Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their gramine salts

Article abstract of DOI:10.1016/j.steroids.2017.07.003

Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be used as starting material in chemical synthesis of compounds with different biological activity strongly depended on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro. The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range. At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydrophobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical calculation.

Full text of DOI:10.1016/j.steroids.2017.07.003

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their
gramine salts
Weronika Kozanecka-Okupnika, Beata Jasiewicza,⁎, Tomasz Pospiesznya, Monika Matuszakb,
Lucyna Mrówczyńskab,⁎
a
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań, Poland
Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Gramine
Nicotine
Bile acids
Human erythrocytes
Membrane partitioning
Haemolysis
Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be
used as starting material in chemical synthesis of compounds with different biological activity strongly depended
on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine
toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine
effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the
haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro.
The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-
MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-
LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range.
At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower
concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane
perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydro-
phobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical
calculation.
1. Introduction
antimicrobial [12–14], antifungal [15,16], antitumor [17] or as drug
carriers [18,19]. The results show that acetyl-derivatives of litocholic
Bile acids are present in the human bile and blood and some of them
have cytotoxic activity [1,2]. Their biological effects strongly depend
on the nature of the chemical structures e.g. hydrophilic ursodeoxy-
cholic acid (UDCA) and its taurine and glycine conjugates protect cells
against apoptosis induced by hydrophobic bile acids [3,4]. Bile acids
and their derivatives have been used for treatment of bile acid defi-
ciency and liver diseases [5] and some of them are TGR5 agonists [6] or
P-glycoprotein (Pgp, ABCB1) inhibitors [7]. Bile acids and their deri-
vatives are also attractive compounds for synthetic chemists because
they have a large, rigid skeleton and possess chemically different polar
hydroxyl groups. It is often necessary to protect this hydroxyl groups
using, for example, acetate or formate as protecting moieties [8–10].
According to Tsemg at al. [11], the formyl groups on these compounds
are quite stable to various reaction conditions. The stability and ready
availability of these compounds make them suitable candidates for use
as starting material in various synthetic schemes. The literature de-
scribes pharmacological applications for bile acids derivatives such as:
acids exhibit significant antibacterial activity and some of them po-
tentiate the effect of antibiotics such as amikacin, gentamicin and
neomycin [20]. Moreover, lithocholic acid and LCA acetate has been
shown to inhibit the proliferation and promote differentiation of human
leukaemia THP-1 cells [21] and co-operation between lithocholic acid
acetate and cotylenin A, shown promising pro-differentiating effects
upon primary human myeloid leukaemia cells in vitro [22]. Considering
all above, we decided to investigate the influence of formyl- and acetyl-
substituents on the cytotoxicity of bile acids (lithocholic: LCA, deoxy-
cholic: DCA and cholic: CA) on human red blood cells (RBC). It is
known that RBC are very convenient systems in the study of the in-
teractions of chemical compounds with the cell membrane. The am-
phiphilic molecules easily incorporated into the membrane of discoid
RBC and can induce their cells shape transformation into echinocytes or
stomatocytes. Depending on the membrane-perturbing activity of
compounds, the membrane-structure alternation may undergo and cell
damage, namely haemolysis, may occur. The number and position of
⁎
Corresponding authors.
E-mail addresses: beatakoz@amu.edu.pl (B. Jasiewicz), lumro@amu.edu.pl (L. Mrówczyńska).
http://dx.doi.org/10.1016/j.steroids.2017.07.003
Received 1 February 2017; Received in revised form 13 June 2017; Accepted 10 July 2017
0039-128X/©2017ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Kozanecka-Okupnik,W.,Steroids(2017),http://dx.doi.org/10.1016/j.steroids.2017.07.003

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
hydroxyl group on the rigid steroidal backbone of bile acids are im-
portant with regard to their cytotoxicity. Depending on the value of the
hydrophobicity index, bile acids can induce stomatocytogenic or echi-
nocytogenic transformation of RBC shape, in the dose-and incubation
time-dependent manner [23,24]. Our previous studies showed that al-
kaloids eg. gramine or nicotine, significantly decrease the capacity of
bile acids to alter the lipid bilayer structure of RBC membrane and
increase the membrane intercalating potency of sterols [25]. Therefore,
the impact of gramine molecule on the cytotoxicity of substituted bile
acids is also discussed. The structures of all new products were con-
firmed by spectral (NMR, FT-IR) analysis, mass spectrometry as well as
PM5 semiempirical calculations. Moreover nicotine salts with formyl-
bile acid were synthesized. The haemolytic activity of all compounds
obtained was studied under physiological conditions in vitro (phosphate
buffer, pH 7.4 at 37 °C).
0.82 (d, 3H, CH3-21), 0.73 (s, 3H, CH3-18).
