Design and synthesis of novel ribofuranose nucleoside analogues as antiproliferative agents: A molecular docking and DFT study

Article abstract of DOI:10.1016/j.molliq.2018.08.009

The new analogues of ribofuranose fused heterocyclic compounds were synthesized a series of diazotization, cyclization, coupling and hydrolysis reactions and structures were characterized by spectroscopic methods. To explain the spectroscopic properties in detail, such as molecular geometric parameters, vibrational wavenumbers, HOMO-LUMO energies, ¹H NMR chemical shift values and electronic transitions, density functional theory (DFT) calculations were used. The structural and spectroscopic data of the molecules in the ground state were calculated by DFT using B3LYP functional with 6-311G(d,p) basis set. Isotropic chemical shifts were calculated using the gauge-invariant atomic orbital (GIAO) method. Furthermore molecular docking (ligand–protein) simulations were performed to obtain antiproliferative activity of the synthesized ribofuranose nucleoside analogues against epidermal growth factor receptor and vascular endothelial growth factor receptor 2. Full fitness score and binding energy length values revealed that studied three compounds can act as potential inhibitor against selected receptors.

Full text of DOI:10.1016/j.molliq.2018.08.009

Accepted Manuscript
Design and synthesis of novel ribofuranose nucleoside analogues
as antiproliferative agents: A molecular docking and DFT study
Çiğdem Karabacak Atay, Tahir Tilki, Bülent Dede
PII:
DOI:
Reference:
S0167-7322(18)32548-0
doi:10.1016/j.molliq.2018.08.009
MOLLIQ 9454
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
17 May 2018
31 July 2018
1 August 2018
Please cite this article as: Çiğdem Karabacak Atay, Tahir Tilki, Bülent Dede , Design
and synthesis of novel ribofuranose nucleoside analogues as antiproliferative agents: A
molecular docking and DFT study. Molliq (2018), doi:10.1016/j.molliq.2018.08.009
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DESIGN AND SYNTHESIS OF NOVEL RIBOFURANOSE
NUCLEOSIDE ANALOGUES AS ANTIPROLIFERATIVE AGENTS: A
MOLECULAR DOCKING AND DFT STUDY
Çiğdem KARABACAK ATAY1, Tahir TİLKİ2, Bülent DEDE2*
1Mehmet Akif Ersoy University, Faculty of Education, Department, of Basic Education, 15030,
Burdur, Turkey
2Süleyman Demirel University, Faculty of Science & Art, Department of Chemistry, 32260, Isparta,
Turkey
*bulentdede@sdu.edu.tr
Corresponding author
Telephone
Fax
: +90 246 2114153
: +90 246 2114399

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Abstract
The new analogues of ribofuranose fused heterocyclic compounds were synthesized a series
of diazotization, cyclization, coupling and hydrolysis reactions and structures were
characterized by spectroscopic methods. To explain the spectroscopic properties in detail,
such as molecular geometric parameters, vibrational wavenumbers, HOMO-LUMO energies,
1H-NMR chemical shift values and electronic transitions, density functional theory (DFT)
calculations were used. The structural and spectroscopic data of the molecules in the ground
state were calculated by DFT using B3LYP functional with 6-311G(d,p) basis set. Isotropic
chemical shifts were calculated using the gauge-invariant atomic orbital (GIAO) method.
Furthermore molecular docking (ligand–protein) simulations were performed to obtain
antiproliferative activity of the synthesized ribofuranose nucleoside analogues against
epidermal growth factor receptor and vascular endothelial growth factor receptor 2. Full
fitness score and binding energy length values revealed that studied three compounds can act
as potential inhibitor against selected receptors.
Keywords: Nucleoside, DFT, molecular docking, antiproliferative, anticancer

