MOLSTRUC Unveiling Structural Features, Chemical Reactivity, and Bioactivity of a Newly Synthesized Purine Derivative through Crystallography and Computational Approaches/ Full article.pdf

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Unveiling Structural Features, Chemical Reactivity, and Bioactivity of a Newly
Synthesized Purine Derivative through Crystallography and Computational
Approaches
Nadeem Abad a,#
, Shafeek Buhlak a,#
, Melek Hajji b,⁎
, Sana Saffour a
, Jihane Akachar a
, Yunus
Kesgun a
, Hanan Al-Ghulikah c
, Essam Hanashalshahaby a
, Hasan Turkez d
, Adil Mardinoglu e,f,⁎
a
Trustlife Labs, Drug Research & Development Center, 34774 Istanbul, Turkiye.
b
Research Unit: Electrochemistry, Materials and Environment, University of Kairouan, 3100
Kairouan, Tunisia.
c
Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University,
P.O. Box 84428, Riyadh 11671, Saudi Arabia.
d
Department of Medical Biology, Faculty of Medicine, Atatürk University, Erzurum, Turkiye.
e
Science for Life Laboratory, KTH-Royal Institute of Technology, SE-17165 Stockholm,
Sweden.
f
Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences,
King's College London, London, SE1 9RT, United Kingdom.
#These authors contributed equally.
*Corresponding authors:
Adil Mardinoglu (adilm@scilifelab.se) & Melek Hajji (melek.hajji@ipeik.u-kairouan.tn)
Revised Manuscript Click here to view linked References

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Abstract
We introduce the synthesis and characterization of a novel purine derivative, 2-amino-6-chloro-
N,N-diphenyl-7H-purine-7-carboxamide. X-ray crystallography was utilized to elucidate its
molecular and crystal structure. A comprehensive crystal packing analysis uncovered a network
of diverse intermolecular interactions, including classical and unconventional hydrogen bonding.
Remarkably, a unique halogen···π (C—Cl···π(ring)) interaction was identified and theoretically
analyzed within a multi-approach quantum mechanics (QM) framework, revealing its lone-pair⋯π
(n→π*) nature. Furthermore, insights into the electronic and chemical reactivity properties are
provided by means of Conceptual Density Functional Theory (CDFT) at wB97X-D/aug-cc-pVTZ
level. The compound's drug-likeness, pharmacokinetics, and toxicology profiles are assessed using
ADMETlab 2.0. Finally, molecular docking simulations were conducted to evaluate its bioactivity
as a potential cyclooxygenase-2 (COX-2) inhibitor. This study significantly advances our
understanding of purine structure and reactivity, offering valuable insights for the development of
targeted purine-based COX-2 inhibitors and anticancer therapeutics.
Keywords: Purines; Crystallographic analysis; Noncovalent interactions; Density functional
theory; ADMET; COX-2 inhibition

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1. Introduction
Purines, a double-ringed heterocycles resulting from the fusion of six-membered pyrimidine ring
and five-membered imidazole ring, play essential roles in cellular processes such as DNA, RNA,
and energy metabolism. Recently, purines have emerged as intriguing candidates for anticancer
research [1–6]. The structures of some anticancer active compounds containing-purines are shown
in Fig. 1. A comprehensive review identified diverse purine-heterocyclic hybrids as scaffolds for
anticancer drugs from the past decade [1]. Ma et al. have synthesized 6-mercapto-purine derivative
(compound 4 in Fig. 1), exhibiting antiproliferative effects against HepG2 and A2780 cancer cells
[2]. Moreover, Attia et al. unveiled a novel class of purine derivatives with potent antiproliferative
activity, particularly compound 4b (identified as 3 in Fig. 1) demonstrated effectiveness against
colon, hepatic, and breast cancer cells [3]. Nageswara Kode et al. disclosed the synthesis and
antitumor activity of a purine-based compound (compound 2 in Fig. 1), exhibiting efficacy against
various cancer cell lines [6]. Furthermore, Salas et al. reported a highly effective 2,6,9-
trisubstituted purine derivative (7h) inducing apoptosis and causing S-phase cell cycle arrest in
HL-60 cells, with notable efficacy in four out of seven tested cancer cell lines [4]. In this context,
motivated by cyclooxygenase-2 (COX-2) overexpression in various cancers, Hassan et al.
designed and synthesized a purine derivative, (compound 1 in Fig. 1), which exhibited central
heterocyclic scaffold bearing two vicinal phenyl and different heteroaryl moieties [5]. The
compound demonstrated potent antitumoral property against leukaemia, ovarian, and breast
carcinoma cell lines, exhibiting both antiproliferative and noteworthy COX-2 inhibitory effects. It
is noteworthy that various studies highlight the correlation of the cyclooxygenase enzyme with
numerous types of cancers [7–9]. For example, COX-2 is overexpressed in pancreatic, breast,
colorectal, stomach, and lung carcinoma [9]. As a result, COX-2 is acknowledged as a pivotal
focus for the advancement of novel anticancer treatments. It is, therefore, highly valuable for the
scientific community to acquire insights into how purine scaffolds, recognized as privileged
structures, might be utilized in forthcoming endeavours for the systematic development of purine-
containing anticancer agents.
This study reports the synthesis of a novel purine derivative, 2-amino-6-chloro-N,N-diphenyl-7H-
purine-7-carboxamide, achieved through a simple reaction of 6-chloro-7H-purin-2-amine with
diphenylcarbamic chloride. We elucidated the molecular and crystal structure of the prepared
derivative using X-ray crystallography. Notably, the structure exhibits a C—Cl···π interactions
between the chlorine atom and the pyrimidine heterocycle ring. These halogen···π interactions,
have frequently observed in diverse systems, contribute significantly to the stability of halogenated
organic compounds, supramolecular assemblies, and solid-state structures [10–14]. Their
importance extends to biomolecules as well [15,16], with documented roles in pyrimidine and
purine-containing systems [17]. In our case, this C—Cl···π interaction offers a valuable
opportunity to gain deeper insights into this stabilizing force through a real-world solid-state
structure. To delve beyond geometric features, we employed multi-approach quantum mechanics
(QM) analysis to elucidate the nature (σ-Hole⋯π or lone pair⋯π) and energetics of this interaction.

