Synthesis, in vitro cytotoxic and apoptotic effects, and molecular docking study of novel adamantane derivatives

Article abstract of DOI:10.1002/ardp.202000256

[4-(Adamantane-1-carboxamido)-3-oxo-1-thia-4-azaspiro[4.4]nonan-2-yl]acetic acid (4a) and [4-(adamantane-1-carboxamido)-8-nonsubstituted/substituted-3-oxo-1-thia-4-azas-piro[4.5]decane-2-yl]acetic acid (4b–g) derivatives were synthesized; their structures were verified by elemental analysis, infrared spectroscopy, ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and mass?spectroscopy data; and their in vitro cytotoxicity activities were investigated against human hepatocellular carcinoma, human prostate adenocarcinoma, and human lung carcinoma cell lines (HepG2, PC-3, and A549, respectively), and a mouse fibroblast cell line (NIH/3T3). All compounds, except?compound 4e, were found as cytotoxic, especially on A549 cells as compared with the other cells (selectivity index = 2.01–11.6). As a further step, the effects of compounds 4a–c on apoptosis induction were tested and the expression of selected apoptosis genes was analyzed. Among the selected compounds, compound 4a induced apoptosis remarkably. Moreover, computational calculations of the binding of compounds 4a–c to the BIR3 domain of the human inhibitor of apoptosis protein revealed ligand–protein interactions at the atomistic level and emphasized the importance of a hydrophobic moiety on the ligands for better binding.

Full text of DOI:10.1002/ardp.202000256

Received: 21 July 2020
Revised: 1 December 2020
Accepted: 5 December 2020
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DOI: 10.1002/ardp.202000256
F U L L P A P E R
Synthesis, in vitro cytotoxic and apoptotic effects, and
molecular docking study of novel adamantane derivatives
Basak Turk‐Erbul1,2 | Ecem F. Karaman3,4 | Gizem N. Duran5 | Mehmet Ozbil6
Sibel Ozden4 | Fusun Goktas1
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1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Istanbul University,
Istanbul, Turkey
Abstract
[4‐(Adamantane‐1‐carboxamido)‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]nonan‐2‐yl]acetic acid (4a)
and [4‐(adamantane‐1‐carboxamido)‐8‐nonsubstituted/substituted‐3‐oxo‐1‐thia‐4‐azas‐
piro[4.5]decane‐2‐yl]acetic acid (4b–g) derivatives were synthesized; their structures
were verified by elemental analysis, infrared spectroscopy, 1H nuclear magnetic re-
sonance (NMR), 13C NMR, and mass spectroscopy data; and their in vitro cytotoxicity
activities were investigated against human hepatocellular carcinoma, human prostate
adenocarcinoma, and human lung carcinoma cell lines (HepG2, PC‐3, and A549, re-
spectively), and a mouse fibroblast cell line (NIH/3T3). All compounds, except compound
4e, were found as cytotoxic, especially on A549 cells as compared with the other cells
(selectivity index = 2.01–11.6). As a further step, the effects of compounds 4a–c on
apoptosis induction were tested and the expression of selected apoptosis genes was
analyzed. Among the selected compounds, compound 4a induced apoptosis remarkably.
Moreover, computational calculations of the binding of compounds 4a–c to the BIR3
domain of the human inhibitor of apoptosis protein revealed ligand–protein interactions
at the atomistic level and emphasized the importance of a hydrophobic moiety on the
ligands for better binding.
2Department of Pharmaceutical Chemistry,
Acibadem Mehmet Ali Aydinlar University,
Istanbul, Turkey
3Department of Pharmaceutical Toxicology,
Biruni University, Istanbul, Turkey
4Department of Pharmaceutical Toxicology,
Istanbul University, Istanbul, Turkey
5Department of Chemistry, Marmara
University, Istanbul, Turkey
6Institute of Biotechnology, Gebze Technical
University, Kocaeli, Turkey
Correspondence
Fusun Goktas, Department of Pharmaceutical
Chemistry, Istanbul University, Beyazıt,
34116 Istanbul, Turkey.
Email: fusung@istanbul.edu.tr
Funding information
Bilimsel Arastirma Projeleri Birimi, Istanbul
Üniversitesi, Grant/Award Number:
TYL‐2017‐25681
K E Y W O R D S
antitumor activity, cytotoxic activity, structure elucidation, synthesis, thiazolidine
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1
INTRODUCTION
of cancer, so induction of apoptosis is thought to be a popular strategy
for killing cancerous cells.[3–6] Therefore, loss of apoptosis provides a
Cancer is one of the leading causes of death worldwide today.[1] Whereas
cancer can be caused due to a genetic change in which a normal cell is
transformed into a malignant cell, cell death escaping is one of the fun-
damental changes in a cell that leads to this malignant transformation.[2]
Drug resistance in cancer treatment is an important factor among all the
factors, leading to ultimate failure. Apoptosis or programmed cell death is
defined by morphological change that causes internal and external
nonpathological cell loss. Apoptosis occurs spontaneously in malignant
tumors and often slows down growth. The role of apoptosis in cancer
development has recently become more attractive for oncologists. An-
tiapoptotic properties or resistant cell death has been proposed as a sign
survival advantage to tumor cells and leads to resistance of cancer cells
for anticancer therapies.[2]
The adamantane and 4‐thiazolidinones rings are crucial components
of bioactive molecules. After the discovery of amantadine as an antiviral
and antiparkinson drug, thousands of adamantane derivatives were
synthesized and tested for biological activities.[7] Up until today,
the studies have shown that adamantane derivatives exhibit various
pharmaceutical effects including antiviral,[8–11] anticancer,[12] anti-
bacterial,[13,14] antifungal,[15,16] anti‐inflammatory,[17,18] trypanocidal,[19]
antiparkinson,[20,21] and 11β‐HSD1 inhibitory activities.[22,23] Moreover,
besides amantadine, some drug molecules containing adamantyl ring are
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Arch Pharm. 2021;354:e2000256.
wileyonlinelibrary.com/journal/ardp
© 2021 Deutsche Pharmazeutische Gesellschaft
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also known, such as adapromine, rimantadine, vildagliptin (antiviral),
saxagliptin, tromantadine (antidiabetic), adapalene (treatment of acne),
bromantane (anxiolytic), memantine (Alzheimer's disease) with their
various pharmacological effects (Figure 1).[24–28] When adamantane is
bound to the scaffold of various structures, this adamantyl moiety in-
creases the cytotoxic effect of the molecule due to its possible lipophilic
character or unique structure.
