Quim. Nova, Vol. 47, No. 5, e-20230131, 1-10, 2024 http://dx.doi.org/10.21577/0100-4042.

20230131

EFFICIENT SYNTHESIS OF 1-(BROMOMETHYL)-3,5-DIMETHOXYBENZENE: X-RAY STRUCTURE,

HIRSHFELD SURFACE ANALYSIS, DFTs, AND MOLECULAR MODELLING INVESTIGATIONS AS
TYROSINASE INHIBITOR

Article
Aamer Saeeda,*, Syeda Abida Ejazb,*, , Mubashir Azizb, Pervaiz Ali Channarc, Laila Sumreend, Rabail Ujane, Tanveer A.
Wanif, Seema Zargarg, Tuncer Hökelekh and Ulrich Flörkei
a
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan
c
Department of Basic sciences and Humanities, Faculty of Information Sciences and Humanities, Dawood University of Engineering
and Technology Karachi, 74800 Karachi, Pakistan
d
Department of Homoeopathic Medical Sciences, Faculty of Medicine & Allied Health Sciences, The Islamia University of
Bahawalpur, 63100 Bahawalpur, Pakistan
e
Dr. M. A. Kazi Institute of Chemistry, University of Sindh, 76080 Jamshoro, Pakistan
f
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, 11451 Riyadh, Saudi Arabia
g
Department of Biochemistry, College of Science, King Saud University, P.O. Box 22452, 11451 Riyadh, Saudi Arabia
h
Department of Physics, Faculty of Engineering, Hacettepe University, 06800 Beytepe-Ankara, Turkey
i
Department Chemie, Fakultät für Naturwissenschaften, Universität Paderborn, Warburgerstraße 100, 33098 Paderborn, Germany

Recebido em 08/06/2023; aceito em 27/09/2023; publicado na web 20/11/2023

The synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene, a crucial intermediate in the formation of various natural products, was
carried out through a straightforward three-step procedure starting from 3,5-dihydroxybenzoic acid. The structure of the synthesized
derivative was fully characterized using a combination of spectroscopic techniques and single crystal X-ray diffraction. The
crystalline sample was found to possess monoclinic symmetry with dimensions a = 8.4054(10) Å, b = 4.4245(6) Å, c = 25.016(3) Å,
volume V = 928.9(2) Å3 and Z = 4.0. In order to assess the inhibitory potential of the synthesized crystal, a comprehensive in-silico
investigation was conducted which involved molecular docking, density function theory (DFT) analysis, and molecular dynamics
(MD) simulation studies. The docking analysis was utilized to determine the binding orientation of the compound with respect to the
target proteins, while DFT analysis was utilized to evaluate the molecule’s reactivity profile. The MD simulation approach was used
to evaluate the structural rearrangements and chemical interactions that occurred during the simulation period. The results of these
studies suggest that the synthesized crystal could potentially serve as a dual inhibitor of RNR and tyrosinase and could be a suitable
candidate for the synthesis of noval anti-cancer agent for the treatment of associated malignancies.

Keywords: benzyl bromide; synthesis; crystal structure; molecular docking; MD simulation.

