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![1. Introduction
Cancer is fundamentally characterized by uncontrolled cell prolif
eration, genomic instability, and the evasion of programmed cell death.
Despite remarkable advances in cancer biology, many solid tumors
remain difficult to treat due to their heterogeneity and adaptive resis
tance mechanisms. Among these, clear cell Renal Cell Carcinoma
(ccRCC) represents a highly aggressive and treatment-resistant malig
nancy. Renal cell carcinoma (RCC) accounts for nearly 90% of kidney
cancers and approximately 4% of all adult malignancies [1]. The clear
cell subtype (ccRCC) constitutes 70–75% of all RCC cases [2]. Because
ccRCC is often asymptomatic in its early stages, over 30% of patients
present with metastatic disease at diagnosis, precluding surgical inter
vention. Despite surgical tumor resection, recurrence occurs in nearly
40% of patients, and the 5-year overall survival remains <10% [3,4].
These statistics highlight the urgent need for new therapeutic strategies
and a deeper molecular understanding of ccRCC pathogenesis [5].
Current therapeutic strategies for ccRCC combine immune checkpoint
inhibitors and targeted therapies, improving patient outcomes but often
leading to the emergence of drug resistance that limits long-term clinical
benefits. Identifying new molecular targets and designing selective in
hibitors remain essential for developing more durable treatment
options.
Computational drug repositioning based on systems biology ap
proaches have a pivotal role in helping to identify potential interactions
between drugs, their targets and even and the link between drugs and
diseases [6,7]. Since pharmacology and safety of the repositioned drugs
have been well-characterized, it can reduce costs and speed up the
development of effective therapies [8,9]. In our previous research that
was performed in our group, we utilized co-expression network analysis
of transcriptomics profiles of ccRCC patients to identify high-priority
hub genes, which resulted in four key genes—BUB1B (BUB1 mitotic
checkpoint serine/threonine kinase B), RRM2 (Ribonucleotide Reduc
tase Regulatory Subunit M2), ASF1B (Anti-Silencing Function 1B His
tone Chaperone), and CCNB2 (Cyclin B2)— that controlled the ccRCC
development were associated with the cell cycle dysfunctions. Further,
TG-101,209 was identified as a drug candidate to regulate BUB1B
expression through drug repositioning. Finally, experimental validation
in the Caki-1 cell line confirmed that TG-101,209 effectively down
regulates BUB1B protein levels and suppresses cell viability, under
scoring its potential as a targeted therapeutic scaffold for ccRCC
management [10].
Pyrimidine, a six-membered aromatic heterocycle with nitrogen
atoms at positions 1 and 3, is a key structural motif in numerous bio
logically active compounds. Its hydrogen-bonding capability, π–π
stacking, and electronic versatility confer broad pharmacological utility
across diverse therapeutic areas. Clinically approved pyrimidine-based
drugs, including 5-fluorouracil, zidovudine, and imatinib, exemplify
the scaffold’s potential in oncology, antiviral, and anti-inflammatory
applications [11,12]. Continued development of pyrimidine de
rivatives and fused heterocycles has enabled the design of molecules
with improved selectivity and multi-target interactions [13,14].
Developing novel therapeutics is a complex, multi-stage endeavour
necessitating the precise design and chemical synthesis of candidates
with superior potency and clinical efficacy. Our previous works identi
fied TG-101,209 as a potential BUB1B inhibitor for ccRCC therapy;
however, structural modifications are necessary to enhance its binding
affinity for the BUB1B and to more effectively promote apoptosis. In
continuation of our efforts to identify effective ccRCC therapeutics, the
present study describes the development and characterization of novel
inhibitors targeting the BUB1B kinase domain [15,16]. This study aimed
to design and evaluate novel TG-101,209 derivatives as modulators of
BUB1B activity. To this end, four derivatives were synthesized by
modifying specific position on the TG-101,209 scaffold, focusing on
enhancing binding efficacy, BUB1B inhibition, and the induction of
apoptosis in Caki-1 cells. These compounds were characterized using an
array of experimental and spectroscopic techniques. Furthermore,
Structure-Activity Relationship (SAR) analysis, molecular docking, and
Molecular Dynamics (MD) simulations were employed to gain a deeper
understanding of how the chemical architecture influences biological
performance. Such an integrated methodology facilitates the identifi
cation of critical functional groups that augment potency, ultimately
supporting the rational design of more effective therapeutic agents.
2. Experimental section
2.1. General information
All reagents were purchased from commercial suppliers and used
without further purification. Reactions were monitored by LC–MS
(Thermo Fisher TSQ Series, Athena C18-WP, 100 Å, 2.1 × 50 mm, 3 μm;
water: methanol with 0.01% formic acid) or by thin-layer chromatog
raphy (TLC). TLC analyses were performed using silica gel pre-coated
aluminum plates (Kieselgel 60, F254, E. Merck, Germany). High-
resolution mass spectra (HRMS) were obtained using a Thermo Fisher
Dionex Ultimate 3000 UHPLC System (Thermo Scientific). Chromato
grams were visualized under ultraviolet light at 254 and 366 nm. 1
H
NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer
in DMSO‑d₆. 13
C NMR spectra were recorded on a Bruker spectrometer
at 126 MHz in deuterated solvents, with the central peak of the solvent
used as the internal standard. Chemical shifts (δ) are reported in parts
per million (ppm) relative to tetramethylsilane (TMS). The following
abbreviations are used to describe signal multiplicities: s (singlet),
d (doublet), t (triplet), q (quartet), qt (quintuplet), and m (multiplet).
Coupling constants (J) are reported in Hertz (Hz). Flash column chro
matography was performed on a PuriFlash XS 520 Plus system (Inter
chim) equipped with a built-in UV detector, ELSD detector, and fraction
collector, using Interchim silica gel columns. The IR spectrum of com
pound 3a was recorded using the diffuse reflectance technique. The
crystal was finely ground, mixed with KBr, and analyzed at room tem
perature over the 4000–400 cm⁻¹ range using a PerkinElmer Frontier FT-
IR spectrometer. The resulting experimental spectrum was employed for
comparison with the theoretically calculated IR frequencies in order to
support the vibrational assignments and validate the optimized molec
ular structure.
2.2. Synthesis
2.2.1. N-cyclobutyl-3-((5-methyl-2-((4-(4-methylpiperazin-1-yl)phenyl)
amino)pyrimidin-4-yl)amino)benzenesulfonamide (3a)
A mixture of intermediate 2 (0.12 g, 0.4 mmol), 3‑bromo-N-cyclo
butylbenzenesulfonamide (0.14 g, 0.48 mmol), Pd2(dba)3 (0.035 g, 0.04
mmol), Xantphos (0.035 g, 0.06 mmol) and cesium carbonate (0.25 g,
0.8 mmol) was suspended in dioxane/DMF (3/1, 8 mL) and heated at
155 ◦
C under the nitrogen atmosphere for 18–24 h, after completion of
the reaction, the reaction mixture was cooled to room temperature and
diluted with DCM (10 mL). The mixture was filtered and the filtrate was
concentrated under vacuum. The residue was purified by column
chromatography (chloroform: methanol = 9: 1) to give the desired
compound 3a (80 mg, yield: 40%) as a light yellow solid. 1
H NMR (500
MHz, DMSO‑d6) δ 8.71 (s, 1H), 8.53 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H),
7.98 – 7.93 (m, 2H), 7.89 (s, 1H), 7.49 – 7.43 (m, 4H), 6.79 (d, J = 9.0
Hz, 2H), 3.63 (m, 1H), 3.03 – 3.00 (m, 4H), 2.46 – 2.44 (m, 4H), 2.22 (s,
3H), 2.11 (s, 3H), 1.92 (m, 2H), 1.80 – 1.74 (m, 2H), 1.49 – 1.41 (m, 2H).