However, homologs of 2‐alkyl/aryl‐substituted 4‐thiazolidinones,
namely spirothiazolidinones, are one of the most studied heterocyclic
systems that show a wide range of biological activities such as anti-
bacterial,[29,30] anticancer,[31,32] antifungal,[33] and antiviral activities.[34]
In light of recent studies about the antitumor, antiproliferative, or
anticancer effects of compounds bearing adamantane ring,[35–40] our
aim is to synthesize several novel spirothiazolidinone compounds
FIGURE 1 Some drug molecules containing the adamantane ring

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bearing adamantane ring (Scheme 1). The structures of all obtained
products were elucidated by spectroscopic methods and were screened
for their in vitro cytotoxic activity against HepG2, PC‐3, A549, and
NIH/3T3 cell lines. Furthermore, the effects of compounds 4a–c on
apoptosis induction were tested and supported with gene expression
results and molecular docking calculations. This investigation can lead
to further design and development of molecules as antitumor agents.
methanol and 98% H2SO4. The methyl ester (3) was obtained with the
reaction of excess hydrazine hydrate under reflux for 15 h without using
any solvent. With the addition of cyclopentanone/cyclohexanone/sub-
stituted cyclohexanone compounds in adamantane‐1‐carbohydrazide, 3
was cyclized with mercaptosuccinic acid in absolute toluene and the
desired derivatives 4a–g were obtained via one‐pot synthesis. Moreover,
the synthesized compounds were characterized via analysis of IR, 1H
nuclear magnetic resonance (NMR), 13C NMR (proton decoupled), 13C
NMR (distortions enhancement by polarization transfer [DEPT]),
2D‐NMR (heteronuclear single‐quantum coherence [HSQC]), and
mass spectroscopy (MS). The atomic numbering of obtained products
applied for NMR analysis is shown in Figure 2.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The IR spectra of compounds 4a–g demonstrated common
characteristic absorption bands at 3496–3111 cm−1 (O–H/N–H),
1707–1616 cm−1 (HO–C═O) 1697–1690 cm−1 (C═O), and
1668–1663 cm−1 (NH–C═O), which provide the evidence for the
The synthesis of the target compounds is shown in Scheme 1. Methyl
adamantane‐1‐carboxylate (2) was synthesized by adamantane‐1‐
carboxylic acid (which is commercially available) in the presence of
SCHEME 1 The synthesis pathway of compounds 4a–g. Compounds: 4b: R = H; 4c: R = CH3; 4d: R = C2H5; 4e: R = C3H7; 4f: R = C(CH3)3; 4g:
R = C6H5. Reagents and conditions: (i) H2SO4, 75°C, 3 h; (ii) hydrazine hydrate, 150°C, 15 h; (iii) anhydrous toluene, 120°C, 16 h

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cycloaddition reaction between azomethine intermediates and
mercaptosuccinic acid.
also confirmed the structure. In mass spectra of 4a, 4c, 4d, and 4f,
[M−H]− molecular ion peaks were observed in the negative ionization
mode, whereas in the positive mode, all characteristic m/z 135 peaks
of adamantane were common.
In the 1H NMR spectra of the compounds, –COOH and –CONH
protons were observed as a singlet at about σ: 12.63–12.54 ppm (1H)
and σ: 9.67–9.59 ppm (1H), respectively. The 1H NMR spectra of
compounds 4a–g displayed doublet of doublets attributed to
(SCHCH2) methine ring protons at 2‐position of the 4‐thiazolidinone
system at about σ: 4.06–3.99 ppm (1H). In the 1H NMR spectra of
compound 4e, –CH2COOH protons were observed as a doublet of
doublets at about σ: 3.01 ppm (1H) and σ: 2.46 ppm (1H).
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2.2
Pharmacology/biology
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2.2.1
Assessment of cytotoxicity
The same resonance spectra of compounds 4a–c, 4d, and 4f
were observed at about σ: 3.05–3.01 ppm (1H, dd) and σ:
2.49–2.41 ppm (5H, m, with dimethyl sulfoxide [DMSO]‐d6). The 1H
NMR spectra of compounds 4a–f showed the resonance of the C2ʹ,
C8ʹ, and C10ʹ protons of adamantane ring at σ: 1.85–1.84 ppm (6H, d)
and the resonance of the C4ʹ, C6ʹ, and C9ʹ protons of adamantane ring
at σ: 1.81–1.60 ppm (m, with 4‐thiazolidinone ring protons).
Furthermore, protons that belong to C3ʹ, C5, and C7ʹ bridgehead
carbons are observed as broad singlet values of 3H integral at δ
1.98–2.01 ppm in a lower field due to the W effect, compared with
other protons connected to adamantane. In the 13C NMR spectra of
compounds 4a–g, the thiazolidinone C2 carbon (σ: 40.49–40.48, with
DMSO‐d6), thiazolidinone C═O carbon (σ: 172.23–172.15 ppm), and
thiazolidinone C5 carbon (σ: 74.80–71.02 ppm) peaks verified the
formation of desired spirothiazolidinone structures. Observed peaks
of acid C═O carbon (σ: 176.92–176.80 ppm), amide C═O carbon
(σ: 168.66–168.57 ppm), and –CH2COOH methylene carbon (σ:
39.30–39.21 ppm) in the 13C NMR spectra supported the cycload-
dition reaction. The 13C NMR (DEPT) spectrum of compound 4b is
also supported by a related interpretation. Moreover, 2D‐NMR
(HSQC) experiment was performed to establish the interfragment
relationship and assign the proton and carbon signals of the proto-
type compound 4c. HSQC (1H‐13C) experiment of representative 4c
The cytotoxic activity of compounds was studied using the MTT
(3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay.
HepG2, PC‐3, A549, and NIH/3T3 cells were exposed to a broad range
of compound concentrations for 24 h. The results of this experiment
and IC50 values of compounds are shown in Table 1. According to
these results, compounds 4a, 4b, and 4c showed a quite high cytotoxic
activity with average IC50 values of 0.15, 0.23, and 0.22 mM, respec-
tively in A549 cells, whereas they exhibited the weakest cytotoxic
effect in NIH/3T3 cells. The cytotoxic activities of the compounds in
cell lines were less potent when compared with doxorubicin. It has
been observed that A549 cells were more sensitive for cytotoxic ac-
tivity among these cell lines. According to our results, we selected
compounds 4a–c for further analysis in A549 cells.
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2.2.2
The effects of compounds 4a, 4b, and 4c on
apoptosis induction
We tested the ability of 4a–c to induce apoptosis by a flow cyto-
metry analysis in A549 cells. The cytograms of bivariate annexin
V/propidium iodide (PI) analysis of A549 cells after treatment with
4a–c are shown in Figure 3. Apoptosis induction was detected at the
rates of 68.86%, 35.04%, and 55.48%, respectively.
