Synthesis of novel dimeric compounds containing triazole using click method and their selective antiproliferative and proapoptotic potential via mitochondrial apoptosis signaling

Article abstract of DOI:10.1007/s00044-020-02510-x

In this study, the main aim was synthesis of dimeric compounds, which contain lithocholic acid and a triazole structure to investigate the selective cellular and molecular antiproliferative and proapoptotic potential of these products in healthy embryonic fibroblast (MEF), cervix cancer (HeLa), and breast cancer (MCF-7) cells. Four ester (5a–d) and five dimeric (6a–d, 7) out of nine novel compounds were obtained. First of all, lithocholic acid was converted to methyl lithocholate and then it was reacted with certain alkynoic acids (a–d) to obtain its alkynoate derivatives (5a–d). Finally, these compounds were converted to dimers (6a–d) by using 2,6-bis(azidomethyl)pyridine via the click method. Our result indicate that, treatment with dimeric compounds can selectively decrease the cell viability and proliferation in cervix cancer HeLa and breast cancer MCF-7 cells, except 7 which caused a strong cytotoxicity on healthy MEF cells. According to MTT assay, Nucblue cell stain and Annexin V/Propodium iodide molecular probe staining, 100 μM concentrations of the dimeric compounds was sufficient in inducing death and apoptotic cell ratio in HeLa and MCF-7 breast cancer cells selectively. In brief, the present study indicates that most effective dimeric compounds are 6a and 6b, which have the highest IC₅₀ (345.8–342.6 μM) value on healthy cell and the lowest IC₅₀ value in both cervix (49.2–36.9 μM) and breast (23.0–66.1 μM) cancer cells especially long-term treatment and which triggers apoptosis pathway specifically.

Full text of DOI:10.1007/s00044-020-02510-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02510-x
ORIGINAL RESEARCH
Synthesis of novel dimeric compounds containing triazole using
click method and their selective antiproliferative and proapoptotic
potential via mitochondrial apoptosis signaling
H. R. Ferhat Karabulut1 Ali Osman Karatavuk1 Hasan Ozyildirim2 Oğuzhan Doğanlar3 Zeynep Banu Doğanlar3
●
●
●
●
Received: 8 November 2019 / Accepted: 15 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
In this study, the main aim was synthesis of dimeric compounds, which contain lithocholic acid and a triazole structure to
investigate the selective cellular and molecular antiproliferative and proapoptotic potential of these products in healthy
embryonic fibroblast (MEF), cervix cancer (HeLa), and breast cancer (MCF-7) cells. Four ester (5a–d) and five dimeric
(6a–d, 7) out of nine novel compounds were obtained. First of all, lithocholic acid was converted to methyl lithocholate and
then it was reacted with certain alkynoic acids (a–d) to obtain its alkynoate derivatives (5a–d). Finally, these compounds
were converted to dimers (6a–d) by using 2,6-bis(azidomethyl)pyridine via the click method. Our result indicate that,
treatment with dimeric compounds can selectively decrease the cell viability and proliferation in cervix cancer HeLa and
breast cancer MCF-7 cells, except 7 which caused a strong cytotoxicity on healthy MEF cells. According to MTT assay,
Nucblue cell stain and Annexin V/Propodium iodide molecular probe staining, 100 µM concentrations of the dimeric
compounds was sufficient in inducing death and apoptotic cell ratio in HeLa and MCF-7 breast cancer cells selectively. In
brief, the present study indicates that most effective dimeric compounds are 6a and 6b, which have the highest IC50
(345.8–342.6 µM) value on healthy cell and the lowest IC50 value in both cervix (49.2–36.9 µM) and breast (23.0–66.1 µM)
cancer cells especially long-term treatment and which triggers apoptosis pathway specifically.
●
●
●
●
Keywords Click chemistry Lithocholic acid HeLa MCF-7 Gene expression
Introduction
click chemistry has developed (Himo et al. 2002, 2005;
Kolb and Sharpless 2003; Malkoch et al. 2005; Rostovtsev
et al. 2002; Morales-Sanfrutos et al. 2008), the synthesis of
1,2,3-triazole derivative heterocyclic compounds and stu-
dies utilizing these compounds have increased. 1,2,3-tria-
zole derivatives attract special interest due to their
biological activity and structural properties in medicine and
material science. In addition to their anticancer activity
(Kádár et al. 2012; Nagesh et al. 2015; Rahman et al. 2017;
Reddy et al. 2018), they are also known to show anti-
microbial (Vatmurge et al. 2008; Yadav et al.,2018), anti-
fungal (Tan et al. 2017; Lima-Neto et al. 2012), antiviral
(Glowacka et al. 2013; Asif 2017), antituberculosis
(Menendez et al. 2011), and anti-HIV (Vasilyeva et al.
2017; Chen et al. 2015) activity. In addition, these 1,2,3-
triazoles are utilized in the dye industry (Duan et al. 2012)
and production of herbicides (Nejma et al. 2018).
Heterocyclic compounds are very important in synthetic
chemistry. Heterocyclic compounds containing the nitrogen
atom especially are widely used. One of the heterocyclic
compound classes is triazole derivative compounds. As
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02510-x) contains supplementary
material, which is available to authorized users.
* H. R. Ferhat Karabulut
fkarabulut@trakya.edu.tr
1
Department of Chemistry, Faculty of Science, Trakya University,
22030 Edirne, Turkey
2
Department of Mathematics and Science Education, Faculty of
Education, Trakya University, 22030 Edirne, Turkey
Bile acids are important natural compounds with
amphiphilic properties due to their skeleton and physiolo-
gical functions (Anandkumar and Rajakumar 2017).
3
Department of Medical Biology, Faculty of Medicine, Trakya
University, 22030 Edirne, Turkey

