A new heterocyclic compound initiates ROS accumulation and cause apoptotic cell death in human bone cancer cells

Article abstract of DOI:10.1016/j.molstruc.2019.03.035

A new heterocyclic compound 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one (1), designed using methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (2) as the starting material, was successfully obtained via multiple synthesis route and finally characterized by fourier transform infrared (FT-IR) spectroscopy, ¹H nuclear magnetic resonance (NMR), and single crystal X-ray crystallography. We investigated its effect on cell viability and proliferation with Cell Counting Kit-8 (CCK8) assay. The results revealed that compound 1 could block the proliferation of Saos-2 bone cancer cells. In addition, Annexin V-FITC/PI assay and Western blot analysis were performed to detect whether compound 1 could induce cell apoptosis. The results indicated that compound 1 could increase the number of apoptotic cells remarkably. Moreover, we examined the impact of compound 1 on ROS generation. Results revealed that compound 1 up-regulated the reactive oxygen species (ROS) genes expression and promoted the accumulation of ROS in Saos-2 cells. Finally, molecular docking has been utilized to study the binding mode of compound 1 with tubulin.

Full text of DOI:10.1016/j.molstruc.2019.03.035

Accepted Manuscript
A new heterocyclic compound initiates ROS accumulation and cause apoptotic
cell death in human bone cancer cells
Guang Lv, Chong-Lan Yin
PII:
S0022-2860(19)30305-9
10.1016/j.molstruc.2019.03.035
MOLSTR 26304
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
19 December 2018
12 March 2019
Please cite this article as: Guang Lv, Chong-Lan Yin, A new heterocyclic compound initiates ROS
accumulation and cause apoptotic cell death in human bone cancer cells, Journal of Molecular
Structure (2019), doi: 10.1016/j.molstruc.2019.03.035
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A new heterocyclic compound initiates ROS accumulation and cause
apoptotic cell death in human bone cancer cells
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Guang Lv , Chong-Lan Yin
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Inner Mongolia University For The Nationlities, Tongliao, Inner Mongolia Autonomous Region, China
Pediatric Internal Medicine, The People’s Hospital of Tongliang District, Chongqing, China
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E-mail: chonglan_yin66@126.com
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Abstract
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A new heterocyclic compound 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one (1), designed
using methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (2) as the starting material, was successfully obtained via
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multiple synthesis route and finally characterized by fourier transform infrared (FT-IR) spectroscopy, H nuclear
magnetic resonance (NMR), and single crystal X-ray crystallography. We investigated its effect on cell viability
and proliferation with Cell Counting Kit-8 (CCK8) assay. The results revealed that compound 1 could block the
proliferation of Saos-2 bone cancer cells. In addition, Annexin V-FITC/PI assay and Western blot analysis were
performed to detect whether compound 1 could induce cell apoptosis. The results indicated that compound 1
could increase the number of apoptotic cells remarkably. Moreover, we examined the impact of compound 1 on
ROS generation. Results revealed that compound 1 up-regulated the reactive oxygen species (ROS) genes
expression and promoted the accumulation of ROS in Saos-2 cells. Finally, molecular docking has been utilized to
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study the binding mode of compound 1 with tubulin.
Keywords: heterocyclic, anti-proliferation, apoptosis, Western blot, ROS, RT-PCR
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1. Introduction
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Although a large amount of chemotherapeutic anticancer agents have been discovered and successfully
applied in the clinical practice, cancer still results in millions of people death and is the most cause of human
deaths in worldwide, especially in the developing and undeveloped countries [1]. The apparent clinical problems
of recent chemotherapeutic drugs such as general toxicity, serious side effects and strong drug resistance have led
to the development of novel anticancer agents based on the nature and artificially synthesized organic compounds
[2]. Recent literatures have revealed that quinazoline derivatives possess high anti-cancer activities, and many of
them are highly selective tyrosine kinase inhibitors (TKI), particularly against epidermal growth factor receptor
(EGFR) which are overexpressed or mutated in most carcinomas and are required for tumor progression [3]. For
instance, Gefitinib is an anilinoquinazoline which is useful in the treatment of certain type of lung cancer that does
not respond to chemotherapy [4-6]. It is the first selective inhibitor of epidermal growth factor receptor’s tyrosine
kinase domain [7, 8]. While 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one (1) which is one of the
most important intermediates in the synthesis of Empagliflozin fascinated us [9]. Thus, much attention has been
devoted to the synthesis, characterization and crystal structure of compound 1 which has never been reported
before [10-12].
