Novel semisynthetic derivatives of bile acids as effective tyrosyl-DNA phosphodiesterase 1 inhibitors

Article abstract of DOI:10.3390/molecules23030679

An Important task in the treatment of oncological and neurodegenerative diseases is the search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the DNA repair system enzymes involved in the removal of DNA damages caused by topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of currently used anticancer drugs. We describe here a new class of semisynthetic small molecule Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening. The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC₅₀ up to 0.29 μM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of the Tdp1 enzyme.

Full text of DOI:10.3390/molecules23030679

molecules
Article
Novel Semisynthetic Derivatives of Bile Acids as
Effective Tyrosyl-DNA Phosphodiesterase
1 Inhibitors
Oksana V. Salomatina 1, Irina I. Popadyuk 1, Alexandra L. Zakharenko 2, Olga D. Zakharova 2,
Dmitriy S. Fadeev 1, Nina I. Komarova 1, Jóhannes Reynisson 3 ID , H. John Arabshahi 3,
Raina Chand 3, Konstantin P. Volcho 1,4, ID , Nariman F. Salakhutdinov 1,4 and Olga I. Lavrik 2,4
*
1
N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, SB RAS, acad. Lavrentjev ave. 9,
Novosibirsk 630090, Russia; ana@nioch.nsc.ru (O.V.S.); popadyuk@nioch.nsc.ru (I.I.P.);
dsf@nioch.nsc.ru (D.S.F.); komar@nioch.nsc.ru (N.I.K); anvar@nioch.nsc.ru (N.F.S.)
Novosibirsk Institute of Chemical Biology and Fundamental Medicine, SB RAS, acad. Lavrentjev ave. 8,
Novosibirsk 630090, Russia; sashaz@niboch.nsc.ru (A.L.Z); isar@niboch.nsc.ru (O.D.Z.);
lavrik@niboch.nsc.ru (O.I.L.)
School of Chemical Sciences, University of Auckland, Auckland 1142, New Zealand;
j.reynisson@auckland.ac.nz (J.R.); j.arabshahi@auckland.ac.nz (H.J.A.); rcha387@aucklanduni.ac.nz (R.C.)
Novosibirsk State University, Pirogova str. 2, Novosibirsk 630090, Russia
2
3
4
*
Correspondence: volcho@nioch.nsc.ru; Tel.: +7-383-3308870
Academic Editor: Derek J. McPhee
Received: 2 March 2018; Accepted: 16 March 2018; Published: 17 March 2018
Abstract: An Important task in the treatment of oncological and neurodegenerative diseases is the
search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1)
is one of the DNA repair system enzymes involved in the removal of DNA damages caused by
topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of
currently used anticancer drugs. We describe here a new class of semisynthetic small molecule
Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening.
The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework
and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies
demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC50 up to
0.29 µM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of
the Tdp1 enzyme.
Keywords: deoxycholic acid; chenodeoxycholic acid; ursodeoxycholic acid; amide; Tdp1 inhibitor;
cancer; tumor; virtual screening; molecular modelling
1. Introduction
Systems that are responsible for repairing DNA damage play a crucial role in maintaining genome
integrity. However, the increased activity of such systems produces significant problems in the
treatment of a variety of immune, neurodegenerative diseases and, especially, cancerous tumors [1–3].
The cytotoxic effects of chemo- and radiotherapy are often based on DNA damage and these methods
are used in clinical practices that treat malignant tumors. The ability of tumor cells to recognize and
repair DNA damage can lead to a resistance to certain anti-cancer medications. Thus, the inhibition
of DNA repair enzymes can increase the effectiveness of several types of antitumor drugs that are in
clinical use [4]. Therefore, the search for inhibitors of the DNA repair system enzymes is one of the
most crucial research areas in medical chemistry today [5–7].
Molecules 2018, 23, 679; doi:10.3390/molecules23030679
www.mdpi.com/journal/molecules

Molecules 2018, 23, 679
2 of 19
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) plays an important role in removing DNA damage
caused by topoisomerase 10 (Top1) inhibitors [
2,10,
as 30-phosphoglycolates and others [ 11]. In addition, Tdp1 is capable of cleaving 50-adducts of
3,8,9]. Tdp1 catalyzes the hydrolysis of adducts
covalently bound to DNA’s 3 -phosphate, including Top1 peptides, Top1–inhibitor complexes, as well
DNA [12]. As a result, Tdp1 counteracts not only drugs aimed at inhibiting Top1 (camptothecins,
indenoisoquinolines), but also topoisomerase II (Top2) inhibitors (etoposide, doxorubicin), bleomycin
and DNA alkylating agents. It is suggested that namely Tdp1 is responsible for the drug resistance of
some cancers [
Tdp1 knock-out models are hypersensitive to camptothecin [13
levels of Tdp1 expression, camptothecin and etoposide cause less DNA damage [17
2
,
6
]. A number of studies supports this hypothesis. For example, it is known that mice
16]. Conversely, in cells with elevated
18]. It has also been
–
,
shown that the suppression of Tdp1 activity makes tumor cells hypersensitive to anticancer drugs with
other mechanisms of action: temozolomide (purine methylation) [19], methyl methanesulfonate
(formation of apurine/apyrimidine sites), bleomycin (single-stranded/double-stranded breaks
with 30-phosphoglycolates), hydrogen peroxide and ionizing radiation (breaks and other types of
damage) [20]. Thus, Tdp1 is considered a promising target enzyme in the development of drugs for
the treatment of oncological diseases [2,21].
Several studies reported the development of specific Tdp1 inhibitors of different classes, such as
diamidines, phosphotyrosine mimetics, indenoisoquinolines and benzopentathiepines as well as
semisynthetic compounds based on natural products (Figure 1) [
available furamidine ( ) (Figure 1), a bisbenzamidine derivative belonging to the diamidines family,
effectively inhibits Tdp1 at low micromolar concentrations both with single- and double-stranded
substrates of DNA [23]. Compound NSC 88915 ) (Figure 1), a conjugate of progesterone and
4-bromobenzenesulfonic acid with relatively high inhibitory characteristics for Tdp1 (IC50 value is
7.7 M), is a representative of phosphotyrosine mimetics [24]. The benzopentathiepines have IC50
values in the range of 0.2–6.0 M. Compound ( ) containing the dibuthylamino group is the most active
(IC50 0.22 M) [25]. Indenoisoquinoline derivatives were discovered as dual Top1-Tdp1 inhibitors
) [11 26] and triple Top1–Tdp1–Tdp2 inhibitors ( ) [27]. The structures of Tdp1 inhibitors based
on natural products are represented by coumarin derivatives ( ) [28], usnic acid derivatives ( ) [29],
and aminoadamantanes containing monoterpene-derived fragments (I) and (J) [30].
3,5,10,11,22–30]. Commercially
A
(
B
µ
µ
C
µ
(D
,
E
,
F
G
H
O
O
O
O
*2TFA
O
H2
N
NH2
NH
H
H
O
A
N
N
N
N
NH
D
Furamidine
G
O
O
Br
O
S
O
R
O
R:
Br
OH
O
O
O
OH
NH
HO
O
N
NH2*HBr
O
O2N
n
O
E
O
H
OH
B
O
NSC 88915
OCH3
H
O
N
HN
S
S
F3
C
N
S
H
S
S
N
N
N
O2N
NH
J
(n-Bu)2N
I
S
O
F
C
O
Figure 1. Structures of reported Tdp1 inhibitors.
This work was aimed at creating and developing a new class of potent inhibitors against Tdp1 and
identifying structure-activity relationships based on the series of bile acids’ derivatives. The proposed
inhibitors of Tdp1 belong to the new chemical class, expanding the arsenal of structurally diverse
inhibitors, thereby increasing the likelihood of successful progress with at least a part of them.

Molecules 2018, 23, 679
3 of 19
2. Results and Discussion
2.1. Virtual Screening
A virtual screen was conducted to identify novel ligands against Tdp1 protein. The crystal
structure (1MU7, 2.0 Å) [31 32] was used for docking against known Tdp1 inhibitors, shown in
,
Figure 1, to observe predicted hydrogen bonding interactions and poses as there is no crystal structure
with a co-crystallized ligand available.
From the natural product library of «InterBioScreen», 9
×
103 compounds were selected for
screening. The GoldScore (GS), ChemScore (CS), Chem Piecewise Linear Potential (ChemPLP),
and Astex Statistical Potential (ASP) scoring functions were used to assess the binding of the ligands.
The scores predicted by these functions reflect the binding energy between the ligands and the protein.
In general, the higher the number produced by the scoring functions, the greater chance enhanced
efficacy is predicted for the ligand. The virtual screen was done in two phases using a relatively low
screening efficiency (30%) intended to weed out compounds that were unlikely to fit the binding
pocket of Tdp1, followed by a more robust (100%) search for the remaining ligands (see Methodology
for further detail). First, all ligands were screened and those with no or weak predicted hydrogen
bonding (<1) were eliminated as well as those with low predicted binding energies (GS < 46, CS < 23
,
ChemPLP < 55 and ASP < 33). This left 998 candidates, which were screened again with a high search
efficiency and again were eliminated based on their binding scores (GS < 50, CS < 25, ChemPLP
< 61 and ASP < 34) and poor hydrogen bonding (<1), resulting in 143 candidates. The remaining
compounds were visually inspected and selected based on the following criteria: at least three of the
four scoring functions agree on a pose; the ability to fill at least one of the two hydrophobic domains,
which made up the binding site and had a predicted hydrogen bonding interaction. The results from
the visual inspection showed most compounds had a predicted hydrogen bonding interaction with
either histidines (263 and 493) or lysines (265 and 495), which were indicated by the literature to be key
interactions for successful binding [5]. Other hydrogen bonding was also seen with Asp 283, 288 and
516, Ser 399, 400, 459 and 518, Tyr 204. Based on the listed criteria, sixteen compounds were selected
for further testing. Of those compounds, 1b, shown in Scheme 1, was found to be active against Tdp1
at the low concentration of 0.3 µM.
2.2. Synthesis and Testing of Bile Acids Tryptamides
Bile acids (BAs), steroidal molecules synthesized from cholesterol, are widespread in nature
and possess a high enantiomeric purity and a broad spectrum of native biological activities
(anti-inflammatory, antiviral, anticancer, immunostimulatory) [33–36]. All of these make BAs
interesting and are perspective starting materials for organic synthesis in order to obtain new
derivatives with new biological properties [37–39].
The commercially available bile acids containing two hydroxy-groups in a steroid framework
(ursodeoxycholic acid (3
α β-dihydroxy-5β-cholan-24-oic acid) UDCA; chenodeoxycholic acid
,7
-cholan-24-oic acid) CDCA, epimer of UDCA at C-7, and deoxycholic acid
β-cholan-24-oic acid) DCA) were chosen as starting compounds (Scheme 1).
(3α
,7
α
-dihydroxy-5
β
(3α
,12
α
-dihydroxy-5
At the first step, we resynthesized compound 1b, and also obtained tryptamides of other bile
acids—chenodeoxycholic acid (compound 2b), and deoxycholic acid (compound 3b) (Scheme 1).
The bile acids were transformed into the target compounds (1b, 2b and 3b) by a four-step synthesis
that included the following stages: (1) acylation of hydroxyl groups in the steroid framework; (2–3)
amidation of bile acid chloride by intermediate interaction with tryptamine; (4) the deprotection of
hydroxyl groups by treatment with KOH in methanol (Scheme 1).