FIGURE 2 Atomic numbering of obtained products applied for nuclear magnetic resonance analysis

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TABLE 1 IC50 (mM) values of the substances obtained by using the MTT test in cancerous and healthy cells
PC‐3
HepG2
a
a
Compound
IC50
SIb
nd
IC50
SIb
4a
nd (>2.55)
2.52 0.1
0.679
4.55
1.06
1.13
1.04
1.88
2.33
1.04
4b
0.41 0.003
1.24 0.05
1.08 0.01
0.45 0.01
0.66 0.02
0.54 0.02
5.86
0.85
0.54
1.31
1.06
1.31
2.12
0.53 0.05
4c
1 0.06
4d
0.51 0.03
0.57 0.02
0.37 0.03
0.31 0.01
4e
4f
4g
Doxorubicin
0.025 0.005
A549
0.051 0.01
NIH/3T3
a
a
SIb
IC50
Compound
IC50
4a
0.15 0.01
0.23 0.02
0.22 0.02
0.24 0.01
0.48 0.02
0.35 0.024
0.28 0.02
0.014 0.003
11.6
10.32
4.77
2.41
1.22
2.01
2.58
3.79
1.71 0.08
2.42 0.08
1.06 0.05
0.58 0.03
0.59 0.05
0.7 0.03
4b
4c
4d
4e
4f
4g
0.71 0.03
0.053 0.007
Doxorubicin
Note: Boldface shows that high cytotoxic activity.
Abbreviations: MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; nd, not determined.
aIC50 = 50% cytotoxic concentration against in vitro tested cells. Data are presented as mean SD.
bSI = selectivity index—IC50 value relative to a normal cell.
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2.2.3
The effects of compounds 4a, 4b, and 4c on
compounds were 4a, 4b, and 4c for A549 in terms of IC50 values. After
exposure to these compounds, the viability of noncancerous cell line,
NIH/3T3, was not generally affected by these compounds. Similar to
our results, Ali et al.[6] observed that the designed new adamantane
derivatives exhibited the lowest IC50 concentrations in A549 cells ac-
cording to the MTT assay.
apoptosis genes
To support our findings, we analyzed the gene expression of selected
apoptosis genes (p53, CASP3, CASP9 BCL2, B, CYCS, AIF, APAF1, and XIAP)
to investigate the potential apoptotic effects of compound 4a–c. In
Figure 4a–c, our data showed that although all compounds altered ex-
pression levels of apoptosis genes significantly, exposure to compound 4a
induced apoptosis more effectively. Expression levels of p53 (1.4‐fold),
CASP3 (1.6‐fold), BAX (4.05‐fold), CYCS (4.42‐fold), and AIF (2‐fold) were
upregulated and expression levels of CASP9 (2.3‐fold), BCL2 (12.5‐fold),
and XIAP (5‐fold) were downregulated after 4a treatments. Besides,
the APAF1 gene expression did not change as compared with the control
group.
According to the MTT results, 4a, 4b, and 4c were selected to assess
the induction of apoptosis in A549 cells by a flow cytometer. Among the
selected compounds, 4a and 4c induced apoptosis significantly at IC50
levels.
It has been reported that many approved cancer treatments
could affect via the intrinsic pathway.[41] The intrinsic pathway (or mi-
tochondrial one) is regulated by BCL2 proteins that lead to the release
of apoptogenic factors and cause the formation of the apoptosome,
including apoptotic protease‐activating factor‐1 (APAF1), cytochrome‐c,
and caspase 9.[42] p53 is one of the best known tumor suppressor
proteins, and besides the induction of apoptosis, it is involved in cell
cycle regulation, development, differentiation, gene amplification, and
cellular senescence.[2] Activated p53 can induce apoptotic cell death
through both the extrinsic and intrinsic pathways.[2,43] The elevated
BAX/BCL2 ratio is regarded as a sign of apoptosis and indicates
The newly synthesized compounds were tested for their ability to
inhibit metabolic activity as an indicator for the viability of cancerous
and noncancerous cell lines. First, the compounds were tested for their
ability to inhibit the viability of HepG2, PC‐3, A549, and NIH/3T3 cell
lines using the MTT assay. According to IC50 values, compounds were
shown to exhibit the most cytotoxic effect on A549 cell line among
cancerous cell lines. Among cancerous cell lines, the most cytotoxic

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mitochondrial dysfunction, which leads to the release of cytochrome‐c
(CYCS). Moreover, this sequential cascade triggers several proteases
including caspase‐1, ‐3, and ‐9.[44‐46] Caspase‐3 (CASP3) also activates
the pathway of several enzymatic events and serves as the promoter of
apoptosis.[47] In the present study, compounds 4a and 4c induced p53
and BAX gene expression and decreased expression levels of BCL2;
however, the BAX/BCL2 ratio was higher after compound 4a exposure.
As a result of this, CYCS and Cas3 gene expression levels were sig-
nificantly upregulated after compound 4a exposure, whereas CASP3
and CASP9 were downregulated after compound 4c exposure in A549
cells. Ali et al.[6] have showed upregulation of p53, BAX, and CASP3 and
downregulation of BCL2 in A549 cells after new adamantane derivative
exposure. We measured the expression levels of APAF1, apoptosis‐
inducing factor (AIF), and X‐linked inhibitor of apoptosis protein (XIAP)
genes as well. XIAP is one of the inhibitors of apoptosis proteins (IAPs),
which blocks the caspase activities.[48]
and compound 4b interacted with a mixture of hydrophobic and
hydrophilic interactions.
IAPs are a family of apoptosis regulators that were upregulated in
various cancer cells. Overexpressions of these proteins maintain the
proliferation of cancer cells and bypassing to several therapeutics for
cancer.[50] In addition, XIAP has been highly reported to be over-
expressed in lung cancer, causing inhibition of apoptosis and treat-
ment resistance.[51,52] Therefore, molecular perspective into XIAP,
which is one of the therapeutic targets for cancer, will be valuable for
the development of drugs for cancer treatment. Our study considered
that 4a–c could be used as a novel potential monovalent Smac mi-
metic due to their high affinity of binding with XIAP which may ex-
hibitthe profile of an XIAP antagonist. As can be seen from the gene
expressions results, decreasing XIAP expression levels could lead to an
increased caspase activity, inducing apoptosis. Hence, antiapoptotic
effects of XIAP have also been decreased due to their cellular amount
reduction, leading to activation of apoptotic genes such as p53, CASP3,
BAX, and CYCS. Hence, this could be the reason that compounds 4a–c
showed a remarkable cytotoxicity against lung cancer cells A549. Due
to the promising biological activities of 4a–c, we also performed the
screening of novel compound candidates for their apoptotic activity
and affinity to XIAP, and it could be suggested that they had potentials
for lung cancer treatment.
After exposure of compound 4a, expression levels of AIF in-
creased and XIAP levels decreased, whereas the APAF1 expression
level did not change. Related with flow analysis, histograms were also
supported with our gene expression results.
