 (m, 9H), 2.50–2.65 (m,
4H, COCH2CH2CO), 3.85 (m, 1H, C(7)H), 3.98 (m, 1H, C(12)H), 4.08–
4.22 (m, 2H, CH2OCO), 4.23–4.36 (m, 2H, CH2OCO), 4.57 (m, 1H,
4.1.25. Benzyl 3a,7a,12a-tris{3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]
propanoyloxy}-5b-cholan-24-oate (29)
The mixture of benzyl cholate (17, 1.01 g, 2.03 mmol), BTEAC
(0.18 g, 0.79 mmol) and CaH2 (0.55 g, 13.06 mmol) in toluene
(15 ml) was mixed with the solution of crude acyl chloride 8
(4.53 g, 11.96 mmol) in toluene (15 ml). The mixture was refluxed
under argon for 3 h. The reaction mixture was poured into aqueous
AcOH (6%) and the aqueous layer was washed with toluene. The
combined organic layers were washed with saturated aqueous
NaHCO3, water, dried with anhydrous MgSO4 and evaporated un-
der reduced pressure. The crude product was purified by Fc (hex-
ane/acetone/AcOH 80:20:1). This provided an oily liquid. Yield:
1.8 g (59%). The product without characterization was used in
the subsequent reaction.
C
(3)H), 5.24 (m, 1H, CHOCO). 13C NMR (62.5 MHz, CDCl3), d: 12.7,
14.2, 17.4, 22.6, 22.8, 23.3, 24.9, 25.0, 26.7, 26.8, 27.6, 28.5, 29.2,
29.3, 29.4, 29.5, 29.6, 30.9, 31.1, 32.0, 34.2, 34.3, 34.5, 34.8, 35.0,
35.2, 35.3, 39.7, 41.4, 42.1, 46.7, 47.2, 62.3, 62.6, 68.4, 69.0, 73.1,
75.0, 171.7 (COOR), 172.0 (COOR), 173.2 (COOR), 173.6 (COOR),
4.1.26. 3
a
,7a
,12a-Tris{3-[2-(2-decanoyloxyethoxy)ethoxycarbonyl]
179.1 (COOH). IR (cmꢀ1):
1732, (CH) 1377,
m
(OH) 3479,
m(CH) 2921, 2853, m(C@O)
propanoyloxy}-5b-cholan-24-oic acid (30)
m
m(CO) 1151. Anal. Calc. for C51H86O12 (891.22):
The solution of benzyl ester 29 (1.81 g, 1.19 mmol) in AcOEt
(25 ml) was stirred with 10% Pd/C (0.19 g) at ambient temperature
under hydrogen for 1.5 h. Then the mixture was filtered through
celite and evaporated under reduced pressure. The crude product
was purified by Fc (hexane/acetone/AcOH 80:20:1). This provided
an oily liquid. Yield: 1.1 g (66%). 1H NMR (250 MHz, CDCl3), d:
0.73 (s, 3H, CH3), 0.83 (d, 3H, CH3, J = 6.02 Hz), 0.88 (m, 9H,
3 ꢁ CH3), 0.91 (s, 3H, CH3), 1.00–2.10 (m, 64H), 2.20–2.40 (m,
8H), 2.55–2.80 (m, 12H, 3 ꢁ COCH2CH2CO), 3.65–3.75 (m, 12H,
3 ꢁ CH2OCH2), 4.19–4.30 (m, 12H, 6 ꢁ CH2OCO), 4.58 (m, 1H,
68.73% C, 9.73% H; found: 67.51% C, 9.88% H. HR-MS: for
C
51H85O12 [MꢀH]ꢀ calculated: 889.6041 m/z; found: 889.6025 m/z.
4.1.29. Benzyl 3a-benzyloxycarboxy-7a,12a-bis{3-[2,3-
bis(decanoyloxy)propyloxycarbonyl]propanoyloxy}-5b-cholan-24-
oate (33)
The mixture of benzyl ester 20 (1.09 g, 1.72 mmol), BTEAC
(0.14 g, 0.61 mmol), CaH2 (0.28 g, 6.65 mmol) and toluene
(20 ml) was mixed with the solution of acyl chloride 13 (3.66 g,
7.05 mmol) in toluene (10 ml), and the resulting mixture was re-
fluxed under argon for 4 h. The mixture was poured into the mix-
ture of aqueous AcOH (3%) and toluene. After shaking the aqueous
layer was washed with toluene. The combined organic layers were
washed with brine (100 ml), dried with anhydrous MgSO4 and
evaporated under reduced pressure. The crude product was puri-
fied by Fc (toluene/AcOEt 15:1 and 25:2). This provided an oily li-
quid. Yield: 1.4 g (50%). 1H NMR (250 MHz, CDCl3), d: 0.69 (s, 3H,
CH3), 0.80 (d, 3H, CH3, J = 5.59 Hz), 0.88 (m, 12H, 4 ꢁ CH3), 0.91
(s, 3H, CH3), 0.95–2.20 (m, 78H), 2.23–2.45 (m, 10H), 2.50–2.75
(m, 8H, 2 ꢁ COCH2CH2CO), 4.05–4.23 (m, 4H, 2 ꢁ CH2OCO), 4.23–
4.36 (m, 4H, 2 ꢁ CH2OCO), 4.46 (m, 1H, C(3)H), 4.93 (m, 1H,
C
(3)H), 4.94 (m, 1H, C(7)H), 5.13 (m, 1H, C
(12)H). 13C NMR
(62.5 MHz, CDCl3), d: 12.3, 14.2, 17.5, 22.6, 22.6, 22.9, 25.0, 25.7,
26.9, 27.4, 28.9, 29.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 30.6,
30.7, 31.4, 32.0, 34.3, 34.5, 34.7, 34.8, 38.0, 41.1, 43.4, 45.3, 47.2,
63.3, 63.7, 63.8, 63.9, 69.1, 69.3, 71.3, 74.6, 75.7, 171.7 (COOR),
171.8 (COOR), 172.0 (COOR), 172.3 (COOR), 172.4 (COOR), 172.5
(COOR), 173.8 (COOR), 173.9 (COOR), 178.8 (COOH). IR (cmꢀ1):
m(CH) 2924, 2855, m(C@O) 1729, m(CH) 1380, m(CO) 1160. Anal.
Calc. for C78H130O23 (1435.85): 65.25% C, 9.13% H; found: 65.20%
C, 9.21% H. HR-MS: for
1433.8925 m/z; found: 1433.8870 m/z.
C
78H129O23 [MꢀH]ꢀ calculated:
C(7)H), 5.10 (m, 3H, PhCH2, C(12)H), 5.14 (s, 2H, PhCH2), 5.25 (m,
4.1.27. Benzyl 3
propanoyloxy}-7
a
a
-{3-[2,3-bis(decanoyloxy)propyloxycarbonyl]
2H, 2 ꢁ CHOCO), 7.30–7.40 (m, 10H, 2 ꢁ Ph). 13C NMR (62.5 MHz,
CDCl3), d: 12.3, 14.2, 17.5, 22.6, 22.8, 22.9, 24.9, 25.0, 25.7, 26.8,
27.4, 28.8, 28.8, 29.0, 29.2, 29.3, 29.4, 29.4, 29.6, 30.9, 31.3, 31.4,
32.0, 34.2, 34.3, 34.5, 34.6, 34.7, 34.8, 38.0, 41.0, 43.5, 45.3, 47.5,
62.3, 62.8, 66.2, 69.0, 69.6, 71.3, 75.8, 78.3, 128.3, 128.4, 128.5,
128.6, 128.7, 128.7, 135.5, 136.3, 154.7 (OCOO), 171.7 (COOR),
171.8 (COOR), 171.9 (COOR), 172.0 (COOR), 172.1 (COOR), 173.0
(COOR), 173.1 (COOR), 173.3 (COOR), 173.4 (COOR), 174.0 (COOR).