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The present work deals with the synthesis and characterization of the title compound 1. The compound 1 was
synthesized by methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (2) as start material obtaining methyl
4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzoate (3), and reduced with Pb/H2 to obtain methyl
2-amino-4-methoxy-5-(3-morpholinopropoxy)benzoate (4), the title compound 1 was prepared after the
cyclization of compound 4 with formamidine acetate (Scheme 1). In our biological study, we evaluated the effect
of compound 1, which inhibited the cell viability and proliferation of Saos-2 cells, but with no cytotoxicity on
human normal cells. Further, our data showed that compound 1 up-regulated the activation of capase-3 and
caspase-9, then inducing Saos-2 cells apoptosis. Moreover, it has been suggested that compound 1 specifically
up-regulated the ROS-related genes expression and initiated the ROS generation in Saos-2 cells. Overall, these
findings highlight a new compound with excellent anti-cancer activity in human cancer treatment.
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O
O
O
N
N
N
HCl
HO
O
COOCH3
NO2
O
O
COOCH3
NO2
O
O
COOCH3
NH2
Cl
Pd/H2
2
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4
HN
NH2
HOAc
O
N
O
O
O
NH
N
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Scheme 1 The synthesis route of compound 1.
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2. Experimental
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2.1 Apparatus and materials
All the reagents were purchased from Beijing Bailingwei Chemical Reagent company (Beijing, China) and
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used as received. IR spectra (400-4000 cm ) were obtained using a Brucker Equinox-55 spectrophotometer. H
NMR spectra were obtained using a Varian Inova-400 spectrometer (at 400 MHz). Mass spectra were obtained
using a micrOTOF-Q II mass spectrometer. The melting points were taken on a XT-4 micro melting apparatus,
and the thermometer was uncorrected.
The human bone cancer Saos-2 cell line (ATCC, CCL-2) and normal human embryonic kidney cell line
HEK-293 (ATCC, CRL-1573) was purchase from American Type Culture Collection (ATCC, Rockville, MD).
The Saos-2 cell line was cultured in ATCC-formulated Eagle's Minimum Essential Medium (Gibco, NY, USA);
the HEK-293 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, USA). Both
culture mediums were supplemented with 100 U/mL Penicillin and Streptomycin solutions (Gibco, Life
Technologies), 10% (v/v) heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 2%
L-glutamine. All the cells were cultured at 37℃ with 5% CO . The culture medium was replaced according to the
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cell growth status.
2.2 Synthesis and characterization of compounds 3, 4 and 1.
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To a slurry of compound 2 (10.0 g, 0.044 mol) and anhydrous potassium carbonate (18.2 g, 0.1317 mol) in
acetonitrile (100 ml), 4-(3-chloropropyl)morpholine hydrochloride (10 g, 0.05 mol) was added and refluxed for
2.5 h. The solvent was distilled under reduced pressure to give residue which was diluted with water (60 ml) and
extracted with ethyl acetate (100 ml) after completion of reaction. The organic layer was distilled under vaccum
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and dried to obtain 13.65 g (87.50%) of the compound 3; m.p. 231-233 C. H NMR (DMSO-d , 400 MHz): δ
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7.62 (s, 1H), 7.31 (s, 1H), 7.07 (s, 1H), 3.90 (s, 3H), 3.79 (s, 3H), 4.18 (t, 2H), 4.17 (s, 2H), 4.16 (s, 3H), 2.37 (d,
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1H), 1.91 (d, 2H). IR (νmax, cm ): 3134, 1695, 1601.
The compound 3 (10 g, 0.0282 mol) was hydrogenated using 10% palladium on carbon (1.0 g) in methanol
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(100 ml) at 3-4 kg/cm at ambient temperature for 5 h. After completion of the reaction, the catalyst was removed
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by filtration. The filtrate was distilled off completely to obtain 8.27 g (90.38%) of compound 4.