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2.3
Molecular docking
In the cytotoxic activity and gene expression test results, com-
pounds 4a–c were determined as the most potent and promising
antitumor agents. IAPs are a family of main apoptosis regulators
and XIAP (X‐chromosome‐linked IAP) is known as the best‐
characterized IAP member.[6,43,49] Therefore, molecular modeling
studies were performed against XIAP (PDB ID: 1G73) protein. 2D
interaction schemes and 3D binding poses are demonstrated in
Figures 5 and 6, respectively. All interactions are listed in
Table 2. As it can be seen from the table, compounds 4a and 4c
interacted with XIAP mostly through hydrophobic interactions
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3
CONCLUSION
The novel adamantyl‐substituted spirothiazolidinone derivatives
were successfully synthesized in the presented study. The structures
were enlightened and confirmed by instrumental techniques. As a
result of the biological assay, all compounds (except for compound
4e) show a selective cytotoxic effect, especially on the A549 cell line.
Compound 4a triggered apoptosis more effectively than other
compounds, which is consistent with gene expression results.
FIGURE 3 Effects of the selected
compounds (at IC50 concentrations) on
apoptosis in A549 cells after 24‐h exposure
determined by staining with Annexin V/
propidium iodide. Data are presented as
mean SD. Statistical analysis was performed
by analysis of variance + Dunnett's post‐hoc
test. Statistically significant changes are
indicated by *p ˂ .05; **p < .001

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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Reagents, starting materials, and solvents were purchased from
Merck and Sigma‐Aldrich. Melting points (mp) were recorded on a
Büchi Melting Point B‐540 device and are uncorrected. 1H and 13C
NMR spectra were obtained by Varian UNITY INOVA 500 MHz
NMR, using DMSO‐d6 as a solvent and tetramethylsilane (TMS) as
internal standard. 2D‐NMR experiments (HSQC) and DEPT 13C NMR
were performed for the interpretation of novel synthesized com-
pounds. The atoms of target compounds were numbered for the
interpretation by NMR, as shown in Figure 1. Atmospheric pressure
chemical ionization (APCI) MS was carried out using an Advion Ex-
pression Compact Mass Spectrometer. Elemental analyses (C, H, N)
were performed by Thermo Finnigan Flash EA 1112 Series device. To
monitor the reaction during the synthesis and to check the purity of
the compounds, thin‐layer chromatography was performed using
Merck pre‐coated with silica gel 60 F254 aluminum sheets (layer
thickness 0.25 mm) and the spots were visualized with the help of
Dragendorff reagent.
The original spectra are provided as Supporting Information.
The InChI codes of the investigated compounds, together with
some biological activity data, are also provided as Supporting
Information.
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4.1.2
Synthesis of methyl adamantane
1‐carboxylate (2)[53]
For synthesis, 98% H2SO4 (8 ml) was added dropwise into a stirred
solution of adamantane‐1‐carboxylic acid (9 g, 0.05 mol) in methanol.
The mixture was heated under reflux for 3 h. During cooling, the
mixture was poured over crushed ice (250 g). Precipitated crystals
were washed with water and 10% NaHCO3 subsequently. Solid
crystals were allowed to dry. Light yellow solid; yield: 90%; mp:
35–36°C; IR ʋmax (cm−1): 2927, 2850 (C–H), 1732 (C═O); 1450, 1425
(C–H); 1238 (C–O).
FIGURE 4 Effects of the selected compounds (at IC50
concentrations) on relative gene expressions (a–c) in A549 cells after
a 24‐h exposure. Data are presented as mean SD. Statistical
analysis was performed by analysis of variance + Dunnett's post‐hoc
test. Statistically significant changes are indicated by
*p < .05; **p < .001
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4.1.3
Synthesis of adamantane 1‐carboxylic acid
These findings indicated that compound 4a could show its antitumor
activity through the induction of apoptosis via intrinsic pathway in
A549 cells. According to the computational calculations, although
compound 4a seemed to have the lowest binding affinity, the dif-
ference of 0.5 kcal/mol does not make the binding less favorable.
Therefore, it is revealed that all three ligands (4a–c) bound to similar
binding pockets with similar binding affinities, making them all pos-
sible drug candidates. Overall, these observations can provide a
promising framework for further design and development of
adamantane‐based compounds with a potential antitumor activity.
hydrazide (3)[54]
The mixture of methyl adamantane‐1‐carboxylate (2; 9.7 g, 0.05 mol)
and excess 98% hydrazine hydrate (15 ml) was stirred under reflux
for 15 h. During cooling, chilled water (150 ml) was added to the
mixture and the separated solid white crystal was filtered off, wa-
shed with cold water, dried, and crystallized from water. White
crystals; yield: 81%; mp: 147–148°C; IR (KBr) ʋ (cm−1): 3332, 3275
(N–H); 2908, 2848 (C–H); 1616 (C═O); 1521 (N–H); 1456, 1367
(C–H). 1H NMR (500 MHz) (DMSO‐d6) δ (ppm): 1.63 (6H, 2d,

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FIGURE 5 2D interaction schemes for compounds (a) 4a, (b) 4b, and (c) 4c. H‐bonding amino acids are represented by green, H‐bonds by
dotted lines, and hydrophobic interaction amino acids are written in black and represented by red half circles
J = 11.7 Hz, adm. C4–H/C6–H/C9–H); 1.75 (6H, d, J = 2.7 Hz, adm.
C2–H/C8–H/C10–H); 1.94 (3H, broad s, adm. C3–H/C5–H/C7–H); 4.10
(2H, s, NH2); 8.67 (1H, s, CONH). 13C (HSQC) NMR (500 MHz)
(DMSO‐d6) δ (ppm): 28.02/28.05/28.11 (3H, adm. C3/C5/C7); 36.53/
36.61/36.68 (adm. C4/C6/C9); 38.98/39.05/39.12 (adm. C2/C8/C10);
39.49 (adm. C1); 176.77 (CONH).
[4‐(Adamantane‐1‐carboxamido)‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]
nonan‐2‐yl]acetic acid (4a)
Brown crystals; yield: 74%; mp: 143°C; IR: 3543, 3241 (O–H/N–H);
2907, 2851 (C–H); 1705, 1694, 1668 (C═O); 1454, 1368 (C–H);
1246 (N–H and C–N). 1H NMR (DMSO‐d6, 500 MHz) δ (ppm):
1.57–1.63 (4H, m, C7–H/C8–H), 1.63–1.78 (8H, m, C6–9–H (ax.), adm.