Molecules 2018, 23, 679
4 of 19
O
O
O
OH
OH
OH
OH
12
7
3
3
3
7
HO
AcO
AcO
OH
HO
OH
a)
HO
DCA
UDCA
CDCA
a)
a)
O
O
O
OH
OH
OH
OAc
OAc
AcO
OAc
AcO
1
2
3
b), c)
b), c)
b), c)
O
O
O
O
NH
NH
OAc
NH
NH
OAc
12
e)
HN
HN
HN
HN
3
HO
OAc
d)
AcO
OAc
AcO
1a
2a
3c
3a
d)
d)
O
O
O
24
O
23
21
NH
NH
NH
NH
20
17
OH
OH
22
16
18
11'
11
19
f)
12
10'
12
13
14
1
2'
8
2
3'
4'
HN
9
HN
HN
15
3
7
10
HN
7
3
3
1'
9'
3
HO
7
OH
5'
HO
5
6
HO
AcO
OH
1b
4
8'
2b
3d
6'
3b
7'
Scheme 1. Synthesis of compound 1a,b, 2a,b and 3a,b that combine the residues of bile acids and
tryptamine: reagent and conditions: (a) Ac2O, DMAP, CH2Cl2 (RT); (b) (COCl)2, DMF, CH2Cl2
(0 ◦C); (c) tryptamine, NEt3, CH2Cl2 (0 ◦C, RT); (d) KOH, MeOH (reflux); (e) KOH, MeOH (RT);
(f) Ac2O, AcOH.
For testing, we used recently designed oligonucleotide biosensors for the real-time detection
of Tdp1 activity based on the ability of Tdp1 to remove fluorophore quenchers from the 30-end of
DNA [27]. The hexadecameric oligonucleotide carried 5(6)-carboxyfluorescein (FAM) at the 50-end
and fluorophore quencher BHQ1 (Black Hole Quencher-1) at the 30-end. Tdp1 inhibitors prevent the
removal of fluorophore quenchers, thus reducing fluorescence intensity.
All the target compounds, 1b
,
2b and 3b, were found to exhibit similar activity (IC50~2.6
µM),
while the activity of the commercial sample of compound 1b was significantly better (IC50 = 0.3
µM)
(Table 1).
Table 1. Tdp1 Inhibitory activity of compounds 1a,b, 2a,b and 3a–d.
1
1
Compound
IC50 µM
Compound
IC50 µM
1a
2a
3a
0.32 ± 0.11
0.38 ± 0.12
0.65 ± 0.16
0.95 ± 0.05
1.2 ± 0.3
1b
2b
3b
3d
2.65 ± 0.3
2.6 ± 0.4
2.7 ± 0.2
3c
0.48 ± 0.04
Fur 2
1
2
IC50 is the concentration that inhibits the activity of the enzyme by 50%. Fur is furamidine (A) (Figure 1).
Analyzing the scheme of the tryptamides synthesis, we proposed that the commercial sample may
contain impurities with protected hydroxyl groups. This is why we also tested intermediates 1a 2a
and 3a containing acetoxy groups. And indeed, the derivatives 1a 2a, and 3a with two acetoxy groups
were able to inhibit Tdp1 within a concentration range IC50 = 0.32–0.65 M. Since we did not observe
,
,
µ

Molecules 2018, 23, 679
5 of 19
a significant difference in the activity of compounds 1a
–3a
,
1b–
3b and DCA is the cheapest bile acid
among those that we used in this work, we focused our further studies on DCA transformations.
Further, in order to study the influence of functional groups and their location in the steroid
framework, we synthesized the tryptamide derivatives of DCA 3c and 3d containing only one acetoxy
group at the 3 or 12 position (Scheme 1). 3-Hydroxy-12-acetoxyderivative 3c was prepared by reaction
of 3,12-diacetoxyderivative, 3a with KOH in MeOH at room temperature, 3-acetoxy-12-hydroxy
derivative 3d was obtained by acylation of the 3-OH-group of compound 3b. The selectivity of these
reactions is explained by the fact that the hydroxyl groups of the steroid framework possess different
reactivity [35
than the activity of dihydroxyderivatives 1b
,
37]. Compounds 3c and 3d have also been tested and their inhibitory activity was better
3b but slightly worse than those of diacetoxyderivatives
–
1a–3a (IC50 = 0.95 and 0.48 µM, respectively) (Table 1).
Thus, we can conclude that the replacement of at least one hydroxy-group on the steroid
framework by an acetoxy group increased the Tdp1 inhibitory activity, while the location of these
functional groups in the steroid framework did not significantly influence the activity of the inhibitors.
2.3. Synthesis and Testing of Deoxycholic Acid Amides
To study the effect of the amide moiety of conjugates on the inhibitory activity, we synthesized
the set of amides by reacting DCA with various amines.
7a , and 8a were obtained with aromatic amines (aniline, p-bromoaniline, p-methylaniline
and 3-aminopyridine) (Scheme 2). Compound 9a was synthesized using aromatic amine
4-(2-aminoethyl)-2,6-bis-t-butylphenol containing C2-linker. Compounds 8a 10a, and 11a were
Compounds 4a,b, 5a,b, 6a,b,
,b
,b
,b,
synthesized by the reaction with aliphatic amines (1-aminoadamantane, N,N-dimethylethanediamine
and 2-morpholinethanamine).
Scheme 2. Synthesis of deoxycholic ac◦id amides 4a
,b–8a,b and 9a–11a: (a) (COCl)2, DMF, CH2Cl2
(0 C); (b) tryptamine, NEt3, CH2Cl2 (0 C, RT); (c) KOH, MeOH (reflux).
◦
The results from the Tdp1 assay for amides of DCA using the FAM-BHQ1 biosensor are shown in
Table 2. Compounds containing acetoxy groups in the steroid framework (index ) were 3–10 fold more
active than the corresponding derivatives containing hydroxyl groups (index ). The derivatives with
either hydrophobic (adamantane derivative 8a, substituted anilines 4a 6a) or weak acidic (indole 3a or
a
b
–

Molecules 2018, 23, 679
6 of 19
phenol 9a moieties) properties were more active than the derivatives with basic properties (aliphatic
amines 10a, 11a, as well as pyridine 8a).
Table 2. Tdp1 Inhibitory activity of compounds 4a,b–8a,b and 9a–11a.
1
1
Compound
IC50 µM
Compound
IC50 µM
4a
5a
6a
7a
8a
0.43 ± 0.13
0.42 ± 0.01
1.00 ± 0.05
4.08 ± 0.08
0.47 ± 0.08
0.29 ± 0.12
>15
4b
5b
6b
7b
8b
6.7 ± 0.7
1.3 ± 0.2
7.6 ± 3.9
>15
2.3 ± 0.4
9a
10a
11a
Fur 2
>15
1.2 ± 0.3
1
2
IC50 is the concentration that inhibits the activity of the enzyme by 50%. Fur is furamidine (A) (Figure 1).
A study of the cytotoxic activity of diacetoxyderivatives on the human mammary adenocarcinoma
(MCF-7) and human colon carcinoma (HCT-116) cells was carried out using the MTT-test. It showed
that the toxicity of the target compounds was absent or was insignificant throughout the range of
studied concentrations (up to 100 µM).
2.4. Molecular Modeling
The twenty-one derivatives of the bile acid were docked against the binding pocket of the Tdp1
crystal structure (PDB ID: 1MU7, resolution 2.0 Å) [32] with good predicted affinity by the scoring
functions used (see Table S1 in the Supplementary Materials). Highly active inhibitors like 3a tend to
have higher scores than less active ligands, as predicted by ChemPLP and GS.
Many studies suggest that blocking the histidine amino acid residues 263 and 493 is
important [28,32]. As can be seen in Figure 2A for the predicted pose of 9a, it denies access to
both of these histidine residues, reducing the enzymatic activity of Tdp1. Only one hydrogen bond is
predicted between 9a and the protein, i.e., Tyr204 and the carbonyl group of the acetate moiety attached
to carbon 12 in the steroid scaffold. A good fit into the binding pocket is observed (see Figure 2B)
with the steroid scaffold occupying the cleft containing His263 and 493. The acetate groups are both
directed towards the aqueous phase and the di-tert-butyl substituted phenol is partially placed in a
shallow lipophilic pocket. In general, a plausible binding mode is generated, which can explain the
excellent efficacy of 9a.
There is a clear trend for enhanced activity with the acetoxy groups as compared to the hydroxyl
moiety when derivatives 1a–8a and 1b–8b are studied (see Tables 1 and 2). The modelling reveals that
the acetoxy group at C3 is pointing into the aqueous phase with both the oxygen atoms able to form
hydrogen bonds with the water molecules, whereas the methyl moiety is adjacent to a lipophilic surface,
as shown in Figure 2B. The hydroxyl group, however, can also form hydrogen bonds with water but
no lipophilic contacts. As for the C12 acetoxy substituent, it forms a hydrogen bond with Tyr204 for
the 1a–8a series, which the hydroxyl does not do for the 1b–8b derivatives. Thus, the lipophilic contact
and hydrogen bond via the acetoxy substituents appear to be important for enhanced binding of the
bile acid derivatives to Tdp1.
The molecular descriptors are given in Table S2 in the Supplementary Materials. Unsurprisingly,
the MW is high with most of the ligand having >500 g mol−1, which is outside drug-like chemical space.
However, none of the ligands are outside the 800 g mol−1 limit, they are within Known Drug Space
(KDI [40], see Table S3 in the Supplementary Materials). All of the bile acids are outside drug-like
chemical space, except 7a, 7b, 10a and 11a in terms of log P. The most active compound 9a has a
predicted log P of 9.0, which is even outside KDI and can explain the high efficacy of this ligand.
The large size and relatively high lipophilicity is not surprising since the bile acids have a steroid core,

Molecules 2018, 23, 679
7 of 19
which is large and greasy. The hydrogen bond donors and acceptors are all within drug-like chemical
space, for the rotatable bonds in a number of ligands (1a, 2a, 3b, 9a, 10a and 11a) are outside this
boundary but within the KDI.
Figure 2. The docked configuration of 9a to the binding site of Tdp1 using the ChemPLP scoring
function. ( ) Hydrogen bond is depicted as a green line between the ligand and the amino acid TYR204.
) The protein surface is rendered. The steroid group is inserted in a lipophilic pocket and the acyl
A
(
B
groups are exposed to the water environment. Red depicts a positive partial charge on the surface,
blue depicts a negative partial charge and grey shows the neutral/lipophilic areas.
3. Materials and Methods
3.1. Chemicals and Reagents
Elemental analyses were determined on an Automatic CHNS-analyser EURO EA3000. Analyses
indicated by the symbols of the elements were within
±
0.4% of the theoretical values. Melting points
were determined on a METTLER TOLEDO FP900 thermosystem and are uncorrected. The element
composition of the products was determined from high-resolution mass spectra recorded on a DFS
(double focusing sector) Thermo Electron Corporation instrument. Optical rotations were measured
1
with a PolAAr 3005 polarimeter. H and 13C-NMR spectra were measured on Bruker spectrometers:
AV-600 (operating frequency 600.30 MHz for 1H and 150.95 MHz for 13C) and DRX-500 (500.13 MHz for
1H and 125.76 MHz for 13C) using CDCl3 solutions of the substances. The chemical shifts were recorded
in δ (ppm) using the δ δ
7.24 of CHCl3 (1H-NMR) and 76.90 (13C-NMR) as internal standards. Chemical
shift measurements are given in ppm and the coupling constants (J) in hertz (Hz). The structure of
the compounds was determined by NMR using the standard one-dimensional and two-dimensional
procedures (1H-1H COSY, 1H-13C HMBC/HSQC, 13C-1H HETCOR/COLOC). The purity of the final
compounds and intermediates for biological testing was confirmed to be more than 95%, as determined
by the HPLC analysis. HPLC analyses were carried out on a MilichromA-02, using a ProntoSIL
120-5-C18 AQ column (BISCHOFF, 2.0
×
75 mm column, grain size 5.0 lm). Mobile phase: Millipore
purified water with 0.1% trifluoroacetic acid with a linear gradient of 0–100% methanol at a flow
◦
rate of 150
µ
L/min at 35 C and UV detection at 210, 220, 240, 260, and 280 nm. Flash column
chromatography was performed with silica gel (Merck, 60–200 mesh). All reactions’ courses were
monitored by TLC analysis using Merck 60 F254 silica gel on aluminum sheets, eluent CHCl3-MeOH
(20:3) or CHCl3-AcOEt (20:3).
Ursodeoxycholic acid (99%), tryptamine (98%), 3-aminopyridine (99%), 1-aminoadamantane
hydrochloride (99%) were purchased from ACROS Organics, chenodeoxycholic acid (>98%) was
purchased from ROTH, deoxycholic acid 99%, oxalyl chloride (98%) were purchased from abcr GmbH
& Co. KG, N1,N1-diethylaminopropan-1,3-diamine (99+%) and 3-morpholinopropan-1-amine (98%)
were purchased from Aldrich. All solvents used in the reactions were previously purified and dried
according to the previously reported procedures.