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Moreover, we employed Conceptual Density Functional Theory (CDFT), molecular electrostatic
potential (MEP), and quantitative "atomic local molecular surface" analysis to enhance our
comprehension of the electronic and chemical reactivity properties of the synthesized compound.
Additionally, in silico investigations were conducted to evaluate its drug-likeness,
pharmacokinetic, and toxicological profiles, utilizing the updated ADMETlab 2.0 integrated
online platform [18]. Finally, the in-silico bioactivity of the synthesized compound concerning its
interactions with the COX-2 enzyme is also assessed through molecular docking approach. We
anticipate that this research will contribute to the structure and reactivity of purines, thereby
advancing our knowledge for development of selective purine-based COX-2 inhibitors and
anticancer therapeutics.
Fig. 1. Some purine-based compounds with potential activity as anticancer.
2. Material and Methods
2.1 Synthesis of the investigated compound: 2-amino-6-chloro-N,N-diphenyl-7H-purine-7-
carboxamide
The title compound 2-amino-6-chloro-N,N-diphenyl-7H-purine-7-carboxamide has been prepared
starting from 200 mg of 6-chloro-7H-purin-2-amine which was stirred with K2CO3 (417.6 mg) and
TBAB (36 mg, 0.11 mmol) in THF for 15 minutes. Subsequently, diphenylcarbamic chloride (462
mg) was introduced into the reaction mixture and stirred for a duration of 12 hours. The
progression of the reaction was monitored using thin-layer chromatography (TLC). At the end of
the reaction, a simple decantation is carried out, then the solvent is evaporated under reduced
pressure, the residue acquired is chromatograph on a column of gel silica using a mixture of hexane
and ethyl acetate as eluent and recrystallized by EtOH and obtained (80%) as white powder.
Eluent (Hexane/ Ethyle acetate (50/50%) Yield=80%, (°F) = 120-122 [(°C) = 48-50], 1H-NMR
(DMSO-d6 400MHz) δ ppm:8.240 (s, 1H, N-CH-N); 7.388-7.47 (m, 10H, CHarom) 7.127 (s, 2H,

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NH2). 13C-NMR (101MHz, DMSO-d6) δppm: 160.203 (C=O), 152.659 (C-Cl), 149.636 (C-NH2),
148.135, 141.676, 140.526, 129.333, 127.535, 126.874, 121.975. LCMS/MS: m/z 365.01 [M+H]+
.
Scheme 1 Synthesis pathway and chemical structure of the investigated compound: 2-amino-6-
chloro-N,N-diphenyl-7H-purine-7-carboxamide.
2.2 Single crystal X-ray diffraction (SCXRD)
The molecular structure of prepared compound was determined using single-crystal X-ray
diffraction. Data were collected at 299.38 K on a Bruker APEX-II CCD diffractometer. Using
Olex2 1.3 [19], the structure was solved with the SHELXT 2014/5 structure solution program [20]
using Intrinsic Phasing and refined with the SHELXL refinement package [21] using Least Squares
minimization. Identification and visualization of weak interactions was made utilizing Platon [22]
and Mercury 4.0 [23]. Additional details on crystal data and structure refinement can be found in
Table 1.
Table 1 Crystal data and details of the structure determination.
CCDC No. 2325198
Empirical formula C18H13ClN6O
Formula weight 364.79
Temperature/K 299.38
Crystal system, Space group Monoclinic, P21/c
a, b, c /Å 9.279(2), 24.458(5), 17.659(4)
α, β, γ /° 90, 93.883(4), 90
Volume/Å3
, Z 3998.3(15), 8
ρcalcg/cm3
, μ/mm-1
1.212, 0.209
F(000) 1504.0
Crystal size/mm3
0.133 × 0.122 × 0.107
Radiation MoKα (λ = 0.71073)
2θ range for data collection/° 2.848 to 54.962

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Index ranges -11 ≤ h ≤ 12, -31 ≤ k ≤ 31, -22 ≤ l ≤ 22
Reflections collected 41070
Independent reflections 9118 [Rint = 0.1039, Rsigma = 0.1146]
Data/restraints/parameters 9118/0/485
Goodness-of-fit on F2
0.918
Final R indexes [I>=2σ (I)] R1 = 0.0583, wR2 = 0.1392
Final R indexes [all data] R1 = 0.1550, wR2 = 0.1768
Largest diff. peak/hole / e Å-3
0.27/-0.23
2.3 Quantum chemical simulations
All density functional theory (DFT) simulations were carried out using GaussView 6.0 and
Gaussian 09, Rev D.01 software [24,25]. The geometry optimizations were performed at
ωB97xD/aug-cc-pVDZ level. The wB97X-D hybrid functional combines long-range corrections
with Grimme's D2 dispersion model [26]. When used in conjunction with the aug-cc-pVXZ
Dunning correlation-consistent polarized valence basis set, it yields accurate results for various
noncovalent interactions, striking a balance between accuracy and computational cost [27–32].
Subsequent single-point energy computations were carried out considering dispersion effects, at
ωB97xD/aug-cc-pVTZ level. All interaction energies were obtained with supermolecule method
and underwent correction for basis set superposition errors (BSSE) through the counterpoise
procedure [33]. The quantum theory of atoms in molecules (QTAIM) [34] and independent
gradient model (IGM) [35] was accomplished, from the DFT-based converged wave functions,
using Multiwfn 3.8 [36] and visualized by VMD 1.9.3 [37]. The electron localization function
(ELF) [38] and the 1D profiles of the electron density (ED) and electrostatic potential (ESP)
functions [39] were calculated in the Multiwfn program. Natural bond orbital (NBO) [40] findings
were obtained using the Gaussian-NBO 3.1 module, implemented in Gaussian program. The
Molecular Electrostatic Potential (MEP) surfaces were performed with GaussView 6.0 at 0.001
a.u. isosurface, combining with quantitative "Atomic local molecular surface" analysis. Global
chemical reactivity indices were predicted through chemical reactivity theory [27]. Technical and
theoretical background of these approaches is extensively detailed in our previous works
[27,29,41].
2.4 ADMET predictions
The drug-likeness and toxicity of the synthesized compound underwent a comprehensive
assessment using "ADMET Evaluation" module of the integrated online platform ADMETlab 2.0
(https://admetmesh.scbdd.com/) [18]. The compound's SMILES string,
“NC1NC(Cl)C2C(N1)N(CN2)C(N(C1CCCCC1) C1CCCCC1)O”, was generated with
ChemDraw Professional 17.1, and served as input. A key feature of ADMETlab is its extensive
coverage, encompassing 75 endpoints across 17 physicochemical, 23 pharmacokinetic, 27

