Wittig reaction (with ethylidene triphenylphosphorane) of oxo-hydroxy derivatives of 5β-cholanic acid: Hydrophobicity, haemolytic potential and capacity of derived ethylidene derivatives for solubilisation of cholesterol

Article abstract of DOI:10.1016/j.steroids.2014.04.018

Bile acid salts are biosurfactants which form mixed micelles with phospholipids in vertebrates. These mixed micelles are suitable for solubilisation of cholesterol. For therapeutic purposes some bile acid salts as sodium ursocholate are used. However, bil

Full text of DOI:10.1016/j.steroids.2014.04.018

Steroids 86 (2014) 16–25
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Steroids
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Wittig reaction (with ethylidene triphenylphosphorane) of oxo-hydroxy
derivatives of 5b-cholanic acid: Hydrophobicity, haemolytic potential
and capacity of derived ethylidene derivatives for solubilisation
of cholesterol
b
a
ˇ b
Mihalj Poša a, , Srdan Bjedov , Ana Sebenji , Marija Sakac
⇑
-
a Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljka 3, 21000 Novi Sad, Serbia
b Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg D. Obradovi´ca 3, 21000 Novi Sad, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Bile acid salts are biosurfactants which form mixed micelles with phospholipids in vertebrates. These
mixed micelles are suitable for solubilisation of cholesterol. For therapeutic purposes some bile acid salts
as sodium ursocholate are used. However, bile acid anions possess low capacity for solubilisation of cho-
lesterol. Thus, synthesis of more hydrophobic and less membranotoxic bile acid derivatives is of the great
interest. In this paper Wittig reaction between ethylidene triphenylphosphorane and different bile acids
oxo derivatives is examined. Wittig reaction of bile acids has not been studied much. C12 oxo group is
inert in this reaction. If Wittig reaction happens on C7 oxo group stereospecifically E ethylidene stereo-
isomer is obtained, while the same reaction on C3 oxo group leads to more reactive not sterospecific prod-
uct. In this paper stereochemical course of investigated Wittig reactions is thoroughly analysed.
Hydrophobicity of derived products is determined over the temperature (T) dependence on retention
coefficients (k) in reversed phase high resolution chromatography. Using method of principle compo-
nents on k = f(T) matrix it is found that values of first principle components best describe hydrophobicity
of analysed bile acids, while the second principal component is responsible for their hydrophilicity. By in
silico molecular descriptors: valence connectivity index of order 3 (X3v) and packing density index (PDI),
linear regression equations are obtained that can be used to predict hydrophobicity (over retention coef-
ficient) of bile acids that belong to set of more congeneric groups. Membranotoxicity is determined by
haemolytic potential. Monoethylidene derivatives of bile acids (in the form of anions) have lower mem-
branotoxicity than deoxycholic acids anion. Sodium salt of deoxycholic acid 7-ethylidene derivative has
11% greater capacity for solubilisation of cholesterol monohydrate than sodium salt of deoxycholic acid.
Ó 2014 Elsevier Inc. All rights reserved.
Received 20 August 2013
Received in revised form 17 April 2014
Accepted 21 April 2014
Available online 10 May 2014
Keywords:
Bile acids
Wittig reaction
Principle component method
Hydrophobicity
Haemolitic potential
Solubilisation of cholesterol
1. Introduction
Bile acid salts show membranotoxicity as they incorporate into
the cell membrane. With the increase in concentration of bile acid
Bile acids synthesised in liver of vertebrates are mainly hydroxy
derivatives of 5-b-cholanic acid [1]. Bile acid salts form micelles
and that is the base for their main physiological role: lipid emulga-
tion in small intestine and transport of lipid components to intes-
tinal epithelium [2]. Bile acid salts form mixed micelles with
phospholipids in bile capillaries (canaliculus). By this cholesterol
is kept in the dissolved form [3]. Besides that, bile acids are syn-
thesised in hepatocytes from cholesterol so they have important
role in regulation of cholesterol excretion [4,5].
salts, after incorporation, membrane destruction and formation of
mixed micelle of bile acid anion and membrane phospholipid hap-
pens [6]. If bile acid anion is more hydrophobic it has greater ten-
dency to form mixed micelle with phospholipids, i.e. higher is its
membranotoxicity [7].
Bile acids (in the form of sodium salts) show promotoric action
in transport of certain drug molecules through lipid barriers as
blood–brain barrier, lipid barrier of nasal and intestinal epithelium
etc. [8–11]. Besides that, bile acid salts form mixed micelles with
anionic surfactants (tween 40, tween 80, triton 100ꢀ etc.) that
can be used as flexible systems for solubilisation of hydrophobic
drugs [12,13]. However, there is usually direct proportionality
between their promotoric action and hydrophobicity of the steroid
⇑
Corresponding author. Tel./fax: +381 21 422 760.
E-mail address: mihaljp@uns.ac.rs (M. Poša).
http://dx.doi.org/10.1016/j.steroids.2014.04.018
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

M. Poša et al. / Steroids 86 (2014) 16–25
17
skeleton, and therefore direct proportionality with membranotox-
icity [6,7]. Thus, for the previous twenty years in biomedical exper-
iments oxo (keto) derivatives of bile acids are examined [14–22].
spectra were recorded on a Bruker Avance III 500 (500 MHz 1H,
125 MHz 13C) apparatus using tetramethylsilane as the internal
standard. HRMS spectra (TOF) were recorded on a 6210 Time-of-
Flight LC/MS Agilent Technologies (ESI+) instrument.
By substitution of
a axial OH group of steroid skeleton with oxo
group, oxygen of the oxo group is shifted towards b side of the ste-
roid skeleton which leads to a lower hydrophobicity of the convex
side of the steroid skeleton of bile acid. Membranotoxicity is also
decreased [1,7,23]. However, with the decrease in hydrophobicity
of bile acid salts, their promotoric action, solubilisation capacity
i.e. micelle and mixed micelle formation capability decreases as
well [7].
