Synthesis and anticancer activity of hybrid molecules based on lithocholic and (5Z,9Z)-tetradeca-5,9-dienedioic acids linked via mono(di,tri,tetra)ethylene glycol and α,ω-diaminoalkane units

Article abstract of DOI:10.3390/ph14020084

For the first time, hybrid molecules were synthesized on the basis of lithocholic and (5Z,9Z)- 1,14-tetradeca-5,9-dienedicarboxylic acids, obtained in two stages using the homo-cyclomagnesiation reaction of 2-(hepta-5,6-diene-1-yloxy)tetrahydro-2H-pyran at the key stage. The resulting hybrid molecules containing 5Z,9Z-dienoic acids are of interest as novel synthetic biologically active precursors to create modern drugs for the treatment of human oncological diseases. The synthesized hybrid molecules were found to exhibit extremely high in vitro inhibitory activity against human topoisomerase I, which is 2-4 times higher than that of camptothecin, a known topoisomerase I inhibitor. Using flow cytometry and fluorescence microscopy, it was first shown that these new molecules are efficient apoptosis inducers in HeLa, U937, Jurkat, K562, and Hek293 cell cultures. In addition, the results of investigations into the effect of the synthesized acids on mitochondria and studies of possible DNA damage in Jurkat tumor cells are also presented.

Full text of DOI:10.3390/ph14020084

pharmaceuticals
Article
Synthesis and Anticancer Activity of Hybrid Molecules Based
on Lithocholic and (5Z,9Z)-Tetradeca-5,9-Dienedioic Acids
Linked via Mono(di,tri,tetra)ethylene Glycol and
α,ω-Diaminoalkane Units
Vladimir A. D’yakonov * , Regina A. Tuktarova, Lilya U. Dzhemileva * , Svetlana R. Ishmukhametova and
Usein M. Dzhemilev
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, pr. Oktyabrya 141, 450075 Ufa, Russia;
regina-tuktarova@yandex.ru (R.A.T.); IshmukhametovaSR@gmail.com (S.R.I.); dzhemilev@anrb.ru (U.M.D.)
*
Correspondence: DyakonovVA@gmail.com (V.A.D.); Dzhemilev@mail.ru (L.U.D.); Tel.: +7-347-284-2750 (V.A.D.)
Abstract: For the first time, hybrid molecules were synthesized on the basis of lithocholic and (5Z,9Z)-
1,14-tetradeca-5,9-dienedicarboxylic acids, obtained in two stages using the homo-cyclomagnesiation
reaction of 2-(hepta-5,6-diene-1-yloxy)tetrahydro-2H-pyran at the key stage. The resulting hybrid
molecules containing 5Z,9Z-dienoic acids are of interest as novel synthetic biologically active pre-
cursors to create modern drugs for the treatment of human oncological diseases. The synthesized
hybrid molecules were found to exhibit extremely high in vitro inhibitory activity against human
topoisomerase I, which is 2–4 times higher than that of camptothecin, a known topoisomerase I
inhibitor. Using flow cytometry and fluorescence microscopy, it was first shown that these new
molecules are efficient apoptosis inducers in HeLa, U937, Jurkat, K562, and Hek293 cell cultures.
In addition, the results of investigations into the effect of the synthesized acids on mitochondria and
studies of possible DNA damage in Jurkat tumor cells are also presented.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: D’yakonov, V.A.;
Tuktarova, R.A.; Dzhemileva, L.U.;
Ishmukhametova, S.R.; Dzhemilev,
U.M. Synthesis and Anticancer
Activity of Hybrid Molecules Based
on Lithocholic and (5Z,9Z)-Tetradeca-
5,9-Dienedioic Acids Linked via
Mono(di,tri,tetra)ethylene Glycol and
α,ω-Diaminoalkane Units.
Keywords: lithocholic acid; cross-cyclomagnesiation; anticancer activity; 5Z,9Z-dienoic acids;
apoptosis; cell signaling
Pharmaceuticals 2021, 14, 84. https://
doi.org/10.3390/ph14020084
1. Introduction
Academic Editors: Jean Jacques
Vanden Eynde and Annie Mayence
Received: 23 December 2020
Accepted: 19 January 2021
Despite the fact that cancer control measures around the world are rapidly devel-
oping and new low-toxic drugs differing in their mechanism of action are being created,
chemotherapy remains a dangerous and difficult treatment with many side effects. One of
the main problems of modern oncology is the fact that tumor cells almost always develop
Published: 23 January 2021
multidrug resistance, which leads to failures in the treatment of cancer [1–3]. Accordingly,
the need for the development of more modern, low-toxic, and selective drugs remains an
urgent task for medicinal chemistry in general.
Flora and fauna are readily available sources of structurally diverse bioactive com-
pounds [4–7]. It is not surprising that chemical modifications have become one of the most
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
promising trends in the search for physiologically active compounds.
Bile acids can serve as attractive objects for synthetic transformations due to their
unique features, such as a chiral and rigid skeleton, as well as the presence of free acidic
and hydroxyl groups [8].
Copyright:
© 2021 by the authors.
Lithocholic acid (LA), one of the main bile acids found in the bile of mammals,
is formed in the intestines under the influence of microflora. In addition, LA and its
derivatives exhibit various biological activities. Many of these compounds are proteasome
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 (https://
creativecommons.org/licenses/by/
4.0/).
regulators [
cholecalciferols and the receptor [11
merases β (pol β) [14], and also have antibacterial [15] and antitumor effects [16–22].
9
,10], activate the vitamin D receptor, and enhance the interaction between
–
13], exhibit inhibitory activity toward DNA poly-
Pharmaceuticals 2021, 14, 84. https://doi.org/10.3390/ph14020084
https://www.mdpi.com/journal/pharmaceuticals

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Our recent studies in the search for new compounds aimed at treating oncological
diseases have shown that the synthesized hybrid molecules based on steroids and cis-
unsaturated acids are apoptosis inducers in Jurkat, K562, U937, HeLa tumor cell lines,
as well as in a specific cell line originally derived from human embryonic kidney cells
(HEK293), and can also inhibit in vitro the supercoiled DNA relaxation induced by topoi-
somerase I [23–26]. In the development of these studies, we prepared conjugates of LA
and (5Z,9Z)-tetradeca-5,9-dienedioic acid, which are linked to each other through ethy-
lene glycol and diaminoalkane spacers of different lengths. The antitumor activity of the
synthesized compounds has also been investigated.
2. Results and Discussion
2.1. Chemistry
The synthesis of target hybrid molecules based on lithocholic acid (LA, 6) and (5Z,9Z)-
tetradeca-5,9-dienedioic acid was performed using two approaches. The first one involves
the 3-O-acylation of acid 6 with a dicarboxylic acid. The second route represents the
modification of the LA carboxyl group at the C-24 position.
(5Z,9Z)-Tetradeca-5,9-diene-1,14-diol 4 and (5Z,9Z)-tetradeca-5,9-dienedioic acid 5
were synthesized employing the previously developed homo-cyclomagnesiation reac-
tion between tetrahydropyran ether of 5,6-hepta-5,6-dien-1-ol 1 and EtMgBr in the pres-
ence of Cp2TiCl2 (5 mol%) as the catalyst [23–27]. Subsequent deprotection of 1,14-bis-
tetrahydropyranyl-5Z,9Z-diene-1,14-diol 3 formed after hydrolysis of magnesacyclopen-
tane 2 in the presence of catalytic amounts of p-toluenesulfonic acid or its oxidation with
Jones reagent led to compounds 4 and 5, respectively (Scheme 1).
Scheme 1. Synthesis of (5Z,9Z)-tetradeca-5,9-diene-1,14-diol 4 and (5Z,9Z)-tetradeca-5,9-dienedioic acid 5. (a) EtMgBr, Mg,
Cp2TiCl2 (5 mol%), diethyl ether. (b) H3O+. (c) n-TsOH, CHCl3, MeOH. (d) H2CrO4/H2SO4, acetone, CH2Cl2.
Condensation of 3-
acid 5 was carried out by a well-known esterification procedure [23] using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) in the presence of N,N-4-
β-lithocholic acid methyl ester 7 with the corresponding fatty
·
dimethylaminopyridine (DMAP) in CH2Cl2 to give 8 with the 82% yield (Scheme 2).
Scheme 2. Synthesis of the LA derivative 8. (a) MeOH, AcCl, rt, 3 h. (b) (5Z,9Z)-tetradeca-5,9-
dienedioic acid 5, EDCl·HCl, DMAP, CH2Cl2.

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Scheme 3 shows the preparation of the LA derivative 11 bearing an acid fragment
in the C-24 position, which includes the esterification reaction between (5Z,9Z)-tetradeca-
5,9-diene-1,14-diol 4 and 3α-O-acetyllithocholic acid 9 and subsequent oxidation of the
resulting compound 10 with Jones reagent.
Scheme 3. Synthesis of the LA derivative 11. (a) AcCl, pyridine, CH2Cl2, DMAP, rt, 16 h. (b) (5Z,9Z)-
Тetradeca-5,9-diene-1,14-diol 4, EDCl·HCl, DMAP, CH2Cl2. (c) Jones reagent, acetone, CH2Cl2.
In order to study the effect of ethylene glycol and diaminoalkane chains on the antitu-
mor activity of the resulting LA derivatives, LA-fatty acid conjugates linked via ethylene
glycol and diaminoalkane units of different lengths were synthesized (Schemes 4 and 5)
The esterification reaction of ethylene glycols with 3 -O-acetyllithocholic acid 9 and 3-
oxocholan-24-oic acid 12 (synthesized as described in [23]), as well as the synthesis of the
target (5Z,9Z)-dienoic acids of LA 15a and 16a occur in the presence of EDC HCl and
DMAP in CH2Cl2. Please see the Supplementary Figures S1–S88.
.
α
–
d
–
d
·
Scheme 4. Synthesis of the LA-fatty acid conjugates linked via ethylene glycol units. (a) Ethylene
glycols of different lengths, EDCl HCl, DMAP, CH2Cl2. (b) (5Z,9Z)-Tetradeca-5,9-dienedioic acid 5,
·
EDCl·HCl, DMAP, CH2Cl2.

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Scheme 5. Synthesis of LA-fatty acid conjugates linked via diaminoalkane units. (a) Oxalyl chloride, CH2Cl2. (b) BOC-
protected diaminoalkanes, diisopropylethylamine, CH2Cl2. (c) Trifluoroacetic acid, CHCl3. (d) (5Z,9Z)-Tetradeca-5,9-
dienedioic acid 5, EDCl·HCl, DMAP, CH2Cl2.
The synthesis of LA-fatty acid conjugates linked via diaminoalkane units of differ-
ent lengths was carried out in several stages. Compounds
9 and 12 bearing the car-
boxyl group were first converted to chloroanhydrides 17 and 18 using oxalyl chloride in
dichloromethane [28]. Chloroanhydrides 17 and 18 without preliminary purification were
involved in reactions with diaminoalkanes to form amides 19a
minal amino group in amides 19a and 20a was protected with the tert-butoxycarbonyl
(BOC) group [29]. After removing the BOC protection from compounds 19a and 20a
with trifluoroacetic acid in the chloroform, amides 21a and 22a were esterified with
according to the described method [23] (Scheme 5).
–d and 20a–d. The free ter-
–d
–d
–d
–d
–d
–d
(5Z,9Z)-tetradeca-5,9-dienedioic acid
5
2.2. Biological Evaluation
2.2.1. Cytotoxic Activity In Vitro
In this work, for the first time, the in vitro antitumor activity for 18 synthesized
compounds ( 11 15a 16a 23a , and 24a ) was assessed on Jurkat, K562, HEK293,
8
,
,
–
d,
–
d,
–d
–d
HeLa, U937 cell lines and normal fibroblasts using Guava Nexin Reagent, Guava Cell Cycle,
and Guava ViaCount (Millipore) reagent kits, including IC50 determination, cell viability
studies, and cell cycle effects using flow cytofluorometry. Table 1 shows the cytotoxic
activity of the synthesized compounds of hybrid molecules based on lithocholic acid
against Jurkat, K562, U937, HeLa tumor cell lines, as well as a specific cell line derived
from human embryonic kidneys cells (HEK293). Incubation time was 48 h.

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Table 1. Cytotoxic activity of compounds 8, 11, 15a–d, 16a–d, 23a–d, and 24a–d (48 h).
Jurkat,
K562,
IC50, µМ
U937,
IC50, µМ
HEK293,
IC50, µМ
HeLa,
IC50, µМ
Fibroblasts, hTopI Inhibition,
Compound
IC50, µМ
1.06 ± 0.11
0.13 ± 0.01
0.01 ± 0.00
0.14 ± 0.01
0.13 ± 0.01
0.06 ± 0.00
0.04 ± 0.00
0.42 ± 0.03
0.09 ± 0.01
0.12 ± 0.01
0.13 ± 0.01
0.32 ± 0.02
0.46 ± 0.04
0.21 ± 0.01
0.14 ± 0.01
0.14 ± 0.02
0.11 ± 0.02
0.08 ± 0.03
0.07 ± 0.01
IC50, µМ
3.96 ± 0.34
0.63 ± 0.05
0.05 ± 0.01
0.73 ± 0.06
0.69 ± 0.05
0.29 ± 0.02
0.21 ± 0.03
1.68 ± 0.15
0.64 ± 0.07
0.61 ± 0.06
0.77 ± 0.08
1.42 ± 0.15
2.11 ± 0.24
1.43 ± 0.12
0.81 ± 0.09
0.96 ± 0.08
0.68 ± 0.06
0.84 ± 0.05
0.69 ± 0.04
µМ
6
8
1.49 ± 0.14
0.56 ± 0.04
0.16 ± 0.01
0.25 ± 0.02
0.23 ± 0.01
0.19 ± 0.02
0.11 ± 0.01
0.64 ± 0.04
0.14 ± 0.01
0.20 ± 0.02
0.21 ± 0.01
0.23 ± 0.01
0.19 ± 0.02
0.22 ± 0.02
0.09 ± 0.03
0.18 ± 0.03
0.09 ± 0.01
0.18 ± 0.06
0.09 ± 0.01
1.71 ± 0.16
0.78 ± 0.06
0.05 ± 0.00
0.17 ± 0.01
0.16 ± 0.02
0.17 ± 0.01
0.09 ± 0.01
0.51 ± 0.05
0.12 ± 0.01
0.16 ± 0.01
0.15 ± 0.01
0.34 ± 0.03
0.26 ± 0.02
0.25 ± 0.02
0.21 ± 0.03
0.24 ± 0.01
0.13 ± 0.01
0.28 ± 0.04
0.09 ± 0.01
1.84 ± 0.17
0.95 ± 0.08
0.21 ± 0.02
0.34 ± 0.03
0.29 ± 0.02
0.21 ± 0.02
0.14 ± 0.01
0.73 ± 0.06
0.16 ± 0.01
0.23 ± 0.02
0.26 ± 0.02
0.53 ± 0.04
0.54 ± 0.03
0.80 ± 0.07
0.98 ± 0.07
0.67 ± 0.05
0.31 ± 0.06
0.58 ± 0.05
0.44 ± 0.03
1.79 ± 0.18
0.92 ± 0.07
0.19 ± 0.02
0.31 ± 0.03
0.37 ± 0.02
0.25 ± 0.02
0.19 ± 0.02
0.78 ± 0.07
0.22 ± 0.02
0.25 ± 0.02
0.24 ± 0.02
0.41 ± 0.04
0.48 ± 0.05
0.38 ± 0.03
0.22 ± 0.01
0.55 ± 0.04
0.22 ± 0.04
0.38 ± 0.07
0.39 ± 0.03
2
0.04
0.01
0.04
0.04
0.02
0.02
1
11
15a
15b
15c
15d
16a
16b
0.06
0.04
0.04
0.08
0.08
0.06
0.04
0.04
0.04
0.04
0.04
16c
16d
23a
23b
23c
23d
24a
24b
24c
24d
DMSO (1%)
Campt.
No dead cells were observed
1.12 ± 0.12
1.54 ± 0.13
1.32 ± 0.12 3.18 ± 0.16
2.21 ± 0.25
7.86 ± 0.69
0.40
The results obtained in the course of studying cytotoxicity of the synthesized deriva-
tives based on lithocholic acid 11 15a 16a 23a , and 24a presented in Table 1
8,
,
–
d,
–
d,
–
d
–d
showed that cell lines are, to varying degrees, sensitive to the action of the investigated
hybrid molecules.
The pronounced heterogeneity of the IC50 values between different tumor cell lines
in vitro is one of the main criteria evidencing in favor of specific antitumor activity rather
than nonspecific toxicity, where the IC50 values similar to each other are obtained in differ-
ent cell lines [30]. The original lithocholic acid 6 and camptothecin, a known inhibitor of hu-
man topoisomerase I, were taken as controls. It was experimentally shown that lithocholic
acid 6 exhibits a cytotoxic effect comparable to Camptothecium in all the cell lines taken into
the study, a significantly lower inhibition activity of the hTopI enzyme. At the same time,
cytotoxicity of the synthesized hybrid molecules increases significantly and exceeds the
cytotoxicity of LA by 2–100 times (Table 1). The Jurkat lymphocytic leukemia cell line was
found to be the most sensitive to the synthesized compounds. Hybrid molecules 11
and 24c exhibited the highest cytotoxic effect against all cancer tumor cell lines used.
In addition, in a series of compounds of hybrid molecules with mono(di,tri,tetra)ethylene
glycol and -diaminoalkane fragments as spacers, the pronounced increase in the cyto-
, 15c,d,
,
d
α,ω
toxicity of molecules with increasing spacer length was observed.
For suspension (Jurkat, K562, and U937) and adhesion (HeLa, Hek293) cell lines,
the index of therapeutic selectivity of cytotoxic action (SI) varies in the range of 2–10 with
respect to normal fibroblasts (Table 1).
New derivatives of 5Z,9Z-dienoic acids based on lithocholic acid have been tested
in vitro for the inhibitory activity of human topoisomerase I (hTopI) by studying the

Pharmaceuticals 2021, 14, 84
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relaxation of supercoiled plasmid DNA under standard conditions. A known inhibitor
of hTopI, camptothecin, was used as a control in all experiments. The inhibitory activity
of hybrid molecules toward hTopI was shown to correlate well with their cytotoxic effect
against tumor cells and varies within 0.01–2 µM (Table 1).
2.2.2. Induction of Apoptosis
Based on the ability of the studied compounds to stimulate cell death at the initial
stages of the investigation, it was decided to use two compound-leads (24b and 24c) to
study apoptosis in the Jurkat tumor cell culture.
The highest percentage of Jurkat cells in the early and late phases of apoptosis was
observed at two concentrations of compound 24b of 0.1 and 0.05
and 83%, respectively (Figure 1, histograms 2 and 3). Compound 24c at a concentration
of 0.1 M was found to be somewhat less active in the induction of apoptosis. The early
µM and constituted 64%
µ
apoptosis was almost 68% in the Jurkat cell line (Figure 1, histogram 6), which is superior
in action to camptothecin.
1
2
5
3
6
4
Figure 1. The Jurkat tumor cell line treated with various concentrations of compounds 24b and 24c
Cells were stained with annexin V/7-AAD and analyzed using flow cytometry. ( 24c (0.02 М);
24c (0.05 М); ( 24c (0.1 М); ( 24d (0.02 М); ( 24d (0.05 М); ( 24d (0.1 М). PerCP:
.
1)
µ
(2)
µ
3)
µ
4)
µ
5)
µ
6)
µ
Peridinin chlorophyll A protein; FITC: Fluorescein isothiocyanate.
2.2.3. Cell Cycle Analysis
Numerous studies show that DNA damage in cells induces the cell cycle arrest at
the checkpoints. In particular, the cytotoxic quinoline alkaloid camptothecin, a well-
known DNA-damaging agent, blocks the cell cycle in many tumor lines due to irreversible
inhibition of topoisomerase I. Analysis of the cell cycle in Jurkat cells after incubation with
compound 24b and the appearance of a hypodiploid DNA peak upon flow cytometry
(Figure 2, histograms 1–3) indicate the inability to arrest the division cycle at checkpoints,
eventually leading to cell death. In this case, the effect of compound 24b on Jurkat cells
differs from the action of camptothecin by a more pronounced blockade of the S-phase.
Thus, compound 24b induces the cell cycle arrest at the G1/S checkpoint.