Molecules 2018, 23, 679
8 of 19
Diacetoxy derivatives of bile acids 1–3 were synthesized according to the known literature
method [41] by treatment of a bile acid, such as ursodeoxycholic (UDCA), chenodeoxycholic (CDCA),
and deoxycholic (DCA) with acetic anhydride in the presence of 4,4-dimethylaminopyridine (DMAP)
in anhydrous dichloromethane with yield 95%.
(a) General procedures for compound 1a–11a
(1)
(2)
Oxalyl chloride (6.0 equiv.) and a few drops DMF were added at 0 ◦C to a solution of
diacetoxy bile acid (1–3) (1.0 equiv.), correspondently, in dry CH2Cl2. The reaction mixture
was stirred for a further 3 h at 0–5 ◦C, diluted with benzene and concentrated in vacuum.
Then CH2Cl2.was added to the reaction mixture to give a solution of bile acid chloride.
◦
The resulting solution bile acid chloride (1.0 equiv.) was added dropwise at 0 C to
a solution of appropriate amine (1.2 equiv.) and NEt3 (1.5 equiv.) in dry CH2Cl2.
The reaction mixture was stirred for a further 18 h at room temperature, diluted with
AcOEt (20 mL) and H2O was added. The organic layer was separated and the aqueous
layer was extracted with AcOEt (2
with brine and dried over calcined MgSO4. The solvent was removed to give an
amorphous solid.
×
30 mL). The combined organic layers were washed
(b) General procedures for compound 1b–8b
A solution of the appropriate amide derivatives (1a–8a) (1 equiv.)—containing acetoxy groups in
the steroid framework—in MeOH was treated with KOH (6 equiv.), refluxed for 2–6 h (monitored
by TLC), concentrated under reduced pressure, diluted with H2O (up to ~50–60 mL), acidified
with 5% HCl solution to pH 3–4, and extracted with mixture CH2Cl2Et2O (1:3 v/v) (3
The combined organic layers were washed with a saturated solution of NaHCO3, brine and dried
over calcined MgSO4. The solvent was removed to give an amorphous solid.
×
30 mL).
3.1.1. N-(20-(1H-Indol-2-yl)-ethyl)-3α,7β-diacetoxy-5β-cholan-24-amide (1a)
Crude product as an amorphous white solid (1.52 g, 97%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 1 (1.20 g, 2.5 mmol) and oxalyl chloride
(1.3 mL, 15.0 mmol) in CH2Cl2 (10 mL)) and tryptamine (0.48 g, 3.0 mmol) with addition of NEt3
(0.6 mL, 3.8 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product was
purified by flash column chromatography (silica gel, 0–5% MeOH in CHCl3) to yield compound 1a
(1.25 g, 80%) as an amorphous white. M.p. 107.6 ◦C [decomposition]. [α2D3] + 42◦ (c 0.20 g/100 mL;
CHCl3). HRMS: m/z calcd for C38H54N2O5: 618.4027; found: 618.4020. Anal. Calcd. For C38H54N2O5:
1
C, 73.75; H, 8.80; N, 4.53; O, 12.93; found C, 73.68; H, 8.76; N, 4.31. H-NMR (CDCl3, 500 MHz):
δ = 8.36
(br s, 1H, N
H-70), 7.09 (dd, 1H, J60,50 = 7.4, H-60), 7.00 (br s, 1H, H-20), 5.65 (m, 1H, C(O)N
4.63 (m, 1H, H-3(
)), 3.57 (m, 2H, CH2-110), 2.95 (m, 2H, CH2-100), 2.00 (s, 3H, CH3-26), 1.96 (s, 3H,
H
-10), 7.57 (d, 1H, J50,60 = 7.7, H-50), 7.35 (d, 1H, J80,70 = 8.0, H-80), 7.18 (dd, 1H, J70,60 = 7.4,
), 4.73 (s, 1H, H-7( )),
H
α
β
CH3-28), 0.93 (s, 3H, CH3-18), 0.85 (d, 3H, J21,20 = 6.2, CH3-21), 0.62 (s, 3H, CH3-19). 13C-NMR (CDCl3,
125 MHz):
δ
= 173.46 (s, C-24), 170.52 (s, C-25), 170.44 (s, C-27), 136.250(s, C-90), 127.150(s, C-40), 121.93
0
0
0
0
(d, C-2 ), 121.93 (d, C-7 ), 119.12 (d, C-5 ), 118.44 (d, C-6 ), 112.60 (s, C-3 ), 111.16 (d, C-8 ), 73.40 (d, C-7),
73.40 (d, C-3), 54.95, 54.75, 43.32, 41.79, 39.70, 39.66, 39.61, 39.12, 35.10, 34.26, 33.74, 33.39, 32.65, 32.65,
31.54, 28.22, 26.15, 25.38, 25.15 (t, C-100), 23.00, 21.66, 21.21, 20.96, 18.25(q, C-21), 11.86(q, C-18).
3.1.2. N-(20-(1H-Indol-2-yl)-ethyl)-3α,7β-dihydroxy-5β-cholan-24-amide (1b)
Crude product as an amorphous white solid (0.55 g, 92%) was prepared from diacetoxy derivative
1a (0.69 g, 1.1 mmol) and KOH (0.37 g, 6.6 mmol) in MeOH (25 mL) according to the general procedure.
An analytically pure sample of 1b (0.22 g, 37%) was obtained by flash column chromatography (silica
gel, 0–5% MeOH in CHCl3). M.p. 114.9 ◦C [decomposition]. [α2D3] + 28◦ (c 0.20 g/100 mL; CHCl3).
HRMS: m/z calcd for C34H50N2O3: 534.3816; found: 534.3813. Anal. Calcd. for C34H50N2O3: C, 76.36;

Molecules 2018, 23, 679
9 of 19
1
H, 9.42; N, 5.24; O, 8.98; found C, 76.43; H, 9.64; N, 4.89. H-NMR (CDCl3, 500 MHz):
δ
= 8.96 (br s,
1H, NH-10), 7.53 (d, 1H, J50,60 = 7.5, H-50), 7.33 (d, 1H, J80,70 = 8.1, H-80), 7.13 (m, 1H, H-70), 7.05 (m, 1H,
H-60), 60.97 (br s, 1H, H-20), 5.97 (m, 1H, C(O)N
H), 3.49 (m, 4H: H-3(β), H-7(α
), CH2-110), 2.91 (m, 2H,
CH2-10 ), 0.87 (s, 3H, CH3-18), 0.82 (d, 3H, J21,20 = 6.0, CH3-21), 0.58 (s, 3H, CH3-19). 13C-NMR (CDCl3,
0 0 0 0
CD3OD, 125 MHz):
δ = 174.00 (s, C-24), 136.17 (s, C-9 ), 127.06 (s, C-4 ), 121.98 (d, C-2 ), 121.73 (d, C-7 ),
119.00 (d, C-50), 118.35 (d, C-60), 112.25 (s, C-30), 111.17 (d, C-80), 71.00 (d, C-3), 70.95 (d, C-7), 55.49,
54.60, 43.46, 43.33, 42.20, 39.88, 39.49, 39.00, 36.85, 36.65, 35.17, 34.69, 33.82, 33.32, 31.68, 29.85, 28.40,
25.01, 23.17 (q, C-19), 20.94, 18.21 (q, C-21), 11.87 (q, C-18).
3.1.3. N-(20-(1H-Indol-2-yl)-ethyl)-3α,7a-diacetoxy-5β-cholan-24-amide (2a)
Crude product as an amorphous white solid (1.40 g, 89%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 2 (1.20 g, 2.5 mmol) and oxalyl chloride
(1.3 mL, 15.0 mmol) in CH2Cl2 (10 mL)) and tryptamine (0.48 g, 3.0 mmol) with addition of NEt3
(0.6 mL, 3.8 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product was
purified by flash column chromatography (silica gel, 0–1% MeOH in CHCl3) to yield compound 2a
(1.10 g, 70%) as an amorphous white. M.p. 98.7 C [decomposition]. [α2D3] + 8◦ (c 0.20 g/100 mL;
◦
CHCl3). HRMS: m/z calcd for C38H54N2O5: 618.4027; found: 618.4023. Anal. Calcd. For C38H54N2O5:
1
C, 73.75; H, 8.80; N, 4.53; O, 12.93; found C, 73.54; H, 8.63; N, 4.55. H-NMR (CDCl3, 500 MHz):
δ = 8.47
(br s, 1H, NH-10), 7.56 (d, 1H, J50,60 = 7.8, H-50), 7.34 (d, 1H, J80,70 = 8.1, H-80), 7.17 (dd, 1H, J70,60 = 7.6,
H-70), 7.09 (dd, 1H, J60,50 = 7.4, H-60), 6.99 (s, 1H, H-20), 5.67 (s, 1H, C(O)N
), 4.84 (s, 1H, H-7( )), 4.55
(m, 1H, H-3(
)), 3.56 (d, 2H, J = 5.8, CH2-110), 2.94 (m, 2H, CH2-100), 2.02 (s, 3H, CH3-26), 2.01 (s, 3H,
H
β
β
CH3-28), 0.89 (s, 3H, CH3-18), 0.86 (d, 3H, J21,20 = 6.0, CH3-21), 0.59 (s, 3H, CH3-19). 13C-NMR (CDCl3,
125 MHz):
δ
= 173.49 (s, C-24), 170.54 (s, C-25), 170.37 (s, C-27), 136.250(s, C-90), 127.160(s, C-40), 121.94
0
0
0
0
(d, C-2 ), 121.89 (d, C-7 ), 119.19 (d, C-5 ), 118.47 (d, C-6 ), 112.66 (s, C-3 ), 111.16 (d, C-8 ), 74.02 (d, C-3),
71.09 (d, C-7), 55.54 (d, C-17), 50.16 (d, C-14), 42.47 (s, C-13), 40.72 (d, C-5),039.63, 39.26, 37.66 (d, C-8),
35.22, 34.68, 34.58, 34.41, 33.83, 33.40, 31.56, 31.12, 27.86, 26.58, 25.13 (t, C-10 ), 23.35, 22.49, 21.46, 21.33,
20.43, 18.17(q, C-21), 11.51 (q, C-18).
3.1.4. N-(20-(1H-Indol-2-yl)-ethyl)-3α,7α-dihydroxy-5β-cholan-24-amide (2b).
Crude product as an amorphous white solid (0.55 g, 92%) was prepared from diacetoxy derivative
2a (0.69 g, 1.1 mmol) and KOH (0.37 g, 6.6 mmol) in MeOH (25 mL) according to the general procedure.
An analytically pure sample of 2b (0.22 g, 37%) was obtained by flash column chromatography (silica
◦
gel, 0–5% MeOH in CHCl3). M.p. 112.9 C [decomposition]. [α2D3] + 5◦ (c 0.20 g/100 mL; CHCl3).
HRMS: m/z calcd for C34H50N2O3: 534.3816; found: 534.3818. Anal. Calcd. for C34H50N2O3: C, 76.36;
1
H, 9.42; N, 5.24; O, 8.98; found C, 75.94; H, 9.06; N, 4.84. H-NMR (CDCl3, 500 MHz):
δ = 8.73 (br s,
1H, NH-10), 7.55 (d, 1H, J50,60 = 7.8, H-50), 7.35 (d, 1H, J80,70 = 7.8, H-80), 7.16 (dd, 1H, J70,60 = 7.0, J70,80
7.8, H-70), 7.08 (dd, 1H, J60,50 = 7.8, H-60), 7.00 (br s, 1H, H-20), 5.80 (br s, 1H, C(O)N
), 3.80 (br s, 1H,
H-7(
)), 3.56 (m, 2H, CH2-110), 3.43 (m, 1H, H-3( )), 2.93 (m, 2H, CH2-100), 0.86 (s, 3H, CH3-18), 0.59
(d, 3H, CH3-21), 0.59 (s, 3H, CH3-19). 13C-NMR (CDCl3, 125 MHz):
=
H
β
β
0
= 173.82 (s, C-24), 136.32 (s, C-9 ),
127.17 (s, C-4 ), 122.12 (d, C-2 ), 121.85 (d, C-7 ), 119.15 (d, C-5 ), 118.48 (d, C-6 ), 112.55 (s, C-3 ), 111.27
(d, C-8 ), 71.79 (d, C-3), 68.33 (d, C-7), 55.50 (d, C-17), 50.20 (d, C-14), 42.44 (s, C-13), 41.29, 39.56, 39.49,
δ
0
0
0
0
0
0
0
39.43, 39.18, 35.27, 35.15, 34.85, 33.31, 32.65, 31.65, 30.86, 30.39, 28.03, 25.14, 23.49, 22.64 (q, C-19), 20.41,
18.22 q, C-21)„ 11.60 (q, C-18).
3.1.5. N-(20-(1H-Indol-2-yl)-ethyl)-3α,12a-diacetoxy-5β-cholan-24-amide (3a)
Crude product as an amorphous white solid (2.1 g, 82%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (2.0 g, 4.1 mmol) and oxalyl chloride
(2.0 mL, 24.6 mmol) in CH2Cl2 (10 mL)) and tryptamine (0.79 g, 4.9 mmol) with addition of NEt3
(0.9 mL, 6.2 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product was
purified by flash column chromatography (silica gel, 0–1% MeOH in CHCl3) to yield compound 3a