Medicinal Chemistry Research
Among the bile acids, the structure with the highest lipo-
philic character is lithocholic acid. Lithocholic acid is
mainly produced by gut microbiota, and effective in reg-
ulating biological events such as activating the vitamin D
receptor (Ahmad and Haeusler 2019). In addition, litho-
cholic acid has an antiproliferative effect by inducing
apoptosis in breast cancer cells (Luu et al. 2018), and has
selective cytotoxicity to childhood brain tumor and human
prostate cancer cells at acceptable concentrations without
affect healthy cells (Goldberg et al. 2011, 2013). In LNCaP
and PC-3 prostate cancer cells, lithocholic acid triggers both
death receptors associated apoptosis signals and caspase-
depended mitochondrial pathways of apoptotic cell death
via decrease in the mitochondrial protein Bcl-2 and clea-
vage of Bax, concomitant with an increase of mitochondrial
outer membrane permeability (Gafar et al. 2016). Qiao et al.
(2001) found that bile acids enhance caspases-3 and -9
cleavage and triggers mitochondrial apoptosis in two dif-
ferent types of colon carcinoma HT29 and HCT-116 cells.
Apoptotic induction with some litocolic acid derivatives via
cleavage of Mcl-1 (Myeloid Cell Leukemia-1) and PARP-1
(Poly(ADP-Ribose)Polymerase-1) as well as the hyper-
phosphorylation of IkBa, an inhibitor of NFkB (nuclear
factor-κB) were reported in KMS-11 human myeloma cell
lines (El Kihel et al. 2008). He et al. (2017) reported that
lithocholic acid derivatives have stronger antitumor activity
than lithocholic acid. There are also various biological
applications of lithocholic acid derivatives as antibacterial
activity (Nascimento et al. 2015), proteasome regulators
(Dang et al. 2012), radiopharmaceutical (Campazzi et al.
1999), and sialyltransferase inhibitors (Abdu-Allhah et al.
2016). Also, the synthesis of dimeric bile acids and dimeric
steroids has been of interest to researchers due to their
different application areas. Jurášek et al. (2013), reacted
cholic acid with propargyl bromide from the carboxylic acid
group to obtain triazole-derived dimers and examined their
hormone receptor activities. Triazole containing dimeric
estrones were synthesized for the synthesis of tetrameric
estrone based macrocyclic compounds (Ramírez-López
et al. 2008).
with different alkynoic acids (a–d) to obtain its terminal
alkynoate derivatives (5a–d). These compounds were con-
verted to dimers (6a–d) by using 2,6-bis(azidomethyl)pyr-
idine via the click method. Then the antiproliferative and
proapoptotic effect of the obtained dimeric compounds at
molecular and cellular levels in the HeLa and MCF-7 cancer
cell lines (6a–d, 7) was examined, and the results were
compared with normal cell MEF. The reason for the use of
bile acid in this study is its biological activity and its ability
to penetrate the cell membrane due to its amphiphilic
properties. The aim was to synthesize the novel dimers and
to investigate the effect of dimers with different carbon
numbers on antiproliferative and proapoptotic activity.
Compound 7 was also synthesized to study the effect of
lithocholic acid on cytotoxic activity.
Materials and methods
Chemistry
All chemical reagents and solvents were purchased from
Merck, Acros, and Sigma-Aldrich and used without any
purification. Solvents were dried with a 3 Å molecular
sieve, activated, and used at analytical grade. Varian Mer-
1
cury Plus 300 MHz was used for H and 13C NMR spectra.
Chemical shift values (δ) are given in parts per million
(ppm). Perkin Elmer Frontier was used for FT-IR spectra.
Waters SYNAPT G1 MS was used for the HR-MS results.
Thin-layer chromatography (TLC) was performed on silica
gel GF254. Silica gel 60 purchased from Merck was used
for the column chromatography.
Synthesis of 2,6-Bis(azidomethyl)pyridine (2)
2,6-Bis(bromomethyl)pyridine (1.0 g, 3.77 mmol) was dis-
solved in 20 mL of DMSO. Sodium azide (0.61 g,
9.43 mmol) was added to the mixture and stirred at room
temperature for 24 h. The reaction was quenched with 30 ml
of ice water. The reaction mixture was extracted with ethyl
acetate. In the organic phase it was washed three times with
40 mL of water and then dried with Na2SO4. The organic
solvent was evaporated in a vacuum. Yield: 91%. IR
Nowadays, cancer is one of the most challenging dis-
eases, which may result in death. According to the litera-
ture, breast cancer was the most widespread type of cancer
in women in 2018, followed by colorectal, lung, and cervix
uteri cancer (WCRF 2018). Therefore, studies on the
development of new cancer drugs have always attracted the
attention of scientists. As mention above, lithocholic acid
and triazole derivative compounds are exhibit biological
activities. For this reason, the novel dimers, containing
lithocholic acid and triazole moieties (6a–d) were synthe-
sized using click method. First of all, lithocholic acid was
converted to methyl lithocholate for the synthesis of dimeric
compounds. Then it was reacted from its 3-OH position
1
(cm−1): 2059. H NMR (300 MHz, CDCl3): δ ppm 7.75
(t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 4.48 (s, 4H).
Synthesis of Methyl 3α-hydroxy-5β-cholane-24-oate (4)
Into the flask placed in an ice bath was added lithocholic acid
(1.17 g, 3.0 mmol) and 20 mL methanol. Acetyl chloride
(1.07 mL, 15 mmol) was added to the solution. The reaction
was stirred for 24 h in an ice bath. Water was added to the
reaction mixture. The resulting precipitate of methyl