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1
2
3
Figure 2. Effect of compound 24b on the cell cycle in Jurkat cells. Cells were analyzed using flow cytofluorometry. (
1) 24c
(0.02 М); ( 24c (0.05 М); ( 24c (0.1 М). Data are presented as the means SEM calculated from results of at least four
µ
2
)
µ
3
)
µ
±
independent experiments. Statistically significant differences from the values in the control are indicated as p < 0.001.
2.2.4. Studying the Effect on Mitochondria
The next step in the analysis of biological activity of the studied compounds was the
analysis of mitochondrial function, since the structure of such compounds suggests the
possibility of their penetration through the mitochondrial membrane and the ability to
uncouple the processes of oxidation and phosphorylation. Cells undergoing apoptosis
due to mitochondrial damage exhibit depolarization of the electrochemical gradient of
the inner mitochondrial membrane, mitochondrial release of apoptogenic molecules and
activation of specific proteases called caspases, protrusion of cytosolic vesicles from the
cell surface and loss of plasma membrane asymmetry, nuclear condensation, and finally
DNA cleavage and rupture of the plasma membrane. A multivariate assessment of apop-
totic markers along with detection of mitochondrial damage makes it possible to clearly
determine the nature of cell death. Cell staining method with three dyes (annexin-Alexa
Fluor488, 7-AAD, and MitoSense Red) allow simultaneously measuring three important
parameters of a cell state, such as changes in mitochondrial potential and externalization of
phosphatidylserine at the cell surface, which, during simultaneous detection, is a reliable
sign of mitochondrial apoptosis. Normal cells with intact mitochondrial membrane poten-
tial exhibit high fluorescence of MitoSense Red, whereas cells in which the mitochondrial
membrane potential is impaired, have much lower fluorescence parameters. Annexin V is a
calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine
usually localized on the inner surface of the cell membrane. Control cells without apoptosis
contain phosphatidylserine on the inner surface of their membrane, while apoptotic cells
show positive green fluorescence due to externalization of phosphatidylserine. DNA in-
tercalator 7-aminoactinomycin (7-AAD), which does not penetrate a living cell, appears
inside the cell only at the late stages of apoptosis upon membrane damage, commonly
observed at late cellular apoptosis and necrosis. Thus, with identical colors during the
cytometric analysis, we observed several cell populations: (1) living cells with intact mito-
chondrial membrane; (2) cells with diffusion membrane potential, but without annexin V
or 7-AAD staining; (3) early apoptotic cells with diffusion membrane potential and annexin
V binding; (4) late apoptotic cells or dead cells; (5) cells with diffusion membrane potential.
We analyzed activity of the synthesized derivatives of lithocholic acid in relation to the
mitochondria of Jurkat tumor cells. The cells were incubated with the tested compounds
for 4 h (Figure 3).

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1
2
3
4
Figure 3. Cont.

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5
6
Figure 3. Detection of changes in mitochondrial membrane potential (∆Ψ) and apoptosis in Jurkat tumor cells treated with the studied
compounds. The cells were treated with the tested compounds at their IC50 concentrations. Incubation time was 4 h. ( ) LA,
; ( 11; ( 15d; ( 24d; ( ) control. Staurosporine at a concentration of 0.8 M. The cells were stained with the following dyes:
MitoSense Red, annexin V—CF488A and 7-AAD.
1
6;
(
2)
8
3)
4)
5)
6
µ
To reveal the ability of the synthesized lithocholic acid derivatives to penetrate the
mitochondrial membrane and uncouple oxidation and phosphorylation thus causing
intramitochondrial stress and initiate mitochondrial apoptosis, compound-leads were
selected not only in terms of cytotoxic activity, but also in terms of pronounced changes
in the kinetics of the cell cycle from various series of hybrid molecules. Compounds
6,
8,
11, 15d, and 24b have been analyzed in detail (Figure 3). All of the above derivatives
of lithocholic acid exhibit the pronounced apoptosis-inducing activity against Jurkat cell
lines, and their effect is comparable to classical mitochondrial apoptosis-inducing agents
such as staurosporine. In this sense, compound 15d exhibited the highest activity with
respect to mitochondrial potential. This compound had an uncoupling effect even to a
slightly greater extent than staurosporine, if we pay attention to the percentage of cells with
damaged mitochondria. In this regard, when exposed to 15d, we observed mitochondrial
damage in 74.71% of the cells, whereas when exposed to staurosporine, the percentage of
such cells was 22.83%. According to our data, lithocholic acid 6 itself belongs to compound-
leads in terms of the effect on mitochondria. The percentage of cells with damaged
mitochondria after treatment with lithocholic acid at a concentration of IC50 was 53.09%.
Other compounds,
quite obviously, is a more extended process in time. For instance, after incubation of
cells with the tested compounds for 4 h, compounds 11, and 24b demonstrated the
percentage of mitochondrial damage of 8.20%, 9.97%, and 22.26%, respectively. Possibly,
compounds 11, and 24b penetrate the mitochondrial membrane somewhat worse or
8, 11, and 24b, also reduce ∆Ψ, and the effect of these compounds,
8,
8
,
the process of uncoupling proceeds more slowly, since these compounds can be the so-
called “soft” uncouplers. Such compounds show themselves as substances that uncouple
oxidation and phosphorylation on the inner mitochondrial membrane by reducing the
mitochondrial potential ∆Ψ. According to Mitchell’s chemiosmotic theory, the factor that
couples oxidation with phosphorylation is the electrochemical proton potential ∆µH+

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which appears on the inner mitochondrial membrane during electron transport. Proton
gradient creating the difference in chemical and electrical potentials, is the source of energy
required for the reaction of ATP formation [31]. Mild uncoupling is a reversible decrease in
the membrane potential of mitochondria, in which there is no significant/physiologically
significant suppression of respiration, damage to the components of the respiratory chain,
or disruption of the physicochemical properties of mitochondrial membranes [32].
2.2.5. Cytochrome C Release from Mitochondria
Cytochrome C is a soluble heme protein localized in the inner space of mitochondria
which plays an irreplaceable role as an electron carrier in oxidative phosphorylation during
respiration, in the transfer of electrons from the cytochrome bc1 complex to cytochrome
oxidase on the inner mitochondrial membrane. During the induction of apoptosis and re-
lated events such as mitochondrial depolarization, cardiolipin peroxidation, cytochrome c,
together with other pro-apoptotic proteins, namely, proCaspase-9, Smac/DIABLO, APF-1
,
and Endo F, is released into the cytosol, signaling the initiation of the intrinsic apoptosis
pathway. When released, cytochrome c activates a caspase cascade thus promoting cell
death. Quantitative and qualitative assessment of cytochrome c release from mitochondria
can be used to characterize the mitochondrial pathway of apoptosis.
The process of the cytochrome C loss after treatment with the tested compound 24d as
compared to staurosporine was analyzed in Jurkat cells (Figure 4). Compound 24d caused
a loss of cytochrome C.
3
1
2
Figure 4. Histograms depicting cells stained using the FlowCellect Cytochrome C Kit. ( ) shows untreated Jurkat cells;
1
(2
) shows cells treated with compound 24d; ( ) shows cells induced with staurosporine (1 µM) for 2 h. The graphs show the
3
downward fluorescence shifts of cytochrome c levels for several cell lines and inducers for the FlowCellect Cytochrome c Kit.
Histogram plots show live cells (negative control) and cells stained with cytochrome c-FITC. Cells stained with cytochrome
c-FITC demonstrate high fluorescence. They are cells with intact mitochondria. Cells which exhibit reduced fluorescence
undergo membrane disruption by the test compound, resulting in a loss of cytochrome C.
2.2.6. Interaction with Histones and Topoisomerase II
For assessing the influence on topoisomerase II of the studied derivatives of hybrid
molecules based on 5Z,9Z-diene acids, the investigation into possible DNA damage in
Jurkat tumor cells was carried out. We had previously shown that various compounds
containing a diene moiety can affect their ability to inhibit topoisomerase II [7,23–27,33–38].
Changes in the concentration of total and phosphorylated proteins H2A.X allow at the
same time confirming the target specificity of the phosphorylation event of this histone
class. Simultaneous detection of unphosphorylated and phosphorylated H2A. X protein
is a sensitive tool for studying the effect of compounds on the genetic apparatus of a cell
and allows one to investigate the relationship between DNA damage and the initiation
of apoptosis. Many chemotherapeutic agents, such as etoposide, damage tumor cells by
stimulating DNA double-strand breaks. Furthermore, the γ-H2A.X level, detected by flow

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cytometry, was shown to correlate with the amount of DNA double-strand breaks and
tumor cell death [39].
After stimulation of Jurkat cells with etoposide for 2 h, a pronounced increase in the
phosphorylated fraction of histone H2A.X was observed (Figure 5). In the experiment,
Jurkat cells were treated with compound 24d, and it was shown that this compound
affects the phosphorylation process of H2A.X insignificantly, virtually not increasing the
population of cells with phosphorylated protein (Figure 5) compared with etoposide as the
reference compound. When cells were exposed to etoposide, the phosphorylated fraction of
the H2A.X protein was determined in all the cells (almost 99%), whereas upon exposure to
compound 24d, only 6.45% of the cells contained phosphorylated protein H2A.X. Therefore,
we can declare that compound 24d virtually does not initiate accumulation of DNA strand
breaks in a cell with subsequent apoptosis activation via the p53-dependent mechanism.
2
1
3
Figure 5. Detection of the phosphorylated H2A.X accumulation in the Jurkat cells exposed to compound 24d com-
pared with etoposide. Incubation with studied compounds was carried out for 2 h. Control (live cells, unstained) ( );
cells treated with compound 24d at its IC50 concentration ( ); cells treated with etoposide at a concentration of 0.2 M/mL
); Q4-2—cells containing phosphorylated histones H2A.X (DNA damage marker—staining with phosphor-specific an-
1
2
µ
(
3
tibodies Anti-phospho-Histone H2A.x (Ser139)–PerCP); Q4-3—cells without staining (live cells); Q4-4—cells containing
non-phosphorylated histones H2A.X (Anti-Histone H2A.X-FITC).
3. Materials and Methods
3.1. Chemistry
Lithocholic acid, 4-dimethylaminopyridine (DMAP), N-[3-(methylamino)propyl]-
N’-ethylcarbodiimide hydrochloride (EDC HCl) were obtained from Sigma-Aldrich and
·
Acros Organics. Dichloromethane was freshly distilled before use. Optical rotations were
measured on a PerkinElmer 341 polarimeter. Melting points were recorded on Stuart
SMP3. IR spectra were recorded on Bruker VERTEX 70V using KBr discs over the range
1
of 400–4000 cm−1. H and 13C NMR spectra were obtained using a Bruker Ascend 500
spectrometer in CDCl3 operating at 500 MHz for 1H and 125 MHz for 13C and a Bruker
AVANCE 400 spectrometer in CDCl3 operating at 400 MHz for 1H and 100 MHz for 13C.
Mass spectra of MALDI TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
Mass Spectrometry)/TOF positive ions (matrix of the sinapic acid) are recorded on a mass
spectrometer Bruker AutoflexTM III Smartbeam. Elemental analyses were measured on a
1106 Carlo Erba apparatus. Individuality and purity of the synthesized compounds were
controlled using TLC on Sorbfil plates; anisic aldehyde in acetic acid was used as a developer.
Column chromatography was carried out on the Acrus silica gel (0.060–0.200 mm).
3.2. Cell Culturing
Cell culturing was carried out following the known procedure [40]. Cells (Jurkat, K562,
U937, HeLa, HEK293, and normal fibroblasts) were purchased from The Health Protection
Agency (HPA) Culture Collections (HPA, Salisbury, UK) and cultured according to the
standard mammalian tissue culture protocols and sterile technique. All cell lines used in
the study were tested and shown to be free of mycoplasma and other viral contamination.

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HeLa and HEK293 cell lines were cultured as monolayers and maintained in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco BRL, Waltham, MA, USA) supplemented with
◦
10% fetal bovine serum and 1% penicillin–streptomycin solution at 37 C in a humidi-
fied incubator under a 5% CO2 atmosphere. The cells were maintained in RPMI 1640
(Jurkat, K562, U937) (Gibco, Waltham, MA, USA) supplemented with 4 mM glutamine,
10% FBS (Sigma, Burlington, MA, USA), and 100 units/mL penicillin–streptomycin (Sigma,
Burlington, MA, USA). All types of cells were grown in an atmosphere of 5% CO2 at
37 ◦C. The cells were subcultured at intervals of 2–3 days. Adherent cells (HeLa, HEK293,
and normal fibroblasts) were suspended using trypsin/EDTA and counted after they had
reached 80% confluency. The cells were then seeded in 24-well plates at 5
×
104 cells per
well and incubated overnight. Jurkat, K562, U937 cells were subcultured at 2-day intervals
with a seeding density of 1 × 105 cells per well in 24-well plates in RPMI with 10% FBS.
3.3. DNA Topoisomerase I Assay
Inhibition of DNA topoisomerase I tests were carried out following the known proce-
dure [40]. The inhibitory activity and the mechanism of inhibition of (5Z,9Z)-5,9-dienoic
acids were determined using the Topoisomerase I Drug Screening Kit TG-1018-2, (Topogen,
Buena Vista, CO, USA) (the tested compound was added before topoisomerase I). The re-
laxation of supercoiled DNA under the action of topoisomerase I was carried out as follows:
the reaction mixture (20 µL) containing 0.25 µg of the DNA plasmid pHOT (TopoGen,
Buena Vista, CO, USA), 1 unit of recombinant topoisomerase I (TopoGen, USA), and the
tested compound was incubated in the buffer (35mM Tris-HCl, рН8.0; 72 mM KCl, 5 mM
MgCl, 5 mM dithiothreitol, 5 mM spermidine, and 0.01% bovine serum albumin) for 30 min
◦
at 37 C using a Biosan thermostat (Riga, Latvia). The tested compound was introduced in
the reaction mixture prior to the addition of the enzyme topoisomerase I. The inhibiting
action on topoisomerase I was monitored using the alkaloid camptothecin (TopoGEN,
Buena Vista, CO, USA). The reaction was terminated by adding sodium dodecyl sulfate
up to a concentration of 1%. After addition of a solution (5 mg/mL) of proteinase K
(Sigma Chemical Co., St. Louis, MO, USA) (1:10), the reaction mixture was incubated for
40 min at 37 ◦C. A 0.1% solution of bromophenol blue (1:10) was added and the samples
were electrophoresed in the absence of ethidium bromide. The reaction products were
separated in a 1% agarose gel (3 V/cm) for 4–6 h. After the electrophoresis, the gels were
treated with a solution of ethidium bromide (0.5 µg/mL). The gels were visualized in the
UV light in a Infinity VX2 1120/Blue X-Press gel documentation system (Vilber Lourmat,
Paris, France). The possible action of the tested compounds on supercoiled DNA was
checked by performing the reaction without topo I, the tested compounds being added in
the same concentrations as in the reaction with the enzyme.
3.4. Cytotoxicity Assay
Cytotoxicity tests were carried out following a known procedure [40]. Viability (live/dead)
assessment was performed by staining cells with 7-AAD (7-aminoactinomycin D) (Biolegend,
San Diego, CA, USA). The cells were seeded in a 24-well plate (1 × 105 cells/well). After treat-
ment (24 h), the cells were harvested, washed 1–2 times with phosphate-buffered saline
(PBS), and centrifuged at 400 g for 5 min. Cell pellets were resuspended in 200 mL of a
flow cytometry staining buffer (PBS without Ca2+ and Mg2+, 2.5% FBS) and stained with
5 µL of a 7-AAD staining solution for 15 min at room temperature in the dark. Samples
were acquired on a NovoCyteTM 2000 FlowCytometry System (ACEA, San Diego, CA,
USA) equipped with a 488 nm argon laser. Detection of 7-AAD emission was done using a
675/30 nm filter in the FL4 channel.
3.5. Viability and Apoptosis
Induction of apoptosis tests was carried out following a known procedure [40]. Apoptosis
was determined by flow cytometric analysis of annexin V and 7-aminoactinomycin D
staining. After treatment (24 h), the cells were harvested, washed 1–2 times with phosphate-