Molecules 2018, 23, 679
10 of 19
◦
(0.94 g, 45%) as an amorphous white. M.p 99.2 C [decomposition]. [α2D3] + 72◦ (c 0.20 g/100 mL;
CHCl3). HRMS: m/z calcd for C38H54N2O5: 618.4027; found: 618.4021. Anal. Calcd. for C38H54N2O5:
C, 73.75; H, 8.80; N, 4.53; O, 12.93; found C, 73.37; H, 8.68; N, 4.18. 1H-NMR (CDCl3, 300 MHz):
δ
= 8.63 (br s, 1H, NH-10), 7.55 (d, 1H, J50,60 = 7.8, H-50), 7.33 (d, 1H, J80,70 = 8.0, H-80), 7.15 (dd, 1H,
J70,60 = 7.2, H-70), 7.06 (dd, 1H, J60,50 = 7.4, H-60), 6.97 (s, 1H, H-20), 5.60 (m, 1H, C(O)N
H), 5.05 (s, 1H,
H-12(β)), 4.67 (m, 1H, H-3(β
)), 3.54 (m, 2H, CH2-110), 2.92 (m, 2H, CH2-100), 2.06 (s, 3H, CH3-26), 2.01
(s, 3H, CH3-28), 0.88 (s, 3H, CH3-18), 0.71 (d, 3H, J21,20 = 6.4, CH3-21), 0.67 (s, 3H, CH3-19). 13C-NM0R
0
(CDCl3, 125 MHz):
δ
= 173.30 (s, C-24), 170.44 (s, C-25), 170.37 (s, C-27), 136.32 (s, C-9 ), 127.25 (s, C-4 ),
122.03 (d, C-20), 121.90 (d, C-70), 119.32 (d, C-50), 118.55 (d, C-60), 112.86 (s, C-30), 111.16 (d, C-80), 75.80
(d, C-12), 74.08 (d, C-3), 49.28, 47.64, 44.89, 41.70, 39.67 (t, C-110), 35.54, 34.67, 34.59, 34.28, 33.90, 33.55,
0
32.14, 31.47, 27.18, 26.75, 26.50, 25.73, 25.52, 25.19 (t, C-10 ), 23.28, 22.92 (q,
21.25 (q, CH3-C(O)O), 17.49 (q, C-21), 12.29 (q, C-18).
CH3-C(O)O), 21.32 (q, C-19),
3.1.6. N-(20-(1H-Indol-2-yl)-ethyl)-3α,12α-dihydroxy-5β-cholan-24-amide (3b)
Crude product as an amorphous white solid (0.35 g, 77%) was prepared from diacetoxy derivative
3a (0.52 g, 0.9 mmol) and KOH (0.3 g, 5.4 mmol) in MeOH (8 mL) according to the general procedure.
An analytically pure sample of 3b (0.20 g, 44%) was obtained by flash column chromatography (silica
gel, 0–8% MeOH in CHCl3). M.p. 117.7 ◦C [decomposition]. [α2D7] + 34◦ (c 0.20 g/100 mL; CH3OH).
1
HRMS: m/z calcd for C34H50N2O3: 534.3816; found: 534.3815. H-NMR (CDCl3, 300 MHz):
δ = 8.63 (br
0
0
0
0
s, 1H, NH-1 ), 7.56 (d, 1H, J50,60 = 7.8, H-5 ), 7.36 (d, 1H, J80,70 = 8.2, H-8 ), 7.17 (m, 1H, H-7 ), 7.08 (m 1H,
0
0
0
H-6 ), 7.00 (br s, 1H, H-2 ), 5.81 (br s, 1H, C(O)NH), 3.90 (s, 1H, H-12(β)), 3.55 (m, 3H: H-3(β), CH2-11 ),
2.93 (m, 2H, CH2-100), 0.89 (d, 3H, J21,20 = 6.0, CH3-21), 0.807 (s, 3H, CH3-18), 0.61 (s, 3H, 0CH3-19).
0
δ = 173.70 (s, C-24), 136.32 (s, C-9 ), 127.23 (s, C-4 ), 122.11 (d, C-2 ), 121.93
13C-NMR (CDCl3, 75 MHz):
0
0
0
0
0
(d, C-7 ), 119.23 (d, C-5 ), 118.54 (d, C-6 ), 112.74 (s, C-3 ), 111.27 (d, C-8 ), 73.03 (d, C-12), 71.59 (d, C-3),
48.10, 46.91, 46.32, 41.91, 39.60 (t, C-11 ), 36.21, 35.83, 35.08, 33.96, 33.50, 33.33, 31.54, 30.26, 28.47, 27.34,
26.98, 25.98, 25.17 (t, C-100), 23.51, 22.99 (q, C-19), 17.27 (q, C-21), 12.61 (q, C-18).
0
3.1.7. N-(20-(1H-Indol-2-yl)-ethyl)-3α-hydroxy-12α-acetoxy-5β-cholan-24-amide (3c)
A mixture of 3a (0.65 g, 1.0 mmol) and KOH (0.17 g, 3.0 mmol) in methanol (5 mL) was stirred at
room temperature (monitored by TLC), concentrated under reduced pressure, diluted with H2O (up to
~15 mL), acidified with 5% HCl solution to pH 3–4, and extracted with mixture CH2Cl2Et2O (1:3 v/v)
(3
×
20 mL). The combined organic layers were washed with saturated solution of NaHCO3, brine and
dried over calcined MgSO4. The solvent was removed to give an amorphous solid 3c (0.50 g, 85%).
An analytically pure sample of 3c (0.45 g, 76%) was obtained by flash column chromatography (silica
gel, 30–55% AcOEt in hexane). M.p. 106.5 ◦C [decomposition]. HRMS: m/z calcd for C36H52N2O4:
1
576.3922; found: 576.3927. H-NMR (CDCl3, 300 MHz):
δ =
= 8.30 (br s, 1H, NH-10), 7.57 (d, 1H, J50,60
7.8, H-50), 7.36 (d, 1H, J80,70 = 8.0, H-80), 7.18 (m, 1H, H-70), 7.09 (m 1H, H-60), 7.09 (br s, 1H, H-20), 5.67
(br s, 1H, C(O)N ), 5.03 (s, 1H, H-12( )), 3.57 (m, 3H: H-3(
), CH2-110), 2.95 (t, 2H, CH2-100, J100,110
6.4), 2.03 (s, 3H, CH3-28), 0.87 (s, 3H, CH3-18), 0.74 (d, 3H, J21,20 = 6.0, CH3-21), 0.66 (s, 3H, CH3-19).
13C-NMR (CDCl3, 75 MHz): = 173.42 (s, C-24), 170.54 (s, C-27), 136.29 (s, C-90), 127.21 (s, C-40),
H
β
β
=
δ
122.03 (d, C-20), 121.92 (d, C-70), 119.32 (d, C-50), 118.54 (d, C-60), 112.80 (s, C-30), 111.17 (d, C-80), 75.84
(d, C-12), 71.56 (d, C-3), 49.25, 47.56, 44.85, 41.83, 39.66 (t, C-110), 36.10, 35.56, 34.84, 34.63, 34.28, 33.88,
33.88, 31.45, 30.29, 27.17, 26.91, 25.81, 25.49, 25.16 (t, C-100), 23.30, 22.96 (q, C-19), 21.32 (q, C-28), 17.47
(q, C-21), 12.29 (q, C-18).
3.1.8. N-(20-(1H-Indol-2-yl)-ethyl)-3α-acetoxy-12α-hydroxy-5β-cholan-24-amide (3d)
To a solution of 3a (0.14 g, 0.22 mmol) in dry CH2Cl2 (10 mL) was added Ac2O (0.03 mL,
0.33 mmol). The reaction mixture was stirred for a further 3 h at room temperature (monitored
by TLC) and added H2O (10 mL). The organic layer was separated and the aqueous layer was extracted
with AcOEt (2
×
30 mL). The combined organic layers were washed with a saturated solution of

Molecules 2018, 23, 679
11 of 19
NaHCO3, brine and dried over calcined MgSO4. The solvent was removed to give an amorphous
solid 3d (0.13 g, quantitative yield). An analytically pure sample of 3d (0.09 g, 69%) was obtained
by flash column chromatography (silica gel, 30–50% AcOEt in hexane). M.p. 196.7–197.1 ◦C. Anal.
Calcd. for C36H52N2O4: C, 74.96; H, 9.09; N, 4.86; O, 11.10; found C, 75.05; H, 8.78; N, 4.65. HRMS:
1
m/z calcd for C36H52N2O4: 576.3922; found: 576.3926. H-NMR (CDCl3, 300 MHz):
δ = 8.49 (br s,
1H, NH-10), 7.56 (d, 1H, J50,60 = 7.8, H-50), 7.35 (d, 1H, J80,70 = 8.0, H-80), 7.17 (m, 1H, H-70), 7.09 (m 1H,
H-60), 6.99 (br s, 1H, H-20), 5.72 (br s, 1H, C(O)N
), 4.68 (m, 1H, H-3( )), 3.92 (s, 1H, H-12( )), 3.56
(m, 2H:, CH2-110, J110,100 = 6.3), 2.94 (t, 2H, CH2-100, J100,110 = 6.3), 1.99 (s, 3H, CH3-26) 0.85-0.92 [6H:
H
β
β
(CH3-21), 0.88 (s, 3H, CH3-18)], 0.62 (s, 3H, CH3-19). 13C-NMR (CDCl3, 75 MHz):
δ
= 173.70 0(s, C-24),
0
0
0
0
170.59 (s, C-25), 136.28 (s, C-9 ), 127.19 (s, C-4 ), 121.97 (d, C-2 ), 121.93 (d, C-7 ), 119.22 (d, C-5 ), 118.49
(d, C-60), 112.69 (s, C-30), 111.19 (d, C-80), 74.17 (d, C-3), 72.93 (d, C-12), 48.10, 47.03, 46.28, 41.67, 39.63
(t, C-110), 35.77, 35.00, 33.94, 33.46, 33.33, 31.99, 31.49, 28.52, 27.28, 26.78, 26.32, 25.84, 25.13 (t, C-100),
23.41, 22.95 (q, C-19), 21.32 (q, C-26), 17.24 (q, C-21), 12.61 (q, C-18).
3.1.9. N-Phenyl-3α,12α-diacetoxy-5β-cholan-24-amide (4a)
Crude product as an amorphous white solid (2.0 g, 87%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (2.0 g, 4.2 mmol) and oxalyl chloride
(2.1 mL, 25.2 mmol) in CH2Cl2 (10 mL)) and aniline (0.45 g, 5.0 mmol) with addition of NEt3 (0.9 mL,
6.3 mmol) in CH2Cl2 (20 mL) according to the general procedure. The crude product was purified by
flash column chromatography (silica gel, CHCl3) to yield compound 4a (1.73 g, 75%) as an amorphous
white. M.p. 97.5 ◦C [decomposition]. [α2D4.1] + 80◦ (c 0.20 g/100 mL; CHCl3). HRMS: m/z calcd for
C34H49NO5: 551.3605; found: 551.3604. Anal. Calcd. for C34H49NO5: C, 74.01; H, 8.95; N, 2.54; O,
14.50; Found C, 73.91; H, 8.88; N, 2.78. 1H-NMR (CDCl3, 300 MHz):
δ
= 7.48 (d, 2H, J = 7.9, H-20,
H-60), 7.38 (s, 1H, N
H
), 7.27 (dd, 2H, J = J = 7.9, H-30, H-50), 7.06 (0dd, 1H, J = 7.4, H-40), 5.06 (s, 1H,
H-12(
β
)), 4.67 (m, 1H, H-3(β)), 2.38 (m, 1H, H-23), 2.19 (m, 1H, H-23 ), 2.08 (s, 3H, CH3-26), 2.00 (s, 3H,
CH3-28), 0.87 (s, 3H, CH3-19), 0.81 (d, 3H, J21,20 = 6.3, CH3-21), 0.69 (s, 3H, CH3-18). 13C MNR (CDCl3,
150 MHz):
δ
= 171.50 (s, C-24), 170.48 (s, C-25), 170.42 (s, C-27), 137.89 (s, C-10), 128.82 (d, C-30), 128.82
(d, C-50), 123.97 (d, C-40), 119.59 (d, C-20), 119.59 (d, C-60), 75.83 (d, C-12), 74.08 (d, C-3), 49.27, 47.71,
44.89, 41.66, 35.51, 34.74, 34.56, 34.24, 33.88, 32.10, 31.33, 27.26, 26.73, 26.48, 25.71, 25.51, 23.29, 22.92
(q, C-19), 21.34 (q, CH3-C(O)O), 21.28 (q, CH3-C(O)O), 17.52 (q, C-21), 12.33(q, C-18).
3.1.10. N-Phenyl-3α,12α-dihydroxy-5β-cholan-24-amide (4b)
Crude product as an amorphous white solid (0.43 g, 77%) was prepared from diacetoxy derivative
4a (0.40 g, 0.7 mmol) and KOH (0.23 g, 4.2 mmol) in MeOH (10 mL) according to the general procedure.
An analytically pure sample of 4b (0.23 g, 68%) was obtained by flash column chromatography (silica
gel, 0–2% MeOH in CHCl3). M.p. 214.2–216.3 ◦C. [α2D5.6] + 35◦ (c 0.20 g/100 mL; CHCl3). HRMS: m/z
calcd for C30H45NO3: 467.3394; found: 467.3388. Anal. Calcd. for C30H45NO3: C, 77.04; H, 9.70; N,
1
2.99; O, 10.26; found C, 77.32; H, 9.39; N, 3.12. H-NMR (CDCl3, 400 MHz):
δ = 8.00 (s, 1H, NH), 7.53
(d, 2H, J = 7.7, H-20, H-60), 7.25 (dd, 2H, J = J = 7.8, H-30, H-50), 7.04 (dd, 1H, J = 7.3, H-40), 3.97 (s,
1H, H-12( )), 3.56 (m, 1H, H-3(
)), 2.38 (m, 1H, H-23), 2.22 (m, 1H, H-230), 0.95 (d, 3H, J21,20 = 6.0,
CH3-21), 0.87 (s, 3H, CH3-19), 0.64 (s, 3H, CH3-18). 13C-NMR (CDCl3, 100 MHz):
= 172.38 (s, C-24),
138.21 (s, C-10), 128.74 (d, C-30), 128.74 (d, C-50), 123.75 (d, C-40), 119.55 (d, C-20), 119.55 (d, C-60), 73.10
(d, C-12), 71.67 (d, C-3), 47.66, 46.41, 46.20, 42.00, 36.19, 35.91, 35.21, 35.03, 34.09, 33.62, 33.45, 30.97,
30.67, 28.15, 27.50, 27.19, 26.09, 23.65, 23.02 (q, C-19), 17.34 (q, C-21), 12.48(q, C-18).
β
β
δ
3.1.11. N-(40-Bromophenyl)-3α,12α-diacetoxy-5β-cholan-24-amide (5a)
Crude product as an amorphous white solid (2.45 g, 92%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (2.0 g, 4.2 mmol) and oxalyl chloride
(2.1 mL, 25.2 mmol) in CH2Cl2 (10 mL)) and p-bromoaniline (0.86 g, 5.0 mmol) with addition of NEt3
(0.9 mL, 6.3 mmol) in CH2Cl2 (20 mL) according to the general procedure. The crude product was