2.2.3. 3α,7α,12α-Triformyloxy-5β-cholan-24-oic acid (4)
Yield 81%, powder, mp 209–211 °C. 1H NMR (500 MHz,
CDCl3,TMS, ppm): δ = 8.15 (s, 1H, 12α-OCHO), 8.10 (s, 1H, 7α-
OCHO), 8.02 (s, 1H, 3α-OCHO), 5.26 (s, 1H, 12β-CH), 5.06 (s, 1H, 7β-
H), 4.71 (m, 1H, 3β-H), 0.93 (s, 3H, CH3-19), 0.83 (d, 3H, CH3-21), 0.75
(s, 3H, CH3-18).
2.2.4. 3α-acetoxy-5β-cholan-24-oic acid (5)
Yield 86%, powder, mp 156–158 °C. 1H NMR (400 MHz, CDCl3,
TMS, ppm): δ = 4.72 (m, 1H, 3β-H), 2.03 (s, 3H, 3α-OCOCH3), 0.93 (s,
3H, 19-CH3), 0.83 (d, 3H, 21-CH3), 0.65 (s, 3H, 18-CH3).
2.2.5. 3α,12α-diacetoxy-5β-cholan-24-oic acid (6)
Yield 30%, powder, mp 125–126 °C. 1H NMR (400 MHz,
CDCl3,TMS, ppm): δ = 4.71 (m, 1H, 12β-H), 3.99 (m, 1H, 3β-H), 2.02
(s, 6H, 3α-OCOCH3), 0.98 (d, 3H, 21-CH3), 0.92 (s, 3H, 19-CH3), 0.69
(s, 3H, 18-CH3).
2. Experimental
2.1. Instrumentation and chemicals
All melting points (mp) were obtained with a Büchi SMP-20 appa-
ratus. 1H NMR spectra were recorded on a Ultrashield spectrometer at
300 MHz with CDCl3 or DMSO-d6 as the solvent and TMS as the internal
standard. Chemical shifts are reported in δ (parts per million) values.
ESI mass spectra were measured on a ZQ Waters Mass Spectrometer.
FT-IR spectra were recorded on Bruker FT-IR IFS 66v/S Spectrometer
(KBr pellets). Analytical thin-layer chromatography (TLC) was carried
out on silica gel plates 60 F254. Detection on TLC was made by the use
of UV light and 10% aqueous H2SO4 (then the plates were heated at
∼120 °C for approximately one minute and allow to cool). All chemi-
cals or reagents used for syntheses were commercially available. PM5
semiempirical calculations were performed using the CAChe Fujitsu
program.
2.2.6. 3α,7α,12α-Triacetoxy-5β-cholan-24-oic (7)
Yield 60%, powder, mp 69–70 °C. 1H NMR (400 MHz, CDCl3, TMS,
ppm): δ = 4.89 (s, 1H, 12β-H), 4.58 (s, 1H, 7β-H), 3.86 (m, 1H, 3β-H),
2.22 (s, 3H, 12α-CH3COO), 2.07 (s, 3H, 7α-CH3COO), 2.03 (s, 3H, 3α-
CH3COO), 0.99 (d, 3H, CH3-21), 0.93 (s, 3H, CH3-19), 0.69 (s, 3H, CH3-
18).
2.3. General synthetic procedure for gramine salts 8–13
The typical and optimum process for preparation of gramine salts is
shown as following: 3α-formyloxy-5β-cholan-24-oic acid (81 mg,
0.2 mmol),
3α,12α-diformyloxy-5β-cholan-24-oic
acid
(90 mg,
0.2 mmol), 3α,7α,12α-triformyloxy-5β-cholan-24-oic acid (98 mg,
0.2 mmol), 3α-acetoxy-5β-cholan-24-oic acid (84 mg, 0.2 mmol),
3α,12α-diacetoxy-5β-cholan-24-oic acid (95 mg, 0.2 mmol), 3α,7α,12α-
triacetoxy-5β-cholan-24-oic acid (107 mg, 0.2 mmol) were dissolved
separately in methanol (in the least volume of solvent). Then gramine
in quality molar ratio was added. The reaction mixture was mixed at
room temperature for 24 h, then the solvent was removed under re-
duced pressure. The crude products were crystallized from methanol.
2.2. General synthetic procedure for formyloxy- and acetoxy-bile acids
The starting formyl- (2–4) and acetyl- (5–7) esters of bile acids were
prepared following the standard procedures. Compounds 2–4 were
obtained according to Nascimento and Li et al. [20,26]. Lithocholic,
deoxycholic or cholic acids were dissolved in formic acid. Next, some
drops of perchloric acid was added. After stirring to 55–60 °C for 24 h,
acetic anhydride was added carefully (to moment when bubbles gas
were observed). The mixture was extracted with diethyl ether. The
extract was washed with 10% NaHCO3, water, brine and dried over
anhydrous Na2SO4. The crude products were obtained from evapora-
tion of solvent under reduced pressure and purification of the residue
over silica gel (CHCl3/MeOH, 100:1). Compounds 5–7 were synthesized
according to Brycki et al. [27,28]. Lithocholic, deoxycholic or cholic
acids were dissolved in anhydrous pyridine. Next, acetic anhydride and
catalytic DMAP was added. After stirring at room temperature for 72 h,
the mixture was extracted with chloroform. The extract was washed
with 0.5 M HCl, 10% NaHCO3, water, brine and dried over anhydrous
Na2SO4. The crude products were obtained from evaporation of solvent
under reduced pressure and purification of the residue over silica gel
(PhCH3/EtOAc, 50:1 for compounds 5 and 6; CHCl3/MeOH, 100:1 for
compound 7).