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buffered saline (PBS), and centrifuged at 400
in 200
Then, 200
to 5
105 cells in 200
room temperature in the dark. At the end of incubation, the cells were analyzed on a
NovoCyteTM2000 FlowCytometry System (ACEA, San Diego, CA, USA).
×
g for 5 min. Cell pellets were resuspended
µ
L of a flow cytometry staining buffer (PBS without Ca2+and Mg2+, 2.5% FBS).
µ
L of the Guava Nexin reagent (Millipore, Bedford, MA, USA) was added
×
µ
L, and the cells were incubated with the reagent for 20 min at
3.6. Cell Cycle Analysis
Cell cycle analysis was carried out following a known procedure [40]. Cell cycle was
analyzed using the method of propidium iodide staining. After treatment (24 h), the cells
were harvested, washed 1–2 times with phosphate-buffered saline (PBS), and centrifuged
at 400
×
g for 5 min. Cell pellets were resuspended in 200 µL of a flow cytometry staining
buffer (PBS without Ca2+ and Mg2+, 2.5% FBS). Then, the cells were plated in 24-well
round bottom plates at a density of 10
and fixed with ice-cold 70% ethanol for 24 h at 0 C. The cells were then washed with
×
105 cells per well, centrifuged at 450
×
g for 5 min,
◦
PBS and incubated with 250 µL of the Guava Cell Cycle Reagent (Millipore, Burlington,
MA, USA) for 30 min at room temperature in the dark. The samples were analyzed on a
NovoCyteTM 2000FlowCytometry System (ACEA, San Diego, CA, USA).
3.7. Mitochondrial Damage
Mitochondrial damage tests were carried out following a known procedure [40]. In our
work, we performed a cytometric assay which allowed multiparametric evaluation of three
cell health markers: change in mitochondrial potential (early apoptosis and cellular stress),
phosphatidylserine expression on the cell surface (late apoptosis), and membrane permeabi-
lization (cell death). Millipore’s FlowCellect
™ MitoDamage Kit’s use of the reagents allows
researchers to obtain information on early and late apoptosis in one simple assay. The cells
◦
were treated with testing compounds at various concentrations and incubated (37 C,
5% CO2) for 4 h. After this time, the cells were dissociated with the Accutase solution,
stained, and analyzed by flow cytometry (NovoCyte Flow Cytometry
™, Burlington, MA,
USA) according to the manufacturer’s protocols (FlowCellect MitoDamage Kit, Merck,
™
and FlowCellect™ Oxidative Stress Characterization Kit, Merck, Burlington, MA, USA).
3.8. Histone H2A.X Analysis
Histone H2A.X analysis was carried out following a known procedure [40]. Cell prepa-
ration for phosphorylation of Histone H2A.X in the Jurkat cells analysis was grown in a
culture flask for 48 h. The medium was changed at 24 h before drug treatment. Phosphoryla-
tion of histone H2A.X was measured with the FlowCellect
Dual Detection Kit (FCCS025153, Millipore, MA, USA). Briefly, isolated and treated cells
were counted and diluted with an assay buffer (5
106 cells/2.5 mL). Paraformaldehyde
™ DNA Damage Histone H2A.X
×
(PFA, Sigma, Burlington, MA, USA) was added at a final concentration of 2% and the cells
◦
were fixed at 37 C for 10 min. The cells were then immediately chilled on ice, centrifuged
washed three times with 0.5% bovine serum albumin (BSA; EMD Bioscience, La Jolla,
CA, USA)–PBS, permeabilized with 70% ethanol in distilled water or 90% methanol in
,
0.5% BSA–PBS, and kept at 4 ◦C for ethanol samples and at
−
20 ◦C for methanol samples
until further staining. After that, the cells were fixed, permeabilized, and incubated with
Anti-phospho-Histone H2A.X (Ser139, Alexa Fluor®555, part number CS208203, Millipore,
Billerica, MA, USA) and anti-H2A.X (PECy5, part number CS209202, Millipore, Billerica,
MA, USA), and then analyzed by flow cytometry.
3.9. Chemical Experimental Data
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectral data and
synthesis method for 2,2’-[(5Z,9Z)-tetradeca-5,9-diene-1,14-diylbis(oxy)]bistetrahydro-2H-
pyran 3 and (5Z,9Z)-tetradeca-5.9-dienedioic acid 5 are described in the literature [23].

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3.9.1. General Procedure for the Synthesis of (5Z,9Z)-Tetradeca-5,9-diene-1,14-diol (4)
To a mixture of methanol (10 mL), CHCl3 (10 mL), and 1,14-tetrahydropyranyl-5Z,9Z-
dien-1,14-diol 3 (0.39 g, 1.0 mmol), catalytic amount◦s of p-toluenesulfonic acid (p-TSA)
were added, and the reaction mixture was stirred at 60 C for 4 h. After this, the solvent was
evaporated, and then the organic extract was washed with a saturated solution of sodium
bicarbonate (3
×
25 mL). The organic layer was dried over MgSO4 and concentrated in
vacuo. The residue was purified by column chromatography using hexane/ethyl acetate
(3:1) as the elution solvent to afford 0.52 g (76% yield) of diol 4.
(5Z,9Z)-Tetradeca-5,9-diene-1,14-diol (4)
Colorless oil, 0.17 g, 76% yield. IR (KBr)
ν
max 3006, 2924, 2852, 1657, 1466, 1408, 1384,
1214, 1165, 1100, 1034, 965, 911, 861, 755, 723, 665 cm−1; H NMR (CDCl3, 500 MHz)
δ
1
5.42–5.34 (4H, m, H-5, H-6, H-9, H-10), 3.57 (4H, t, J = 6.5 Hz, H-1, H-14), 2.07–2.01 (8H, m,
H-4, H-7, H-8, H-11), 1.58–1.52 (4H, m, H-2, H-13), 1.43–1.36 (4H, m, H-3, H-12); 13C NMR
(CDCl3, 125 MHz)
δ 129.9 (C-6, C-9), 129.5 (C-5, C-10), 62.6 (C-1, C-14), 32.3 (C-2, C-13),
27.4 (C-7, C-8), 26.9 (C-4, C-11), 25.8 (C-3, C-12).
3.9.2. Reaction of Methyl Ester of Lithocholic Acid (7) with (5Z,9Z)-Tetradeca-5.9-dienedioic
Acid (5)
Lithocholic acid derivative
8 was synthesized according to a modified literature
procedure [26]. To a solution of methyl ester of lithocholic acid (0.39 g, 1.0 mmol) in CH2Cl2
(50 mL), (5Z,9Z)-tetradeca-5.9-dienedioic acid 4 (0.51 g, 2.0 mmol) was added, followed
by EDC HCl (0.48 g, 2.5 mmol) and DMAP (18 mg, 0.15 mmol) under argon. The mixture
·
was stirred at room temperature for 12 h until the reaction was complete (TLC monitoring,
hexane/ethyl acetate). The mixture was diluted with H2O (10 mL) and the CH2Cl2 layer
was separated, dried over MgSO4, and concentrated. The crude product was purified by
column chromatography (silica gel) using hexane/ethyl acetate (5:1) as the elution solvent
to afford compound 8.
(5Z,9Z)-14-{[(3α,5β)-24-Methoxy-24-oxocholan-3-yl]oxy}-14-oxotetradeca-5,9-dienoic
acid (8)
Colorless waxy solid, 0.42 g, 67% yield. [
2929, 2867, 1735, 1709, 1449, 1384, 1326, 1307, 1243, 1165, 1100, 1022, 802, 722 cm−1
1H NMR (CDCl3, 500 MHz)
5.43–5.35 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.79–4.71 (1H,
α] ν
21 + 30.3 (c 0.36, CHCl3); IR (KBr)
max
D
;
δ
m, H-3), 3.68 (3H, s, O-CH3), 2.37 (2H, t, J = 7.5 Hz, H-2’), 2.35-1.02 (28H, m), 2.29 (2H, t,
J = 7.5 Hz, H-13’), 2.12–2.06 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.73–1.67 (4H, m, H-3’, H-12’),
0.94 (3H, s, H-19), 0.92 (3H, d, J = 6.4 Hz, H-21), 0.66 (3H, s, H-18); 13C NMR (CDCl3,
125 MHz)
δ 178.8 (C-1’), 174.9 (C-24), 173.5 (C-14’), 130.4, 130.1 (C-6’, C-9’), 129.1, 128.8 (C-
5’, C-10’), 74.3 (C-3), 56.5 (C-14), 55.9 (C-17), 51.5 (O-CH3), 42.7 (C-13), 41.9 (C-5), 40.4 (C-9),
40.1 (C-12), 35.8 (C-8), 35.4 (C-20), 35.0 (C-1), 34.6 (C-10), 34.2 (C-13’), 33.3 (C-2’), 32.3 (C-4),
31.1 (C-23), 31.0 (C-22), 28.2 (C-16), 27.4, 27.3 (C-7’, C-8’), 27.0 (C-6), 26.7 (C-2), 26.6 (C-11’),
26.5 (C-4’), 26.3 (C-7), 25.0 (C-12’), 24.6 (C-3’), 24.2 (C-15), 23.3 (C-19), 20.8 (C-11), 18.3 (C-21),
12.0 (C-18); anal. calcd for C39H62O6: C, 74.72; H, 9.97; found C, 74.67; H, 9.92.
3.9.3. Reaction of 3α-Acetoxy-5β-cholan-24-oic Acid with (5Z,9Z)-Tetradeca-5,9-diene-
1,14-diol (4)
Lithocholic acid derivative 10 was synthesized according to a modified literature pro-
cedure [38]. To a solution of 3
(50 mL), (5Z,9Z)-tetradeca-5,9-diene-1,14-diol (0.46 g, 2.0 mmol) was added, followed by
EDC HCl (0.48 g, 2.5 mmol) and DMAP (18 mg, 0.15 mmol) under argon. The mixture
α-acetoxy-5β-cholan-24-oic acid (0.42 g, 1.0 mmol) in CH2Cl2
·
was stirred at room temperature for 12 h until the reaction was complete (TLC monitoring,
hexane/ethyl acetate). The mixture was diluted with H2O (10 mL) and the CH2Cl2 layer
was separated, dried over MgSO4, and concentrated. The crude product was purified by
column chromatography (silica gel) using hexane/ethyl acetate (5:1) as the elution solvent
to afford compound 10.

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(5Z,9Z)-14-Hydroxytetradeca-5,9-dien-1-yl (3α,5β)-3-(acetyloxy)cholan-24-oate (10)
Colorless waxy solid, 0.44 g, 71% yield. [
19 + 25.0 (c 0.94, CHCl3); IR (KBr)
2933, 2865, 1736, 1451, 1382, 1363, 1242, 1166, 1028, 911, 811, 756 cm−1; 1H NMR (CDCl3,
500 MHz) 5.40–5.31 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.73–4.66 (1H, m, H-3), 4.05 (2H, t,
α
]
ν
max
D
δ
J = 6.8 Hz, H-1’), 3.62 (2H, t, J = 6.4 Hz, H-14’), 2.35-1.00 (28H, m), 2.11–2.03 (8H, m, H-4’,
H-7’, H-8’, H-11’), 2.01 (3H, s, COCH3), 1.68–1.65 (2H, m, H-2’), 1.57–1.51 (2H, m, H-13’),
1.44–1.36 (4H, m, H-3’, H-12’), 0.92 (3H, s, H-19), 0.90 (3H, d, J = 6.4 Hz, H-21), 0.63 (3H,
s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.4 (C-24), 170.6 (COCH3), 129.9, 129.7, 129.6,
129.4 (C-5’, C-6’, C-9’, C-10’), 74.4 (C-3), 64.2 (C-1’), 62.7 (C-14’), 56.5 (C-14), 55.9 (C-17),
42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.3 (C-20), 35.0 (C-1), 34.6 (C-10),
32.4 (C-13’), 32.2 (C-4), 31.3 (C-23), 31.0 (C-22), 28.2 (C-2’, C-16), 27.4, 27.3 (C-7’, C-8’),
27.0 (C-2), 26.9, 26.8 (C-4’, C-11’), 26.6 (C-6), 26.3 (C-7), 25.9 (C-3’), 25.8 (C-12’), 24.2 (C-15),
23.3 (C-19), 21.4 (COCH3), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C40H66O5: C,
76.63; H, 10.61; found C, 76.51; H, 10.53.
3.9.4. Oxidation of the Esterification Product 10 with Jones Reagent
To a solution of compound 10 (0.44 g, 0.7 mmol) in acetone (25 mL) and CH2Cl2 (5 mL)
at room temperature, Jones reagent (0.75 mL) was added dropwise. The reaction mixture
was stirred at room temperature for 0.5 h, then quenched with water (10 mL), concentrated
under reduced pressure to remove the excess of acetone and CH2Cl2, and the aqueous layer
was extracted with ethyl acetate (3
and concentrated in vacuo. The residue was purified by column chromatography using
×
20 mL). The organic layer was dried over MgSO4
hexane/ethyl acetate (3:1) as the elution solvent to afford dienoic acid 11.
(5Z,9Z)-14-{[(3
α
,5
β
)-3-(Acetyloxy)-24-oxocholan-24-yl]oxy}tetradeca-5,9-dienoic Acid (11
)
Colorless waxy solid, 0.37 g, 83% yield. [
α] ν
23 + 26.4 (c 0.66, CHCl3); IR (KBr)
max
D
2931, 2866, 1736, 1709, 1450, 1382, 1363, 1242, 1167, 1096, 1027, 980, 803, 726 cm−1; 1H NMR
(CDCl3, 500 MHz)
δ 5.44–5.34 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.76–4.70 (1H, m, H-3),
4.07 (2H, t, J = 6.4 Hz, H-14’), 2.39–2.33 (2H, m, H-2’), 2.35-1.02 (28H, m), 2.13–2.05 (8H,
m, H-4’, H-7’, H-8’, H-11’), 2.04 (3H, s, COCH3), 1.73–1.67 (2H, m, H-3’), 1.66–1.61 (2H, m,
H-13’), 1.47–1.41 (2H, m, H-12’), 0.93 (3H, s, H-19), 0.92 (3H, d, J = 6.4 Hz, H-21), 0.65 (3H,
s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 179.2 (C-1’), 174.5 (C-24), 170.8 (COCH3), 130.4,
129.7 (C-6’, C-9’), 129.6, 128.8 (C-5’, C-10’), 74.4 (C-3), 64.3 (C-14’), 56.5 (C-14), 56.0 (C-17),
42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.4 (C-20), 35.0 (C-1), 34.6 (C-10),
33.4 (C-2’), 32.2 (C-4), 31.3 (C-23), 31.0 (C-22), 28.2 (C-13’, C-16), 27.3 (C-7’, C-8’), 27.0 (C-2),
26.8, 26.5 (C-4’, C-11’), 26.6 (C-6), 26.3 (C-7), 25.9 (C-12’), 24.6 (C-3’), 24.2 (C-15), 23.3 (C-19),
21.5 (COCH3), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C40H64O6: C, 74.96; H,
10.06; found C, 74.84; H, 9.99.
3.9.5. General Procedure for the Synthesis of Ethylene Glycol Derivatives of
3a-Acetoxy-5β-cholan-24-oic Acid and 3-Oxo-cholan-24-oic Acid 13a–d, 14a–d
To a solution of 3
α
-acetoxy-5β-cholan-24-oic acid 9 or 3-oxo-cholan-24-oic acid 12
(1.0 mmol) in CH2Cl2 (50 mL), ethylene glycol (2.5 mmol) was added, followed by EDC
·
HCl
(0.58 g, 3.0 mmol) and DMAP (18 mg, 0.15 mmol) under argon. The mixture was stirred at
room temperature for 16 h until the reaction was complete (TLC monitoring, hexane/ethyl
acetate). The mixture was diluted with H2O (10 mL) and the CH2Cl2 layer was separated,
dried over MgSO4, and concentrated. The crude product was isolated by column chro-
matography (silica gel) using hexane/ethyl acetate (1:1) as the elution solvent for 13a
14a and hexane/ethyl acetate (1:3) for 13b–d, 14b–d.
,
2-Hydroxyethyl (3α,5β)-3-(Acetyloxy)cholan-24-oate (13a)
◦
White crystals, 0.35 g, 76% yield. MP 130–132 C, [
α
]
D
19 + 24.9 (c 1.05, CHCl3); IR (KBr)
ν
3016, 2938, 2868, 1736, 1449, 1381, 1363, 1243, 1170, 1086, 1027, 981, 949, 887, 756, 667,
max
615 cm−1; 1H NMR (CDCl3, 500 MHz)
δ 4.75–4.69 (1H, m, H-3), 4.23–4.19 (2H, m, CH2O),

