Synthesis of new cisplatin derivatives from bile acids

Article abstract of DOI:10.3390/molecules25030655

A series of bile acid derived 1,2- and 1,3-diamines as well as their platinum(II) complexes were designed and synthesized in hope to get a highly cytotoxic compound by the combination of two bioactive moieties. All complexes obtained were subjected to cytotoxicity assays in vitro and some hybrid molecules showed an expected activity.

Full text of DOI:10.3390/molecules25030655

molecules
Article
Synthesis of New Cisplatin Derivatives from
Bile Acids
1
,
1,
1
, Lucie Rárová 2,
Barbara Seroka * , Zenon Łotowski *, Agnieszka Hryniewicka
3
,
1
1,†
Rafal R. Sicinski *, Aneta M. Tomkiel and Jacek W. Morzycki
1
Faculty of Chemistry, University of Białystok, K. Ciołkowskiego 1K, 15-245 Białystok, Poland;
aga_h@uwb.edu.pl (A.H.); a.tomkiel@uwb.edu.pl (A.M.T.); morzycki@uwb.edu.pl (J.W.M.)
Laboratory of Growth Regulators, Faculty of Science, Palacký University, and Institute of Experimental
Botany of the Czech Academy of Sciences, Šlechtitel u˚ 27, CZ-78371 Olomouc, Czech Republic;
lucie.rarova@upol.cz
Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
Correspondence: barbaraseroka05@gmail.com (B.S.); zlch@uwb.edu.pl (Z.Ł.);
rasici@chem.uw.edu.pl (R.R.S.)
2
3
*
†
Dedicated to Professor Hector F. DeLuca (University of Wisconsin Madison, USA) on the occasion of his
9
0th birthday.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Rafal R. Sicinski
Received: 15 November 2019; Accepted: 2 February 2020; Published: 4 February 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A series of bile acid derived 1,2- and 1,3-diamines as well as their platinum(II) complexes
were designed and synthesized in hope to get a highly cytotoxic compound by the combination of
two bioactive moieties. All complexes obtained were subjected to cytotoxicity assays in vitro and
some hybrid molecules showed an expected activity.
Keywords: bile acids; lithocholic acid; deoxycholic acid; hyodeoxycholic acid; cholic acid; steroidal
diamines; platinum(II) complexes
1
. Introduction
Cisplatin is one of the most effective chemotherapeutic agents used in the treatment of many
human cancers [1]. Unfortunately, its therapeutic use is limited due to harmful side effects, in particular
nephrotoxicity [
2,
3
]. Although many anticancer platinum complexes have been developed so far [ ],
4
there is still a need to find more active and more selective drugs. Combining of two bioactive molecules
as a way to improve the biological properties of the starting compounds is a common practice in
medical chemistry [5]. In addition, a natural compound that is designed to form a hybrid molecule
together with cisplatin should be a good transporter for transferring the drug to cancer cells. Binding
biomolecules to drugs always causes a drastic change in the structure of the carrier as well as in the
structure of the pharmacologically active compound. We recently used this idea to design steroid-based
cisplatin derivatives according to the general formula shown in Figure 1 [6,7].
As a result of our previous work, steroidal cisplatin analogs were obtained: two series of
cholestane-based Pt(II) complexes derived from cholesterol (
from 16-dehydropregnenolone acetate [ ]. However, the most promising was cisplatin derivative
based on lithocholic acid (LCA) [ ]. Although the complex showed only moderate cytotoxicity (IC50
M) in the initial test against the HeLa cervical cancer cell line, it proved to be non-toxic to normal
1–4) and a complex 5 (Figure 2) synthesized
6
6
7
6
28 µ
fibroblasts. This selectivity opens the way for modification of this complex to be more active while
maintaining a lack of toxicity to healthy cells.
Molecules 2020, 25, 655; doi:10.3390/molecules25030655
www.mdpi.com/journal/molecules

Molecules 2020, 25, 655
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Figure 1. General model of steroid-based cisplatin derivatives (n = 1 or 2).
Figure 2. Steroid-based cisplatin derivatives [6,7].
The biological functions of bile acids depend on their chemical nature and polarity [
8]. Both
LCA and deoxycholic acid (DCA) are hydrophobic but show different toxicity [
9]. Bajaj et al. [10]
have reported that DCA having two hydroxyl groups shows weak interactions with cell membranes
compared to highly hydrophobic LCA. The hydration barrier between cell membranes and DCA does
not favor the interactions that cause poor uptake in mammalian cells. In contrast, more hydrophobic
LCA can easily penetrate cell membranes. In addition, the varied activity of DCA and LCA may also
result from their different binding to cellular receptors [11–13]. Lithocholic acid exhibits high antitumor
activity [14 15], is involved in human and animal carcinogenesis [16], can selectively kill neuroblastoma
,
cell lines [17], and selectively induces apoptosis in prostate [18] and colon cancer cells [19]. LCA elicits
apoptosis by activation both extrinsic and intrinsic pathways without entering to cancer cells. It has
been suggested that target of cancerous tissue is most likely localized at the surface of the plasma
membrane and is either activated or deactivated by LCA in order to elicit apoptosis [18].
Bile acids directly activate various intracellular ligand-activated nuclear receptors, such as the
farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR), and cell surface
G protein-coupled receptors (GPCRs), such as the G protein-coupled bile acid receptor (TGR5 and
Gpbar-1). FXR and PXR are highly expressed in tissues that are exposed to bile acids, including
the liver and the intestine, whereas VDR is widely expressed in most tissues. TGR5 is highly
expressed along the intestinal tract, with the highest expression found in the ileum and colon. It is
also expressed in nontraditional bile acid target organs including white and brown adipose, spleen,
kidney, pancreas, lung, macrophages, and the central nervous system [20]. Moreover, some receptors
found to be overexpressed in cancer cell lines, e.g., GPCRs are overexpressed in lung, prostate, breast,
and neuroblastomas. This fact is exploited in targeting drugs to cancerous tissues and the tumor
microenvironment [21]. For this reason, bile acids have become important carriers of drugs in medicinal

Molecules 2020, 25, 655
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chemistry [22]. Furthermore, the combination of a pharmacologically active compound with bile acid
makes it possible to exploit the natural resorption and can lead to better uptake of pharmaceuticals [23].
Paschke et al. have reported synthesis and biological evaluation of Pt(II)-based cholic acid derivatives,
which consist of three functional parts—a transport fragment, a spacer and a biologically active drug
component: cisplatin [24] or carboplatin [25]. Some other anticancer Pt(II)-based bile acid derivatives
are known, e.g. Bamet-R1, Bamet-R2, Bamet-UD1, or Bamet-UD2, in which platinum is bound to the
glycocholic or ursodeoxycholic acids [26–30]. Bartoli et al. have reported another example of Pt(II)
complex of cholic acid, in which functional groups present in the steroidal A ring coordinate with
platinum [31]. All the above-mentioned complexes contain an ester linkage between the steroid moiety
and platinum-based drug, which guarantees the release of the cisplatin-like metabolite. The complex
6
is, to the best of our knowledge, the first Pt(II) complex with stable linkage of LCA and cisplatin,
based on carbon–carbon bond. This approach ensures that LCA is capable of playing double role:
as a carrier and anticancer co-drug. A spacer between cisplatin and biomoiety is needed because it
separates the active parts of both molecules so that they can be recognized and act independently.
Such an arrangement assures that their biological properties are preserved [32] and also some synergic
effects can be expected.
Taking into account the promising cytotoxic properties of the cisplatin analog
6 [7], as well as the
chemical nature of bile acids, we designed new hybrid platinum moieties. We synthesized two series
of platinum complexes with a different distance between the amino groups based on four bile acids:
three hydrophobic (LCA, DCA, hyodeoxycholic) and one hydrophilic (cholic acid). The goal of our
research is to find the most active and most selective compound as well as to compare the activity of
different bile acid derivatives.
2
. Results and Discussion
2
.1. Chemistry
First attempts to synthesize steroidal analogs of cisplatin employed the synthetic paths that
we previously developed [
commercially available LCA (7a) is shown in Scheme 1. Firstly, the hydroxyl group at C-3 was
protected as TBDMS ether ( ). For the conversion of carboxylic acid to corresponding amine, and
then to 1,2- and 1,3-diamines, the multi-step transformation of the side chain was carried out. The
methylation of the carboxylic group with methanol/HCl was followed by LiAlH reduction of methyl
6]. The strategy for the 1,2-diamine and 1,3-diamine synthesis from
8
4
ester
, which was further transformed into azide 12 with sodium azide in DMF [33]. Deprotection
of the hydroxyl group at C-3 allowed to obtain 13 in a good overall yield (all steps were fairly
9 to primary alcohol 10. Then the hydroxyl group at C-24 was converted into methanesulfonate
1
1
efficient). The Staudinger reaction of azide 13 with triphenylphosphine yielded aminosteroid 14
In the next step an introduction of 2-cyanoethyl or cyanomethyl chains to 14 was attempted. This
would allow for an extension of the side chain with simultaneous introduction of the second nitrogen
.
atom. The alkylation of 14 with bromoacetonitrile in the presence of Na CO in ethanol afforded
2
3
2
4-(N-cyanomethyl)-aminocholan-3
α
-ol (15a). Alternatively, 14 reacted with acrylonitrile to afford the
-ol (16a). The final step of the synthesis was
0
addition product—24-(N-2 -cyanoethyl)-aminocholan-3
α
the reduction of nitrile functional groups with lithium aluminum hydride to give products 17a and 18a
in high yields.
0
The synthesis allowed to obtain 24-(2 -amino-ethylamino)-cholan-3
α
-ol
(
17a
)
and
0
2
4-(3 -amino-propylamino)-cholan-3
α
-ol (18a) in satisfactory total yields. However, a large number of
steps due to necessary protection of functional groups prompted us to look for a shorter method of
synthesis of these compounds.