2.3.1. Gramine-3α-formyloxy-5β-cholan-24-oic acid salt (8)
Yield 97%, powder, mp 32 °C. 1H NMR (300 MHz, CDCl3, TMS,
ppm): δ = 10.43 (s, 1H, ^N+-H), 8.74 (s, 1H, NH), 8.04 (s, 1H, 3α-
OCHO), 7.52 (d, 1H, 7′-H), 7.40 (d, 1H, 4′-H), 7.08–7.18 (m, 3H, 2′-H,
6′-H, 5′-H), 4.88–4.80 (m, 1H, 3β-H), 4.14 (s, 2H, 10′-H), 2.53 (s, 6H,
N+(CH3)2), 0.92 (bs, 3H, CH3-19), 0.89 (bs, 3H, CH3-21), 0.62 (bs, 3H,
CH3-18). ESI-MS: m/z 787 [C48H80O6+Cl]−, 751 [C48H80O6-H]−, 579
[M+H]+
,
551 [C35H54N2O3+H]+
,
304 [C20H22N3]+
,
175
[C11H15N2]+, 130 [C9H8N]+. FT-IR (KBr) νmax: 3214, 2933, 2865,
1720, 1631, 1565, 1448, 1377, 1341.
2.3.2. Gramine-3α,12α-diformyloxy-5β-cholan-24-oic acid salt (9)
Yield 99%, powder, mp 135 °C. 1H NMR (300 MHz, CDCl3, TMS,
ppm): δ = 10.37 (s, 1H, ^N+-H), 8.73 (s, 1H, NH), 8.10 (s, 1H, 12α-
OCHO), 8.03 (bs, 1H, 3α-OCHO), 7.52 (d, 1H, 7′-H), 7.39 (d, 1H, 4′-H),
7.09–7.19 (m, 3H, 2′-H, 6′-H, 5′-H), 5.23 (bs, 1H, 12β-H), 4.86–4.79 (m,
1H, 3β-H), 4.15 (s, 2H, 10′-H), 2.53 (s, 6H, N+(CH3)2), 0.91 (s, 3H,
CH3-19), 0.82 (bs, 3H, CH3-21), 0.70 (bs, 3H, CH3-18). ESI-MS: m/z 895
2.2.1. 3α-formyloxy-5β-cholan-24-oic acid (2)
Yield 84%, powder, mp 128–130 °C. 1H NMR (500 MHz, CDCl3,
TMS, ppm): δ = 8.01 (s, 1H, 3α-OCHO), 4.82 (m, 1H, 3β-H), 0.90 (s,
3H, CH3-19), 0.89 (br, 3H, CH3-21), 0.62 (s, 3H, CH3-18).
[C52H80O12-H]−
,
867 [C51H80O11-H]−
,
,
839 [C50H80O10-H]−
,
447
130
[C26H40O6-H]−
,
419 [C25H40O5-H]−
175 [C11H15N2]+
,
[C9H8N]+. FT-IR (KBr) νmax: 3187, 2935, 2867, 1719, 1631, 1568,
2.2.2. 3α,12α-Diformyloxy-5β-cholan-24-oic acid (3)
1449, 1377, 1180.
Yield 40%, powder, mp 170–171 °C. 1H NMR (500 MHz,
CDCl3,TMS, ppm): δ = 8.12 (s, 1H, 12α-OCHO), 8.02 (s, 1H, 3α-
OCHO), 5.23 (s, 1H, 12β-H), 4.83 (m, 1H, 3β-H), 0.91 (s, 3H, CH3-19),
2.3.3. Gramine-3α,7α,12α-triformyloxy-5β-cholan-24-oic acid salt (10)
Yield 98%, powder, mp 70–73 °C. 1H NMR (300 MHz, CDCl3, TMS,
2

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
ppm): δ = 9.69 (s, 1H, ^N+-H), 8.69 (s, 1H, NH), 8.14 (s, 1H, 7α-
OCHO), 8.10 (s, 1H, 12α-OCHO), 8.04 (bs, 1H, 3α-OCHO), 7.57 (d, 1H,
7′-H), 7.40 (d, 1H, 4′-H), 7.36 (s, 1H, 2′-H), 7.11–7.20 (m, 2H, 6′-H, 5′-
H), 5.26 (bs, 1H, 12β-H), 5.06 (bs, 1H, 7β-H), 4.88–4.80 (m, 1H, 3β-H),
4.17 (s, 2H, 10′-H), 2.57 (s, 6H, N+(CH3)2), 0.92 (s, 3H, CH3-19), 0.84
(bs, 3H, CH3-21), 0.73 (s, 3H, CH3-18). ESI-MS: m/z 491 [C27H40O8-
H]−, 304 [C20H22N3]+, 175 [C11H15N2]+, 130 [C9H8N]+. FT-IR (KBr)
νmax: 3402, 2932, 2868, 1717, 1585, 1458, 1382, 1181.