13
C NMR (126 MHz, DMSO‑d6) δ 158.93, 158.38, 156.23, 145.68,
141.67, 140.70, 133.29, 129.07, 125.29, 120.08, 120.01, 119.16,
115.96, 105.39, 54.74, 49.04, 47.61, 45.78, 30.62, 14.55, 13.51. MS
(ESI): m/z 508. 17 [M+H]+
. HRMS (ESI): calcd for C26H33N7O2S
[M+H]+
508.24892; found 508.24854.
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![2.2.2. 5-methyl-N4
-(5-methyl-4-(4-(trifluoromethyl)phenyl)pyrimidin-2-
yl)-N2
-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine (3b)
A mixture of intermediate 2 (0.15 g, 0.48 mmol), 2‑chloro-5-methyl-
4-(4-(trifluoromethyl)phenyl)pyrimidine (0.12 g, 0.44 mmol),
Pd2(dba)3 (0.04 g, 0.048 mmol), Xantphos (0.08 g, 0.13 mmol) and
cesium carbonate (0.3 g, 0.9 mmol) was suspended in dioxane/DMF (3/
1, 5 mL) and heated at 155 ◦
C under the nitrogen atmosphere for 20 h,
after completion of the reaction, the reaction mixture was cooled to
room temperature and diluted with CHCl3 (15 mL). The mixture was
filtered and the filtrate was concentrated under vacuum. The residue
was purified by column chromatography (chloroform: methanol = 9: 1)
to give the desired compound 3b (35 mg, yield: 15%) as a yellow solid.
1
H NMR (500 MHz, DMSO‑d6) δ 9.33 (s, 1H), 8.86 (s, 1H), 8.63 (s, 1H),
8.04 (s, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.50 (d, J
= 9.0 Hz, 2H), 6.48 (d, J = 8.9 Hz, 2H), 2.91 – 2.89 (m, 4H), 2.42 – 2.39
(m, 4H), 2.31 (s, 3H), 2.19 (s, 3H), 2.09 (s, 3H). 13
C NMR (126 MHz,
DMSO‑d6) δ 163.31, 160.51, 158.43, 158.38, 158.20, 157.99, 145.49,
141.83, 133.37, 130.00, 129.65, 129.39, 129.35, 127.46, 125.31,
125.28, 125.25, 125.22, 123.13, 121.27, 119.49, 115.85, 108.77, 54.78,
49.20, 45.88, 16.08, 13.78. MS (ESI): m/z 535. 26 [M+H]+
. HRMS (ESI):
calcd for C28H29F3N8 [M+H]+
535.25400; found 535.25366.
2.2.3. N-cyclopentyl-3-((5-methyl-2-((4-(4-methylpiperazin-1-yl)phenyl)
amino)pyrimidin-4-yl)amino)benzenesulfonamide (3c)
A mixture of intermediate 2 (0.15 g, 0.5 mmol), 3‑bromo-N-cyclo
pentylbenzenesulfonamide (0.17 g, 0.55 mmol), Pd2(dba)3 (0.03 g,
0.035 mmol), Xantphos (0.04 g, 0.075 mmol) and cesium carbonate
(0.33 g, 1.0 mmol) was suspended in Dioxane/DMF (3/1, 8 mL) and
heated at 155 ◦
C under the nitrogen atmosphere for 20 h, after
completion of the reaction, the reaction mixture was cooled to room
temperature and diluted with DCM (15 mL). The mixture was filtered
and the filtrate was concentrated under vacuum. The residue was pu
rified by column chromatography (chloroform: methanol = 9: 1) to give
the desired compound 3c (50 mg, yield: 19%) as a white solid. 1
H NMR
(500 MHz, DMSO‑d6) δ 8.73 (s, 1H), 8.54 (s, 1H), 8.04 (s, 1H), 7.89 (s,
1H), 7.66 (d, J = 7.0 Hz, 2H), 7.52 – 7.46 (m, 4H), 6.81 (d, J = 9.1 Hz,
2H) 3.44 – 3.40 (m, 1H), 3.03 – 3.01 (m, 4H), 2.46 – 2.44 (m, 4H), 2.22
(s, 3H), 2.11 (s, 3H), 1.63 – 1.60 (m, 2H), 1.54 – 1.53 (m, 2H), 1.37 –
1.33 (m, 4H). 13
C NMR (126 MHz, DMSO‑d6) δ 159.09, 158.53, 156.33,
145.84, 141.78, 140.86, 133.45, 129.24, 125.28, 120.23, 120.20,
119.36, 116.10, 105.57, 54.93, 54.65, 49.21, 45.97, 32.68, 23.01,
13.70. MS (ESI): m/z 522. 19 [M+H]+
. HRMS (ESI): calcd for
C27H35N7O2S [M+H]+
522.26457; found 522.26392.
2.2.4. 3-((5-methyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)
pyrimidin-4-yl)amino)-N-phenylbenzenesulfonamide (3d)
A mixture of intermediate 2 (0.1 g, 0.3 mmol), 3‑bromo-N-phenyl
benzenesulfonamide (0.11 g, 0.36 mmol), Pd2(dba)3 (0.02 g, 0.02
mmol), Xantphos (0.03 g, 0.045 mmol) and cesium carbonate (0.2 g, 0.6
mmol) was suspended in dioxane/DMF (3/1, 8 mL) and heated at 155 ◦
C
under the nitrogen atmosphere for 24 h, after completion of the reaction,
the reaction mixture was cooled to room temperature and diluted with
DCM (10 mL). The mixture was filtered and the filtrate was concentrated
under vacuum. The residue was purified by column chromatography
(chloroform: methanol = 9: 1) to give the desired compound 3d (55 mg,
yield: 30%) as a white solid. 1
H NMR (500 MHz, DMSO‑d6) δ 8.69 (s,
1H), 8.53 (s, 1H), 8.20–8.19 (m, 1H), 7.89 (s, 1H), 7.47–7.43 (m, 4H),
7.24 − 7.20 (m, 3H), 7.12–7.11 (m, 2H), 7.02 – 6.99 (m, 2H), 6.82–6.80
(m, 2H), 3.03 (m, 4H), 2.45 (m, 4H), 2.22 (s, 3H), 2.09 (s, 3H). 13
C NMR
(126 MHz, DMSO‑d6) δ 158.94, 158.50, 156.42, 145.90, 140.98,
139.92, 137.98, 133.33, 129.31, 125.69, 124.06, 120.32, 120.23,
120.02, 119.41, 116.09, 105.62, 54.92, 49.20, 45.98, 13.65. MS (ESI):
m/z 530. 23 [M+H]+
HRMS (ESI): calcd for C28H31N7O2S [M+H]+
530.23327; found 530.23260.
2.3. Biological assays
2.3.1. Cell culture, cell viability, and cytotoxicity assays
The human clear cell renal cell carcinoma (ccRCC) cell line Caki-1
was purchased from CLS Cell Lines Service (Germany). Cells were
cultured in McCoy’s 5A (Modified) medium (16,600,082, Thermo Fisher
Scientific, USA) supplemented with 10% fetal bovine serum (FBS,
F7524, Sigma-Aldrich) and 1% penicillin–streptomycin (P4333, Sigma-
Aldrich). MRC-5 human lung fibroblast cells were maintained in high-
glucose DMEM (D0822, Sigma-Aldrich) supplemented with 10% FBS
and 1% P/S. The tested compounds were initially dissolved in 100%
DMSO to prepare stock solutions and subsequently diluted in the culture
medium to the final working concentrations, ensuring that the final
DMSO concentration remained below 0.1% (v/v). During the incubation
period, no visible precipitation or degradation was observed in the assay
medium across the tested concentration range. For MTT and LDH assays,
2 × 10⁴ Caki-1 cells per well were seeded into 96-well plates in triplicate.
Cells were treated with serial concentrations (30, 10, 3, 1, 0.3, 0.1, and
0.03 µM) for 48 h. Cell viability was assessed using the MTT assay kit
(M6494, Thermo Fisher Scientific), while cytotoxicity was evaluated
with the LDH assay kit (ab65393, Abcam), following the manufacturers’
protocols. Absorbance was measured with a microplate reader (Hidex
Sense Meta Plus).