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toxicologic, and 13 medicinal chemistry parameters. Additionally, the user-friendly interface
enables a rapid and nuanced assessment of the ADMET profile, providing detailed numerical
predictions, supported with color-coded decision states (green: excellent, yellow: medium, red:
poor) and a radar plot considering 13 key properties.
2.5 In silico docking experiments
The crystallographic data for the cyclooxygenase-2 (COX-2) enzyme was accessed from the
Protein Data Bank (PDB ID: 3LN1) [42]. The ligands and one monomer of COX-2 were prepared
for docking via standard methods using AutoDock Tools 1.5.6 [43]. The docking was carried out
using a flexible ligand docking approach with AutoDock Vina 1.2.0 [44]. A three-dimensional
affinity grid (52×40×40) with a grid point spacing of 0.375 Å was positioned around the COX-2
active site. The best-ranked conformation complex was analyzed using PyMOL 2.5 [45] assisted
by ChatGPT-PyMOL Plugin [46], and through LigPlot+ V.2.2.8 program [47].
3. Results and Discussion
3.1 Structural analysis
The asymmetric unit comprises two independent molecules characterized by differences in the
planarity of their central cores and the orientations of substituents (Fig. 2). The purine moiety
containing Cl2 exhibits slight non-planarity, evident in the dihedral angle of 1.55(16) between the
constituent rings, with a total puckering amplitude (Q) of 0.033(3) Å. In the second molecule, the
corresponding angle and total puckering amplitude are 0.94(15)° and 0.037(3) Å, respectively.
Furthermore, the C4-N5-C6-N6 torsion angle is 140.5(3)°, while the C20-N11-C24-N12 angle is
-138.9(3)°. Additionally, the (C7/C8/C9/C10/C11/C12) and (C13/C14/C15/C16/C17/C18)
benzene rings are inclined to each other by 83.55(18)°, whereas the corresponding angle in the
second molecule is 73.48(17)°. All bond lengths, however, remain within expected ranges.
Fig. 2. ORTEP-Illustration of the asymmetric unit of the crystal structure, showing two
independent molecules with their labelling scheme and 50% probability ellipsoids. Atom colours:
white (H), grey (C), blue (N), red (O) and green (Cl).

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In the crystal, the two molecules within the asymmetric unit form connections through C5—
H5···O2 and N7—H7B···O1 hydrogen bonds (Fig. S1). Crystal packing analysis reveals a
network of diverse intermolecular interactions that stabilize the compound's structure (Table 2),
which are categorized as: classical hydrogen bonding (N1—H1A···N2 and N1—H1B···O2),
unconventional weak hydrogen bonds (C10—H10···N8 and C23—H23···O1), C/N–H···π(ring)
interactions (N1—H1B···π, C32—H32···π and C34—H34··· π). Although aromatic rings are
present, no true π-π stacking interactions were observed due to all inter-ring plane distances
exceeding the typical limit of 3.8 Å [48,49]. The closest centroid separation was 4.188(2) Å,
exceeding this threshold, so these interactions were not included in Table 2. Interestingly, a
halogen⋯π(ring) interaction, C2—Cl1⋯π(pyrimidine), was observed, offering an additional
stabilizing force (see Table 2, Fig. 3). The Cl⋯π (centroid) distance is 3.556 Å. The distances
between Cl and the nearest atom from pyrimidine ring (3.237 Å for Cl1⋯C19) is shorter than the
sum of the van der Waals radii of the involved atoms (Cl = 1.75 Å, C = 1.70 Å). The angles
surrounding chlorine (C2–Cl–centroid = 141.18°, C2–Cl–(π-plane) = 71.82 °) deviate from the
anticipated directional characteristics of classical halogen bonds. The nature and energetics of this
halogen⋯π interaction are explored further in subsequent sections using quantum mechanics DFT-
based approaches.
Fig. 3. Illustration of intermolecular halogen⋯π(ring) interaction, C2—Cl1⋯π(pyrimidine) within
the crystalline structure. Interaction between Cl1 atom and centroid of pyrimidine ring is depicted
by dashed magenta lines. Atom colours: white (H), grey (C), blue (N), red (O) and green (Cl).
Table 2 Geometric parameters (A, °) of potential noncovalent interactions.
Hydrogen Bonding:
D—H…A a
D—H H…A D…A D—H…A
N1—H1A···N2 (i)
0.78(4) 2.38(4) 3.153(4) 174(4)
N1—H1B···O2 (ii)
0.93(3) 2.48(3) 3.345(4) 155(3)
C10—H10···N8 (iii)
0.93 2.58 3.444(5) 154