Molecules 2020, 25, 655
4 of 15
Scheme 1. Synthesis of 1,2- and 1,3-diamines from lithocholic acid (LCA).
Starting materials for an alternative synthesis of these bidentate ligands were bile acids, namely
lithocholic acid (7a), deoxycholic acid (7b), hyodeoxycholic acid (7c), and cholic acid (7d). The
structures of these compounds along with their designed modifications are shown in Scheme 2 and
Table 1.
Scheme 2. Synthesis of cisplatin derivatives from different bile acids.

Molecules 2020, 25, 655
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Table 1. Starting bile acids, their synthesized derivatives and target platinum(II) complexes.
Acids
Lithocholic acid 7a (R =R =R =H)
Esters
19a
Amides
Amines
Complexes
n = 2, 20a
n = 3, 21a
n = 2, 17a
n = 3, 18a
n = 1, 22a
n = 2, 23a
1
2
3
Deoxycholic acid 7b
R =R =H; R =OH)
n = 2, 20b
n = 3, 21b
n = 2, 17b
n = 3, 18b
n = 1, 22b
n = 2, 23b
1
9b
9c
9d
(
1
2
3
Hyodeoxycholic acid 7c (R =OH;
n = 2, 20c
n = 3, 21c
n = 2, 17c
n = 3, 18c
n = 1, 22c
n = 2, 23c
1
1
R =R =H)
2
3
Cholic acid 7d
R =H; R =R =OH)
n = 2, 20d
n = 3, 21d
n = 2, 17d
n = 3, 18d
n = 1, 22d
n = 2, 23d
1
(
1
2
3
The aim of these chemical transformations of bile acids was to introduce a strongly chelating
group into the side chain of steroid. Since amines are ligands of choice for transition metals, a series
of eight steroidal diamines derived from four different bile acids were designed. The two amino
groups placed at the side chain terminal should be relatively close to each other to make sure that
the configuration at the platinum atom in the complex is cis (it is known that the trans isomers are
biologically inactive). In the first step of synthesis the carboxylic group was activated as a methyl ester
by treatment of bile acids 7a
hydrochloric acid. At the second step, esters 19a
–
d
with methanol in the presence of catalytic amounts of concentrated
were subjected to aminolysis with an excess of
–
d
ethylenediamine or propane-1,3-diamine in methanolic solution. The reactions were rather slow—they
◦
were completed at 65 C in four days. In this way, two series of amides were obtained in high (about
90%) yields: 20a–d and 21a–d, respectively. The last step of the ligand synthesis consisted of the
reduction of the amides with lithium aluminum hydride under relatively harsh conditions (3 days at
reflux in THF). Since the obtained diamines strongly bound to aluminum ions, a special procedure was
elaborated for the product isolation. The method consisted of treatment of the reaction mixture with
a small amount of aqueous sodium hydroxide. Then water was removed with anhydrous sodium
sulfate and all inorganic material was filtered off. The products in both series, 1,2-diamines (17a
,3-diamines (18a ), were obtained in satisfactory (62–73%) yields. These steroidal ligands were used
in the next step for the complexation reaction with potassium tetrachloroplatinate according to the
–d) and
1
–d
known procedure [34]. The platinum complexes 22a
–
d
and 23a
–
d
were characterized by IR, MS and
195
−1
Pt NMR spectra. The IR spectra of complexes displayed sharp bands in the range 3223–3091 cm
assigned to N-H stretching characteristic for coordination. The mass spectra showed a characteristic
1
94
195
196
195
pattern resulting from platinum isotope distribution (i.e., Pt, Pt and Pt). Considering that Pt
yields relatively narrow signals and has a wide chemical shift range, the recorded NMR spectra of
195
platinum complexes confirm both their purity and structure. The observed chemical shifts of Pt
signals in the spectra of the synthesized complexes 22a and 23a lie within the range characteristic
for complexes of the type PtN Cl . Thus, the chemical shifts of the former compounds are very similar
–
d
–d
2
2
to these of substituted ethylenediamine complexes (ca.
−
2360 ppm) reported in the literature [35
]
whereas values of the latter resemble this found [36] in the cis-Pt(MeNH ) Cl complex (
δ
−
2222 ppm).
2
2
2
2
.2. Biological Evaluation
All new platinum(II) complexes were screened for cytotoxic activity against following tumor cell
lines: T-lymphoblastic leukemia (CEM), epithelial breast carcinoma (MCF7), and cervical carcinoma
cell lines (HeLa) (Table 2). A control of cytotoxicity was done with normal human fibroblasts (BJ)
and human umbilical vein endothelial cells (HUVEC). The cytotoxicity of all substances in BJ and
HUVEC was very similar. Lithocholic acid-based complex 23a proved to be the most active among
all tested compounds. Its sensitivity against HeLa cell line (7.0
11.4 M). However, the toxicity of 23a against normal human cells (BJ and HUVEC) was similar
to that of cisplatin. It should be noticed that lithocholic acid-based complex 22a and deoxycholic
µM) was better than that of cisplatin
(
µ