2.3.2. Western blot and CETSA analysis
To evaluate BUB1B protein expression, 4 × 10⁵ Caki-1 cells were
seeded in 6-well plates and treated with 10 µM of the compounds for 24
h. Protein lysates were prepared using CelLytic M (C2978, Sigma-
Aldrich). For CETSA experiments, 5 × 10⁵ Caki-1 cells were treated
with 20 µM of each compound for 1 h. After PBS washing, cells were
subjected to heat shock at 45 ◦
C for 3 min using a MultiTherm Shaker
(H5000-HC-E, Benchmark Scientific) at 300 rpm. Proteins were
extracted through three freeze–thaw cycles (liquid nitrogen and 25 ◦
C),
followed by centrifugation at 20,000 × g for 20 min at 4 ◦
C. SDS–PAGE
was performed using Mini-PROTEAN® TGX™ Precast Gels (4561,086,
Bio-Rad), and proteins were transferred onto 0.2 μm PVDF membranes
(1704,158, Bio-Rad) using a Trans-Blot® Turbo™ Transfer System (Bio-
Rad). Membranes were incubated overnight at 4 ◦
C with primary anti
bodies: Anti-BUB1B (ab254326, Abcam), PARP (95,425, Cell Signaling),
active Caspase-9 (ab2324, Abcam), active Caspase-3 (ab32042, Abcam),
and GAPDH (ab8245, Abcam). After washing, membranes were incu
bated with HRP-conjugated secondary antibodies (Goat anti-Rabbit IgG-
HRP, ab205718; Goat anti-Mouse IgG-HRP, ab6789; Abcam) for 1 h at 4
◦
C. Protein bands were visualized using an enhanced chem
iluminescence substrate (WBLUF0500, Merck) and imaged with
ImageQuant™ LAS 500 (GE Healthcare).
2.3.3. Flow cytometry analysis of the cell cycle
Caki-1 cells (4 × 10⁵ per well) were treated with 10 µM of the
compounds for 48 h in 6-well plates. Cells were fixed with 70% ethanol,
stained with 10 µg/mL propidium iodide (PI), and analyzed using a BD
FACS Canto II flow cytometer (PE-A channel). A total of 20,000 cells per
sample were measured, and data were analyzed using BD FACSDiva 9.0
software.
2.3.4. BUB1B IC₅₀ enzymatic assay
The BUB1B kinase activity assay for IC₅₀ determination was per
formed by Reaction Biology. Test compounds were prepared at ten
concentrations (100 µM, 30 µM, 10 µM, 3 µM, 1 µM, 300 nM, 100 nM, 30
nM, 10 nM, and 3 nM). Each reaction contained 25 µL of assay buffer (70
mM HEPES–NaOH, pH 7.5; 3 mM MgCl₂; 3 mM MnCl₂; 3 µM Na₃VO₄; 1.2
mM DTT; 50 μg/mL PEG20000), 10 µL of 0.3 µM [γ-³³P]-ATP, 5 µL of
compound solution, and 10 µL of 9.5 nM recombinant BUB1B. The
mixtures were incubated at 30 ◦
C for 60 min, and the reactions were
terminated with 50 µL of 2% (v/v) H₃PO₄. Plates were washed twice with
0.9% (w/v) NaCl, and the incorporation of ³³Pi was quantified using a
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![microplate scintillation counter (MicroBeta, Wallac).
2.4. Single-crystal X-ray diffraction
The crystal structure of compound 3a was determined using single-
crystal X-ray diffraction (SC-XRD). Data was collected on a Bruker
APEX II three-circle diffractometer. Using Olex2 [17], the structure was
solved with the olex2.solve [18] structure solution program using
Charge Flipping and refined with the XL [19] refinement package using
Least Squares minimization. The crystallographic information files (CIF)
are deposited in the Cambridge Crystallographic Data Centre (CCDC),
and are directly accessible at https://www.ccdc.cam.ac.uk/structures/
(CCDC numbers: 2377,633). Software packages Mercury (version
2022.3.0) [20] and Platon (version 1.17) [21] were employed to identify
and visualize noncovalent interactions. The asymmetric unit was drawn
using ORTEP-3 (version 2023.1) [22]. Additional details on crystal data
and structure refinement can be found in Table 1.
2.5. Hirshfeld surfaces
Hirshfeld surface analysis was performed using CrystalExplorer21
[23] to quantify intermolecular interactions stabilizing the crystal
packing. CIF structural data were used to generate Hirshfeld surfaces
and 2D fingerprint plots, revealing the nature and distribution of
atom–atom contacts. Surfaces were mapped with the normalized contact
distance (dnorm), displayed with transparency and a red–blue scale
(–0.141 to +1.400 a.u.) to highlight close contacts and potential weak
hydrogen bonds. Fingerprint plots were generated at 0.5 a.u. isovalue,
over a 1–3 Å range, and filtered by element. The void surface, repre
senting the procrystal electron density, was visualized at 0.002 e au⁻³ for
the full unit cell.
2.6. Energy frameworks
Energy lattice and energy framework analyses were performed using
the Tonto program implemented within CrystalExplorer21. Molecular
clusters within a 3.8 Å radius were generated, and interaction energy
was derived using the B3LYP/6–31G(d,p) level of theory. The tube di
mensions were calibrated using a scale factor of 100. The default scale
factors for the energy framework calculations were set to Kele= 1.057,
Kpol= 0.740, Kdis= 0.871, Krep= 0.618, with a cut-off value of 5 kJ/mol.
2.7. Quantum chemical calculations
All density functional theory (DFT) calculations were performed
using the Gaussian 09 software package [24] in conjunction with the
GaussView 6.0.16 program [25]. The experimental X-ray crystal struc
ture served as the initial geometry for all computational work. All cal
culations were performed in the gas phase. For geometry optimization,
vibrational frequency analysis, and the calculation of global chemical
reactivity descriptors—including Frontier Molecular Orbitals (HOMO,
LUMO) and the Molecular Electrostatic Potential (MEP)—calculations
were performed at the B3LYP/6–311++G(d,p) level of theory [26,27].
The absence of imaginary frequencies confirmed each optimized struc
ture as a true energy minimum. The MEP surface was generated from the
resulting wavefunction and visualized on the 0.001 a.u. electron density
isosurface using GaussView 6.0.16. For the analysis of noncovalent in
teractions, the Independent Gradient Model based on Hirshfeld partition
(IGMH) was employed [28,29]. To accurately describe dispersion forces,
geometry optimizations of selected dimers were performed using the
dispersion-corrected ωB97X-D functional [30,31] with the aug-cc-pVDZ
basis set [32]. Higher-accuracy single-point energy calculations were
subsequently carried out using the aug-cc-pVTZ basis set. This approach
has proven reliable for characterizing intermolecular interactions in
organic molecular crystals [33–35]. The IGMH analysis was conducted
on the obtained DFT-wavefunctions using Multiwfn 3.8 [36,37]. The 3D
δginter
isosurfaces (isovalue = 0.01 a.u.) were visualized with VMD 1.9.3
[38], and the corresponding 2D scatter plots were generated with
Gnuplot 6.0.4 [39]. Due to the lack of solid-state structures for 3b-3d,
the detailed DFT analysis is intentionally restricted to 3a and is meant
as an in-depth characterization of the lead rather than a full
quantum-chemical SAR.