2.1. 3(EZ)-Ethylidene-7,12-dioxo-5b-cholanoic acid (4a,b) and 3(EZ),
7(E)-diethylidene-12-oxo-5b-cholanoic acid (5a,b)
Ethyltriphenyl phosphonium bromide (2.3170 g; 6.24 mmol)
was added to flask and dried under high vacuum for 5 h. After fill-
ing with argon, t-BuOK (0.4636 g; 4.13 mmol) was added followed
by dry THF (30 mL). Entire mixture was heated to reflux under
argon for 15 min, during which time the mixture turned orange
as the ylide formed. Solution of (1) (0.4995 g; 1.24 mmol) in dry
THF (12 mL) was added to refluxing solution, which was then stir-
red at reflux for 3.5 h. After cooling to room temperature, reaction
was stopped by adding HCl 1:1 (5 mL, pH 1), and aqueous solution
was extracted with EtOAc (3 ꢀ 10 mL). The combined organic
extracts were dried over Na2SO4, filtered, and then the solvent
was removed in vacuo. Flash column chromatography (toluene/
EtOAc 9:1) of crude product yielded pure mixture of E and Z iso-
mers of compound (4) (0.0911 g, 15%, in the form of oil), and pure
mixture of E and Z isomers of compound (5) (0.2248 g, 38.5%, in the
form of oil).
The aim of this paper is examination of reactivity (stereoselec-
tivity and stereospecificity) of bile acid oxo derivatives in Wittig
reaction with ethylydene triphenylphosphorane (Fig. 1). There
are only few published Wittig reactions of steroidal skeleton, espe-
cially of bile acids, and olefination was successful only at C3 car-
bonyl group [24,25]. Substitution of oxo group with ethylidene
group can change hydrophobicity of bile acid steroid skeleton. It
is desirable to obtain derivatives with oxo group (effect of lowering
of hydrophobicity) and ethylidene group (effect of rising of hydro-
phobicity) in their steroidal skeleton. For derived ethylidene deriv-
atives hydrophobicity is examined with reversed phase liquid
chromatography of high resolution (RPHPLC) so temperature
dependence of retention coefficients is determined. The other goal
of this paper is to connect hydrophobicity of obtained derivatives
with in silico molecular descriptors, i.e. to find relation between
experimental (chromatographic) variables and in silico descriptors.
Membranotoxicity (membranolytic activity) of bile acids (in anio-
nic form) in this paper is expressed (determined) with haemolytic
potential (lysis of erythrocytes) [7]. The purpose is to examine if
hydrophobicity determines haemolytic potential in the group of
bile acid derivatives which do not belong only to one congeneric
group. Also, the goal is to determine cholesterol solubilisation
capacity for newly synthesised bile acids ethylidene derivatives.
2.1.1. Spectroscopic data for mixture of (4a) and (4b)
1H NMR (500 MHz, DMSO-d6, ppm): 0.75 (d, J = 5.6 Hz, 3H, H
from C21 methyl group), 0.99 (s, 3H, H from C18 methyl group),
1.25 (s, 3H, H from C19 methyl group), 1.50 (d, J = 6.5 Hz, CH3 from
ethylidene), 5.15–5.05 (m, 1H, CH from ethylidene). 13C NMR
(125 MHz, DMSO-d6, ppm): 11.41(C18), 12.43 and 12.45 (CH3 from
ethylidene), 18.65 (C21), 21.98, 22.26 and 22.34 (C19), 24.64, 27.21,
29.34, 30.33, 30.37, 31.12, 34.99, 36.05, 36.41, 36.43, 36.87, 37.69,
38.15, 38.17, 44.42, 44.48, 44.92, 45.10, 45.32, 46.46, 47.27, 47.90,
47.94, 51.61, 56.23, 115.75 and 115.86 (CH from ethylidene on C3),
137.79 and 137.92 (C3), 174.81(C24), 209.90 and 209.94 (C7),
212.31 (C12). HRMS: calculated for C26H38O4 (M+H)+: 415.28429;
measured: 415.28292. IR (cmꢁ1): 843, 1701, 1716, 3446.
2. Materials and methods
Bile acids for Wittig reaction: 3,7,12-trioxo-5b-cholanoic acid
(dehydrocholic acid, 1) was obtained from Sigma–Aldrich (99%),
2.1.2. Spectroscopic data for mixture of (5a) and (5b)
3a-Hydroxy-7,12-dioxo-5b-cholanoic acid (2), and
3
a,12a
-
1H NMR (500 MHz, DMSO-d6, ppm): 0.74 (d, J = 5.6 Hz, 3H, C21
methyl group), 1.01 (s, 3H, C18 methyl group), 1.12 (s, 3H, C19
methyl group), 1.47–1.53 (m, 3H, CH3 from ethylidene), 5.04–
5.10 (m, 1H, CH from ethylidene on C3), 5.20–5.25 (m, 1H, CH from
ethylidene on C7). 13C NMR (125 MHz, DMSO-d6, ppm): 11.46 (C18),
12.45 and 12.89 and 12.95 (CH3 from ethylidene), 18.56 (C21),
22.22, 22.96 (C19), 23.05, 25.44, 27.06, 27.90, 30.30, 30.71, 31.11,
31.16, 31.39, 34.99, 36.33, 36.89, 37.26, 37.28, 37.76, 37.89,
39.01, 39.17, 39.34, 39.51, 39.67, 39.84, 40.01, 41.92, 45.43,
45.76, 46.29, 46.34, 46.59, 53.20, 56.31, 114.76 and 114.89 (CH
dihydroxy-7-oxo-5b-cholanoic acid (3) were synthesised as
described previously [1]. Other analysed bile acids: deoxycholic
(9), chenodeoxycholic (8), are obtained from Alfa Aesar (99%). Each
bile acid was recrystallised from the mixture of methanol/
water = 3:2 for three times and once from the ethanol. Flash chro-
matography was performed on silica gel 60 (0.04–0.063 mm,
Merck). Melting points were determined using a Nagema Boeitus
melting point apparatus and the results are uncorrected. IR spectra
were recorded on a Nexus 670 FT-IR spectrometer. 1H and 13C NMR
Fig. 1. Reaction scheme of olefination of analysed oxo and hydroxyl derivative of 5b-cholanic acid.

18
M. Poša et al. / Steroids 86 (2014) 16–25
from ethylidene on C3), 115.10 and 115.19 (CH from ethylidene on
C7), 137.89 (C7), 138.99 and 139.03 (C3), 174.79 (C24), 213.68 (C12).
HRMS: calculated for C28H42O3 (2M+H)+: 853.63407; measured:
853.63382. IR (cmꢁ1): 757, 1708, 2931.
2.5. Determination of haemolytic potential
Erythrolysis was determined from citrate rabbit blood accord-
ing to Bowe et al. [11].