Molecules 2020, 25, 655
6 of 15
acid-based derivatives: 22b and 23b showed a moderate cytotoxic activity. This result is consistent
with that obtained for complex ]. Taking into account lack of activity of cholic and hyodeoxycholic
acids-based complexes at studied concentrations (against all tested cell lines IC50 > 50 M) we can
6 [7
µ
suppose that modifications of bile acids should be focused on the most lipophilic lithocholic acid
derivatives. The results presented here provide an inspiration for further studies in this area.
Table 2. IC50 (µM, in DMF) values ± S.D. for the Pt(II)-complexes after 72 h of treatment.
Cells IC50 (µM)
Complexes
CEM
MCF7
HeLa
BJ
HUVEC
2
2
2
2
2a *
40.2 ± 2.6
3.6 ± 0.1
37.1 ± 6.8
31.6 ± 3.5
>50
>50
>50
>50
>50
3a
2b
3b
[7]
7.0 ± 0.5
20.5 ± 3.1
23.3 ± 4.7
>50
7.7 ± 1.7 ◦
7.0 ± 0.2
35.5 ± 5.3
36.2 ± 4.3
28.0 ± 4.7
11.4 ± 3.8
6.3 ± 0.8
36.6 ± 5.1
42.5 ± 3.2
>50
6.9 ± 0.6
38.2 ± 0.6
34.4 ± 5.6
>50
6
cisplatin
0.8 ± 0.1
6.9 ± 0.9
5.5 ± 1.1
*
test done at temperature 37 C due to solubility problems.
3
. Materials and Methods
3
.1. General
NMR spectra were recorded with an Avance II 400 spectrometer (Bruker, Fällanden, Switzerland)
1
13
195
operating at 400 MHz ( H), 100 MHz ( C) or 86 MHz ( Pt) using CDCl and CD OD solutions (with
3
3
TMS as the internal standard) or DMF in case of Pt(II) complexes. Infrared spectra were recorded using
Attenuated Total Reflectance (ATR) as solid samples with a Series II Magna-IR 550 FT-IR spectrometer
(
(
Nicolet, Madison, WI, USA). Mass spectra were obtained at 70 eV with an AMD-604 spectrometer
Agilent, Santa Clara, NJ, USA) and Accurate-Mass Q-TOF LC/MS 6530 spectrometers with electrospray
ionization (ESI). The reaction products were isolated by column chromatography, performed using
0–230 mesh silica gel (J.T. Baker, Phillipsburg, NJ, USA). Bile acids were purchased from TCI EUROPE
7
N.V. Compound 13 was synthesized according to literature procedure [33] starting from lithocholic
acid (7a) in 77% total yield.
3
.2. Chemical Synthesis
3
.2.1. Reduction of 24-azidocholan-3α-ol (13)
To a solution of azide 13 (0.49 g, 1.27 mmol) in THF (20 mL), triphenylphosphine (1 g, 3.81 mmol,
3
eq) and water (0.5 mL) were added. The resulting mixture was stirred at ambient temperature
for 20 h. The reaction mixture was filtered through an anhydrous Na SO and then solvents were
2
4
evaporated under reduced pressure. The residue was subjected to a silica gel column chromatography
1
(
MeOH/CH Cl 1:9) to give 24-aminocholan-3
α
-ol (14) (0.44 g, 97%) as a white solid. H NMR (400 MHz,
2
2
methanol-d4)
0
δ: 3.56 (m, 1H, H-3β), 2.62 (m, 2H, H-24), 0.94 (d, 3H, J = 6.4 Hz, H-21), 0.93 (s, 3H, H-19),
.66 (s, 3H, H-18); 13C NMR (100 MHz, methanol-d ) δ: 71.6 (CH), 57.0 (CH), 56.6 (CH), 43.0 (C), 42.6
4
(
CH), 42.3 (CH ), 40.9 (CH), 40.6 (CH ), 36.3 (CH ), 36.3 (CH), 36.0 (CH), 35.7 (CH ), 34.9 (C), 33.4
2 2 2 2
(
CH ), 30.3 (CH ), 29.6 (CH ), 28.6 (CH ), 27.5 (CH ), 26.8 (CH ), 24.5 (CH ), 23.5 (CH ), 21.1 (CH ),
2 2 2 2 2 2 2 3 2
1
8.7 (CH ), 12.1 (CH ).
3 3
3
.2.2. Reaction of 24-aminocholan-3α-ol (14) with bromoacetonitrile
A solution of aminosteroid 14 (81 mg, 0.22 mmol) in absolute ethanol (10 mL) was treated
with a drop of bromoacetonitrile (1.8
carbonate (28.5 mg, 0.27 mmol, 1.2 eq). The solution was stirred at 55 C for 4 h, cooled to room
temperature, concentrated under reduced pressure and purified by a silica gel column chromatography.
µ
L, 0.27 mmol, 1.2 equiv.) in presence of anhydrous sodium
◦

Molecules 2020, 25, 655
7 of 15
2
4-(N-Cyanomethyl)aminocholan-3
α
-ol (15a) was obtained as a white solid in 61% (55 mg) yield using
◦
1
MeOH/CH Cl (1:9) for elution. M.p. 111–113 C (MeOH/CH Cl ); H NMR (400 MHz, CDCl )
δ
: 3.61
), 3.59 (s, 2H, CH -CN), 2.69 (m, 2H, H-24), 0.9129 (d, 3H, J = 6.4 Hz, H-21), 0.9133 (s, 3H,
2
2
2
2
3
(
m, 1H, H-3
β
2
H-19), 0.64 (s, 3H, H-18); 13C NMR (100 MHz, CDCl3)
δ
: 117.8 (CN), 71.7 (CH), 56.5 (CH), 56.1 (CH),
4
9.4 (CH ), 42.6 (C), 42.0 (CH), 40.4 (CH), 40.1 (CH ), 37.3 (CH ), 36.4 (CH ), 35.8 (CH), 35.6 (CH), 35.3
2
2
2
2
(
(
CH ), 34.5 (C), 33.2 (CH ), 30.5 (CH ), 28.3 (CH ), 27.1 (CH ), 26.4 (CH ), 26.0 (CH ), 24.2 (CH ), 23.3
2 2 2 2 2 2 2 2
−1
CH ), 20.8 (CH ), 18.6 (CH ), 12.0 (CH ); IR (ATR, cm
) ν: 3311, 2926, 2857, 2238, 1454; HRMS calcd
3
2
3
3
+
for C H N O 401.3532 [M + H] , found: 401.3530.
26
45
2
3
.2.3. Reaction of 24-aminocholan-3α-ol (14) with acrylonitrile
Acrylonitrile (2.2 L, 0.33 mmol, 1.5 eq) was added to a solution of 24-aminocholan-3α-ol 14 (80
µ
mg, 0.22 mmol, 1.0 eq) in absolute ethanol (10 mL). The reaction mixture was then stirred at room
temperature for 30 min and at reflux for 1 h, cooled to room temperature, then evaporated under
0
reduced pressure to give the crude product. 24-(N-2 -Cyanoethyl)-aminocholan-3
α
-ol (16a) was purified
by a silica gel column chromatography with MeOH/CH Cl (1:24) elution affording product as a white
2
2
◦
1
solid in 92% (85 mg) yield. M.p. 92–93 C (MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ: 3.60 (m, 1H,
2
2
3
H-3
β
), 2.92 (t, 2H, J = 6.6 Hz, CH -CN), 2.58 (m, 2H, H-24), 2.52 (t, 2H, J = 6.6 Hz, NH-CH ), 0.91 (s, 3H,
2
2
13
H-19), 0.90 (d, 3H, J = 5.9 Hz, H-21), 0.63 (s, 3H, H-18); C NMR (100 MHz, CDCl3)
δ: 118.6 (CN), 71.6
(
CH), 56.4 (CH), 56.1 (CH), 49.7 (CH ), 45.0 (CH ), 42.6 (C), 42.0 (CH), 40.4 (CH), 40.1 (CH ), 36.4 (CH ),
2 2 2 2
3
2
3
5.8 (CH), 35.6 (CH), 35.3 (CH ), 34.5 (C), 33.3 (CH ), 30.5 (CH ), 28.3 (CH ), 27.1 (CH ), 26.5 (CH ),
2 2 2 2 2 2
−1
6.4 (CH ), 24.1 (CH ), 23.3 (CH ), 20.7 (CH ), 18.6 (CH ), 18.6 (CH ), 11.9 (CH ); IR (ATR, cm ) ν:
2
2
3
2
2
3
3
+
301, 2926, 2856, 2246, 1453, 1368; HRMS calcd for C H N O 415.3688, [M + H] , found: 415.3892.
27
47
2
3
2
.2.4. Reduction of 24-(N-cyanomethyl)-aminocholan-3α-ol (15a) and
0
4-(N-2 -cyanoethyl)-aminocholan-3α-ol (16a)
To a solution of nitrile 15a (40 mg, 0.10 mmol) in anhydrous diethyl ether (10 mL) was added lithium
aluminum hydride (15 mg, 0.4 mmol, 4 eq). The reaction mixture was stirred at room temperature
for 10 min and at reflux for 3 h. Then, the mixture was cooled to room temperature and water was
added dropwise followed by 20% aqueous sodium hydroxide. The resultant slurry was stirred for 15
min and anhydrous Na SO was added. The stirring was continued for an additional 15 min. Solid
2
4
materials were washed with diethyl ether and removed by filtration. The filtrate was concentrated
under reduced pressure. The crude product was purified by a silica gel column chromatography with
0
MeOH/CH Cl /NH aq (50:50:3) elution affording 24-(2 -amino-ethylamino)-cholan-3
α
-ol (17a) as a white
: 3.54 (m, 1H,
), 2.79 (t, 2H, J = 5.9 Hz, N-CH ), 2.70 (t, 2H, J = 5.9 Hz, N-CH ), 2.58 (m, 2H, H-24), 0.97 (d, 3H, J
2
2
3
◦
1
solid in 82% (33 mg) yield. M.p. 107–110 C (MeOH); H NMR (400 MHz, methanol-d )
δ
4
H-3
6.5 Hz, H-21), 0.95 (s, 3H, H-19), 0.70 (s, 3H, H-18); C NMR (100 MHz, methanol-d4) δ: 72.4 (CH),
8.0 (CH), 57.6 (CH), 52.1 (CH ), 51.1 (CH ), 43.9 (C), 43.5 (CH), 41.9 (CH), 41.6 (CH ), 41.3 (CH ), 37.2
β
2
2
1
3
=
5
2
2
2
2
(
(
2
CH), 37.18 (CH ), 37.1 (CH), 36.5 (CH ), 35.7 (C), 34.7 (CH ), 31.2 (CH ), 29.4 (CH ), 28.4 (CH ), 27.7
2 2 2 2 2 2
−
CH ), 26.9 (CH ), 25.3 (CH ), 24.0 (CH ), 22.0 (CH ), 19.2 (CH ), 12.5 (CH ); IR (ATR, cm ) ν: 3303,
2 2 2 3 2 3 3
1
+
925, 2856, 1453, 1368; HRMS calcd for C H N O 405.3845 [M + H] , found: 405.3863.
26 49 2
0
4-(3 -Amino-propylamino)-cholan-3α-ol (18a) was prepared in a similar way from 16a using
2
MeOH/CH Cl /NH aq (4:1:01) for elution. The pure product 18a was obtained as a white solid
2
2
3
◦
1
in 80% (69 mg) yield. M.p. 92–94 C (MeOH/CH Cl ); H NMR (400 MHz, MeOD)
δ
: 3.54 (m, 1H,
2
2
H-3
6.5 Hz, H-21), 0.95 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
8.0 (CH), 57.6 (CH), 51.1 (CH ), 48.2 (CH ), 43.9 (C), 43.5 (CH), 41.9 (CH), 41.6 (CH ), 40.5 (CH ),
β), 2.73 (t, 2H, J = 7.1 Hz, N-CH ), 2.67 (t, 2H, J = 7.3 Hz, N-CH ), 2.58 (m, 2H, H-24), 0.97 (d, 3H, J
2
2
13
=
δ: 72.4 (CH),
5
2
2
2
2
3
7.2 (CH), 37.18 (CH ), 37.1 (CH), 36.5 (CH ), 35.7 (C), 34.7 (CH ), 32.2 (CH ), 31.2 (CH ), 29.4 (CH ),
2 2 2 2 2 2
2
cm
8.4 (CH ), 27.7 (CH ), 26.7 (CH ), 25.3 (CH ), 23.9 (CH ), 22.0 (CH ), 19.2 (CH ), 12.5 (CH ); IR (ATR,
2 2 2 2 3 2 3 3
−1
+
) ν: 3272, 2925, 2856, 1453, 1367; HRMS calcd for C H N O 419.4001 [M + H] , found: 419.4068.
27 51 2