2.4.3. Nicotine-3α,7α,12α-triformyloxy-5β-cholan-24-oic acid salt (16)
Yield 89%, brown oil. 1H NMR (300 MHz, DMSO-d6, TMS, ppm): δ
8.59 (1H, ArH), 8.50 (1H, ArH); 7.73 (1H, ArH); 7.42 (1H, ArH); 2.09
(N-CH3, s), 8.14 (s, 1H, 7α-OCHO), 8.10 (s, 1H, 12α-OCHO), 8.04 (bs,
1H, 3α-OCHO), 0.91 (s, 3H, CH3-19), 0.86 (bs, 3H, CH3-21), 0.72 (s,
3H, CH3-18). ESI-MS: m/z 627 [C36H55O7N2]+, 491 [C27H40O8-H]−
,
163 [C10H15N]+
.
2.3.4. Gramine-3α-acetoxy-5β-cholan-24-oic acid salt (11)
2.5. Human erythrocyte membrane perturbing activity of compounds
studied
Yield 98%, powder, mp 50-55 °C. 1H NMR (300 MHz, CDCl3, TMS,
ppm): δ = 9.14 (s, 1H, ^N+-H), 8.60 (s, 1H, NH), 7.59 (d, 1H, 7′-H),
7.41 (d, 1H, 4′-H), 7.28 (s, 1H, 2′-H), 7.11–7.21 (m, 2H, 6′-H, 5′-H),
4.76–4.68 (m, 1H, 3β-H), 4.02 (s, 2H, 10′-H), 2.47 (s, 6H, N+(CH3)2),
2.03 (s, 3H, 3α-CH3COO), 0.92 (bs, 6H, CH3-21, CH3-19), 0.63 (s, 3H,
CH3-18). ESI-MS: m/z 835 [C52H84O8-H]−, 453 [C26H42O4+Cl]−, 417
2.5.1. Erythrocyte preparation
Freshly human erythrocytes (RBC) suspensions were obtained from
the blood bank. RBC were washed three times (3000 rpm/10 min/
+4 °C) in phosphate buffered saline (PBS, pH 7.4) supplemented with
10 mM glucose. After washing, RBC were suspended in the buffer at
1.65x109 cells/mL. Cells were stored at +4 °C and used within 5 h.
[C26H42O4-H]−, 304 [C20H22N3]+, 175 [C11H15N2]+, 130 [C9H8N]+
FT-IR (KBr) νmax: 3215, 2936, 2866, 1735, 1562, 1448, 1379, 1361,
.
1244.
2.3.5. Gramine-3α,12α-diacetoxy-5β-cholan-24-oic acid salt (12)
Yield 80%, powder, mp 50–55 °C. 1H NMR (300 MHz, CDCl3, TMS,
ppm): δ = 12.36 (s, 1H, ^N+-H), 9.56 (s, 1H, NH), 7.60 (d, 1H, 7′-H),
7.37 (d, 1H, 4′-H), 7.23 (s,1H, 2′-H), 7.02–7.16 (m, 2H, 6′-H, 5′-H),
4.81–4.75 (m, 1H, 3β-H), 4.15 (s, 1H, 12β-H), 3.93 (s, 2H, 10′-H), 2.41
(s, 6H, N+(CH3)2), 2.03 (s, 6H, 3α-CH3COO, 12α-CH3COO), 0.90 (bs,
3H, CH3-21), 0.89 (bs, 3H, CH3-19), 0.66 (s, 3H, CH3-18). ESI-MS: m/z
951 [C56H88O12-H]−, 475 [C28H44O6-H]−, 304 [C20H22N3]+, 175
[C11H15N2]+, 130 [C9H8N]+. FT-IR (KBr) νmax: 3183, 2944, 2869,
1734, 1631, 1563, 1449, 1378, 1378.