,12 -dihydroxy-5b-cholan-24-oate (31)
a
Acyl chloride 13 (3.84 g, 7.39 mmol) dissolved in CH2Cl2 (40 ml)
was slowly added to the solution of benzyl cholate (17, 6.32 g,
12.68 mmol) and DMAP (0.92 g, 7.53 mmol) in CH2Cl2 (60 ml) at
0 °C (ice bath). Then the mixture was warmed to ambient temper-
ature and stirred for 1.5 h. The mixture was poured into the mix-
ture of aqueous AcOH (3%) and CH2Cl2. After shaking aqueous
layer was separated and washed with CH2Cl2. The combined organ-
ic layers were washed with brine, dried with anhydrous MgSO4
and evaporated under reduced pressure. The crude product was
purified by gradient Fc (hexane/acetone 10:1 to 4:1). This provided
an oily liquid. Yield: 2.2 g (30%). 1H NMR (250 MHz, CDCl3), d: 0.59
(s, 3H, CH3), 0.81 (m, 6H, 2 ꢁ CH3), 0.83 (s, 3H, CH3), 0.90 (d, 3H,
CH3, J = 5.54 Hz), 0.95–2.00 (m, 49H), 2.05–2.43 (m, 9H), 2.43–
2.60 (m, 4H, COCH2CH2CO), 3.77 (m, 1H, C(7)H), 3.90 (m, 1H,
4.1.30. 7
a
,12a-Bis{3-[2,3-bis(decanoyloxy)propyloxycarbonyl]
propanoyloxy}-3
a-hydroxy-5b-cholan-24-oic acid (34)
The solution of HCOONH4 (0.44 g, 6.98 mmol) in MeOH (30 ml)
and 10% Pd/C (0.14 g) were added to the solution of ester 33
(1.35 g, 0.84 mmol) in AcOEt (40 ml), and the mixture was stirred
under argon for 3.5 h. Then the mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by Fc (hexane/acetone/AcOH 80:30:1). This provided an
oily liquid. Yield: 1.0 g (86%). 1H NMR (250 MHz, CDCl3), d: 0.70
(s, 3H, CH3), 0.80 (d, 3H, CH3, J = 5.95 Hz), 0.85 (m, 12H, 4 ꢁ CH3),
0.88 (s, 3H, CH3), 0.90–2.20 (m, 79H), 2.23–2.37 (m, 10H), 2.55–
2.75 (m, 8H, 2 ꢁ COCH2CH2CO), 3.45 (m, 1H, C(3)H), 4.05–4.22 (m,
4H, 2 ꢁ CH2OCO), 4.23–4.35 (m, 4H, 2 ꢁ CH2OCO), 4.90 (m, 1H,
C(12)H), 4.02–4.16 (m, 2H, CH2OCO), 4.18–4.30 (m, 2H, CH2OCO),
4.51 (m, 1H, C(3)H), 5.04 (2H, PhCH2), 5.18 (m, 1H, CHOCO), 7.22–
7.32 (m, Ph). 13C NMR (62.5 MHz, CDCl3), d: 12.6, 14.2, 17.4, 22.6,
22.8, 23.2, 24.9, 25.0, 26.7, 26.8, 27.5, 28.5, 29.1, 29.2, 29.4, 29.5,
31.0, 31.4, 32.0, 34.2, 34.3, 34.5, 34.8, 35.0, 35.2, 39.7, 41.3, 42.1,
46.6, 47.3, 62.3, 62.5, 66.2, 68.2, 69.0, 72.9, 74.9, 128.2, 128.3,
128.6, 136.2, 171.6 (COOR), 172.0 (COOR), 173.1 (COOR), 173.5
(COOR), 174.1 (COOR).
C
(7)H), 5.09 (m, 1H, C(12)H), 5.18–5.31 (m, 2H, 2 ꢁ CHOCO). 13C
NMR (62.5 MHz, CDCl3), d: 12.3, 14.2, 17.5, 22.6, 22.7, 22.9, 24.9,

L. Coufalová et al. / Steroids 78 (2013) 435–453
449
25.0, 25.5, 27.3, 28.9, 29.1, 29.2, 29.4, 29.5, 29.6, 30.4, 30.6, 30.8,
31.4, 32.0, 34.1, 34.3, 34.3, 34.7, 35.0, 37.9, 39.0, 41.0, 43.4, 45.2,
47.3, 62.2, 62.3, 62.6, 62.7, 62.8, 68.9, 71.5, 71.6, 75.9, 76.0, 171.7
(COOR), 172.0 (COOR), 173.1 (COOR), 173.4 (COOR), 173.5 (COOR),
The crude product was purified by gradient Fc (toluene/AcOEt
100:0 to 0:100). Yield: 4.5 g (37%). 1H NMR (250 MHz, CDCl3), d:
0.66 (s, 3H, CH3), 0.80–0.92 (m, 15H, 5 ꢁ CH3), 0.95–2.50 (m,
64H), 2.58 (m, 2H), 2.70 (m, 2H), 2.90 (m, 2H), 3.83 (m, 1H,
173.6 (COOR), 179.2 (COOH). IR (cmꢀ1):
2854, (C = O) 1732, (CH) 1378,
m
(OH) 3476,
m(CH) 2923,
C(7)H), 3.96 (m, 1H, C(12)H), 4.62 (m, 1H, C(3)H), 5.11 (2H, PhCH2),
m
m
m
(CO) 1148. Anal. Calc. for
7.30–7.38 (m, 5H, Ph). 13C NMR (62.5 MHz, CDCl3), d: 11.9, 12.3,
12.7, 12.9, 13.1, 17.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.7, 21.2, 22.6,
22.7, 22.9, 23.3, 24.6, 24.9, 26.7, 26.8, 27.5, 28.1, 28.5, 29.1, 29.7,
31.0, 31.2, 31.4, 32.8, 32.9, 34.6, 34.8, 35.0, 35.2, 35.3, 37.4, 37.5,
37.6, 37.7, 39.5, 39.7, 41.3, 42.2, 46.7, 47.7, 66.2, 68.3, 73.0, 74.9,
75.2, 117.5, 123.1, 125.1, 126.9, 128.3, 128.4, 128.7, 136.3, 140.6,
149.5, 171.1 (COOR), 171.8 (COOR), 174.1 (COOR).
C
78H132O19 (1373.87): 68.19% C, 9.68% H; found: 66.89% C, 9.75%
H. HR-MS: for C78H131O19 [MꢀH]ꢀ calculated: 1371.9285 m/z;
found: 1371.9222 m/z.