Compound 4 (8.0 g, 0.0247 mol) was added to formamidine acetate (2.68 g, 0.0257 mol) in methanol (64 ml)
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and heated at 50-60 C for 6 h. The reaction mass was cooled to room temperature and stirred for 3 h after
completion of reaction. The resulting solid was filtered, washed with methanol (10 ml) and dried to obtain 6.44 g
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(81.73%) of the title compound 1; m.p. 267-269 C H NMR (DMSO-d , 400 MHz): δ 12.06 (s, 1H), 7.96 (s, 1H),
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7.45 (s, 1H), 7.12 (s, 1H), 4.11 (t, 2H), 3.91 (s, 3H), 3.58 (t, 4H), 3.58 (t, 4H), 1.92 (t, 1H). HRMS (ESI ): m/z:
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calcd for C H N O : 343.1508 [M+Na ]; found: 343.1532. IR (ν , cm ): 2909, 1639, 1607.
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2.3 Crystal structure determination
Suitable single crystal of compound 1 (obtained by slow volatilization of its CH Cl solution) was carefully
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selected under optical microscope and glued on thin glass fibers. The single crystal X-ray diffraction study was
performed on the Oxford Xcalibur E diffractometer. The data collection was carried out using the CrysAlisPro
software [13]. Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK
scaling algorithm. The SHELXS program based on the direct method was used to acquire the structural model and
then modified via the SHELXL program [14]. All non-hydrogen atoms of 1 were refined anistropically, and all
the hydrogen atoms attached to carbon atoms were fixed at their ideal positions. Pertinent crystal data and
structural refinement results for compound 1 were summarized in Table 1.
Table 1 Crystal data and structure refinements for compound 1.
Formula
C H N O
16 22 3 4
Mr
320.36
Crystal system
Space group
a / Å
Monoclinic
P2 /c
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19.7641 (15)
8.7452 (6)
9.2099 (8)
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b / Å
c / Å
α / °
β / °
93.800 (7)
90
γ / °
V / Å3
1588.3 (2)
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Z
Crystal size / mm3
Dcalc / g·cm-3
μ(Mo Kα) / mm-1
θ range / °
Reflections collected
0.48 × 0.43 × 0.18
1.340
0.097
2.065 to 25.345
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No. unique data [R(int)] 2901 [0.0300]
No. data with I ≥ 2σ(I)
1997
R1
0.0509
0.1529
1867419
ωR (all data)
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2.4 Cell proliferation assay
The Cell Counting Kit-8 assay (Dojindo, Japan) was carried out to test the viability and proliferation of the
human bone cancer Saos-2 cell line and human embryonic kidney HEK-293 cells treated with compound 1 at
serious of concentrations for 24 h. This assay was conducted as manufacturer’s protocol [15]. For preparation, the
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Saos-2 and HEK-293 cells were planted in 96-well plates at a density of 1×10 cells per well and incubated at 37℃
with 5% humidified CO . When the cells grow into the logarithmic growth phase with confluence of 70%-80%,
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serious concentrations of compound 1 (1, 2, 4, 8, 10, 20, 40, 80, 100 μM) was added into wells and incubated for
24 h at 37℃, 5% CO . After that, the culture medium was removed and the cells were washed with pre-clod PBS
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3 times. 10 μL per well of CCK-8 (Sigma) in 100 μL medium without FBS was added to the cells and the 96-well
plates were incubated for another 2 hours in the dark. The cell viability was then assessed by measuring the
absorbance of each well at 450 nm using a multifunctional microplate reader (BioTek, Winooski, VT, USA). The
cell viability curves were calculated and plotted according to the absorbance values. All experiments were
repeated three times independently. The IC values were calculated using SPSS version 22.0.