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3.84–3.80 (2H, m, CH2OH), 2.42-1.02 (28H, m), 2.03 (3H, s, COCH3), 0.93 (3H, s, H-19),
0.92 (3H, d, J = 6.4 Hz, H-21), 0.65 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.7 (C-24),
170.7 (COCH3), 74.4 (C-3), 65.9 (CH2O), 61.2 (CH2OH), 56.5 (C-14), 55.9 (C-17), 42.7 (C-13),
41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.4 (C-20), 35.0 (C-1), 34.6 (C-10), 32.2 (C-4),
31.1 (C-23), 30.9 (C-22), 28.2 (C-16), 27.0 (C-6), 26.6 (C-2), 26.3 (C-7), 24.2 (C-15), 23.3 (C-19),
21.5 (COCH3), 20.8 (C-11), 18.2 (C-21), 12.0 (C-18); anal. calcd for C28H46O5: C, 72.69; H,
10.02; found C, 72.57; H, 9.96.
2-(2-Hydroxyethoxy)ethyl (3α,5β)-3-(Acetyloxy)cholan-24-oate (13b)
White crystals, 0.38 g, 74% yield. MP 72–74 ◦C, [
α
]
D
+ 17.0 (c 0.82, CHCl3); IR (KBr)
19
ν
2938, 2868, 1736, 1449, 1381, 1363, 1243, 1171, 1132, 1066, 1028, 982, 949, 889, 755,
max
666, 616 cm−1; H NMR (CDCl3, 500 MHz)
δ 4.63–4.57 (1H, m, H-3), 4.15–4.12 (2H, m,
1
CH2O), 3.65–3.57 (4H, m, CH2O, CH2OH), 3.51–3.48 (2H, m, CH2O), 2.71 (1H, br s, OH),
2.32-0.90 (28H, m), 1.91 (3H, s, COCH3), 0.83 (3H, s, H-19), 0.81 (3H, d, J = 6.4 Hz, H-21),
0.55 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.0 (C-24), 170.4 (COCH3), 74.2 (C-3),
72.4 (CH2O), 69.0 (CH2O), 63.2 (CH2O), 61.5 (CH2OH), 56.4 (C-14), 55.9 (C-17), 42.6 (C-13),
41.8 (C-5), 40.3 (C-9), 40.0 (C-12), 35.7 (C-8), 35.2 (C-20), 34.9 (C-1), 34.5 (C-10), 32.1 (C-4),
30.9 (C-23), 30.8 (C-22), 28.1 (C-16), 26.9 (C-6), 26.5 (C-2), 26.2 (C-7), 24.1 (C-15), 23.2 (C-19),
21.3 (COCH3), 20.7 (C-11), 18.2 (C-21), 11.9 (C-18); anal. calcd for C30H50O6: C, 71.11; H,
9.95; found C, 71.01; H, 9.86.
2-[2-(2-Hydroxyethoxy)ethoxy]ethyl (3α,5β)-3-(Acetyloxy)cholan-24-oate (13c)
Colorless waxy solid, 0.39 g, 71% yield. [
2938, 2867, 1735, 1635, 1450, 1384, 1243, 1171, 1124, 1066, 1026, 945, 880, 805, 722 cm−1
1H NMR (CDCl3, 500 MHz)
4.66–4.60 (1H, m, H-3), 4.17–4.15 (2H, m, CH2O), 3.64–3.58
α] ν
19 + 17.0 (c 0.82, CHCl3); IR (KBr)
max
D
;
δ
(8H, m, CH2O, CH2OH), 3.56–3.53 (2H, m, CH2O), 2.84 (1H, br s, OH), 2.35-0.92 (28H, m),
1.95 (3H, s, COCH3), 0.86 (3H, s, H-19), 0.84 (3H, d, J = 6.5 Hz, H-21), 0.58 (3H, s, H-18);
13C NMR (CDCl3, 125 MHz)
δ 174.1 (C-24), 170.5 (COCH3), 74.3 (C-3), 72.5 (CH2O), 70.5,
70.3 (CH2O), 69.1 (CH2O), 63.2 (CH2O), 61.6 (CH2OH), 56.4 (C-14), 55.9 (C-17), 42.7 (C-13),
41.8 (C-5), 40.3 (C-9), 40.1 (C-12), 35.7 (C-8), 35.3 (C-20), 34.9 (C-1), 34.5 (C-10), 32.2 (C-4),
31.0 (C-23), 30.8 (C-22), 28.1 (C-16), 26.9 (C-6), 26.6 (C-2), 26.3 (C-7), 24.1 (C-15), 23.3 (C-19),
21.4 (COCH3), 20.8 (C-11), 18.2 (C-21), 11.9 (C-18); anal. calcd for C32H54O7: C, 69.78; H,
9.88; found C, 69.61; H, 9.85.
2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethyl (3
Colorless waxy solid, 0.41 g, 70% yield. [
α
,5
β
)-3-(Acetyloxy)cholan-24-oate (13d
)
α
]
19 + 21.8 (c 0.64, CHCl3); IR (KBr)
ν
max
D
2938, 2867, 1735, 1636, 1450, 1381, 1243, 1171, 1124, 1066, 1028, 949, 914, 887, 845, 803, 733,
1
661, 615 cm−1; H NMR (CDCl3, 500 MHz)
δ 4.70–4.64 (1H, m, H-3), 4.20–4.18 (2H, m,
CH2O), 3.71–3.59 (12H, m, CH2O, CH2OH), 3.57–3.55 (2H, m, CH2O), 2.87 (1H, br s, OH),
2.35-0.95 (28H, m), 1.98 (3H, s, COCH3), 0.89 (3H, s, H-19), 0.87 (3H, d, J = 6.5 Hz, H-21),
0.60 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.2 (C-24), 170.6 (COCH3), 74.3 (C-3),
72.6 (CH2O), 70.6, 70.5, 70.3, (CH2O), 69.2 (CH2O), 63.3 (CH2O), 61.6 (CH2OH), 56.4 (C-14),
55.9 (C-17), 42.7 (C-13), 41.8 (C-5), 40.6 (C-9), 40.1 (C-12), 35.7 (C-8), 35.3 (C-20), 34.9 (C-1),
34.5 (C-10), 32.2 (C-4), 31.1 (C-23), 30.8 (C-22), 28.1 (C-16), 26.9 (C-6), 26.6 (C-2), 26.3 (C-7),
24.1 (C-15), 23.3 (C-19), 21.4 (COCH3), 20.8 (C-11), 18.2 (C-21), 12.0 (C-18); anal. calcd for
C34H58O8: C, 68.65; H, 9.83; found C, 68.55; H, 9.74.
2-Hydroxyethyl (5β)-3-Oxocholan-24-oate (14a)
◦
19
White crystals, 0.32 g, 77% yield. MP 134–136 C, [
IR (KBr) 2936, 2865, 1734, 1713, 1446, 1381, 1299, 1249, 1179, 1084, 1035, 946, 887,
756, 667, 615 cm−1; H NMR (CDCl3, 500 MHz)
(2H, m, CH2OH), 2.87 (1H, br s, OH), 2.70-1.02 (28H, m), 0.96 (3H, s, H-19), 0.87 (3H, d,
J = 6.5 Hz, H-21), 0.62 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
213.6 (C-3), 174.7 (C-24),
65.9 (CH2O), 60.9 (CH2OH), 56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.7 (C-13), 42.3 (C-4),
α
]
+ 27.0 (c 0.79, CHCl3);
D
ν
max
1
δ
4.16–4.10 (2H, m, CH2O), 3.77–3.73
δ

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40.6 (C-9), 39.9 (C-12), 37.1 (C-2), 36.9 (C-1), 35.5 (C-8), 35.3 (C-20), 34.8 (C-10), 31.1 (C-23),
30.8 (C-22), 28.1 (C-16), 26.6 (C-6), 25.7 (C-7), 24.1 (C-15), 22.6 (C-19), 21.1 (C-11), 18.3 (C-21),
12.0 (C-18); anal. calcd for C26H42O4: C, 74.60; H, 10.11; found C, 74.51; H, 10.02.
2-(2-Hydroxyethoxy)ethyl (5β)-3-Oxocholan-24-oate (14b)
Colorless waxy solid, 0.35 g, 75% yield. [
α
]
19 + 20.0 (c 0.81, CHCl3); IR (KBr)
ν
max
D
−1
2934, 2866, 1735, 1714, 1451, 1379, 1298, 1254, 1173, 1132, 1072, 966, 946, 889, 769, 615 cm
;
1H NMR (CDCl3, 500 MHz)
δ
4.24–4.20 (2H, m, CH2O), 3.74–3.67 (4H, m, CH2O, CH2OH),
3.60–3.58 (2H, m, CH2O), 2.71-1.03 (28H, m), 0.99 (3H, s, H-19), 0.90 (3H, d, J = 6.5 Hz, H-21),
0.66 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
213.4 (C-3), 174.2 (C-24), 72.3 (CH2O),
δ
69.1 (CH2O), 63.3 (CH2O), 61.7 (CH2OH), 56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.7 (C-13),
42.3 (C-4), 40.7 (C-9), 40.0 (C-12), 37.2 (C-2), 36.9 (C-1), 35.5 (C-8), 35.3 (C-20), 34.9 (C-10),
31.1 (C-23), 30.9 (C-22), 28.1 (C-16), 26.9 (C-6), 25.7 (C-7), 24.1 (C-15), 22.6 (C-19), 21.2 (C-11),
18.3 (C-21), 12.1 (C-18); anal. calcd for C28H46O5: C, 72.69; H, 10.02; found C, 72.56; H, 9.97.
2-[2-(2-Hydroxyethoxy)ethoxy]ethyl (5β)-3-Oxocholan-24-oateb (14c)
Colorless waxy solid, 0.37 g, 73% yield. [
2930, 2866, 1735, 1714, 1619, 1450, 1383, 1352, 1298, 1255, 1173, 1124, 1104, 1068, 945, 805,
530 cm−1; 1H NMR (CDCl3, 500 MHz)
4.20–4.18 (2H, m, CH2O), 3.69–3.61 (8H, m, CH2O,
CH2OH), 3.56–3.54 (2H, m, CH2O), 2.72 (1H, br s, OH), 2.67-1.03 (28H, m), 0.97 (3H, s, H-19),
0.87 (3H, d, J = 6.5 Hz, H-21), 0.63 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
213.2 (C-3),
α] ν
17 + 20.5 (c 0.89, CHCl3); IR (KBr)
max
D
δ
δ
174.1 (C-24), 72.5 (CH2O), 70.5, 70.3 (CH2O), 69.1 (CH2O), 63.2 (CH2O), 61.6 (CH2OH),
56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.7 (C-13), 42.3 (C-4), 40.6 (C-9), 39.9 (C-12), 37.1 (C-2),
36.9 (C-1), 35.5 (C-8), 35.3 (C-20), 34.8 (C-10), 31.0 (C-23), 30.8 (C-22), 28.1 (C-16), 26.6 (C-6),
25.7 (C-7), 24.1 (C-15), 22.6 (C-19), 21.1 (C-11), 18.2 (C-21), 12.0 (C-18); anal. calcd for
C30H50O6: C, 71.11; H, 9.95; found C, 71.06; H, 9.89.
2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethyl (5β)-3-Oxocholan-24-oate (14d)
Colorless waxy solid, 0.39 g, 70% yield. [
α
]
18 + 17.2 (c 0.99, CHCl3); IR (KBr)
ν
max
D
−1
2931, 2866, 1734, 1714, 1619, 1451, 1383, 1298, 1259, 1173, 1125, 1105, 945, 887, 801, 531 cm
;
1H NMR (CDCl3, 500 MHz)
δ
4.19–4.16 (2H, m, CH2O), 3.70–3.60 (12H, m, CH2O, CH2OH),
3.56–3.54 (2H, m, CH2O), 2.83 (1H, br s, OH), 2.67-1.03 (28H, m), 0.96 (3H, s, H-19), 0.87 (3H,
d, J = 6.5 Hz, H-21), 0.63 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
213.2 (C-3), 174.1 (C-24),
δ
72.5 (CH2O), 70.6, 70.5, 70.3, (CH2O), 69.2 (CH2O), 63.3 (CH2O), 61.6 (CH2OH), 56.4 (C-14),
55.9 (C-17), 44.3 (C-5), 42.7 (C-13), 42.3 (C-4), 40.6 (C-9), 39.9 (C-12), 37.1 (C-2), 36.9 (C-1),
35.5 (C-8), 35.3 (C-20), 34.8 (C-10), 31.0 (C-23), 30.8 (C-22), 28.1 (C-16), 26.6 (C-6), 25.7 (C-7),
24.1 (C-15), 22.6 (C-19), 21.1 (C-11), 18.2 (C-21), 12.0 (C-18); anal. calcd for C32H54O7: C,
69.78; H, 9.88; found C, 69.72; H, 9.83.
3.9.6. Reaction of Ethylene Glycol Derivatives of 3a-Acetoxy-5β-cholan-24-oic Acid and
3-Oxo-cholan-24-oic Acid 13a–d, 14a–d with (5Z,9Z)-Tetradeca-5,9-dienedioic Acid (5)
Lithocholic acid derivative 15a
fied literature procedure [26]. To a solution of ethylene glycol derivatives of 3
-cholan-24-oic acid 13a or 3-oxo-cholan-24-oic acid 14a (1.0 mmol) in CH2Cl2
(50 mL), (5Z,9Z)-tetradeca-5.9-dienedioic acid ( ) (0.51 g, 2.0 mmol) was added, followed
by EDC HCl (0.48 g, 2.5 mmol) and DMAP (18 mg, 0.15 mmol) under argon. The mixture
–
d
and 16a
–
d
was synthesized according to a modi-
α
-acetoxy-
5
β
–
d
–d
5
·
was stirred at room temperature for 16 h until the reaction was complete (TLC monitoring,
hexane/ethyl acetate). The mixture was diluted with H2O (10 mL) and the CH2Cl2 layer
was separated, dried over MgSO4, and concentrated. The crude product was purified by
column chromatography (silica gel) using hexane/ethyl acetate (2:1) as the elution solvent
to afford compounds 15a–d or 16a–d.

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(5Z,9Z)-14-(2-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]oxy}ethoxy)-14-oxotetradeca-
5,9-dienoic acid (15a)
Colorless waxy solid, 0.44 g, 64% yield. [α] ν
19 + 15.9 (c 0.76, CHCl3); IR (KBr)
max
D
3065, 2937, 2865, 1947, 1735, 1679, 1450, 1385, 1244, 1141, 1105, 1026, 870, 803, 750, 690,
613 cm−1; 1H NMR (CDCl3, 500 MHz)
δ
5.45–5.32 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.75–4.71
(1H, m, H-3), 4.41–4.39 (4H, m, CH2O), 2.40-1.02 (28H, m), 2.39–2.33 (4H, m, H-2’, H-13’),
2.13–2.05 (8H, m, H-4’, H-7’, H-8’, H-11’), 2.04 (3H, s, COCH3), 1.74–1.67 (4H, m, H-3’,
H-12’), 0.94 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.66 (3H, s, H-18); 13C NMR (CDCl3,
125 MHz)
δ 178.7 (C-1’), 174.1 (C-24), 173.6 (C-14’), 170.7 (COCH3), 130.4, 130.3 (C-6’, C-9’),
128.9 (C-5’, C-10’), 74.4 (C-3), 62.1 (CH2O), 62.0 (CH2O), 56.5 (C-14), 56.0 (C-17), 42.7 (C-13),
41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.3 (C-20), 35.0 (C-1), 34.6 (C-10), 33.5 (C-13’),
33.2 (C-2’), 32.2 (C-4), 31.1 (C-23), 30.9 (C-22), 28.2 (C-16), 27.3 (C-7’, C-8’), 27.0 (C-6),
26.6 (C-4’, C-11’), 26.5 (C-2), 26.3 (C-7), 24.8 (C-12’), 24.6 (C-3’), 24.2 (C-15), 23.3 (C-19),
21.5 (COCH3), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C42H66O8: C, 72.17; H,
9.52; found C, 72.11; H, 9.45.
(5Z,9Z)-14-[2-(2-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]oxy}ethoxy)ethoxy]-14-
oxotetradeca-5,9-dienoic Acid (15b)
Colorless waxy solid, 0.46 g, 63% yield. [
3063, 2938, 2863, 1947, 1734, 1679, 1451, 1383, 1244, 1139, 1103, 1026, 870, 801, 750, 693,
614 cm−1; 1H NMR (CDCl3, 500 MHz)
5.42–5.31 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.72–4.68
α] ν
19 + 20.0 (c 0.95, CHCl3); IR (KBr)
max
D
δ
(1H, m, H-3), 4.23–4.20 (4H, m, CH2O), 3.70–3.67 (4H, m, CH2O), 2.40-0.95 (28H, m), 2.36–
2.30 (4H, m, H-2’, H-13’), 2.10–2.03 (8H, m, H-4’, H-7’, H-8’, H-11’), 2.01 (3H, s, COCH3),
1.73–1.66 (4H, m, H-3’, H-12’), 0.91 (3H, s, H-19), 0.89 (3H, d, J = 6.5 Hz, H-21), 0.63 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 178.8 (C-1’), 174.2 (C-24), 173.7 (C-14’), 170.7 (COCH3),
130.3, 130.2 (C-6’, C-9’), 128.9, 128.8 (C-5’, C-10’), 74.4 (C-3), 69.0 (CH2O), 63.2 (CH2O),
56.5 (C-14), 55.9 (C-17), 42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.3 (C-20),
35.0 (C-1), 34.6 (C-10), 33.5 (C-13’), 33.3 (C-2’), 32.2 (C-4), 31.1 (C-23), 30.9 (C-22), 28.2 (C-
16), 27.3, 27.2 (C-7’, C-8’), 26.9 (C-6), 26.6 (C-11’), 26.5 (C-2, C-4’), 26.3 (C-7), 24.8 (C-12’),
24.6 (C-3’), 24.2 (C-15), 23.3 (C-19), 21.4 (COCH3), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal.
calcd for C44H70O9: C, 71.12; H, 9.50; found C, 71.01; H, 9.43.
(5Z,9Z)-14-{2-[2-(2-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]oxy}ethoxy)ethoxy]ethoxy}-
14-oxotetradeca-5,9-dienoic Acid (15c)
Colorless waxy solid, 0.48 g, 61% yield. [
3065, 2938, 2867, 1946, 1736, 1681, 1454, 1381, 1245, 1100, 1027, 981, 873, 802, 735, 698,
615 cm−1; 1H NMR (CDCl3, 500 MHz)
5.45–5.30 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.77–
α] ν
19 + 15.5 (c 0.85, CHCl3); IR (KBr)
max
D
δ
4.72 (1H, m, H-3), 4.26–4.23 (4H, m, CH2O), 3.74–3.71 (4H, m, CH2O), 3.69–3.67 (4H, m,
CH2O), 2.40-1.00 (28H, m), 2.39–2.33 (4H, m, H-2’, H-13’), 2.13–2.06 (8H, m, H-4’, H-7’, H-8’,
H-11’), 2.04 (3H, s, COCH3), 1.74–1.67 (4H, m, H-3’, H-12’), 0.94 (3H, s, H-19), 0.92 (3H, d,
J = 6.5 Hz, H-21), 0.65 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 178.3 (C-1’), 174.3 (C-
24), 173.8 (C-14’), 170.7 (COCH3), 130.4, 130.2 (C-6’, C-9’), 128.9 (C-5’, C-10’), 74.4 (C-3),
70.5 (CH2O), 69.3 (CH2O), 63.3 CH2O), 56.5 (C-14), 56.0 (C-17), 42.7 (C-13), 41.9 (C-5),
40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.4 (C-20), 35.0 (C-1), 34.6 (C-10), 33.6 (C-13’), 33.2 (C-
2’), 32.2 (C-4), 31.1 (C-23), 30.9 (C-22), 28.2 (C-16), 27.3 (C-7’, C-8’), 27.0 (C-6), 26.6 (C-4’,
C-11’), 26.5 (C-2), 26.3 (C-7), 24.8 (C-12’), 24.6 (C-3’), 24.2 (C-15), 23.3 (C-19), 21.5 (COCH3),
20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C46H74O10: C, 70.20; H, 9.48; found C,
70.13; H, 9.43.
(5Z,9Z)-14-{2-[2-(2-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]oxy}ethoxy)ethoxy]ethoxy}-
14-oxotetradeca-5,9-dienoic Acid (15d)
Colorless waxy solid, 0.50 g, 61% yield. [
2929, 2868, 1736, 1619, 1451, 1383, 1363, 1243, 1142, 1100, 1028, 802, 755, 673, 614 cm−1
1H NMR (CDCl3, 500 MHz)
5.44–5.33 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.76–4.70 (1H,
m, H-3), 4.26–4.23 (4H, m, CH2O), 3.74–3.71 (4H, m, CH2O), 3.69–3.67 (8H, m, CH2O),
α] ν
19 + 14.0 (c 0.90, CHCl3); IR (KBr)
max
D
;
δ