4.1.31. Benzyl 3a,7a,12a-tris{3-[2,3-bis(decanoyloxy)propyloxycarbonyl]
propanoyloxy}-5b-cholan-24-oate (35)
The mixture of benzyl ester 17 (0.86 g, 1.72 mmol), BTEAC
(0.22 g, 0.97 mmol), CaH2 (0.45 g, 10.69 mmol) and toluene
(15 ml) was mixed with the solution of acyl chloride 13 (5.17 g,
9.96 mmol) in toluene (10 ml), and the resulting mixture was re-
fluxed under argon for 3 h. The reaction mixture was poured into
the mixture of aqueous AcOH (6%) and toluene. After shaking the
aqueous layer was washed with toluene. The combined organic
layers were washed with saturated aqueous NaHCO3 and water,
dried with anhydrous MgSO4 and evaporated under reduced pres-
sure. The crude product was purified by gradient Fc (hexane/ace-
tone 20:1 to 5:1). This provided an oily liquid. Yield: 1.6 g (48%).
1H NMR (250 MHz, CDCl3), d: 0.70 (s, 3H, CH3), 0.80 (d, 3H, CH3,
J = 5.53 Hz), 0.88 (m, 21H, 7 ꢁ CH3), 0.95–2.15 (m, 106H), 2.20–
2.45 (m, 14H), 2.50–2.80 (m, 12H, 3 ꢁ COCH2CH2CO), 4.08–4.24
(m, 6H, 3 ꢁ CH2OCO), 4.24–4.37 (m, 6H, 3 ꢁ CH2OCO), 4.58 (m,
1H, C(3)H), 4.93 (m, 1H, C(7)H), 5.07–5.15 (m, 3H, PhCH2, C(12)H),
5.20–5.33 (m, 3H, 3 ꢁ CHOCO), 7.31–7.38 (m, 5H, Ph).
4.1.34. (+/ꢀ)-7
a,12a-Dihydroxy-3a-(3-{[2,5,7,8-tetramethyl-2-
(4,8,12-trimethyltridecyl)chroman-6-yl]oxycarbonyl}propanoyloxy)-
5b-cholan-24-oic acid (38)
To the solution of HCOONH4 (1.20 g, 19.03 mmol) and benzyl
ester 37 (4.50 g, 4.45 mmol) in AcOEt (50 ml) and MeOH
(150 ml) 10% Pd/C (0.54 g) was added and the mixture was stir-
red under argon for 5.5 h. The mixture was filtered through cel-
ite and evaporated under reduced pressure. The crude product
was purified by gradient Fc (CH2Cl2/MeOH/AcOH 100:5:1 and
100:10:1). This provided an oily liquid. Yield: 3.3 g (81%). 1H
NMR (250 MHz, CDCl3), d: 0.69 (s, 3H, CH3), 0.80–0.92 (m, 15H,
5 ꢁ CH3), 0.95–2.50 (m, 64H), 2.58 (m, 2H), 2.71 (m, 2H), 2.91
(m, 2H), 3.85 (m, 1H, C(7)H), 3.99 (m, 1H, C(12)H), 4.62 (m, 1H,
C
(3)H). 13C NMR (62.5 MHz, CDCl3), d: 11.9, 12.3, 12.7, 13.1,
17.5, 19.7, 19.8, 19.9, 20.7, 21.2, 22.6, 22.7, 22.8, 23.3, 24.0,
24.6, 24.9, 26.8, 26.8, 27.6, 28.1, 28.5, 29.1, 29.7, 30.9, 31.1,
31.2, 32.8, 32.9, 34.6, 34.8, 35.0, 35.3, 37.4, 37.5, 37.6, 37.7,
39.5, 39.7, 41.3, 42.2, 46.7, 47.3, 68.4, 73.1, 74.9, 75.2, 117.5,
123.1, 125.1, 126.9, 140.6, 149.5, 171.2 (COOR), 171.9 (COOR),
4.1.32. 3a,7a,12a-Tris{3-[2,3-bis(decanoyloxy)propyloxycarbonyl]
propanoyloxy}-5b-cholan-24-oic acid (36)
To the solution of benzyl ester 35 (1.54 g, 0.79 mmol) in AcOEt
(20 ml) 10% Pd/C (0.13 g) was added and the mixture was stirred
under hydrogen for 30 min. The mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by gradient Fc (hexane/acetone/AcOH 80:5:1 and
80:10:1). This provided an oily liquid. Yield: 1.0 g (70%). 1H NMR
(250 MHz, CDCl3), d: 0.72 (s, 3H, CH3), 0.78–0.94 (m, 24H,
8 ꢁ CH3), 0.95–2.15 (m, 106H), 2.20–2.45 (m, 14H), 2.50–2.80 (m,
12H, 3 ꢁ COCH2CH2CO), 4.05–4.23 (m, 6H, 3 ꢁ CH2OCO), 4.23–
4.36 (m, 6H, 3 ꢁ CH2OCO), 4.57 (m, 1H, C(3)H), 4.92 (m, 1H,
179.1 (COOH). IR (cmꢀ1):
(C@O) 1732, 1709, (CH) 1456,
Calc. for
m(OH) 3476,
m(CH) 2923, 2866,
m
m
m
(CO) 1144, 1072, 1022. Anal.
C
57H92O9 (921.33): 74.31% C, 10.06% H; found:
73.69% C, 10.58% H. HR-MS: for C57H91O9 [M–H]- calculated:
919.6663 m/z; found: 919.6680 m/z.
4.1.35. Benzyl 3a-benzyloxycarboxy-7a,12a-bis(3-{[2,5,7,8-
tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-
yl]oxycarbonyl}propanoyloxy)-5b-cholan-24-oate (39)
C
(7)H), 5.12 (m, 1H, C(12)H), 5.18–5.33 (m, 3H, 3 ꢁ CHOCO). 13C
The mixture of ester 20 (1.00 g, 1.58 mmol), BTEAC (0.16 g,
0.70 mmol), CaH2 (0.26 g, 6.18 mmol) and toluene (20 ml) was
mixed with the solution of acyl chloride 16 (2.81 g, 5.12 mmol)
in toluene (12 ml) and refluxed under argon for 3 h. The reaction
mixture was poured into the mixture of aqueous AcOH (3%) and
toluene. After shaking the aqueous layer was washed with tolu-
ene. The combined organic layers were washed with brine, dried
with anhydrous MgSO4 and evaporated under reduced pressure.