2.8. Molecular docking
Molecular docking studies were carried out using Glide XP [40]. The
2D structures of the compounds were drawn with ChemDraw and con
verted to 3D geometries using LigPrep [41] with the OPLS_4 force field
to generate energy-minimized conformations and relevant tautomeric,
stereochemical, and ionization variants. All possible ionizable (proto
nation/deprotonation) states as well as tautomeric forms in the pH range
of 7.0 ± 2.0 were generated using the Epik function of LigPrep module
[42]. The crystal structure of the human BUB1 kinase domain (PDB ID:
6F7B, 2.00 Å resolution) was obtained from the Protein Data Bank and
prepared in Maestro using the Protein Preparation Wizard [43]. Bond
orders, formal charges, and missing hydrogens were corrected; incom
plete amide groups were refined; protonation states were adjusted at
physiological pH; and all water molecules were removed. Energy
minimization was performed using the OPLS_4 force field. The receptor
grid was generated in the Glide module with the grid box centered on the
active site at x = 10.07 Å, y = –31.06 Å, z = –15.57 Å, accommodating
ligands up to 16 Å. Docking was performed in extra precision (XP) mode
with flexible ligand sampling. The Glide scoring function (G-score),
incorporating van der Waals, Coulombic, lipophilic, hydrogen-bonding,
buried polar, rotatable bond, and site penalty terms, was used to eval
uate binding affinities. Default XP parameters were applied, including a
van der Waals scaling factor of 0.80 and a partial charge cutoff of 0.15e.
The alignment between the native conformation of the co-
crystallized ligand and the conformation predicted by the docking
simulation resulted in RMSD value of 0.5741 Å. This value is well within
the widely accepted threshold for docking validation. This result dem
onstrates that the docking protocol effectively reproduces the experi
mental binding mode, ensuring the reliability of the subsequent
calculations.
2.9. Molecular dynamics (MD) simulation
Molecular dynamics simulations were performed using the Desmond
package [44] to evaluate the structural stability and conformational
flexibility of the BUB1 kinase domain–ligand complex. The crystal
structure of BUB1B in complex with BAY-1816,032 (PDB ID: 6F7B) was
Table 1
Crystal data and details of the structure determination for compound 3a.
CCDC Number 2377,633
Chemical formula C26H33N7O2S
Formula weight (g/mol) 507.65
Crystal system/Space group Triclinic, P-1
a, b, c (Å) 11.080(2), 15.267(2), 18.038(2)
α, β, γ (◦
) 108.373(2), 98.529(2), 97.246(2)
Volume (Å3
) / Z 2814.9(6) / 4
Dcalc (g. cm− 3
) / μ(mm− 1
) 1.198 / 0.150
F(000) 1080
Crystal size (mm3
) 0.15 × 0.21 × 0.35
θ (◦
) 1.4 − 27.5
Index ranges − 14, 14 / − 19, 19 / − 23, 23
Reflections measured 35,710
Independent/obs. reflections 12,888/6959
Data/parameters 12,888/653
Goodness-of-fit on F2
0.91
Final R indices 0.0554
wR2 0.1547
Rint 0.089
Largest diff. peak/hole (e.Å− 3
) 0.49/− 0.55
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![obtained from the Protein Data Bank. Since no PDB ID currently exists
for BUB1B in complex with the ligand (holo-structure), we selected this
specific PDB ID (6F7B) file based on the high sequence conservation and
similar domain organization between BUB1 and BUB1B, as well as
previously reported findings in the literature [45]. The complex was
placed at the center of an orthorhombic simulation box filled with SPC
water molecules, maintaining a 10 Å buffer from the box edges. Na⁺ and
Cl⁻ ions were added to neutralize the system. Simulations were per
formed under isothermal–isobaric (NPT) conditions at 310 K and 1 atm,
using the Nose–Hoover thermostat for temperature coupling and the
Martyna–Tobias–Klein barostat for pressure control. After system
equilibration, production MD was conducted with trajectories saved
every 200 ps. Stability and convergence were analyzed by monitoring
the root-mean-square deviation (RMSD) of the protein backbone and
ligand throughout the simulation.
3. Results and discussion
3.1. Chemistry
Intermediate 2 was obtained in good yield by reacting 2‑chloro-5-
methylpyrimidin-4-amine with 4-(4-methylpiperazin-1-yl)aniline in
acetic acid at 100 ◦
C for 3 h. Subsequent Buchwald–Hartwig cross-
coupling using Pd₂(dba)₃ as catalyst, Xantphos as ligand, and Cs₂CO₃
as base in a dioxane/DMF mixture at 155 ◦
C for 18–24 h afforded the
target compounds 3a–3d (Scheme 1). Overall, the reaction sequence
confirms the efficiency of the adopted synthetic strategy in delivering
the targeted products. Compared with reported routes for analogous
pyrimidine derivatives, this approach employs readily available starting
materials and a modular Pd-catalyzed C–N coupling that enables late-
stage diversification of the pyrimidine core with good functional
group tolerance. The main limitations are the requirement for palladium
catalysis, elevated reaction temperatures, and relatively long reaction
times, which may impact scalability. Compound 3a was selected as a
representative example for detailed spectroscopic characterization. The
FT-IR spectrum of 3a exhibits characteristic vibrations consistent with
the proposed structure (Figure S2A, Supplementary Information).
N–H asymmetric and symmetric stretching vibrations appeared at 3360
and 3245 cm⁻¹, respectively, while aromatic C–H stretching occurred in
the range 3064–3164 cm⁻¹. The –CH₃ and –CH₂– groups showed
stretching vibrations at 2979/2842 cm⁻¹ and 2942/2798 cm⁻¹, respec
tively, with CH₂ scissoring at 1472 cm⁻¹. Asymmetric and symmetric
S=O stretches were observed at 1291 and 1155 cm⁻¹, SO₂ scissoring and
Scheme 1. Reagents and conditions: a) 4-(4-methylpiperazin-1-yl)aniline, acetic acid, 100 ◦
C for 3 h; b) Pd2(dba)3, Xantphos, cesium carbonate, Dioxane/DMF, 155
◦
C for 20 h, 3‑bromo-N-cyclobutylbenzenesulfonamide for 3a, 2‑chloro-5-methyl-4-(4-(trifluoromethyl)phenyl)pyrimidine for 3b, 3‑bromo-N-cyclo
pentylbenzenesulfonamide for 3c, 3‑bromo-N-phenylbenzenesulfonamide for 3d.
Fig. 1. ORTEP-illustration of the asymmetric unit in the X-ray crystal structure
of compound 3a, with 50% probability ellipsoids. Atom colours: grey (C), blue
(N), red (O), and yellow (S).
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![3.2.2. Crystal packing
The crystal structure indicated a network of several noncovalent
interactions stabilizing the compound in its solid state. These in
teractions encompass classical N—H⋅⋅⋅O and N—H⋅⋅⋅N hydrogen bonds,
unconventional C—H⋅⋅⋅O hydrogen bonds, and C—H⋅⋅⋅π(ring) in
teractions [46]. As depicted in Fig. 2, two strong N—H⋅⋅⋅O hydrogen
bonds (dH…O = 2.21/2.46 Å) form between the oxygen atoms (O1/O4) of
sulfonamide groups and the H-atoms (H(N6)/H(N13)) of amine groups.
Likewise, the nitrogen atoms (N5/N11) of pyrimidine moieties accept
H-atoms (H(N3)/H(N10)) from amine groups, resulting in two short
N—H⋅⋅⋅N hydrogen bonds (dH…N = 2.08/2.35 Å). Additionally, the
molecular packing is reinforced by weak unconventional C—H⋅⋅⋅O
hydrogen bonding (dH⋅⋅⋅O = 2.57 Å) involving the sulfonamide oxygen
atom (O1) and hydrogen atoms of phenyl groups (Fig. 3). Furthermore,
adjacent molecules within the crystalline structure are linked through
C—H⋅⋅⋅π interactions, exemplified by two symmetric C42—H42⋅⋅⋅π(Cg)
interactions as shown in Fig. 3 (dH⋅⋅⋅Cg = 2.84 Å, where Cg is the centroid
of the pyrimidine ring).
3.3. Hirshfeld surfaces, fingerprint plots, and voids analyses
To elucidate the molecular arrangement within the crystal lattice,
Hirshfeld surface analysis was performed. This computational technique
enabled the identification and quantification of intermolecular in
teractions [47,48]. The Hirshfeld surface, color-coded using the dnorm
property (red for shorter contacts, white for contacts at van der Waals
radii, and blue for longer contacts), is presented in Fig. 4. This visual
representation clearly indicates the presence of classical N—H⋅⋅⋅O and
N—H⋅⋅⋅N hydrogen bonds. Notably, the findings corroborate well with
X-ray observations, as the shortest H-bond N3—H3⋅⋅⋅N13 exhibits the
largest and brightest red spots between the interacting atoms.