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N7—H7B···O1 1.00(5) 2.21(5) 3.165(4) 161(4)
C5—H5···O2 0.93 2.40 3.235(4) 149
C23—H23···O1(iv)
0.93 2.43 3.293(4) 155
π-(ring) interactions:
C/N—H…Cg H…Cg C/N…Cg C—H…Cg
N1—H1B···Cg1 3.0(3) 3.600(4) 124(2)
C32—H32···Cg2 2.77 3.620(3) 153
C34—H34···Cg3 2.96 3.730(4) 141
C—X…Cg H…Cg C—X, π b
C—X…Cg
C2—Cl1···Cg4 3.556(2) 71.82 141.18(1)
a
A = acceptor, D = donor. b
C—X, π is the angle of the C-Cl bond with the π-plane. Cg = centroid of the rings: Cg1,
Cg2, Cg3 and Cg4 are the centroids of the rings (C20-C21-N10-C23-N11), (C13-C14-C15-C16-C17-C18), (C1-N2-
C2C3-C4-N3) and (C19-N8-C20-C21-C22-N9), respectively. Symmetry codes: (i): 2-x,1-y,1-z (ii): 1+x,y,z (iii): x,1/2-
y,1/2+z (iv): -1+x,y,z
3.2 Quantum mechanical investigations on noncovalent interactions
Quantum chemical approaches are widely used to quantify and visualize noncovalent interactions
within different molecular systems [50–55]. Herein, we conducted a comprehensive quantum
mechanics study, primarily focused on exploring the energetics and nature (σ-Hole⋯π or lone
pair⋯π) of the crystallographically observed halogen⋯π interaction. This study utilized
dispersion-corrected density functional theory (DFT-D) at the ωB97xD/aug-cc-pVTZ level. To
achieve this, a dimeric sub-structure was retrieved from the crystallographic structure and partially
optimized. The optimization involved all hydrogen atoms while keeping the positions of other
atoms frozen. This approach allowed the assessment of the interaction in its solid-state
configuration, avoiding geometric rearrangement and ensuring a converged wavefunction.
Initially, a Quantum Theory of 'Atoms-in-Molecules' (QTAIM) analysis was performed, and the
resulting molecular graph is illustrated in Fig. 4. The interaction between C2—Cl1⋯π(pyrimidine)
is evident, with a bond critical point (BCP) and bond path connecting Cl1 and the C19 atom of the
pyrimidine ring. The calculated electron density (ρ) and Laplacian (∇2
ρ) values of 0.0044 a.u. and
0.0284 a.u., respectively, indicate a closed-shell type interaction. Subsequently, Independent
Gradient Model (IGM) analysis was conducted, revealing an extended green IGM isosurface in
the contact region, signifying the weak dispersive character of this interaction (Fig. 4).

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Fig. 4. (Above) QTAIM molecular graph showing the halogen⋯π(ring) intermolecular interaction:
Small yellow spheres and magenta lines represent bond critical points and bond paths, respectively.
Electron density (ρ) and Laplacian (∇2
ρ) of electron density values at bond critical point (BCP),
are provided in a.u. (Below) IGM-graph: isosurfaces are shown in green colour (Isovalue = 0.01
a.u.). Atom colours: white (H), silver (C), red (O), blue (N) and green (Cl).
Furthermore, the interaction energy for the homodimer was computed, accounting for the basis set
superposition error (BSSE) through counterpoise correction. The resulting ∆EBSSE value was -3.69
kcal/mol (Fig. 5). To further explore the characteristics of this intermolecular pattern, an
examination was conducted using Natural Bond Orbital (NBO) analysis, considering orbital
interaction and charge-transfer concepts. Fig. 5 displays an NBO diagram showing donor-acceptor
orbital interaction along with second-order perturbation energy (E(2)
). Notably, the C2—Cl1⋯π
interaction is characterized by LP1(Cl1)→ π*(N9–C19) electronic delocalization, with a
stabilization E1 value of 0.69 kcal/mol. This orbital interaction corresponds to a lone-pair⋯π
interaction, with the lone pair bonding orbital on the Cl atom acting as the donor orbital (Lewis
base). Moreover, the analysis revealed the presence of a π(N9-C19)→σ*(C2–Cl1) interaction with
a very low stabilization energy value (E(2)
) of 0.07 kcal/mol. In this case, the π(N9-C19) bonding
orbital in pyrimidine functions as the donor orbital (Lewis base), suggesting a σ-hole interaction
character. The E1 value being larger than the E2 value, along with the absence of LP(N) → σ(Cl–
C) electronic delocalization, signifies that the investigated interaction is likely a lone-pair⋯π (n
→ π*) interaction. This observation supports the QTAIM results, which do not show evidence of
a direct contact (bond path and bond critical point) between the Cl atom and neighbouring N atoms.

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Fig. 5. (Above) Interaction energy (∆EBSSE) for the halogen⋯π(ring) intermolecular interaction
computed at ωB97xD/aug-cc-pVTZ level, with counterpoise-correction for basis set superposition
error (BSSE). (Below) NBO-diagram displaying donor–acceptor orbital scheme. Arrow indicates
the electron transfer direction. Stabilization energy (E(2)
) in kcal/mol. Atom colours: white (H),
silver (C), red (O), blue (N) and green (Cl).
The nature of the Cl⋯π(ring) interaction was further investigated electrostatically using the
Molecular Electrostatic Potential (MEP) analysis. MEP surface helps identify potential sites for
noncovalent bonding by highlighting regions of electron richness (negative potential) and electron
deficiency (positive potential) around the molecule. It can also assess the size and strength of the
σ-holes [56]. Fig. 6 shows the MEP surface for the fully optimized monomer structure calculated
at the ωB97xD/aug-cc-pVTZ level. As observed, the chlorine atom (Cl1) exhibits a low positive
MEP value (+2.059 kcal/mol) opposite the C-Cl bond, indicating an electron-deficient area
corresponding to the Cl σ-hole. This suggests a low propensity for Cl1 to act as a σ-hole donor in
noncovalent interactions. Conversely, the electron belt (σ-lump) around Cl1 displays a negative
MEP value (-11.587 kcal/mol). On the other hand, the C19 atom has a positive MEP value (+3.603
kcal/mol), albeit lower in magnitude. This indicates its suitability to act as an electron acceptor
(electrophilic site). Therefore, the MEP analysis suggests an electrostatically favorable, but likely
weak, interaction where the electron-rich region of Cl1 interacts with the electron-deficient area at
C19. However, MEP analysis alone cannot conclusively determine the interaction nature and the
philicity of the interacting centers.