Molecules 2020, 25, 655
8 of 15
3
.2.5. Esterification of bile acids 7a–7d
A solution of lithocholic acid 7a (3 g, 8 mmol) in methanol (200 mL) was treated with two drops of
concentrated hydrochloric acid and the mixture was stirred at ambient temperature for 48 h. After this
time, sodium bicarbonate was added to neutralize the reaction mixture and the solvent was evaporated.
The residue was extracted with methylene chloride (3
sulfate. The organic solvent was evaporated and the residue was filtered through a silica gel column
×
100 mL) and dried over anhydrous sodium
with AcOEt/hexane (3:17) as an eluent. The pure methyl lithocholate (19a) was obtained as a white solid
1
in 96% (3 g) yield. H NMR (400 MHz, CDCl )
δ
: 3.66 (s, 3H, O-CH ), 3.62 (m, 1H, H-3
β
), 2.35 and 2.22
3
3
13
(
(
(
(
each m, 1H and 1H, H-23), 0.92 (s, 3H, H-19), 0.91 (d, 3H, J = 6.1 Hz, H-21), 0.64 (s, 3H, H-18); C NMR
100 MHz, CDCl3) : 174.7 (C=O), 71.5 (CH), 56.3 (CH), 55.8 (CH), 51.3 (CH ), 42.6 (C), 41.9 (CH), 40.3
CH), 40.0 (CH ), 36.2 (CH ), 35.7 (CH), 35.24 (CH ), 35.2 (CH), 34.4 (C), 30.9 (CH ), 30.8 (CH ), 30.3
δ
3
2
2
2
2
2
CH ), 28.0 (CH ), 27.1 (CH ), 26.3 (CH ), 24.0 (CH ), 23.2 (CH ), 20.7 (CH ), 18.1 (CH ), 11.9 (CH ).
2
2
2
2
2
3
2
3
3
Methyl deoxycholate (19b) was prepared in a similar way from deoxycholic acid using MeOH/CH Cl
2
2
1
(
(
1:19) for elution. The pure product 19b was obtained as a white solid in 98% (1.52 g) yield. H NMR
400 MHz, CDCl3) δ: 3.98 (m, 1H, H-12β), 3.68 (s, 3H, O-CH ), 3.61 (m, 1H, H-3β), 2.38 and 2.24 (each
3
13
m, 1H and 1H, H-23), 0.97 (d, 3H, J = 6.3 Hz, H-21), 0.91 (s, 3H, H-19), 0.68 (s, 3H, H-18); C NMR (100
MHz, CDCl3) : 174.7 (C=O), 73.0 (CH), 71.6 (CH), 51.4 (CH ), 48.1 (CH), 47.1 (CH), 46.4 (C), 42.0 (CH),
δ
3
3
2
6.3 (CH ), 35.9 (CH), 35.2 (CH ), 35.16 (CH), 34.0 (C), 33.5 (CH), 31.1 (CH ), 30.8 (CH ), 30.3 (CH ),
2 2 2 2 2
8.5 (CH ), 27.4 (CH ), 27.1 (CH ), 26.1 (CH ), 23.6 (CH ), 23.1 (CH ), 17.2 (CH ), 12.6 (CH ).
2
2
2
2
2
3
3
3
Methyl hyodeoxycholate (19c) was prepared in a similar way from hyodeoxycholic acid with
MeOH/CH Cl (1:19) elution. The product 19c was obtained as a white solid in 97% (5 g) yield.
2
2
1
H NMR (400 MHz, CDCl3)
δ: 4.07 (m, 1H, H-6β), 3.68 (s, 3H, O-CH ), 3.61 (m, 1H, H-3β), 2.37 and
3
2
.23 (each m, 1H and 1H, H-23), 0.924 (d, 3H, J = 6.3 Hz, H-21), 0.918 (s, 3H, H-19), 0.65 (s, 3H, H-18);
1
3
C NMR (100 MHz, CDCl3)
δ: 174.6 (C=O), 71.3 (CH), 67.8 (CH), 56.1 (CH), 55.9 (CH), 51.4 (CH3),
4
8.4 (CH), 42.7 (C), 39.9 (CH ), 39.8 (CH), 35.8 (C), 35.6 (CH ), 35.3 (CH), 34.7 (CH), 34.6 (CH ), 31.0
2
2
2
(
CH ), 30.8 (CH ), 29.9 (CH ), 29.1 (CH ), 28.0 (CH ), 24.1 (CH ), 23.5 (CH ), 20.7 (CH ), 18.1 (CH ),
2
2
2
2
2
2
3
2
3
1
1.9 (CH3).
Methyl cholate (19d) was obtained in a similar way from cholic acid as a white solid in 91% (4.6 g) yield
1
using AcOEt/hexane (9:1) for elution. H NMR (400 MHz, CDCl )
δ
: 3.99 (m, 1H, H-12
), 0.99 (d, 3H, J = 6.3 Hz, H-21), 0.91 (s, 3H, H-19), 0.70 (s,
: 174.7 (C=O), 72.8 (CH), 71.6 (CH), 68.1 (CH), 51.3 (CH3),
6.7 (CH), 46.2 (C), 41.33 (CH), 41.3 (CH), 39.3 (CH), 39.1 (CH ), 35.2 (CH), 35.1 (CH ), 34.6 (CH ), 34.6
β), 3.87 (m, 1H,
3
H-7β), 3.68 (s, 3H, -O-CH ), 3.47 (m, 1H, H-3β
3
H, H-18); 13C NMR (100 MHz, CDCl3)
δ
3
4
2
2
2
(
(
C), 30.9 (CH ), 30.7 (CH ), 30.2 (CH ), 28.0 (CH ), 27.4 (CH ), 26.0 (CH), 23.0 (CH ), 22.3 (CH ), 17.1
2 2 2 2 2 2 3
CH ), 12.2 (CH ).
3
3
3
.2.6. Reaction of methyl esters 19a–d with ethylenediamine
Methyl lithocholate (19a) (1 g, 2.6 mmol) was dissolved in methanol (5 mL), then ethylenediamine
◦
(
1.55 mL, 23.4 mmol, 9 eq) was added. The reaction mixture was stirred at 65 C for
9
6 h. Methanol and the lion’s share of ethylenediamine were removed under reduced pressure.
0
N-(2 -Aminoethyl)-lithocholamide (20a) was purified by a silica gel column chromatography with
MeOH/NH aq (20:0.07) elution affording product as a white solid in 90% (0.96 g) yield. M.p.
3
◦
1
1
72–174 C (MeOH); H NMR (400 MHz, methanol-d )
δ: 3.54 (m, 1H, H-3
β), 3.23 (t, 2H, J = 6.4 Hz,
4
CO-NH-CH ), 2.71 (t, 2H, J = 6.4 Hz, CH -NH ), 2.26 and 2.11 (each m, 1H and 1H, CH -CO-NH), 0.97
2
2
2
2
13
(
d, 3H, J = 6.5 Hz, H-21), 0.95 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100 MHz, methanol-d4) δ:
1
4
2
77.1 (C=O), 72.4 (CH), 57.9 (CH), 57.4 (CH), 43.9 (C), 43.5 (CH), 43.0 (CH ), 42.0 (CH ), 41.9 (CH),
2 2
1.5 (CH ), 37.2 (CH), 37.18 (CH ), 36.9 (CH), 36.5 (CH ), 35.7 (C), 34.1 (CH ), 33.3 (CH ), 31.2 (CH ),
2
2
2
2
2
2
9.3 (CH ), 28.4 (CH ), 27.7 (CH ), 25.3 (CH ), 24.0 (CH ), 22.0 (CH ), 18.9 (CH ), 12.5 (CH ); IR (ATR,
2
2
2
2
3
2
3
3