The crude product was purified by Fc (toluene/AcOEt/AcOH
200:5:1). This provided an oily liquid. Yield: 1.5 g (57%). 1H
NMR (250 MHz, CDCl3), d: 0.68 (s, 3H, CH3), 0.75 (d, 3H, CH3,
J = 5.95 Hz), 0.81–0.93 (m, 27H, 9 ꢁ CH3), 0.95–2.40 (m, 94H),
2.49–2.62 (m, 4H, 2 ꢁ CH2Ar), 2.65–3.10 (m, 8H, 2 ꢁ COCH2CH2-
CO), 4.43 (m, 1H, C(3)H), 4.95 (m, 1H, C(7)H), 5.06 (4H, 2 ꢁ PhCH2-
O), 5.14 (m, 1H, C(12)H), 7.29–7.37 (m, 10H, 2 ꢁ Ph). 13C NMR
(62.5 MHz, CDCl3), d: 11.9, 12.0, 12.2, 12.3, 13.1, 17.5, 19.6,
19.7, 19.8, 19.9, 20.7, 21.2, 22.6, 22.8, 22.9, 24.6, 25.0, 25.8,
26.8, 27.2, 28.1, 28.8, 28.9, 29.1, 29.4, 29.5, 30.7, 31.2, 32.8,
32.9, 34.4, 34.6, 34.7, 37.4, 37.5, 37.6, 37.7, 37.9, 39.5, 41.0,
43.5, 45.3, 47.6, 66.2, 69.5, 71.3, 75.2, 75.8, 78.2, 117.4, 117.5,
123.0, 123.1, 125.2, 126.9, 128.3, 128.4, 128.5, 128.6, 128.6,
128.7, 135.4, 136.2, 140.6, 140.7, 149.5, 149.6, 154.6, 170.8
(COOR), 171.0 (COOR), 171.9 (COOR), 172.0 (COOR), 173.9
(COOR).
NMR (62.5 MHz, CDCl3), d: 12.3, 14.2, 17.5, 22.7, 22.8, 22.9, 24.9,
25.0, 28.8, 28.9, 29.0, 29.1, 29.2, 29.3, 29.4, 29.6, 30.6, 30.7, 31.4,
32.0, 34.2, 34.3, 34.5, 34.8, 34.9, 38.0, 41.1, 43.5, 45.3, 47.3, 62.2,
62.3, 62.7, 62.8, 68.9, 71.3, 74.6, 75.8, 171.6 (COOR), 171.7 (COOR),
171.9 (COOR), 172.0 (COOR), 172.1 (COOR), 173.0 (COOR), 173.1
(COOR), 173.2 (COOR), 173.3 (COOR), 173.4 (COOR), 173.5 (COOR),
178.6 (COOH). IR (cmꢀ1):
1378, (CO) 1148. Anal. Calc. for C105H178O26 (1856.52): 67.93%
m(CH) 2923, 2854, m(C@O) 1733, m(CH)
m
C, 9.66% H; found: 67.07% C, 10.05% H. HR-MS: for C105H177O26
[MꢀH]ꢀ calculated: 1854.2528 m/z; found: 1854.2402 m/z.
4.1.33. Benzyl (+/ꢀ)-7
a,12a-dihydroxy-3a-(3-{[2,5,7,8-tetramethyl-
2-(4,8,12-trimethyltridecyl)chroman-6-
yl]oxycarbonyl}propanoyloxy)-5b-cholan-24-oate (37)
Acyl chloride 16 (7.96 g, 14.49 mmol) dissolved in CH2Cl2
(20 ml) was slowly added to the solution of benzyl cholate (17,
6.03 g, 12.1 mmol) and DMAP (1.80 g, 14.73 mmol) in CH2Cl2
(40 ml) at 0 °C (ice bath). The mixture was stirred at this tempera-
ture for 20 min. Then the mixture was warmed to ambient temper-
ature and poured into aqueous AcOH (6%). After shaking the
aqueous layer was washed with CH2Cl2. The combined organic lay-
ers were washed with saturated aqueous NaHCO3, water, dried
with anhydrous MgSO4 and evaporated under reduced pressure.

450
L. Coufalová et al. / Steroids 78 (2013) 435–453
4.1.36. 3
a
-Hydroxy-7
a
,12
a
-bis(3-{[2,5,7,8-tetramethyl-2-(4,8,12-
25.9, 26.9, 27.3, 28.1, 28.6, 29.0, 29.1, 29.5, 30.5, 30.8, 31.2, 32.8,
32.9, 34.5, 34.7, 34.8, 37.4, 37.5, 37.6, 37.7, 38.0, 39.5, 41.0, 43.5,
45.3, 47.5, 71.3, 74.6, 75.2, 75.8, 117.5, 123.1, 125.0, 125.1, 126.8,
140.6, 149.5, 170.9 (COOR), 171.0 (COOR), 171.1 (COOR), 171.8
trimethyltridecyl)chroman-6-yl]oxycarbonyl}propanoyloxy)-5b-
cholan-24-oic acid (40)
The mixture of ester 39 (1.34 g, 0.808 mmol), HCOONH4 (0.42 g,
6.66 mmol), AcOEt (40 ml), MeOH (30 ml) and 10% Pd/C (0.11 g)
was stirred under argon for 2 h. The mixture was filtered through
celite and evaporated under reduced pressure. The crude product
was purified by gradient Fc (CH2Cl2/MeOH/AcOH 300:5:1). This
provided an oily liquid. Yield: 1.0 g (85%). 1H NMR (250 MHz,
CDCl3), d: 0.73 (s, 3H, CH3), 0.79 (d, 3H, CH3, J = 6.17 Hz), 0.82–
0.92 (m, 27H, 9 ꢁ CH3), 0.95–2.40 (m, 95H), 2.50–2.65 (m, 4H,
2 ꢁ CH2Ar), 2.72–3.50 (m, 8H, 2 ꢁ COCH2CH2CO), 3.38 (m, 1H,
(COOR), 171.8 (COOR), 179.4 (COOH). IR (cmꢀ1):
m
(CH) 2924,
2866,
m(C@O) 1753, 1731, 1708, m(CH) 1377, m(CO) 1137. Anal.
Calc. for C123H196O17 (1946.86): 75.88% C, 10.15% H; found:
74.83% C, 10.36% H. HR-MS: for C123H195O17 [MꢀH]ꢀ calculated:
1944.4394 m/z; found: 1944.4269 m/z.
4.2. Lipophilicity determination and calculation of molecular
descriptors
C
(3)H), 4.96 (m, 1H, C(7)H), 5.15 (m, 1H, C
(12)H). 13C NMR
(62.5 MHz, CDCl3), d: 11.9, 12.0, 12.2, 12.3, 13.3, 17.5, 19.6, 19.7,
19.8, 19.9, 20.0, 20.7, 21.2, 22.6, 22.7, 22.8, 23.0, 24.6, 24.9, 25.6,
27.3, 28.1, 28.8, 28.9, 29.0, 29.5, 29.6, 30.4, 30.5, 30.8, 31.2, 31.5,
32.8, 32.9, 34.4, 34.7, 35.1, 37.4, 37.5, 37.6, 37.7, 38.1, 39.0, 39.5,
41.1, 43.4, 45.3, 47.5, 71.6, 71.6, 75.2, 76.0, 117.5, 117.6, 123.1,
125.1, 125.2, 126.9, 140.6, 140.7, 149.6, 171.0 (COOR), 171.1
RM values were determined from the RP-18 TLC measurements.
The solution of a compound in CH2Cl2 was spotted on a TLC plate
(TLC silica gel 60 RP-18 F254s, 20 ꢁ 20 cm, Merck), 1.5 cm from
the edge. The volatiles were carefully evaporated, and the plate
was developed by MeOH/AcOH 100:1 solvent mixture for 2.5 h.
After drying, the plate was sprayed for the detection by solution
of Ce(SO4)2 in H2SO4 and heated. RM data were obtained from equa-
tion: RM = log(1/Rfꢀ1). RM values can be used as the lipophilicity
index converted to log P scale [48].