Molecules 2018, 23, 679
12 of 19
purified by flash column chromatography (silica gel, CHCl3) to yield compound 5a (2.04 g, 77%) as
an amorphous white. M.p. 109.3 ◦C [decomposition]. [α2D4.3] + 62◦ (c 0.20 g/100 mL; CHCl3). HRMS:
m/z calcd for C34H4879BrNO5: 629.2710; found: 629.2701. Anal. Calcd. for C34H48BrNO5: C, 64.75; H,
1
7.67; Br, 12.67; N, 2.22; O, 12.68; found C, 65.10; H, 7.75; Br, 13.00; N, 2.35. H-NMR (CDCl3, 600 MHz):
δ
= 7.43 (s, 1H, NH), 7.38 (m, 4H, H-20, H-30, H-50, H-60), 5.03 (s, 1H, H-12(
β
)), 4.67 (m, 1H, H-3(
β)),
2.37 (m, 1H, H-23), 2.18 (m, 1H, H-230), 2.07 (s, 3H, CH3-26), 2.01 (s, 3H, CH3-28), 0.87 (s, 3H, CH3-19),
0.80 (d, 3H, J21,20 = 6.4, CH3-21), 0.69 (s, 3H, CH3-18). 13C-NMR (CDCl3, 150 MHz):
170.51 (s, C-25), 170.44 (s, C-27), 136.96 (s, C-1 ), 131.76 (d, C-3 ), 131.76 (d, C-5 ), 121.15 (d, C-2 ), 121.15
(d, C-6 ), 116.49 (s, C-4 ), 75.82 (d, C-12), 74.08 (d, C-3), 49.27, 47.70, 44.90, 41.65, 35.50, 34.72, 34.56, 34.53,
δ
= 171.55 (s, C-24),
0
0
0
0
0
0
34.24, 33.88, 32.10, 31.23, 27.25, 26.72, 26.48, 25.70, 25.52, 23.28, 22.93 (q, C-19), 21.36 (q,
21.30 (q, CH3-C(O)O), 17.53 (q, C-21), 12.33 (q, C-18).
CH3-C(O)O),
3.1.12. N-(40-Bromophenyl)-3α,12α-dihydroxy-5β-cholan-24-amide (5b)
Crude product as an amorphous white solid (1.20 g, 81%) was prepared from diacetoxy derivative
5a (1.75 g, 2.7 mmol) and KOH (0.91 g, 16.2 mmol) in MeOH (10 mL) according to the general procedure.
An analytically pure sample of 5b (1.17 g, 79%) was obtained by flash column chromatography (silica
gel, 0.5–3% MeOH in CHCl3). M.p. 229.4 ◦C [decomposition]. [α2D6.8] + 25◦ (c 0.20 g/100 mL; CHCl3).
HRMS: m/z calcd for C30H4479BrNO3: 545.2499; found: 545.2491. Anal. Calcd. for C30H44BrNO3: C,
65.92; H, 8.11; Br, 14.62; N, 2.56; O, 8.78; found C, 65.57; H, 7.90; Br, 14.62; N, 2.63. Anal. Calcd. for
C30H44BrNO3: C, 65.92; H, 8.11; Br, 14.62; N, 2.56; O, 8.78; found C, 65.57; H, 7.90; Br, 14.62; N, 2.63.
1H-NMR (CDCl3, CD3OD, 400 MHz):
J = 7.8, H-30, H-50), 3.86 (s, 1H, H-12(
0.90 (d, 3H, J21,20 = 5.7, CH3-21), 0.81 (s, 3H, CH3-19), 0.57 (s, 3H, CH3-18). 13C-NMR (CDCl3, CD3OD,
δ
= 9.00 (s, 1H, N
H
), 7.38 (d, 2H, J = 7.8, H-20, H-60), 7.29 (d, 2H,
β
β
)), 3.47 (m, 1H, H-3(
)), 2.29 (m, 1H, H-23), 2.15 (m, 1H, H-230),
100 MHz):
δ
= 173.09 (s, C-24), 137.37 (s, C-10), 131.42 (d, C-30), 131.42 (d, C-50), 121.14 (d, C-20), 121.14
(d, C-60), 115.99 (s, C-40), 72.88 (d, C-12), 71.19 (d, C-3), 47.77, 46.23, 46.11, 41.77, 35.78, 35.65, 35.00,
34.97, 33.85, 33.29, 33.25, 31.10, 29.60, 28.20, 37.25, 26.85, 25.91, 23.45, 22.79 (q, C-19), 16.88 (q, C-21),
12.37 (q, C-18).
3.1.13. N-(p-Tolyl)-3α,12α-diacetoxy-5β-cholan-24-amide (6a)
Crude product as an amorphous white solid (1.2 g, quantitative yield) was obtained from acid
chloride intermediate (preparing by reaction diacetoxy derivative 3 (1.0 g, 2.1 mmol) and oxalyl
chloride (1.1 mL, 12.6 mmol) in CH2Cl2 (10 mL)) and p-toluidine (0.27 g, 2.5 mmol) with addition of
NEt3 (0.5 mL, 3.1 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product
was purified by flash column chromatography (silica gel, CHCl3) to yield compound 6a (1.0 g, 84%) as
an amorphous white. M.p. 94.8 ◦C [decomposition]. [α2D5.8] + 71◦ (c 0.20 g/100 mL; CHCl3). HRMS:
m/z calcd for C35H51NO5: 565.3762; found: 565.3759. Anal. Calcd. for C35H51NO5: C, 74.30; H, 9.09; N,
1
2.48; O, 14.14; found C, 74.00; H, 8.72; N, 2.50. H-NMR (CDCl3, 600 MHz):
δ
= 7.65 (s, 1H, N
H
), 7.35 (d,
)), 2.34
2H, J = 8.4, H-20, H-60), 7.05 (d, 2H, J = 8.3, H-30, H-50), 5.04 (s, 1H, H-12(
β)), 4.65 (m, 1H, H-3(β
(m, 1H, H-23), 2.25 (s, 3H, CH3-70), 2.15 (m, 1H, H-230), 2.05 (c, 3H, CH3-26), 1.99 (s, 3H, CH3-28), 0.85
(s, 3H, CH3-19), 0.78 (d, 3H, J21,20 = 6.3, CH3-21), 0.67 (s, 3H, CH3-18). 13C-NMR (CDCl3, 150 MHz):
δ
= 171.49 (s, 0C-24), 170.43 (s, C-25), 170.36 (s, C-27), 135.39 (s, C-10), 133.40 (s, C-40), 129.18 (d, C-30),
0
0
129.18 (d, C-5 ), 119.70 (d, C-2 ), 119.70 (d, C-6 ), 75.79 (d, C-12), 74.02 (d, C-3), 49.21, 47.67, 44.81, 41.59,
35.44, 34.69, 34.49, 34.38, 34.17, 33.81, 32.04, 31.33, 27.18, 26.66, 26.42, 25.64, 25.44, 23.22, 22.86 (q, C-19),
21.27 (q, CH3-C(O)O), 21.21 (q, CH3-C(O)O), 20.64 (q, CH3-70), 17.45 (q, C-21), 12.25 (q, C-18).
3.1.14. N-(p-Tolyl)-3α,12α-dihydroxy-5β-cholan-24-amide (6b)
Crude product as an amorphous white solid (0.19 g, 98%) was prepared from diacetoxy derivative
6a (0.23 g, 0.4 mmol) and KOH (0.13 g, 2.4 mmol) in MeOH (10 mL) according to the general procedure.
An analytically pure sample of 6b (0.14 g, 72%) was obtained by flash column chromatography (silica
gel, 0–5% MeOH in CHCl3). M.p. 117.5 ◦C [decomposition]. [α2D6.3] + 33◦ (c 0.20 g/100 mL; CHCl3).