2.2. 3a-Hydroxy-12-oxo-7(E)-ethylidene-5b-cholanoic acid (6)
2.6. Determination of bile salt cholesterol solubilising capacity
The compound (6) was prepared according to the method
described above. Crude product was purified by flash column chro-
matography (toluene/EtOAc 1:1), and compound (6) was obtained
(11%, mp 189–191 °C after crystallisation from acetone/hexane
1:1), as fine white needles.
Solubilisation of cholesterol by sodium salts of analysed bile
acids was determined using method of Poša et al. [8]. The amount
of dissolved cholesterol from water solution was determined by
enzymatic method with cholesterol oxidase (Reanal, Budapest),
measuring absorbance at 500 nm on Agilent 8453 spectrophotom-
eter, using distilled water as blank. The bile salt solubilising capac-
ity (CChm) is expressed as the number of moles of dissolved
cholesterol monohydrate per mole of bile acid (mole fraction of
dissolved cholesterol).
1H NMR (500 MHz, DMSO-d6, ppm): 0.72 (d, J = 5.9 Hz, 3H from
C21 methyl group), 0.97 (m, 2H), 0.98 (s, 3H, from C18 methyl
group), 1.09 (s, 3H, from C19 methyl group), 1.50 (d, J = 6.6 Hz,
3H, from ethylidene methyl group), 3.35 (m, 1H, from C3), 5.19
(q, J = 6.8 Hz, 1H, CH from ethylidene). 13C NMR (125 MHz,
DMSO-d6, ppm): 11.73 (C18), 13.18 (CH3 from ethylidene), 18.82
(C21), 23.06 (C19), 25.77 (C15), 27.36, 30.12, 30.54, 31.32, 31.59,
34.73, 35.26, 36.17, 36.53, 38.11 (C11), 42.24, 43.93, 45.70, 47.05
(C9), 53.82 (C14), 56.69, 69.40 (C3), 115.09 (CH from ethylidene),
138.40 (C7), 175.20 (C24), 214.71 (C12). HRMS: calculated for
2.7. Molecular descriptor and data treatment
The bile acid molecule structures were preoptimised with the
Molecular Mechanics Force Field procedure included in Hyper-
chem version 7.5, and the resulting geometries were further sub-
mitted to the semi-empirical method Parametric Method-3 using
the Fletcher–Reeves algorithm and a gradient norm limit of
0.009 kcal/Å. Using Dragon 6.0 software next molecular descriptors
were generated: unsaturation count (Uc), unsaturation index (Ui),
hydrophilic factor (Hy), Ghose–Crippen molar refractivity (AMR),
topological polar surface area using O polar contributions (TPSA),
Moriguchi octanol–water partition coefficient (MlogP), squared
Moriguchi octanol–water partition coefficient (MlogP2), Ghose–
Crippen octanol–water partition coefficient (ALogP), squared
Ghose–Crippen octanol–water partition coefficient (ALogP2), total
surface area from P_VSA-like descriptors (SAtot), surface area of
acceptor atoms from P_VSA-like descriptors (SAacc), surface area
of donor atoms from P_VSA-like descriptors (SAdon), McGowan
volume (Vx), van der Waals volume from McGowan volume
(VvdwMG), van der Waals volume from Zhao–Abraham–Zissimos
equation (VvdwZAZ), packing density index (PDI), Verhaar Fish
base-line toxicity from MLOGP (BLTF96), valence connectivity
index of order 0 (X0v), valence connectivity index of order 1
(X1v), valence connectivity index of order 2 (X2v) and valence con-
nectivity index of order 3 (X3v) [27]. For calculating principle com-
ponents, NIPALS [28] algorithm Statistica 8 software was used.
C
26H40O4 (M+NH4)+: 434.32649; measured: 434.32439. IR
(cmꢁ1): 3376, 2931, 1705, 753.
2.3. 3a,12a-Dihydroxy-7(E)-ethylidene-5b-cholanoic acid (7)
The compound (7) was prepared according to the method
described for (4a,b) and (5a,b). Crude product was purified by flash
column chromatography (CH2Cl2/acetone 5:1), and (7) was
obtained as colourless oil (8.2%).
1H NMR (500 MHz, DMSO-d6, ppm): 0.61 (s, 3H, H from C18
methyl group), 0.92 (d, J = 6.5 Hz, 3H, H from C21 methyl group),
0.97 (s, 3H, H from C19 methyl group), 1.52 (d, J = 6.6 Hz, 3H, CH3
from ethylidene), 3.30–3.40 (m, 1H, H from C3 CHOH group), 3.82
(s, 1H, H from C12 CHOH group), 5.23 (q, J = 6.5 Hz, 1H, CH from
ethylidene). 13C NMR (125 MHz, DMSO, ppm): 12.71 (C18), 12.88
(CH3 from ethylidene), 17.17 (C21), 23.37 (C19), 24.71 (C15), 27.18,
28.59 (C11), 30.09, 31.02, 31.41, 31.49, 34.76, 34.87, 35.19, 35.81,
36.14, 40.63, 42.45, 44.64, 45.65, 46.17, 69.54 (C3), 70.32 (C12),
113.63 (CH from ethylidene), 139.63 (C7), 175.32 (C24). HRMS: cal-
culated for C26H42O4 (2M+H)+: 837.62390; measured: 837.62277.
IR (cmꢁ1): 756, 1709, 2869, 2940, 3402.