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2.5 Western blot detection
Western blot analysis of all samples was performed as described previously in detail [16]. In brief, the Saos-2
cancer cells were divided into different groups and exposed to different concentrations of compound 1 for 24 h
and collected. Then, the protein from cells was extracted with RIPA buffer (1% NP-40, 1% sodium dodecyl
sulfate (SDS), 150 mM NaCl, 25 mM Tris-HCl (pH 7.6), 1% deoxycholic acid sodium salt, 1% PMSF) for 30 min
on the ice, and then quantified with BCA Protein Assay Kit (Beyotime Biotechnology) according to the protocol.
The total proteins in Saos-2 cells were separated by sodium dodecylsulfate–polyacrylamide SDS-PAGE gel
electrophoresis and electrophoretically transferred to a 0.22 mm polyvinylidene fluoride (PVDF) membrane
(Millipore, Bedford, MA, USA). The membranes were incubated with 5% nonfat milk solution for blocking
non-specific binding for 2 h at room temperature and incubated with primary antibody at 4℃ overnight. After
washing twice with PBST, the membranes were incubated with appropriate secondary antibody conjugated with
horseradish peroxidase for 2 h at room temperature. Images were captured using the ChemiDoc™ Touch Imaging
System (Bio-Rad, Hercules, California, USA). The Quantity One software (Bio-Rad, Hercules, California, USA)
was used to quantify the density of bands.
2.6 Cell apoptosis analysis
For Saos-2 cell apoptosis analysis, the Annexin V-FITC/PI apoptosis assay (BD Biosciences, San Jose, CA,
USA) was carried out following the manufacturer’s instructions [17]. Before experiment, the Saos-2 cells were
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seeded in 6-well plates (2×10 cells/well) at 37℃ in 5% CO humidified atmosphere overnight. The cells were
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divided into four groups and treated with or without compound 1 (1×IC50 and 3×IC ) for 24 h. The dimethyl
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sulfoxide (0.1% DMSO) and poptosis inducer kit (Beyotime, Shanghai, China) were used as the negative and
positive control, respectively. Then, the cells were harvested with 0.25% w/v trypsin followed by washing with 4℃
phosphate buffered saline three times. 500 μL Annexin V binding buffer was added to re-suspend the Saos-2 cells
followed by 5ꢀμL Annexin V-FITC and 5ꢀμL propidium iodide (PI) addition for 15 min incubation at 37℃ in the
dark. The stained cells in each group were detected with (FACScan, BD, NJ, USA) at an excitation wavelength of
488ꢀnm and emission wavelengths of 525 and 625ꢀnm. The results were analyzed using flow cytometry
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(FACSCalibur, BD Biosciences, USA). Each experiment was performed in triplicate independently.
2.7 Intracellular ROS detection
The intracellular ROS accumulation in Saos-2 cells after treated with or without compound 1 were measured
by 2’,7’-dichloro fluorescin diacetate (DCFH-DA) (Sigma) staining method according to the manufacturer’s
instructions [18]. The 2’,7’-dichloro fluorescin diacetate (DCFH-DA) could be converted into 2’,7’-dichloro
fluorescin (DCF), which is usually used as an indicator of intracellular ROS generation. The Saos-2 cells were
divided into four groups and treated with or without compound 1 (1×IC50 and 3×IC ) for 24 h. The dimethyl
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sulfoxide (0.1% DMSO) and poptosis inducer kit (Beyotime, Shanghai, China) were used as the negative and
positive control, respectively. The florescent values in each group were detected with flow cytometer (BD Via,
New Jersey, USA) at an excitation wavelength of 488ꢀ/ 530 nm and analyzed using FlowJo7.6 software. All
experiment was performed at least 3 times.
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2.8 qRT-PCR assay
The mRNA expressions of ROS related genes in Saos-2 cells after treated with compound 1 was measured
using quantitative reverse transcription polymerase chain reaction (qRT-PCR) according to the protocol [19].
Total RNA in Saos-2 cancer cells were extracted using TRIzol™ Plus RNA Purification Kit (Invitrogen, Carlsbad,
CA, USA) according to the instruction. The measurement of total RNA quality was conducted on a Nano-100
Micro Spectrophotometer. The cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, Cambridge, MA, USA). The PCRs were conducted using the qRT-PCR miRNA Detection
Kit (Invitrogen): 95℃ for 15 min, followed by 40 cycles of denaturation at 95℃ for 5 s, annealing at 55℃ for
30 s and extension at 72℃ for 30s. The relative expression level of genes was determined according to the
2-ΔΔCt method as previously described. Each experiment was performed in triplicate.