Molecules 2020, 25, 655
9 of 15
−
1
+
cm )
ν
: 3340, 3266, 3080, 2925, 2857, 1649, 1552; HRMS calcd for C H N O 419.3638 [M + H] ,
26
47
2
2
found: 419.3621.
0
N-(2 -Aminoethyl)-deoxycholamide (20b) was purified by a silica gel column chromatography with
MeOH/NH aq (40:0.07) elution affording product as a white solid in 90% (0.48 g) yield. M.p. 110–112
3
◦
1
C (MeOH); H NMR (400 MHz, methanol-d )
δ: 3.96 (m, 1H, H-12
β), 3.52 (m, 1H, H-3β), 3.23 (t,
4
2
H, J = 6.4 Hz, CO-NH-CH ), 2.71 (t, 2H, J = 6.4 Hz, CH -NH ), 2.26 and 2.13 (each m, 1H and 1H,
2
2
2
13
CH -CO-NH), 1.02 (d, 3H, J = 6.3 Hz, H-21), 0.93 (s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz,
2
methanol-d4)
δ: 177.2 (C=O), 74.0 (CH), 72.5 (CH), 49.3 (CH), 48.1 (CH), 47.6 (C), 43.6 (CH), 42.9 (CH2),
4
2.0 (CH ), 37.5 (CH), 37.2 (CH ), 36.9 (CH), 36.4 (CH ), 35.3 (C), 34.8 (CH), 34.1 (CH ), 33.3 (CH ), 31.1
2
2
2
2
2
(
(
CH ), 29.9 (CH ), 28.7 (CH ), 28.4 (CH ), 27.5 (CH ), 24.9 (CH ), 23.7 (CH ), 17.7 (CH ), 13.2 (CH ); IR
2 2 2 2 2 2 3 3 3
ATR, cm ) ν: 3293, 2923, 2862, 1633, 1544; HRMS calcd for C H N O 435.3587 [M + H] , found:
26 47 2 3
−1
+
4
35.3579.
0
N-(2 -Aminoethyl)-hyodeoxycholamide (20c) was purified by a silica gel column chromatography with
MeOH/NH aq (20:0.07) elution affording product as a white solid in 87% (0.93 g) yield. M.p. 102–104
3
◦
1
C (MeOH); H NMR (400 MHz, methanol-d )
δ: 4.01 (m, 1H, H-6β), 3.51 (m, 1H, H-3β), 3.23 (t,
4
2
H, J = 6.4 Hz, CO-NH-CH ), 2.71 (t, 2H, J = 6.4 Hz, CH -NH ), 2.26 and 2.11 (each m, 1H and 1H,
2
2
2
13
CH -CO-NH), 0.97 (d, 3H, J = 6.5 Hz, H-21), 0.93 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100 MHz,
2
methanol-d4)
δ
: 177.1 (C=O), 72.4 (CH), 68.6 (CH), 57.6 (CH), 57.4 (CH), 49.9 (CH), 44.0 (C), 43.0 (CH2),
4
3
2.0 (CH ), 41.4 (CH ), 41.3 (CH), 36.9 (CH), 36.86 (CH ), 36.8 (C), 36.2 (CH), 35.6 (CH ), 34.1 (CH ),
2
2
2
2
2
3.2 (CH ), 31.1 (CH ), 30.0 (CH ), 29.2 (CH ), 25.3 (CH ), 24.1 (CH ), 21.9 (CH ), 18.9 (CH ), 12.5
2
2
1
2
2
2
3
2
3
−
(
CH ); IR (ATR, cm )
ν
: 3281, 3085, 2928, 2860, 1643, 1546; HRMS calcd for C H N O 435.3587 [M
3
+
26 47
2
3
+
H] , found: 435.3577.
0
N-(2 -Aminoethyl)-cholamide (20d) was purified by a silica gel column chromatography with
MeOH/NH aq (100:0.9) elution affording product as a white solid in 92% (0.98 g) yield. M.p.
3
◦
1
1
3
3
35–137 C (MeOH/H O); H NMR (400 MHz, methanol-d )
δ
: 3.95 (m, 1H, H-12
β), 3.80 (m, 1H, H-7β),
2
4
.37 (m, 1H, H-3
H, J = 6.3 Hz, H-21), 0.92 (s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
β), 3.24 (t, 2H, J = 6.3 Hz, CO-NH-CH ), 2.73 (t, 2H. J = 6.3 Hz, CH -NH ), 1.03 (d,
2
2
2
1
3
δ: 177.3
(
C=O), 74.0 (CH), 72.9 (CH), 69.0 (CH), 48.0 (CH), 47.5 (C), 43.2 (CH), 43.0 (CH), 42.8 (CH ), 42.0 (CH ),
2 2
4
2
3
1.0 (CH), 40.5 (CH ), 36.9 (CH), 36.5 (CH ), 35.9 (CH ), 35.9 (C), 34.1 (CH ), 33.3 (CH ), 31.2 (CH ),
2 2 2 2 2 2
−1
9.6 (CH ), 28.7 (CH ), 27.9 (CH), 24.2 (CH ), 23.2 (CH ), 17.7 (CH ), 13.0 (CH ); IR (ATR, cm ) ν:
2
2
2
3
3
3
+
348, 3289, 2924, 2864, 1642, 1547; HRMS calcd for C H N O 451.3536 [M + H] , found: 451.3567.
26
47
2
4
3
.2.7. Reaction of methyl esters 19a–d with propane-1,3-diamine
Methyl lithocholate (19a) (0.8 g, 2 mmol) was dissolved in methanol (4 mL), then
propane-1,3-diamine (1.2 mL, 14 mmol, 7 eq) was added. The reaction mixture was stirred at
◦
6
5 C for 72 h. The solvent and the lion’s share of propane-1,3-diamine were removed under reduced
pressure. The crude product was purified by a silica gel column chromatography. Elution with
0
MeOH/NH aq (20:0.07) afforded N-(3 -aminopropyl)-lithocholamide (21a) as a white solid in 82% (0.73
3
◦
1
g) yield. M.p. 73–75 C (MeOH); H NMR (400 MHz, methanol-d )
δ
: 3.54 (m, 1H, H-3β), 3.23 (t,
4
2
H, J = 6.8 Hz, CO-NH-CH ), 2.65 (t, 2H, J = 7.0 Hz, CH -NH ), 2.23 and 2.10 (each m, 1H and 1H,
2
2
2
13
CH -CO-NH), 0.96 (d, 3H, J = 6.6 Hz, H-21), 0.95 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100
2
MHz, methanol-d4)
δ: 176.9 (C=O), 72.4 (CH), 57.9 (CH), 57.4 (CH), 43.9 (C), 43.5 (CH), 41.9 (CH), 41.5
(
CH ), 39.7 (CH ), 37.6 (CH ), 37.2 (CH), 37.2 (CH ), 36.9 (CH), 36.5 (CH ), 35.7 (C), 34.1 (CH ), 33.4
2
2
2
2
2
2
(
1
4
CH ), 33.37 (CH ), 31.2 (CH ), 29.3 (CH ), 28.4 (CH ), 27.7 (CH ), 25.3 (CH ), 23.9 (CH ), 21.9 (CH ),
2 2 2 2 2 2 2 3 2
−
8.9 (CH ), 12.5 (CH ); IR (ATR, cm ) ν: 3351, 2927, 2857, 1638, 1550; HRMS calcd for C H N O
3 3 27 49 2 2
1
+
33.3794 [M + H] , found: 433.3785.
0
N-(3 -Aminopropyl)-deoxycholamide (21b) was purified by a silica gel column chromatography with
MeOH/NH aq (20:0.07) elution affording product as a white solid in 85% (0.47 g) yield. M.p. 130–132
3