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1. Introduction
Cancer is the most important health problem in the 21st century. The incidence of cancer in
many countries has even caught heart disease. Cancer is the name of the illness that is caused
by the deterioration of some organs or tissue cells in one day by going out of control and
tending to irregularity in a harmonious state of our body which is governed by strict control
mechanisms. Today, cancer has become a common disease that can be caught for the life of
one of every three people today. The number of cancer-affected patients is increasing all over
the world. Therefore, the studies on the synthesis of new and effective anticancer agents are
very important and increasing. Modified nucleosides have attracted interest in recent years
because the showing interesting biological properties such as antiviral and anticancer agents.
Nucleoside analogues are among the first chemotherapeutic agents used in cancer treatment.
Finding new drugs is a very expensive and time-consuming process. Although there are too
many candidate molecules with potency to be drug, the experiments that have to be performed
for each cause the drug cost to increase and the prolongation of the duration of the drug to be
taken [1-4].
At this point, the molecular docking method is at the forefront. In structure-based drug design,
molecular docking is the most common and has been widely used since the early 1980s [5].
The molecular docking approach can be used to model the interaction between a small
molecule and a protein at the atomic level, which allow us to characterize the behaviour of
small molecules in the binding site of target proteins as well as to elucidate fundamental
biochemical processes [6].

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The protein bound by the epidermal growth factor on the surface of some cells causes the
cells to divide. Since the epidermal growth factor is present at high levels abnormally on
many cancer cell surfaces, these cells may be severely disrupted in the presence of epidermal
growth factor. Activation of receptors by growth factors in the proliferation of tumour cells is
an important step. Epidermal growth factor receptor (EGFR) inhibitors are highly effective
agents in the treatment of metastatic epithelial cancers [7,8].
The vascular endothelial growth factor (VEGF) family of peptide growth factors and
receptors are key regulators of tumour growth and metastatic dissemination [9]. Vascular
endothelial growth factor receptor 2 (VEGFR2) is a type V receptor tyrosine kinase mainly
known to be expressed in vascular endothelial cells and encoded by the KDR (kinase insert
domain receptor) gene [10]. VEGFR2 is expressed in many but not all vascular endothelial
cells from early fetal development, and it is more highly expressed in neovascular tumour
endothelia than in normal endothelia [11,12]. Recently, the emergence of tyrosine kinase
inhibitors with VEGFR2 specificity, such as sunitinib, sorafenib and cabozantinib, and
VEGFR2 blocked with bevacizumab (analogous to herceptin in breast cancer) with a VEGF
neutralizing antibody, suggests that these inhibitors may be effective against VEGFR2 [13-
16]. Eventually blocking VEGFR2 signalling pathway has become an attractive approach for
the treatment of cancers [17,18].
Based on the above consideration we synthesized novel analogues including both the
ribofuranose and the heterocyclic base starting from nitro aniline derivatives to clarify the
importance of sugar moiety. Quantum chemical calculations were carried out using the
density functional theory (DFT) and experimental results were compared with the theoretical
ones. To understand the interactions of synthesized nucleoside analogues at active sites of

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epidermal growth factor receptor (PDB ID: 1M17) and vascular endothelial growth factor
receptor 2 (PDB ID: 2XIR), molecular docking studies were also performed and reported in
this paper.
2. Experimental
2.1. Experimental equipment
 Chemicals: purchased from Merck and Aldrich Chemical Company and used without
further purification.
FT-IR analysis: Shimadzu IR Prestige-21 Fourier Transforminfrared (FT-IR)
spectrophotometer (on a KBr disk)
Nuclear magnetic resonance (1H-NMR) analysis: Bruker Ultrashield Superconducting 400
MHz
Ultraviolet–visible (UV–vis) absorption analysis: Shimadzu UV-1601 double beam
spectrophotometer
Melting points: Electrothermal 9100 melting point apparatus
2.2. Synthesis of nucleosides 5a-c
o-, m-, p-nitro substitute aniline compounds 1a-c (0.01 mol) was dissolved in hydrochloric
acid and the solution was cooled to 0–5 oC. Then, sodium nitrite (0.01 mol) in water (20 mL)
o
was added to this solution at 0–5 C. The resulting diazonium solution was added to a stirred
solution of ethylacetoacetate little by little; therewithal solid sodium acetate was added to
maintain the pH at 7-8. This mixture was stirred at 0-5 oC for 4 h. The resulting solid 2a-c was
filtered, washed with cold water three times and dried. 0.005 mol solid 2a-c and hydrazine
hydrate were heated under reflux in ethanol for 8 h and the reaction mixture was saturated
with water. The resulting solid product 3a-c was filtrated and crystallized from DMF-H2O.