2.9 Statistical analysis
Statistical analysis was performed using the SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). All data
were expressed as means ± standard deviation (SD) from three independent experiments. Comparisons between
two groups were made with Student’s t-test, and differences among more than three groups were tested by
one-way ANOVA with Prism Software (version 6.07, GraphPad Software Inc, San Diego, CA). The differences
were considered significant at *p < 0.05, **p < 0.01 and ***p < 0.005.
3.0 Simulation Details
The structure of compound 1 was optimized by density functional theory (DFT) using a B3LYP/6-31G(d)
basis set. The structure optimization and energy calculations were performed with the GAUSSIAN 09
program.
Autodock Vina v1.2 has been utilized to study the binding mode of compound 1 with tubulin. The tubulin
was used as downloaded without further modification from the protein data bank (PDB ID: 1AS0). The geometry
structures of compounds A and B were optimized through quantum chemistry calculations by Gaussian 09,
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B3LYP theory of level along with 6-31g basic set were used. The AutoDockTools v1.5.6 has been adopted to
transfer 1AS0 and optimized structures of A and B to autodock vina input files. Only polar hydrogens on these
structures are considered. The center coordinates of search grid of tubulin were set to 38.15, -26.99 and 6.18,
respectively, the length of the search grid was 15. All parameters needed by autodock vina are used as default if
not mentioned specifically. The calculated results were analyzed and visualized by PyMoL v1.8.6.
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3. Results and Discussion
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3.1 Molecular structure
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The structure of the compound C H N O (1) was determined by X-ray diffraction crystal structural
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analysis. The crystal parameters of 1 are listed following: a = 19.7641(15) Å, b = 8.7452(6) Å, c = 9.2099(8) Å, β
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= 93.800(7)°. V = 1588.4(2) Å , Z = 4, Dc = 1.340 g/cm . The result reveals that the compound 1 belongs to the
monoclinic crystal system with the space groups of P2 /c. Its molecular structural unit is shown in Fig. 1. In the
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structural unit, the -C H NO and -C H N O groups are linked to the -C H O group. In the structural unit, one
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-OCH group is connected to the phenyl group [C(3)-C(8)]. In this crystal structure, the bond distances of C-C,
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C-N and C-O are in the scopes of 1.370(3)-1.517(4) Å, and 1.291(3)-1.458(3) Å, 1.228(2)-1.435(2) Å,
respectively. The related bond distances are in their normal scopes.
Fig. 2 represents the crystal packing structure of the titled compound along the b axis. In its crystal packing
structure, the two kinds of hydrogen bonding C-H…O and N-H…N could be found among their molecular units.
The detailed information of hydrogen bonding is given in Table 2. By means of the two types of hydrogen
bonding interactions, a two-dimensional layered structure is resulted.
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Fig. 1 Molecular structure unit for compound 1.
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Fig. 2 Crystal packing structure of compound 1 along the b axis.
Table 2 The hydrogen bonds for compound 1.
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D–H…A
D–H
H…A
1.9800
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D…A
D–H…A
174.00
Symmetry code
1-x, 1/2+y, 1/2-z
1-x, -1/2+y, 1/2-z
N1–H1…N2
C2–H2…O1
0.8600
0.9300
2.840 (2)
3.260 (3)
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3.2 Compound 1 inhibited viability of Saos-2 cancer cells
Effect of compound 1 on the viability and proliferation of Saos-2 cells was investigated with CCK8 cell
proliferation assay with the oxaliplatin as the positive control. To find the adequate dose of compound 1, we tested
the effect of compound 1 on the viability of Saos-2 cells at various doses ranging from 1 to 80 μM. As results
shown in Fig. 3A, the viability of Saos-2 cells treated with compound 1 was significantly reduced compared to the
oxaliplatin, and this inhibitory effect shown a dose-dependent relationship. Unexpectedly, compound 1 has no
inhibitory effect on normal human cells, HEK-293 (Fig. 3B). The IC values were calculated with SPSS 22.0 and
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listed in Table 3. Compound 1 has the IC50 value of 1.8±0.1 µM exhibiting extraordinary anti-proliferation
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activity on human bone cancer cells. We show two doses of 1×IC and 3×IC for the subsequent experiments.