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2.40–1.00 (28H, m), 2.39–2.33 (4H, m, H-2’, H-13’), 2.13–2.05 (8H, m, H-4’, H-7’, H-8’, H-
11’), 2.04 (3H, s, COCH3), 1.74–1.67 (4H, m, H-3’, H-12’), 0.94 (3H, s, H-19), 0.92 (3H, d,
J = 6.5 Hz, H-21), 0.65 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 177.3 (C-1’), 174.3 (C-
24), 173.8 (C-14’), 170.7 (COCH3), 130.3, 130.2 (C-6’, C-9’), 128.9 (C-5’, C-10’), 74.4 (C-3),
70.6 (CH2O), 70.5 (CH2O), 69.32 (CH2O), 63.4 (CH2O), 56.5 (C-14), 56.0 (C-17), 42.7 (C-13),
41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.4 (C-20), 35.0 (C-1), 34.6 (C-10), 33.6 (C-13’),
33.1 (C-2’), 32.3 (C-4), 31.1 (C-23), 30.9 (C-22), 28.2 (C-16), 27.3 (C-7’, C-8’), 27.0 (C-6),
26.6 (C-2, C-11’), 26.5 (C-4’), 26.3 (C-7), 24.8 (C-12’), 24.6 (C-3’), 24.2 (C-15), 23.3 (C-19),
21.5 (COCH3), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C48H78O11: C, 69.37; H,
9.46; found C, 69.30; H, 9.39.
(5Z,9Z)-14-(2-{[(5β)-3,24-Dioxocholan-24-yl]oxy}ethoxy)-14-oxotetradeca-5,9-dienoic
Acid (16a)
Colorless waxy solid, 0.42 g, 65% yield. [
3063, 2938, 2865, 1953, 1729, 1679, 1443, 1385, 1239, 1125, 1105, 1011, 875, 803, 750, 690,
615 cm−1; 1H NMR (CDCl3, 500 MHz)
5.44–5.33 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.31–
α] ν
19 + 16.9 (c 0.73, CHCl3); IR (KBr)
max
D
δ
4.26 (4H, m, CH2O), 2.73-1.06 (28H, m), 2.39–2.32 (4H, m, H-2’, H-13’), 2.14–2.06 (8H, m,
H-4’, H-7’, H-8’, H-11’), 1.74–1.67 (4H, m, H-3’, H-12’), 1.02 (3H, s, H-19), 0.92 (3H, d,
J = 6.5 Hz, H-21), 0.69 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 178.3 (C-1’),
174.0 (C-24), 173.6 (C-14’), 130.3 (C-6’, C-9’), 128.9 (C-5’, C-10’), 62.1 (CH2O), 62.0 (CH2O),
56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.8 (C-13), 42.3 (C-4), 40.7 (C-9), 40.0 (C-12), 37.2 (C-2),
36.9 (C-1), 35.5 (C-8), 35.3 (C-20), 34.9 (C-10), 33.5 (C-13’), 33.2 (C-2’), 31.1 (C-22), 30.9 (C-23),
28.2 (C-16), 27.3 (C-7’, C-8’), 26.6 (C-6), 26.5 (C-4’, C-11’), 25.8 (C-7), 24.8 (C-12’), 24.6 (C-3’),
24.2 (C-15), 22.6 (C-19), 21.2 (C-11), 18.3 (C-21), 12.1 (C-18); anal. calcd for C40H62O7: C,
73.36; H, 9.54; found C, 73.29; H, 9.50.
(5Z,9Z)-14-[2-(2-{[(5β)-3,24-Dioxocholan-24-yl]oxy}ethoxy)ethoxy]-14-oxotetradeca-5,9-
dienoic Acid (16b)
Colorless waxy solid, 0.44 g, 64% yield. [
3066, 2938, 2863, 1951, 1731, 1675, 1441, 1385, 1235, 1120, 1101, 1011, 875, 801, 750, 693,
615 cm−1; 1H NMR (CDCl3, 500 MHz)
5.45–5.33 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.26–
α] ν
19 + 17.9 (c 0.78, CHCl3); IR (KBr)
max
D
δ
4.22 (4H, m, CH2O), 3.74–3.69 (4H, m, CH2O), 2.73-1.06 (28H, m), 2.39–2.32 (4H, m, H-2’,
H-13’), 2.14–2.06 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.74–1.66 (4H, m, H-3’, H-12’), 1.03 (3H,
s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.69 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ
213.6 (C-3), 178.3 (C-1’), 174.2 (C-24), 173.8 (C-14’), 130.3, 130.2 (C-6’, C-9’), 128.9 (C-5’,
C-10’), 69.1 (CH2O), 63.3 (CH2O), 56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.8 (C-13), 42.3 (C-4),
40.7 (C-9), 40.0 (C-12), 37.2 (C-2), 37.0 (C-1), 35.5 (C-8), 35.3 (C-20), 34.9 (C-10), 33.6 (C-13’),
33.2 (C-2’), 31.1 (C-22), 30.9 (C-23), 28.2 (C-16), 27.3 (C-7’, C-8’), 26.6 (C-6), 26.5 (C-4’,
C-11’), 25.8 (C-7), 24.8 (C-12’), 24.6 (C-3’), 24.2 (C-15), 22.6 (C-19), 21.2 (C-11), 18.3 (C-21),
12.1 (C-18); anal. calcd for C42H66O8: C, 72.17; H, 9.52; found C, 72.07; H, 9.45.
(5Z,9Z)-14-{2-[2-(2-{[(5β)-3,24-Dioxocholan-24-yl]oxy}ethoxy)ethoxy]ethoxy}-14-oxotetradeca-
5,9-dienoic Acid (16c)
Colorless waxy solid, 0.45 g, 61% yield. [
2929, 2866, 1736, 1712, 1619, 1452, 1383, 1298, 1245, 1173, 1143, 1106, 1044, 965, 860, 731,
530 cm−1; 1H NMR (CDCl3, 500 MHz)
5.40–5.31 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.24–4.20
α] ν
18 + 12.5 (c 0.93, CHCl3); IR (KBr)
max
D
δ
(4H, m, CH2O), 3.69–3.66 (4H, m, CH2O), 3.65–3.63 (4H, m, CH2O), 2.70-1.04 (28H, m), 2.37–
2.30 (4H, m, H-2’, H-13’), 2.13–2.03 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.71–1.65 (4H, m, H-3’,
H-12’), 0.99 (3H, s, H-19), 0.90 (3H, d, J = 6.5 Hz, H-21), 0.66 (3H, s, H-18); 13C NMR (CDCl3,
125 MHz)
δ 213.7 (C-3), 178.5 (C-1’), 174.2 (C-24), 173.7 (C-14’), 130.3, 130.2 (C-6’, C-9’),
128.9, 128.8 (C-5’, C-10’), 70.5 (CH2O), 69.3 (CH2O), 63.3 (CH2O), 56.4 (C-14), 55.9 (C-17)
,
44.3 (C-5), 42.7 (C-13), 42.3 (C-4), 40.7 (C-9), 40.0 (C-12), 37.1 (C-2), 36.9 (C-1), 35.5 (C-8),
35.3 (C-20), 34.9 (C-10), 33.6 (C-13’), 33.3 (C-2’), 31.1 (C-23), 30.8 (C-22), 28.1 (C-16), 27.3,
27.2 (C-7’, C-8’), 26.6 (C-6), 26.5 (C-4’, C-11’), 25.7 (C-7), 24.8 (C-12’), 24.6 (C-3’), 24.1 (C-15),

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22.6 (C-19), 21.2 (C-11), 18.3 (C-21), 12.1 (C-18); anal. calcd for C44H70O9: C, 71.12; H, 9.50;
found C, 71.02; H, 9.44.
(5Z,9Z)-14-{2-[2-(2-{[(5β)-3,24-Dioxocholan-24-yl]oxy}ethoxy)ethoxy]ethoxy}-14-oxotetradeca-
5,9-dienoic Acid (16d)
Colorless waxy solid, 0.48 g, 60% yield. [α] ν
18 + 10.4 (c 0.75, CHCl3); IR (KBr)
max
D
2929, 2866, 1735, 1715, 1619, 1456, 1384, 1298, 1246, 1172, 1143, 1104, 1041, 947, 859, 825,
1
732, 530 cm−1; H NMR (CDCl3, 500 MHz)
δ 5.45–5.34 (4H, m, H-5’, H-6’, H-9’, H-10’),
4.26–4.23 (4H, m, CH2O), 3.74–3.69 (4H, m, CH2O), 3.68–3.66 (8H, m, CH2O), 2.74-1.06
(28H, m), 2.40–2.33 (4H, m, H-2’, H-13’), 2.14–2.05 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.75–1.67
(4H, m, H-3’, H-12’), 1.03 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.69 (3H, s, H-18);
13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 177.4 (C-1’), 174.3 (C-24), 173.9 (C-14’), 130.3,
130.2 (C-6’, C-9’), 128.9 (C-5’, C-10’), 70.6 (CH2O), 70.5 (CH2O), 69.22 (CH2O), 63.4 (CH2O),
56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.8 (C-13), 42.4 (C-4), 40.7 (C-9), 40.1 (C-12), 37.2 (C-2),
37.0 (C-1), 35.5 (C-8), 35.3 (C-20), 34.9 (C-10), 33.6 (C-13’), 33.1 (C-2’), 31.1 (C-23), 30.9 (C-22),
28.2 (C-16), 27.4, 27.3 (C-7’, C-8’), 26.6 (C-6), 26.5 (C-4’, C-11’), 25.8 (C-7), 24.8 (C-12’),
24.6 (C-3’), 24.2 (C-15), 22.7 (C-19), 21.2 (C-11), 18.3 (C-21), 12.1 (C-18); anal. calcd for
C46H74O10: C, 70.20; H, 9.48; found C, 70.11; H, 9.41.
3.9.7. General Procedure for the Synthesis of BOC-Protected Diaminoalkane Derivatives of
3a-Acetoxy-5β-cholan-24-oic Acid and 3-Oxo-cholan-24-oic acid 19a–d, 20a–d
Oxalyl chloride (2 mL) was added to a solution of 9 or 12 (1.2 mmol) in CH2Cl2
(20 mL) and stirred at room temperature for 24 h excluding air humidity, affording the
crude 24-acylchloride 17 or 18. After evaporation to dryness, another portion of CH2Cl2
(10 mL) was added, followed by diisopropylethylamine (0.5 mL, 3.0 mmol) and tert-butyl
(2-aminoalcane)carbamate (1.8 mmol), stirred at room temperature for an additional 16 h,
then evaporated, and the residue was purified by chromatography on silica gel using
hexane/ethyl acetate (1:2) as the mobile phase, affording the product 19a–d or 20a–d.
(3α,5β)-24-({2-[(tert-Butoxycarbonyl)amino]ethyl}amino)-24-oxocholan-3-yl Acetate (19a)
◦
19
White crystals, 0.60 g, 90% yield. MP 118–120 C, [
IR (KBr) 2934, 2867, 1719, 1696, 1649, 1450, 1382, 1365, 1244, 1172, 1028, 755, 665,
610 cm−1; 1H NMR (CDCl3, 500 MHz)
α
]
+ 32.4 (c 0.76, CHCl3);
D
ν
max
δ
6.62 (1H, br s, NH), 5.31 (1H, t, J = 6.5 Hz, NH),
4.69–4.63 (1H, m, H-3), 3.32–3.28 (2H, m, CH2N), 3.23–3.17 (2H, m, CH2N), 2.22-0.95 (28H,
m), 1.98 (3H, s, CH3CO), 1.39 (9H, s, CH3), 0.88 (3H, s, H-19), 0.86 (3H, d, J = 6.5 Hz, H-21),
0.59 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.4 (C-24), 170.6 (CH3CO), 156.9 (COO),
79.4 (C(CH3)3), 74.4 (C-3), 56.4 (C-14), 56.0 (C-17), 42.7 (C-13), 41.8 (C-5), 40.5 (CH2N),
40.4 (C-9), 40.3 (CH2N), 40.1 (C-12), 35.7 (C-8), 35.5 (C-20), 34.9 (C-1), 34.5 (C-10), 33.5 (C-23),
32.2 (C-4), 31.7 (C-22), 28.4 (CH3), 28.2 (C-16), 26.9 (C-6), 26.6 (C-2), 26.3 (C-7), 24.1 (C-15),
23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21), 12.0 (C-18); anal. calcd for C33H56N2O5:
C, 70.68; H, 10.06; found C, 70.59; H, 9.98.
(3
α
,5
β
)-24-({4-[(tert-Butoxycarbonyl)amino]butyl}amino)-24-oxocholan-3-yl Acetate (19b)
White crystals, 0.61 g, 87% yield. MP 88–90 ◦C, [
α
]
D
+ 26.9 (c 0.86, CHCl3); IR (KBr)
21
−1
ν
max 2932, 2866, 1736, 1696, 1649, 1534, 1449, 1383, 1364, 1244, 1172, 1027, 754, 666, 608 cm
;
1H NMR (CDCl3, 500 MHz)
δ 6.21 (1H, br s, NH), 4.84 (1H, br s, NH), 4.66–4.60 (1H, m,
H-3), 3.21–3.16 (2H, m, CH2N), 3.08–3.03 (2H, m, CH2N), 2.19-0.93 (28H, m), 1.97 (3H, s,
CH3CO), 1.48–1.44 (4H, m, CH2), 1.37 (9H, s, CH3), 0.86 (3H, s, H-19), 0.85 (3H, d, J = 6.5 Hz
H-21), 0.58 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
173.7 (C-24), 170.6 (CH3CO),
,
δ
156.1 (COO), 79.4 (C(CH3)3), 74.3 (C-3), 56.4 (C-14), 56.0 (C-17), 42.7 (C-13), 41.8 (C-5),
40.4(C-9), 40.1 (C-12, CH2N), 39.0 (CH2N), 35.7 (C-8), 35.5 (C-20), 34.9 (C-1), 34.5 (C-10),
33.5 (C-23), 32.2 (C-4), 31.8 (C-22), 28.4 (CH3), 28.2 (C-16), 27.6 (CH2), 26.9 (C-6), 26.6 (C-2,
CH2), 26.3 (C-7), 24.1 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18);
anal. calcd for C35H60N2O5: C, 71.39; H, 10.27; found C, 71.32; H, 10.21.

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(3
α
,5
β
)-24-({6-[(tert-Butoxycarbonyl)amino]hexyl}amino)-24-oxocholan-3-yl Acetate (19c
)
Colorless waxy solid, 0.61 g, 84% yield. [
α] ν
21 + 26.9 (c 0.86, CHCl3); IR (KBr)
max
D
2935, 2863, 1734, 1696, 1645, 1534, 1447, 1383, 1363, 1244, 1169, 1027, 753, 665, 608 cm−1
;
1H NMR (CDCl3, 500 MHz)
δ 5.84 (1H, br s, NH), 4.66–4.62 (1H, m, H-3), 4.64 (1H, br s,
NH), 3.20–3.15 (2H, m, CH2N), 3.08–3.02 (2H, m, CH2N), 2.21-0.95 (28H, m), 1.99 (3H, s,
CH3CO), 1.48–1.40 (4H, m, CH2), 1.39 (9H, s, CH3), 1.31–1.25 (4H, m, CH2), 0.88 (3H, s,
H-19), 0.87 (3H, d, J = 6.5 Hz, H-21), 0.59 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ
173.6 (C-24), 170.6 (CH3CO), 156.1 (CO), 78.9 (C(CH3)3), 74.4 (C-3), 56.5 (C-14), 56.1 (C-17),
42.7 (C-13), 41.9 (C-5), 40.4(C-9), 40.1 (C-12, CH2N), 39.1 (CH2N), 35.8 (C-8), 35.5 (C-20),
35.0 (C-1), 34.6 (C-10), 33.6 (C-23), 32.2 (C-4), 31.9 (C-22), 29.9 (CH2), 29.5 (CH2), 28.4 (CH3),
28.2 (C-16), 26.9 (C-6), 26.6 (C-2), 26.3 (C-7), 26.2 (CH2), 26.1 (CH2), 24.2 (C-15), 23.3 (C-19),
21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21), 12.0 (C-18); anal. calcd for C37H64N2O5: C, 72.04; H,
10.46; found C, 71.96; H, 10.41.
tert-Butyl 4-[(3
α
,5
β
)-3(acetyloxy)-24-oxocholan-3-yl]piperazine-1-carboxylate Acetate (19d)
21
Colorless waxy solid, 0.58 g, 83% yield. [
α
]
+ 25.0 (c 0.82, CHCl3); IR (KBr)
D
ν
2933, 2866, 1734, 1698, 1649, 1455, 1419, 1381, 1364, 1285, 1242, 1168, 1127, 1028,
max
997, 863, 754, 665, 614 cm−1; 1H NMR (CDCl3, 500 MHz)
δ 4.72–4.66 (1H, m, H-3), 3.58–
3.54 (2H, m, CH2N), 3.44–3.36 (6H, m, CH2N), 2.38-0.96 (28H, m), 2.01 (3H, s, CH3CO),
1.45 (9H, s, CH3), 0.93 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.63 (3H, s, H-18);
13C NMR (CDCl3, 125 MHz)
δ 172.3 (C-24), 170.6 (CH3CO), 154.5 (COO), 80.2 (C(CH3)3),
74.3 (C-3), 56.5 (C-14), 56.1 (C-17), 45.4 (CH2N), 42.7 (C-13), 41.9 (C-5), 41.3 (CH2N),
40.4 (C-9), 40.1 (C-12), 35.8 (C-8), 35.6 (C-20), 35.0 (C-1), 34.6 (C-10), 32.2 (C-4), 31.4 (C-23),
30.3 (C-22), 28.4 (CH3), 28.3 (C-16), 26.9 (C-6), 26.6 (C-2), 26.3 (C-7), 24.2 (C-15), 23.3 (C-19),
21.4 (CH3CO), 20.8 (C-11), 18.5 (C-21), 12.0 (C-18); anal. calcd for C35H58N2O5: C, 71.63; H,
9.96; found C, 71.58; H, 9.91.
tert-Butyl (2-{[(5β)-3,24-dioxocholan-24-yl)amino]ethyl}carbamate (20a)
17
White waxy solid, 0.55 g, 89% yield. [
α
]
D
+ 19.0 (c 0.88, CHCl3); IR (KBr)
ν 2934,
max
1
2867, 1707, 1652, 1531, 1454, 1366, 1269, 1252, 1172, 1028, 754, 665, 531 cm−1; H NMR
(CDCl3, 500 MHz) 6.36 (1H, br s, NH), 5.07 (1H, br s, NH), 3.37–3.33 (2H, m, CH2N), 3.29–
3.26 (2H, m, CH2N), 2.72-1.05 (28H, m), 1.44 (9H, s, CH3), 1.02 (3H, s, H-19), 0.92 (3H, d,
J = 6.5 Hz, H-21), 0.68 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
213.5 (C-3), 174.3 (C-24),
δ
δ
156.9 (COO), 79.6 (C(CH3)3), 56.4 (C-14), 56.0 (C-17), 44.3 (C-5), 42.8 (C-13), 42.4 (C-4),
40.7 (C-9, CH2N), 40.3 (CH2N), 40.1 (C-12), 37.2 (C-2), 37.0 (C-1), 35.5 (C-8, C-20), 34.9 (C-10),
33.5 (C-23), 31.7 (C-22), 28.4 (CH3), 28.2 (C-16), 26.6 (C-6), 25.8 (C-7), 24.1 (C-15), 22.6 (C-19),
21.2 (C-11), 18.4 (C-21), 12.1 (C-18); anal. calcd for C31H52N2O4: C, 72.05; H, 10.14; found C,
72.00; H, 10.09. MALDI TOF: m/z 539.387 ([M + Na]+, calcd 539.382), 555.362 ([M + K]+,
calcd 555.356).
tert-Butyl Butyl (4-{[(5β)-3,24-Dioxocholan-24-yl)amino]butyl}carbamate (20b)
16
White waxy solid, 0.55 g, 85% yield. [
α
]
+ 19.3 (c 0.97, CHCl3); IR (KBr) ν
max
D
2929, 2865, 1711, 1650, 1536, 1452, 1379, 1269, 1252, 1173, 756, 666 cm−1; 1H NMR (CDCl3,
500 MHz) 6.11 (1H, br s, NH), 4.71 (1H, br s, NH), 3.27–3.23 (2H, m, CH2N), 3.14–3.10
(2H, m, CH2N), 2.71-1.05 (28H, m), 1.49–1.44 (4H, m, CH2), 1.43 (9H, s, CH3), 1.01 (3H,
s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.67 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ
δ
213.4 (C-3), 173.9 (C-24), 156.2 (COO), 79.2 (C(CH3)3), 56.4 (C-14), 56.0 (C-17), 44.3 (C-5),
42.8 (C-13), 42.4 (C-4), 40.7 (C-9, CH2N), 40.1 (C-12), 39.2 (CH2N), 37.2 (C-2), 37.0 (C-1),
35.5 (C-8, C-20), 34.9 (C-10), 33.5 (C-23), 31.9 (C-22), 28.4 (CH3), 28.2 (C-16), 27.7 (CH2),
26.6 (C-6, CH2), 25.8 (C-7), 24.1 (C-15), 22.6 (C-19), 21.2 (C-11), 18.4 (C-21), 12.1 (C-18);
anal. calcd for C33H56N2O4: C, 72.75; H, 10.36; found C, 72.69; H, 10.33. MALDI TOF:
m/z 689.415 ([M + Na]+, calcd 689.512), 705.377 ([M + K]+, calcd 705.486).