Medicinal Chemistry Research
lithocholate was filtered off and dried without further pur-
ification. Yield: 98%. White crystal. mp: 128.9–129.2 °C (lit.
28.4, 27.2, 26.9, 26.6, 24.4, 23.6, 21.1, 18.5, 14.7, 12.3.
HR-MS (ESI): m/z 493.3298 [M+Na]+ (calcd for
C30H46O4Na; 493.3294).
1
mp :129 °C). IR (cm−1): 3475, 1728. H NMR (300 MHz,
CDCl3): δ ppm 3.47–3.77 (m, 4H), 2.13–2.45 (m, 2H),
1.00–2.02 (m, 27H), 0.87–0.97 (m, 6H), 0.62–0.70 (m, 3H).
13C NMR (75 MHz, CDCl3): δ ppm 174.7, 71.6, 56.3, 55.8,
51.4, 42.6, 42.0, 40.3, 40.0, 35.7, 35.2, 35.3, 34.4, 30.9, 30.8,
30.4, 28.0, 27.1, 26.3, 24.1, 23.3, 20.7, 18.1, 11.9.
Methyl 3α-(5-hexynoyloxy)-5β-cholane-24-oate (5c)
Yield: 76%. White crystal. Rf: 0.18. mp: 66–67 °C. IR
(cm−1): 3289.5, 2120, 1733.6. 1H NMR (300 MHz, CDCl3)
δ ppm 4.49–4.94 (m, 1H), 3.67 (br. s, 3H), 2.42 (t, J =
7.32 Hz, 2H), 2.16–2.39 (m, 4H) 1.01–2.05 (m, 29H)
0.86–0.98 (m, 6H) 0.65 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ ppm 175.0, 172.8, 83.6, 74.6, 69.3, 56.7, 56.2,
51.7, 43.0, 42.1, 40.6, 40.3, 36.0, 35.6, 35.3, 34.8, 33.6,
32.5, 31.2, 28.4, 27.2, 26.9, 26.5, 24.4, 24.0, 23.6, 21.1,
18.5, 18.1, 12.3. HR-MS (ESI): m/z 507.3471 [M+Na]+
(calcd for C31H48O4Na; 507.3450).
General procedure for esterification of methyl
lithocholate by alkynoic acids (5a–d)
Alkynoic acid (2.56 mmol) (a-d) was dissolved in CH2Cl2
(20 mL). DCC/DMAP solution was prepared by dissolving
DCC (2.56 mmol) and DMAP (2.56 mmol) in CH2Cl2
(20 mL). To a solution of methyl lithocholate (1.28 mmol)
in 20 mL of CH2Cl2 that was cooled to 0 °C was added half
of an alkynoic acid solution and this was followed by the
dropwise addition of half a DCC/DMAP solution. The
mixture was stirred at 0 °C for 5 min, then the rest of the
acid solution was quickly added, followed by the dropwise
addition of the rest of the DCC/DMAP solution.
The reaction mixture was stirred 10 min for 5a and 1 h for
5a–d at 0 °C, after which the reaction was quenched with
water. The organic phase was separated. The water phase
was extracted twice with 20 mL of CH2Cl2. The organic
phases were collected and dried over MgSO4. The organic
phase was evaporated in a vacuum. Residue was purified
using column chromatography in Ethyl acetate/n-Hexane
(1/20).
Methyl 3α-(6-heptynoyloxy)-5β-cholane-24-oate (5d)
Yield: 77%. White crystal. Rf: 0.18. mp: 96–98 °C. IR (cm−1):
1
3265.8, 1737.3, 1723.9. H NMR (300 MHz, CDCl3): δ ppm
4.66–4.89 (m, 1H), 3.68 (s, 3H), 2.16–2.48 (m, 6H), 0.85–2.11
(m, 37H), 0.66 (br. s., 3H). 13C NMR (75 MHz, CDCl3): δ
ppm 175.1, 173.2, 84.3, 74.5, 68.8, 56.7, 56.2, 51.8, 43.0,
42.1, 40.6, 40.4, 36.0, 35.6, 35.3, 34.8, 34.4, 32.5, 31.3, 31.2,
28.4, 28.1, 27.3, 26.9, 26.6, 24.4, 24.4, 23.6, 21.1, 18.5, 18.4,
12.3. HR-MS (ESI): m/z 499.3793 [M+H]+ (calcd for
C32H51O4; 499.3787).
General procedure for synthesis of dimer (6a–d)
Methyl 3α-propynoyloxy-5β-cholane-24-oate (5a)
2,6-Bis(azidomethyl)pyridine (0.38 mmol) was dissolved in
20 mL of DMF, followed by the addition of CuSO4.5H2O
(0.08 mmol), sodium ascorbate (0.15 mmol) and the ester
derivative compound (5a–d) (0.78 mmol). The solution was
stirred at 40 °C for 24 h. The reaction was quenched by adding
water. The precipitated solids were filtered off and purified by
column chromatography in CH3OH/CH2Cl2 (1/20).
Yield: 19%. White crystal. Rf: 0.17. mp:176–178 °C. IR
(cm−1): 3241.2, 2114.6, 1730.0, 1709.2. 1H NMR
(300 MHz, CDCl3): δ ppm 4.71–5.00 (m, 1H), 3.68 (s, 3H),
2.87 (s, 1H), 2.15–2.46 (m, 2H), 1.69–2.03 (m, 7H),
1.00–1.68 (m, 19H), 0.87–0.99 (m, 6H), 0.65 (s, 3H). 13C
NMR (75 MHz, CDCl3): δ ppm 175.0, 152.5, 75.4, 74.3,
56.7, 56.2, 51.7, 43.0, 42.1, 40.6, 40.4, 36.0, 35.6, 35.2,
34.8, 32.2, 31.3, 31.2, 28.4, 27.2, 26.6, 26.5, 24.4, 23.5,
21.1, 18.5, 12.3. HR-MS (ESI): m/z 465.2983 [M+Na]+
(calcd for C28H42O4Na; 465.2981).
2,6-Bis[methyl 5β-cholane-24-oate-3α-yl-formate-1-yl)-1H-
1,2,3-triazole-1-yl)- methyl] pyridine (6a)
Yield: 86%. White crystal. Rf: 0.74. mp: 130–132 °C. IR
(cm−1): 1718.6, 1542.0, 1335.9. 1H NMR (300 MHz,
CDCl3): δ ppm 8.22 (br. s., 2H), 7.73 (t, J = 7.5 Hz, 1H),
7.19 (d, J = 7.6 Hz, 2H), 5.69 (br. s., 4H), 4.88–5.21
(m, 2H), 3.67 (br. s., 6H), 2.15–2.50 (m, 4H), 0.76–2.15
(m, 64H), 0.65 (br. s., 6H). 13C NMR (75 MHz, CDCl3): δ
ppm 175.0, 160.4, 154.3, 141.4, 139.2, 128.3, 122.4, 75.9,
56.7, 56.2, 55.6, 51.7, 43.0, 42.2, 40.5, 40.4, 36.0, 35.6,
35.3, 34.9, 32.4, 31.3, 31.2, 28.4, 27.3, 26.8, 26.5, 24.4,
23.5, 21.0, 18.5, 12.3. HR-MS (ESI): m/z 1074.7012
[M+H]+ (calcd for C63H92N7O8 1074.7007).
Methyl 3α-(4-pentynoyloxy)-5β-cholane-24-oate (5b)
Yield: 79%. White crystal. Rf: 0.18. mp:120–122 °C. IR
(cm−1): 3308.1, 2121, 1732.5. 1H NMR (300 MHz,
CDCl3): δ ppm 4.68–4.85 (m, 1H), 3.68 (s, 3H), 2.47–2.58
(m, 4H), 2.14–2.45 (m, 2H), 0.99–2.03 (m, 27H), 0.87–0.97
(m, 6H), 0.65 (s, 3H). 13 C NMR (75 MHz, CDCl3): δ ppm
175.0, 171.5, 82.8, 75.0, 69.2, 56.7, 56.2, 51.7, 43.0, 42.1,
40.7, 40.4, 36.0, 35.6, 35.2, 34.8, 33.9, 32.5, 31.3, 31.2,