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Fig. 3 Compound 1 inhibits the viability and proliferation of Saos-2 cells in a dose-dependent manner. The
Saos-2 cell viability curves plotted by CCK8 assay after treated with serious doses of compound 1 (1, 2, 4, 8, 10,
20, 40, 80 μM) for 24 h (A); The human normal cell line HEK-293 was treated with compound 1 at the same
concentrations, and the cell viability was assayed by CCK-8 assay (B). Data are represented as mean ± SD of
three independent experiments performed in triplicate.
Table 3 IC value of compound 1 (µM)*
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Cell/ drug Compound 1 Oxaliplatin
Saos-2
1.8±0.1
4.6±0.3
HEK-293
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*The IC50 values of compound 1 on Saos-2 and HEK-293 cells. The Oxaliplatin was used in as the positive
control. The IC50 values were calculated according the CCK-8 results presented in Fig. 3. Mean ± SD of three
independent experiments performed in triplicate are shown.
3.3 Compounds induce cell apoptotic cell death
Most anti-cancer drugs inhibit the viability and proliferation of cancer cells via inducing the production of
apoptosis. To investigate whether apoptosis also accounted for the cytotoxicity of compound 1 on Saos-2 cell
death, the Annexin V-FITC/ PI double staining assay and western blot were both carried out to detect the
apoptosis rate of Saos-2 cancer cells after treated with compound 1 at indicated concentration for 24 h. As the
results shown in Fig. 4A, after incubated with compound 1, the treatment groups exhibited a higher apoptosis rate
than the negative control group, and showed a similar effect with positive group. The Saos-2 cancer cells showed
a dose-dependent sensitivity to compound 1, which induce the apoptosis to 84.59±2.9% and 96.39±3.1% at the
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concentration of 1×IC and 3×IC , respectively. Complimentary assessment of apoptosis using the western blot
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assay under identical treatment conditions also showed that the key markers of cell apoptosis cleaved caspase-3
and cleaved caspase-9 was elevated remarkable after treated with compound 1 and showed a dose relationship
(Fig. 4B). The above results indicate that compound 1 could increase the apoptosis of Saos-2 cancer cells.
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(A)
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Fig. 4 Compound 1 promotes the apoptosis of Saos-2 cacner cells. Upon reaching 70% -80% confluence, the
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Saos-2 cells were treated with indicated doses of compound 1 (1×IC50 and 3×IC ) for 24 h. The cell apoptosis
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was analyzed with Annexin V‑FITC Apoptosis Detection kit combined with flow cytometry (A); Western blot
analysis was performed to inspect the expression levels of cleaved caspase-3 and cleaved caspase-9 in Saos-2
cells. GAPDH was used to confirm equal amount of proteins loaded in each lane (B).
3.4 Compound 1 induces ROS generation in Saos-2 cells
The previous studies prompt us that ROS plays an essential role in cell apoptosis induced by various
anti-cancer agents. Therefore, we speculated compound 1 may lead to increased ROS and induce the Saos-2
cancer cell apoptosis in the same way. Then, we further explored whether compound 1 could influence the
accumulation of ROS in Saos-2 cells with ROS assay and RT-PCR. As result shown in Fig. 5A, the intracellular
ROS level was significantly increased in compound 1 treatment group and showed a dose-dependent relationship.
The ROS-related genes expression measured by RT-PCR also confirmed this, which was up-regulated by
compound 1 obviously (Fig. 5B).
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(A)
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(B)
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Fig. 5 Compound 1 initiated ROS generation in Saos-2 cells. The Saos-2 cancer cells were incubated with
compound 1 at indicated concentrations for 24 h and cellular ROS was measured by flow cytometry. Each group
was repeated three times (A); The expression of ROS related genes in Saos-2 cells after treated with compound 1
was measured by RT-PCR (B).