C4ʹ–H, C6ʹ–H/C9ʹ–H); 1.84 (6H, d, J = 2.7 Hz, adm. C2ʹ–H/C8ʹ–H/
C10ʹ–H); 1.98 (3H, broad s, adm. C3ʹ–H/C5ʹ–H/C7ʹ–H); 2.06–2.28 (2H,
m, C6–9–H (eqv.)); 2.49 (5H, m, with DMSO‐d6 –CHa–COOH); 3.01
(1H, dd, J = 16.9; 4.0 Hz, –CHb–COOH) 4.05 (1H, dd, J = 10.4; 4.0 Hz,
C2–H); 9.59 (1H, s, CONH); 12.56 (1H, s, COOH). 13C NMR (DMSO‐
d6, 500 MHz) δ (ppm): 23.30 (cyclopentilidene C7/C8); 27.94/27.97
(adm. C3ʹ/C5ʹ/C7ʹ); 36.38/36.45/36.52 (adm. C4ʹ/C6ʹ/C9ʹ); 38.68 (cy-
clopentilidene C6/C9); 38.77/38.82/38.88 (adm. C2ʹ/C8ʹ/C10ʹ); 40.26
(C1ʹ); 40.48 (DMSO‐d6 and C2); 39.00 (CH2–COOH); 74.80 (cyclo-
pentilidene C5); 168.58 (CONH); 172.15 (C3); 176.80 (COOH); anal.
calcd. for C20H28N2O4S·H2O: C, 58.51; H, 7.37; N, 6.82. Found: C,
58.47; H, 7.07; N, 6.70.
|
4.1.4
General procedure for the synthesis of
spirothiazolidinone derivatives bearing the
adamantine ring (4a–g)
Cyclopentanone/4‐(substituted/substituted) cyclohexanone com-
pound (0.015 mol) was added into the solution of adamantane
1‐carboxylic acid hydrazide (0.005 mol) in 30 ml of anhydrous
toluene. The reaction mixture was heated under reflux using a
Dean–Stark trap for 2 h. After adding 2‐mercaptosuccinic acid
(0.015 mol), the reaction mixture was heated for
a further
13–14 h. Anhydrous toluene was removed under reduced pres-
sure and the excess acid was neutralized with a saturated sodium
bicarbonate solution. After cooling, the product formed was fil-
tered off, washed with water, and purified by crystallization from
the appropriate solvent.
[4‐(Adamantane‐1‐carboxamido)‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]
decan‐2‐yl]acetic acid (4b)
White powder; yield: 41%; mp: 156–158°C; IR: 3465, 3115 (O–H/
N–H); 2905, 2851 (C–H); 1707, 1690, 1666 (C═O); 1520 (N–H);
FIGURE 6 Binding sites for compounds (a) 4a, (b) 4b, and (c) 4c. Hydrophobic amino acids are represented by blue, hydrophilic residues by
red, ligand carbon atoms by cyan, and hydrogen atoms by white. Structural Zn2+ ion is represented by a purple sphere

TURK‐ERBUL ET AL.
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TABLE 2 Binding sites for compounds 4a, 4b, and 4c with
interaction types for each amino acid on XIAP‐BIR3 and binding
affinities on each site are provided
[4‐(Adamantane‐1‐carboxamido)‐8‐methyl‐3‐oxo‐1‐thia‐4‐azaspiro‐
[4.5]decan‐2‐yl]acetic acid (4c)
White crystals; yield: 33%; mp: 181–182°C; IR: 3489, 3118 (O–H/
N–H); 2904, 2851 (C–H); 1705, 1689, 1664 (C═O); 1515 (N–H);
1452, 1368 (C–H); 1238 (N–H and C–N). 1H NMR (DMSO‐d6,
500 MHz) δ (ppm): 0.85 (3H, t, J = 6.3 Hz, –CH3); 1.01–1.28 (3H, m,
sty.C8–H, sty.C7–9‐ax‐H); 1.61–1.80 (12H, m, adm. C4ʹ–H/C6ʹ–H/
C9ʹ–H and sty. C6–H/C10–H/C7‐eqv‐H and C9‐eqv‐H); 1.85 (6H, d,
J = 2.4 Hz, adm. C2ʹ–H/C8ʹ–H/C10ʹ–H); 1.98 (3H, broad s, adm. C3ʹ–H/
C5ʹ–H/C7ʹ–H); 2.45 (5H, m, DMSO‐d6 and –CHa–COOH); 3.01 (1H,
dd, J = 16.9; 3.8 Hz, –CHb–COOH) 3.99 (1H, dd, J = 10.5; 3.9 Hz,
C2–H); 9.59 (1H, s, CONH); 12.60 (1H, s, COOH). 13C NMR (DMSO‐
d6, 500 MHz) δ (ppm): 22.21 (CH3); 27.91/28.06 (cyclohex. C7/C9);
31.04 (cyclohex. C8); 27.97/28.00 (adm. C3ʹ/C5ʹ/C7ʹ); 31.04 (C8);
36.40/36.48/36.55 (adm. C4ʹ/C6ʹ/C9ʹ); 38.61 (cyclohex. C6/C10);
38.75/38.81/38.87 (adm. C2ʹ/C8ʹ/C10ʹ); 39.23 (CH2–COOH); 40.28
(C1ʹ); 40.49 (DMSO‐d6 and C2); 71.30 (cyclohex. C5); 168.64
(CONH); 172.20 (C3); 176.81 (COOH); MS APCI(−) m/z (%): 419 [M
−H]−, MS APCI(+) m/z (%): 135 (100). Anal. calcd. for
Interacting
Binding affinity
Interaction type (kcal/mol)
Compound residues
4a
Ala263
Asp264
Tyr265
Phe301
Tyr329
Glu332
Gln333
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H‐bonding
−6.9
Hydrophobic
H‐bonding
4b
Ala263
Asp264
Tyr265
Arg268
Arg286
Phe301
His302
Tyr329
Glu332
H‐bonding
Hydrophobic
Hydrophobic
H‐bonding
−7.2
H‐bonding
Hydrophobic
Hydrophobic
H‐bonding
C22H32N2O4S·H2O: C, 60.25; H, 7.81; N, 6.39. Found: C, 60.35; H,
Hydrophobic
7.36; N, 6.23.