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tert-Butyl (6-{[(5β)-3,24-Dioxocholan-24-yl)amino]hexyl}carbamate (20c)
14
Colorless waxy solid, 0.58 g, 85% yield. [
α
]
+ 14.0 (c 0.95, CHCl3); IR (KBr)
D
1
ν
2932, 2864, 1710, 1649, 1539, 1454, 1378, 1271, 1251, 1173, 754, 666 cm−1; H NMR
max
(CDCl3, 500 MHz)
δ 5.95 (1H, br s, NH), 4.68 (1H, br s, NH), 3.20–3.16 (2H, m, CH2N),
3.08–3.04 (2H, m, CH2N), 2.68-1.02 (28H, m), 1.47–1.40 (4H, m, CH2), 1.39 (9H, s, CH3),
1.30–1.26 (4H, m, CH2), 0.97 (3H, s, H-19), 0.89 (3H, d, J = 6.5 Hz, H-21), 0.64 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.4 (C-3), 173.6 (C-24), 156.1 (COO), 78.9 (C(CH3)3),
56.4 (C-14), 56.0 (C-17), 44.3 (C-5), 42.7 (C-13), 42.3 (C-4), 40.7 (C-9), 40.2 (CH2N), 40.0 (C-12),
39.1 (CH2N), 37.2 (C-2), 36.9 (C-1), 35.5 (C-8, C-20), 34.8 (C-10), 33.6 (C-23), 31.8 (C-22),
29.9 (CH2), 29.5 (CH2), 28.4 (CH3), 28.2 (C-16), 26.6 (C-6), 26.2 (CH2), 26.1 (CH2), 25.7 (C-7),
24.1 (C-15), 22.6 (C-19), 21.2 (C-11), 18.4 (C-21), 12.1 (C-18); anal. calcd for C35H60N2O4:
C, 73.38; H, 10.56; found C, 73.29; H, 10.53. MALDI TOF: m/z 595.475 ([M + Na]+, calcd
595.445), 611.439 ([M + K]+, calcd 611.419).
tert-Butyl 4-(3,24-Dioxocholan-24-yl)piperazine-1-carboxylate (20d)
Colorless waxy solid, 0.53 g, 83% yield. [
2932, 2865, 1699, 1647, 1455, 1419, 1365, 1285, 1253, 1168, 1129, 1026, 997, 754, 665 cm−1
1H NMR (CDCl3, 500 MHz)
3.60–3.55 (2H, m, CH2N), 3.48–3.40 (6H, m, CH2N), 2.73–1.06
(28H, m), 1.48 (9H, s, CH3), 1.02 (3H, s, H-19), 0.95 (3H, d, J = 6.5 Hz, H-21), 0.69 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
213.4 (C-3), 172.3 (C-24), 154.6 (COO), 80.3 (C(CH3)3),
α] ν
21 + 16.6 (c 0.86, CHCl3); IR (KBr)
max
D
;
δ
δ
56.4 (C-14), 56.1 (C-17), 45.5 (CH2N), 44.3 (C-5), 42.8 (C-13), 42.4 (C-4), 41.3 (CH2N),
40.7 (C-9), 40.1 (C-12), 37.2 (C-2), 37.0 (C-1), 35.6 (C-8), 35.5 (C-20), 34.9 (C-10), 31.4 (C-22),
30.4 (C-23), 28.4 (CH3), 28.3 (C-16), 26.6 (C-6), 25.8 (C-7), 24.2 (C-15), 22.7 (C-19), 21.2 (C-11),
18.5 (C-21), 12.1 (C-18); anal. calcd for C33H54N2O4: C, 73.02; H, 10.03; found C, 72.93; H,
9.99. MALDI TOF: m/z 565.379 ([M + Na]+, calcd 565.398), 581.346 ([M + K]+, calcd 581.372).
3.9.8. General Procedure for Removing BOC-Protected Groups
Trifluoroacetic acid (3.4 mL) was added to a solution of 19a–d or 20a–d (1.0 mmol) in
CHCl3 (20 mL), and the mixture was stirred at room temperature for 1 h. The solvent was
then evaporated under reduced pressure, and the residue was purified by chromatography
on silica gel using CHCl3/MeOH (10:1) as the mobile phase, affording the product 21a
or 22a–d.
–d
(3α,5β)-24-[(2-Aminoethyl)amino]-24-oxocholan-3-yl Acetate (21a)
19
White crystals, 0.45 g, 98% yield. MP 34–36 ◦C, [
α
]
D
+ 23.9 (c 0.69, CHCl3); IR (KBr)
ν
2931, 2868, 1681, 1541, 1449, 1383, 1364, 1247, 1203, 1181, 1138, 1028, 981, 837, 799,
max
756, 666, 615 cm−1; 1H NMR (CDCl3, 500 MHz)
δ 8.11 (2H, br s, NH2), 7.55 (1H, br s, NH),
4.73–7.68 (1H, m, H-3), 3.56–3.46 (2H, m, CH2NH), 3.15–3.06 (2H, m, CH2NH2), 2.27–1.02
(28H, m), 2.01 (3H, s, CH3CO), 0.92 (3H, s, H-19), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.63 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 176.6 (C-24), 170.7 (CH3CO), 74.4 (C-3), 56.4 (C-14),
56.1 (C-17), 42.7 (C-13), 41.8 (C-5), 40.4 (C-9), 40.1 (C-12), 40.0 (CH2NH2), 37.3 (CH2NH),
35.8 (C-8), 35.6 (C-20), 35.0 (C-1), 34.6 (C-10), 33.2 (C-23), 32.2 (C-4), 31.6 (C-22), 28.2 (C-16),
27.0 (C-6), 26.6 (C-2), 26.3 (C-7), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11),
18.2 (C-21), 11.9 (C-18); anal. calcd for C28H48N2O3: C, 73.00; H, 10.50; found C, 72.95;
H, 10.46.
(3α,5β)-24-[(4-Aminobutyl)amino]-24-oxocholan-3-yl Acetate (21b)
17
White solid, 0.48 g, 98% yield. [
α
]
+ 18.0 (c 0.89, CHCl3); IR (KBr)
ν
2937,
max
D
2868, 1678, 1647, 1550, 1448, 1381, 1363, 1245, 1203, 1179, 1135, 1028, 836, 799, 755, 722,
665 cm−1; 1H NMR (CDCl3, 500 MHz)
δ
7.97 (2H, br s, NH2), 6.77 (1H, br s, NH), 4.73–4.68
(1H, m, H-3), 3.23–3.17 (2H, m, CH2NH), 2.99–2.93 (2H, m, CH2NH2), 2.25-1.00 (28H, m),
2.02 (3H, s, CH3CO), 1.72–1.66 (2H, m, CH2), 1.58–1.52 (2H, m, CH2), 0.93 (3H, s, H-19),
0.90 (3H, d, J = 6.5 Hz, H-21), 0.64 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ
174.9
(C-24), 170.6 (CH3CO), 74.4 (C-3), 56.5 (C-14), 56.1 (C-17), 42.7 (C-13), 41.9 (C-5), 40.4 (C-9),

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40.2 (C-12), 39.4 (CH2NH2), 38.6 (CH2NH), 35.8 (C-8), 35.6 (C-20), 35.0 (C-1), 34.6 (C-10),
33.4 (C-23), 32.3 (C-4), 31.6 (C-22), 28.2 (C-16), 27.0 (C-6), 26.6 (C-2), 26.4 (C-7), 26.2 (CH2),
24.4 (CH2), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal.
calcd for C30H52N2O3: C, 73.72; H, 10.72; found C, 73.67; H, 10.69.
(3α,5β)-24-[(6-Aminohexyl)amino]-24-oxocholan-3-yl Acetate (21c)
24
White solid, 0.50 g, 97% yield. [
α
]
+ 17.3 (c 0.96, CHCl3); IR (KBr)
ν
2937,
max
D
2866, 1680, 1644, 1548, 1448, 1380, 1363, 1244, 1202, 1179, 1137, 1041, 979, 933, 909, 835,
799, 755, 722, 667, 549 cm−1; 1H NMR (CDCl3, 500 MHz)
δ
7.97 (2H, br s, NH2), 6.41 (1H,
br s, NH), 4.73–4.68 (1H, m, H-3), 3.18–3.15 (2H, m, CH2NH), 2.95–2.90 (2H, m, CH2NH2),
2.26–0.96 (28H, m), 2.02 (3H, s, CH3CO), 1.70–1.65 (2H, m, CH2), 1.50–1.45 (2H, m, CH2),
1.36–1.29 (4H, m, CH2), 0.93 (3H, s, H-19), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.64 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 174.5 (C-24), 170.6 (CH3CO), 74.4 (C-3), 56.5 (C-14),
56.1 (C-17), 42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.2 (C-12), 39.7 (CH2NH2), 39.2 (CH2NH),
35.8 (C-8), 35.6 (C-20), 35.0 (C-1), 34.6 (C-10), 33.5 (C-23), 32.2 (C-4), 31.9 (C-22), 28.9 (CH2),
28.2 (C-16), 27.0 (C-6, CH2), 26.6 (C-2), 26.3 (C-7), 25.9 (CH2), 25.6 (CH2), 24.2 (C-15),
23.3 (C-19), 21.5 (CH3CO), 20.8 (C-11), 18.3 (C-21), 12.0 (C-18); anal. calcd for C32H56N2O3:
C, 74.37; H, 10.92; found C, 74.32; H, 10.89.
(3α,5β)-24-Oxo-24-piperazin-1-ylcholan-3-yl Acetate (21d)
24
White solid; 0.47 g, 97% yield. [
α
]
+ 20.5 (c 0.99, CHCl3); IR (KBr)
ν
2938,
max
D
2867, 1727, 1674, 1447 1431, 1383, 1363, 1246, 1202, 1180, 1134, 1028, 833, 799, 755, 722, 666,
564 cm−1; 1H NMR (CDCl3, 500 MHz)
δ
10.02 (1H, br s, NH), 4.71–4.67 (1H, m, H-3), 3.88–
3.77 (4H, m, CH2N), 3.22–3.17 (4H, m, CH2NH), 2.38-0.98 (28H, m), 2.00 (3H, s, CH3CO),
0.91 (3H, s, H-19), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.63 (3H, s, H-18); 13C NMR (CDCl3,
125 MHz)
δ 172.3 (C-24), 170.6 (CH3CO), 74.4 (C-3), 56.4 (C-14), 55.9 (C-17), 43.4 (CH2NH),
42.7 (C-13), 42.3 (CH2N), 41.8 (C-5), 40.4 (C-9), 40.1 (C-12), 38.2 (CH2N), 35.8 (C-8),
35.5 (C-20), 35.0 (C-1), 34.5 (C-10), 32.2 (C-4), 31.1 (C-22), 29.9 (C-23), 28.3 (C-16), 26.9 (C-6),
26.6 (C-2), 26.3 (C-7), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21),
12.0 (C-18); anal. calcd for C30H50N2O3: C, 74.03; H, 10.35; found C, 73.96; H, 10.32.
(5β)-N-(2-Aminoethyl)-3-oxocholan-24-amide (22a)
Colorless waxy solid, 0.41 g, 98% yield. [
2927, 2866, 1678, 1547, 1454, 1379, 1339, 1256, 1202, 1181, 1136, 836, 799, 755 cm−1; 1H NMR
(CDCl3, 500 MHz) 8.27 (2H, br s, NH2), 7.67 (1H, br s, NH), 3.54–3.48 (2H, m, CH2NH),
3.15–3.10 (2H, m, CH2NH2), 2.71-1.04 (28H, m), 1.00 (3H, s, H-19), 0.90 (3H, d, J = 6.5 Hz
H-21), 0.67 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
212.1 (C-3), 175.7 (C-24), 56.3 (C-14),
α] ν
19 + 17.0 (c 0.88, CHCl3); IR (KBr)
max
D
δ
,
δ
56.0 (C-17), 44.3 (C-5), 42.7 (C-13), 42.2 (C-4), 40.6 (C-9), 40.0 (C-12, CH2NH2), 37.2 (C-2),
36.9 (C-1, CH2NH), 35.5 (C-8, C-20), 34.8 (C-10), 32.9 (C-23), 31.5 (C-22), 28.1 (C-16),
26.6 (C-6), 25.7 (C-7), 24.1 (C-15), 22.4 (C-19), 21.1 (C-11), 18.1 (C-21), 11.8 (C-18); anal. calcd
for C26H44N2O2: C, 74.95; H, 10.64; found C, 74.89; H, 10.60.
(5β)-N-(4-Aminobutyl)-3-oxocholan-24-amide (22b)
Colorless waxy solid, 0.43 g, 97% yield. [
2936, 2867, 1682, 1646, 1557, 1542, 1456, 1379, 1339, 1268, 1202, 1179, 1134, 835, 799, 753,
722 cm−1; 1H NMR (CDCl3, 500 MHz)
8.14 (2H, br s, NH2), 6.79 (1H, br s, NH), 3.23–
α] ν
14 + 11.0 (c 0.98, CHCl3); IR (KBr)
max
D
δ
3.20 (2H, m, CH2NH), 2.99–2.95 (2H, m, CH2NH2), 2.73-1.05 (28H, m), 1.73–1.69 (2H, m,
CH2), 1.59–1.55 (2H, m, CH2), 1.03 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.68 (3H,
s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 174.9 (C-24), 56.4 (C-14), 55.9 (C-17),
44.2 (C-5), 42.8 (C-13), 42.3 (C-4), 40.7 (C-9), 40.0 (C-12), 39.4 (CH2NH2), 38.5 (CH2NH),
37.2 (C-2), 36.9 (C-1), 35.5 (C-8, C-20), 34.9 (C-10), 33.2 (C-23), 31.8 (C-22), 28.2 (C-16),
26.6 (C-6), 26.2 (CH2), 25.8 (C-7), 24.5 (CH2), 24.2 (C-15), 22.6 (C-19), 21.2 (C-11), 18.3 (C-21),
12.1 (C-18); anal. calcd for C28H48N2O2: C, 75.63; H, 10.88; found C, 75.57; H, 10.83. MALDI
TOF: m/z 445.377 ([M + H]+, calcd 445.379).