INTRODUCTION of dark spots on the skin, also known as age spots, which can progress
to more serious forms of skin cancer such as melanoma. Therefore,
Ribonucleotide reductase is an essential enzyme that plays a tyrosinase activity is considered a basic factor in the development of
significant role in synthesis of DNA and repair. It is present in all skin cancer and its regulation is an important target for the prevention
living organisms and is responsible for converting ribonucleotides, the and treatment of skin cancer.5 Both enzymes can serve as promising
building blocks of RNA, into their respective deoxyribonucleotides, targets for treatment of cancer and associated malignancies.
which are the building blocks of DNA. The importance of The halogenated bromophenols have significant medicinal values
ribonucleotide reductase lies in its ability to regulate the pool including antifungal, microbial, cytotoxicity and enzyme inhibition
of deoxyribonucleotides, which are used as substrates for DNA properties.6 The 1-(bromomethyl)-3,5-dimethoxybenzene has been
synthesis. Defects in ribonucleotide reductase have found to link widely used as a key starting material in the synthesis of various
with various diseases, including cancer and other DNA damage- naturally occurring isocoumarins and 3,4-dihydroisocoumarins,
related disorders.1 Understanding the mechanism and regulation of having dioxygenation at carbon 6 and 8 due to the fact that they are
ribonucleotide reductase is crucial for developing new therapeutic biogenetically obtained from the acetate-polymalonate pathway. For
strategies to treat such diseases.2,3 Aberrant expression of ribonucleotide example, 6-methoxymellein,7 diaporthin,8 fusamarin,9 peniolactol,10
reductase (RNR) protein may lead to development of metastasis. cytogenin,11 scorzocreticin,11 feralolide,12 and dihydroisocoumarins
Similarly, tyrosinase enzyme catalyzes the melanin formation and from Ononis natrix,12 are well-known compounds. In addition, it has
responsible for skin pigmentation.4 Tyrosinase is an enzyme that plays been used in the total synthesis of a naturally occurring substance
an essential role in the development of skin cancer. It is involved in denbinobin, which is chemically 1,4-phenanthrenequinone and it
the production of melanin pigment that imparts color to the skin, hair, inhibits the replication of HIV-1,13 and cytotoxic compounds which
and eyes. In normal skin, tyrosinase is regulated and controlled, but include Leucetta-derived 2-aminoimidazoles, naamidine H and
in skin cancer, its activity can become unregulated, leading to the naamine G,14 9-tetrahydrocannabinol, which is naturally obtained
overproduction of melanin. This excess melanin results in the formation from the Cannabis sativa (marijuana).15 Although its crystal structure
is already known from powder diffraction analysis,16 therefore as we
*e-mail: abida.ejaz@iub.edu.pk; aamersaeed@yahoo.com were successful in growing single crystals, we have re-determined the
2 Saeed et al. Quim. Nova

structure from single crystal data. Furthermore, the inhibition profile with Uiso = 1.2 Ueq(C) or 1.5Ueq(Cmethyl) and C−H distances ranging
of the synthesized crystal against RNR and tyrosinase enzymes was from 0.95 to 0.99 Å. H(Cmethyl) was allowed to spin but not tipped,
determined through comprehensive in-silico investigations including according to the rules. Table 1 contains selected bond geometries,
DFT analysis, molecular docking, and MD simulation studies. while Table 2 has more refinement data on these bond geometries.
The DFT studies were performed to evaluate the physicochemical In the case of CCDC, the deposit number is 1884625.
properties and electronic density of the compounds, and the molecular
docking approach was adopted to determine the in-silico binding Hirshfeld surface analysis
orientation of the compound against the selected targets. The stability
of the top-ranked pose was then evaluated through molecular dynamic In this study a high-resolution structural analysis was performed19
simulation studies. These findings of current study highlight the for the crystal structure of title compound using Crystal Explorer 17.520
potential of the synthesized crystal as a dual inhibitor of RNR and in an attempt to discover the intermolecular association present in
tyrosinase and lay the foundation for further studies towards its the crystal structure. The investigation was performed to find the
development as a therapeutic agent. There are several studies2,3 that stacking interactions along with the position of atoms in short
have investigated the mechanisms of action for inhibitors of RNR contacts having hydrogen bonding potential, as well as the quantity of
and tyrosinase, which are both potential targets for the treatment of these interactions. It is possible to identify places that are especially
skin cancer. One study proposed a transmetalative approach using a significant for intermolecular interactions using the normalized
Ti(IV) chelator to inhibit both RNR R1 and R2 subunits, suggesting contact distance, abbreviated as dnorm. The white colored surface
that this approach could have a dual inhibitory effect on RNR and on the HS plotted over dnorm, represents interactions with distances
tyrosinase. Another study4 investigated the mode of action of an equal to the total sum of van der Waals radii, while the red and blue
antiproliferative compound and found that RNR is considered one colored surfaces are indication of interactions with comparatively
of the main targets of TSCs, yet there may be additional targets that shorter bond lengths (in close contact) or longer (in distinct contact)
contribute to their efficacy. Additionally, some compounds such as than the van der Waals radii.
monobenzone have been shown to inhibit tyrosinase and induce
melanosome autophagy, which could provide a potential mechanism Computational studies
of action for dual inhibition of RNR and tyrosinase. However, further
research is needed to fully understand the mechanism of action and Density functional theory (DFT)
potential efficacy of dual inhibition of RNR and tyrosinase for the Density functional theory is a computational tool for computing
treatment of skin cancer. the ground state geometries of the selected compounds. The ground
state geometries were optimized with DFT calculations by means of
EXPERIMENTAL the B3LYP functional including STO-3G basis set.21,22 The STO‑3G
basis set is a minimal basis set used in quantum chemistry. It is used to
Melting point were determined using a MelTemp MPD apparatus. represent molecular orbitals in terms of atomic orbitals. The STO-3G
FTIR spectrum was taken on a single beam Nicolet IR 100 (Fourier basis set uses 3 primitive Gaussian functions to describe the valence
transform). 1H NMR and 13C NMR spectra were monitored in electrons of an atom. It is a simple and computationally efficient basis
CDCl3 using 300 MHz Bruker DPX spectrometer. MS analysis set that is commonly used for small to medium sized molecules. In
was performed by instrument LC system Agilent 1200 series and this current study, Gaussian 09W software23 was used for calculating
LECO‑183 CHNS analyzer was utilized for elemental analysis. DFT. The resultant check files were interpreted in Gauss View 6.0.24,25

Synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene (4) Molecular docking

In order to ascertain the chemical basis of the inhibitory
Phosphorus tribromide (2.60 mL, 7.44 g, 0.027 mol) was added mechanism, it is crucial to interpret the binding orientation of the
dropwise to a container having solution of 3,5-dimethoxybenzyl ligand inside biological targets. A molecular docking technique was
alcohol (3) (0.027 mol, 6.64 g) in 50 mL of dry benzene and the used to evaluate the way that produced crystals bound to the intended
reaction mixture was stirred for 4 h at room temperature. After proteins. AutoDock suite was used for the molecular docking.26 The
completion of reaction as monitored by the analytical TLC using biological targets were retrieved from the Protein Data Bank using
solvent system n-hexane ethyl acetate (8:2), the reaction mixture the PDB IDs 2BQ127 and 4OUA28 for the ribonucleotide reductase
was transferred into ice-cold water, stirred vigorously. Ethyl acetate and the tyrosinase enzyme, respectively (https://www.rcsb.org/). In
was added, and the organic layer was separated. The organic phase the beginning, undesirable water molecules and hetero atoms were
was successively washed with water, 5% sodium bicarbonate dried removed using MOE’s preliminary preparation wizard. Then, at the
using anhydrous magnesium sulphate, and then rotary evaporated to sharpest energy gradient, both biological targets were successively
leave a white solid. Recrystallization from petroleum ether afforded reduced in energy and protonated in 3D. MMFF94x force field was
the pure product as colorless prisms. (7.2 g, or 88%). also used to include gasteiger charges. After the protein synthesis
has been completed, the MOE site finder utility was used to choose
Structure determination of 1-(bromomethyl)-3,5-dimethoxy the active site’s amino acid residue. After preliminary energy
benzene (4) minimization through ChemDraw 3D,29 the synthesized crystal was
converted into the desired format for docking purposes. Using London
The data were recorded at 130(2) K on a Bruker AXS Smart dG as a scoring function, the output structure was docked at specific
APEX CCD diffractometer16 utilizing MoK rays; 8110 reflections amino acid residues with triangular matcher placement.30 For each
were recorded at an angle of 1.63 < q < 27.88°.17 Indirect techniques,18 biological target, the rigid docking was employed to enhance the
full-matrix refinement with least squares,17 on F2 and 111 parameters reliability of the docking studies.31 Furthermore, docking protocol
for 2208 specific intensities I > 2σ(I), after excluding hydrogen atoms was validated, ligand molecule against each protein was redocked
all the remaining atoms were refined anisotropically, H atoms from within the active pocket. The RMSD values of less than 2 Å between
method of difference,18 fine-tuned Fourier maps on idealized places regenerated and native pose were considered as validated protocol.
Vol. 47, No. 5 Efficient synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene 3