4c
Pro257
Ala263
Tyr265
Arg268
Arg286
Phe301
His302
Tyr329
Glu332
Hydrophobic
H‐bonding
−7.4
[4‐(Adamantane‐1‐carboxamido)‐8‐ethyl‐3‐oxo‐1‐thia‐4‐azaspiro‐
[4.5]decan‐2‐yl]acetic acid (4d)
Hydrophobic
H‐bonding
White crystals; yield: 81%; mp: 156–157°C; IR: 3481, 3117 (O–H/
N–H); 2902, 2852 (C–H); 1705, 1689, 1667 (C═O); 1519 (N–H);
1443, 1367 (C–H); 1245 (N–H and C–N). 1H NMR (DMSO‐d6,
500 MHz) δ (ppm): 0.84 (3H, t, J = 7.4 Hz, –CH2CH3); 0.96–1.13 (3H,
m, sty.C8–H, sty. C7–9‐ax‐H); 1.13–1.28 (2H, m, –CH2CH3); 1.62–1.80
(12H, m, adm. C4ʹ‐H/C6ʹ‐H/C9ʹ‐H and sty. C6–H/C10–H/C7‐eqv‐H and
C9‐eqv‐H); 1.85 (6H, d, J = 2.4 Hz, adm. C2ʹ‐H/C8ʹ‐H/C10ʹ‐H); 1.98
(3H, broad s, adm. C3ʹ–H/C5ʹ–H/C7ʹ–H); 2.45 (5H, m, DMSO‐d6 and
–CHa–COOH); 3.01 (1H, dd, J = 16.9; 3.8 Hz, –CHb–COOH); 3.99
(1H, dd, J = 10.5; 3.9 Hz, C2–H); 9.59 (1H, s, CONH); 12.57 (1H, s,
COOH). 13C NMR (DMSO‐d6, 500 MHz) δ (ppm): 11.83 (–CH2CH3);
29.06 (–CH2CH3); 29.51/29.59 (cyclohex. C7/C9); 37.57 (cyclohex.
C8); 27.97/28.00 (adm. C3ʹ/C5ʹ/C7ʹ); 36.40/36.48/36.55 (adm. C4ʹ/C6ʹ/
C9ʹ); 38.61 (cyclohex. C6/C10); 38.75/38.81/38.87 (adm. C2ʹ/C8ʹ/C10ʹ);
39.24 (CH2–COOH); 40.28 (C1ʹ); 40.48 (DMSO‐d6 and C2); 71.60
(cyclohex. C5); 168.62 (CONH); 172.20 (C3); 176.79 (COOH); MS
APCI(−) m/z (%): 433 [M−H]−, MS APCI(+) m/z (%) 135 (100). Anal.
calcd. for C23H34N2O4S·H2O: C, 61.03; H, 8.02; N, 6.19. Found: C,
61.09; H, 7.57; N, 6.14.
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
1446, 1369 (C–H); 1249 (N–H and C–N). 1H NMR (DMSO‐d6,
500 MHz) δ (ppm): 0.98–1.05 (1H, broad q, sty.C8‐ax‐H); 1.32–1.43
(2H, m, sty.C7–9‐ax‐H); 1.53 (1H, d, J = 12.7 Hz, sty.C8‐eqv‐H);
1.60–1.78 (12H, m, adm. C4ʹ–H/C6ʹ–H/C9ʹ–H and sty.C6–H/C10–H/
C7‐eqv‐H and C9‐eqv‐H); 1.85 (6H, d, J = 2.4 Hz, adm. C2ʹ–H/C8ʹ–H/
C10ʹ–H); 1.98 (3H, broad s, adm. C3ʹ–H/C5ʹ–H/C7ʹ–H); 2.46 (5H, m,
DMSO‐d6 and –CHa–COOH); 3.01 (1H, dd, J = 16.9; 3.8 Hz,
CHb–COOH) 3.99 (1H, dd, J = 10.4; 3.9 Hz, C2–H); 9.59 (1H, s,
CONH); 12.60 (1H, s, COOH). 13C NMR (DMSO‐d6, 500 MHz) δ
(ppm): 23.19/23.52 (cyclohex. C7/C9); 24.51 (cyclohex. C8); 27.97/
28.00 (adm. C3ʹ/C5ʹ/C7ʹ); 36.41/36.48/36.55 (adm. C4ʹ/C6ʹ/C9ʹ); 38.63
(cyclohex. C6/C10); 38.76/38.82/38.88 (adm. C2ʹ/C8ʹ/C10ʹ); 39.21
(CH2–COOH); 40.29 (C1ʹ); 40.48 (DMSO‐d6 and C2); 71.44 (cyclohex.
C5); 168.57 (CONH); 172.19 (C3); 176.84 (COOH); anal. calcd. for
[4‐(Adamantane‐1‐carboxamido)‐3‐oxo‐8‐propyl‐1‐thia‐4‐azaspiro‐
[4.5]decan‐2‐yl]acetic acid (4e)
White powder; yield: 93%; mp: 148–149°C; IR: 3482, 3110 (O–H/N–H);
2908, 2849 (C–H); 1705, 1690, 1667 (C═O); 1519 (N–H); 1442, 1367
(C–H); 1242 (N–H and C–N). 1H NMR (DMSO‐d6, 500 MHz) δ (ppm):
0.85 (3H, t, J = 7.3 Hz, –CH2CH2CH3); 0.97–1.17 (5H, m, sty.C8–H,
sty.C7–9‐ax‐H and –CH2CH2CH3); 1.19–1.31 (2H, m, –CH2CH2CH3);
1.60–1.81 (12H, m, adm. C4ʹ–H/C6ʹ–H/C9ʹ–H and sty. C6–H/C10–H/C7‐
eqv‐H and C9‐eqv‐H); 1.85 (6H, d, J = 2.3 Hz, adm. C2ʹ–H/C8ʹ–H/C10ʹ–H);
C
21H30N2O4S·H2O: C, 59.41; H, 7.60; N, 6.60. Found: C, 59.43; H,
7.50; N, 6.57. 13C NMR (DEPT) (500 MHz; DMSO‐d6/TMS) δ (ppm):
23.11/23.51 (C7/C9); 24.49 (C8); 27.51 (adm. C3ʹ/C5ʹ/C7ʹ); 35.99
(adm. C4ʹ/C6ʹ/C9ʹ); 37.48 (cyclohex. C6/C10); 38.32 (adm. C2ʹ/C8ʹ/
C10ʹ); 38.67 (CH2–COOH); 39.79 (C1ʹ); 39.95 (cyclohex. C2); 70.93
(cyclohex. C5); 168.07 (CONH); 171.68 (C3); 176.34 (COOH).

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TURK‐ERBUL ET AL.
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|
Pharmacological/biological assays
1.98 (3H, broad s, adm. C3ʹ‐H/C5ʹ–H/C7ʹ–H); 2.46 (1H, dd, J = 10.5;
6.4 Hz, –CHa–COOH); 3.01 (1H, dd, J = 16.9; 3.7 Hz, CHb–COOH); 3.99
(1H, dd, J = 10.5; 3.9 Hz, C2–H); 9.59 (1H, s, CONH); 12.61 (1H, s,
COOH). 13C NMR (DMSO‐d6, 500 MHz) δ (ppm): 14.65, –CH2CH2CH3);
27.97/28.00 (adm. C3ʹ/C5ʹ/C7ʹ); 29.06, –CH2CH2CH3); 29.59/29.51
(cyclohex. C7/C9); 36.40/36.48/36.55 (adm. C4ʹ/C6ʹ/C9ʹ); 38.61 (cyclo-
hex. C6/C10); 37.56 (cyclohex. C8); 38.74/38.81/38.87 (adm. C2ʹ/C8ʹ/
C10ʹ); 38.87 (–CH2CH2CH3); 39.24 (CH2–COOH); 40.28 (C1ʹ); 40.48
(DMSO‐d6 and C2); 71.60 (cyclohex. C5); 168.62 (CONH); 172.20 (C3);
176.81 (COOH); anal. calcd. for C24H36N2O4S·H2O: C, 61.77; H, 8.21;
N, 6.00. Found: C, 61.87; H, 7.76; N, 5.99.