Conversely, the longest, and thus weakest, H-bond N6—H6⋅⋅⋅O4 is
manifested by smaller and less bright red dots. Quantitative information
regarding intermolecular contacts was obtained from the 2D fingerprint
plots (Fig. 5). Hydrogen bonding contributes 12.9% (H⋅⋅⋅O) and 8.7%
(H⋅⋅⋅N) to the overall interactions, respectively. Furthermore, C—H⋅⋅⋅π
(ring) interactions, represented by H⋅⋅⋅C contacts, account for 15.1%.
Consistent with crystallographic results, C⋅⋅⋅C and C⋅⋅⋅N contacts are
very weak at 1.1% and 0.9%, respectively, confirming the absence of
significant π-stacking interactions.
Fig. 5. Two-dimensional fingerprint plots, illustrating the individual contributions of intermolecular noncovalent interactions within the crystal structure of
compound 3a.
Fig. 6. Crystal void surface within the crystal structure of compound 3a
derived from procrystal electron density analysis. The total void volume is
795.58 ų, corresponding to 28.26% of the unit cell volume.
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![To evaluate the crystal packing efficiency and potential structur
e–property relationships, a void analysis was performed based on iso
surfaces of the procrystal electron density (Fig. 6) [49]. The total void
volume was calculated to be 795.58 ų, corresponding to 28.26% of the
unit cell volume, indicating the presence of appreciable empty space
within the crystal lattice. Despite this relatively large void fraction, the
crystal structure remains effectively stabilized by directional intermo
lecular interactions, particularly the N–H⋅⋅⋅N and N/C–H⋅⋅⋅O hydrogen
bonds that dominate the primary packing motifs. This apparent coex
istence of significant void space and strong pairwise interactions suggest
Fig. 8. Independent gradient model based on Hirshfeld partition (IGMH) analysis of noncovalent interaction motifs in selected dimers D1–D3. Three-dimensional
δginter
isosurfaces (0.01 a.u.) coloured by sign(λ₂)ρ are shown together with the corresponding atomic-pair indices (δGpair
) and percentage contributions. The asso
ciated two-dimensional scatter plots of δginter
versus sign(λ₂)ρ are provided for each dimer. Atom colour scheme: H (white), C (grey), N (blue), O (red), S (yellow).
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
9](https://image.slidesharecdn.com/trsm005-a06-260615081048-9bb4de92/85/Novel-pyrimidine-derivatives-as-potent-BUB1B-inhibitors-for-clear-cell-renal-cell-carcinoma-From-synthesis-and-characterization-to-docking-insights-and-therapeutic-validation-9-320.jpg)
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![an anisotropic packing arrangement, in which densely hydrogen-bonded
regions alternate with void-rich zones supported mainly by weaker
dispersion interactions. Although no guest species are present in the
current structure, the presence of accessible voids may indicate a po
tential for host–guest inclusion or structural adaptability under external
stimuli, which could be of interest for future investigations.
3.4. Lattice energies and energy framework analyses
Energy Frameworks offer a valuable tool for visualizing the supra
molecular architecture of molecular crystal structures. We calculated
the lattice energy and associated energy frameworks to evaluate the
stability, strength, and extent of pairwise interactions within the crys
talline structure of compound 3a. Table 2 presents the decomposed
lattice energies, categorized into coulombic, polarization, dispersion,
and repulsion components. Fig. 7 illustrates the energy frameworks,
with Eele, Edis, and Etot represented by red, green, and blue cylinders,
respectively. These cylinders signify the relative strengths of molecular
packing in different directions. Energies between molecular pairs are
depicted as cylinders connecting the centroids of molecular pairs, with
the cylinder radius proportional to the interaction energy magnitude.
The calculated total energy is − 359.4 kJ/mol. The electrostatic, polar
ization, dispersion, and repulsion energies are − 187.9 kJ/mol, − 54.2
kJ/mol, − 422.5 kJ/mol, and 305.2 kJ/mol, respectively. The dispersion
interaction energy significantly dominates over other interactions. For
this compound, dispersion energies contribute more to the total energy
than Coulombic energies. While the partial sum of dispersion energies
slightly exceeds that of electrostatic energies, the electrostatic energy
framework exhibits stronger interactions, as evidenced by thicker, larger
connecting cylinders. A more detailed inspection of the pairwise inter
action energies listed in Table 2 reveals that the most stabilizing Etot
contributions correspond to molecular pairs engaged in the head-to-tail
hydrogen-bonded dimers described in the crystal structure section. In
particular, the strongest interaction (Etot = − 31.8 kJ/mol, R = 10.92 Å)
is associated with the dimer sustained by cooperative N–H⋅⋅⋅N and
N–H⋅⋅⋅O hydrogen bonds between symmetry-related molecules. These
hydrogen-bonded dimers give rise to the thickest Etot cylinders
observed in the energy framework (Fig. 7), confirming that directional
electrostatic interactions dominate the primary stabilization pathways
within the crystal. Secondary Etot contributions, with interaction en
ergies in the range of − 25 to − 47 kJ/mol, originate from molecular pairs
linked by weaker N–H⋅⋅⋅O, C–H⋅⋅⋅O, and C–H⋅⋅⋅π interactions. These
contacts generate thinner cylinders in the energy framework and act as
auxiliary stabilizing forces that reinforce the three-dimensional packing.
In general, the topology of the energy framework directly mirrors the
hierarchy of noncovalent interactions identified crystallographically,
demonstrating that the crystal packing of compound 3a is governed by a
limited number of energetically dominant hydrogen-bonded dimers
complemented by a network of weaker dispersive contacts.
3.5. Independent gradient model based on Hirshfeld partition (IGMH)
study of noncovalent interactions
To further elucidate the nature and relative strength of the weak
noncovalent interactions identified in the crystal structure, the inde
pendent gradient model based on Hirshfeld partition (IGMH) approach
was employed. This advanced real-space method represents an
improved version of the original independent gradient model (IGM), as
it incorporates Hirshfeld atomic partitioning, thereby enabling a quan
titative decomposition of interactions into atomic and fragment contri
butions [28,29]. By relying explicitly on wavefunction information,
IGMH provides a more physically grounded description than the pro
molecular IGM, avoiding artificially inflated (“bulgy”) isosurfaces.
Moreover, it overcomes key limitations of conventional NCI (RDG)
analysis by clearly separating intra- and inter-fragment interactions. For
Fig. 9. (A) Optimized molecular structure of compound 3a, (B) HOMO and LUMO orbitals, and (C) molecular electrostatic potential (MEP) surface (B3LYP/
6–311++G(d,p)).
Table 3
HOMO and LUMO energies and global reactivity descriptors of
compound 3a calculated at the B3LYP/6–311++G(d,p) level.
Parameters include energy gap (ΔE), electronegativity (χ),
chemical potential (μ), global hardness (η), softness (S), and
electrophilicity index (ω), providing insight into the compound’s
chemical stability and reactivity.
Parameter Value
EHOMO (eV) –5.2659
ELUMO (eV) –1.3943
ΔE (ELUMO–EHOMO, eV) 3.8711
Electronegativity, χ (eV) 3.3301
Chemical potential, μ (eV) –3.3301
Global hardness, η (eV) 1.9358
Softness, S (eV⁻¹) 0.2583
Electrophilicity index, ω (eV) 2.8643
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![N3–H3⋅⋅⋅N11 interaction in dimer D1 exhibits a distinct blue region
embedded within the green envelope, reflecting a relatively stronger
attractive interaction. These observations are further supported by the
corresponding 2D scatter plots of δginter
versus sign(λ₂)ρ, which display
interaction-specific peaks located in negative sign(λ₂)ρ regions charac
teristic of hydrogen bonding. Notably, the most intense peak corre
sponds to dimer D1, for which δginter
reaches approximately 0.04 a.u.,
confirming the stronger nature of the N–H⋅⋅⋅N hydrogen bond relative to
the N/C–H⋅⋅⋅O interactions present in the structure.