Log P values (i.e., the logarithm of the partition coefficient for
octanol/water) were predicted using ACD/LogP DB software
(ACD/Labs, ver. 12.01, Advanced Chemistry Development, Inc., Tor-
onto, ON, Canada, 2010). Log S values (as ACD/Labs aqueous log S at
pH 7.4) were calculated by ACD/LogS DB software (ver. 12.01, Ad-
vanced Chemistry Development, Inc., Toronto, ON, Canada, 2010).
ACD/Solubility DB (ver. 12.01, Advanced Chemistry Development,
Inc., Toronto, ON, Canada, 2010) is a program that calculates aque-
ous solubility values at any pH under the standard conditions (and
zero ionic strength). The accuracy of calculations (according to the
vendor) for simple structures is usually better than 0.2–0.5 loga-
rithmic units (for complex structures it is better than 0.5–1.0 log-
arithmic units). Solubility is not derived from log P and takes into
account not only the pH (solubility as a function of pH) but com-
pares the fragmental estimations with experimental material from
ca 6000 compounds databased. Molar volume (MV [cm3]) was cal-
culated by means of ACD/ChemSketch (ACD/Labs, ver. 12.01, Ad-
vanced Chemistry Development, Inc., Toronto, ON, Canada, 2010).
Polar surface area (PSA [Å2]) was calculated using CambrigeSoft-
ware ChemBio3D (ver. 12, CambridgeSoft, Cambridge, MA, USA).
All the results are shown in Table 1.
(COOR), 171.9 (COOR), 178.8 (COOH). IR (cmꢀ1):
2865, (C@O) 1730, (CO) 1140, 1109, 1072. Anal. Calc. for
m(CH) 2923,
m
m
C
90H144O13 (1434.10): 75.38% C, 10.12% H; found: 74.87% C,
10.59% H. HR-MS: for C90H143O13 [MꢀH]ꢀ calculated:
1432.0529 m/z; found: 1432.0514 m/z.
4.1.37. Benzyl (+/ꢀ)-3
a,7a,12a-tris(3-{[2,5,7,8-tetramethyl-2-(4,8,12-
trimethyltridecyl)chroman-6-yl]oxycarbonyl}propanoyloxy)-5b-
cholan-24-oate (41)
The mixture of benzyl cholate (17, 1.00 g, 2.01 mmol), BTEAC
(0.18 g, 0.79 mmol), CaH2 (0.51 g, 12.11 mmol) and toluene
(20 ml) was mixed with the solution of acyl chloride 16 (6.65 g,
12.11 mmol) in toluene (10 ml) and refluxed under argon for
3.5 h. The mixture was poured into the mixture of aqueous AcOH
(3%) and toluene. After shaking the aqueous layer was washed with
toluene. The combined organic layers were washed with brine,
dried with anhydrous MgSO4 and evaporated under reduced pres-
sure. The crude product was purified by flash chromatography on
silica gel (toluene/AcOEt 20:1). This provided an oily liquid. Yield:
3.5 g (86%). 1H NMR (250 MHz, CDCl3), d: 0.68 (s, 3H, CH3), 0.76 (d,
3H, CH3, J = 5.99 Hz), 0.80–0.93 (m, 39H, 13 ꢁ CH3), 0.95–2.40 (m,
129H), 2.50–3.05 (m, 18H, 3 ꢁ COCH2CH2CO, 3 ꢁ CH2Ar), 4.58 (m,
1H, C(3)H), 4.94 (m, 1H, C(7)H), 5.07 (2H, PhCH2), 5.15 (m, 1H,
C
(12)H), 7.29–7.35 (m, 5H, Ph). 13C NMR (62.5 MHz, CDCl3), d:
12.0, 12.3, 12.4, 13.1, 17.5, 19.6, 19.7, 19.8, 19.9, 20.7, 21.2, 22.7,
22.8, 22.9, 24.6, 25.0, 25.9, 27.0, 28.1, 28.7, 29.0, 29.2, 29.5, 30.8,
31.3, 32.8, 32.9, 34.5, 34.7, 34.8, 37.5, 37.6, 37.7, 37.8, 38.0, 39.5,
41.1, 43.6, 45.4, 47.7, 66.2, 71.4, 74.6, 75.2, 75.9, 123.1, 125.0,
125.1, 126.9, 128.3, 128.4, 128.7, 136.3, 140.7, 149.6, 170.9 (COOR),
171.1 (COOR), 171.2 (COOR), 171.8 (COOR), 171.9 (COOR), 173.9
(COOR).
4.3. In vitro screening of penetration enhancing effect and sample
analysis
Skin samples were obtained from porcine ear. Full thickness
dorsal skin was cut in fragments and stored at ꢀ20 °C until utilized.
Skin samples were slowly thawed (at 4 °C overnight and then at
ambient temperature) before each experiment. The penetration
enhancing effect of newly synthesized target compounds was eval-
uated in vitro, using a vertical Franz diffusion cell (SES – Analytical
Systems, GmbH, Germany), with a donor surface area of 0.635 cm2
and receptor volume of 5.2 ml. The skin was mounted between the
donor and receptor compartments of the Franz diffusion cell with
the epidermal side up. The receptor compartment was filled with
phosphate buffered saline (pH 7.4) and was maintained at
37 0.5 °C, while using circulating water bath. The receptor com-
partment content was continuously stirred using a magnetic stir-
ring bar. The skin was kept in contact with the receptor phase
for 0.5 h prior to the experiment. The donor samples were prepared
by dissolving the tested enhancer (1 mg) in propylene glycol
(0.5 ml), and the solution of theophylline (5 mg) in water (0.5 ml)
was added. This mixture was shaken vigorously and then sonicated
for 10 min at 40 °C, then this stable system (dissolved theophylline
4.1.38. (+/ꢀ)-3
a,7a,12a-Tris(3-{[2,5,7,8-tetramethyl-2-(4,8,12-
trimethyltridecyl)chroman-6-yl]oxycarbonyl}propanoyloxy)-5b-
cholan-24-oic acid (42)
The solution of HCOONH4 (0.42 g, 6.66 mmol) in MeOH (30 ml)
and 10% Pd/C (0.10 g) were added to the solution of benzyl ester 41
(3.15 g, 1.55 mmol) in AcOEt (50 ml), and the mixture was stirred
under argon for 1.5 h. Then the mixture was filtered through celite
and evaporated under reduced pressure. The crude product was
purified by Fc (hexane/acetone/AcOH 80:10:1). This provided an
oily liquid. Yield: 2.6 g (87%). 1H NMR (250 MHz, CDCl3), d: 0.73
(s, 3H, CH3), 0.79 (d, 3H, CH3, J = 6.07 Hz), 0.83–0.95 (m, 39H,
13 ꢁ CH3), 1.00–2.40 (m, 129H), 2.50–3.05 (m, 18H, 3 ꢁ COCH2CH2-
CO, 3 ꢁ CH2Ar), 4.59 (m, 1H, C(3)H), 4.97 (m, 1H, C(7)H), 5.18 (m, 1H,
C
(12)H). 13C NMR (62.5 MHz, CDCl3), d: 11.9, 12.2, 12.4, 13.1, 17.5,
19.6, 19.7, 19.8, 19.9, 20.0, 20.7, 21.2, 22.7, 22.8, 22.9, 24.6, 24.9,

L. Coufalová et al. / Steroids 78 (2013) 435–453
451
in enhancer emulsion) was applied to the skin surface and the do-
4.5. In vitro cytotoxicity/anti-proliferative assay
nor compartment of the cell was covered by ParafilmÒ. The control
samples were prepared in the same manner without enhancers.