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(5β)-N-(6-Aminohexyl)-3-oxocholan-24-amide (22c)
Colorless waxy solid, 0.45 g, 97% yield. [
21 + 15.5 (c 0.82, CHCl3); IR (KBr)
α
]
ν
max
D
−1
2935, 2865, 1682, 1638, 1557, 1542, 1456, 1380, 1268, 1202, 1180, 1135, 835, 799, 755, 722 cm
;
1H NMR (CDCl3, 500 MHz)
δ
8.07 (2H, br s, NH2), 6.42 (1H, br s, NH), 3.19–3.16 (2H, m,
CH2NH), 2.95–2.91 (2H, m, CH2NH2), 2.70-1.05 (28H, m), 1.69–1.65 (2H, m, CH2), 1.49–1.46
(2H, m, CH2), 1.37–1.31 (4H, m, CH2), 1.03 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21),
0.69 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 174.4 (C-24), 56.4 (C-14),
55.9 (C-17), 44.3 (C-5), 42.8 (C-13), 42.3 (C-4), 40.7 (C-9), 40.1 (C-12), 39.6 (CH2NH2),
39.1 (CH2NH), 37.2 (C-2), 36.9 (C-1), 35.5 (C-8, C-20), 34.9 (C-10), 33.4 (C-23), 31.9 (C-22),
29.0 (CH2), 28.2 (C-16), 27.1 (CH2), 26.6 (C-6), 25.9 (CH2), 25.8 (C-7), 25.6 (CH2), 24.1 (C-15),
22.6 (C-19), 21.2 (C-11), 18.3 (C-21), 12.1 (C-18); anal. calcd for C30H52N2O2: C, 76.22; H,
11.09; found C, 76.17; H, 11.05. MALDI TOF: m/z 511.362 ([M + K]+, calcd 511.367).
(5β)-24-Oxo-24-piperazin-1-ylcholan-3-one (22d)
17
Colorless waxy solid, 0.42 g, 96% yield. [
α
]
+10.0 (c 0.89, CHCl3); IR (KBr) ν
max
D
2934, 2866, 2498, 1681, 1443 1378, 1203, 1181, 1135, 1026, 835, 800, 753, 721, 667 cm−1
1H NMR (CDCl3, 500 MHz)
9.59 (1H, br s, NH), 3.91–3.77 (4H, m, CH2N), 3.28–3.22 (4H,
m, CH2NH), 2.71–1.04 (28H, m), 1.02 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.68 (3H,
s, H-18); 13C NMR (CDCl3, 125 MHz)
213.8 (C-3), 172.8 (C-24), 56.4 (C-14), 55.8 (C-17),
;
δ
δ
44.2 (C-5), 43.5 (CH2NH), 42.8 (C-13), 42.3 (C-4), 42.3, 38.4 (CH2N), 40.7 (C-9), 40.0 (C-12),
37.2 (C-2), 36.9 (C-1), 35.5 (C-8, C-20), 34.8 (C-10), 31.0 (C-22), 29.8 (C-23), 28.2 (C-16),
26.6 (C-6), 25.7 (C-7), 24.1 (C-15), 22.6 (C-19), 21.2 (C-11), 18.4 (C-21), 12.0 (C-18); anal. calcd
for C28H46N2O2: C, 75.97; H, 10.47; found C, 75.95; H, 10.46. MALDI TOF: m/z 465.341 ([M
+ Na]+, calcd 465.346), 481.301 ([M + Na]+, calcd 481.319).
3.9.9. Reaction of Diaminoalkane Derivatives of 3a-Acetoxy-5β-cholan-24-oic Acid and
3-Oxo-cholan-24-oic Acid 21a–d, 22a–d with (5Z,9Z)-Tetradeca-5,9-dienedioic Acid (5)
Lithocholic acid derivative 23a
fied literature procedure [41]. To a solution of diaminoalkane derivatives of 3
-cholan-24-oic acid 21a or 3-oxo-cholan-24-oic acid 22a (1.0 mmol) in CH2Cl2
(50 mL), (5Z,9Z)-tetradeca-5,9-dienedioic acid ( ) (0.51 g, 2.0 mmol) was added, followed
by EDC HCl (0.48 g, 2.5 mmol) and DMAP (18 mg, 0.15 mmol) under argon. The mixture
–
d
and 24a
–
d
was synthesized according to a modi-
α
-acetoxy-
5
β
–
d
–d
5
·
was stirred at room temperature for 12 h until the reaction was complete (TLC monitoring,
hexane/ethyl acetate). The mixture was diluted with H2O (10 mL) and the CH2Cl2 layer
was separated, dried over MgSO4, and concentrated. The crude product was purified by
column chromatography (silica gel) using CHCl3/MeOH (10:1) as the elution solvent to
afford compounds 23a–d or 24a–d.
(5Z,9Z)-14-[(2-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]amino}ethyl)amino]-14-
oxotetradeca-5,9-dienoic Acid (23a)
Colorless waxy solid, 0.47 g, 68% yield. [
2929, 2866, 1735, 1647, 1546, 1449, 1379, 1363, 1243, 1065, 1028, 756, 666, 608 cm−1; 1H NMR
(CDCl3, 500 MHz) 7.08 (1H, br s, NH), 6.85 (1H, br s, NH), 5.43–5.33 (4H, m, H-5’, H-6’ H-9’,
α] ν
19 + 16.1 (c 0.76, CHCl3); IR (KBr)
max
D
δ
,
H-10’), 4.73–4.68 (1H, m, H-3), 3.39–3.33 (4H, m, CH2NH), 2.37–1.00 (28H, m), 2.37–2.34
(2H, m, H-2’), 2.23–2.19 (2H, m, H-13’), 2.10–2.04 (8H, m, H-4’, H-7’, H-8’, H-11’), 2.03 (3H,
s, CH3CO), 1.73–1.66 (4H, m, H-3’, H-12’), 0.93 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21),
0.64 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 176.2 (C-1’), 175.1 (C-24), 174.5 (C-14’),
170.7 (CH3CO), 130.2, 130.1 (C-6’, C-9’), 129.1 (C-5’, C-10’), 74.4 (C-3), 56.5 (C-14), 56.0 (C-17),
42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 39.9, 39.7 (CH2NH), 35.9 (C-13’), 35.8 (C-8),
35.5 (C-20), 35.0 (C-1), 34.6 (C-10), 33.5 (C-23), 33.1 (C-2’), 32.2 (C-4), 31.8 (C-22), 28.2 (C-16),
27.7, 27.5 (C-7’, C-8’), 27.3 (C-11’), 27.0 (C-6), 26.7 (C-4’), 26.6 (C-2), 26.3 (C-7), 25.6 (C-12’),
24.7 (C-3’), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21), 12.1 (C-18); anal.
calcd for C42H68N2O6: C, 72.37; H, 9.83; found C, 72.30; H, 9.79; MALDI TOF: m/z 719.428
([M + Na]+, calcd 719.498), 735.508 ([M + K]+, calcd 735.471).

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(5Z,9Z)-14-[(4-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]amino}butyl)amino]-14-
oxotetradeca-5,9-dienoic Acid (23b)
Colorless waxy solid, 0.49 g, 68% yield. [
2932, 2864, 1734, 1647, 1541, 1456, 1383, 1363, 1243, 1165, 1029, 800, 753 cm−1; 1H NMR
(CDCl3, 500 MHz) 6.49 (1H, br s, NH), 6.11 (1H, br s, NH), 5.47–5.35 (4H, m, H-5’, H-6’
α] ν
19 + 13.5 (c 0.37, CHCl3); IR (KBr)
max
D
δ
,
H-9’, H-10’), 4.73–4.68 (1H, m, H-3), 3.30–3.25 (4H, m, CH2NH), 2.36 (2H, t, J = 7.2 Hz, H-2’),
2.30-1.00 (28H, m), 2.24 (2H, t, J = 7.2 Hz, H-13’), 2.11–2.05 (8H, m, H-4’, H-7’, H-8’, H-11’),
2.04 (3H, s, CH3CO), 1.74–1.66 (4H, m, H-3’, H-12’), 1.57–1.51 (4H, m, CH2), 0.94 (3H, s,
H-19), 0.92 (3H, d, J = 6.8 Hz, H-21), 0.65 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ
176.1 (C-1’), 174.6 (C-24), 174.3 (C-14’), 170.7 (CH3CO), 130.3, 130.1 (C-6’, C-9’), 129.2 (C-5’,
C-10’), 74.4 (C-3), 56.5 (C-14), 56.1 (C-17), 42.7 (C-13), 41.9 (C-5), 40.4 (C-9), 40.2 (C-12),
39.9 (C-25, C-28), 36.1 (C-13’), 35.8 (C-8), 35.5 (C-20), 35.0 (C-1), 34.6 (C-10), 33.6 (C-23),
33.3 (C-2’), 32.3 (C-4), 31.9 (C-22), 28.2 (C-16), 27.6, 27.5 (C-7’, C-8’), 27.0 (C-6), 26.9, 26.8 (C-4’
,
C-11’), C-26, C-27), 26.6 (C-2), 26.3 (C-7), 25.9 (C-12’), 24.8 (C-3’), 24.2 (C-15), 23.3 (C-19),
21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21), 12.1 (C-18); anal. calcd for C44H72N2O6: C, 72.89; H,
10.01; found C, 72.88; H, 9.98; MALDI TOF: m/z 747.440 ([M + Na]+, calcd 747.529), 763.422
([M + K]+, calcd 763.503).
(5Z,9Z)-14-[(4-{[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]amino}hexyl)amino]-14-
oxotetradeca-5,9-dienoic Acid (23c)
Colorless waxy solid, 0.48 g, 65% yield. [
2933, 2864, 1734, 1646, 1549, 1455, 1379, 1363, 1243, 1163, 1111, 1046, 979, 933, 909, 729, 668,
549 cm−1; 1H NMR (CDCl3, 500 MHz)
6.36 (1H, br s, NH), 6.12 (1H, br s, NH), 5.40–5.32
α] ν
19 + 15.3 (c 0.95, CHCl3); IR (KBr)
max
D
δ
(4H, m, H-5’, H-6’, H-9’, H-10’), 4.72–4.68 (1H, m, H-3), 3.20 (4H, q, J = 6.5 Hz, CH2NH),
2.31 (2H, t, J = 7.5 Hz, H-2’), 2.27-1.00 (28H, m), 2.19 (2H, J = 7.5 Hz, H-13’), 2.11–2.03 (8H, m,
H-4’, H-7’, H-8’, H-11’), 2.00 (3H, s, CH3CO), 1.69–1.64 (4H, m, H-3’, H-12’), 1.49–1.45 (4H,
m, CH2), 1.33–1.28 (4H, m, CH2), 0.90 (3H, s, H-19), 0.89 (3H, d, J = 6.8 Hz, H-21), 0.61 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 176.5 (C-1’), 174.3 (C-24), 173.9 (C-14’), 170.7 (CH3CO),
130.3, 130.2 (C-6’, C-9’), 129.1 (C-5’, C-10’), 74.4 (C-3), 56.5 (C-14), 56.0 (C-17), 42.7 (C-13),
41.9 (C-5), 40.4 (C-9), 40.1 (C-12), 39.1, 39.0 (CH2NH), 36.1 (C-13’), 35.8 (C-8), 35.5 (C-20),
35.0 (C-1), 34.6 (C-10), 33.6 (C-23), 33.5 (C-2’), 32.2 (C-4), 31.9 (C-22), 29.3, 29.2 (CH2),
28.2 (C-16), 27.5, 27.4 (C-7’, C-8’), 26.9 (C-6), 26.8 (C-11’), 26.6 (C-2), 26.4 (C-4’), 26.3 (C-7),
25.9 (C-12’, CH2), 24.8 (C-3’), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.4 (C-21),
12.0 (C-18); anal. calcd for C46H76N2O6: C, 73.36; H, 10.17; found C, 73.30; H, 10.12; MALDI
TOF: m/z 775.579 ([M + Na]+, calcd 775.560), 791.556 ([M + K]+, calcd 791.534).
(5Z,9Z)-14-{4-[(3α,5β)-3-(Acetyloxy)-24-oxocholan-24-yl]piperazin-1-yl}-14-oxotetradeca-
5,9-dienoic Acid (23d)
Colorless waxy solid, 0.46 g, 64% yield. [
2935, 2866, 1732, 1647, 1432, 1379, 1363, 1243, 1179, 1026, 983, 888, 753, 666, 615 cm−1; 1H
NMR (CDCl3, 500 MHz) 5.40–5.30 (4H, m, H-5’, H-6’, H-9’, H-10’), 4.67 (1H, m, H-3),
α] ν
19 + 18.0 (c 0.94, CHCl3); IR (KBr)
max
D
δ
3.62–3.56 (4H, m, CH2N), 3.47–3.43 (4H, m, CH2N), 2.33–2.28 (4H, m, H-2’, H-13’), 2.38–
0.96 (28H, m), 2.10–2.01 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.99 (3H, s, CH3CO), 1.68–1.64
(4H, m, H-3’, H-12’), 0.90 (3H, d, J = 6.8 Hz, H-21), 0.89 (3H, s, H-19), 0.61 (3H, s, H-18);
13C NMR (CDCl3, 125 MHz)
δ 176.8 (C-1’), 172.8 (C-24), 172.4 (C-14’), 170.7 (CH3CO),
130.3, 130.1 (C-6’, C-9’), 129.1, 128.9 (C-5’, C-10’), 74.4 (C-3), 56.4 (C-14), 55.9 (C-17), 45.6,
45.4 (CH2N), 42.7 (C-13), 41.8 (C-5), 41.6, 41.3 (CH2N), 40.4 (C-9), 40.1 (C-12), 35.7 (C-8),
35.6 (C-20), 34.9 (C-1), 34.5 (C-10), 33.3 (C-13’), 32.6 (C-2’), 32.3 (C-4), 31.3 (C-22), 30.2 (C-23),
28.3 (C-16), 27.4, 27.3 (C-7’, C-8’), 26.9 (C-6), 26.7 (C-4’), 26.6 (C-2), 26.4 (C-11’), 26.3 (C-7),
25.2 (C-3’), 24.7 (C-12’), 24.2 (C-15), 23.3 (C-19), 21.4 (CH3CO), 20.8 (C-11), 18.5 (C-21),
12.0 (C-18); anal. calcd for C44H70N2O6: C, 69.34; H, 9.26; found C, 69.29; H, 9.20; MALDI
TOF: m/z 745.544 ([M + Na]+, calcd 745.513), 761.515 ([M + K]+, calcd 761.487).

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(5Z,9Z)-14-[(2-{[(5β)-3,24-Dioxocholan-24-yl]amino}ethyl)amino]-14-oxotetradeca-5,9-
dienoic Acid (24a)
Colorless waxy solid, 0.44 g, 67% yield. [α] ν
16 + 11.9 (c 0.91, CHCl3); IR (KBr)
max
1
D
2929, 2865, 1713, 1651, 1550, 1455, 1379, 1257, 1165, 1150, 1076, 1023, 754, 665 cm−1; H
NMR (CDCl3, 500 MHz) δ 7.04 (1H, br s, NH), 6.85 (1H, br s, NH), 5.44–5.30 (4H, m, H-5’,
H-6’, H-9’, H-10’), 3.38–3.34 (4H, m, CH2NH), 2.70-1.05 (28H, m), 2.38–2.34 (2H, m, H-2’),
2.22–2.18 (2H, m, H-13’), 2.10–2.04 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.70–1.64 (4H, m, H-3’,
H-12’), 1.01 (3H, s, H-19), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.67 (3H, s, H-18); 13C NMR
(CDCl3, 125 MHz) δ 213.6 (C-3), 176.3 (C-1’), 175.6 (C-24), 175.0 (C-14’), 130.2, 130.1 (C-6’,
C-9’), 129.2, 129.0 (C-5’, C-10’), 56.4 (C-14), 55.9 (C-17), 44.3 (C-5), 42.7 (C-13), 42.4 (C-4),
40.7 (C-9), 40.0 (C-12), 39.7, 39.6 (CH2NH), 37.2 (C-2), 36.9(C-1), 35.9 (C-13’), 35.5 (C-8,
C-20), 34.9 (C-10), 33.4 (C-23), 33.2 (C-2’), 31.8 (C-22), 28.2 (C-16), 27.6, 27.5 (C-7’, C-8’),
26.7 (C-11’), 26.6 (C-6), 26.4 (C-4’), 25.8 (C-7), 25.7 (C-12’), 24.7 (C-3’), 24.2 (C-15), 22.6 (C-19),
21.2 (C-11), 18.4 (C-21), 12.1 (C-18); anal. calcd for C40H64N2O5: C, 73.58; H, 9.88; found
C, 73.54; H, 9.84; MALDI TOF: m/z 675.307 ([M + Na]+, calcd 675.482), 691.276 ([M + K]+,
calcd 691.482).
(5Z,9Z)-14-[(4-{[(5β)-3,24-Dioxocholan-24-yl]amino}butyl)amino]-14-oxotetradeca-5,9-
dienoic Acid (24b)
Colorless waxy solid, 0.45 g, 66% yield. [α] ν
17 + 15.0 (c 0.77, CHCl3); IR (KBr)
max
1
D
2932, 2865, 1712, 1645, 1551, 1446, 1379, 1230, 1181, 1149, 1077, 1022, 754, 666 cm−1; H
NMR (CDCl3, 500 MHz) δ 6.54 (1H, br s, NH), 6.25 (1H, br s, NH), 5.42–5.33 (4H, m, H-5’,
H-6’, H-9’, H-10’), 3.28–3.24 (4H, m, CH2NH), 2.72-1.05 (28H, m), 2.37–2.33 (2H, m, H-2’),
2.22–2.18 (2H, m, H-13’), 2.11–2.04 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.71–1.66 (4H, m, H-3’,
H-12’), 1.55–1.51 (4H, m, CH2), 1.02 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.68 (3H, s,
H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 176.2 (C-1’), 174.5 (C-24), 174.2 (C-14’),
130.3, 130.1 (C-6’, C-9’), 129.2 (C-5’, C-10’), 56.4 (C-14), 56.0 (C-17), 44.3 (C-5), 42.8 (C-13),
42.4 (C-4), 40.7 (C-9), 40.1 (C-12), 39.1, 39.0 (CH2NH), 37.2 (C-2), 36.9 (C-1), 36.1 (C-13’),
35.5 (C-8, C-20), 34.9 (C-10), 33.6 (C-23), 33.4 (C-2’), 31.9 (C-22), 28.2 (C-16), 27.6, 27.5 (C-7’,
C-8’), 26.9, 26.8 (CH2), 26.7 (C-11’), 26.6 (C-6), 26.4 (C-4’), 25.9 (C-12’), 25.8 (C-7), 24.7 (C-3’),
24.2 (C-15), 22.6 (C-19), 21.2 (C-11), 18.4 (C-21), 12.1 (C-18); anal. calcd for C42H68N2O5:
C, 74.07; H, 10.06; found C, 74.01; H, 10.02; MALDI TOF: m/z 681.472 ([M + H]+, calcd
681.521), 703.444 ([M + Na]+, calcd 703.503), 719.407 ([M + K]+, calcd 719.477).
(5Z,9Z)-14-[(6-{[(5β)-3,24-Dioxocholan-24-yl]amino}hexyl)amino]-14-oxotetradeca-5,9-
dienoic Acid (24c)
Colorless waxy solid, 0.45 g, 64% yield. [
α
]
23 + 11.0 (c 0.75, CHCl3); IR (KBr)
ν
max
D
−1
2929, 2861, 1713, 1645, 1551, 1454, 1378, 1260, 1181, 1149, 1103, 1077, 1022, 991, 801, 755 cm
;
1H NMR (CDCl3, 500 MHz)
δ
6.16 (1H, br s, NH), 5.88 (1H, br s, NH), 5.47–5.34 (4H, m,
H-5’,H-6’, H-9’, H-10’), 3.28–3.24 (4H, m, CH2NH), 2.73-1.05 (28H, m), 2.38–2.33 (2H, m,
H-2’), 2.25–2.21 (2H, m, H-13’), 2.12–2.05 (8H, m, H-4’, H-7’, H-8’, H-11’), 1.74–1.67 (4H, m,
H-3’, H-12’), 1.53–1.48 (4H, m, CH2), 1.38–1.33 (4H, m, CH2), 1.03 (3H, s, H-19), 0.94 (3H, d,
J = 6.5 Hz, H-21), 0.69 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 176.5 (C-1’),
174.2 (C-14’, C-24), 130.2, 130.1 (C-6’, C-9’), 129.1 (C-5’, C-10’), 56.4 (C-14), 56.0 (C-17),
44.3 (C-5), 42.8 (C-13), 42.4 (C-4), 40.7 (C-9), 40.1 (C-12), 39.1, 38.9 (CH2NH), 37.2 (C-2),
37.0 (C-1), 36.2 (C-13’), 35.5 (C-8, C-20), 34.9 (C-10), 33.6 (C-23), 33.4 (C-2’), 31.9 (C-22), 29.3,
29.1 (CH2), 28.2 (C-16), 27.6, 27.5 (C-7’, C-8’), 26.8 (C-11’), 26.6 (C-6), 26.4 (C-4’), 26.0, 25.8,
(C-7, C-12’, CH2), 24.8 (C-3’), 24.2 (C-15), 22.7 (C-19), 21.2 (C-11), 18.4 (C-21), 12.1 (C-18);
anal. calcd for C44H72N2O5: C, 74.53; H, 10.24; found C, 74.48; H, 10.19; MALDI TOF: m/z
709.589 ([M + H]+, calcd 709.552), 731.571 ([M + Na]+, calcd 731.534), 747.541 ([M + K]+,
calcd 747.508).