To provide a quantitative assessment, the interfragment interactions
were further decomposed into atomic-pair contributions using the
IGMH-derived atomic-pair index (δGpair
), calculated on a high-quality
grid. This descriptor quantitatively reflects the contribution of a given
atomic pair to the total δginter
between the interacting fragments. As
shown in Fig. 6, the δGpair
values and their percentage contributions
clearly identify N3–H3⋅⋅⋅N11 as the strongest interaction, with a δGpair
value of 0.071 a.u., accounting for 9.43% of the total interfragment
interaction. Additionally, based on these quantitative results, the
hydrogen bonds in the crystal can be ranked in descending order of
strength as follows: N–H⋅⋅⋅N > N–H⋅⋅⋅O > C–H⋅⋅⋅O. Remarkably, this
hierarchy is fully consistent with the corresponding H⋅⋅⋅acceptor dis
tances derived from the X-ray crystal structure. Finally, the presence of a
weak C–H⋅⋅⋅π interaction is also detected, associated with the
H42A⋅⋅⋅N12 contact (D3), which exhibits a δGpair
value of 0.028 a.u.
(1.90%), confirming its minor yet non-negligible contribution to the
overall stabilization of the crystal packing.
3.6. DFT calculations on chemical reactivity
DFT computations at the B3LYP/6–311++G(d,p) level were per
formed to evaluate the electronic properties and chemical reactivity of
compound 3a. Frontier Molecular Orbitals (FMOs), including the
Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccu
pied Molecular Orbital (LUMO), provide insight into the electron-
donating and electron-accepting tendencies of the molecule. The en
ergy gap between HOMO and LUMO (ΔE) serves as a metric for chemical
reactivity, with larger gaps indicating higher kinetic stability and lower
reactivity [50]. The optimized molecular geometry of compound 3a is
shown in Fig. 9A. RMSD for experimental and optimized structures of 3a
over heavy atoms is 1.0574 Å was obtained, which is well within the
widely accepted threshold. The bond length and angle values calculated
using DFT methods were compared with the corresponding experi
mental values, as summarized in Table S1. An excellent agreement was
observed between the theoretical and experimental data, with a corre
lation coefficient (R2
) of 0.99 for both parameters (for example the
calculated dihedral angles for C23–N7–S1–O1, C20–C19–S1–O1, and
C18–C19–S1–O2 at the B3LYP/6–311++G(d,p) level are 177.61◦
,
155.99◦
, and − 157.80◦
, respectively, showing excellent correlation with
the corresponding experimental X-ray values of 177.21◦
, 159.96◦
, and
− 153.14◦
). The DFT-calculated HOMO and LUMO energies are –5.2659
eV and –1.3943 eV, respectively, yielding a HOMO-LUMO gap of 3.8711
eV, indicative of a chemically hard and stable molecule. HOMO electron
density is primarily localized on the pyrimidine, phenyl, and piperazine
rings, while LUMO density resides on the cyclobutene, benzenesulfo
namide, and pyrimidine rings (Fig. 9B). Next, Molecular Electrostatic
Potential (MEP) analysis was performed to visualize electron density
distribution and identify reactive sites. The MEP map (Fig. 9C) is
color-coded: red represents electron-rich regions (potential nucleophilic
sites), blue indicates electron-poor regions (potential electrophilic sites),
and green corresponds to non-polar or neutral areas. In compound 3a,
red regions surround the sulfonyl oxygen atoms (S=O), suggesting
probable electrophilic interaction sites, while hydrogen atoms on the
pyrimidine moiety occupy blue regions, prone to nucleophilic attack.
These features provide insight into potential protein-ligand interactions
and biological activity. Moreover, global reactivity descriptors were
calculated using Koopmans’ theorem, including electronegativity (χ),
chemical potential (μ), global hardness (η), softness (S), and electro
philicity index (ω), as summarized in Table 3. For compound 3a, the
high ΔE (3.8711 eV) and η (1.9358 eV) indicate significant stability and
low polarizability, while the electrophilicity index (ω = 2.8643 eV) re
flects the molecule’s electron-accepting ability. The low chemical soft
ness (S = 0.2583 eV⁻¹) further confirms molecular hardness and low
reactivity.
These findings are further corroborated by both experimental data
Table 4
Cell cycle populations analysed by PI-stained FACS analysis.
Control BAY1816032 TG-
101,209
3a 3d 3b 3c
G0/
G1
69.3 73.1 56.6 53.9 52.3 89.4 50.9
S 10.1 7.0 1.4 1.8 1.9 1.4 1.2
G2/
M
16.5 15.9 37.4 39.5 42.5 6.6 45.2
Fig. 11. BUB1B target validation, A. IC50 values of compounds to BUB1B enzymatic activity. B. Cellular Thermal Shift Assay (CETSA). Western blot analysis showing
thermal shift of BUB1B soluble protein by the compounds (top). Protein band intensities were measured and plotted as a histogram. * P < 0.05.
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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![properties, thereby warranting further preclinical evaluation.
CRediT authorship contribution statement
Negar Parvizi: Writing – review & editing, Writing – original draft,
Methodology, Investigation, Data curation. Mohamed El Hafi: Writing
– review & editing, Writing – original draft, Methodology. Melek Hajji:
Writing – original draft, Methodology, Formal analysis, Data curation.
Younos Bouzian: Writing – original draft, Methodology. Woonghee
Kim: Writing – review & editing, Writing – original draft, Validation,
Methodology, Formal analysis, Data curation. Mine Subaşioğlu:
Writing – review & editing, Writing – original draft, Methodology,
Investigation, Formal analysis, Data curation. Mehmet Ozcan: Writing
– review & editing, Writing – original draft, Validation, Methodology,
Formal analysis, Data curation. Hanan Al-Ghulikah: Formal analysis,
Data curation. Hasan Turkez: Supervision, Conceptualization. Adil
Mardinoglu: Supervision, Project administration, Funding acquisition,
Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal re
lationships, which may be considered as potential competing interests: A
patent application ‘NEW COMPOUNDS AND TREATMENT OF RENAL
CELL CARCINOMA’ has been filed and licensed to WO 2023/166,153
A1.
Acknowledgments
This work was financially supported by the Trustlife Therapeutics
and Knut and Alice Wallenberg Foundation (No: 72254). We would like
to thank Prof. Turan Öztürk at Istanbul Technical University for
providing access to the EDINBURGH INSTRUMENTS DS5 UV–Vis
Spectrophotometer in his laboratory, where the UV–Vis absorption
measurements were carried out. Also we would like to thank Aybüke
Gizem Yılmazbaş for her assistance in sample preparation for the me
dium stability tests
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.molstruc.2026.146659.
Data availability
Data will be made available on request.
References
[1] R.L. Siegel, K.D. Miller, H.E. Fuchs, A. Jemal, Cancer statistics, CA Cancer J. Clin.
71 (2021) 7–33, https://doi.org/10.3322/caac.21654.
[2] B. Shuch, A. Amin, A.J. Armstrong, J.N. Eble, V. Ficarra, A. Lopez-Beltran,
G. Martignoni, B.I. Rini, A. Kutikov, Understanding pathologic variants of renal cell
carcinoma: distilling therapeutic opportunities from biologic complexity, Eur. Urol.
67 (2015) 85–97, https://doi.org/10.1016/j.eururo.2014.04.029.
[3] H. Koul, J.S. Huh, K.O. Rove, L. Crompton, S. Koul, R.B. Meacham, J.F. Kim,
Molecular aspects of renal cell carcinoma: a review, Am. J. Cancer Res. 1 (2001).
www.ajcr.us/ISSN:2156-6976/ajcr0000020.