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Compounds 3a-c were heated under reflux for 12 h in hexamethyldisilazane with (NH4)2SO4.
The solution was concentrated and in anhydrous 1,2-dichloroethane, residue was dissolved.
Trimethylsilyl
trifluoromethanesulfonate
and
1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-
ribofuranose were added to this solution. This mixture was stirred for 24 h at room
temperature, washed three times, dried with sodium sulfate, filtered and evaporated.
Recrystallization from DMF-H2O gave crystals of dye 4a-c. Obtained compounds 4a-c were
dissolved in methanol–ammonia solution and for a week stirred at room temperature. Finally,
to get the nucleosides 5a-c, the mixture was evaporated and purified by crystallization from
DMF-H2O. (5a: Red solid, yield 76%, m.p. 96-98oC, 5b: Yellow solid, yield 73%, m.p. 85-
87oC, 5c: Orange solid, yield 67%, m.p. 87-89oC). The general route for the synthesis of
nucleosides is shown in Figure 1.
2.3. Computational details
Calculations were carried out using Gaussian 09 and visualizations were performed by
GaussView 5.0.9 program packages [19,20]. The molecular structure of the synthesized
compounds in the ground state were optimized by the DFT method using B3LYP functional
with 6-311G(d,p) basis set [21,22]. The simulated UV-vis spectra were performed at the time
dependent density functional theory (TD-DFT) using B3LYP level with same basis set
[23,24]. Contribution percentages of the excitations were calculated using GaussSum 3.0
1
program [25]. H-NMR shielding constants were calculated by applying gauge-invariant
atomic orbital (GIAO) method [26,27].
2.4. Molecular Docking Studies
The interactions of synthesized molecules with epidermal growth factor receptor (PDB ID:
1M17) and vascular endothelial growth factor receptor 2 (PDB ID: 2XIR) were analysed

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using docking simulation. Modelling was performed on SwissDock web server using EADock
DSS algorithm [28]. In the molecular docking studies, the optimized geometries from the
theoretical calculations of the ligands were used. High resolution crystal structures of EGFR
(PDB ID: 1M17) and VEGFR2 (PDB ID: 2XIR) were obtained from protein data bank
(https://www.rcsb.org). All images in molecular docking studies were drawn with the UCSF
Chimera package [29].
3. Results and Discussion
3.1. Spectral characteristics
Tautomerism is called the balance of compounds that can be converted into one to another.
The most well-known tautomerism is the keto-enol tautomerism. The two forms can be
transformed into one another by the action of alpha hydrogen and the displacement of the
bonding electrons. Tautomeric forms of synthesized nucleosides 5a-c were presented in
Figure 2.
The FTIR spectra of nucleosides 5a-c showed aliphatic (Alip.-H) band at 2960-2920 cm−1,
aromatic (Ar-H) band at 3190-3160 cm−1, azo (N=N) band at 1570-1490 cm−1, asymmetric N-
O band at 1620-1610 cm−1, symmetric N-O band at 1450 cm−1 and C-O band at 1250-1220
cm−1. As corresponding B3LYP/6-311G(d,p) calculations, aliphatic (Alip.-H) band at 2919-
2918 cm−1, aromatic (Ar-H) band at 3122-3095 cm−1, azo (N=N) band at 1546–1471 cm−1,
asymmetric N-O band at 1586-1585 cm−1, symmetric N-O band at 1417-1416 cm−1 and C-O
band at 1211-1123 cm−1 were recorded. Experimentally and theoretically FT-IR spectra of
nucleoside 5a was shown in Figure 3 and nucleosides 5b-c were given in Figure S1