Molecular dynamics simulations from the mean benzene plane, which are 0.0123(17), 0.0173(18),
Molecular dynamics (MD) simulations were performed using −0.0076(7), −0.0086(15), and −0.0135(16) Å. The torsion angles
Desmond software on a protein-ligand complex. The simulation associated with the methoxy groups are nearly planar. The bond
spanned 100 ns in the TIP3P solvent model. The OPLS3 forcefield32 length between C1−C2 was 1.49 Å were as bond angle C6−O2−C9
was chosen for the simulation under periodic boundary conditions.33 was 117.83. The detailed bond angle, bond length and torsion angles
The complex was placed in an orthorhombic solvation box, filled is tabulated in Table 1.
with TIP3P water molecules.34 NaCl counter ions, at 0.15 mol L-1 The molecule is connected through various hydrogen bonds
concentration, were added for neutralization. Steric clashes were forming centro symmetric dimers stacked along the b-axis and
removed with 2000 steps of energy minimization using the steepest forms ring motifs (R22 (8)) around the molecules. Intermolecular
descent method. System equilibration occurred in an NPT ensemble at C3−H…O1 bonds are formed between the dimers, which results in
300 K temperature and 1.01 bar pressure. Short-range van der Waals a two-dimensional network extending down the c-axis (Figure 1).
interactions had a 10-angstrom cutoff. To ensure constant conditions,
a Martyna-Tobias-Klein barostat and Nose-Hoover thermostat were
employed.35 Trajectories of the simulation were saved every 100 ps.
The entire MD run for each protein-ligand complex lasted 100 ns.
For motion equations, a time step of 2 fs was used. The particle mesh
Ewald method36 ensured accurate electrostatic interactions.37 These
simulations provided insights into the molecular behavior of the
biological target and the ligand molecule.

RESULTS AND DISCUSSION

Synthesis
Figure 1. Hydrogen bonds on the a-axis of the crystal packing; hydrogen
The compound 1-(bromomethyl)-3,5-dimethoxybenzene (4) was atoms that are not involved are deleted
produced in two phases using the technique indicated in Scheme 1
with a modest modification of a previously reported method. Description of crystal structure of 1-(bromomethyl)-3,5-dimethoxy
Treatment with dimethylsulfate in dry acetone and anhydrous benzene
potassium carbonate as a mild base resulted in the formation of This study conspicuously illustrates the structure of our title
3,5-dimethoxymethylbenzoate (2) from 3,5-dihydroxybenzoic compound in single crystal, as established using spectroscopic data,
acid (1). The FTIR spectrum at 1732 cm-1 revealed a distinctive is consistent with the structure of the compound discovered earlier
(C=O) stretching absorption, which was confirmed. In order to get by other means. Table 2 provides experimental characteristics,
3,5-dimethoxyphenylmethanol, the methyl ester was reduced with including crystal data, data collection and data refinement, as well
lithium aluminum hydride in dry THF (3). In the analysis of the as the findings of the experiment, and is followed by a summary of
1
H NMR spectrum the signal at 3.06 ppm confirms the presence of the experiment’s results. It was observed that crystal was colorless
hydroxyl group. The reaction of 3,5-dimethoxybenzyl alcohol with having molecular formula of C9H11BrO2. The shape of crystal was
phosphorous tribromide in dry benzene at room temperature resulted monoclinic with P21/c space group. The dimensions of crystal
in the formation of 1-(bromomethyl)-3,5-dimethoxybenzene (4). The was as follows: a = 8.4054(10), b = 4.4245(6), c = 25.016(3) Å,
lack of a wide signal at 3.06 ppm in the 1H NMR spectrum showed β = 93.213(2)°, V = 928.9(2) Å3, Z = 4, ρ = 1.652 mg cm-3,
that alcohol had been converted to bromide. μ = 4.38 mm-1, F(000) = 464.
In Figure 2, a clear depiction of the crystal structure, along
Crystal structure of 1-(bromomethyl)-3,5-dimethoxy benzene with numbering of atoms that were used to put it together is given.
The aromatic ring is almost perfectly coplanar with the atoms Within the benzene ring A (C2−C7), the atoms O1, O2, C1, C8, and
O1, O2, C1, C8, and C9. There are five different displacements C9 are separated from the mean plane by distances of −0.0473(1),