Medicinal Chemistry Research
2,6-Bis[methyl 5β-cholane-24-oate-3α-yl-propanoate-3-yl)-
1H-1,2,3-triazole-1-yl)-methyl] pyridine (6b)
was allowed to warm to room temperature and the solvent
removed under vacuum. The residue was purified using
column chromatography with methanol. Yield 64%.
Creamy crystal. Rf: 0.71. mp: 162–164 °C. IR (cm−1):
3401.0, 1703.0, 1566.9, 1353.1. 1H NMR (300 MHz, D2O):
δ ppm 7.65–8.06 (m, 3H), 7.20 (d, J = 7.6 Hz, 2H), 5.66
(br. s., 4H), 3.54 (br. s., 4H). 13C NMR (75 MHz, D2O): δ
ppm 178.4, 154.4, 143.9, 139.9, 125.1, 122.6, 54.7, 34.2.
HR-MS (ESI): m/z 380.1086 [M+Na]+ (calcd for
C15H15N7O4Na 380.1083).
Yield 89%. White crystal. Rf: 0.41. mp: 75–77 °C. IR (cm−1):
1731.0, 1720.4, 1542.0; 1356.9. 1H NMR (300 MHz,
CDCl3): δ ppm 7.65 (t, J = 7.6 Hz, 1H), 7.49 (s, 2H), 7.06
(d, J = 7.6 Hz, 2H), 5.61 (s, 4H), 4.60–4.81 (m, 2H), 3.67
(s, 6H), 3.06 (t, J = 7.0 Hz, 4H), 2.72 (t, J = 6.9 Hz, 4H),
2.08–2.45 (m, 4H), 0.75–2.03 (m, 64H), 0.64 (s, 6H). 13C
NMR (75 MHz, CDCl3): δ ppm 175.0, 172.5, 155.1, 147.3,
138.8, 122.2, 121.8, 74.8, 56.7, 56.2, 55.4, 51.7, 43.0, 42.1,
40.6, 40.3, 36.0, 35.6, 35.2, 34.8, 34.2, 32.5, 31.3, 31.2,
28.4, 27.3, 26.9, 26.5, 24.4, 23.6, 21.3, 21.1, 18.5, 12.3.
HR-MS (ESI): m/z 1130.7664 [M+H]+ (calcd for
C67H100N7O8 1130.7633).
Cells and chemicals
Two cancer cell lines, human cervix adenocarcinoma HeLa
(ATCC® CCL-2™) and human breast adenocarcinoma
MCF7 (ATCC® HTB-22™), and one healthy cell mouse
embryonic fibroblast MEF (CF-1) (ATCC® SCRC-1040™)
were obtained from ATCC (USA). The whole experiment
was carried out in DMEM: F-12 Medium (ATCC® 30-
2006™), including heat-inactivated 10% FBS (Multicell)),
2 mM glutamine (Gibco-Life Technologies), and 1% Pen-
Strep (Multicell). The cancerous and healthy cells were
cultured at 37 °C and 5% CO2. Cell culture experiments
were started for all cells in the 5th passage and finished in
the 20th. The MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-
2,5-Diphenyltetrazolium Bromide], cell grade ultrapure
water, NucBlue™ Live ReadyProbes™ Reagent, a Tali®
Apoptosis Kit—Annexin V Alexa Fluor® 488 and Propi-
dium Iodide cell apoptosis kit, the PureLink® RNA Mini
Kit, a High Capacity cDNA Reverse Transcription Kit and
Power SYBR® Green Master Mix were purchased from
Thermo Fisher Scientific.
2,6-Bis[methyl 5β-cholane-24-oate-3α-yl-butanoate-4-yl)-
1H-1,2,3-triazole-1-yl)-methyl] pyridine (6c)
Yield 84%. White crystal. Rf: 0.44. mp: 76–78 °C. IR
1
(cm−1): 1730.5, 1553, 1379. H NMR (300 MHz, CDCl3):
δ ppm 7.68 (t, J = 7.8 Hz, 1H), 7.45 (s, 2H), 7.09 (d,
J = 7.6 Hz, 2H), 5.62 (s, 4H), 4.63–4.81 (m, 2H), 3.67 (s,
6H), 2.79 (t, J = 7.3 Hz, 4H), 2.12–2.45 (m, 9H), 0.73–2.10
(m, 67H), 0.65 (s, 6H). 13C NMR (75 MHz, CDCl3): δ ppm
175.0, 173.0, 155.1, 148.1, 138.8, 121.8, 74.6, 56.7, 56.2,
55.4, 51.7, 43.0, 42.1, 40.6, 40.3, 36.0, 35.6, 35.3, 34.8,
34.3, 32.5, 31.3, 31.2, 28.4, 27.3, 26.9, 26.5, 25.2, 25.0,
24.4, 23.6, 21.1, 18.5, 12.3. HR-MS (ESI): m/z 1158.7972
[M+H]+ (calcd for C69H104N7O8 1158.7946).
2,6-Bis[methyl 5β-cholane-24-oate-3α-yl-petanoate-5-yl)-
1H-1,2,3-triazole-1-yl)-methyl] pyridine (6d)
Cell proliferation assay (MTT)
Yield 87%. White crystal. Rf: 0.38. mp: 70–72 °C. IR
1
(cm−1): 1729.9, 1554, 1378. H NMR (300 MHz, CDCl3):
The effect on cell viability of dimeric compounds (6a–d, 7)
δ ppm 7.67 (t, J = 7.6 Hz, 1H), 7.43 (s, 2H), 7.06 (d,
J = 7.6 Hz, 2H), 5.62 (s, 4H), 4.59–4.84 (m, 2H), 3.67 (s,
6H), 2.76 (br. s., 4H), 2.12–2.46 (m, 8H), 0.77–2.04 (m,
72H), 0.64 (s, 6H). 13C NMR (75 MHz, CDCl3): δ ppm
175.0, 173.3, 155.2, 148.6, 138.8, 121.8, 121.7, 74.5, 56.7,
56.2, 55.4, 51.7, 43.0, 42.1, 40.6, 40.3, 36.0, 35.6, 35.3,
34.8, 34.6, 32.5, 31.3, 31.2, 29.0, 28.4, 27.3, 26.9, 26.5,
25.6, 24.7, 24.4, 23.6, 21.1, 18.5, 12.3. HR-MS (ESI): m/z
1186.8267 [M+H]+ (calcd for C71H108N7O8 1186.8259).
was assayed by
a
MTT test. Homo sapience
adenocarcinoma-type cell lines, HeLa and MCF7, and
normal cells MEF were used in the experiment. The cervix
cancer HeLa (3000 cells/well), breast cancer MCF7
(5000 cells/well) and embryonic fibroblast MEF (2000
cells/well) were seeded into 96-well cell culture-type plates
and were incubated at 37 °C for 12 h in a humidified
incubator containing 5% CO2. After an incubation period in
cell culture media to permit cell attachment, the control
group treated vehicle (1% DMSO + ultrapure water) and
experimental groups were treated with serial sixfold dilu-
tions (15.62–500 μM) of dimeric compounds (6a–d, 7)
diluted in cell culture grade ultrapure water. Both the
vehicle control and experimental groups were incubated for
up to 48 h, and six replicates were conducted for each 24
and 48 h time-point. When the time was completed, a 20 μl
MTT solution was pipetted using a micropipette in each
Synthesis of Pyridine-2,6-diylbis(methylene)bis(1H-
1,2,3-triazole-1,4-diyl)diacetic acid (7)
2,6-bis(azidomethyl)pyridine (0.38 mmol), CuSO4.5H2O
(0.08 mmol), sodium ascorbate (0.15 mmol), and 3-
butynoic acid (0.95 mmol) were dissolved in 20 mL of
DMF and stirred at 40 °C for 24 h. After 24 h, the reaction

Medicinal Chemistry Research
well (5 mg/ml concentration) and the plates were incubated
for 4 h at 37 °C. The blue formazan crystals were liquefied
in ultrapure DMSO (200 μl/well) and were used for dis-
solving the blue formazan, which was then evaluated at
490 nm with a spectrophotometer (Multiscan Go Microplate
Reader, Thermo Scientific, USA). IC50 values were calcu-
lated by the Probit analysis options of SPSS 20 software.
GATCTGAATTCTGGTGTATGAGA-3′), apoptotic pro-
tease activating factor1 (APAF 1:F:5′-GATATGGAATG
TCTCAGATGGCC-3′, R:5′-GGTCTGTGAGGACTCCC
CA-3′) and caspase 3 (Casp 3: F:5′-GGTATTGAGAC
AGACAGTGG-3′,R:5′-CATGGGATCTGTTTCTTTGC-3′)
genes in plated MEF, HeLa, and MCF7 cells treated with
EDS were analyzed by qRT-PCR using the Power SYBR®
Green Master Mix (Life Technologies, USA) on an ABI
Quant Studio 5 Real-Time PCR system using the following
PCR condition: 1 cycle of 2 min at 50 °C and 10 min at 95 °C
followed by 40 cycles of denaturation at 95 °C for 15 s,
annealing and extension at 60 °C for 1 min.
Molecular fluorescent apoptosis detection assay
We used NucBlue® Live ReadyProbes® Reagent to observe
cell nucleus morphology for apoptosis stimulation. For this
purpose, 24-well plates were divided into six groups for
each cell, and cells were seeded on the well surface at 2–6 ×
104 cells/well. After 24 h, the control and experiment cells
were treated with vehicle and 100 μM of dimeric com-
pounds (6a–d, 7), respectively. After 48 h of incubation,
live cells in DMEM:F12 medium were labeled with four
drops of NucBlue® Live ReadyProbes® Reagent (Thermo
Scientific ready). After 30 min, all cells were washed in
duplicate with PBS and were observed and imaged at 40×
magnification on a Zeiss Axio Vert.A1 fluorescent micro-
scope with a DAPI filter.
The rate of apoptotic and nectoric cells treated with
derivatives was evaluated by a Tali™ Image-Based Cyt-
ometer assay using Annexin V and PI (Tali® Apoptosis Kit;
Life Technologies) according to the manufacturer’s
instructions with minor modification (Guclu et al. 2018).
Live, dead (PI+) and apoptotic cells (Annexin V+, and
Annexin V+: PI+) were counted and calculated by Tali®
Image-Based Cytometer software with apoptosis options.
Statistical analysis
The IC50 values were calculated with Probit analyses using
SPSS 20 software. The mRNA expressions were calculated
using the comparative cycle threshold (2−ΔΔCt) method and
given as the relative fold change compared with the control
and normalized with GAPDH mRNA expression. In addi-
tion, differences in the leave, death and apoptotic cell count
derive from the Tali assay and the relative fold change
levels of gene expressions were compared using one-way
analysis of variance (oneway-ANOVA) with Duncan’s
separation of means test using SPSS 20 software (p ≤ 0.05).
Results and discussion
Chemistry
In our study, 2,6-bis(azidomethyl)pyridine (2) used as a
linker was synthesized with a yield of 91% in according to
the procedure (Narayanaswamy et al. 2011) in the literature
(Fig. 1). The peak of azide at 2059 cm−1 in the IR spectrum
confirmed the accuracy of the structure.
Methyl lithocholate was obtained for the synthesis of
terminal alkyne derivative esters to be used in the click
reaction. For this purpose, lithocholic acid (3) was reacted
with acetyl chloride in methanol and converted to methyl
lithocholate (4) at 98% yield.
For obtaining their terminal alkyne derivatives, this
compound (4) was reacted with propynoic acid (a), 4-
pentynoic acid (b), 5-hexynoic acid (c), and 6-heptynoic
acid (d) by using DCC/DMAP methods (Fig. 2). Ester
derivatives (5a–d) afforded 19, 79, 76, and 77% yield,
Gene expression assay
The gene expression assay was isolated from three cell lines
cultured in a 25 cm2 flask using the PureLink® RNA Mini
Kit (Life Technologies, USA), according to the manu-
facturer’s instructions. The RNA concentrations were
measured using Optizen NanoQ (Korea). For synthesis of
the first strand of cDNA using a High Capacity cDNA
Reverse Transcription Kit (Life Technologies, USA),
cDNA synthesis was performed using the thermal cycler
Applied Biosystems® Veriti® (Step 1:25 °C, 10 min; Step 2:
37 °C, 120 min; Step 3: 85 °C, 5 min). The cDNA was
stored at −20 °C for the qRT-PCR analysis. Gene expres-
sion levels of tumor suppressor (P53; F:5′-CAC
GAGCGCTGCTCAGATAGC-3′, R: 5′-ACAGGCACAA
ACACGCACAAA-3′), B-cell lymphoma 2 (BCL2; F:5′-A
TGTGTGTGGAGAGCGTCAA-3′, R:5′-ACAGTTCCAC
AAAGGCATCC-3′), BCL2 associated X (BAX: F:5′-T
TCATCCAGGATCGAGCAGA-3′, R:5′-GCAAAGTAGA
AGGCAACG-3′), cytochrome complex (Cyt-C: F:5′-AGT
GGCT AGAGTGGTCATTCATTTACA-3′, R:5′-TCAT
Fig. 1 Synthesis of 2,6-Bis(azidomethyl)pyridine (2)