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Fig. 6. Molecular Electrostatic Potential (MEP) surface calculated at the ωB97xD/aug-cc-pVTZ
level. The MEP is visualized on a 0.001 a.u. electron density isosurface. MEP values (in kcal/mol)
were determined using the Multiwfn program.
To further understand the nature of the interaction, we performed an integrated Electron
Localization Function (ELF)/Quantum Theory of Atoms in Molecules (QTAIM) analysis. This
approach allows us to locate regions of shared and unshared electron pairs, visualize bond paths at
the interaction site, and determine the philicity (electron-donating/accepting character) of
interacting atoms [56,57]. Fig.7 presents a combined ELF/QTAIM map projected onto a plane
formed by C2, Cl1, and C19 atoms. For the Cl1···C19 interaction, the bond path passes through
the edge of the lone pair on Cl and the area with a low ELF value around the C atom. This confirms
that the contact cannot be classified as σ-hole-based halogen bonding. Additionally, it suggests the
weak nucleophilic character of the chloride anion (Cl1) and the weak electrophilic character of the
C19 center. These observations corroborate the findings obtained previously from the MEP
analysis.
To complement our understanding of the philicity of the interacting atoms, we performed a
combined electron density (ED)/electrostatic potential (ESP) study [39]. This analysis compares
the minima of ED and ESP along the bond path, allowing us to differentiate electron-
donor/acceptor sites [58,59]. In polar noncovalent interactions, the ESP minimum shifts towards
the electron-donating (nucleophilic) center, while the ED minimum shifts towards the electron-
accepting (electrophilic) site. Fig. 7 shows the one-dimensional ESP minimum along the
Cl1···C19 line located closer to the electron-donating chloride anion (Cl1). Conversely, the ED
minimum is located closer to the C19 atom. Interestingly, the ED and ESP minima are almost
superimposed, suggesting a weak polar interaction. Based on these observations, we can conclude

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that the interaction is a weak lone-pair⋯π interaction, with the C atom acting as an electrophile
and the Cl centre acting as a nucleophile.
Fig. 7. (Above) Electron localization function (ELF) projection for the C—Cl⋯π(pyrimidine)
interaction, with plotted contour lines (black, step is 0.05), bond path (magenta line), BCP (green
dot). (Below) 1D profiles of the electron density (ED, black line) and electrostatic potential (ESP,
red line) functions plotted along the Cl1···C19 bond path.. The arrow points to the electrophilic
site provider.

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3.3 Chemical reactivity study
This section investigates the chemical reactivity behaviours of our compound. Initially, global
reactivity indices were predicted using Conceptual Density Functional Theory (CDFT) at the
ωB97xD/aug-cc-pVTZ level. The molecule containing Cl1 was fully optimized and utilized for
the current analysis (see Fig. 8a). The shapes and energies of the HOMO and LUMO frontier
orbitals were obtained and depicted in Fig. 8b. The electronic chemical potential (μ) was estimated
as follows: μ ≈ 1/2(ELUMO + EHOMO) ≈ 1/2(I + A), where I and A indicate the ionization potential
(I = −EHOMO) and the electron affinity (A = −ELUMO). The chemical potential (μ) was found to be
−3.994 eV. Similarly, the chemical hardness (η) is given by η ≈ (ELUMO − EHOMO) ≈ (I − A) and
was determined to be 4.661 eV. Furthermore, the title molecule exhibited a global electrophilicity
index (ω = μ2
/2η) value of 1.711 eV. Accordingly, it can be categorized as a strong electrophile
[29-31]. The global nucleophilicity (N) was obtained from the HOMO energies: N = EHOMO −
EHOMO(TCE), where tetracyanoethylene (TCE) is the reference with a HOMO energy equal to
−9.12 eV. The title compound is classified as moderate nucleophile in polar organic reactions, as
it exhibits a nucleophilicity value of 2.795 eV, falling within the range of 2.0 to 3.0 eV [29-31].
Additionally, we assessed local reactivity by identifying potential sites for electrophilic and
nucleophilic attacks. For this purpose, we employed a comprehensive approach that involved
visualizing the color-coded Molecular Electrostatic Potential (MEP) surface and conducting
quantitative "Atomic local molecular surface" analysis. The three-dimensional MEP surface
(isosurface = 0.001 a.u) is depicted in Fig. 8c. Red regions indicate negative sites that attract
electrophiles, while blue regions represent positive sites favoured by nucleophiles. The most
negative sites are N4-atom of the imidazole moiety (-23.972 kcal/mol), O-atom of the carbonyl
group (-21.062 kcal/mol), and N2-atom of the pyrimidine subunit (-17.677 kcal/mol). Conversely,
the H-atoms of the (-NH2) group bonded to pyrimidine unit exhibit the highest ESP values
(+18.673 kcal/mol for H1B and +15.501 kcal/mol for H1A), making them preferred targets for
nucleophilic attacks. Positive regions are also observed over the H5 hydrogen atom of imidazole
(+14.359 kcal/mol) and the H-atom H12 of the benzene ring (+14.052 kcal/mol). In conclusion,
the chemical reactivity analysis revealed that the N4, O1, and N2 atoms are susceptible to
electrophilic attack, while the H-atoms of the (-NH2) group and the H5 and H12 hydrogen atoms
are potential targets for nucleophiles.

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Fig. 8. (a) Optimized geometry structure with atom numbering scheme. (b) Electron density
distribution (isosurface= 0.02 a.u.) and energy levels of the HOMO and LUMO. Orbital energies
are shown in eV. (c) Views of the molecular electrostatic potential (MEP) surface calculated at the
ωB97xD/aug-cc-pVTZ level, visualized on the 0.001 a.u. electron density isosurface. Maximum
and minimum MEP values (in kcal/mol) were determined using Multiwfn 3.8.
3.4 ADMET properties
We have evaluated the physicochemical, ADME (absorption, distribution, metabolism, and
excretion) and toxicity properties of the studied compound using the ADMETlab 2.0.
Physicochemical and medicinal chemistry profiles: Fig. 9 showcases the radar plot summarizing
13 key physicochemical properties of our molecule (blue line) compared to the reference optimal
range (yellow and red). Remarkably, our compound falls entirely within the pink "drug-like" zone,
suggesting promising physicochemical profile. With a molecular weight of 386.260 g/mol, falling
within optimal range (100~600), it demonstrates an ideal size for pharmacokinetic considerations.
Key molecular parameters such as volume (375.640), hydrogen acceptors (nHA: 7), hydrogen
donors (nHD: 6), and rotatable bonds (nRot: 4) indicate favourable features for interactions and
flexibility. The presence of 4 rings, a maximum ring size of 9, and 8 heteroatoms showcase
structural diversity crucial for pharmaceutical success. Notably, LogS (-1.559), logP (1.586), and
logD (2.528) within optimal ranges support promising pharmaceutical potential, emphasizing the
compound's water solubility, lipophilicity, and distribution characteristics. Importantly, the title
compound also exhibits a promising medicinal chemistry profile by adhering to four key drug-
likeness rules, namely Lipinski Rule, Pfizer Rule, GSK Rule, and the Golden Triangle.
Furthermore, the synthetic accessibility score (SAscore) of 5.009 is considered low (SAscore < 6),
suggesting favorable ease of synthesis.