[4] A. Sánchez-Gastaldo, E. Kempf, A. González Del Alba, I. Duran, Systemic treatment
of renal cell cancer: a comprehensive review, Cancer Treat. Rev. 60 (2017) 77–89,
https://doi.org/10.1016/j.ctrv.2017.08.010.
[5] K.A. Lawson, W.M. Linehan, De-clear cell differentiated renal cell carcinoma — a
new therapeutic target, Nat. Rev. Urol. 22 (2025) 73–74, https://doi.org/10.1038/
s41585-024-00911-w.
[6] A. Mardinoglu, J. Nielsen, Systems medicine and metabolic modelling, J. Intern.
Med. 271 (2012) 142–154, https://doi.org/10.1111/j.1365-2796.2011.02493.x.
Fig. 17. The ligand TG-10,129 property trajectory of the BUB1B complex with TG-10,129 during the 200 ns simulation.
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
17](https://image.slidesharecdn.com/trsm005-a06-260615081048-9bb4de92/85/Novel-pyrimidine-derivatives-as-potent-BUB1B-inhibitors-for-clear-cell-renal-cell-carcinoma-From-synthesis-and-characterization-to-docking-insights-and-therapeutic-validation-17-320.jpg)
- 18 / 18
![[7] A. Mardinoglu, B.Ø. Palsson, Nat. Rev. Genet genome-scale models in human
metabologenomics, Nat. Rev. Genet. 26 (2025) 123–140, https://doi.org/10.1038/
s41576-024-00768-0.
[8] A. Mardinoglu, J. Nielsen, The impact of systems medicine on human health and
disease, Front. Physiol. 7 (2016), https://doi.org/10.3389/fphys.2016.00552.
[9] A. Mardinoglu, J. Boren, U. Smith, M. Uhlen, J. Nielsen, Systems biology in
hepatology: approaches and applications, Nat. Rev. Gastroenterol. Hepatol. 15
(2018) 365–377, https://doi.org/10.1038/s41575-018-0007-8.
[10] X. Li, K. Shong, W. Kim, M. Yuan, H. Yang, Y. Sato, H. Kume, S. Ogawa, H. Turkez,
S. Shoaie, J. Boren, J. Nielsen, M. Uhlen, C. Zhang, A. Mardinoglu, Prediction of
drug candidates for clear cell renal cell carcinoma using a systems biology-based
drug repositioning approach, EBioMedicine 78 (2022) 103963, https://doi.org/
10.1016/j.ebiom.2022.103963.
[11] Z. Kilic-Kurt, N. Ozmen, F. Bakar-Ates, Synthesis and anticancer activity of some
pyrimidine derivatives with aryl urea moieties as apoptosis-inducing agents,
Bioorg. Chem. 101 (2020) 104028, https://doi.org/10.1016/j.
bioorg.2020.104028.
[12] V. Sharma, N. Chitranshi, A.K. Agarwal, Significance and biological importance of
pyrimidine in the microbial world, Int. J. Med. Chem. 2014 (2014) 1–31, https://
doi.org/10.1155/2014/202784.
[13] V. Finger, M. Kufa, O. Soukup, D. Castagnolo, J. Roh, J. Korabecny, Pyrimidine
derivatives with antitubercular activity, Eur. J. Med. Chem. 246 (2023) 114946,
https://doi.org/10.1016/j.ejmech.2022.114946.
[14] T.J. Yeh, L.P. Chan, H.T. Tsai, C.M. Hsu, S.F. Cho, M.R. Pan, Y.C. Liu, C.J. Huang,
C.W. Wu, J.S. Du, H.C. Wang, The overall efficacy and outcomes of metronomic
tegafur-uracil chemotherapy on locally advanced head and neck squamous cell
carcinoma: a real-world cohort experience, Biology 10 (2021) 168, https://doi.
org/10.3390/biology10020168.
[15] Y. Bouzian, M. El Hafi, N. Parvizi, W. Kim, M. Subaşioğlu, M. Ozcan, H. Turkez,
A. Mardinoglu, Design and evaluation of novel inhibitors for the treatment of clear
cell renal cell carcinoma, Bioorg. Chem. 151 (2024) 107597, https://doi.org/
10.1016/j.bioorg.2024.107597.
[16] M. El Hafi, Y. Bouzian, N. Parvizi, W. Kim, M. Subaşioğlu, M. Ozcan, H. Turkez,
A. Mardinoglu, Synthesis and biological assessment of BUB1B inhibitors for the
treatment of clear cell renal cell carcinoma, Eur. J. Med. Chem. 285 (2025)
117247, https://doi.org/10.1016/j.ejmech.2025.117247.
[17] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, OLEX2 :
a complete structure solution, refinement and analysis program, J. Appl.
Crystallogr. 42 (2009) 339–341, https://doi.org/10.1107/S0021889808042726.
[18] L.J. Bourhis, O.V. Dolomanov, R.J. Gildea, J.A.K. Howard, H. Puschmann, The
anatomy of a comprehensive constrained, restrained refinement program for the
modern computing environment – Olex2 dissected, Acta Crystallogr, A Found Adv.
71 (2015) 59–75, https://doi.org/10.1107/S2053273314022207.
[19] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr. C.
Struct. Chem. 71 (2015) 3–8, https://doi.org/10.1107/S2053229614024218.
[20] C.F. Macrae, I. Sovago, S.J. Cottrell, P.T.A. Galek, P. McCabe, E. Pidcock,
M. Platings, G.P. Shields, J.S. Stevens, M. Towler, P.A. Wood, Mercury 4.0: from
visualization to analysis, design and prediction, J. Appl. Crystallogr. 53 (2020)
226–235, https://doi.org/10.1107/S1600576719014092.
[21] A.L. Spek, Structure validation in chemical crystallography, Acta Crystallogr. D. 65
(2009) 148–155, https://doi.org/10.1107/S090744490804362X.
[22] L.J. Farrugia, WinGX and ORTEP for Windows: an update, J. Appl. Crystallogr. 45
(2012) 849–854, https://doi.org/10.1107/S0021889812029111.
[23] P.R. Spackman, M.J. Turner, J.J. McKinnon, S.K. Wolff, D.J. Grimwood,
D. Jayatilaka, M.A. Spackman, CrystalExplorer : a program for Hirshfeld surface
analysis, visualization and quantitative analysis of molecular crystals, J. Appl.
Crystallogr. 54 (2021) 1006–1011, https://doi.org/10.1107/
S1600576721002910.
[24] J. and others Frisch, MJea and Trucks, GW03 and Schlegel, HB e al and Scuseria,
GE and Robb, MA and Cheeseman, JR and Montgomery Jr, JA and Vreven, TKKN
and Kudin, KN and Burant, Gaussian 03, revision C. 02, (2004).
[25] R. Dennington, T.A. Keith, J.M. Millam, GaussView Version 6, Semichem Inc,
Shawnee Mission KS, 2016.
[26] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange,
J. Chem. Phys. 98 (1993) 5648–5652, https://doi.org/10.1063/1.464913.
[27] R. Krishnan, J.S. Binkley, R. Seeger, J.A. Pople, Self-consistent molecular orbital
methods. XX. A basis set for correlated wave functions, J. Chem. Phys. 72 (1980)
650–654, https://doi.org/10.1063/1.438955.
[28] T. Lu, Q. Chen, Independent gradient model based on Hirshfeld partition: a new
method for visual study of interactions in chemical systems, J. Comput. Chem. 43
(2022) 539–555, https://doi.org/10.1002/jcc.26812.
[29] T. Lu, Visualization analysis of covalent and noncovalent interactions in real space,
Angew. Chem., Int. Ed. 64 (2025), https://doi.org/10.1002/anie.202504895.
[30] S. Grimme, J. Antony, S. Ehrlich, H. Krieg, A consistent and accurate ab initio
parametrization of density functional dispersion correction (DFT-D) for the 94
elements H-Pu, J. Chem. Phys. 132 (2010), https://doi.org/10.1063/1.3382344.