Medicinal Chemistry Research
Fig. 2 Esterification of lithocholic acid
Fig. 3 Dimerization of lithocholic acid derivatives via click reaction
respectively. In the esterification reaction with propynoic
acid, it was found that the yield was lower than the other
compounds (5b–d) because of Michael-type additions. The
structure of the compounds was confirmed by NMR, IR and
(5a–d) were reacted with compound 2 in the presence of
copper sulfate and sodium ascorbate to form dimers (6a–d)
containing a 1,2,3-triazole skeleton (Fig. 3). Final products
(6a–d) were afforded at 86, 89, 84, and 87% yield,
1
1
mass spectroscopy. In the H NMR spectrum, the shift of
respectively. In the H-NMR spectrum, the presence of CH
C3 hydrogen from 3.47–3.77 ppm to 4.5–5.0 ppm indicates
that alkynoate esters (5a–d) had occurred. Unlike the others,
the alkyne proton of compound 5a was observed at 2.86
ppm. In the 13C-NMR spectra of the obtained compounds,
different from the starting material, the formation of new
carbonyl peaks of 152 and 172 ppm for 5a and 5b–d,
respectively, confirms the structure. In addition, peaks at 75
and 83 ppm, which belong to alkyne carbons, were
observed for 5b–d in the 13C-NMR spectrum. In the IR
spectrum, an ester carbonyl peak at 1730 cm−1 and alkyne
peak at 3240–3300 cm−1 confirmed the structure. Apart
from this spectral analysis, HR-MS analyses were carried
out for the 5a–d compounds and their structures were
confirmed with a difference of 0.2: 0.4: 2.1 and 0.6 mDa
when their molecular weight is compared. The esterification
reaction of methyl lithocholate with 3-butynoic acid resul-
ted in allene derivative ester, instead of its butynoate esters.
Finally, the dimers (6a–d) were obtained using the click
method. For this purpose, the alkyne-derived compounds
protons of the triazole ring at 8.2 ppm for 6a and 7.4 ppm
for 6b–d, also the shifting of the pyridine-bounded
methylenes from 4.48 to 5.6 ppm indicates the formation
of the triazole structure. In the 13C-NMR spectrum, the
carbons of the triazole (6a–d) in addition to the pyridine and
carbonyl peaks in the starting materials were observed at
about 122 and 138 ppm. In the infrared spectrum of 6a–d,
the disappearance of the peaks of N3 at 2059 cm−1 in the
azide compound and peak of 3240–3300 cm−1 in the
alkynoate ester confirmed the structure. The mass differ-
ences between theoretically-calculated and experimental
data were 0.5; 3.1; 2.6 and 0.8 mDa, respectively for the
compounds (6a–d). Spectral data confirmed that the tar-
geted compounds were successfully synthesized.
Furthermore, in order to find out the effect of the pre-
sence of lithocholic acid moiety on anticancer activity
compound 7, which does not contain lithocholic acid, it was
synthesized and evaluated (Fig. 4). For this purpose, 2,6-Bis
(azidomethyl)pyridine was converted to dimer (7) via click