Scheme 1. Synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene

Table 1. Bonding parameters including bond lengths and bond angles

Bond length (Å) Bond angle (°) Torsion angle

Br1−C1 1.988(2) Br1−C1−C2 110.75(17) C5−C6−O2−C9 3.1(4)
C1−C2 1.498(3) C4−O1−C8 117.82(18) C5−C4−O1−C8 −3.6(3)
O1−C4 1.372(3) C6−O2−C9 117.83(19) C3−C2−C1−Br1 −104.8(2)
O1−C8 1.427(3)
O2−C9 1.428(3)
O2−C6 1.368(3)
4 Saeed et al. Quim. Nova

Table 2. Various parameters of crystal structure refinement for compound 4 centrosymmetric dimers (which include R22 (8) ring motifs) (Figure 1).
As a consequence of these dimers a parallel two-dimensional network
Molecular formula C9H11BrO2
is created. There is very little amount of interaction present between
Molecular weight 231.09 C and H. The most significant interactions to be mindful of in crystal
Temperatute (K) 130 packing are hydrogen bonding along with van der Waals forces, which
Wavelength (Å) 0.71073 are both formed by the crystal packing. Between the two variables,
Crystal typea Monoclinic there is no indication of a statistically significant interaction, which
is expressed as a correlation.
Space group P21/c
Size of unit cell (Å) a = 8.4054(10) Table 3. Geometry of intermolecular hydrogen bonds
b = 4.4245(6)
D−H (Å) H…A (Å) D…A (Å) D−H…A (°)
c = 25.016(3)
β = 93.213(2) C3−H3a…O1 a
0.95 2.50 3.398(3) 157.0
Cubic volume (Å ) 3
928.9(2) C7−H7a…O2b 0.95 2.53 3.460(3) 166.8
Z b
4 Plane of symmetry (rotation). Plane of symmetry (translation).
a b

Calculated density (mg m ) -3

1.652
Coefficient of absorptionc (mm-1) 4.38 Surface analysis using the Hirshfeld method
The comprehensive Hirshfeld analysis of synthesized crystal was
F (000)d 464
executed on Crystal explorer 17.2. It was observed that the oxygen
Size of crystal (mm ) 3
0.50 × 0.07 × 0.06 atoms H3A and H7A play significant role as donors and acceptors in
q range for data collectione (°) 1.6-27.9 the dominant C–H…O hydrogen bonding and is evident by the sharp
Range of index −11 ≤ h ≤ 11, −5 ≤ k ≤ 5, red color spots nearby these oxygen atoms; in the same way when
−30 ≤ l ≤ 32 electrostatic potential is mapped the positive and negative potential
Collected reflections 8110 on the Hirshfeld surface (HS) is indicated by the blue and red regions
as shown in Figure 3.
Independent reflections 2208 [R(int) = 0.030]
Refining method Full-matrix least-squares on F2
Data/restraints/parameters 2208/0/111
Goodness-of-fit on F
f 2
1.037
Final R indices R1 = 0.0314
[I > 2σ(I)] wR2 = 0.0720
R indices (all data) R1 = 0.0442
wR2 = 0.0765
Highest difference peak and hole (e Å−3) 0.63/−0.25
a
Indicates the crystal system or structure of the compound. bRepresents the
number of formula units per unit cell. cRefers to the measure of how much a
material absorbs radiation. dRepresents the sum of the structure factors for all
atoms in the unit cell. eSpecifies the range of angles (in degrees) over which
the data was collected. fRepresents the measure of how well the refined model
fits the experimental data.