Samples (0.5 ml) of the receptor phase were withdrawn at seven
pre-determined time intervals over 24 h (1, 2, 4, 6, 8, 12 and
24 h) and the cell was refilled with an equivalent amount of fresh
buffer solution. A minimum of five determinations was performed
using skin fragments from a minimum of 2 animals for each com-
pound. The samples were immediately analyzed by HPLC.
Dulbecco’s modified Eagle’s medium (DMEM),
L
-glutamine,
trypsin, penicillin and streptomycin were purchased from Sigma
(MO, USA); foetal bovine serum (FBS) and Calcein AM from Invitro-
gen (CA, USA). The cell lines used for screening, T-lymphoblastic
leukemia CEM, breast adenocarcinoma MCF7, and human foreskin
fibroblasts BJ, were obtained from the American Type Culture Col-
lection (Manassas, VA, USA). Cells were cultured in DMEM medium
(Sigma, MO, USA). All media used were supplemented with 10%
Analysis of samples for theophylline content was performed
using an Agilent 1200 series HPLC system, equipped with a diode
array detection (DAD) system, a quaternary model pump and an
automatic injector (Agilent Technologies, Germany). Data acquisi-
tion was performed using ChemStation chromatography software.
heat-inactivated foetal bovine serum, 2 mM L-glutamine, 10000 U
penicillin and 10 mg/ml streptomycin. The cell lines were main-
tained under standard cell culture conditions at 37 °C and 5% CO2
in a humid environment. Cells were subcultured twice or three
times a week using the standard trypsinization procedure. Suspen-
sions with approximately 1.0 ꢁ 105 cells/ml (0.5 ꢁ 105 cells/ml for
BJ) were distributed in 96-well microtiter plates, and after 12 h of
stabilization the tested compounds were added at the desired con-
centrations in Lutrol F 127. Control cultures were treated with Lu-
trol F 127 alone, and the final concentration of Lutrol F 127 in the
reaction mixture never exceeded 0.5%. In most cases six serial 3-
fold dilutions of the test substances were added at time zero in
A Waters SymmetryÒ C8
ford, MA, USA) chromatographic column was used. A mixture of
MeCN (HPLC grade, 50.0%) and H2O (HPLC – Mili-Q Grade, 50.0%)
was used as a mobile phase. The total flow of the column was
5
l
m, 4.6 ꢁ 250 mm (Waters Corp., Mil-
0.5 ml/min, injection 10 ll, column temperature 25 °C and sample
temperature 10 °C. The detection wavelength of 280 nm was cho-
sen. The retention time (tR) of theophylline was 5.07 0.05 min.
The cumulative amounts of theophylline that penetrated
through the skin into the receptor compartment (
l
g/cm2), were
20
concentration in the wells was 37
Calcein AM solution (2 M, Molecular Probes, Invitrogen, CA,
l
l aliquots to the microtiter plate wells, and the highest final
corrected for sample removal, and plotted against time (h). An
l
M. After incubation for 72 h,
approximately linear dependence was found (R2 P 0.98), and stea-
l
dy state fluxes (
l
g/cm2/h) were calculated using the linear region
USA) was added, and the cells were incubated for a further hour.
The fluorescence from viable cells was then quantified, using a Flu-
oroskan Ascent fluorometer (Labsystems, Finland). The percentage
of surviving cells in each well was calculated by dividing the inten-
sity of the fluorescence signals from the exposed wells by the
intensity of signals from control wells and multiplying by 100.
These ratios were then used to construct dose-response curves
from which IC50 values were calculated, and the results obtained
for the respective compounds are shown in Table 2.
of the plots. Enhancement ratios (ERs) were calculated as ratios
of theophylline flux with and without the enhancer. The results
are summarized in Table 2.
4.4. PAMPA experiments
The penetration enhancing effect of newly synthesized final
compounds was evaluated in vitro, using a vertical PAMPA (Parallel
Artificial Membrane Permeability Assay) system (BD GentestTM-
Pre-Coated PAMPA Plate System, 96 wells, http://www.bdbeuro-
pe.com). The donor samples were prepared by dissolving the
4.6. Statistical analysis
tested enhancer (16.9 lmol) and theophylline (83.3 lmol) in etha-
All experiments were carried out fivefold (ERs data for skin),
fourfold (PAMPA) or threefold (anti-proliferative activity). Data
were expressed as means SD. Differences were evaluated by
one-way analysis of the variance (ANOVA) test completed by the
Bonferroni’s multicomparison test (ORIGIN PRO7). The differences
were considered significant at P = 0.05.
nol (0.3 ml) and water (2.7 ml). This mixture was shaken vigor-
ously for 30 s and then sonicated for 10 min at 40 °C. The control
samples were prepared in the same manner without enhancers.
As a receptor phase carbonate buffer saline (pH 7.4) was used.
About 0.5 h before the experiment the PAMPA system was taken
out from the freezer and warm up to the ambient temperature.
The receptor phase (200 ll/well) was pipetted into the upper wells.
The donor phase (stable system of dissolved theophylline in en-
hancer emulsion/microsuspension) was pipetted into the lower
Acknowledgments
ones (300
l
l/well). After 5 h 10
l
l of the receptor phase was taken
l). A
This study was supported by the Czech Science Foundation (the
Grant Agency of the Czech Republic) in projects no. P304/11/2246,
304/10/1951 and P503/11/0616, by grant no. ED0007/01/01 Centre
of the Region Hana for Biotechnological and Agricultural Research,
by the Ministry of Education of the Czech Republic MSM6046
137305 and by Sanofi-Aventis Pharma Slovakia.
from each well and mixed with physiological solution (990
minimum of four determinations was performed. The samples
l
were stored at ꢀ18 °C until analyzed by HPLC.
Analysis of samples for theophylline content was performed,
using a Waters Alliance 2695 XE HPLC separation module and a
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA). A Waters SymmetryÒ C8
5
l
m, 4.6 ꢁ 250 mm (Waters
References
Corp., Milford, MA, USA) chromatographic column was also used.
The HPLC separation process was monitored by Empower™ 2
Chromatography Data Software, Waters 2009 (Waters Corp., Mil-
ford, MA, USA). The mixture of MeCN (HPLC grade, 50.0%) and
H2O (HPLC – Mili-Q Grade, 50.0%) was used as a mobile phase.
[1] Junghanns JUAH, Muller RH. Nanocrystal technology, drug delivery and clinical
applications. Int J Nanomed 2008;3:295.
[2] Payghan SA, Bhat M, Savla A, Toppo E, Purohit S. Solubility of active
pharmaceutical ingredients (API) has always been a concern for formulators,
since inadequate aqueous solubility may hamper development of parenteral
The total flow of the column was 0.5 ml/min, injection 10 ll, col-
products
and
limit
bioavailability
of
oral
products.