Molecules 2018, 23, 679
13 of 19
HRMS: m/z calcd for C31H47NO3: 481.3551; found: 481.3553. Anal. Calcd. for C31H47NO3: C, 77.29;
1
H, 9.83; N, 2.91; O, 9.96; found C, 77.60; H, 9.74; N, 2.99. H-NMR (CDCl3, 300 MHz):
δ
= 7.89 (s, 1H,
N
H
), 7.40 (d, 2H, J = 8.2, H-20, H-60), 7.05 (d, 2H, J = 8.1, H-30, H-50), 3.96 (s, 1H, H-12(
)), 2.90 (br s, 2H, OH), 2.26 (s, 3H, CH3-7 ), 0.96 (d, 3H, J21,20 = 6.3, CH3-21), 0.87 (s, 3H, CH3-19),
β
)), 3.57 (m, 1H,
0
H-3(
β
0.64 (s, 3H, CH3-18). 13C-NMR (CDCl3, 75 MHz): δ = 172.18 (s, C-24), 135.61 (s, C-10), 133.34 (d, C-40),
129.23 (d, C-30), 129.23 (d, C-50), 119.70 (d, C-20), 119.70 (d, C-60), 73.13 (d, C-12), 71.65 (d, C-3), 47.80,
46.40, 46.37, 41.96, 36.19, 35.89, 35.18, 35.02, 34.05, 33.67, 33.43, 31.11, 30.43, 28.25, 27.46, 27.11, 26.07,
23.63, 22.99 (q, C-19), 20.71 (q, CH3-70), 17.34 (q, C-21), 12.53 (q, C-18).
3.1.15. N-(Pyridin-3-yl)-3α,12α-diacetoxy-5β-cholan-24-amide (7a)
Crude product as an amorphous white solid (1.34 g, quantitative yield) was obtained from acid
chloride intermediate (preparing by reaction diacetoxy derivative 3 (1.0 g, 2.1 mmol) and oxalyl
chloride (1.1 mL, 12.6 mmol) in CH2Cl2 (10 mL)) and 3-aminopyridine (0.24 g, 2.5 mmol) with addition
of NEt3 (0.5 mL, 3.1 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product
was purified by flash column chromatography (silica gel, 0–5% MeOH in CHCl3) to yield compound
7a (0.72 g, 62%) as an amorphous white. M.p. 161.2-161.7 ◦C. [α2D6] + 75◦ (c 0.20 g/100 mL; CHCl3).
HRMS: m/z calcd for C33H48N2O5: 552.3558; found: 552.3563. Anal. Calcd. for C33H48N2O5: C, 71.71;
1
H, 8.75; N, 5.07; O, 14.47; found C, 71.47; H, 8.52; N, 5.16. H-NMR (CDCl3, 600 MHz):
δ = 8.54 (d, 1H,
J = 2.3, H-20), 8.30 (d, 1H, J = 4.6, H-40), 8.23 (s, 1H, N
4.1, H-50), 5.07 (s, 1H, H-12( )), 2.44 (m, 1H, H-23), 2.25 (m, 1H, H-230), 2.09 (s,
)), 4.68 (m, 1H, H-3(
3H, CH3-26), 2.02 (s, 3H, CH3-28), 0.89 (s, 3H, CH3-19), 0.80 (d, 3H, J21,20 = 5.8, CH3-21), 0.70 (s, 3H,
H
), 8.20 (d, 1H, J = 8.6, H-60), 7.26 (dd, 1H, J = J =
β
β
CH3-18). 13C-NMR (CDCl3, 150 MHz):
δ
= 172.30 (s,0C-24), 170.53 (s0, C-25), 170.50 (s, C-27), 144.68
0
0
0
(d, C-4 ), 140.71 (d, C-6 ), 135.07 (s, C-3 ), 127.06 (d, C-2 ), 123.64 (d, C-5 ), 75.80 (d, C-12), 74.07 (d, C-3),
49.22, 47.65, 44.86, 41.60, 35.45, 34.71, 34.28, 34.20, 33.85, 32.06, 31.16, 29.53, 27.23, 26.68, 26.45, 25.67,
25.50, 23.26, 22.91 (q, C-19), 21.36 (q, CH3-C(O)O), 21.30 (q, CH3-C(O)O), 17.49 (q, C-21), 12.30 (q, C-18).
3.1.16. N-(Pyridin-3-yl)-3α,12α-dihydroxy-5β-cholan-24-amide (7b)
Crude product as an amorphous white solid (0.44 g, 88%) was prepared from diacetoxy derivative
7a (0.59 g, 1.1 mmol) and KOH (0.37 g, 6.6 mmol) in MeOH (10 mL) according to the general procedure.
An analytically pure sample of 7b (0.22 g, 44%) was obtained by flash column chromatography (silica
gel, 1–5% MeOH in CHCl3). M.p. 114.6 ◦C [decomposition]. [α2D7.1] + 35◦ (c 0.20 g/100 mL; CH3OH).
HRMS: m/z calcd for C29H44N2O3: 468.3347; found: 468.3344. Anal. Calcd. for C29H44N2O3: C,
1
74.32; H, 9.46; N, 5.98; O, 10.24; found C, 73.98; H, 9.44; N, 5.70. H-NMR (CDCl3, MeOH, 400 MHz):
0
0
0
δ
= 9.53 (s, 1H, N
H
), 8.45 (s, 1H, H-2 ), 8.24 (d, 1H, J = 7.9, H-4 ), 8.14 (d, 1H, J = 4.2, H-6 ), 7.23 (m, 1H,
)), 3.48 (m, 1H, H-3(
)), 2.36 (m, 1H, H-23), 2.22 (m, 1H, H-230), 0.93 (d, 3H,
21,20 = 5.8, CH3-21), 0.82 (s, 3H, CH3-19), 0.59 (s, 3H, CH3-18). 13C-NMR (CDCl3, MeOH, 100 MHz):
H-50), 3.88 (s, 1H, H-12(
J
β
β
δ
= 173.77 (s, C-24), 143.07 (d, C-40), 139.74 (d, C-60), 135.97 (s, C-30), 127.69 (d, C-20), 123.87 (d, C-50),
72.91 (d, C-12), 71.19 (d, C-3), 47.84, 46.20, 46.14, 41.79, 35.77, 35.68, 35.00, 34.98, 33.88, 33.27, 33.10,
31.04, 29.62, 28.24, 27.28, 26.85, 25.93, 23.48, 22.82 (q, C-19), 16.89 (q, C-21), 12.41 (q, C-18).
3.1.17. N-(10-Adamantyl)-3α,12α-diacetoxy-5β-cholan-24-amide (8a)
Crude product as an amorphous white solid (0.73 g, 95%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (0.60 g, 1.3 mmol) and oxalyl chloride
(0.6 mL, 7.8 mmol) in CH2Cl2 (10 mL)) and 1-aminoadamantane (prepared in situ from of
1-aminoadamantane hydrochloride (0.26 g, 1.4 mmol) and NEt3 (0.02 mL, 0.14 mmol)), with addition
of NEt3 (0.3 mL, 2.0 mmol) in CH2Cl2 (10 mL) according to the general procedure. The crude product
was purified by flash column chromatography (silica gel, CHCl3) t◦o yield compound 8a (0.36 g, 47%)
◦
as an amorphous white. M.p. 120.7 C. [decomposition]. [α2D6.1] + 68 (c 0.20 g/100 mL; CHCl3). HRMS:
m/z calcd for C38H59NO5: 609.4388; found: 609.4383. Anal. Calcd. for C38H59NO5: C, 74.84; H, 9.75;
1
N, 2.30; O, 13.12; found C, 74.76; H, 9.83; N, 2.41. H-NMR (CDCl3, 600 MHz):
δ = 5.07 (s, 1H, H-12(β)),

Molecules 2018, 23, 679
14 of 19
5.03 (dd, 1H, J = 5.0, N
H
), 4.66 (m, 1H, H-3(
β
)), 2.05 (s, 3H, CH3-26), 2.02 (br s, 3H, H-30, H-50, H-80),
1.99 (s, 3H, CH3-28), 1.94 (s, 6H, CH2-20, CH2-60, CH2-90), 1.63 (m, 6H, CH2-40, CH2-70, CH2-100), 0.86
(s, 3H, CH3-19), 0.76 (d, 3H, J21,20 = 6.5, CH3-21), 0.68 (s, 3H, CH3-18). 13C-NMR (CDCl3, 150 MHz):
δ
= 172.31 (s, C-24), 170.39 (s, C-25), 170.31 (s, C-27), 75.80 (d, C-12), 74.04 (d, C-3), 51.56, 49.27, 47.67,
44.87, 41.68, 41.55 (t, C-20, C-60, C-90), 36.22 (t, C-40, C-70, C-100), 35.53, 34.64, 34.57, 34.53, 34.25, 33.88,
32.11, 31.49, 29.29 (d, C-30, C-50, C-80), 27.22, 26.74, 26.47, 25.71, 25.49, 23.28, 22.91 (q, C-19), 21.30
(q, CH3-C(O)O), 21.23 (q, CH3-C(O)O), 17.53 (q, C-21), 12.29 (q, C-18).
3.1.18. N-(10-Adamantyl)-3α,12α-dihidroxy-5β-cholan-24-amide (8b)
Crude product as an amorphous white solid (0.17 g, quantitative yield) was prepared from
diacetoxy derivative 8a (0.20 g, 0.3 mmol) and KOH (0.10 g, 1.8 mmol) in MeOH (5 mL) according
to the general procedure. An analytically pure sample of 8b (0.12 g◦, 71%) was obtained by flash
◦
column chromatography (silica gel, 1–5% MeOH in CHCl3). M.p. 300.8 C [decomposition]. [α2D7] +23
(c 0.20 g/100 mL; CH3OH). HRMS: m/z calcd for C34H55NO3: 525.4177; found: 525.4173. Anal. Calcd.
1
for C34H55NO3: C, 77.66; H, 10.54; N, 2.66; O, 9.13; found C, 77.92; H, 10.29; N, 2.79. 0 H-N0MR (CDCl3,
0
CD3OD, 400 MHz): δ0= 3.79 (s, 1H, H-12(
0.74 (s, 3H, CH3-19), 0.51 (s, 3H, CH3-18). 13C-NMR (CDCl3, CD3OD, 100 MHz):
β
)), 3.38 (m, 1H, H-3(
β
)), 1.90 (br s, 3H, H-3 , H-5 , H-8 ), 1.82
0
0
0
0
0
(s, 6H, CH2-2 , CH2-6 , CH2-9 ), 1.51 (s, 6H, CH2-4 , CH2-7 , CH2-10 ), 0.81 (d, 3H, J21,20 = 6.3, CH3-21),
δ = 173.90 (s, C-24),
72.67 (d, C-12), 71.00 (d, C-3), 51.41, 47.64, 46.39, 46.01, 41.72, 40.95 (t, C-20, C-60, C-90), 35.91 (t, C-40,
C-70, C-100), 35.57, 35.54, 34.97, 34.89, 34.74, 33.12, 31.45, 29.38, 29.01 (d, C-30, C-50, C-80), 28.09, 27.19,
26.74, 25.80, 23.34, 22.64 (q, C-19), 16.66 (q, C-21), 12.23 (q, C-18).
00
0
0
0
3.1.19. N-(2 -(3 ,5 -Di-tret-buthyl-4 -hydroxyphenyl)-ethyl)-3α,12α-diacetoxy-5β-cholan-24-amide (9a)
Crude product as an amorphous white solid (0.29 g, 90%) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (0.20 g, 0.40 mmol) and oxalyl chloride
(0.2 mL, 2.5 mmol) in CH2Cl2 (10 mL)) and 2-(30,50-di-tret-buthyl-40-hydroxyphenyl)ethan-1-amine
(0.11 g, 0.44 mmol) with addition of NEt3 (0.1 mL, 0.66 mmol) in CH2Cl2 (10 mL) according to the
general procedure. The crude product was purified by flash column chromatography (silica gel,
0–2% MEOH in CHCl3) to ◦yield compound 9a (0.09 g, 28%) as an amorphous white. M.p. 98.7 ◦C.
[decomposition]. [α2D3.5] +66 (c 0.20 g/100 mL; CH3OH). HRMS: m/z calcd for C44H69O6N1: 707.5119;
found: 707.5122. Anal. Calcd. for C44H69NO6: C, 74.64; H, 9.82; N, 1.98; O, 13.56; found C, 74.38; H,
1
9.86; N, 2.11. H-NMR (CDCl3, 300 MHz):
δ
= 6.94 (s, 2H, H-20, H-60), 5.48 (m, 1H, NH), 5.09 (s, 1H,
OH), 5.04 (s, 1H, H-12(
(s, 3H, CH3-26), 2.00 (s, 3H, CH3-28), 0.87 (s, 3H, CH3-19), 0.77 (d, 3H, J21,20 = 6.1, CH3-21), 0.69 (s,
β)), 4.67 (m, 1H, H-3(β
)), 3.43 (m, 2H, CH2-160), 2.69 (m, 2H, CH2-150), 2.06
3H, CH3-18). 13C-NMR (CDCl3, 75 MHz):
δ
= 173.030(s, C-24), 170.40 (s, C-25), 170.30 (s, C-27), 152.21
0
0
0
0
0
(s, C-4 ), 135.94 (s, C-3 ), 135.94 (s, C-5 ), 129.31 (s, C-1 ), 125.18 (d, C-2 ), 125.18 (d, C-6 ), 75.77 (d, C-12),
74.04 (d, C-3), 49.26, 47.58, 44.86, 41.67, 40.79, 35.52, 35.49, 34.74, 34.57, 34.24, 34.14, 33.88, 33.59, 32.11,
31.53, 30.17 (q, C(CH3)3), 27.25, 26.73, 26.48, 25.70, 25.49, 23.26, 22.92 (q, C-19), 21.31 (q, CH3-C(O)O),
21.23 (q, CH3-C(O)O), 17.49 (q, C-21), 12.28 (q, C-18).
3.1.20. N-(30,30-Diethylaminopropyl)-3α,12α-diacetoxy-5β-cholan-24-amide (10a)
Crude product as an amorphous yellow (2.2 g, 92%) was obtained from acid chloride intermediate
(preparing by reaction diacetoxy derivative 3 (2.0 g, 4.2 mmol) and oxalyl chloride (2.1 mL, 25.2 mmol)
in CH2Cl2 (10 mL)) and N1,N1-diethylaminopropan-1,3-diamine (0.65 g, 5.0 mmol) with addition of
NEt3 (0.9 mL, 6.3 mmol) in CH2Cl2 (20 mL) according to the general procedure. The crude product
was purified by flash column chromatography (silica gel, 0–20% MeOH in CHCl3) to yield compound
◦
◦
10a (1.70 g, 67%) as an amorphous yellow. M.p. 45.8 C [decomposition]. [α2D4.8] + 75 (c 0.20 g/100 mL;
CHCl3). HRMS: m/z calcd for C35H60N2O5: 588.4497; found: 588.4495. Anal. Calcd. for C35H60N2O5:
1
C, 71.39; H, 10.27; N, 4.76; O, 13.59; found C, 71.44; H, 10.23; N, 4.79. H-NMR (CDCl3, 400 MHz):
δ
= 7.44 (m, 1H, NH), 4.99 (s, 1H, H-12(β)), 4.60 (m, 1H, H-3(β
)), 3.23 (ddd, 2H, J = J = J = 5.6, CH2-10),