Figure 3. 3D views of the 1-(bromomethyl)-3,5-dimethoxybenzene. The first

view (A) shows the 1-(bromomethyl)-3,5-dimethoxybenzene plotted over
dnorm ranging from −0.1668 to 1.1141 a.u, while the second view (B) dis-
plays the compound V/S electrostatic potential energy using STO-3G basis
set using Hatree fock calculations. Finally, the third view (C) shows the
1-(bromomethyl)-3,5-dimethoxybenzene V/S shape-index

The positive electrostatic potential (hydrogen-bond donors)

which is represented by the blue region, and the negative electrostatic
potential (hydrogen-bond acceptors) is represented by the red areas
Figure 2. Molecular geometry of 1-(bromomethyl)-3,5-dimethoxybenzene near the hydrogen atoms H3A and H7A indicating their roles as the
with ellipsoidal displacement at 50% negative electrostatic potential (hydrogen-bond acceptors). As long
as there are no red or blue triangles, the π…π interactions are very
−0.0051(1), −0.0452(1), −0.0132(1), 0.0402(19), −0.0132(1), low (HS). An example of the HS shape-index is existence of two
0.0402(19), and 0.0402(19) Å. This resulted in the benzene ring A adjoining red and blue triangles are present, which allows you to see
being coplanar with the atoms O1, O2, C1, C8, and C9. how stacking is occurring. Because the title chemical has no π…π
Table 3 illustrates how intermolecular C−HBnz …OMeth interactions with other molecules, it is clear from Figure 4 that it does
hydrogen bonds (Bnz = benzene and Meth = methoxy), which not interact with other molecules. If you look at the two-dimensional
are formed in the crystal structure between molecules holding plot of the structure, it is also possible to detect the intermolecular
all molecules together. All molecules are brought together by interactions that exist between the molecules.
Vol. 47, No. 5 Efficient synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene 5