<http://
umn temperature 25 °C and sample temperature 10 °C. The detec-
tion wavelength of 280 nm was chosen. The retention time (tR) of
theophylline was 5.07 0.05 min. Enhancement ratios (ERs) were
calculated as ratios of theophylline flux with and without the en-
hancer. The results are summarized in Table 2.
www.pharmainfo.net/reviews/potential-solubility-drug-discovery-and-
development/>.
[3] Kerns EH, Li D. Drug-like properties: concept, structure design and
methods. San Diego: Elsevier; 2008.
[4] Hofmann AF, Mysels KJ. Bile salts as biological surfactants. Colloid Surf B
1988;30:145.

452
L. Coufalová et al. / Steroids 78 (2013) 435–453
[5] Kim KH, Singh BN. Drug delivery – oral route. In: Swarbrick J, Boylan JC,
[35] Sayyed-Ahmad A, Lichtenberger LM, Gorfe AA. Structure and dynamics of
cholic acid and dodecylphosphocholine-cholic acid aggregates. Langmuir
2010;26:13407.
editors. Encyclopedia of Pharmaceutical Technology. New York: Marcel
Dekker; 2002. p. 899–900.
[6] Smith EW, Maibach HI. Percutaneous Penetration Enhancers. 2nd ed. Boca
Raton: CRC Press; 2006.
[36] Brychtova K, Jampilek J, Opatrilova R, Raich I, Csollei J. Synthesis, physico-
chemical properties and penetration activity of alkyl-6-(2,5-dioxopyrrolidin-
[7] Sharma P, Chawla HPS, Panchagnula R. The role of sorption promoters in
increasing the bioavailability of drugs in oral preparations. Drugs Fut
1999;24:1221.
[8] Mrozek L, Dvorakova L, Mandelova Z, Rarova L, Rezacova A, Placek L, Opatrilova
R, Dohnal J, Paleta O, Kral V, Drasar P, Jampilek J. Investigation of new acyloxy
derivatives of cholic acid and their esters as drug absorption modifiers.
Steroids 2011;76:1082.
[9] Reddy SM, Sinha VR, Reddy DS. Novel oral colon-specific drug delivery systems
for pharmacotherapy of peptide and nonpeptide drugs. Drugs Today
1999;35:537.
1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates
penetration enhancers. Bioorg Med Chem 2010;18:73.
as
potential
transdermal
[37] Brychtova K, Opatrilova R, Raich I, Kalinowski DS, Dvorakova L, Placek L, Csollei
J, Richardson DR, Jampilek J. Investigating the activity of 2-substituted alkyl-6-
(2,5-dioxopyrrolidin-1-yl)hexanoates as skin penetration enhancers. Bioorg
Med Chem 2010;18:8556.
[38] Brychtova K, Dvorakova L, Opatrilova R, Raich I, Kacerova S, Placek L,
Kalinowski DS, Richardson DR, Jampilek J. Investigation of substituted 6-
aminohexanoates as skin penetration enhancers. Bioorg Med Chem
2012;20:86.
[10] Michael S, Thole M, Dillmann R, Fahr A, Drewe J, Fricker G. Improvement of
[39] Franz TJ. Percutaneous absorption on the relevance of in vitro data. J Invest
Dermatol 1975;64:190.
intestinal peptide absorption by
a
synthetic bile acid derivative,
cholylsarcosine. Eur J Pharm Sci 2000;10:133.
[40] Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening:
parallel artificial membrane permeation assay in the description of passive
absorption processes. J Med Chem 1998;41:1007.
[11] Cernea S, Kidron M, Wohlgelernter J, Modi P, Raz I. Comparison of
pharmacokinetic and pharmacodynamic properties of single-dose oral
insulin spray and subcutaneous insulin injection in healthy subjects using
the euglycemic clamp technique. Clin Ther 2004;26:2084.
[12] Sharma P, Varma MVS, Chawla HPS, Panchagnula R. Relationship between
lipophilicity of BCS class III and IV drugs and the functional activity of peroral
absorption enhancers. Farmaco 2005;60:870.
[13] Mikov M, Fawcett JP, Kuhajda K, Kevresan S. Pharmacology of bile acids and
their derivatives: absorption promoters and therapeutic agents. Eur J Drug
Metab Pharmacokinet 2006;31:237.
[14] Kim SK, Lee DY, Lee E, Lee YK, Kim CY, Moon HT, Byun Y. Absorption study of
deoxycholic acid-heparin conjugate as a new form of oral anti-coagulant. J
Control Release 2007;120:4.
[15] Mikov M, Al-Salami H, Golocorbin-Kon S, Skrbic R, Raskovic A, Fawcett JP. The
influence of 3alpha,7alpha-dihydroxy-12-keto-5beta-cholanate on gliclazide
pharmacokinetics and glucose levels in a rat model of diabetes. Eur J Drug
Metab Pharmacokinet 2008;33:137.
[16] Yang L, Zhang H, Fawcett JP, Mikov M, Tucker IG. Effect of bile salts on the
transport of morphine-6-glucuronide in rat brain endothelial cells. J Pharm Sci
2011;100:1516.
[41] Avdeef A, Tsinman O. PAMPA – a drug absorption in vitro model 13. Chemical
selectivity due to membrane hydrogen bonding: in combo comparisons of
HDM-, DOPC-, and DS-PAMPA models. Eur J Pharm Sci 2006;28:43.
[42] Tam KY, Velicky M, Dryfe RAW. The importance of and different approaches to
permeability determination. In: Mandic Z, editor. Physico-chemical methods
in drug discovery and development. Zagreb: IAPC Publishing; 2012. p. 121–64.
[43] Anelli LP, Lattuada L, Uggeri F. One-pot mitsunobu-staudinger preparation of
3-aminocholan-24-oic acid esters from 3-hydroxycholan-24-oic acid esters.
Synth Commun 1998;28:109.
[44] Larsson U, Carlson R. Synthesis of amino acids with modified. Principal
properties 3: sulfur-containing amino acids. Acta Chem Scand 1994;48:511.
[45] Fourneau EJ. Amino alcohols. Derivatives of glycerol and phenol ethers. Pharm
Chim 1910;1:97.
[46] Cunningham C, Wenzel SW, Decker C, Seidling JR. (Kimberly-Clark Worldwide,
Inc.). Tain-discharging and removing system. WO 2009133518 A2, 05.11.2009.
[47] Delaire S, Adao A, Desmurs JR, Gelo-Pujic M, Saint-Jalmes L, Kassem T. (Rhodia
Chimie
& Chanel Parfums Beaute). Polyphenol-based bioprecursor. WO
2006134282 A1, 21.12.2006.
[17] Yang L, Fawcett JP, Ostergaard J, Zhang H, Tucker IG. Mechanistic studies of the
effect of bile salts on rhodamine 123 uptake into RBE4 cells. Mol Pharm
2012;9:29.
[18] Duchateau GSMJE, Zuidema J, Merkus FWHM. Bile salts and intranasal drug
absorption. Int J Pharm 1986;31:196.
[19] Zhou XH, Po ALW. Effects of cholic acid and other enhancers on the
bioavailability of insulin from a subcutaneous site. Int J Pharm 1991;69:29.
[20] Barry BW. Modern methods of promoting drug absorption through the skin.
Mol Aspects Med 1991;12:195.