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Fig. 9. ADMETLab 2.0 radar plot visually summarizes the compound's key physicochemical
properties, emphasizing its favorable drug-likeness profile. Endpoint abbreviations are provided
in Supplementary Information.
ADME (absorption, distribution, metabolism, and excretion) analysis: Absorption characteristics
of the analysed compound are reported in Table S1. The absorption profile indicates optimal
MDCK permeability (2.4 x 10-06
) and negligeable P-glycoprotein inhibition (0.006). However,
challenges are anticipated for Pgp-substrate endpoints, Human Intestinal Absorption (HIA), and
human oral bioavailability (F20% and F30%). Furthermore, the Caco-2 cell permeability value (-
5.884 cm/s; acceptable range > -5.15) falls slightly above the desired cut-off, presenting an
opportunity for targeted optimization. The distribution profile underscores favourable outcomes,
with a satisfactory Plasma Protein Binding (PPB) value of 65.932% (< 90%). The Volume of
Distribution (VD) at 0.781 L/kg signifies an optimal distribution volume, meeting the criterion for
a proper VD within the predicted range of 0.04-20 L/kg. The Blood-Brain Barrier (BBB)
Penetration value of 0.116 cm/s falls within the excellent and optimal range, highlighting the
compound's potential effectiveness and its ability to penetrate the blood-brain barrier. Similarly,
the Fraction Unbound (Fu) value of 50.98% (≥ 5%) is within the excellent range, indicating a
suitable fraction of the compound that is unbound and available for distribution. The metabolism
profile demonstrates predominantly favourable outcomes (see Table S2), with no predicted
inhibition or substrate activity for CYP1A2, CYP2C19, CYP2C9, and CYP3A4. However, a high
predicted probability value (0.852) of being a CYP2D6 substrate is observed, suggesting a
potential area for optimization, despite a low probability of CYP2D6 inhibition. In the excretion
assessment, while the short half-life (T1/2) of 0.265 hours is favourable for efficient elimination,
the lower clearance rate (CL) at 3.252 mL/min/kg indicates a potential challenge in the drug's

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clearance. This suggests the need for attention and further exploration in optimizing the clearance
aspects of the drug.
Toxicity evaluation: In the toxicity evaluation, most endpoints present optimal values, indicating
a favourable safety profile (refer to Table S3). The compound exhibits negligible side effects
concerning the hERG (Human ether-a-go-go related gene), and low risks for Human
Hepatotoxicity (H-HT), Drug-Induced Liver Injury (DILI), Rat Oral Acute Toxicity, and
Carcinogenicity. However, caution is advised for AMES Toxicity and Respiratory Toxicity,
suggesting potential genotoxicity and respiratory concerns, respectively. Furthermore, FDAMDD
shows a relatively high value (0.975), indicating a potential issue with the maximum recommended
daily dose. In Toxicophore Rules, the compound demonstrates a generally favourable profile, with
no alerts in Acute Toxicity, SureChEMBL, and FAF-Drugs4 Rules. However, specific alerts
linked to the Cl-C fragment raise concerns, particularly in Genotoxic and NonGenotoxic
Carcinogenicity, Skin Sensitization, Aquatic Toxicity, and NonBiodegradability Rules. These
alerts underscore areas requiring focused evaluation and optimization to comprehensively
understand and mitigate potential risks associated with the Cl-C fragment.
3.5 Molecular docking analysis
We conducted a molecular docking study to explore the potential inhibitory activity of the
synthesized purine derivative against the cyclooxygenase-2 (COX-2) enzyme (PDB ID: 3LN1).
COX-2, a dimeric isozyme that is a key target for anti-inflammatory drugs, also shows elevated
expression in various cancers, making it a significant target for new anticancer agents. The docking
protocol's reliability was validated by successfully re-docking the crystallized ligand celecoxib.
Superimposition of the native and newly docked conformations (Fig. 10) showed an RMSD of
0.89 Å, confirming accurate reproduction of the native conformation.
Fig. 10. Alignment of celecoxib in the native co-crystal (yellow) and the re-docked pose
(magenta), RMSD = 0.89 Å.

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The investigated compound exhibited a good binding affinity (-9.1 kcal/mol), comparable to that
of the standard celecoxib (-9.3 kcal/mol), as evidenced by the docking energy score. The
compound's noncovalent binding scheme involves both polar and nonpolar interactions (Fig. 11).
It forms two short H-bonds: One between the O-atom of the carboxamide group and the nitrogen
atom of the imidazole ring of Histidine (His342) amino acid. Another between the H-atoms of the
(-NH2) group bonded to the pyrimidine subunit and the oxygen atom from Serine (Ser339) amino
acid. LigPlot analysis revealed several stabilizing hydrophobic contacts between the purine moiety
and residues Thr79, His75, and Pro500. Additionally, nonpolar aliphatic interactions occur
between the phenyl rings and a region formed by Gln179, Asp501, and Pro177 (Fig. S2).
Fig. 11. 3D-Visualizations of the optimal docked pose - (Above) Cartoon depiction illustrating the
binding of the purine derivative ligand (carbon-magenta capped stick) within the active site cavity
of the COX-2 receptor, with hydrogen bonds displayed as yellow dashed lines. (Below) Surface
representation, highlighting polar contacts in red and nonpolar aliphatic contacts in green.
4. Conclusion
In conclusion, this research successfully synthesized and characterized the novel purine derivative,
2-amino-6-chloro-N,N-diphenyl-7H-purine-7-carboxamide. X-ray crystallographic analysis
revealed a diverse array of intermolecular interactions in its crystal packing, including classical
and unconventional H-bonds, C—H···π interactions, and a unique C—Cl···π(ring) interaction.
Quantum chemical calculations confirmed the distinctive nature of the C—Cl···π(ring) interaction
as a lone-pair⋯π (n→π*) halogen interaction, differing from the common σ-hole-based halogen