Medicinal Chemistry Research
Fig. 4 Synthesis of pyridine-2,6-
diylbis(methylene)bis(1H-1,2,3-
triazole-1,4-diyl) diacetic acid
(7) via click reaction
reaction. From the 1H-NMR spectrum of the obtained
compound, the shift of the pyridine-bound methylenes from
4.48 to 5.66 ppm and the presence of methylene protons
adjacent to the carbonyl carbon at 3.54 ppm indicates that
the triazole structure has been formed. In the 13C-NMR
spectra of Compound 7, pyridine and carbonyl peaks
were observed at 178.4, 154.4, 143.9, 139.9, 125.1, and
122.6 ppm in addition to the two carbon peaks of the tria-
zole ring. In the IR spectrum, disappearance of the azide
peak at 2089 cm−1 in compound 2 and the difference of 0.3
mDa in the mass analysis confirm that the structure was
obtained.
death mechanisms and their selective capability against
cancer cells, using a molecular probe staining method
detected by image-based cytometry. In this assay, phos-
phatidylserine, located at the cytoplasmic side of the cell
membrane in living cells, is migrated to the outer membrane
during apoptosis. Phosphatidylserine on the outer mem-
brane was stained with Annexin V and this apoptotic cell
was green irradiated. The propodium iodide (PI) irradiated
red signals and stained only death cells (Luchetti et al. 2006;
Salucci et al. 2014). Although cell cultures provide impor-
tant data for drug development studies, it is often necessary
to use a higher dose in human tissue to fight cancer, in order
to overcome the defense mechanisms of the organism. In
cancer treatments, the most important condition for deter-
mining the drug dose is for the drug to have the ability to
destroy cancerous tissue, without damaging healthy cells.
Therefore, we used a 100 µM dose, which is higher than that
of the determined IC50 doses induce a significant death in
cancer cells without damaging healthy cells. Our results
demonstrate that dimeric compounds selectively trigger
both apoptotic and necrotic cell death in both cervix and
breast cancer cells in a dose-dependent manner, as indicated
by the increase in Annexin V and propodium iodide posi-
tive cells incubated with dimeric compounds (Fig. 7). Our
analysis showed a 10.8–44.5% (in HeLa) and 13.5–41.8%
(in MCF-7) increase in Annexin V+ cells (apoptotic cells)
and a maximum increase of 34.4% (in HeLa) and 24.6% (in
MCF-7) PI+ cells (necrotic cells) following 100 µM of
dimeric compound treatment for 48 h. Furthermore, this
effect was selective, as healthy embryonic fibroblast cells
remained unaffected by this treatment at the same con-
centrations and time-points (Fig. 7). The selectivity of
dimeric compounds on cancer cells was further confirmed
by the Nucblue Live staining, which was indicated by
nuclear condensation and cell morphology. Since this assay
indicates that dimeric compounds were effective at a
100 µM dose in only cancerous cells, these compounds are
thought to have a therapeutic potential, as they can be used
reliably even at high doses without losing their selectivity
(Fig. 8).
The purities of steran ring-containing dimeric com-
pounds were verified by qNMR analysis and found to be in
the range of 96.14 to 99.93%. Purity analysis for compound
7 was carried out by using HPLC and found to be 100%.
Biological evaluations
The first step in comprehending the effect of dimeric
compounds (6a–d, 7) in this study was to assess the effect
of dimeric compounds on healthy and cancer cell viability.
Following the application with increasing concentration of
dimeric compounds at 24 and 48 h timings, cells were
incubated with MTT test solution, which is processed into a
violet crystal product by live cells by dynamic metabolism.
We determined the effectiveness of the dimeric compounds
in decreasing the viability of both cervix and breast ade-
nocarcinoma cells (HeLa and MCF-7) without affecting
healthy cells, except at high doses. This effect was dose
dependent. A noticeable effect of lithocholic acid and its
derivatives is cytotoxic, anticancer characteristics on the
cancer cell line (He et al. 2017; Hryniewicka et al. 2018).
Indeed, dimeric compounds had a significant effect on the
viability and proliferation of both cancerous lines in our
panel. In HeLa cells, treatment with 26.07–62.69 µM
dimeric compounds for 24 h was sufficient to cause most
cells to kill (Fig. 5); whereas in other cancer cell lines,
significant effects required higher doses of dimeric com-
pounds (32.75–208.87 µM) (Fig. 5) or longer exposure
(Fig. 6). The MEF cell line was affected significantly less
than the HeLa and MCF-7 cells by the same treatment dose
and time. Results of the MTT test and IC50 values of the
dimeric compounds on our panel are shown in Figs 5 and 6.
To further evaluate the anticancer properties of the
dimeric compounds, we investigated their role in cellular
Intrinsic apoptosis or mitochondrial apoptosis signaling
is a novel aim of several cancer therapy regimes including
irradiation gamma chemotherapy, immunotherapy, and
alternative medicine. For this reason, we analyzed gene
expression belonging to both antiapoptotic and proapoptotic
genes together with
a
tumor suppressor on the

Medicinal Chemistry Research
Fig. 5 Cell viability in MEF; HeLa and MCF-7 cells treated with six
different doses of ergosterol derivatives obtained by serial dilution
starting from a 500 µM dose for 24 h. All data are given as the mean
values of percentages for each group SE. n = 6. MTT data at 24 h
were used to calculate IC50 values using Probit analyses
mitochondrial apoptotic process in cervix and breast cancer
cell lines to evaluate the effect of dimeric compound
treatments on apoptotic cell death in HeLa and MCF-7.
The proapoptotic activity of tumor suppressor P53 has
been recognized as apoptotic regulating the proapoptotic
Puma, Noxa, and Bax proteins (Green and Kroemer 2004;
Fulda and Debatin 2006). The expression of tumor sup-
pressor P53 gene significantly increased only in 6b (20.9-
fold) and 6c (14.5-fold) treated HeLa cells and 6a (41.83-
fold), 6b (2.35 fold), 6c (4.1-fold), 6d (23.3-fold) and
treated MCF-7 cells, compared with the vehicle-treated
control group (Fig. 5). Bcl-2 is an important apoptosis
inhibitor protein and plays an important role in the protec-
tion of mitochondrial membrane potential. Its over-
expression strongly prevents start of the apoptosis process
in cells (Porter and Janicke 1999). The decrease in the ratio

Medicinal Chemistry Research
Fig. 6 Cell viability in MEF; HeLa and MCF-7 cells treated with six
different doses of ergosterol derivatives obtained by serial dilution
starting from a 500 µM dose for 48 h. All data are given as the mean
values of percentages for each group SE. n = 6. MTT data at 48 h
were used to calculate IC50 values using Probit analyses
of Bcl-2 to BAX regulated by elevated BAX expression and
Bcl-2 inhibition is an important initial factor of apoptosis
(Li et al. 2013). BAX is a proapoptotic protein and its over-
expression causes degradation of mitochondrial membrane
potential and pore permeability (Fulda and Debatin 2006;
Decaudin et al. 1998). A damaged mitochondrial membrane
permits the release of Cytochrome-c to the cell cytosol, and
this protein integrates with the APAF-1; then caspase 9
joins this complex and apoptosomes occur (Bratton et al.
2001; Cain et al. 2000). Apoptosome shows a high level of
interest in caspase proteins. In this case, many caspase
molecules are activated by this structure and an apoptosis
cycle is completed.
Our results demonstrated that in HeLa cells while the
cellular inhibitor of apoptosis gene Bcl-2 significantly
expressed only the 6c-treated group, other dimeric

Medicinal Chemistry Research
Fig. 7 Percentage result of live, dead and apoptotic cells stained with
Annexin V/ propidium iodide as determined using a Tali image-based
cytometer. All data are given as mean values of percentages for each
group SE. n = 6. An asterisk indicates significantly different values
compared with MEF cells analyzed by one-way ANOVA and Duncan
test (p ≤ 0.05). Double asterisks indicate significantly different values
compared with other cancerous cell lines (T-test: p ≤ 0.05)
compound-treated groups were placed at the same statistical
level as the control. However, the Bcl-2/Bax ratio sig-
nificantly decreased in 6a- and 6d-treated HeLa cells. In the
MCF-7 cell line, dimeric compounds inhibited Bcl-2 and
significantly triggered BAX expression in all experimental
groups, except 7 treatment. A significant increase in the
expression of Cyt-c was observed in 6a and 6d (1.7–4.8-
fold) treated HeLa cells and almost all treated groups
(4.11–46.5-fold) in MCF-7 cells. In the last step, the
apoptosis biomarker Casp-3 overexpressed in both 6a (2.0-
fold) and 6d (2.4-fold) treated HeLa cells and all the
dimeric compound-treated group (2.7–51.2-fold) of breast
cancer MCF-7 cells, except for the 6c treatment (Fig. 9).
Conclusions
In this study, four esters (5a–d) and five dimers (6a–d, 7)
out of nine novel compounds were synthesized. The struc-
ture of the obtained compounds was confirmed by FTIR,
1H-NMR, 13C-NMR, and HRMS methods and their purity
was determined by qNMR and HPLC. These dimeric
compound’s anticancer activities investigated against HeLa
and MCF-7 cancer cell line and analyzed gene expression
level on tumor suppressing cells for learning both anti-
apoptotic and proapoptotic pathway. Biologic activity test
results showed that the dimeric compounds synthesized in
this study can selectively decrease the cell viability and
proliferation in cervix cancer HeLa and breast cancer MCF-
7 cells. However, in particular, the 7 derivative caused
strong cytotoxicity in normal MEF cells. Therefore, we
Fig. 8 Apoptosis induction of 100 µM dimeric compounds treatment in
MEF, HeLa, and MCF-7 cells. Representative images of Nucblue live
cell staining were shown blue color. Apoptotic cells exhibited mor-
phological changes in the nuclei typical of apoptosis