[31] J.-D. Chai, M. Head-Gordon, Long-range corrected hybrid density functionals with
damped atom–atom dispersion corrections, Phys. Chem. Chem. Phys. 10 (2008)
6615, https://doi.org/10.1039/b810189b.
[32] T.H. Dunning, Gaussian basis sets for use in correlated molecular calculations. I.
The atoms boron through neon and hydrogen, J. Chem. Phys. 90 (1989)
1007–1023, https://doi.org/10.1063/1.456153.
[33] H. Mtiraou, A. Ghabi, H. Al-Ghulikah, M.A. Habib, M. Hajji, Structural and
chemical reactivity insights of a benzimidazolidinone-based N-heterocycle: a
multiapproach quantum-chemical study, Chem. Heterocycl. Compd. N. Y 60
(2024) 611–616, https://doi.org/10.1007/s10593-025-03384-w.
[34] M. Hajji, A. Haouas, N. Abad, T. Guerfel, The unconventional noncovalent
interactions control: crystallographic and theoretical analyses of the crystalline
structure of 1,1′-(1-Chloro-4-methoxyphenyl)dibenzene as a case study,
ChemistrySelect 8 (2023), https://doi.org/10.1002/slct.202302624.
[35] S. Ibrahim, A. Ghabi, N. Amiri, H. Mtiraoui, M. Hajji, R. Bel-Hadj-Tahar,
M. Msaddek, Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked
benzimidazolone derivatives: DFT calculations and biological activities, Monatsh.
Chem. 152 (2021) 523–535, https://doi.org/10.1007/s00706-021-02764-0.
[36] T. Lu, F. Chen, Multiwfn: a multifunctional wavefunction analyzer, J. Comput.
Chem. 33 (2012) 580–592, https://doi.org/10.1002/jcc.22885.
[37] T. Lu, A comprehensive electron wavefunction analysis toolbox for chemists,
Multiwfn, J. Chem. Phys. 161 (2024), https://doi.org/10.1063/5.0216272.
[38] W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics, J. Mol.
Graph. 14 (1996) 33–38, https://doi.org/10.1016/0263-7855(96)00018-5.
[39] T. Williams, C. Kelley, Gnuplot: An Interactive Plotting Program (version 6.0.4),
(2025). http://gnuplot.info/.
[40] Y. Yang, K. Yao, M.P. Repasky, K. Leswing, R. Abel, B.K. Shoichet, S.V. Jerome,
Efficient exploration of chemical space with docking and deep learning, J. Chem.
Theory. Comput. 17 (2021) 7106–7119, https://doi.org/10.1021/acs.
jctc.1c00810.
[41] L. Schrödinger, LigPrep (Version Release 2023-4), Schrödinger, LLC, New York,
NY, 2023.
[42] J.C. Shelley, A. Cholleti, L.L. Frye, J.R. Greenwood, M.R. Timlin, M. Uchimaya,
Epik: a software program for pK(a) prediction and protonation state generation for
drug-like molecules, J. Comput. Aided. Mol. Des. 21 (2007) 681–691, https://doi.
org/10.1007/s10822-007-9133-z.
[43] 2023 Schrödinger Release 2023-3: Protein Preparation Wizard; Epik, Schrödinger,
LLC, New York, NY, Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger,
LLC, New York, NY, 2023.
[44] K.J. Bowers, E. Chow, H. Xu, R.O. Dror, M.P. Eastwood, B.A. Gregersen, F.
D. Sacerdoti, Scalable algorithms for molecular dynamics simulations on
commodity clusters, in: Paper presented at the Proceedings of the 2006 ACM/IEEE
Conference on Supercomputing 84, 2016, https://doi.org/10.1145/
1188455.1188544.
[45] V.M. Bolanos-Garcia, T.L. Blundell, BUB1 and BUBR1: multifaceted kinases of the
cell cycle, Trends. Biochem. Sci. 36 (2011) 141–150, https://doi.org/10.1016/j.
tibs.2010.08.004.
[46] M. Hajji, A. Haouas, N. Abad, T. Guerfel, The unconventional noncovalent
interactions control: crystallographic and theoretical analyses of the crystalline
structure of 1,1′-(1-Chloro-4-methoxyphenyl)dibenzene as a case study,
ChemistrySelect 8 (2023), https://doi.org/10.1002/slct.202302624.
[47] M.A. Spackman, D. Jayatilaka, Hirshfeld surface analysis, Cryst. Eng. Comm. 11
(2009) 19–32, https://doi.org/10.1039/B818330A.
[48] N. Hajji, N. Abad, M.H. Habib, S.M.H. Elmgirhi, T. Guerfel, Computational
chemistry methods for modelling non-covalent interactions and chemical
reactivity— an overview, J. Indian Chem. Soc. 98 (2021) 100208, https://doi.org/
10.1016/j.jics.2021.100208.
[49] M.J. Turner, J.J. Mckinnon, D. Jayatilaka, M.A. Spackman, Visualisation and
characterisation of voids in crystalline materials, Cryst. Eng. Comm. 13 (2011)
1804–1813, https://doi.org/10.1039/C0CE00683A.
[50] M. Lamsayah, M. Khoutoul, A. Takfaoui, R. Touzani, High liquid–liquid extraction
selectivity of Fe(II) and Pb(II) with TD-DFT theoretical calculations of long chain
acid pyrazole- and triazole-based ligands, Cogent. Chem. 2 (2016) 1230359,
https://doi.org/10.1080/23312009.2016.1230359.
N. Parvizi et al. Journal of Molecular Structure 1374 (2026) 146659
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Novel pyrimidine derivatives as potent BUB1B inhibitors for clear cell renal cell carcinoma: From synthesis and characterization to docking insights and therapeutic validation
A B S T R A C T Clear cell Renal Cell Carcinoma (ccRCC) is the most common subtype of renal malignancy and remains a major clinical challenge. This challenge underscores the urgent need to identify novel molecules with potential as therapeutic targets in ccRCC. Therefore, in this study, four novel pyrimidine-based derivatives of TG-101,209 (3a-d) were designed and synthesized as potential inhibitors of BUB1B (Budding Uninhibited by Benzimidazoles 1 Mitotic Checkpoint Serine/Threonine Kinase B), a mitotic checkpoint kinase involved in ccRCC pathogenesis. Complete structural characterization of all synthesized compounds was achieved using Fouriertransform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H and 13C NMR), and mass spectrometry (MS). The crystalline architecture and intermolecular interactions of compound 3a were further elucidated by single-crystal X-ray diffraction. Theoretical computations were performed using Density Functional Theory (DFT) at the B3LYP/6–311++G(d,p) level of theory to investigate the electronic and structural properties of the most active compound. The computed IR spectrum of compound 3a showed excellent agreement with experimental data, supporting the structural findings from X-ray analysis. Hirshfeld surfaces (HS) analyses were carried out to visualize the intermolecular interactions in the crystal packing of 3a. This analysis highlighted key N—H⋅⋅⋅O and N—H⋅⋅⋅N hydrogen bonds, which were found to be in excellent agreement with the experimental single-crystal X-ray diffraction data. Multiple noncovalent interactions (N–H⋅⋅⋅O/N, C–H⋅⋅⋅O, C–H⋅⋅⋅π) were identified in the solid state and quantitatively examined using Independent Gradient Model based on Hirshfeld partition (IGMH) analysis, confirming N–H⋅⋅⋅N as the strongest interaction. In vitro cytotoxicity assays on Caki-1 cells revealed that compounds 3a, 3c, and 3d exerted significant antiproliferative effects. Compound 3a exhibited superior BUB1B inhibition compared to the other derivatives, eliciting a robust apoptotic response as evidenced by enhanced PARP cleavage and caspase activation. Molecular docking and dynamics studies revealed stable and favorable binding affinity of the compound 3a within the BUB1B active site, consistent with experimental observations. The combined experimental and computational findings indicate that these pyrimidine derivatives, particularly compound 3a, act as promising BUB1B inhibitors with potential therapeutic relevance in ccRCC therapy.