Molecules 2018, 23, 679
15 of 19
2.41 (ddd, 6H, J = J = J = 7.0, CH2-30, CH2-40, CH2-60), 2.00 (s, 3H, CH3-26), 1.94 (s, 3H, CH3-28), 0.81
(s, 3H, CH3-19), 0.71 (d, 3H, J21,20 = 6.2, CH3-21), 0.63 (s, 3H, CH3-18). 13C-NMR (CDCl3, 100 MHz):
δ
= 172.83 (s, C-24), 170.27 (s, C-25), 170.20 (s, C-27), 75.64 (d, C-12), 73.89 (d, C-3), 52.49, 49.14, 47.44,
0
0
46.54 (t, C-4 ), 46.54 (t, C-6 ), 44.69, 41.52, 39.72, 35.35, 34.61, 34.42, 34.09, 33.73, 33.49, 31.94, 31.55, 27.10,
26.59, 26.33, 25.56, 25.35, 25.12, 23.15, 22.80 (q, C-19), 21.20 (q,
17.32 (q, C-21), 12.13 (q, C-18), 11.60 (q, C-50), 11.60 (q, C-70).
CH3-C(O)O), 21.10 (q, CH3-C(O)O),
3.1.21. N-(30-Morpholinopropyl)-3α,12α-diacetoxy-5β-cholan-24-amide (11a).
Crude product as an amorphous solid (2.5 g, quantitative yield) was obtained from acid chloride
intermediate (preparing by reaction diacetoxy derivative 3 (2.0 g, 4.2 mmol) and oxalyl chloride (2.1 mL,
25.2 mmol) in CH2Cl2 (10 mL)) and 3-morpholinopropan-1-amine (0.72 g, 5.0 mmol) with addition of
NEt3 (0.9 mL, 6.3 mmol) in CH2Cl2 (20 mL) according to the general procedure. The crude product was
purified by successive flash column chromatographies: (1) silica gel, 0–1% MeOH in CHCl3; (2) silica
gel, 15–40% AcOEt in n-hexane, to yield compound 11a (1.75 g, 69%) as an amorphous white solid. M.p.
61.0 ◦C [decomposition]. [α2D5] + 78◦ (c 0.20 g/100 mL; CHCl3). HRMS: m/z calcd for C35H58N2O6:
602.4289; found: 602.4287. Anal. Calcd. for C35H58N2O6: C, 69.73; H, 9.70; N, 4.65; O, 15.92 found C,
69.75; H, 9.73; N, 4.62; 1H-NMR (CDCl3, 400 MHz):
4.60 (m, 1H, H-3(
)), 3.61 (dd, 4H, J = J = 4.2, CH2-50, CH2-60), 3.23 (ddd, 2H, J = J = J = 5.9, CH2-10),
2.36 (m, 6H, CH2-30, CH2-40, CH2-70), 2.00 (s, 3H, CH3-26), 1.94 (s, 3H, CH3-28), 0.81 (s, 3H, CH3-19),
δ = 6.81 (dd, 1H, J = 5.0, NH), 4.98 (s, 1H, H-12(β)),
β
0.72 (d, 3H, J21,20 = 6.3, CH3-21), 0.63 (s, 3H, CH3-18). 13C-NMR (CDCl3, 100 MHz):
δ = 172.92 (s, C-24),
170.27 (s, C-25), 170.19 (s, C-27), 75.59 (d, C-12), 73.87 (d, C-3), 66.72 (t, C-50, C-60), 57.47 (t, C-30), 53.40
(t, C-40, C-70), 49.11, 47.46, 44.69, 41.50, 38.86 (t, C-10), 35.33, 34.60, 34.41, 34.06, 33.71, 33.50, 31.92,
31.49, 27.12, 26.69, 26.57, 26.31, 25.54, 25.34, 24.75, 23.13, 22.80 (q, C-19), 21.20 (q,
(q, CH3-C(O)O), 17.34 (q, C-21), 12.15 (q, C-18).
CH3-C(O)O), 21.09
3.2. Tdp1 Assay
The recombinant Tdp1 was purified to homogeneity by chromatography on Ni-chelating resin
and phosphocellulose P11 as described [42 43] using the plasmid pET 16B-Tdp1 kindly provided by
,
Dr. K.W. Caldecott (University of Sussex, United Kingdom).
Real-Time Detection of Tdp1 Activity. The Tdp1 activity measurements were carried out as
described [25]. Briefly, Tdp1-biosensor with a final concentration of 50 nM was incubated in a volume
of 200 µL containing a Tdp1 buffer (50 mM Tris-HCl pH8.0, 50 mM NaCl, 7 mM β-mercaptoethanol)
supplemented with purified 1.3 nM Tdp1 and various concentrations of the inhibitor in a 1.5% DMSO
solution. Fluorescence measurements (Ex485/Em520 nm) were carried out during the linear phase
of reaction (from 0 to 8 min) every 55 s. The reactions were incubated at a constant temperature of
26 ◦C in a POLARstar OPTIMA fluorimeter, BMG LABTECH, GmbH. The influence of compounds
was evaluated by comparing the fluorescence increase rate in the presence of compounds to that of
the DMSO control wells. The data were imported into the MARS Data Analysis 2.0 program (BMG
LABTECH), and IC50 values (concentration of a compound required to reduce the enzyme activity by
50%) were calculated. Tdp1-biosensor 50-(5,6 FAM-aac gtc agg gtc ttc c-BHQ1)-30 was synthesized in
the Laboratory of Biomedical Chemistry, Institute of Chemical Biology and Fundamental Medicine,
Novosibirsk, Russia.
IC50 values are listed in Tables 1 and 2.
3.3. Analysis of Cytotoxicity
Cytotoxicity analysis of the compounds was carried out on the cell lines MCF-7 (human breast
adenocarcinoma) and HCT-116 (human colon carcinoma). Compound-induced cell death was assessed
using a standard MTT test [44]. MCF-7 and HCT-116 cells (~2000 cells per well) were incubated for 24 h
◦
at 37 C in an IMDM medium containing 10% (v/v) fetal bovine serum, 40
µg/mL gentamicin,
100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25
µg/mL amphotericin in a humidified

Molecules 2018, 23, 679
16 of 19
atmosphere (5% CO2). After the formation of a 50% monolayer, the test preparations in DMSO
were added to the culture medium (the volume of the added reagents was 1/100 of the total volume
of the culture medium, the volume of DMSO was 1% of the final volume), and the growth of the cell
culture was observed for three days. Control cells were grown in the presence of 1% DMSO.
The toxicity of the compounds was absent or was insignificant throughout the range of
concentrations studied (up to 100 µM).
3.4. Virtual Screening and Molecular Modelling
The compounds were screened/docked to the crystal structure of Tdp1 (PDB ID: 1MU7, resolution
2.0 Å) [32], which was obtained from the Protein Data Bank (PDB) [45,46]. The SciGress FJ 2.6 program
was used to prepare the crystal structure for docking [47], i.e., hydrogen atoms were added,
and the co-crystallized tungsten(VI)ion was removed as well as the crystallographic water molecules.
The SciGress software suite was also used to build the inhibitors and the MM2 force field was used
to optimize the structures [48]. The center of the binding pocket was defined as the position of the
hydrogen atom of His263 and the ND1 nitrogen formed a coordination bond with the tungsten ion
(x = 8.312, y = 12.660, z = 35.452) with 10 Å radius. For the initial screen, 30% search efficiency was
used with ten runs per compound. For the second phase (re-dock) and the molecular modelling,
100% efficiency was used in conjunction with fifty docking runs. The basic amino acids lysine and
arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be
deprotonated. The GoldScore (GS) [49], ChemScore (CS) [50,51], Chem Piecewise Linear Potential
(ChemPLP) [52], and Astex Statistical Potential (ASP) [53] scoring functions were implemented to
validate the predicted binding modes and relative energies of the ligands using the GOLD v5.4 software
suite [49]. The virtual screen was conducted with of 9.2
InterBioScreen natural product collection. The Marvin Sketch software package was used to calculate
the molecular descriptors of the compounds [54].
×
103 molecular entities obtained from the
4. Conclusions
Thus, the amides obtained from deoxycholic acid and containing tryptamine (compound
1a
bis-t-butylphenol (compound 9a) fragments demonstrated high inhibition activity against Tdp1 with
IC50 values in the range 0.29–0.47 M. It should also be noted that these compounds have a more
–3a), p-bromoaniline (compound 5a), 1-aminoadamantane (compound 8a) and 4-(2-aminoethyl)-2,6-
µ
pronounced activity than the reference substance furamidine. The compounds demonstrated low
cytotoxicity which may simplify their use as a component of “cocktails” with known anticancer drugs.
Thus, the compounds can be considered as a basis for the development of new promising agents for
the combined chemotherapy of oncological diseases.
Obviously, the use of metabolically unstable acetoxy group makes the successful transition to
in vivo experiments unlikely. Therefore, the purpose of our further research will be the modification of
the steroid scaffold by metabolically stable functional groups and the study of their effect on inhibitory
activity against Tdp1.
Supplementary Materials: Supplementary materials are available online: virtual screening & molecular modeling
data and copies of 1H-NMR and 13C-NMR spectra of all compounds.
Acknowledgments: This work was supported by the Russian Science Foundation (Grant 16-13-10074).
The authors would like to acknowledge the Multi-Access Chemical Service Center SB RAS for spectral and
analytical measurements.
Author Contributions: All the authors together conceived and planned the research, discussed the results,
and wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 679
17 of 19
References
1.
Lu, J.Y.D.; Su, P.; Barber, J.E.M.; Nash, J.E.; Le, A.D.; Liu, F.; Wong, A.H.C. The neuroprotective effect of
nicotine in Parkinson’s disease models is associated with inhibiting PARP-1 and caspase-3 cleavage. PeerJ
2017, 5, e3933. [CrossRef] [PubMed]
2.
3.
4.
Dexheimer, T.S.; Antony, S.; Marchand, C.; Pommier, Y. Tyrosyl-DNA phosphodiesterase as a target for
anticancer therapy. Anticancer Agents Med. Chem. 2008, 8, 381–389. [CrossRef] [PubMed]
Laev, S.S.; Salakhutdinov, N.F.; Lavrik, O.I. Tyrosyl-DNA phosphodiesterase inhibitors: Progress and
potential. Bioorg. Med. Chem. 2016, 24, 5017–5027. [CrossRef] [PubMed]
Jdey, W.; Thierry, S.; Russo, C.; Devun, F.; Al Abo, M.; Noguiez-Hellin, P.; Sun, J.S.; Barillot, E.; Zinovyev, A.;
Kuperstein, I.; et al. Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of
AsiDNA and PARP Inhibitors. Clin. Cancer Res. 2017, 23, 1001–1011. [CrossRef] [PubMed]
Huang, S.N.; Pommier, Y.; Marchand, C. Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors. Expert Opin.
Ther. Pat. 2011, 21, 1285–1292. [CrossRef] [PubMed]
Beretta, G.L.; Cossa, G.; Gatti, L.; Zunino, F.; Perego, P. Tyrosyl-DNA phosphodiesterase 1 targeting for
modulation of camptothecin-based treatment. Curr. Med. Chem. 2010, 17, 1500–1508. [CrossRef] [PubMed]
Geenen, J.J.J.; Linn, S.C.; Beijnen, J.H.; Schellens, J.H.M. PARP Inhibitors in the Treatment of Triple-Negative
Breast Cancer. Clin. Pharmacokinet. 2017, 1–11. [CrossRef] [PubMed]
Pommier, Y. Topoisomerase I inhibitors: Camptothecins and beyond. Nat. Rev. Cancer 2006, 6, 789–802.
[CrossRef] [PubMed]
5.
6.
7.
8.
9.
Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. DNA topoisomerases and their poisoning by anticancer and
antibacterial drugs. Chem. Biol. 2010, 17, 421–433. [CrossRef] [PubMed]
10. Kawale, A.S.; Povirk, L.F. Tyrosyl-DNA phosphodiesterases: Rescuing the genome from the risks of
relaxation. Nucleic Acids Res. 2018, 46, 520–537. [CrossRef] [PubMed]
11. Nguyen, T.X.; Morrell, A.; Conda-Sheridan, M.; Marchand, C.; Agama, K.; Bermingham, A.; Stephen, A.G.;
Chergui, A.; Naumova, A.; Fisher, R.; et al. Synthesis and biological evaluation of the first dual tyrosyl-DNA
phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors. J. Med. Chem. 2012, 55, 4457–4478. [CrossRef]
[PubMed]
12. Borda, M.A.; Palmitelli, M.; Verón, G.; González-Cid, M.; de Campos Nebel, M. Tyrosyl-DNA-
phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes
in human cells. Mutat. Res. 2015, 781, 37–48. [CrossRef] [PubMed]
13. El-Khamisy, S.F.; Katyalm, S.; Patel, P.; Ju, L.; McKinnon, P.J.; Caldecott, K.W. Synergistic decrease of DNA
single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin. DNA Repair 2009, 8,
760–766. [CrossRef] [PubMed]
14. Das, B.B.; Antony, S.; Gupta, S.; Dexheimer, T.S.; Redon, C.E.; Garfield, S.; Shiloh, Y.; Pommier, Y. Optimal
function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK. EMBO J.
2009, 28, 3667–3680. [CrossRef] [PubMed]
15. Katyal, S.; El-Khamisy, S.F.; Russell, H.R.; Li, Y.; Ju, L.; Caldecott, K.W.; McKinnon, P.J. TDP1 facilitates
chromosomal single-strand break repair in neurons and is neuroprotective in vivo. EMBO J. 2007, 26,
4720–4731. [CrossRef] [PubMed]
16. Hirano, R.; Interthal, H.; Huang, C.; Nakamura, T.; Deguchi, K.; Choi, K.; Bhattacharjee, M.B.; Arimura, K.;
Umehara, F.; Izumo, S.; et al. Spinocerebellar ataxia with axonal neuropathy: Consequence of a Tdp1
recessive neomorphic mutation. EMBO J. 2007, 26, 4732–4743. [CrossRef] [PubMed]
17. Barthelmes, H.U.; Habermeyer, M.; Christensen, M.O.; Mielke, C.; Interthal, H.; Pouliot, J.J.; Boege, F.;
Marko, D. TDP1 overexpression in human cells counteracts DNA damage mediated by topoisomerases I
and II. J. Biol. Chem. 2004, 279, 55618–55625. [CrossRef] [PubMed]
18. Nivens, M.C.; Felder, T.; Galloway, A.H.; Pena, M.M.; Pouliot, J.J.; Spencer, H.T. Engineered resistance to
camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate
synthase. Cancer Chemother. Pharmacol. 2004, 53, 107–115. [CrossRef] [PubMed]
19. Alagoz, M.; Wells, O.S.; El-Khamisy, S.F. TDP1 deficiency sensitizes human cells to base damage via distinct
topoisomerase I and PARP mechanisms with potential applications for cancer therapy. Nucleic Acids Res.
2014, 42, 3089–3103. [CrossRef] [PubMed]