Molecules 2020, 25, 655
10 of 15
◦
1
C (MeOH); H NMR (400 MHz, methanol-d )
δ: 3.96 (m, 1H, H-12
β), 3.52 (m, 1H, H-3β), 3.23 (t,
4
2
H, J = 6.8 Hz, CO-NH-CH ), 2.65 (t, 2H, J = 7.0 Hz, CH -NH ), 2.25 and 2.10 (each m, 1H and 1H,
2 2 2
13
CH -CO-NH), 1.02 (d, 3H, J = 6.4 Hz, H-21), 0.93 (s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz,
2
methanol-d4)
δ: 177.0 (C=O), 74.0 (CH), 72.5 (CH), 49.3 (CH), 48.1 (CH), 47.6 (C), 43.6 (CH), 39.7 (CH2),
3
7.6 (CH ), 37.5 (CH), 37.2 (CH ), 36.9 (CH), 36.4 (CH ), 35.3 (C), 34.8 (CH), 34.1 (CH ), 33.4 (CH ), 33.3
2
2
2
2
2
(
1
CH ), 31.1 (CH ), 29.9 (CH ), 28.7 (CH ), 28.4 (CH ), 27.5 (CH ), 24.9 (CH ), 23.7 (CH ), 17.7 (CH ),
2 2 2 2 2 2 2 3 3
−
1
3.2 (CH ); IR (ATR, cm
)
ν
: 3295, 2921, 2860, 1634, 1546; HRMS calcd for C H N O 449.3743 [M +
3
27 49
2
3
+
H] , found: 449.3804.
0
N-(3 -Aminopropyl)-hyodeoxycholamide (21c) was purified by a silica gel column chromatography. Elution
◦
with MeOH/NH aq (20:0.07) afforded product as a white solid in 89% (0.98 g) yield. M.p. 89–91 C
MeOH); H NMR (400 MHz, methanol-d )
3
1
(
δ
: 4.01 (m, 1H, H-6β), 3.50 (m, 1H, H-3β), 3.23 (t, 2H, J = 6.8
4
Hz, CO-NH-CH ), 2.65 (t, 2H, J = 7.0 Hz, CH -NH ), 2.23 and 2.10 (each m, 1H and 1H, CH -CO-NH),
2
2
2
2
13
0
.97 (d, 3H, J = 6.5 Hz, H-21), 0.93 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
δ:
1
76.9 (C=O), 72.4 (CH), 68.6 (CH), 57.6 (CH), 57.4 (CH), 49.9 (CH), 44.0 (C), 41.4 (CH ), 41.3 (CH), 39.7
2
(
(
(
CH ), 37.6 (CH ), 36.9 (C), 36.85 (CH), 36.8 (CH ), 36.2 (CH), 35.6 (CH ), 34.1 (CH ), 33.4 (CH ), 33.3
2 2 2 2 2 2
CH ), 31.1 (CH ), 30.0 (CH ), 29.2 (CH ), 25.3 (CH ), 24.1 (CH ), 21.9 (CH ), 18.8 (CH ), 12.5 (CH ); IR
2
2
2
2
2
3
2
3
3
−1
+
ATR, cm ) ν: 3282, 2930, 2864, 1642, 1543; HRMS calcd for C H N O 449.3743 [M + H] , found:
27 49 2 3
4
49.3758.
0
N-(3 -Aminopropyl)-cholamide (21d) was purified by a silica gel column chromatography with
MeOH/NH aq (20:0.07) elution affording product as a white solid in 89% (0.49 g) yield. M.p.
3
◦
1
9
8–101 C (AcOEt/MeOH); H NMR (400 MHz, methanol-d )
δ: 3.95 (m, 1H, H-12
β), 3.80 (m, 1H,
4
H-7
β
), 3.37 (m, 1H, H-3
β), 3.23 (t, 2H, J = 6.8 Hz, CO-NH-CH ), 2.68 (t, 2H, J = 7.0 Hz, CH -NH ), 1.03
2
2
2
13
(d, 3H, J = 6.3 Hz, H-21), 0.92 (s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz, methanol-d4) δ:
1
77.1 (C=O), 74.0 (CH), 72.9 (CH), 69.0 (CH), 48.0 (CH), 47.5 (C), 43.2 (CH), 43.0 (CH), 41.0 (CH), 40.5
(
(
(
CH ), 39.5 (CH ), 37.5 (CH ), 36.9 (CH), 36.5 (CH ), 35.9 (CH ), 35.9 (C), 34.1 (CH ), 33.4 (CH ), 32.9
2 2 2 2 2 2 2
CH ), 31.2 (CH ), 29.6 (CH ), 28.7 (CH ), 27.9 (CH), 24.2 (CH ), 23.2 (CH ), 17.7 (CH ), 13.0 (CH ); IR
2
2
2
2
2
3
3
3
−
1
ATR, cm ) ν: 3344, 3208, 3093, 2925, 2862, 1625, 1561; HRMS calcd for C H N O 465.3692 [M +
27 49 2 4
+
H] , found: 465.3672.
3
.2.8. Reduction of amides 20a–d and 21a–d
◦
To a cooled (4 C) suspension of N-(2-aminoethyl)-lithocholamide (20a) (0.5 g, 1.2 mmol) in
anhydrous THF (30 mL) lithium aluminum hydride (228 mg, 6 mmol, 5 eq) was added. The reaction
mixture was stirred at reflux for 72 h. Then it was cooled to room temperature and water (0.1 mL)
was added dropwise, followed by 20% aqueous sodium hydroxide (0.2 mL). The resultant slurry
was stirred for 15 min, and then anhydrous sodium sulfate was added. Stirring was continued for
an additional 15 min. Solid materials were removed by filtration, washed with THF and the filtrate
concentrated under reduced pressure.
This general work-up procedure cannot be applied for hyodeoxycholic acid amide derivatives
(20c or 21c) due to an easy degradation of products under described conditions. In these cases a
routine extractive work-up (decomposition of hydride with a large excess of water and extraction with
dichloromethane) was used.
0
2
4-(2 -Amino-ethylamino)-cholan-3
α
-ol (17a) was purified by a silica gel column chromatography with
MeOH/CH Cl /NH aq (50:50:3) elution affording product as a white solid in 68% (0.33 g) yield.
2
2
3
0
2
4-(3 -Amino-propylamino)-cholan-3
α-ol (18a) was purified by a silica gel column chromatography with
MeOH/CH Cl /NH aq (4:1:01) elution affording product as a white solid in 62% (0.15 g) yield.
2
2
3
0
2
4-(2 -Amino-ethylamino)-cholane-3
α,12α-diol (17b) was purified by a silica gel column chromatography
with MeOH/NH aq (20:0.07) elution affording product as a white solid in 67% (65 mg) yield. M.p.
3
◦
0–72 C (MeOH); H NMR (400 MHz, methanol-d ) δ: 3.96 (m, 1H, H-12β), 3.52 (m, 1H, H-3β), 2.80 (t,
4
1
7