2.5.2. In vitro haemolytic assay
RBC (1.65 × 108 cells/mL, ∼1.5% haematocrit) were incubated in
phosphate-buffered saline (PBS, pH 7.4) supplemented with 10 mM
glucose and containing tested compounds (in the concentration range
from 0 mg/mL to 0.1 mg/mL) for 60 min at 37 °C in a shaking water
bath. RBC incubated in PBS only were taken as the control. Controls and
sample tests were run in triplicate and the experiments were repeated
four times with RBC from different donors. After incubation, the RBC
suspensions were centrifuged (3000 rpm/10 min/+4 °C) and the de-
gree of haemolysis was estimated by measuring the absorbance of the
supernatant at λ = 540 nm. The results were expressed as percentage
(%) of haemolysis. Haemolysis 0% was taken as the absorbance of the
supernatant of erythrocyte suspensions in PBS in the absence of tested
compounds (control sample) and the total haemolysis (100%) was de-
termined when PBS was replaced by cold distilled water. The values are
2.3.6. Gramine-3α,7α,12α-triacetoxy-5β-cholan-24-oic acid salt (13)
Yield 92%, powder, mp 90 °C. 1H NMR (300 MHz, CDCl3, TMS,
ppm): δ = 12,01 (s, 1H, ^N+-H), 10.06 (s, 1H, NH), 7.55 (d, 1H, 7′-H),
7.37 (d, 1H, 4′-H), 7.29 (s,1H, 2′-H), 7.07–7.17 (m, 2H, 6′-H, 5′-H), 5.08
(bs, 1H, 12β-H), 4.90 (bs, 1H, 7β-H), 4.61–4.53 (m, 1H, 3β-H),4.00 (s,
2H, 10′-H), 2.46 (s, 6H, N+(CH3)2), 2.10 (s, 3H, 12α-CH3COO), 2.08 (s,
3H, 7α-CH3COO), 2.05 (s, 3H, 3α-CH3COO), 0.90 (s, 3H, CH3-19), 0.83
(d, 3H, CH3-21), 0.69 (s, 3H, CH3-18). ESI-MS: m/z 569
the mean
SD of four independent experiments.
2.5.3. Light microscope studies of erythrocytes shape transformation
The cells were fixed with 5% paraformaldehyde plus 0.01% glu-
taraldehyde for 30 min at RT in the dark. After washing in PBS, RBC
were settled on poly-L-lysine-treated (0.1 mg/mL, 10 min, RT) cover
glasses and mounted on 80% glycerol. The cover slips were sealed with
nail polish. A large number of RBC in several separate experimental
samples were studied.
[C30H46O8+Cl]−
,
533[C30H46O8-H]−
,
175 [C11H15N2]+
,
130
[C9H8N]+. FT-IR (KBr) νmax: 3386, 2945, 2871, 1732, 1732, 1562,
1439, 1377, 1249.
2.4. General synthetic procedure for nicotine salts 14–16
A mixture of (−)-(S)-nicotine (81 mg, 0.5 mmol) and 3α-formyloxy-
5β-cholan-24-oic acid (203 mg, 0.5 mmol), 3α,12α-diformyloxy-5β-
cholan-24-oic acid (225 mg, 0.5 mmol), 3α,7α,12α-triformyloxy-5β-
cholan-24-oic acid (245 mg, 0.5 mmol) was stirred in methanol (10 mL)
at room temperature for 20 h. After completion of the reaction as in-
dicated by TLC the solvent was removed under reduced pressure.
2.5.4. Scanning electron microscope studies of erythrocytes shape
transformation
Erythrocytes fixed with 5% PFA plus 0.01% glutaraldehyde (as in
Section 2.5.3) were post-fixed with 0.1% glutaraldehyde for 1 h at room
temperature (RT). Cells were washed by exchanging of supernatant
with PBS. The samples were gently vortexed and cells were fixed again
with 2% glutaraldehyde for another hour at RT. After washing as above,
cells were post-fixed with 1% OsO4 for 30 min at RT. The supernatant
was exchange with PBS and samples were very gently vortexed. Fixed
erythrocytes were dehydrated in a series of ethanol solution (50%, 60%,
70%, 80%, 90%, 95%, and 100%), gold-sputtered, and examined in a
EVO 40 (ZEISS, Germany) scanning electron microscope.
2.4.1. Nicotine-3α-formyloxy-5β-cholan-24-oic acid salt (14)
Yield 95%, brown oil. 1H NMR (300 MHz, DMSO-d6, TMS, ppm): δ
8.55 (1H, ArH), 8.46 (1H, ArH); 7.71 (1H, ArH); 7.35 (1H, ArH); 2.06
(N-CH3, s), 8.05 (s, 1H, 3α-OCHO), 0.91 (bs, 3H, CH3-19), 0.88 (bs, 3H,
CH3-21), 0.61 (bs, 3H, CH3-18). ESI-MS: m/z 539 [C34H55O3N2]+, 403
[C25H39O4]−, 375 [C24H39O3]−, 163 [C10H15N]+
.
2.4.2. Nicotine-3α,12α-diformyloxy-5β-cholan-24-oic acid salt (15)
Yield 86%, brown oil. 1H NMR (300 MHz, DMSO-d6, TMS, ppm): δ
8.54 (1H, ArH), 8.45 (1H, ArH); 7.71 (1H, ArH); 7.35 (1H, ArH); 2.07
(N-CH3, s), 8.10 (s, 1H, 12α-OCHO), 8.03 (bs, 1H, 3α-OCHO), 0.90 (s,
3H, CH3-19), 0.82 (bs, 3H, CH3-21), 0.72 (bs, 3H, CH3-18). ESI-MS: m/z
2.5.5. Statistical analysis
The results were presented as mean value
standard deviation
(SD) for four independent experiments. A paired t-test was used to
compare the every two suitable compounds. Statistical significance was
defined as p < 0.05.