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(Supplementary data, Appendix A). Vibrational frequency values of nucleosides 5a-c were
given in Table 1, along with experimental and theoretical data. Small deviations were
observed owing to the vibration bands complexity when the results were compared.
The linear regressions between the experimental and theoretical frequencies of nucleosides
(5a-c) were displayed in Figure 4. The obtained equality by using the method of DFT/B3LYP
for 5a-c were y= 0.9858x (R2=0.998), y= 0.9772x (R2=0.9976), y= 0.98x (R2=0.9998),
respectively. These results showed us that the experimentally obtained frequency values were
in agreement with the theoretically calculated frequency values.
1H-NMR spectra of nucleosides 5a-c showed broad peaks at 11.63-11.52 ppm (OH) of
pyrazole and 4.71-3.79 ppm (OH) of ribofuranose, respectively. The other δ values of 2.50-
2.08 ppm (pyrazole, CH3), 5.29-5.08 ppm (ribofuranose, -CH), 5.53-5.51 ppm (ribofuranose, -
1
CH2) and 8.50-7.35 ppm (aromatic H) were recorded. H-NMR spectra of nucleoside 5a was
given in Figure 5 and nucleosides 5b-c were illustrated in Figure S2 (Supplementary data,
Appendix A). Calculated by B3LYP/6-311G(d,p), chemical shift δ values of 2.37-2.33 ppm
(pyrazole, CH3), 5.69-4.02 ppm (ribofuranose, -CH), 4.12–4.11 ppm (ribofuranose, -CH2) and
8.97-7.68 ppm (aromatic H) were obtained. Nucleosides 5a-c showed peaks at 5.59-5.44 ppm
(OH) of pyrazole and 1.67-0.63 ppm (OH) of ribofuranose. It is observed that the theoretical
1
and experimental H-NMR values are in harmony with each other except for –OH protons.
This can be explained as follows: due to the tautomerization of the synthesized nucleosides,
the protons of -OH in the theoretical studies may resonate in different regions. Calculated and
experimental 1H-NMR values of nucleosides 5a-c are presented in Table 2.

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The UV spectrum of nucleosides 5a-c measured in five different solvents depending on
polarities at various concentrations (106-108 M) between 300 and 700 nm. Nucleoside 5a gave
double maxima in all solvents. When the first maximums were compared, it is observed that
the wavelengths did not change much. The most bathochromic shift at the wavelengths of the
second maxima was in DMF. According to the λmax in chloroform, hypochromic shift was
observed in methanol. Nucleoside 5b except for acetic acid, gave double maxima. Second
maxima values of 5b in DMF were shifted bathochromically, according to the λmax of the
others. Nucleoside 5c gave single maxima in methanol, acetic acid, chloroform and double
maxima in DMSO and DMF. Bathochromic shifts were the largest in DMSO and DMF when
compared with others. Absorption spectrums of nucleosides 5a-c were shown in Fig. 6.
Electronic transition wavelengths of the nucleosides 5a-c were calculated for all solvents
using the TD-DFT method with B3LYP/6-311G (d,p) level. Calculated and experimentally
obtained λmax (nm) values were listed in Table 3. The calculated λmax values of nucleoside 5a-
c gave three maxima in all solvents and no significant change was observed. Excitation
energies, oscillator strengths and absorbance of the nucleosides were also calculated in the
DMF solvent using the TD-DFT method with same level of theory (Table 4-6). In electronic
transitions of nucleosides 5b and 5c, the major contributions for each transition were
calculated to occur only between one orbital couple. In the 5a nucleosides, three or two orbital
couples contributed to each electronic transition. The highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO) are known as boundary
molecular orbitals and are the most important orbitals in molecular systems. Four important
frontier molecular orbitals (HOMO-1, HOMO, LUMO and LUMO+1) were selected for each
nucleosides and given in Figure 7-9. As can be seen from the diagrams, the frontier molecular
orbitals were usually located over the benzene and pyrazole rings of the nucleosides 5a-c. On