Molecules 2020, 25, 655
11 of 15
2
H, J = 5.9 Hz, N-CH ), 2.71 (t, 2H, J = 5.9 Hz, N-CH ), 2.59 (m, 2H, H-24), 1.03 (d, 3H, J = 6.5 Hz,
2
2
13
H-21), 0.93 (s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
δ: 74.1 (CH), 72.6 (CH),
5
1.9 (CH ), 51.1 (CH ), 49.8 (CH), 48.3 (CH), 47.5 (C), 43.6 (CH), 41.2 (CH ), 37.5 (CH), 37.2 (CH ), 37.1
2 2 2 2
(
CH), 36.4 (CH ), 35.3 (C), 34.8 (CH), 34.7 (CH ), 31.1 (CH ), 29.9 (CH ), 28.8 (CH ), 28.4 (CH ), 27.5
2
2
2
2
2
2
−
1
(
CH ), 26.9 (CH ), 24.9 (CH ), 23.7 (CH ), 18.0 (CH ), 13.2 (CH ); IR (ATR, cm )
ν
: 3287, 2924, 2858,
2
2
2
3
3
3
+
1
452, 1369; HRMS calcd for C H N O 421.3794 [M + H] , found: 421.3794.
26 49 2 2
0
4-(3 -Amino-propylamino)-cholane-3α,12α-diol (18b) was purified by a silica column chromatography
2
with MeOH/NH aq (100:1) elution affording product as a white solid in 65% (63 mg) yield. M.p. 64–66
3
◦
1
C (MeOH); H NMR (400 MHz, methanol-d )
δ: 3.96 (m, 1H, H-12
β), 3.52 (m, 1H, H-3β), 2.69 (t, 2H, J
4
=
7.0 Hz, N-CH ), 2.64 (t, 2H, J = 7.4 Hz, N-CH ), 2.56 (m, 2H, H-24), 1.02 (d, 3H, J = 6.4 Hz, H-21), 0.93
2
2
13
(s, 3H, H-19), 0.71 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
δ: 74.1 (CH), 72.6 (CH), 51.3 (CH2),
4
9.9 (CH), 48.3 (CH), 48.26 (CH ), 47.5 (C), 43.6 (CH), 40.6 (CH ), 37.5 (CH), 37.2 (CH ), 37.1 (CH), 36.4
2 2 2
(
(
1
CH ), 35.3 (C), 34.8 (CH), 34.7 (CH ), 32.9 (CH ), 31.1 (CH ), 29.9 (CH ), 28.8 (CH ), 28.4 (CH ), 27.5
2
2
2
2
2
2
2
−
1
CH ), 27.0 (CH ), 24.9 (CH ), 23.7 (CH ), 18.0 (CH ), 13.2 (CH ); IR (ATR, cm )
ν
: 3278, 2925, 2858,
2
2
2
3
3
3
+
454, 1369; HRMS calcd for C H N O 435.3951 [M + H] , found: 435.3946.
27 51 2 2
0
4-(2 -Amino-ethylamino)-cholane-3α,6α-diol (17c) was purified by a silica gel column chromatography
2
with MeOH/NH aq (25:0.2) elution affording product as a white solid in 69% (167 mg) yield. M.p.
3
◦
1
7
2–74 C (MeOH); H NMR (400 MHz, methanol-d )
δ
: 4.01 (m, 1H, H-6
β), 3.51 (m, 1H, H-3β), 2.75 (t,
4
2
H, J = 5.9 Hz, N-CH ), 2.65 (t, 2H, J = 5.9 Hz, N-CH ), 2.55 (m, 2H, H-24), 0.97 (d, 3H, J = 6.5 Hz,
2
2
13
H-21), 0.93 (s, 3H, H-19), 0.70 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
δ: 72.4 (CH), 68.6 (CH),
5
7.7 (CH), 57.6 (CH), 52.7 (CH ), 51.3 (CH ), 49.9 (CH), 44.0 (C), 41.7 (CH ), 41.4 (CH ), 41.3 (CH), 37.1
2
2
2
2
(
CH), 36.9 (C), 36.8 (CH ), 36.2 (CH), 35.6 (CH ), 34.7 (CH ), 31.1 (CH ), 30.0 (CH ), 29.3 (CH ), 27.1
2
2
2
2
2
2
−
1
(
CH ), 25.3 (CH ), 24.1 (CH ), 21.9 (CH ), 19.2 (CH ), 12.5 (CH ); IR (ATR, cm )
ν
: 3285, 2927, 2857,
2
2
3
2
3
3
+
1
453, 1368; HRMS calcd for C H N O 421.3794 [M + H] , found: 421.4040.
26
49
2
2
0
2
4-(3 -Amino-propylamino)-cholane-3
α
,6
α-diol 18c was purified by a silica gel column chromatography
with MeOH/NH aq (100:1) elution affording product as a white solid in 73% (106 mg) yield. M.p.
3
◦
1
6
4–66 C (MeOH); H NMR (400 MHz, methanol-d )
δ
: 4.01 (m, 1H, H-6
β), 3.51 (m, 1H, H-3β), 2.72 (t,
4
2
H, J = 7.1 Hz, N-CH ), 2.66 (t, 2H, J = 7.3 Hz, N-CH ), 2.57 (m, 2H, H-24), 0.97 (d, 3H, J = 6.4 Hz,
2
2
13
H-21), 0.93 (s, 3H, H-19), 0.69 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
δ: 72.4 (CH), 68.6 (CH),
5
7.7 (CH), 57.5 (CH), 51.2 (CH ), 49.9 (CH), 48.2 (CH ), 44.0 (C), 41.4 (CH ), 41.3 (CH), 40.5 (CH ), 37.1
2
2
2
2
(
(
2
CH), 36.9 (C), 36.8 (CH ), 36.2 (CH), 35.6 (CH ), 34.7 (CH ), 32.4 (CH ), 31.1 (CH ), 30.0 (CH ), 29.3
2 2 2 2 2 2
−
CH ), 26.8 (CH ), 25.3 (CH ), 24.1 (CH ), 21.9 (CH ), 19.2 (CH ), 12.4 (CH ); IR (ATR, cm ) ν: 3275,
2 2 2 3 2 3 3
1
+
926, 2858, 1454, 1369; HRMS calcd for C H N O 435.3951 [M + H] , found: 435.3927.
27
51
2
2
0
2
4-(2 -Amino-ethylamino)-cholane-3
α
,7α
,12α
-triol 17d was purified by a silica gel column chromatography
with MeOH/ CH Cl /NH aq (5:5:0.7) elution affording product as a white solid in 67% (0.32 g) yield.
2
2
3
◦
1
M.p. 107–111 C (MeOH); H NMR (400 MHz, methanol-d )
δ: 3.96 (m, 1H, H-12
β), 3.80 (m, 1H, H-7β),
4
3
1
7
.37 (m, 1H, H-3
.03 (d, 3H, J = 6.5 Hz, H-21), 0.92 (s, 3H, H-19), 0.72 (s, 3H, H-18); C NMR (100 MHz, methanol-d4)
4.1 (CH), 72.9 (CH), 69.1 (CH), 52.4 (CH ), 51.2 (CH ), 48.3 (CH), 47.5 (C), 43.2 (CH), 43.0 (CH), 41.5
β), 2.77 (t, 2H, J = 5.9 Hz, N-CH ), 2.68 (t, 2H, J = 6.1 Hz, N-CH ), 2.57 (m, 2H, H-24),
2
2
13
δ:
2
2
(
(
CH ), 41.0 (CH), 40.5 (CH ), 37.2 (CH), 36.5 (CH ), 35.9 (C), 35.88 (CH ), 34.7 (CH ), 31.2 (CH ), 29.6
2 2 2 2 2 2
−1
CH ), 28.8 (CH ), 27.9 (CH), 27.1 (CH ), 24.2 (CH ), 23.2 (CH ), 18.0 (CH ), 13.0 (CH ); IR (ATR, cm
)
2
2
2
2
3
3
3
+
ν: 3328, 2925, 2854, 1467, 1369; HRMS calcd for C H N O 437.3743 [M + H] , found: 437.3765.
26
49
2
3
0
2
4-(3 -Amino-propylamino)-cholane-3
α,7α,12α-triol 18d was purified by a silica gel column
chromatography with MeOH/CH Cl /NH aq (5:5:1) elution affording product as a white solid in 65%
2
2
3
◦
1
(
(
(
0.63 g) yield. M.p. 105–108 C (MeOH); H NMR (400 MHz, methanol-d )
δ
: 3.96 (m, 1H, H-12
β), 3.80
4
m, 1H, H-7
m, 2H, H-24), 1.04 (d, 3H, J = 6.5 Hz, H-21), 0.92 (s, 3H, H-19), 0.72 (s, 3H, H-18); C NMR (100 MHz,
: 74.0 (CH), 72.9 (CH), 69.0 (CH), 50.9 (CH ), 48.2 (CH), 48.0 (CH ), 47.4 (C), 43.2 (CH),
β), 3.37 (m, 1H, H-3β), 2.79 (t, 2H, J = 7.1 Hz, N-CH ), 2.75 (t, 2H, J = 7.3 Hz, N-CH ), 2.65
2
2
13
methanol-d4)
δ
2
2