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3.5 Molecular Docking
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The molecular autodocking method is widely used for investigating the possible/potential interactions
between the drug molecules and the protein, generally the protein molecule 1AS0 will be chosen as the
benchmark. Here the synthesized antivirus drug molecule 1 which has shown potential for disease therapy
observed from experiment is simulated by molecular autodocking with the aim of providing further evidences.
The structure obtained through the X-ray (Fig. 6) is firstly optimized by the density functional theory (DFT),
because the molecule is in its active state when it is traveling within the organics, while the structure given by the
experiment is in the crystal state which has lots of constrains by the surrounding molecules. Table 4 listed the
summaried structural parameters, the biggest deviation comes from the dihedrals which is about 6.3 degree for
C9-C10-C11-N3, while bond lengths and angles are predicted quite precisely. After obtaining the active molecule,
the autodocking simulation has been performed, the hetero-atoms on the drug molecule could form polar
interactions with the polar hydrogen on the tested protein 1AS0. We found that the oxygen atom on the
morpholine ring has the polar interaction with residue ARG-242, surprisingly, rest hetero-atoms like the oxygen
atom on the methoxy group, the oxygen atom from the ether bond and from the carbonyl group, and all the
nitrogen atoms don’t contribute into any polar interactions with surrounding residues, such as LEU-232,
ASN-149, ARG-144, ASP-150, ALA-152, LYS-288, etc. The most binding mode is shown in Fig. 6, from which
we can see that the corresponding binding energy and the length of the polar interaction are -7.1 kcal/mol and 2.3
angstrom, respectively. Such a short length indicates that the polar interaction is really strong, which means that
the proposed drug molecule could interact with the organics actively, and has good agreement with experiment.
Table 4 Comparison between experimental and simulated structure parameters of the selected bond lengths, bond
angles and dihedrals of compound 1.
Parameter
X-ray
Calculation
Bond length (Å)
1
1
.227
.385
C1-O1
C1-N1
C2-N2
C7-O4
C6-O2
C11-N3
1.225
1.405
1.290
1.356
1.357
1.464
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1
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.360
.367
.458
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C13-O3
1.420
Angle/Dihedrals (°)
1
1
21.3
17.2
O1-C1-N1
120.7
118.0
118.1
112.4
179.0
178.7
58.3
C7-O4-C16
1
1
1
1
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16.9
11.4
71.7
77.8
4.6
C6-O2-C9
C9-C10-C11
C6-O2-C9-C10
O2-C9-C10-C11
C9-C10-C11-N3
N3-C12-C13-O3
-57.7
-57.4
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Fig. 6 The binding mode between the proposed molecule (stick) and surrounding residues from protein 1AS0
which has the lowest binding energy -7.1 kcal/mol, the corresponding length of the polar interaction is 2.3
angstrom.
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4. Conclusion
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In the present study, we synthesized compound 1 with a completely novel structure for the first time, and we
also demonstrated for the first time that compound 1 has excellent anti-cancer activity for human bone cancer
treatment. The previous researches have proved that compound 1 could reduce the viability and proliferation of
Saos-2 cells, compared with no cytotoxicity on normal human cells. In this study, the Annexin V-FITC/PI
apoptosis assay and ROS measurement was further conducted to explore the manner of Saos-2 cell death. The
results indicated that compound 1 induce the Saos-2 cancer cell death via a ROS mediated apoptosis pathway. In
conclusion, based on the above results, compound 1 has a potential to be the anti-cancer agent for human bone
cancer treatment. Finally, from molecular docking we can see that compound 1 have good potential in the
antivirus area, which is in good agreement with experimental observations.
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. A new heterocyclic compound was successfully obtained via multiple synthesis route.
. We investigated its effect on cell viability and proliferation with Cell Counting Kit-8 (CCK8) assay.
. Annexin V-FITC/PI assay and Western blot analysis were performed to detect whether compound 1 could
induce cell apoptosis.
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. We examined the impact of compound 1 on ROS generation.
. Molecular docking has been utilized to study the binding mode of compound 1 with tubulin.