2.4. Reverse phase HPLC method
3. Results and discussions
The HPLC system Agilent 1100 Series, equipped with degasser,
binary pump, automatic injector and DAD detector with software
system for data processing Agilent Chem Station was used and
the analyses were performed on a reversed-phase C-18 column:
3.1. Reactions of Wittig olefination of bile acid oxo derivatives
Wittig olefination (with: ethylidenetriphenylposphorane) of
3,7,12-trioxo-5b-cholanoic acid (1) gives mixture of 3(EZ)-ethyli-
dene-7,12-dioxo-5b-cholanoic acid (4) and 3(EZ),7(E)-diethylid-
ene-12-oxo-5b-cholanoic acid (5). Monoethylidene product (4)
consists of equimolar amounts of E and Z isomers, and diethylidene
product (5) is consisted of equimolar amounts of 3E,7E- and
3Z,7E-isomers. We have found that 7,12-dioxolithocholic acid (2)
regio and stereoselectively gives 7(E)-ethylidene product (6), while
7-oxodeoxycholic acid (3) in same reaction conditions yields ster-
eoselective product (7) (Fig. 1). Butylidenephosphorane failed to
react in the same reaction conditions. Stereochemistry on C7 was
determined using various NMR experiments which supports E con-
figuration. In ROESY spectrum of 5, 6 and 7 vinylic hydrogen of C7
ethylidene, has cross peaks that originate from through space
Eclipse Plus C18 (250 mm ꢀ 3 mm, 5
lm, 250 Å) column (Zorbax
SD). The mobile phase was 0.01 M phosphate buffer/metha-
nol = 70:130 (v/v) maintained at pH 7 and the injection volume
was 10 lL. Solutions of bile acids and their derivatives in mobile
phase were prepared in concentration of 1 mg/ml. All separations
were performed isocratically at a flow rate of 1 ml/min and a col-
umn temperature changing from 20 to 45 °C. The detection was
performed at 210 nm [26]. The HPLC capacity (retention) factor
(k) was calculated from the eluted peak retention time (t):
tx ꢁ t0
k ¼
t0
where tx and t0 are the retention times of the bile acids and the
unretained solvent front respectively.
interaction with
materials).
aH on C14 and aH on C15. (Fig. 2 and Supporting

M. Poša et al. / Steroids 86 (2014) 16–25
19
Fig. 2. ROESY spectrum of molecules (6).
Regiochemistry and stereoselectivity of Wittig reactions for
examined bile acid oxo derivatives can be explained if we observe
molecular graph of cholanoic acid and its subgraph Gs with C2 axis
of symmetry [1]. By C2 axis of symmetry mutual relative position of
steroid skeleton oxo groups and appropriate substructures of the
steroid ring system can be defined. So, in molecular graph of
3,7,12-trioxo-5b-cholanoic acid (1) C7 oxo group is in cis position
related to C15 and C16 atoms of steroid skeletons D ring, in relation
to C2 axis of symmetry (of subgraph Gs) as referent system, while
acid (1) that environment of C7 oxo group is analogue with envi-
ronment of keto group of unsymmetrical ketones (or aldehydes),
while surrounding of C3 oxo group of molecule (1) and its first vic-
inal C atoms corresponds to structure of symmetric ketones.
If resonantly unstabilised ylide is used (as in this paper) for
asymmetric ketones (aldehydes) in Wittig reaction usually Z olefin
is obtained (thermodynamically the most stable product). This is a
result of relatively high energy content of ethylidene triphenyl-
phosphorane, so transition state for formation of oxophosphetane
is early and has twisted orientation in order to lower steric repul-
sive interactions between ylide phenyl groups and the rest of the
ketone, respectively ylide methyl group and substituent from the
ketone [29–32]. As mentioned before in Wittig olefination reaction
(with ethylidenetriphenylphosphorane) from 7-oxodeoxycholic
acid (3) stereoselectively 7(E)-ethylidene deoxycholic acid is
formed (7). In order to obey Bürgi–Dunitz angle of ylide attack as
anionic nucleophile, for spatial reasons attack on C7 oxo group is
possible only from the b side of the steroid skeleton (Supporting
materials). This results in twisted cyclic transition state with E con-
C12 oxo group is in a cis position with C17 side chain (the same ref-
erent system). Oxo group on C3 carbon coincides with C2 axis of
symmetry (Fig. 3). Also, it can be seen from molecular graph of bile
figuration (if ylide attack from the opposite
a side would result in Z
configuration transit state) (Fig. 4. trans-NP1). However, since ylide
has carbanion character, in nucleophilic attack of triphenylphos-
phorane both possible enantiomeric structures participate
(Fig. 4). In attach from the b side of the steroid skeleton it is possi-
ble to have transition state of oxophosphetane derivative in cis
configuration (Fig. 4. cis-NP1). In this transition state there are spa-
tial repulsive interactions between mutually sinclinal (sc) methyl
group (from ylide) and D ring of the steroid skeleton (Fig. 4. cis-
NP1). However, due to rigidity of steroid skeleton it is impossible
to relocate D ring from sc position (it is possible in asymmetric ali-
phatic ketones with free rotation). So, olefination of C7 oxo group of
molecule (3) with unstabilised triphenylphosphorane leads to
twisted transition state of oxophosphetane with the lowest possi-
ble spatial repulsive interactions and E configuration, which is con-
figuration of formed olefin bond of the molecule (7). Derivative (7)
with E olefin is thermodynamically more stable product than the
Fig. 3. Definition of referent element: C2 axis of symmetry of molecular subgraph Gs
of cholanic acid.

20
M. Poša et al. / Steroids 86 (2014) 16–25
Fig. 4. Possible transition states towards b attack of the steroid skeleton C7 carbonyl
group with ethylidene triphenylphosphorane.
Fig. 5. Conformational analysis E and Z stereoisomers 3-ethylidene-7,12-dioxo-5b-
hypothetical Z derivative (Supporting materials). So, due to rigid
configuration of steroid skeleton, i.e. cis position of C7 oxo group
and steroid skeleton D ring (molecular graph of bile acid Fig. 3),
contrary to literature data that confirm that asymmetric ketones
cholanic acid (4).
configuration of ethylidene groups of derivatives (6) and (7) while
C12 oxo group does not participate in analysed Wittig reaction.
Unreactivity of analysed oxo derivatives in Wittig reaction with
butylidenephosphorane confirms importance of the previous ste-
reochemical analysis since for this voluminous group, in proper
Newman projection formula, even if hydrogen is in sc or sp posi-
tion to propyl group repulsive interactions happen.
(aldehyes) give mainly olefin derivatives of
Z configuration
[30,31], here thermodynamically more stable olefin derivative
with E configuration is formed.
Regioselectivity of Wittig reaction with ethylidene triphenyl-
phosphorane for 7,12-dioxolitocholic acid (2) is a consequence of
the spatial ecraning of C12 oxo group with C17 side chain (Support-
ing materials).
In olefination of oxo groups of trioxo derivatives (1) equimolar
mixture of Z and E 3-ethyilidene derivatives is obtained (4). Oxo
group on C3 carbon of the steroid overlap with direction of C2 axis
of symmetry of subgraph Gs (i.e. axis of symmetry passes through
C3 oxo group, so it behaves as oxo group of symmetric ketones).