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the other hand, it was determined that the ribofuranose skeletons did not fully contribute to
HOMO and LUMO. The calculated HOMO energies for the nucleosides 5a, 5b and 5c were -
5.913, -6.077 and -6.178 eV, respectively while the LUMO energies were -2.173, -2.206 and -
2.641 eV, respectively. The energy gap between HOMO and LUMO characterizes the
molecular chemical stability [30]. The HOMO-LUMO energy ranges of the synthesized
nucleosides were calculated to be 3.744, 3.871 and 3.537 eV, respectively. Moving from these
values, it was thought that 5b was the hardest and stable molecule among synthesized
nucleosides. It is also expected that the nucleoside 5c with the least HOMO-LUMO energy
gap will be more reactive in chemical reactions.
3.2. Molecular structure
Geometry optimization of the synthesized nucleosides 5a-c were done using B3LYP
functional with 6-311G(d,p) basis set in gas phase. The fully optimized geometries of the 5a,
5b and 5c including numbering atoms are shown in Figure 10, 11 and 12, respectively. The
values of the calculated bond lengths (Å), bond angles (o) and dihedral angles (o) are given in
Table 7. In all three nucleosides, the shortest bond length was observed between the N12 and
N13 atoms forming the azo group. The C-N bond between the benzene and the nitro group in
the 5c molecule was somewhat shorter than the other nucleosides. The calculated single bond
length of C10-N12 for 5a, 5b and 5c was found as 1.412, 1.419 and 1.417 Å, respectively
which indicated that the shortest benzene-azo bond was located in molecule 5a. As can be
seen from Table 4, the bond lengths between atoms in pyrazole and ribofuranose structures
were approximately the same for all three nucleosides. When the bond angles between the
atoms in the nucleosides were examined, it was observed that the O-N-O bond angle in the
nitro group of the compound 5a was slightly larger, while the azo group was always
connected to the benzene group at the same angle. The calculated value of the O26-N25-C11-

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C10, O42-N41-C6-C11 and O42-N41-C7-C6 dihedral angle for the 5a, 5b and 5c between
benzene ring and oxygen atoms of nitro group was found as -142.2o, -179.9o, -179.9o,
respectively. This indicates that the benzene ring and the nitro group were not lying in the
same plane in molecule 5a. The same observation was observed between benzene and azo
groups. The dihedral angle of C11-C10-N12-N13 was 35.7o in the 5a molecule, while it was
calculated as -0.5o and -0.2o in 5b and 5c, respectively, indicating that the benzene ring and
the azo group in the 5a molecule were not lying in the same plane. Furthermore in the
synthesized nucleosides the dihedral angles of C18-C19-C23-O24 were calculated to be
around 170o, indicating that the -CH2OH group was present in the same plane with the
ribofuranose ring in all three nucleosides.
3.3. Molecular electrostatic potential (MEP)
The surface emerging in the molecular electrostatic potential (MEP) diagram gives
information on molecular size, shape, charge distribution and electrostatic potential value.
These diagrams are important in determining the locations of the electrophilic and
nucleophilic reactions in the molecule. In the MEP maps, the red parts represent the
electronically rich regions of the molecule, while the blue parts represent the electronically
poor regions. The molecular electrostatic potential (MEP) surfaces of the synthesized
nucleosides 5a-c were calculated using the B3LYP level of DFT using 6-311G(d,p) basis set,
and the 3D maps are given in Figure 13-15.
When the MEP surfaces of the compounds were examined, it was seen that the regions with
the highest electron density in the three molecules were around the oxygen atoms in the nitro
group, which had a high electronegativity effect. Electron density was also observed on the
oxygen atom in the ribofuranose ring. There is also some negative charge on the azo groups of

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the molecules, while the azo group with the most electron-rich region is in the 5a molecule.
The blue regions in the molecules are on hydrogen atoms that are directly connected to the
electronegative oxygen atoms. Thus, the regions with the lowest electron density in the
synthesized nucleosides are around O-H groups attached to the pyrazole and ribofuranose
rings.
3.4. Molecular docking studies
Investigation of protein and ligand interaction plays an important role in the design of
structurally based drugs. In order to examine the antiproliferative activities of synthesized
nucleoside analogues 5a-c, a detailed intermolecular interaction between the ligands and
biological targets were analyzed through application of molecular docking studies on the
crystal structure of epidermal growth factor receptor (PDB ID: 1M17) and vascular
endothelial growth factor receptor 2 (PDB ID: 2XIR). Binding energy, full fitness score and
hydrogen bond location with length were found and results were given in Table 8 and 9.
These results showed that binding energy range of all compounds was between -6.35 and -
10.35 kcal/mol and number of hydrogen bonds range was 2-5. The docking results showed
that synthesized compounds fit well in the binding cavity of EGFR (Figure 16, 17) and
VEGFR2. When binding energies were examined, it was determined that the interaction of all
three ligands synthesized with EGFR was stronger than VEGFR2. The strongest interaction
between (5a-c)- EGFR was in the 5b- EGFR pair which has lowest interaction energy of -
10.35 kcal/mol. This most stable docked pose showed five hydrogen bonding interactions
with Leu 838, Glu 673, Glu 673, Ala 674, Ser 744 in the active site of EGFR.
The amino acid Glu 673 had formed two hydrogen bonding using OE2 and OE1 atoms with
2.746 and 1.713 Å bond lengths, respectively. Another hydrogen bond was observed at a