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bond. DFT-based chemical reactivity analysis identified the compound as a potent electrophile and
a moderately reactive nucleophile in polar organic reactions, highlighting potential attack sites for
both electrophilic and nucleophilic reactions. The compound exhibited a promising drug-like
profile with favourable ADMET characteristics, though challenges, particularly related to the –
(C—Cl) fragment, necessitate further optimization to address potential toxicity risks. The studied
purine derivative showcased promise as a COX-2 inhibitor, displaying a favourable binding
affinity of -9.1 kcal/mol, similar to the standard inhibitor celecoxib (-9.3 kcal/mol), and
establishing stabilizing interactions within the COX-2 active site, including two strong hydrogen
bonds. Further studies, including in vitro and in vivo experiments, are essential to validate its
potential and explore its anti-inflammatory and anticancer properties. This research lays the
groundwork for future investigations into this novel purine scaffold, contributing to the
development of potential therapeutic agents.
Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
AM and HT are the founder and shareholder of Molius Therapeutics. The other authors declare
no competing interests.
Acknowledgments
The authors extend their appreciation to Princess Nourah bint Abdulrahman University
Researchers Supporting Project number (PNURSP2024R95), Princess Nourah bint Abdulrahman
University, Riyadh, Saudi Arabia.
The authors gratefully acknowledge the financial support from Molius Therapeutics and The Knut
and Alice Wallenberg Foundation (No.72254).
Supporting Information
CCDC 2325198 contains the supplementary crystallographic data for this paper. These data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44
1223 336033).

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https://doi.org/10.1021/acs.inorgchem.1c01591.

• Synthesis of novel nitrogen-rich purine-based heterocyclic compound
• Structural analysis through X-ray crystallography
• Multi-approach quantum mechanics (QM) analysis of noncovalent interactions
• Conceptual Density Functional Theory (CDFT) study of chemical reactivity
• ADMET and In silico docking simulations on bioactivity behaviours
Highlights (for review)

1
Electronic supplementary information (ESI)
Unveiling Structural Features, Chemical Reactivity, and Bioactivity of a Newly
Synthesized Purine Derivative through Crystallography and Computational
Approaches
Nadeem Abad a,#
, Shafeek Buhlak a,#
, Melek Hajji b,⁎
, Sana Saffour a
, Jihane Akachar a
, Yunus
Kesgun a
, Hanan Al-Ghulikah c
, Essam Hanashalshahaby a
, Hasan Turkez d
, Adil Mardinoglu e,f,⁎
a
Trustlife Labs, Drug Research & Development Center, 34774 Istanbul, Turkiye.
b
Research Unit: Electrochemistry, Materials and Environment, University of Kairouan, 3100 Kairouan, Tunisia
c
Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428,
Riyadh 11671, Saudi Arabia
d
Department of Medical Biology, Faculty of Medicine, Atatürk University, Erzurum, Turkiye
e
Science for Life Laboratory, KTH-Royal Institute of Technology, SE-17165 Stockholm, Sweden.
f
Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King's College
London, London, SE1 9RT, United Kingdom
#These authors contributed equally.
*Corresponding authors:
Adil Mardinoglu (adilm@scilifelab.se) & Melek Hajji (melek.hajji@ipeik.u-kairouan.tn)
Table of contents
Item No. Content Page No.
1 Fig. S1 The two molecules within the asymmetric unit form connections
through C5—H5…O2 and N7—H7B···O1 hydrogen bonds
2
2 Fig. S2 2D-LigPlot+ illustrations of the noncovalent interaction network
between the studied purine ligand and COX-2 enzyme.
2
3
Table S1 Absorption characteristics of the analysed compound.
3
4 Table S2 The metabolism of studied compound by enzymes from the human
cytochrome P450 group.
4
5
Table S3 Toxicity characteristics of the of studied compound.
4
6
Fig. S3. 1H NMR spectrum of the studied compound
5
7
Fig. S4. 13C NMR spectrum of the studied compound
6
8 Fig. S5. LCMS spectrum of the studied compound 6
Supplementary Material Click here to access/download;Supplementary
Material;Electronic supplementary information (ESI).pdf

2
Fig. S1 The two molecules within the asymmetric unit form connections through
C5—H5…O2 and N7—H7B···O1 hydrogen bonds
Fig. S2 2D-LigPlot+ illustrations of the noncovalent interaction network between the
studied purine ligand and COX-2 enzyme. Hydrogen bonding is shown with dashed green
lines (contact distances in Å). Amino acid residues are labelled.

3
Endpoint abbreviations for physicochemical and medicinal chemistry profiles
(Figure 7 in main article):
MW: Molecular weight; nRig: number of rigid bonds; fChar: formal charge; nHet: number
of heteroatoms; MaxRing: number of atoms in the biggest ring; nRing number of rings;
nRot: number of rotatable bonds; TPSA: topological polar surface area; nHD: number of
hydrogen bond donors; nHA: number of hydrogen bond acceptor; LogD: logP at
physiological pH 7.4; LogS: log of the aqueous solubility; and LogP: log of the
octanol/water partition coefficient.
Table S1 Absorption characteristics of the analysed compound. For the Pgp-inh/sub, HIA,
and F endpoints, the probability (from 0 to 1) of falling within specified target ranges is
provided. Values are categorized as follows: 0–0.3: excellent; 0.3–0.7: medium; 0.7–1.0:
poor.
Property Value Unit
Caco-2 Permeability -5.884 log cm/s
MDCK Permeability 2.4 x 10-6 cm/s
Pgp-inhibitor 0.006 −
Pgp-substrate 0.924 −
HIA 0.962 −
F20% 0.862 −
F30% 0.831 −

4
Table S2 The metabolism of studied compound by enzymes from the human cytochrome
P450 group. Provided values donate the probability (from 0 to 1) of being a
substrate/inhibitor of considered enzymes. Values are categorized as follows: Category
0: non-substrate/non-inhibitor; Category 1: substrate/inhibitor.
Enzyme Probability
CYP1A2 inhibitor 0.004
CYP1A2 substrate 0.018
CYP2C19 inhibitor 0.053
CYP2C19 substrate 0.106
CYP2C9 inhibitor 0.001
CYP2C9 substrate 0.03
CYP2D6 inhibitor 0.03
CYP2D6 substrate 0.852
CYP3A4 inhibitor 0
CYP3A4 substrate 0.022
Table S3 Toxicity characteristics of the of studied compound. Provided values donate the
probability (from 0 to 1) of being toxic. Values are categorized as follows: 0–0.3: excellent;
0.3–0.7: medium; 0.7–1.0: poor.
Property Probability
hERG Blockers 0.107
H-HT 0.329
DILI 0.023
AMES Toxicity 0.831
Rat Oral Acute Toxicity 0.182
FDAMDD 0.975
Skin Sensitization 0.097
Carcinogenicity 0.162
Eye Corrosion 0.003
Eye Irritation 0.008
Respiratory Toxicity 0.766