447 [C26H40O6-H]−
, , , 163
419 [C25H40O5-H]− 175 [C11H15N2]+
[C10H15N]+
.
3

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of gramine salts 8–13 and nicotine salts 14–16.
3. Results and discussion
Table 1
Heat of formation (HOF) [kcal/mol] of bile acids (LCA, DCA, CA), its formyl (2–4) and
acetyl (5–7) derivatives as well as their salts (8–1 3) and the length [Å] of intramolecular
bonds between O⋯H⋯N.
3.1. Chemistry
Bile acids were converted to their corresponding formyl derivatives
by heating with formic acid [10,20]. The acetyl derivatives of bile acids
were prepared following the standard Ac2O/pyr/DMAP procedure [29].
A mixture of the cholic acids and gramine (or nicotine) in methanol
gave salts (8–16) with the total yields 80–99%. The general synthetic
method for the preparation of newly obtained salts is outlined in
Scheme 1.
Compound
HOF [kcal/mol]
ΔHOF [kcal/mol]
Length [Å] O⋯H⋯N
LCA
DCA
CA
2
3
4
5
6
7
8
−235.0878a
−277.2635a
−316.9693a
−271.7707
−348.9356
−424.7801
−276.4870
−367.4560
−448.4233
−229.4106
−306.7766
−382.7269
−238.4242
−325.3092
−410.1348
–
–
–
–
–
–
–
–
–
–
–
−36.6829
−71.6721
−107.8108
−41.3992
−90.1925
−131.4540
42.3601
42.1590
42.0532
38.0628
42.1468
38.2885
Structures of all salts were confirmed by their 1H NMR and ESI MS
spectra analyses. The 1H NMR spectra of 8–13 besides characteristic
signals in the range of 0.62–0.73 ppm (CH3-18), 0.89–0.92 ppm (CH3-
19) and at 0.82–0.92 ppm (CH3-21) shows signals attributed to HCOO−
and CH3COO− groups at about 8 ppm and 2 ppm, respectively. In the
spectra of these compounds characteristic singlets in the range of
2.41–2.57 ppm were assigned to the N-methyl protons and singlets at
3.93–4.17 ppm and 9.14–12.36 ppm to C10′-H and ^N+-H protons,
respectively. In the 1H NMR spectra of nicotine salts (14–16) the two
protons of pyridine ring being at α position to the nitrogen atom are
largely deshielded and appear at about 8.5 ppm. The signal of N-me-
thylene protons appear at about 2 ppm. The salts obtained gave mass
spectra with the m/z values corresponding to fragmentation of acid
molecule and gramine or nicotine. In ESI MS spectra of gramine salts, in
positive ion mode the signal of indole fragment (m/z = 130) is ob-
served, whereas in nicotine salts the signal of nicotinium cation at m/
z = 163 is present. The absorption in the 3150–3250 cm−1 region (for
8–13) corresponds to the ν(^N+H…O) band which is a typical ab-
sorbance for hydrogen bond with an N…O distance about 3 Å.
–
1.72/1.45
1.76/1.44
1.77/1.43
1.61/1.46
1.68/1.46
1.63/1.47
9
10
11
12
13
ΔHOF = HOFformyl/acetyl
– HOFbile
acids(LCA, DCA, CA).
derivatives(2–7)
ΔHOF = HOFsalts(8–10) – HOFformyl
ΔHOF = HOFsalts(11–13) – HOFacetyl
derivatives(2–4).
derivatives(5–7).
a
See [25].
triformyloxy-5β-cholan-24-oic acid (4) as well as 3α-acetoxy-5β-
cholan-24-oic acid (5), 3α,12 α-diacetoxy-5β-cholan-24-oic acid (6),
3α,7α,12α-triacetoxy-5β-cholan-24-oic acid (7) have lower values of
HOF than the acids with unsubstituted hydroxyl groups [25]. This fact
can be explained by the greater stability by ester groups in isolated
molecules and also caused by difficulty to form intramolecular hy-
drogen bonds, which can be formed in the case of unsubstituted hy-
droxyl groups. Furthermore, the increase of the number of free hy-
droxyl groups causes the increase of the HOF values [25]. The presence
of ester group at 3α, 7α as well as 12 α position increases the stability of
the compounds (2–7), and as a result of their conjugates with gramine
(8–13). In addition, values of HOF decreases with increasing size of
substituents in the reactive parts of steroid skeleton. The salts of formyl
and acetyl-derivatives of lithocholic, deoxycholic and cholic acids are
shown in Fig. 1. The lowest value of HOF for all salts was observed for
3.2. Theoretical calculations
The final heats of formation (HOF) for the bile acids (LCA, DCA, CA)
and their formyl- (2–4) and acetyl- (5–7) derivatives as well as formyl-
(8–10) and acetyl- (11–13) salts are presented in Table 1. The esters
derivatives of bile acids: 3α-formyloxy-5β-cholan-24-oic acid (2),
3α,12α-diformyloxy-5β-cholan-24-oic acid (3), 3α,7 α,12 α-
4

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
Fig. 1. Molecular models of gramine salts with formyl
(8–10) and acetyl (11–13) derivatives of bile acids calcu-
lated by the PM5 method.