Medicinal Chemistry Research
MCF7
*
Fig. 9 Relative fold change
qRT-PCR analysis of apoptosis
pathway genes in IC50 dose of
-dimeric compounds treated
MEF, HeLa, and MCF-7 cell
lines for 48 h. All data were
normalized with GAPDH
expression and given as relative
to the control
(control = 1); n = 4. An asterisk
indicates significantly different
values were analyzed by one-
way ANOVA and Duncan test
(p ≤ 0.05)
HeLa
5
4
3
2
1
0
150
100
50
*
6a
6a
*
*
*
*
*
*
*
0
30
20
10
0
5
4
3
2
1
0
*
6b
6b
*
*
*
*
*
*
20
15
10
5
6
4
6c
6c
*
*
*
*
*
*
2
*
0
0
6
4
2
0
30
20
10
0
6d
6d
*
*
*
*
*
*
*
*
10
8
10
8
7
7
*
*
6
6
4
4
*
*
*
2
2
0
0
consider that the use of this substance in cancer treatments
is limited. In addition, other derivatives showed a very low
cytotoxic effect on healthy cells. In this present study, we
report that the use of 100 µM concentrations of the dimeric
compounds was sufficient in selectively inducing death and
an apoptotic cell ratio in HeLa and MCF-7 breast cancer

Medicinal Chemistry Research
cells. This indicates that 6a, 6b, and 6d can act directly to
induce apoptosis in both HeLa and MCF-7 cancer cells at
relatively low IC50 levels, unlike other derivatives for HeLa
cells. These findings highlights that the 6a derivative has
potent antiproliferative and proapoptotic properties, in
addition to 6d. In conclusion, our results indicate that the
obtained dimeric compounds are selective in inducing
apoptotic cell death in breast cancer cells by targeting the
p53-induced mitochondrial pathway. The same mechanism
is observed only in 6a, 6b, and 6d derivative-treated HeLa
cells at 48 h. The effect of the number of carbon atoms on
anticancer activity of the synthesized compounds was
investigated but no significant effect was observed. In
addition, although the presence of lithocholic acid moiety
did not elevated programmed cell death on HeLa and MCF-
7 cancer cell lines, it induced cytotoxic effect on normal cell
MEF. Finally, we consider that the most effective dimeric
compounds are 6a and 6b, which have the highest IC50
value on healthy cells and the lowest IC50 value in both
cervix and breast cancer cells, especially in long-term
treatment, and which specifically trigger the apoptosis
pathway.
rhenium(I) carbonyl complexes of natural bile acids and deriva-
tives. Inorg Chim Acta 286:46–54
Chen J, Liu C-F, Yang C-W, Zeng C-C, Liu W, Hu L-M (2015)
Design and synthesis of 5-chloro-2-hydroxy-3-triazolylbenzoic
acids as HIV integrase inhibitors. Med Chem Res 24:2950–2959
Dang Z, Jung K, Qian K, Lee K-H, Huang L, Chen C-H (2012)
Synthesis of lithocholic acid derivatives as proteasome regulators.
ACS Med Chem Lett 3:925–930
Decaudin D, Marzo I, Brenner C, Kroemer G (1998) Mitochondria in
chemotherapy-induced apoptosis: a prospective novel target of
cancer therapy. Int J Oncol 12:141–193
Duan T, Fan K, Fu Y, Zhong C, Chen X, Peng T, Qin J (2012)
Triphenylamine-based organic dyes containing a 1,2,3-triazole
bridge for dye-sensitized solar cells via a ‘Click’ reaction. Dyes
Pigm 94:28–33
El Kihel L, Clément M, Bazin MA, Descamps G, Khalid M, Rault S
(2008) New lithocholic and chenodeoxycholic piper-
azinylcarboxamides with antiproliferative and pro-apoptotic
effects on human cancer cell lines. Bioorg Med Chem
16:8737–8744
Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis
pathways in anticancer chemotherapy. Oncogene 25:4798–4811
Gafar AA, Draz HM, Goldberg AA, Bashandy MA, Bakry S, Khalifa
MA, Sanderson JT (2016) Lithocholic acid induces endoplasmic
reticulum stress, autophagy and mitochondrial dysfunction in
human prostate cancer cells. PeerJ 4:e2445
Glowacka IE, Balzarini J, Wróblewski AE (2013) The synthesis,
antiviral, cytostatic and cytotoxic evaluation of a new series of
acyclonucleotide analogues with a 1,2,3-triazole linker. Eur J
Med Chem 70:703–722
Acknowledgements This study was supported by Trakya University
Scientific Research Projects Unit in projects number TUBAP 2015/72.
Goldberg AA, Beach A, Davies GF, Harkness TA, Leblanc A,
Titorenko VI (2011) Lithocholic bile acid selectively kills neu-
roblastoma cells, while sparing normal neuronal cells. Oncotarget
2:761–782
Compliance with ethical standards
Goldberg AA, Titorenko VI, Beach A, Sanderson JT (2013) Bile acids
induce apoptosis selectively in androgen-dependent and -inde-
pendent prostate cancer cells. PeerJ 1:e122
Conflict of interest The authors declare that they have no conflict of
interest.
Green DR, Kroemer G (2004) The pathophysiology of mitochondrial
cell death. Science 305:626–629
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Guclu H, Doganlar ZB, Gurlu VP, Ozal A, Dogan A, Turhan MA,
Doganlar O (2018) Effects of cisplatin-5-fluorouracil combina-
tion therapy on oxidative stress, DNA damage, mitochondrial
apoptosis, and death receptor signalling in retinal pigment epi-
thelium cells. Cutan Ocul Toxicol 37:291–304
He XL, Xing Y, Gu X-Z, Xiao J-X, Wang Y-Y, Yi Z, Qiu W-W
(2017) The synthesis and antitumor activity of lithocholic acid
and its derivatives. Steroids 125:54–60
Himo F, Demko ZP, Noodleman L, Sharpless KB (2002) Mechanisms
of tetrazole formation by addition of azide to nitriles. J Am Chem
Soc 124:12210–12216
Himo F, Lovell T, Hilgraf R, Rostovtsev VV, Noodleman L, Sharpless
KB, Fokin VV (2005) Copper(I)-catalyzed synthesis of azoles.
DFT study predicts unprecedented reactivity and intermediates. J
Am Chem Soc 127:210–216
Hryniewicka A, Łotowski Z, Seroka B, Witkowski S, Morzycki JW
(2018) Synthesis of a cisplatin derivative from lithocholic acid.
Tetrahedron 74:5392–5398
Jurášek M, Dzubák P, Sedlák D, Dvoráková H, Hajdúch M, Bartunek
P, Drašar P (2013) Preparation, preliminary screening of new
types of steroid conjugates and their activities on steroid recep-
tors. Steroids 78:356–361
Kádár Z, Molnár J, Schneider G, Zupkó I, Frank É (2012) A facile
‘click’ approach to novel 15β-triazolyl-5α-androstane derivatives,
and an evaluation of their antiproliferative activities in vitro.
Bioorg Med Chem 20:1396–1402
References
Abdu-Allhah HHM, Chang TT, Li W-S (2016) Synthesis of B- and C-
ring-modified lithocholic acid analogues as potential sialyl-
transferase inhibitors. Steroids 112:54–61
Ahmad TR, Haeusler RA (2019) Bile acids in glucose metabolism and
insulin signalling-mechanisms and research needs. Nat Rev
Endocrinol 15:701–712
Anandkumar D, Rajakumar P (2017) Synthesis and anticancer activity
of bile acid dendrimers with triazole as bridging unit through
click chemistry. Steroids 125:37–46
Asif M (2017) Pharmacological activities of Triazole analogues as
antibacterial, antifungal, antiviral agents. Pharm Sci Asia
44:59–74
Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M,
Alnemri ES, Cohen GM (2001) Recruitment, activation and
retention of caspases-9 and-3 by Apaf-1 apoptosome and asso-
ciated XIAP complexes. EMBO J 20:998–1009
Cain K, Bratton SB, Langlais C, Walker G, Brown DG, Sun XM,
Cohen GM (2000) Apaf-1 oligomerizes into biologically active∼
700-kDa and inactive∼ 1.4-MDa apoptosome complexes. J Biol
Chem 275:6067–6070
Campazzi E, Cattabriga M, Marvelli L, Marchi A, Rossia R, Pieragnoli
MR, Fogagnolo M (1999) Organometallic radiopharmaceuticals:

Medicinal Chemistry Research
Kolb HC, Sharpless KB (2003) The growing impact of click chemistry
on drug discovery. Drug Discov Today 8:1128–1136
Qiao D, Stratagouleas ED, Martinez JD (2001) Activation and role of
mitogen-activated protein kinases in deoxycholic acid-induced
apoptosis. Carcinogenesis 22:35–41
Rahman M, Mohammad Y, Fazili KM, Bhat KA, Ara T (2017)
Synthesis and biological evaluation of novel 3-O-tethered tria-
zoles of diosgenin as potent antiproliferative agents. Steroids
118:1–8
Ramírez-López P, de la Torre MC, Montenegro HE, Asenjo M, Sierra
MA (2008) A straightforward synthesis of tetrameric estrone-
based macrocycles. Org Lett 10:3555–3558
Reddy VG, Bonam SR, Reddy TS, Akunuri R, Naidu VGM, Nayak
VL, Bhargava SK, Kumar HMS, Srihari P, Kamal A (2018)
4β-amidotriazole linked podophyllotoxin congeners: DNA
topoisomerase-IIα inhibition and potential anticancer agents for
prostate cancer. Eur J Med Chem 144:595–611
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002) A step-
wise huisgen cycloaddition process: copper(i)-catalyzed regiose-
lective “ligation” of azides and terminal alkynes. Angew Chem
Int Ed 41:2596–2599
Salucci S, Burattini S, Battistelli M, Baldassarri V, Curzi D, Valmori
A, Falcieri E (2014) Melatonin prevents chemical-induced hae-
mopoietic cell death. Int J Mol Sci 15:6625–6640
Tan W, Li Q, Gao Z, Qiu S, Dong F, Guo Z (2017) Design, synthesis
of novel starch derivative bearing 1,2,3-triazolium and pyr-
idinium and evaluation of its antifungal activity. Carbohydr
Polym 157:236–243
Vasilyeva SV, Shtil AA, Petrova AS, Balakhnin SM, Achigecheva
PY, Stetsenko DA, Silnikov VN (2017) Conjugates of phos-
phorylated zalcitabine and lamivudine with SiO2 nanoparticles:
synthesis by CuAAC click chemistry and preliminary assessment
of anti-HIV and antiproliferative activity. Bioorg Med Chem
25:1696–1702
Li SZ, Zhang HH, Zhang JN, Zhang ZY, Zhang XF, Zhang XD, Du RL
(2013) ALLN hinders HCT116 tumor growth through Bax-
dependent apoptosis. Biochem Biophys Res Commun 437:325–330
Lima-Neto RG, Cavalcante NNM, Srivastava RM, Mendonça Jr FJB,
Wanderley AG, Neves RP, dos Anjos JV (2012) Synthesis of
1,2,3-triazole derivatives and in vitro antifungal evaluation on
candida strains. Molecules 17:5882–5892
Luchetti F, Canonico B, Curci R, Battistelli M, Mannello F, Papa S,
Falcieri E (2006) Melatonin prevents apoptosis induced by UV-B
treatment in U937 cell line. J Pineal Res 40:158–167
Luu TH, Bard J-M, Carbonnelle D, Chaillou C, Huvelin J-M, Bobin-
Dubigeon C, Nazih H (2018) Lithocholic bile acid inhibits lipo-
genesis and induces apoptosis in breast cancer cells. Cell Oncol
41:13–24
Malkoch M, Thibault RJ, Drockenmuller E, Messerschmidt M, Voit B,
Russell TP, Hawker CJ (2005) Orthogonal approaches to the
simultaneous and cascade functionalization of macromolecules
using click chemistry. J Am Chem Soc 127:14942–14949
Menendez C, Gau S, Lherbet C, Rodriguez F, Inard C, Pascad MR,
Baltas M (2011) Synthesis and biological activities of triazole
derivatives as inhibitors of InhA and antituberculosis agents. Eur
J Med Chem 46:5524–5531
Morales-Sanfrutos J, Ortega-Munoz M, Lopez-Jaramillo J, Hernandez-
Mateo F, Santoyo-Gonzalez F (2008) Synthesis of calixarene-
based cavitands and nanotubes by click chemistry. J Org Chem
73:7768–7771
Nagesh HN, Suresh N, Prakash GVSB, Gupta S, Rao JV, Sekhar
KVGC (2015) Synthesis and biological evaluation of novel
phenanthridinyl piperazine triazoles via click chemistry as anti-
proliferative agents. Med Chem Res 24:523–532
Narayanaswamy N, Maity D, Govindaraju T (2011) Reversible fluor-
escence sensing of Zn2+ based on pyridine-constrained bis(triazole-
linked hydroxyquinoline) sensor. Supramol Chem 23:703–709
Nascimento PGG, Lemos TLG, Almeida MCS, Souza JMO, Bizerra
AMC, Santiago GMP, Costa JGM, Coutinho HDM (2015)
Lithocholic acid and derivatives: antibacterial activity. Steroids
104:8–15
Nejma AB, Znati M, Daich A, Othman M, Lawson AM, Jannet HB
(2018) Design and semisynthesis of new herbicide as 1,2,3-triazole
derivatives of the natural maslinic acid. Steroids 138:102–107
Porter AG, Janicke RU (1999) Emerging roles of caspase-3 in apop-
tosis. Cell Death Differ 6:99–104
Vatmurge NS, Hazra BG, Pore VS, Shirazi F, Chavan PS, Deshpande
MV (2008) Synthesis and antimicrobial activity of β-lactam-bile
acid conjugates linked via triazole. Bioorg Med Chem Lett
18:2043–2047
WCRF (2018), World Cancer Research Fund. American Institute for
Cancer Research. Worldwide Cancer Data. https://www.wcrf.org/
dietandcancer/cancer-trends/worldwide-cancer-data Accessed 24
January 2019
Yadav P, Lal K, Kumar L, Kumar A, Kumar A, Paul AK, Kumar R
(2018) Synthesis, crystal structure and antimicrobial potential of
some fluorinated chalcone-1,2,3-triazole conjugates. Eur J Med
Chem 155:263–274