4.2
|
4.2.1
Cell culture and cytotoxicity
The cytotoxicity of the newly synthesized compounds was evaluated
by the MTT assay using the human hepatocellular carcinoma
(HepG2, HB‐8065™), human prostate adenocarcinoma (PC‐3,
CRL‐1435™), human lung carcinoma (A549, CCL‐185™), and mouse
fibroblast cell lines (NIH/3T3, CRL‐1658™). Cell lines were obtained
from the American Type Culture Collection (ATCC). Cells were
incubated in Dulbecco's modified Eagle's medium/Nutrient Mixture
F‐12 (DMEM/F‐12; Wisent Bioproducts) with 10% fetal bovine ser-
um and antibiotics (1% penicillin, 1% streptomycin) in 95% O2
and 5% CO2 at 37°C. The monolayer cells grown to 80–85% con-
fluency were detached with trypsin. The MTT assay evaluates the
viability of cells by the reduction of yellow tetrazolium salt (MTT) via
a mitochondrial‐dependent reaction to form an insoluble purple
formazan crystal.[55,56] Furthermore, 1 × 104 cells in 100 μl medium
in each well were seeded into a 96‐well plate and incubated at
37°C (5% CO2) for 24 h to allow for cell attachment. After 24 h, the
cells were treated with serial concentrations of the test compounds
in the range of 31.25–1000 µg/ml. Test compounds were dissolved in
DMSO. After dilutions, cells in each well were treated with six dif-
ferent concentrations of each compound. The medium containing no
sample (growth control), 0.1% sodium dodecyl sulfate (positive
control), and 1% DMSO (solvent control) served as the controls.
Doxorubicin was used as a positive control in the concentration
range of 3.125–100 µM. For all concentrations, it was tested in tri-
plicates and each test was repeated twice. Assay and evaluations of
results were performed as described previously.[57]
[4‐(Adamantane‐1‐carboxamido)‐8‐tert‐butyl‐3‐oxo‐1‐thia‐4‐
azaspiro[4.5]decan‐2‐yl]acetic acid (4f)
White powder; yield: 69%; mp: 180°C; IR: 3496, 3119 (O–H/N–H);
2907, 2848 (C–H); 1695, 1663, 1522 (N–H); 1443, 1367 (C–H);
1240 (N–H and C–N). 1H NMR (DMSO‐d6, 500 MHz) δ (ppm): 0.83
(9H, s, –C(CH3)3); 0.85–0.96 (1H, m, sty.C8‐ax‐H); 1.08–1.26 (2H, m,
C7–9‐ax‐H); 1.61–1.80 (12H, m, adm. C4ʹ‐H/C6ʹ‐H/C9ʹ‐H and
sty.C6–H/C10–H/C7–9‐eqv‐H); 1.85 (6H, d, 2,09 Hz, adm. C2ʹ–H/
C8ʹ–H/C10ʹ–H); 1.98 (3H, broad s, adm. C3ʹ–H/C5ʹ–H/C7ʹ–H); 2.46
(5H, m, DMSO‐d6 and –CHa–COOH); 3.01 (1H, dd, J = 16.9; 3.7 Hz,
–CHb–COOH); 3.99 (1H, dd, J = 10.5; 4.0 Hz, C2–H); 9.59 (1H, s,
CONH); 12.54 (1H, s, COOH). 13C NMR (DMSO‐d6, 500 MHz) δ
(ppm): 24.02/24.32 (cyclohex. C7/C9); 27.75 (C(CH3)3); 27.97/28.00
(adm. C3ʹ/C5ʹ/C7ʹ); 32.42 (C(CH3)3); 36.40/36.48/36.55 (adm. C4ʹ/C6ʹ/
C9ʹ); 38.62 (cyclohex. C6/C10); 38.75/38.81/38.85 (adm. C2ʹ/C8ʹ/C10ʹ);
39.24 (CH2–COOH); 40.27 (C1ʹ); 40.49 (DMSO‐d6 and C2); 46.11
(cyclohex. C8); 71.48 (cyclohex. C5); 168.62 (CONH); 172.21 (C3);
176.80 (COOH); MS APCI(−) m/z (%): 461 [M−H]−, MS APCI(+) m/z
(%): 135 (100); anal. calcd. for C25H38N2O4S·H2O: C, 62.47; H, 8.39;
N, 5.83. Found: C, 62.54; H, 7.99; N, 5.70.
|
4.2.2
Analysis of apoptosis by flow cytometry
[4‐(Adamantane‐1‐carboxamido)‐8‐phenyl‐3‐oxo‐1‐thia‐4‐azaspiro‐
[4.5]decan‐2‐yl]acetic acid (4g)
Apoptosis detection was evaluated using APC Annexin V Apoptosis
Detection Kit with PI (BioLegend Inc.) by flow cytometry (Acea Novocyte
1000) in A549 cells according to manufacturer's instructions. In brief,
A549 cells were seeded on a 24‐well plate at a density of 5 × 104 cells/
well for 24 h at 37°C for adherence and then treated with IC50 of 4a–c
compounds. Following a 24‐h treatment, the cells were trypsinized,
washed twice in cold phosphate‐buffered saline, and resuspended in
binding buffer. Then, the cells were incubated in the dark with 5 μl of
Annexin V‐FITC and 5 µl of PI for 15 min. After incubation, 400 µl of
annexin‐binding buffer was added, and the samples were analyzed by
flow cytometry. The experiment was repeated three times and the data
presented are the average values (%) compared with the control group.