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distance of 2.159 Å with Leu 838 and H atom of CH-OH moiety. Finally the amino acids Ala
674 (2.409 Å) and Ser 744 (2.901 Å) had formed hydrogen bonding with oxygen atoms of
CH-OH and nitro groups, respectively. Full fitness scores of the studied molecules were found
to be between 1671.46 and 2284.53. The good inhibition activity of 5b compared to 5a and 5c
was further confirmed by full fitness score. The calculated full fitness score for interaction 5b-
EGFR is 2284.53. The designed and synthesized compounds 5a-c showed good activity and
this was confirmed by the mode of orientation of these molecules with targets. Among the
compounds 5a-c, 5b was considered as the best compound into the binding site of EGFR with
the five hydrogen bonding, lowest binding energy (-10.35 kcal/mol) and highest full fitness
score 2284.53.
4. Conclusion
Modified nucleosides have attracted interest in recent years because the showing interesting
biological properties [31-33]. So, the design and synthesis of a three novel ribofuranose
1
nucleosides 5a-c were demonstrated in this report. Compounds were characterized by H-
NMR, FT-IR and UV-Vis spectroscopy. The molecular structural parameters, vibrational
frequencies and chemical shifts of the fundamental modes, MEP, HOMO-LUMO analysis of
the synthesized molecules were determined from DFT method utilizing 6-311G(d,p) basis set.
It was found that theoretical spectroscopic results were in a good agreement with those of the
experiments. Furthermore, the molecular docking simulations were performed using
epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2
(VEGFR2) to predict antiproliferative activity of the synthesized nucleosides. All three
compounds studied inhibited both EGFR and VEGFR2, while the best interaction was
observed between 5b-EGFR with 5 hydrogen bonds and -10.35 kcal/mol binding energy.
Based on the results of the molecular docking studies, nucleoside 5b was found to have a very

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high potential to inhibit the EGFR. These results can be very useful in the development of
potential anticancer drugs.

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Figure Captions
Figure 1. The general route for the synthesis of nucleosides 5a-c.
Figure 2. Tautomeric forms of the 5a-c nucleosides.
Figure 3. Experimentally (upper) and theoretically (lower) FT-IR spectra of the 5a
nucleoside.
Figure 4. The linear regression between the experimental and theoretical frequencies of the
5a (uppermost), 5b (middle) and 5c (undermost) nucleosides.
Figure 5. 1H-NMR spectrum of the 5a nucleoside.
Figure 6. The experimentally absorption spectrum of 5a nucleoside.
Figure 7. Frontier molecular orbitals with energy values of the 5a nucleoside calculated at
B3LYP/6-311G(d,p) level
Figure 8. Frontier molecular orbitals with energy values of the 5b nucleoside calculated at
B3LYP/6-311G(d,p) level
Figure 9. Frontier molecular orbitals with energy values of the 5c nucleoside calculated at
B3LYP/6-311G(d,p) level
Figure 10. Optimized geometric structure of the 5a nucleoside performed by B3LYP/6-
311G(d,p) level
Figure 11. Optimized geometric structure of the 5b nucleoside performed by B3LYP/6-
311G(d,p) level
Figure 12. Optimized geometric structure of the 5c nucleoside performed by B3LYP/6-
311G(d,p) level
Figure 13. Molecular electrostatic potential map of the 5a nucleoside calculated at B3LYP/6-
311G(d,p) level.

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Figure 14. Molecular electrostatic potential map of the 5b nucleoside calculated at B3LYP/6-
311G(d,p) level.
Figure 15. Molecular electrostatic potential map of the 5c nucleoside calculated at B3LYP/6-
311G(d,p) level.
Figure 16. Interacting mode of the 5b nucleoside with EGFR (PDB ID: 1M17)
Figure 17. The binding of the 5b nucleoside in the cavity of EGFR (PDB ID: 1M17)

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.
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y = 0.9772x
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Figure 5.

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Figure 6.

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Figure 7.