Also, introduced olefin bond of ethylidene derivative (4) overlaps
axis of symmetry of subgraph Gs. Thus, local steric environment
of ethylidene methyl group (as transit states of oxophosphetane
derivative) does not differ for Z and E geometrical isomers (i.e.
diastereoisomers) (Fig. 5). Beside derivative (4) in olefination pro-
cess of molecule (1) 3,7-diethyilidene derivative is obtained as well
(5) for which configuration on C7 ethylidene group is identical to
3.2. Hydrophobicity and haemolytic potential of observed bile acids
ethylidene derivatives
Hydrophobicity (lipophilicity) of analysed molecules (Table 1)
is determined over dependence between retention coefficients
(RPHPLC) and temperature. In Table 1 (data matrix – retention
coefficients) principal component method is applied. In plane of
principle component values PC1 – PC2 analysed bile acids form
three groups, and two molecules do not belong to any of groups
(Fig. 6). According to biplot analysis, the most important values
in sense of grouping of analysed molecules is retention coefficient
measured on 30 °C (k30), since the line of this variable forms the

M. Poša et al. / Steroids 86 (2014) 16–25
21
Table 1
Retention coefficients (k) on different temperatures.
Molecules
t/°C
20
25
30
35
40
45
1
2
3
4
5
6
7
8
9
0.3011
0.5031
1.1056
4.8151
7.8062
4.3506
7.4603
5.1537
6.9986
0.3035
0.4526
1.0733
4.7548
7.575
0.3009
0.3691
1.0208
4.3701
7.1725
4.0713
5.3368
3.8174
5.6770
0.2986
0.349
0.9934
3.916
6.9221
3.8073
4.9215
3.2818
4.7446
0.2953
0.3336
0.8701
3.3116
6.5494
3.4752
4.311
0.2771
0.3151
0.8378
3.4675
6.4312
2.9976
3.8362
2.6238
4.1587
4.524
6.2299
4.4632
6.4363
2.8723
4.2247
0.6
0.4
7
9
8
0.2
I
III
0.0
2
4
6
1
3
-0.2
-0.4
II
increase hydrophobicity
5
-0.6
-4
-3
-2
-1
0
1
2
3
4
PC1 (score): 98.48%
Fig. 6. Grouping of examined bile acids in the plane of principle components
(score).
smallest angle with PC1 axis (PC axis according to which analysed
bile acids best separate, Fig. 7). Group I is formed by starting oxo
derivatives (1,2 and 3).
Group II is formed by ethylidene derivatives (4) and (6). For
these molecules it is typical that beside one ethylidene group they
Fig. 8. Position of C3 OH group related to the steroid skeleton mean plane = SSMP
(SSMP(A) = mean plane of steroid skeleton in the region A of the ring; MP = mean
plane of cyclohexane).
posses two
a equatorial O atoms bonded to steroid skeleton. In
cycloxehane (i.e. ring A) would be parallel with the bond: steroid
carbon (C1 or C13)-carbon (C18 or C19) angular methyl group), con-
cerning that rings A and B are cis connected, follows that C3 axis of
ring A (when cis connected to ring B) is rotated for 60° (actually the
whole A ring) in regard to axis of the bond: steroid carbon–carbon
of angular methyl group, which leads to parallel orientation of C3
OH group with axial C7 and C12 OH groups of cholic acid. In normal
phase thin layer chromatography (NPTLC) equatorial C3 OH group
behaves as axial C7 and C12 OH groups of cholic acid (by oxidation
of each of three steroid OH groups of cholic acid Rf value of
obtained derivatives increases) [23]. Nevertheless, in RPHPLC
experiment, i.e. over molecule adsorption on hydrophobic station-
ary phase, C3 OH group behaves as equatorial OH group. In isolated
A ring equatorial OH groups form angle of 30° with the mean plane
(MP) of the steroid skeleton and of cyclohexane and this angle does
not changes when A ring is rotated for 60° (i.e. while A and B rings
of the steroid skeleton mutually connect, Fig. 8). This means that in
steroid skeleton of ethylidene derivative (6) equatorial C3 OH
group as well as C12 oxo group regarded to the steroid skeleton
derivative (4) these are O atoms of C7 and C12 oxo groups, and in
derivative (6) O atom of C12 oxo group and C3 hydroxyl group.
However, C3 OH group of derivative (6) is parallelwith the bond:
steroid carbon (C1 or C13)-carbon (C18 or C19) of angular methyl
group (Fig. 8), as well as are parallel axial OH groups from C7 and
C12 carbon of cholic acid. Although C3 OH group is equatorial in iso-
lated ring A of steroid skeleton (Fig. 8. i.e. if C3 axis of symmetry of
k40
k35
7
8
2
4
6
3
1
5
9
k30
mean plane (SSMP) form angle of 30° (from the
a side) so they
k25
can stabilise water molecules by hydrogen bonds partially from b
side of the steroid skeleton (bile acids connect to hydrophobic sta-
tionary phase over their convex, b side of the steroid skeleton
[1,7]). This changes the equilibrium of bonding of bile acid
(adsorption) for hydrophobic phase to desorption, i.e. lowers
hydrophobicity of the molecule. According to number of equatorial
k20
k45
PC1
Fig. 7. Biplot analysis.

22
M. Poša et al. / Steroids 86 (2014) 16–25
O atoms and number of ethylidene groups, molecules (4) and (6)
should have the same hydrophobicity. However, from k30 it follows
that ethylidene derivative (4) is slightly more hydrophobic than
ethylidene derivative (6) (Table 1). Difference in their hydropho-
bicity can be interpreted in differences of ecraning of equatorial
O atoms of steroid skeleton and in different positions of ethylidene
group regarded to SSMP. For derivative (4) C12 oxo group is steri-
cally ecraned (formation of hydrogen bonds with water molecules
from hydration sheath is partially prevented) with C17 side chain
(according to proper Newman projection formula C12 oxo group
and C17 carbon with the side chain are in mutual synperiplanar
position) the same is with C12 oxo group of derivative (6). C7 car-
bon with oxo group of molecule (4) is sterically ecraned as well
as with C15 methylene group of the D ring of the steroid skeleton
(according to proper Newman projection formula C7 carbon with
oxo group is in synclinal position to C15 methylene group. i.e. C7
oxo group and D ring are in cis position regarded to C2 axis of sub-
graph Gs). However equatorial C3 OH group of derivative (6) is not
sterically ecraned (in proper Newman projection formulas towards
C3 OH group there are no synclinal or synperiplanar steric volumi-
nous groups), thus it more efficiently influences stabilisation of
water molecules from hydration sheath, so derivative (6) is less
hydrophobic than derivative (4). Ethylidene group (CH3–CH@) in
a position C3 (derivative (4)) can be formally considered as substi-
tution of O atom of b equatorial oxo group (O=) with CH3–CH frag-
ment, thus group CH3–CH@ occupies b equatorial position (i.e.