[21] Carelli V, Di Colo G, Nannipieri E, Serafini MF. Bile acids as enhancers of steroid
penetration through excised hairless mouse skin. Int J Pharm 1993;89:81.
[22] Hadgraft J. Pharmaceutical skin penetration enhancers. New York: Marcel
Dekker; 1993.
[48] Mannhold R, Dross K, Sonntag C. Estimation of lipophilicity by reversed-phase
thin-layer chromatography. In: Pliska V, Testa B, van de Waterbeemd H,
editors. Lipophilicity in drug action and toxicology. Weinheim: VCH; 1996. p.
141–56.
[49] Dewitte RS. Understanding polyelectrolytes. Modern Drug Discov 2004;7:41.
[50] Japertas P, Didziapetris R, Petrauskas A. Fragmental methods in the analysis of
biological activities of diverse compound sets. Mini-Rev Med Chem
2003;3:797–808.
[51] Ni N, El-Sayed MM, Sanghvi T, Yalkowsky SH. Estimation of the effect of NaCl
on the solubility of organic compounds in aqueous solutions. J Pharm Sci
2000;89:1620.
[52] Katz M, Ben-Shlush I, Kolusheva S, Jelinek R. Rapid colorimetric screening of
drug interaction and penetration through lipid barriers. Pharm Res
2006;23:580.
[23] Lassmann-Vague V, Raccah D. Alternatives routes of insulin delivery. Diabetes
Metab 2006;32:513.
[53] Fang JY, Tsai TH, Hung CF, Wong WW. Development and evaluation of the
essential oil from Magnolia fargesii for enhancing the transdermal absorption
of theophylline and cianidanol. J Pharm Pharmacol 2004;56:1493.
[54] Sloan KB, Beall HD, Taylor HE, Getz JJ, Villaneuva R, Nipper R, Smith K.
[24] Posa M, Kevresan S, Mikov M, Cirin-Novta V, Kuhajda K. Effect of cholic acid
and its keto derivatives on the analgesic action of lidocaine and associated
biochemical parameters in rats. Eur J Drug Metab Pharmacokinet 2007;32:109.
[25] Hassan N, Ahad A, Ali M, Ali J. Chemical permeation enhancers for transbuccal
drug delivery. Exp Opin Drug Deliv 2010;7:97.
Transdermal delivery of theophylline from alcohol vehicles. Int
1998;171:185.
J Pharm
[26] Jampilek J, Brychtova K. Azone analogues: classification, design, and
transdermal penetration principles. Med Res Rev 2012;32:907.
[27] Hofmann AF, Roda A. Physicochemical properties of bile acids and their
relationship to biological properties: an overview of the problem. J Lipid Res
1984;25:1477.
[55] Evrard D, Touitou E, Kolusheva S, Fishov Y, Jelinek R. A new colorimetric assay
for studying and rapid screening of membrane penetration enhancers. Pharm
Res 2001;18:943.
[56] Williams AC, Barry BW. Urea analogues in propylene glycol as penetration
enhancers in human skin. Int J Pharm 1989;56:43.
[28] Araki YI, Lee S, Sugihara G, Furuichi M, Yamashita S, Ohseto F. New cationic
surfactants derived from bile acids: synthesis and thermodynamic and
biophysicochemical properties such as membrane perturbation and protein
solubilizing abilities. Colloid Surf B 1996;8:81.
[29] Hishikawa Y, Sada K, Watanbe R, Miyata M, Hanabusa K. A novel class of
organogelator based on n-isopropylcholamide and the first observation of
fibrous colloidal aggregates. Chem Lett 1998;1998:795.
[30] Valenta C, Nowack E, Bernkop-Schnurch A. Deoxycholate-hydrogels: novel
drug carrier systems for topical use. Int J Pharm 1999;185:103.
[31] Willemen HM, Vermonden TA, Marcelis TM, Sudholter EJR. N-cholyl amino
[57] Yamane MA, Williams AC, Barry BW. Terpene penetration enhancers in
propylene glycol/water co-solvent systems: effectiveness and mechanism of
action. J Pharm Pharmacol 1995;47:978.
[58] Jacobi U, Kaiser M, Toll R, Mangelsdorf S, Audring H, Otberg N, Sterry W,
Lademann J. Porcine ear skin: an in vitro model for human skin. Skin Res
Technol 2007;13:19.
[59] Herkenne C, Naik A, Kalia YN, Hadgraft J, Guy RH. Pig ear skin ex vivo as a
model for in vivo dermatopharmacokinetic studies in man. Pharm Res
2006;23:1850.
[60] Meyer W, Schwarz K, Neurand KT. The skin of domestic mammals as a model
for the human skin, with special reference to the domestic pig. Curr Probl
Dermatol 1978;7:39.
acid alkyl esters
2001;2001:2329.
– a novel class of organogelators. Eur J Org Chem
[32] Willemen HM, Vermonden T, Marcelis ATM, Sudholter EJR. Alkyl derivatives of
cholic acid as organogelators: one-component and two-component gel.
Langmuir 2002;18:7102.
[61] Wilkinson SC, Maas WJM, Nielsen JB, Greaves LC, Sandt JJM, Williams FM.
Interactions of skin thickness and physicochemical properties of test
compounds in percutaneous penetration studies. Int Arch Occup Environ
Health 2006;79:405.
[33] Dukh M, Saman D, Kroulik J, Cerny I, Pouzar V, Kral V, Drasar P. Metal
coordination as
a
tool for controlling the self-assembling and gelation
[62] Brain KR, Walters KA. Molecular modeling of skin permeation enhancement by
chemical agents. In: Walters KA, Hadgraft J, editors. Pharmaceutical skin
penetration enhancement. New York: Marcel Dekker; 1993. p. 389–416.
[63] Suzuki H, Sunada H. Mechanistic studies on hydrotropic solubilization of
nifedipine in nicotinamide solution. Chem Pharm Bull 1998;46:125.
properties of novel type cholic amide-phenanthroline gelating agent.
Tetrahedron 2003;59:4069.
[34] Valkonen A, Lahtinen M, Virtanen E, Kikkonen S, Kolehmainen E. Bile acid
amidoalcohols: simple organogelators. Biosens Bioelectron 2004;20:1233.

L. Coufalová et al. / Steroids 78 (2013) 435–453
453
[64] Da Silva RC, Spitzer M, Da Silva LHM, Loh W. Investigations on the mechanism
of aqueous solubility increase caused by some hydrotropes. Thermochim Acta
1999;328:161.
[65] Devinsky F, Kopecka-Leitmanova A, Sersen F, Balgavy P. Cut-off effect in
antimicrobial activity and in membrane perturbation efficiency of the
homologous series of N,N-dimethylalkylamine oxides. J Pharm Pharmacol
1990;42:790.
[66] Balgavy P, Devinsky F. Cut-off effects in biological activities of surfactants. Adv
Coll Interface Sci 1996;66:23.
[67] Sarapuk J, Kubica K. Cut-off phenomenon. Cell Mol Biol Lett 1998;3:261.
[68] Kralova K, Sersen F, Devinsky F, Lacko I. Photosynthesis-inhibiting effects of
cationic biodegradable gemini surfactants. Tenside Surfactants Deter 2010;47:
288.