(8)
(9)
(10)
(11)
(12)
(13)
salts derived from cholic acid: 10 and 13. In these salts, the number of
formyl or acetyl groups in the steroid skeleton lowers the value of HOF.
This effect is the same as in the case of bile acids containing a sub-
stituted hydroxyl group. Simultaneously bile acids having a substituted
hydroxyl groups in positions 3α,7α and/or 12α (compounds 2–7) have
lower values HOF respect to the corresponding salts (compounds 8–13).
This follows from the fact that in the salts proton is delocalized between
the oxygen atoms of carboxyl group and angular nitrogen atom of
gramine. Furthermore the rings of gramine molecule are always or-
iented in perpendicular to the bile acid skeleton. The distances between
the proton and quaternary nitrogen atom as well as the proton and
oxygen atom of carboxylate anion are 1.43–1.45 Å and 1.72–1.77 Å for
formyl salts: 8–10 and 1.46–1.47 Å and 1.61–1.68 Å for acetyl salts:
11–13, respectively. This result is in a very good agreement with other
findings as well as with our previous calculations [25].
similar to the original cholic acid. The acetyl-derivative of cholic acid
(7) was not active as membrane perturbing agents as the original cholic
acid at the all concentrations studied.
As shown in Fig. 2, gramine significantly decreased the membrane-
perturbing activity of hydrophobic bile acids derivatives. However,
both formyl- and acetyl-derivatives of lithocholic acid (8, 11) were an
exception at the highest concentration (0.1 mg/mL). The possible ex-
planation could be the extremely high haemolytic activity of this most
hydrophobic bile acid. Therefore, the addition of gramine to formyl-
and acetyl-derivatives of lithocholic acid was not efficient factor to
reduce their cytotoxicity activity. These results are in line with our
previous findings regarding the haemolytic activity of LCA and DCA
salts with gramine [25]. Similarly to the natural bile acids, the formyl-
and acetyl-derivatives of lithocholic and deoxycholic acids were echino-
and/or stomatocytogenic agents, depending on their hydrophobicity
and concentration. Gramine did not influence the efficiency of bile
acids esters for their typical structure- and dose-dependent echinocytic
or stomatocytogenic RBC shape transformation. Representative results
of erythrocyte stomatocytic shape transformation observed for acetyl-
lithocholic acid (5) and its gramine derivative (11) (at concentration
equal 0.001 mg/mL) are presented in Fig. 3.
To study the influence of other alkaloids on the membrane per-
turbing activity of bile acids we synthesized formyl-derivatives of li-
thocholic, deoxycholic and cholic acids with nicotine salts, namely
compound 14, 15 and 16. Our previous results showed that nicotine
decreased the membrane interacting potential of bile acids depending
on their hydrophobicity [26]. In the present study, nicotine decreased
haemolytic activity of formyl-derivatives of bile acids, depending on
their hydrophobicity and the concentration used. Namely, there was a
more than 92% decrease in haemolysis, from 98,5% to 5,76% at the
concentration 0.1 mg/mL of nicotine salt with formyl-cholic acid salt
(16), with respect to that achieved for formyl-cholic acid alone (4).
Similarly, there was a 95% decrease in haemolysis, namely from 97.5%
3.3. Haemolytic activity of formyl- and acetyl-cholic acids and their
gramine salts
The haemolytic activity of bile acids and their gramine salts was
studied with a concentration in the range of 0.001 to 0.1 mg/mL
(Fig. 2). Lithocholic acid, a monohydroxy bile acid, characterized the
highest haemolytic activity as the most hydrophobic compound stu-
died. Dihydroxydeoxycholic acid and trihydroxycholic acid, as more
hydrophilic acids, were almost no membrane-perturbing agents at the
used concentration rage. As shown in Fig. 2 the haemolytic activity of
formyl- and acetyl-lithocholic (2, 5) and deoxycholic (3, 6) acids were
significantly higher (p < 0.05) in comparison with their native forms
at the whole concentration range. However, the differences were the
most pronounced at their highest concentrations (0.05 and 0.1 mg/mL).