forms angle of 30° with SSMP on convex b side of the steroid skel-
eton). Similarly for derivative (6) ethylidene group on C7 can be
Fig. 9. Association of bile acids on hydrophobic stationary phase: prevention of
hydration of L7 hydrocarbon side of steroid skeleton by mutual association
(HHS = hydrated hydrophobic surface).
switched towards SSMP, so in this molecule regarded to the other
analysed ethylidene derivatives hydrophobic surface of the convex
b side of the steroid skeleton is largely increased.
On the plot (Fig. 10) of weight coefficients (loadings) PC1 and
PC2 (calculated from the matrix of molecular descriptors from
the category: molecular properties and connectivity – programme
package Dragon 6.0 and two colons of principle components values
of analysed obtained from temperature dependence on retention
coefficients: PC1-t and PC2-t) molecular descriptor vectors are pre-
sented [28]. Four group of variables exist (Fig. 10) based on the
angle between their vectors. In group I between PC1-t and Morig-
uchies partition coefficient (MlogP) i.e. Ghose-Crippen partition
coefficient (AlogP) there is a good positive correlation (vector
PC1-t is placed between vector of descriptors MlogP and AlogP).
Also, between connectivity indices (X1v, X2x i X3v) and PC1-t i.e.
formally considered as substitution of oxygen atom of C7
rial oxo group with CH3–CH fragment, which means that CH3–CH@
group will have equatorial orientation, i.e. it will form the angle
of 30° with SSMP but from concave side of the steroid skeleton.
a equato-
a
a
According to that, for derivative (4) C3 ethylidene group is for 60°
more switched towards b side of the steroid skeleton than C7 eth-
ylidene group of derivative (6).
Third group (III) is formed of deoxycholic acid (9) and 7-ethyl-
idendeoxycholic acid (7) (Fig. 6). For both molecules the presence
of two OH group on identical positions on steroid skeleton is com-
mon. According to k30 values (retention coefficients measured on
30 °C, from biplot analysis (Fig. 7), best represent obtained groups
of analysed molecules in PC1–PC2 plot) deoxycholic acid is slightly
more hydrophobic than ethylinede derivative (7). During the
adsorption process on hydrophobic stationary phase, bile acids
(for stationary phase) are bonded over their b side of the steroid
skeleton. However, between adsorbed bile acids additional hydro-
phobic interactions are possible [1] over their hydrocarbon lateral
sides (for example in deoxycholic acid it is their lateral side where
is C7 carbon – L7 side) (Fig. 9). By introduction of ethylidene group
in C7 position of steroid skeleton of deoxycholic acid hydrophobic
surface is increased on the b side of the steroid skeleton since
1.0
Uc
VvdwMG
II
0.5
0.0
BLTF96
VvdwZAZ
X0v
AMR
Vx
PDI
introduced ethylidene group is in
a equatorial position, i.e. forms
SAdon
SAacc
angle of 30° with SSMP. Hydrophobicity of derivative (7) should
be higher than hydrophobicity of deoxycholic acid. However, as
ethylidene group of derivative (7) is located on L7 side of the ste-
roid skeleton, it can be assumed that introduced ethylidene group
on stationary phase sterically disturb association of molecules over
L7 lateral sides of the steroid skeleton, i.e. decreases hydrophobic-
ity of derivative (7) regarded to deoxycholic acid (Fig. 9). Henode-
oxycholic acid (8) is less hydrophobic than deoxycholic acid (9)
and 7-ethylidendeoxycholic acid (7) (Fig. 6 and Table 1) since its
C7 axial OH group is sterically less ecraned with D ring of the ste-
roid skeleton than C12 axial OH group (in molecules (7) and (9))
which is ecraned with C17 conformationally motile side chain of
the steroid skeleton.
MlogP2
X1v
TPSA(NO)
MlogP
PC1-t
X3v
X2v
III
AlogP2
I
AlogP
-0.5
-1.0
PC2-t
Hy
SAtot
-1.0
-0.5
0.0
PC1(loading): 69.42%
0.5
1.0
On PC1–PC2 plot most hydrophobic molecule is diethylidene
derivative (5), since its derivative both ethylidene groups are
Fig. 10. Relation between molecular descriptors and PC1-t and PC2-t.

M. Poša et al. / Steroids 86 (2014) 16–25
23
in silico partition coefficients (MlogP and AlogP) there is a good
8
7
6
5
4
3
2
1
0
positive correlation. Descriptors (vectors) from groups I and II
are mutually orthogonal (i.e. there is no correlation between
them), which means that descriptors of groups I and II for analysed
molecules explain different structural characterisations. Also, mol-
ecule descriptors of the group III are orthogonal to descriptors
from the group I. PC2-t, which is in the group III best correlate with
hydrophilic factor (Hy) (PC1-t and PC2-t are mutually orthogonal
i.e. explain different information in the matrix of dependence of
retention coefficients (Table 1)). According to that, values (score)
PC1-t best explain hydrophobicity of analysed bile acids, while val-
ues PC2-t best explain hydrophilicity of analysed molecules. By
that retention coefficients of bile acids are decomposed on hydro-
phobic and hydrophilic part (since PC1-t have significantly higher
eigenvalue than PC2-t, it means that retention coefficients are
mainly determined with hydrophobicity of a molecule). Molecular
descriptors of the group IV (mainly) form angle of 180° with
descriptors of the group I, i.e. mutually are inverse (negative corre-
lation) (Fig. 10).
5
9
8
7
4
6
3
2
1
95%confidence
8 9
0
1
2
3
4
5
6
7
Predicted Values
Fig. 11. Predicted values of k30 according to Eq. (2) in a function of experimental
values k30
.