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(5Z,9Z)-{4-[(5β)-3,24-Dioxocholan-24-yl]piperazin-1-yl}-14-oxotetradeca-5,9-dienoic
Acid (24d)
Colorless waxy solid, 0.43 g, 63% yield. [
2931, 2864, 1715, 1647, 1434, 1363, 1245, 1221, 1180, 1077, 1019, 802, 753 cm−1; 1H NMR
(CDCl3, 500 MHz) 5.44–5.31 (4H, m, H-5’, H-6’, H-9’, H-10’), 3.66–3.60 (4H, m, CH2N),
α] ν
18 + 13.1 (c 0.95, CHCl3); IR (KBr)
max
D
δ
3.52–3.45 (4H, m, CH2N), 2.40-0.96 (28H, m), 2.39–2.33 (4H, m, H-2’, H-13’), 2.14–2.03 (8H,
m, H-4’, H-7’, H-8’, H-11’), 1.72–1.66 (4H, m, H-3’, H-12’), 1.02 (3H, s, H-19), 0.95 (3H, d,
J = 6.8 Hz, H-21), 0.69 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 213.6 (C-3), 176.7 (C-1’),
172.6 (C-14’, C-24), 130.4, 130.2 (C-6’, C-9’), 129.1, 128.9 (C-5’, C-10’), 56.4 (C-14), 55.9 (C-17),
45.6, 45.4 (CH2N), 44.3 (C-5), 42.8 (C-13), 42.3 (C-4), 41.7, 41.6 (CH2N), 40.4 (C-9), 40.0 (C-12),
37.2 (C-2), 36.9 (C-1), 35.6 (C-8), 35.5 (C-20), 34.9 (C-10), 33.2 (C-2’), 32.6 (C-13’), 31.3 (C-22),
30.3 (C-23), 28.3 (C-16), 27.5, 27.4 (C-7’, C-8’), 26.8 (C-4’), 26.6 (C-6), 26.4 (C-11’), 25.8 (C-7),
25.3 (C-3’), 24.7 (C-12’), 24.2 (C-15), 22.6 (C-19), 21.2 (C-11), 18.5 (C-21), 12.1 (C-18); anal.
calcd for C42H66N2O5: C, 74.29; H, 9.80; found C, 74.23; H, 9.77; MALDI TOF: m/z
679.503 ([M + H]+, calcd 679.497), 701.477 ([M + Na]+, calcd 701.497), 717.443 ([M + K]+,
calcd 717.497).
4. Conclusions
In summary, for the first time, we synthesized hybrid molecules being synthetic analogs
of natural 5Z,9Z-dienoic acids on the basis of lithocholic acid and (5Z,9Z)-1,14-tetradeca-5,9-
dienedicarboxylic acid obtained in two stages using the homo-cyclomagnesiation reaction
of 2-(hepta-5,6-diene-1-yloxy)tetrahydro-2H-pyran at the key stage. It was shown that
all the synthesized hybrid molecules exhibited high cytotoxicity against a panel of five
tumor cell lines (Jurkat, K562, HEK293, HeLa, and U937) as compared with the original
lithocholic acid and also, possibly, to a small extent affected topoisomerase II, initiated
mitochondrial apoptosis in Jurkat tumor cells, thus causing loss of cytochrome C and
activation of caspases. The studied derivatives of lithocholic acid induced G1/S cell
cycle arrest, which indicates their high antitumor activity and allows considering these
compounds as promising drug candidates.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824
7/14/2/84/s1, 1H NMR and 13C NMR spectra of all compounds 8, 11, 13–16, 21–24.
Author Contributions: Conceptualization, V.A.D., L.U.D., and U.M.D.; methodology, R.A.T.; vali-
dation, R.A.T., V.A.D., and L.U.D.; formal analysis, S.R.I.; investigation, L.U.D.; writing—original
draft preparation, V.A.D. and L.U.D.; writing—review and editing, V.A.D. and L.U.D.; visualization,
V.A.D.; supervision, V.A.D. and L.U.D.; project administration, V.A.D.; funding acquisition, L.U.D.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Russian Science Foundation, grant number 20-64-47019.
The structural studies of the synthesized compounds were performed with the use of Collective
Usage Centre “Agidel” at the Institute of Petrochemistry and Catalysis of the Russian Academy
of Sciences (RAS). The anticancer activity studies of the synthesized compounds were performed
at the Laboratory of Molecular Design and Biological Screening of Candidate Substances for the
Pharmaceutical Industry at the Institute of Petrochemistry and Catalysis of the RAS. Partly (in terms
of the production of synthesized compounds for carrying out biological activity) the work partially
was done within approved plans for research projects at the IPC RAS State Registration No. AAAA-
A19-119022290008-6 (2019–2021) and AAAA-A19-119022290007-9 (2019–2021).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
or in the decision to publish the results.

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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Bukowski, K.; Kciuk, M.; Kontek, R. Mechanisms of Multidrug Resistance in Cancer Chemotherapy. Int. J. Mol. Sci. 2020, 21, 3233.
[CrossRef] [PubMed]
Nooter, K.; Stoter, G. Molecular mechanisms of multidrug resistance in cancer chemotherapy. Pathol. Res. Pract. 1996, 192,
768–780. [CrossRef]
Patel, N.H.; Rothenberg, M.L. Multidrug resistance in cancer chemotherapy. Invest. New Drugs. 1994, 12, 1–13. [CrossRef]
[PubMed]
Kikuchi, H.; Yuan, B.; Hu, X.; Okazaki, M. Chemopreventive and anticancer activity of flavonoids and its possibility for clinical
use by combining with conventional chemotherapeutic agents. Am. J. Cancer Res. 2019, 9, 1517–1535.
Lichota, A.; Gwozdzinski, K. Anticancer Activity of Natural Compounds from Plant and Marine Environment. Int. J. Mol. Sci.
2018, 19, 3533. [CrossRef]
Ye, Q.; Liu, K.; Shen, Q.; Li, Q.; Hao, J.; Han, F.; Jiang, R.-W. Reversal of Multidrug Resistance in Cancer by Multi-Functional
Flavonoids. Front. Oncol. 2019, 9, 487. [CrossRef]
D’yakonov, V.A.; Dzhemileva, L.U.; Dzhemilev, U.M. Natural Compounds with bis-Methylene-Interrupted Z-Double Bonds:
Plant Sources, Strategies of Total Synthesis, Biological Activity, and Perspectives. Phytochem. Rev. 2020. [CrossRef]
Hofmann, A.F.; Hagey, L.R. Bile acids: Chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol. Life Sci.
2008, 65, 2461–2483. [CrossRef]
Dang, Z.; Lin, A.; Ho, P.; Soroka, D.; Lee, K.-H.; Huang, L.; Chen, C.-H. Synthesis and proteasome inhibition of lithocholic acid
derivatives. Bioorg. Med. Chem. Lett. 2011, 21, 1926–1928. [CrossRef]
10. Dang, Z.; Jung, K.; Qian, K.; Lee, K.-H.; Huang, L.; Chen, C.-H. Synthesis of Lithocholic Acid Derivatives as Proteasome
Regulators. ACS Med. Chem. Lett 2012, 3, 925–930. [CrossRef]
11. Ishizawa, M.; Matsunawa, M.; Adachi, R.; Uno, S.; Ikeda, K.; Masuno, H.; Shimizu, M.; Iwasaki, K.; Yamada, S.; Makishima, M.
Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia. J. Lipid Res. 2008, 49,
763–772. [CrossRef] [PubMed]
12. Adachi, R.; Honma, Y.; Masuno, H.; Kawana, K.; Shimomura, I.; Yamada, S.; Makishima, M. Selective activation of vitamin D
receptor by lithocholic acid acetate, a bile acid derivative. J. Lipid Res. 2005, 46, 46–57. [CrossRef] [PubMed]
13. Cheng, J.; Fang, Z.Z.; Kim, J.H.; Krausz, K.W.; Tanaka, N.; Chiang, J.Y.; Gonzalez, F.J. Intestinal CYP3A4 protects against lithocholic
acid-induced hepatotoxicity in intestine-specific VDR-deficient mice. J. Lipid Res. 2014, 55, 455–465. [CrossRef] [PubMed]
14. Mizushina, Y.; Kasai, N.; Miura, K.; Hanashima, S.; Takemura, M.; Yoshida, H.; Sugawara, F.; Sakaguchi, K. Structural Relationship
of Lithocholic Acid Derivatives Binding to the N-Terminal 8-kDa Domain of DNA Polymerase
10677. [CrossRef] [PubMed]
β. Biochemestry 2004, 43, 10669–
15. do Nascimento, P.G.G.; Lemo, T.L.G.; Almeida, M.C.S.; de Souza, J.M.O.; Bizerra, A.M.C.; Santiago, G.M.P.; da Costa, J.G.M.;
Coutinho, H.D.M. Lithocholic acid and derivatives: Antibacterial activity. Steroids 2015, 104, 8–15. [CrossRef]
16. Schneider, H.; Fiander, H.; Harrison, K.A.; Watson, M.; Burton, G.W.; Arya, P. Inhibitory potency of lithocholic acid analogs and
other bile acids on glucuronosyltransferase activity in a colon cancer cell line. Bioorg. Med. Chem. Lett. 1996, 6, 637–642. [CrossRef]
17. Vogel, S.M.; Bauer, M.R.; Joerger, A.C.; Wilcken, R.; Brandt, T.; Veprintsev, D.B.; Rutherford, T.J.; Fersht, A.R.; Boeckler, F.M.
Lithocholic acid is an endogenous inhibitor of MDM4 and MDM2. Proc. Natl. Acad. Sci. USA 2012, 109, 16906–16910. [CrossRef]
18. Agarwal, D.S.; Anantaraju, H.S.; Sriram, D.; Yogeeswari, P.; Nanjegowda, S.H.; Mallu, P.; Sakhuja, R. Synthesis, characterization
and biological evaluation of bile acid-aromatic/heteroaromatic amides linked via amino acids as anti-cancer agents. Steroids 2016
107, 87–97. [CrossRef]
,
19. Bjedov, S.; Jakimov, D.; Pilipovic´, A.; Poša, M.; Sakacˇ, M. Antitumor activity of newly synthesized oxo and ethylidene derivatives
of bile acids and their amides and oxazolines. Steroids 2017, 120, 19–25. [CrossRef]
20. He, X.-L.; Xing, Y.; Gu, X.-Z.; Xiao, J.-X.; Wang, Y.-Y.; Yi, Z.; Qiu, W.-W. The synthesis and antitumor activity of lithocholic acid
and its derivatives. Steroids 2017, 125, 54–60. [CrossRef]
21. Samadi, M.; Nury, T.; Khalafi-Nezhad, A.; Lizard, G. Protecting group-free radical decarboxylation of bile acids: Synthesis of
novel steroidal substituted maleic anhydrides and maleimides and evaluation of their cytotoxicity on C6 rat glioma cells. Steroids
2017, 125, 124–130. [CrossRef] [PubMed]
22. Goldberg, A.A.; Beach, A.; Davies, G.F.; Harkness, T.A.; Leblanc, A.; Titorenko, V.I. Lithocholic bile acid selectively kills
neuroblastoma cells, while sparing normal neuronal cells. Oncotarget 2011, 2, 761–782. [CrossRef] [PubMed]
23. D’yakonov, V.A.; Dzhemileva, L.U.; Tuktarova, R.A.; Makarov, A.A.; Islamov, I.I.; Mulyukova, A.R.; Dzhemilev, U.M. Catalytic
cyclometallation in steroid chemistry III: Synthesis of steroidal derivatives of 5Z,9Z-dienoic acid and their human topoisomerase
I inhibitory activity. Steroids 2015, 102, 110–117. [CrossRef] [PubMed]
24. D’yakonov, V.A.; Tuktarova, R.A.; Dzhemileva, L.U.; Ishmukhametova, S.R.; Yunusbaeva, M.M.; Ramazanov, I.R.; Dzhemilev,
U.M. Novel Hybrid Molecules on the Basis of Steroids and (5Z,9Z)-Tetradeca-5,9-diene-1,14-dioic Acid: Synthesis, Anti-Cancer
Studies and Human Topoisomerase I Inhibitory Activity. Anti-Cancer Agents Med. Chem. 2017, 17, 1126–1135. [CrossRef]
25. D’yakonov, V.A.; Tuktarova, R.A.; Dzhemileva, L.U.; Ishmukhametova, S.R.; Yunusbaeva, M.M.; Dzhemilev, U.M. Catalytic
cyclometallation in steroid chemistry V: Synthesis of hybrid molecules based on steroid oximes and (5Z,9Z)-tetradeca-5,9-
dienedioic acid as potential anticancer agents. Steroids 2018, 138, 14–20. [CrossRef] [PubMed]

Pharmaceuticals 2021, 14, 84
29 of 29
26. D’yakonov, V.A.; Tuktarova, R.A.; Dzhemileva, L.U.; Ishmukhametova, S.R.; Yunusbaeva, M.M.; Dzhemilev, U.M. Catalytic
cyclometallation in steroid chemistry VI: Targeted synthesis of hybrid molecules based on steroids and tetradeca-5Z,9Z-diene-
1,14-dicarboxylic acid and study of their antitumor activity. Steroids 2018, 138, 6–13. [CrossRef]
27. D’yakonov, V.A.; Makarov, A.A.; Andreev, E.N.; Dzhemileva, L.U.; Dzhemilev, U.M. Catalytic cycloalumination of 1,2-dienes in
the total synthesis of natural grenadamide and lyngbyoic acid. Russ. Chem. Bull. 2020, 69, 386–389. [CrossRef]
28. Mohammadkhani, L.; Heravi, M.M. Oxalyl Chloride: A Versatile Reagent in Organic Transformations. ChemistrySelect 2019, 4,
6309–6337. [CrossRef]
29. Green, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis; Wiley: New York, NY, USA, 1999; pp. 518–525.
30. Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique, 4th ed.; Wiley: Hoboken, NJ, USA, 2000; p. 600.
31. Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 1961
191, 144–148. [CrossRef]
,
32. Lyamzaev, K.G.; Sumbatyan, N.V.; Nesterenko, A.M.; Kholina, E.G.; Voskoboynikova, N.; Steinhoff, H.J.; Mulkidjanian, A.Y.;
Chernyak, B.V. MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation. Oxid Med.
Cell. Longev. 2019, 9710208. [CrossRef]
33. D’yakonov, V.A.; Makarov, A.A.; Dzhemileva, L.U.; Makarova, E.Kh.; Khusnutdinova, E.K.; Dzhemilev, U.M. The facile synthesis
of the 5Z,9Z-dienoic acids and their topoisomerase I inhibitory activity. Chem. Commun. 2013, 49, 8401–8403. [CrossRef] [PubMed]
34. D’yakonov, V.A.; Dzhemileva, L.U.; Makarov, A.A.; Mulyukova, A.R.; Baev, D.S.; Khusnutdinova, E.K.; Tolstikova, T.G.;
Dzhemilev, U.M. Stereoselective Synthesis of 11-Phenylundeca-5Z,9Z-dienoic Acid and Investigation of its Human Topoisomerase
I and IIα Inhibitory Activity. Bioorg. Med. Chem. Lett. 2015, 25, 2405–2408. [CrossRef] [PubMed]
35. D’yakonov, V.A.; Dzhemileva, L.U.; Makarov, A.A.; Mulyukova, A.R.; Baev, D.S.; Khusnutdinova, E.K.; Tolstikova, T.G.;
Dzhemilev, U.M. nZ,(n+4)Z-Dienoic Fatty Acids: A New Method for the Synthesis and Inhibitory Action on Topoisomerase I and
Iiα. Med. Chem. Res. 2016, 25, 30–39. [CrossRef]
36. D’yakonov, V.A.; Dzhemileva, L.U.; Dil’mukhametova, L.К.; Dzhemilev, U.М. Synthesis of new Cu complex based on natural
5Z,9Z-eicosadienoic acid-effective topoisomerase I inhibitor and cytotoxin against the cisplatin-resistant cell line. ACS Omega
2019, 4, 17581–17587. [CrossRef]
37. D’yakonov, V.A.; Dzhemileva, L.U.; Dzhemilev, U.M. Advances in the Chemistry of Natural and Semisynthetic Topoisomerase
I/II Inhibitors. Stud. Nat. Prod. Chem. 2017, 54, 21–86. [CrossRef]
38. Makarov, A.A.; Dzhemileva, L.U.; Salimova, A.R.; Makarova, E.Kh.; Ramazanov, I.R.; D’yakonov, V.A.; Dzhemilev, U.M.
New Synthetic Derivatives of Natural 5Z,9Z-Dienoic Acids: Stereoselective Synthesis and Study of the Antitumor Activity.
Bioorg. Chem. 2020, 104, 104303. [CrossRef]
39. Muslimovic, A.; Ismail, I.H.; Gao, Y.; Hammarsten, O. An optimized method for measurement of gamma-H2AX in blood
mononuclear and cultured cells. Nat. Protoc. 2008, 3, 1187–1193. [CrossRef]
40. Dzhemileva, L.U.; D’yakonov, V.A.; Islamov, I.I.; Yunusbaeva, M.M.; Dzhemilev, U.M. New 1Z,5Z-diene macrodiolides: Catalytic
synthesis, anticancer activity, induction of mitochondrial apoptosis, and effect on the cell cycle. Bioorg. Chem. 2020, 99, 103832.
[CrossRef]
41. Bildziukevich, U.; Vida, N.; Rárová, L.; Kolárˇ, M.; Šaman, D.; Havlícˇek, L.; Drašar, P.; Wimmer, Z. Polyamine derivatives of
betulinic acid and β-sitosterol: A comparative investigation. Steroids 2015, 100, 27–35. [CrossRef]