Interestingly, at concentration equal to 0.1 mg/mL, formyl derivative of
cholic acid (4) was as effective as lithocholic (2, 5) and deoxcholic (3,
6) acids derivatives. At lower concentration (0.05–0.001 mg/mL), the
membrane perturbing activity of formyl derivative of cholic acid was
5

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
Fig. 2. The haemolytic activity of: bile acids (LCA, DCA, CA), their formyl-(2–4) and acetyl-(5–6) forms, their gramine salts with formyl-(8–10) and acetyl-(11–13) bile acids. The results
are presented as mean SD (n = 4). *p < 0.05.
to 2.34% at the concentration 0.01 mg/mL of nicotine salt with formyl-
lithocholic acid (14), with respect to that achieved for formyl-litho-
cholic acid alone (2). All new synthesized nicotine salts with formyl-bile
acids induced RBC shape alterations, similar to those observed for bile
acids alone (results not shown). However, at the concentration
0.01 mg/mL of nicotine salts with formyl-bile acids, RBC did not un-
dergo any shape transformation. Therefore, it can be concluded, that
different alkaloids, namely gramine and nicotine, decrease significantly
the membrane interacting activity of formyl-bile acids.
acids molecule increases the values of TPSA and number of hydrogen-
bond acceptors (up 3 relative to the parent acids). A high PSA and a
large number of hydrogen bond acceptors has been associated with the
poor membrane permeability due to the additional desolvation energy
required to break hydrogen bonds when moving from an aqueous en-
vironment to the lipid membrane. Modification of deoxycholic and
cholic acids reduces the number of hydrogen-bond donors particularly
in the case of the compounds 4 and 7 which may affect their biological
activity.
To explain quantitative structure-haemolytic activity relationships
of the bile acids and their derivatives (2–7), physicochemical calcula-
tions were conducted using Molinspiration Property Calculator,
v2016.10. The physicochemical properties of molecules, such as, their
lipophilicities and polar surface areas (PSAs) play important roles in
determining biological responses, and are commonly used to study the
structure activity relationships of bioactive molecules. This physio-
chemical parameters of bile acids and their derivatives 2–7 are listed in
Table 2.
Low TPSA (< 75) has been associated with an increased risk of
adverse events due to non-specific toxicity, particularly when combined
with high lipophilicity. Therefore, the high haemolytic activity of li-
thocholic acid can be explain by its high lipophilicity (LogP > 5) and
low TPSA. The introduction of formyl- and acetyl-substituents to bile
4. Conclusions
PM5 calculations performed for formyl and acetyl derivatives of bile
acids indicate that the number of hydroxyl groups in the steroid ske-
leton influences the value of HOF. The higher number of unsubstituted
or substituted hydroxyl groups reduced the HOF and consequently
stability of the salt. The consequence of both formyl- and acetyl-mod-
ifications of hydrophobic bile acids, namely lithocholic and deoxy-
cholic, was the increase of their membrane-perturbing activity. On the
other hand, the haemolytic activity of acetyl-derivatives of hydrophilic
cholic acid was no different from its original form; however, the formyl-
derivative of cholic acid was as effective as the formyl-derivatives of
lithocholic and deoxycholic acids. Although the presence of formyl- and
Fig. 3. Human erythrocytes incubated (A) with acetyl-lithocholic acid (5) and (B) with its gramine derivative (11) at 0.001 mg/mL (60 min, 37 °C). (C) discocytes (control cells),
Stomatocytes (cupe cells) are dominated cell type in A and B. Insets in A and B: stomatocytes as observed under a scanning electron microscope. Scal bars represent 10 μm in A and B,
1 μm in C and 2 μm in both insets.
6

W. Kozanecka-Okupnik et al.
Steroidsxxx(xxxx)xxx–xxx
Table 2
Molinspiration calculations of the molecular properties of bile acids and their derivatives 2–7.
Compound
MW (g/mol)1
Natoms2
LogP3
TPSA4
OH-NH interact5
OeN interact6
Nrotb7
Volume8
LCA
DCA
CA
2
3
4
5
6
7
376.58
392.58
408.58
436.59
464.60
492.61
450.62
492.65
534.60
27
28
29
31
33
35
32
35
38
5.16
4.25
3.33
3.75
4.18
4.60
4.04
4.74
5.44
57.53
77.75
97.98
104.06
110.14
116.21
104.06
110.14
116.21
2
3
4
3
2
1
3
2
1
3
4
5
6
7
8
6
7
8
4
4
4
6
8
10
6
8
10
389.59
397.63
405.68
425.63
445.58
465.53
442.19
478.70
515.21
1
Molecular weight.
Number of non hydrogen atoms.
2
3
4
5
6
7
8
Calculated octanol/water partition coefficients.
Topological polar surface areas.
Number of hydrogen-bond donors (OH and NH groups).
Number of hydrogen-bond acceptors (O and N atoms).
Number of rotatable bonds.
Molecular volume.
(2013) 321–326.
acetyl-substituents in bile acids increases their membrane-perturbing
activity, this feature can be reduced or eliminated in the presence of
different alkaloids e.g. gramine or nicotine. Because the cell membrane
serves as a barrier in every type of cell, therefore, it can be concluded
that alkaloids may reduce the cytotoxicity of membrane-active agents.
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tivity of cholic acid derivatives with three permanent ionic head groups, Angew.
Chem. Int. Ed. 41 (2002) 4275–4277.
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Synthesis, in vitro antifungal activity and mechanism of action of four sterol hy-
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[17] D. Brossard, L. El Kihel, M. Clément, W. Sebbahi, M. Khalid, C. Roussakis, S. Rault,
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.steroids.2017.07.003.
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7