Large correlation (small angle between variables) between in
silico descriptors and PC1-t (which represents extracted hydropho-
bicity of analysed bile acids from temperature dependence on
retention coefficients) in group I suggests that hydrophobicity of
analysed bile acids (retention coefficient, k30) can be predicted
with molecular descriptor from the group mentioned above. This
is very important since analysed group of bile acids is not homog-
enous congeneric group but the group of three different classes of
bile acids (oxo derivatives, ethylidene derivatives and bile acids
with OH group in steroid skeleton). Usually linear dependence
between hydrophobicity and molecular descriptors has worse sta-
tistical indicators if studied group of molecules is not a one
(homogenous) group. Due to small number of molecules in ana-
lysed group (n = 9), and in order to avoid overfitting, for obtaining
functional dependence between k30 and in silico molecular descrip-
tors linear regression with one independent variable (descriptor)
i.e. Hansch Fujita principle (molecules/descriptors >5) is applied
[33]. For k30 linear equation with the best statistical parameters
is equation which independent variable is connectivity index
(X3v) (descriptor from the group I, Fig. 10):
(OH, oxo and ethylidene) so, in PDI descriptor for these molecules
ecraning effect of C12 OH is not contained.
Haemolytic potential represents ability of a molecule (usually
surfactant) to destroy membrane of the erythrocyte. Usually, if
some surface active molecule is more hydrophobic it destroys
membrane (i.e. extracts phospholipids by which it forms mixed
micelle) [6]. Haemolytic potential can be quantified with concen-
tration of analysed bile acid sodium salt when 50% of the erythro-
cyte is destroyed (Lys50) and concentration when 100%
erythrocyte is destroyed (Lys100) [7]. If values Lys50 and Lys100
are lower haemolytic potential is higher i.e. higher is membrano-
toxicity of a molecule. From analysed ethylidene derivatives of
molecules with two ethylidene groups (5) (in anionic form) due
to their lower solubility in physiological solution it is not possible
to determine their haemolytic potential. Other analysed ethylidene
derivatives have lower Lys50 and Lys100 values than deoxycholic
acid (9) (anionic form) (Table 2). Deoxycholic acid is the most
hydrophobic (9) of all analysed bile acids, and deoxycholic anions
are well known to destroy cell membrane which is probably conse-
quence of formation of micelles with higher structural anisotropy
then other bile acid anions form (deoxycholic acid anions form gels
as well) [1,2]. Introduction of one ethylidene group on L7 lateral
side of steroid or in the position C3 of steroid certainly increases
haemolytic potential of ethlylidene derivatives with respect to
starting oxo derivatives, but for derivatives (4) and (6) haemolytic
potential is 5 to 6 times lower than for deoxycholate, while for 7-
ethylidene deoxycholate (7), which is formally derivative of deoxy-
cholate, haemolytic potential is decreased for 22% (Lys50) and 28%
(Lys100) related to haemolytic potential of anion (9).
k30 ¼ ꢁ73:04ðꢂ12:59Þ þ 7:49ðꢂ1:31ÞX3
v
ð1Þ
n = 9; R = 0.9171; F = 37.053; p < 0.0005; sd = 1.05; q2CV ¼ 0:8281.
Since there is a negative correlation between molecular descrip-
tors from the groups I and IV (angles between proper descriptor
vectors are mainly 180°, Fig. 10), i.e. descriptors from the group
IV are inverse with bile acids hydrophobicity. However, descriptors
from the group IV explain similar steroid skeleton structural vari-
ations as PC1-t (group I) just in opposite direction. Thus, functional
dependence between k30 and descriptors of the group IV is deter-
mined. Equation for k30 with best statistical parameters is equation
in which independent variable is molecular descriptor: packing
density index (PDI).
Table 2
k30 ¼ ꢁ12:76ðꢂ23:02Þ ꢁ 3:22ðꢂ0:39ÞPDI
ð2Þ
Haemolytic potential and and the bile salt cholesterol monohydrate solubilising
capacity.
n = 9; R = 0.9514; F = 66.85; p < 0.00008; sd = 0.81; q2CV ¼ 0:8624.
Both Eqs. (1) and (2) have good predictive power, since q2CV ꢃ 5
(q2CV is obtained by leave one out cross validation method). On
Fig. 11 dependence between experimental values k30 and predicted
k30 values according to Eq. (2) are presented. It can be seen from
Fig. 11 that, between all analysed bile acids retention coefficients,
predicted value for deoxycholic acid (9) deviates the most from
experimental value. For deoxycholic acid steric ecraning of C12
hydroxyl group with C17 side chain is not contained in molecular
descriptor PDI, while in other molecules with C12 OH or oxo group
there is a higher number of functional groups on steroid skeleton
Molecules
CChm/mol/molꢁ1 BS
Lys50 (mM)
Lys100 (mM)
1
2
3
4
5
6
7
8
9
0.000
30 4.30
56.00 7.00
27.50 2.50
12.50 0.75
n.d.
15 1.0
2.95 35
3.15 0.40
2.30 0.15
175 38.50
115.00 25.00
63.25 7.65
16.00 1.20
0.0007 0.0001
0.0018 0.0002
0.058 0.03
n.d.
0.061 0.002
0.078 0.003
0.062 0.002
0.070 0.004
19 1.50
6.10 0.20
6.25 0.50
5.00 0.50
n. d = not defined; n (number of repetitions) = 5.

24
M. Poša et al. / Steroids 86 (2014) 16–25
Molecular descriptors as MlogP, MlogP2 and AlogP i.e. descrip-
(Fig. 9). Similarly, in process of solubilisation of cell membrane
(haemolytic potential) bile salts saturate membrane (saturation)
[6], and their convex (hydrophobic) side of steroid skeleton is in
interaction with hydrocarbon chains of lecithin (corresponds to
stationary phase in RPHPLC experiment), while lateral sides of
the steroid skeleton can interact with steroid skeleton of the other
steroid molecule (in saturation phase) or with hydrocarbon
remains of phospholipid fat acids. Ethylidene group of derivative
(7) hinders lateral intermolecular interactions in hydrophobic
environment (stationary phase RPHPL and saturation of cell mem-
brane) (Fig. 9), so derivative (7) has slightly lower hydrophobicity
and haemolytic potential than sodium deoxycholate (9). Higher
capacity for solubilisation of cholesterol monohydrate of derivative
(7) than of (9) is a consequence of the fact that bile salts form
micelles consisting of only several building units (unlike classic
surfactants, for example sodium dodecylsulphate) [35,36], so in
such micelles only hydrophobicity of convex sides of steroid skel-
eton is expressed. Molecule of cholesterol incorporates in hydro-
phobic domain of the micelle so if b side of the steroid skeleton
possess larger surface area, as it is in ethylidene derivative (7)
(related to (9)), there is a higher probability of incorporation of
hydrophobic guest. In in vivo system capacity for solubilisation of
cholesterol significantly increases comparing to values from
Table 2. since in bile canaliculus bile acid salts form mixed micelles
with phospholips (mainly lecithin) with significantly larger hydro-
phobic domain than micelle of bile salts have [37].