5
Fig. S3. 1H NMR spectrum of the studied compound

6
Fig. S4. 13C NMR spectrum of the studied compound
Fig. S5. LCMS spectrum of the studied compound

checkCIF/PLATON report
You have not supplied any structure factors. As a result the full set of tests cannot be run.
THIS REPORT IS FOR GUIDANCE ONLY. IF USED AS PART OF A REVIEW PROCEDURE FOR
PUBLICATION, IT SHOULD NOT REPLACE THE EXPERTISE OF AN EXPERIENCED
CRYSTALLOGRAPHIC REFEREE.
No syntax errors found. CIF dictionary Interpreting this report
Datablock: 23gtu253_gbm_w003_0m
Bond precision: C-C = 0.0051 A Wavelength=0.71073
Cell: a=9.279(2) b=24.458(5) c=17.659(4)
alpha=90 beta=93.883(4) gamma=90
Temperature: 299 K
Calculated Reported
Volume 3998.4(15) 3998.3(15)
Space group P 21/c P 1 21/c 1
Hall group -P 2ybc -P 2ybc
Moiety formula C18 H13 Cl N6 O [+ solvent] C18 H13 Cl N6 O
Sum formula C18 H13 Cl N6 O [+ solvent] C18 H13 Cl N6 O
Mr 364.79 364.79
Dx,g cm-3 1.212 1.212
Z 8 8
Mu (mm-1) 0.209 0.209
F000 1504.0 1504.0
F000’ 1505.66
h,k,lmax 12,31,22 12,31,22
Nref 9150 9118
Tmin,Tmax 0.973,0.978 0.651,0.746
Tmin’ 0.973
Correction method= # Reported T Limits: Tmin=0.651 Tmax=0.746
AbsCorr = NONE
Data completeness= 0.997 Theta(max)= 27.481
R(reflections)= 0.0583( 3724)
wR2(reflections)=
0.1768( 9118)
S = 0.918 Npar= 485
ChekCIF Click here to access/download;Supplementary
Material;ChekCIF.pdf

The following ALERTS were generated. Each ALERT has the format
test-name_ALERT_alert-type_alert-level.
Click on the hyperlinks for more details of the test.
Alert level C
PLAT026_ALERT_3_C Ratio Observed / Unique Reflections (too) Low .. 41% Check
PLAT230_ALERT_2_C Hirshfeld Test Diff for C31 --C36 . 6.0 s.u.
PLAT245_ALERT_2_C U(iso) H7A Smaller than U(eq) N7 by 0.017 Ang**2
PLAT331_ALERT_2_C Small Aver Phenyl C-C Dist C7 --C12 . 1.37 Ang.
PLAT331_ALERT_2_C Small Aver Phenyl C-C Dist C31 --C36 . 1.37 Ang.
PLAT340_ALERT_3_C Low Bond Precision on C-C Bonds ............... 0.00507 Ang.
PLAT420_ALERT_2_C D-H Bond Without Acceptor N7 --H7A . Please Check
Alert level G
PLAT432_ALERT_2_G Short Inter X...Y Contact Cl1 ..C19 . 3.24 Ang.
1-x,1-y,1-z = 3_666 Check
PLAT606_ALERT_4_G Solvent Accessible VOID(S) in Structure ........ ! Info
PLAT868_ALERT_4_G ALERTS Due to the Use of _smtbx_masks Suppressed ! Info
PLAT933_ALERT_2_G Number of HKL-OMIT Records in Embedded .res File 33 Note
PLAT941_ALERT_3_G Average HKL Measurement Multiplicity ........... 4.5 Low
0 ALERT level A = Most likely a serious problem - resolve or explain
0 ALERT level B = A potentially serious problem, consider carefully
7 ALERT level C = Check. Ensure it is not caused by an omission or oversight
5 ALERT level G = General information/check it is not something unexpected
0 ALERT type 1 CIF construction/syntax error, inconsistent or missing data
7 ALERT type 2 Indicator that the structure model may be wrong or deficient
3 ALERT type 3 Indicator that the structure quality may be low
2 ALERT type 4 Improvement, methodology, query or suggestion
0 ALERT type 5 Informative message, check

It is advisable to attempt to resolve as many as possible of the alerts in all categories. Often the minor
alerts point to easily fixed oversights, errors and omissions in your CIF or refinement strategy, so
attention to these fine details can be worthwhile. In order to resolve some of the more serious problems
it may be necessary to carry out additional measurements or structure refinements. However, the
purpose of your study may justify the reported deviations and the more serious of these should
normally be commented upon in the discussion or experimental section of a paper or in the
"special_details" fields of the CIF. checkCIF was carefully designed to identify outliers and unusual
parameters, but every test has its limitations and alerts that are not important in a particular case may
appear. Conversely, the absence of alerts does not guarantee there are no aspects of the results needing
attention. It is up to the individual to critically assess their own results and, if necessary, seek expert
advice.
Publication of your CIF in IUCr journals
A basic structural check has been run on your CIF. These basic checks will be run on all CIFs
submitted for publication in IUCr journals (Acta Crystallographica, Journal of Applied
Crystallography, Journal of Synchrotron Radiation); however, if you intend to submit to Acta
Crystallographica Section C or E or IUCrData, you should make sure that full publication checks are
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Publication of your CIF in other journals
Please refer to the Notes for Authors of the relevant journal for any special instructions relating to CIF
submission.
PLATON version of 06/07/2023; check.def file version of 30/06/2023

Datablock 23gtu253_gbm_w003_0m - ellipsoid plot

CIF
Click here to access/download
Mol Files
CIF.cif

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
Declaration of Interest Statement

MOLSTRUC Unveiling Structural Features, Chemical Reactivity, and Bioactivity of a Newly Synthesized Purine Derivative through Crystallography and Computational Approaches/ Full article.pdf

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