Molecules 2018, 23, 679
18 of 19
20. Murai, J.; Huang, S.Y.; Das, B.B.; Dexheimer, T.S.; Takeda, S.; Pommier, Y. Tyrosyl-DNA phosphodiesterase
1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells.
J. Biol. Chem. 2012, 287, 12848–12857. [CrossRef] [PubMed]
21. Cortes Ledesma, F.; El Khamisy, S.F.; Zuma, M.C.; Osborn, K.; Caldecott, K.W. A human 50-tyrosyl DNA
phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature 2009, 461, 674–678. [CrossRef]
[PubMed]
22. Comeaux, E.; van Waardenburg, R.C. Tyrosyl-DNA phosphodiesterase I resolves both naturally and
chemically induced DNA adducts and its potential as a therapeutic target. Drug Metab. Rev. 2014, 46,
494–507. [CrossRef] [PubMed]
23. Antony, S.; Marchand, C.; Stephen, A.; Thibaut, L.; Agama, K.; Fisher, R.; Pommier, Y. Novel high-throughput
electrochemiluminescent assay for identification of human tyrosyl–DNA phosphodiesterase (Tdp1) inhibitors
and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1. Nucleic Acids Res. 2007, 35,
4474–4484. [CrossRef] [PubMed]
24. Dexheimer, T.; Gediya, L.; Stephen, A.; Weidlich, I.; Antony, S.; Marchand, C.; Interthal, H.; Niklaus, M.;
Fisher, R.; Njar, V.; et al. 4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and related
novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors. J. Med. Chem. 2009, 52,
7122–7131. [CrossRef] [PubMed]
25. Zakharenko, A.; Khomenko, T.; Zhukova, S.; Koval, O.; Zakharova, O.; Anarbaev, R.; Lebedeva, N.;
Korchagina, D.; Komarova, N.; Vasiliev, V. Synthesis and biological evaluation of novel tyrosyl-DNA
phosphodiesterase 1 inhibitors with a benzopentathiepine moiety. Bioorg. Med. Chem. 2015, 9, 2044–2052.
[CrossRef] [PubMed]
26. Nguyen, T.X.; Abdelmalak, M.; Marchand, C.; Agama, K.; Pommier, Y.; Cushman, M. Synthesis and biological
evaluation of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines as potential dual topoisomerase I
(Top1)-tyrosyl-DNA phosphodiesterase I (TDP1) inhibitors. J. Med. Chem. 2015, 58, 3188–3208. [CrossRef]
[PubMed]
27. Wang, P.; Elsayed, M.; Plescia, C.; Ravji, A.; Redon, C.; Kiselev, E.; Marchand, C.; Zeleznik, O.; Agama, K.;
Pommier, Y.; et al. Synthesis and biological evaluation of the first triple inhibitors of human topoisomerase
1, tyrosyl-DNA phosphodiesterase 1 (Tdp1), and tyrosyl-DNA phosphodiesterase 2 (Tdp2). J. Med. Chem.
2017, 60, 3275–3288. [CrossRef] [PubMed]
28. Khomenko, T.; Zakharenko, A.; Odarchenko, T.; Arabshahi, H.; Sannikova, V.; Zakharova, O.; Korchagina, D.;
Reynisson, J.; Volcho, K.; Salakhutdinov, N.; et al. New inhibitors of tyrosyl-DNA phosphodiesterase I
(Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties. Bioorg. Med. Chem. 2016, 24, 5573–5581.
[CrossRef] [PubMed]
29. Zakharenko, A.; Luzina, O.; Koval, O.; Nilov, D.; Gushchina, I.; Dyrkheeva, N.; ˇSvedas, V.; Salakhutdinov, N.;
Lavrik, O. Tyrosyl-DNA phosphodiesterase 1 inhibitors: Usnic acid enamines enhance the cytotoxic effect of
camptothecin. J. Nat. Prod. 2016, 79, 2961–2967. [CrossRef] [PubMed]
30. Ponomarev, K.Y.; Suslov, E.V.; Zakharenko, A.L.; Zakharova, O.D.; Rogachev, A.D.; Korchagina, D.V.;
Zafar, A.; Reynisson, J.; Nefedov, A.; Volcho, K.P.; et al.
Aminoadamantanes Containing
Monoterpene-derived Fragments as Potent Tyrosyl-DNA phosphodiesterase 1 Inhibitors. Bioorg. Chem. 2018
,
76, 392–399. [CrossRef] [PubMed]
31. Davies, D.R.; Interthal, H.; Champoux, J.J.; Hol, W.G.J. Crystal Structure of a Transition State Mimic for Tdp1
Assembled from Vanadate, DNA, and a Topoisomerase I-Derived Peptide. Chem. Biol. 2003, 10, 139–147.
[CrossRef]
32. Davies, D.R.; Interthal, H.; Champoux, J.J.; Hol, W.G.J. Insights into Substrate Binding and Catalytic
Mechanism of Human Tyrosyl-DNA Phosphodiesterase (Tdp1) from Vanadate and Tungstate-inhibited
Structures. J. Mol. Biol. 2002, 324, 917–932. [CrossRef]
33. Hofmann, A.F.; Hagey, L.R. Key discoveries in bile acid chemistry and biology and their clinical applications:
History of the last eight decades. J. Lipid Res. 2014, 55, 1553–1595. [CrossRef] [PubMed]
34. Russell, D.W. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 2003, 72,
137–174. [CrossRef] [PubMed]
35. Navacchia, M.L.; Marchesi, E.; Mari, L.; Chinaglia, N.; Gallerani, E.; Gavioli, R.; Capobianco, M.L.; Perrone, D.
Rational Design of Nucleoside–Bile Acid Conjugates Incorporating a Triazole Moiety for Anticancer
Evaluation and SAR Exploration. Molecules 2017, 22, 1710. [CrossRef] [PubMed]

Molecules 2018, 23, 679
19 of 19
36. Li, X.; Zhao, T.; Cheng, D.; Chu, C.; Tong, S.; Yan, J.; Li, Q.-Y. Synthesis and Biological Activity of Some Bile
Acid-Based Camptothecin Analogues. Molecules 2014, 19, 3761–3776. [CrossRef] [PubMed]
37. Popadyuk, I.I.; Salomatina, O.V.; Salakhutdinov, N.F. Modern approaches to modification of bile acids for
the synthesis of compounds possessing valuable physicochemical and biological properties. Russ. Chem. Rev.
2017, 86, 388–443. [CrossRef]
38. Sharma, R.; Long, A.; Gilmer, J.F. Advances in bile acid medicinal chemistry. Curr. Med. Chem. 2011, 18,
4029–4052. [CrossRef] [PubMed]
39. Davis, A.P. Bile Acid Scaffolds in Supramolecular Chemistry: The Interplay of Design and Synthesis.
Molecules 2007, 12, 2106–2122. [CrossRef] [PubMed]
40. Zhu, F.; Logan, G.; Reynisson, J. Wine Compounds as a Source for HTS Screening Collections. A Feasibility
Study. Mol. Inf. 2012, 31, 847–855. [CrossRef] [PubMed]
41. Brossard, D.; Lechevrel, M.; El Kihel, L.; Quesnelle, C.; Khalid, M.; Moslemi, S.; Reimund, J.M. Synthesis
and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon
adenocarcinoma cell lines. Eur. J. Med. Chem. 2014, 86, 279–290. [CrossRef] [PubMed]
42. Interthal, H.; Pouliott, J.J.; Champoux, J.J. The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the
phospholipase D superfamily. Proc. Natl. Acad. Sci. USA 2001, 98, 12009–12014. [CrossRef] [PubMed]
43. Lebedeva, N.A.; Rechkunova, N.I.; Lavrik, O.I. AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.
FEBS Lett. 2011, 585, 683–686. [CrossRef] [PubMed]
44. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
45. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E.
The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235–242. [CrossRef] [PubMed]
46. Berman, H.; Henrick, K.; Nakamura, H. Announcing the worldwide Protein Data Bank. Nat. Struct. Mol. Biol.
2003, 10, 980. [CrossRef] [PubMed]
47. Fijitsu Limited. SciGress Ultra v. F.J 2.6; Fijitsu Limited: Tokyo, Japan, 2000.
48. Allinger, N.L. Conformational analysis. 130. MM2. A hydrocarbon force field utilizing V1 and V2 torsional
terms. J. Am. Chem. Soc. 1977, 99, 8127–8134. [CrossRef]
49. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and validation of a genetic algorithm
for flexible docking. J. Mol. Biol. 1997, 267, 727–748. [CrossRef] [PubMed]
50. Eldridge, M.D.; Murray, C.W.; Auton, T.R.; Paolini, G.V.; Mee, R.P. Empirical scoring functions: I.
The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor
complexes. J. Comput.-Aided Mol. Des. 1997, 11, 425–445. [CrossRef] [PubMed]
51. Baxter, C.A.; Murray, C.W.; Clark, D.E.; Westhead, D.R.; Eldridge, M.D. Flexible Docking Using Tabu Search
and an Empirical Estimate of Binding Affinity. Proteins Struct. Funct. Bioinform. 1998, 33, 367–382. [CrossRef]
52. Korb, O.; Stützle, T.; Exner, T.E. Empirical scoring functions for advanced protein-ligand docking with
PLANTS. J. Chem. Inf. Model. 2009, 49, 84–96. [CrossRef] [PubMed]
53. Mooij, W.T.M.; Verdonk, M.L. General and targeted statistical potentials for protein-ligand interactions.
Proteins Struct. Funct. Bioinform. 2005, 61, 272–287. [CrossRef] [PubMed]
54. ChemAxon-Marvin. Version 15.7.20.0. 2015. Available online: http://www.chemaxon.com (accessed on
16 March 2018).
Sample Availability: Samples of the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).