tors which are directly connected to molecules hydrophobicity
[34], chromatographic parameters of reversed phase HPLC: PC1-t
and k30 are in good correlation to haemolytic potential (Table 3,
with Lys50 and Lys100, mentioned descriptors are in negative cor-
relation since values of Lys50 and Lys100 are higher if haemolytic
potential is smaller). Valence connectivity index (X3v) also shows
good correlation with haemolytic potential. Over equation (1)
X3v is in functional dependence with retention capacity k30 i.e.
with hydrophobic surface of the molecule. According to that, for
describing membranotoxicity of bile acid anions in a group where
is a big structural difference i.e. molecules do not belong to the
same congeneric group; molecule descriptors which are directly
or indirectly connected with hydrophobic surface of steroid on
its convex b side (PC1-t and k30) can be used. These are molecular
descriptors from I and IV group in the plane of principle compo-
nent weights PC1 and PC2 (Fig. 10).
Sodium salt of cholic acid three oxo derivative (1) does not
show power for solubilisation of cholesterol monohydrate, while
sodium salt of 7,12- dioxo derivative of litocholic acid (2) and
sodium salt of 7-oxo derivative of deoxycholic acid posses capacity
for solubilisation of cholesterol monohydrate (CChm) whose values
does not exceed 3% of the value of capacity for solubilisation of
cholesterol monohydrate of sodium chenodeoxycholate (8) and
sodium deoxycholate (9) (Table 2). This is consistent with our pre-
vious studies of solubilisation of cholesterol monohydrate with
sodium salt of bile acids [8]. Sodium salt of novel ethylidene deriv-
atives (4) and (6) statistically do not differ according to CChm values
(they belong to the same distribution). Based on PC1 values they
have a common hydrophobicity. Their CChm values are 80% of CChm
value of sodium deoxycholate (9). Sodium salt of 7-ethylidene
derivative of deoxycholic acid (7) has 11% higher value than CChm
of sodium deoxycholate (9). However, deoxycholic acid (9) has
higher hydrophobicity and higher haemolytic potential than ethyl-
idene derivative (7) (Tables 1 and 2). Hydrophobicity expressed
over retention coefficient in reversed phase chromatography is
ability of some bile acid to bond to hydrophobic stationary phase
over b side of the steroid skeleton and ability of lateral (side) inter-
molecular hydrophobic interactions on stationary phase as well
Reliable correlation between CChm values and molecular
descriptors i.e. retention parameters cannot be obtained since from
the starting group of bile acids (n = 9 molecules) for derivative (5)
CChm cannot be determined since it is not soluble in water, while
derivative (1) does not show capacity for solubilisation of choles-
terol (Table 2). Remain group of molecules (n = 7) is binary group,
in which oxo derivatives (2) and (3) have negligible CChm values
comparing to CChm values of ethylidene derivatives.
4. Conclusions
For analysed bile acid oxo derivatives oxo group in a position
C12 cannot be substituted with ethylene group in Wittig reaction.
Wittig reaction of ethylidene triphenylphosphorane and appropri-
ate bile acid oxo derivative in a position C7 of steroid gives stereo-
specifically E ethylidene derivative (steric control: vicinity of ring
D). Stereospecificity does not exist during introduction of ethyli-
dene group in a position C3. Reactivity of oxo groups in Wittig reac-
tion decreases in the next order: C3 > C7 and C12 is inert.
Hydrophobicity of analysed group of bile acids can be presented
with values of principle components PC1 which are calculated
from the temperature dependence on retention coefficients. For
principle components PC1 the highest influence has retention coef-
ficients k30. Hydrophobicity of molecule increases with the rise in
the number of ethylidene group in the steroid. Haemolytic poten-
tial (membranotoxicity) of monoethylidene derivatives is lower
than membranotoxicity of deoxycholic acid. Haemolytic potential
is in a good correlation with molecular descriptors that can be con-
nected with hydrophobicity of the steroid. Capacity of ethylidene
derivative (7) for solubilisation of cholesterol monohydrate is
10% higher than capacity of sodium deoxycholate (9).
Table 3
Correlations between haemolytic potential and molecular descriptors i.e. retention
coefficient.
Lys50
0.5326
ꢁ0.3819
ꢁ0.4081
0.7477
ꢁ0.7844
ꢁ0.7774
ꢁ0.5808
0.2855
ꢁ0.3869
ꢁ0.4758
ꢁ0.4759
ꢁ0.3130
0.2918
Lys100
Uc
Hy
AMR
0.5653
ꢁ0.5138
ꢁ0.5494
0.7240
ꢁ0.8970
ꢁ0.8743
ꢁ0.7991
0.1700
ꢁ0.5402
ꢁ0.6384
ꢁ0.6384
ꢁ0.4546
0.4406
TPSA(NO)
MlogP
MlogP2
AlogP
AlogP2
SAtot
SAacc
SAdon
Vx
VvdwMG
VvdwZAZ
PDI
0.8321
0.8286
0.8986
0.8962
BLTF96
X0v
X1v
X2v
X3v
PC1-t
PC2-t
k30
0.8285
0.8996
ꢁ0.3501
ꢁ0.5862
ꢁ0.6988
ꢁ0.7720
ꢁ0.8742
ꢁ0.6715
ꢁ0.8745
ꢁ0.4902
ꢁ0.7558
ꢁ0.8813
ꢁ0.9139
ꢁ0.8906
ꢁ0.5847
ꢁ0.8917
Acknowledgements
This study was supported by the Ministry of Science and Tech-
nological Development of the Republic of Serbia (Project No.
172021). The work was also financially supported by the Provincial
Secretariat for Science and Technological Development, AP Vojvo-
dina, Republic of Serbia, Grant No. 114-451-2113/2013.
Marked correlations are significant at p < .05000; n (number of molecules) = 8.

M. Poša et al. / Steroids 86 (2014) 16–25
25
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