White powder; yield: 61%; mp: 174–175°C; IR: 3489, 3111 (O–H/
N–H); 2908, 2849 (C–H); 1697, 1667, 1522 (N–H); 1442, 1367
(C–H); 1242 (N–H and C–N). 1H NMR (DMSO‐d6, 500 MHz) δ
(ppm): 1.54–1.74 (9H, m, adm. C4ʹ–H/C6ʹ–H/C9ʹ–H/C7‐eqv‐H and
C9‐eqv‐H); 1.77–1.94 (12H, m, adm. C2ʹ–H/C8ʹ–H/C10ʹ–H;
sty.C6–H/C10–H, C7‐9‐ax‐H); 2.01 (3H, broad s, adm. C3ʹ–H/
C5ʹ–H/C7ʹ–H); 2.41–2.55 (6H, m, DMSO‐d6 ile sty.C8‐ax‐H and
–CHa–COOH); 3.05 (1H, dd, J = 1.9; 3.7 Hz, –CHb–COOH); 4.06
(1H, dd, J = 10.5; 3.8 Hz, C2–H); 9.67 (1H, s, CONH); 12.63 (1H, s,
COOH). 13C NMR (DMSO‐d6, 500 MHz) δ (ppm): 27.99/28.01
(adm. C3ʹ/C5ʹ/C7ʹ); 30.74/31.19 (cyclohex. C7/C9); 36.43/36.50/
36.57 (adm. C4ʹ/C6ʹ/C9ʹ); 38.63 (cyclohex. C6/C10); 38.79/38.84/
38.90 (adm. C2ʹ/C8ʹ/C10ʹ); 39.30 (CH2–COOH); 40.31 (C1ʹ); 40.48
(DMSO‐d6 and C2); 41.97 (cyclohex. C8); 71.02 (cyclohex. C5);
126.63 C4(Ph); 127.98, C2(Ph)/C6(Ph); 128.88/128.74 C3(Ph)/
C5(Ph); 146.37 C1(Ph); 168.66 (CONH); 172.23 (C3); 176.92
(COOH); anal. calcd. for C27H34N2O4S·H2O: C, 64.77; H, 7.25; N,
5.60. Found: C, 64.88; H, 6.90; N, 5.45.
|
4.2.3
Quantitative real‐time polymerase chain
reaction analysis of apoptosis genes
A549 cells were treated with the IC50 concentrations of the se-
lected 4a–c compounds. After incubation for 24 h, total RNA was

TURK‐ERBUL ET AL.
11 of 13
|
TABLE 3 Primers used in real‐time
polymerase chain reaction analysis of
selected apoptosis genes and the
Gene
Primer sequences (5ʹ–3ʹ)
Ta (°C)
References
p53
F: AGAGTCTATAGGCCCACCCC
R: GCTCGACGCTAGGATCTGAC
61
[6]
corresponding annealing temperatures
CASP3
CASP9
BCL2
BAX
F: GCTATTGTAGGCGGTTGT
R: TGTTTCCCTGAGGTTTGC
53
57
66
61
53
59
61
55
48
[6]
F: ACCAGAGATTCGCAAACCAG
R: TCACCAAATCCTCCAGAACC
[58]
[6]
F: TGTGGCCCAGATAGGCACCCAG
R: ACTTCGCCGAGATGTCCAGCCAG
F: ACCAAGAAGCTGAGCGAGTATC
R: ACAAAGATGGTCACGGTCTGCC
[6]
CYCS
AIF
F: CTTACACAGCCGCCAATA
R: CTTCTTCTTAATGCCGACAA
[59]
[58]
[60]
[61]
[62]
F: TGCTGGTGGACATGAAGGAC
R: TTTGGCGAACCCTGTCTCC
APAF1
XIAP
F: CCTCCAAAAACCCAGCCAAC
R: TCCAGGACCCTGGGGATTTC
F: ACTCTACTACACAGGTATTGG
R: TCAGAACTCACAGCATCAG
β‐Actin
F: AACTACCTTCAACTCCAT
R: TGATCTTGATCTTCATTGTG
|
extracted from control and 4a‐, 4b‐, and 4c‐treated groups in
A549 cell lines using a High Pure RNA Isolation Kit (Roche Life
Sciences) according to the instructions provided by the manu-
facturer. Complementary DNA (cDNA) was synthesized using the
mixture of anchored oligo(dT) and random hexamer primers by
Transcriptor First Strand cDNA Synthesis Kit (Roche Life
Sciences). Gene expression levels of apoptosis genes including
p53, CASP3, CASP9, BCL2, BAX, CYCS, AIF, APAF1, and XIAP were
measured using BioLine SensiFast™ Syber® No‐Rox Kit on
LightCycler® 480 Instrument II (Roche Life Science). The primer
sequences and their annealing temperatures of the genes are
provided in Table 3 (Sentromer DNA Technologies). The gene
expression analysis and evaluations of results for all genes were
performed as described previously.[57]
4.3
Molecular calculations
Modeling
|
4.3.1
BIR3 domain of human XIAP was obtained from Smac bound X‐ray
structure with 2.0 Å resolution (PDB ID: 1G73).[63] Smac and water
molecules were removed, whereas Zn2+ ion was retained. This XIAP
structure was used as an initial structure for the relaxation of the
protein through molecular dynamics (MD) simulations.
Compounds 4a–c were sketched using Marvin Sketch 15.12.14
software.[64] Geometry optimization of the compounds was per-
formed using YASARA Structure software[65] with NOVA force
field.[66] These structures were used for molecular docking.
|
4.3.2
Molecular docking
|
4.2.4
Statistical analysis
The equilibrated XIAP protein structure was obtained from 20 ‐ns‐long
MD simulation, whose details are discussed below. Then, rigid receptor
and flexible ligand docking were performed on YASARA Structure
software, which utilized AutoDock Vina software.[67] Docking space
(grid) was limited to a searching space, which was created with 8.0 Å
distance around all atoms of the protein, yielding a cubic box with
dimensions 54 × 54 × 54 Å. Binding affinities reported in the paper were
obtained from molecular docking calculations.
Results of cytotoxicity, apoptosis assay, and gene expression
analysis levels were represented as mean SD. Statistical
analysis was performed by analysis of variance, followed
by Dunnett's multiple comparison test using “SPSS version
21.0 for Windows,” statistical program (IBM Analytics).
p < .05 and p < .001 were selected as the levels of
significance.

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4.3.3
Molecular dynamics simulations
[17] A. A. Kadi, N. R. El‐Brollosy, O. A. Al‐Deeb, E. E. Habib,
T. M. Ibrahim, A. A. El‐Emam, Eur. J. Med. Chem. 2007, 42, 235.
https://doi.org/10.1016/j.ejmech.2006.10.003
MD simulations of ligands bound to XIAP protein were performed
using YASARA2 force field, as implemented in the YASARA Struc-
ture software. The box was filled with single point charge water
molecules.[68] The protein–ligand complex was placed into a box
with dimensions of 54 × 54 × 54 Å. The boundary of the simulated
box was conditioned in the periodic form. The water density was set
at 0.997 g/ml at a temperature of 303 K. The simulation was run
for 20 ns.
[18] V. S. Georgiev. US Patent 4,549,014, 1985.
[19] I. Papanastasiou, A. Tsotinis, N. Kolocouris, S. R. Prathalingam,
J. M. Kelly, J. Med. Chem. 2008, 51, 1496. https://doi.org/10.1021/
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ACKNOWLEDGMENT
The present work was supported by the Istanbul University Scientific
Research Projects (Project No: TYL‐2017‐25681).
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
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http://orcid.org/0000-0003-2412-2232
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