Molecules 2020, 25, 655
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4
3.0 (CH), 41.0 (CH), 40.5 (CH ), 40.3 (CH ), 37.1 (CH), 36.5 (CH ), 35.9 (CH ), 35.9 (C), 34.6 (CH ), 31.2
2 2 2 2 2
(
1
CH ), 31.0 (CH ), 29.6 (CH ), 28.8 (CH ), 27.9 (CH), 26.4 (CH ), 24.2 (CH ), 23.2 (CH ), 18.0 (CH ),
2 2 2 2 2 2 3 3
−
1
3.0 (CH ); IR (ATR, cm
)
ν
: 3352, 2925, 2860, 1454, 1371; HRMS calcd for C H N O 451.3900 [M +
3
27 51
2
3
+
H] , found: 451.3932.
3
.2.9. General procedure for preparation of complexes from K PtCl and bile acid-derived diamines
2 4
1
7a–d and 18a–d
The solution of potassium tetrachloroplatinate (51 mg, 0.12 mmol) in DMF (1.5 mL) and
distilled water (1.5 mL) were added to a solution of 24-(2-amino-ethylamino)-cholan-3 -ol
17a) (50 mg, 0.12 mmol) in DMF (1.5 mL). The resulting mixture was stirred in the dark
α
(
for 3 days. Then, the solvent was evaporated and the residue was stirred vigorously in a
saturated aqueous potassium chloride solution (4 mL) for 20 min. The resulting suspension
was filtered, washed with water, and dried in a desiccator over phosphorus pentoxide for 1 day.
0
0
cis-Dichloro-24-[(N-2 -aminoeth-1 -yl-
(
κ
N)aminocholan-3
α
-ol-
κ
N]platinum(II) (22a) was obtained in 76%
−
1
195
63 mg) yield. IR (ATR, cm ) ν: 3445, 3197, 3122, 2926, 2857, 1585, 1451, 1371, 1028; Pt NMR (86
MHz, DMF) δ: -2336.6; HRMS calcd for C H Cl N O Pt [M + Na] : 693.2703 found: 693.2654.
1
96
+
26
48
2
2
0
0
cis-Dichloro-24-[(N-3 -aminoprop-1 -yl-
κN)aminocholan-3α-ol-κN]platinum(II) (23a) was prepared in a
−
1
similar way. The product 23a was obtained in 80% (65 mg) yield. IR (ATR, cm )
091, 2926, 2857, 1605, 1450, 1372, 1030;
C H Cl N O Pt [M + Na] : 707.2860 found: 707.2840.
ν
: 3473, 3223,
195
3
Pt NMR (86 MHz, DMF)
δ:
−
2264.3; HRMS calcd for
196
+
27
50
2
2
0
0
cis-Dichloro-24-[(N-2 -aminoeth-1 -yl-
κN)aminocholane-3α,12α-diol-κN]platinum(II) (22b) was prepared
−1
in a similar way. The product 22b was obtained in 67 % (54 mg) yield. IR (ATR, cm )
124, 2926, 2861, 1651, 1452, 1374, 1032;
ν
: 3425, 3196,
195
3
Pt NMR (86 MHz, DMF)
δ:
−
2335.2; HRMS calcd for
196
+
C H Cl N O
2
Pt [M + Na] : 709.2652 found: 709.2644.
26
48
2
2
0
0
cis-Dichloro-24-[(N-3 -aminoprop-1 -yl-
κN)aminocholane-3α,12α-diol-κN]platinum(II) (23b) was prepared
−
1
in a similar way. The product 23b was obtained in 78% (62 mg) yield. IR (ATR, cm )
147, 2924, 2860, 1595, 1446, 1375, 1039;
ν
: 3420, 3196,
195
3
Pt NMR (86 MHz, DMF)
δ:
−
2265.6; HRMS calcd for
196
+
C H Cl N O
2
Pt [M + Na] : 723.2809 found: 723.2785.
27
50
2
2
0
0
cis-Dichloro-24-[(N-2 -aminoeth-1 -yl-
κN)aminocholane-3α,6α-diol-κN]platinum(II) (22c) was prepared in
−
1
a similar way. The product 22c was obtained in 74% (60 mg) yield. IR (ATR, cm )
133, 2929, 2860, 1627, 1453, 1373, 1031;
ν
: 3393, 3198,
195
3
Pt NMR (86 MHz, DMF)
δ:
−
2336.1; HRMS calcd for
196
+
C H Cl N O
2
Pt [M + Na] : 709.2652 found: 709.2644.
26
48
2
2
0
0
cis-Dichloro-24-[(N-3 -aminoprop-1 -yl-
κN)aminocholane-3α,6α-diol-κN]platinum(II) (23c) was prepared
−1
in a similar way. The product 23c was obtained in 69% (55 mg) yield. IR (ATR, cm )
140, 2929, 2860, 1594, 1452, 1373, 1034;
ν
: 3423, 3205,
195
3
Pt NMR (86 MHz, DMF)
δ:
−
2264.6; HRMS calcd for
196
+
C H Cl N O
2
Pt [M + Na] : 723.2809 found: 723.2785.
27
50
2
2
0
0
cis-Dichloro-24-[(N-2 -aminoeth-1 -yl-
κ
N)aminocholane-3
α
,7α
,12α
-triol-
κ
N]platinum(II)
(
22d
)
ν
was
: 3424,
−
1
prepared in a similar way. The product 22d was obtained in 81% (65 mg) yield. IR (ATR, cm
203, 3126, 2922, 2861, 1586, 1454, 1372, 1030; Pt NMR (86 MHz, DMF)
)
195
3
δ:
−
2334.9; HRMS calcd for
196
+
C H Cl N O
3
Pt [M + Na] : 725.2601 found: 725.2579.
26
48
2
2
0
0
cis-Dichloro-24-[(N-3 -aminoprop-1 -yl-
κ
N)aminocholane-3
α
,7α
,12α
-triol-
κ
N]platinum(II)
(
23d
)
was
: 3412,
−
1
prepared in a similar way. The product 23d was obtained in 61% (48 mg) yield. IR (ATR, cm
211, 3138, 2926, 2863, 1604, 1453, 1374, 1035; Pt NMR (86 MHz, DMF)
)
ν
195
3
δ:
−
2257.8; HRMS calcd for
196
+
C H Cl N O
3
Pt [M + Na] : 739.2758 found: 739.2723.
27
50
2
2

Molecules 2020, 25, 655
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3
.3. Cytotoxicity Assay
Cisplatin derivatives were evaluated for their cytotoxic activities in vitro in following cell lines
derived from various tumors: T-lymphoblastic leukemia (CEM), epithelial breast carcinoma (MCF7)
and cervical carcinoma cell lines (HeLa) (all from ECACC, UK) and also in normal human fibroblasts
(
BJ; from ATCC, USA). Human umbilical vein endothelial cells (HUVEC) were a kind gift from Prof
Jitka Ulrichov (Medical Faculty, Palack University, Olomouc, Czech Republic). HUVEC were grown
á
ý
in ECPM medium (Provitro, Berlin, Germany). Six serial 3-fold dilutions in triplicate of each drug in
DMF was administered in every cell line for 72 h. Alamar Blue dye was used for staining of living cells
only. IC50 values (50% inhibitory concentrations) were calculated as the proportions of surviving cells.
A detailed procedure for the cytotoxicity assay was described previously, except for using Alamar Blue
dye instead of Calcein AM [37]. Briefly, the percentage of surviving cells in each well was calculated
by dividing the intensity 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, i.e., concentrations of the respective compounds that were lethal to
50% of the tumor cells, were calculated. The results obtained from cytotoxicity assays are shown in
Table 2 (mean values ± SD obtained from three independent experiments performed in triplicate).
4
. Conclusions
In summary, we have developed two new synthetic routes to the derivatives of bile acids possessing
,2- and 1,3-diamino moieties in the side chain. In the first synthesis the key transformation was
1
bromoacetonitrile alkylation of the primary amine or its addition to acrylonitrile. The crucial step of
the second strategy was monoamidation of a methyl ester with diamine. These methodologies provide
simple, convenient and efficient routes to the synthesis of various steroidal diamines. Comparison of
these strategies, taking as an example transformation of lithocholic acid (
has proved that the second, significantly shorter synthetic route affords the final products in higher
17a) or similar (18a) yield. The described herein protocol for the preparation of diamines can be also
7) into diamines 17a and 18a,
(
applied in a large scale synthesis. The platinum complexes obtained from the diamines showed a
moderate cytotoxicity but their selectivity against human cancer cells was unsatisfactory.
Author Contributions: Performed the experiments and wrote original draft: B.S., Z.Ł. and A.H. Evaluated
biological activity: L.R. Reviewed and edited: R.R.S. Collected and analyzed MS data: A.M.T. Conceptualization:
J.W.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Science Centre, Poland, Grant OPUS 8, 2014/15/B/ST5/02129.
Biological testing was supported by the European Regional Development Fund—Project ENOCH (No.
CZ.02.1.01/0.0/0.0/16_019/0000868).
Acknowledgments: The authors would like to thank Eva Hrdli cˇ ková for excellent technical assistance and Dr.
Leszek Siergiejczyk for NMR measurements.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds: 17a
–d (1,2-diamines), 18a–d (1,3-diamines), 22a–d and 23a–d
[
platinum(II) complexes] are available from the authors.
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