Figure 4A-4D depict the intermolecular interactions between Table 4. Bond lengths between selected atoms (Å)
hydrogen and oxygen, illustrating the comprehensive insights gained O1…C3a 3.398 (3) H7A…O2d 2.53
from the Hirshfeld surface analysis regarding these interactions. H8A…O1e 2.86
H8A…C3e 2.93
H1A…C2b 2.94 H8A…C4e 2.75
H1A…C7b 2.94 H8A…C5 2.78
H1A…H7A 2.55 H8B…H8Cf 2.42
H1B…H3A 2.34 H8C…C5 2.69
H1B…O1a 2.77 H9A…C5 2.76
H3A…O1a 2.50 H9C…O2e 2.82
H9C…C5 2.72
H9C…C6e 2.92
Br1…H1a 2.50
H5A…C8 2.51 Br1…H1b 2.50
H5A…C9 2.52 Br1…H3a 3.89
H5A…H8A 2.36 Br1…H7a 3.42
H5A…H8C 2.23
H5A…H9A 2.31
H5A…H9C 2.29
Figure 4. 3D representation of Hirshfeld surface with interatomic parameters a
The atom O1 is related to the atom C3 by a symmetry operation rotation.
b
Plane of symmetry (translational). cPlane of symmetry (rotational). dPlane
The compound’s HS analysis data is presented in Table 4. of symmetry (reflections). ePlane of symmetry (rotation). fPlane of symmetry
By merging de and di values, a comprehensive overview of the (translational).
intermolecular interactions within the crystal can be visualized in a
two-dimensional fingerprint plot, as illustrated in Figure 5. Figure 5a in the fingerprint plot that has been represented into C…C contacts
depicts two-dimensional plot, whereas Figures 5b-5h depict the (Figure 5f) and is used to visualize the contributing 2.1% to overall
fingerprint plots divided into, H−Br/BrH, H−O/O−H, C−C, O−O, packing of crystal as a spoon-like spike with the tip at de = di ~ 1.75 Å,
H−H, H−C/C−H and C−Br/BrC contacts38 also how much each one which is the resulting from weak interatomic C…C contacts (Table 4).
contributed to the Hirshfeld surface.38 In Figure 5b according to Each of the Hirshfeld surface representations in Figures 5a-5d are
the high hydrogen content of the molecule, the 40.3% benefaction plotted with the function dnorm to show how individual intermolecular
to the HS surface may be observed as widely dispersed regions of interactions, as well as their quantitative contributions, are depicted.
high density in the fingerprint plot delineated into H…H interactions These representations illustrate the molecular linkages, as well as
because of high hydrogen atoms in the molecule. their quantitative contributions are given above. With the use of the
The pair of practically splitted spikes with the ends at Hirshfeld surface analysis, we can confirm that interactions between
de + di ~ 2.28 Å due to the short interatomic H…H interactions. The the H−atoms are important in the creation of the packing. As a result
presence of H…Br/Br…H contacts in the structure, which account for of several of H/H, H/O/OH, and H/C/CH contacts, it explains the
24.2% of the overall HS contribution, have an asymmetric distribution probable importance of hydrogen bonding and van der Waals forces
of contact sites, as shown in the Figure below (Figure 5c). Consider in the packing of crystal.
the case of the Br…H interactions in Figure 5c, which contribute
more (15.8%) than their HBr counterparts (8.4%) and are shown as Molecular docking studies
a spikes pair with the tips at de + di ~ 3.09 Å. The weak interatomic
hydrogen bonding between Br and H results in a short interatomic Molecular docking employs computational methods to predict
hydrogen bonding between Br and H. how well a ligand binds to its protein target, making it essential for
In Figure 5d, the weak C–H interaction present within the crystal identifying potential inhibitors of bio-targets like RNR and tyrosinase.
forms the characteristic wings pair similar to forceps that results in RNR, for instance, is an enzyme that is vital to DNA synthesis by
the fingerprint plot describing the H…C/H…C contacts (Figure 5d) converting ribonucleotides to deoxyribonucleotides. It is composed
to get an asymmetric points distribution, with C…H interactions of two dissimilar subunits i.e., R1 containing polythiols and R2
accounting for a greater proportion of the total (8.9%) compared containing free tyrosyl radical and non-heme iron site. Overexpression
to the H–C portion (7.8%). As a result, when the molecule tips are of RNR has been observed in many cancers, which has led to the
present at de + di ~ 2.63 Å, the combined H/C interactions provides development of RNR inhibitors as potential anti-cancer agents.
16.7% of the total molecular HS area (Table 4). Similarly, tyrosinase is responsible for melanin synthesis, which is
The H…O/O…H connections in the structure (Figure 5e), which essential for skin pigmentation. Overexpression of tyrosinase has been
account for 15.0% of the overall HS contribution, exhibit a similar observed in melanoma, a type of skin cancer, and tyrosinase inhibitors
asymmetric distribution of contact sites to the H…O/O…H connections have been developed as potential anti-cancer agents for melanoma.
in the structure, as well. H…O/O…H interactions account for a bigger Choosing both RNR and tyrosinase as biotargets for molecular
proportion of the total (8.1%) than their H…H counterparts (6.8%), docking has a rationale since their inhibition may have synergistic
as seen by a spike pair with their tops at de + di ~ 2.34 Å which are effects in cancer treatment. For instance, melanoma has been found
the result of weak interatomic H…O/H…H contacts in Figure 5e to be resistant to chemotherapy. By combining a tyrosinase inhibitor
(Table 4). In the following step, the symmetrical points distribution with an RNR inhibitor, it may increase the efficacy of treatment.
6 Saeed et al. Quim. Nova

Figure 5. The 2D graphs of 1-(bromomethyl)-3,5-dimethoxybenzene showing interactions and bonding distances (a) which are separated into (b) H…H (40.3%),
(c) H…Br/Br…H (24.2%), (d) H…C/C…H (16.7%), (e) H…O/O…H (15.0%), (f) C…C (2.1%), (g) O…O (1.0 %), (h) C…Br/Br…C (0.6%)

Molecular docking studies targeting both biotargets can help identify in the docked conformation of (4)’s interactions with the RNR
potential drug candidates that can be tested for their effectiveness in protein. Through hydrogen bonds, compound 4 was interacting with
combination therapy for cancer treatment. significant amino acid residues. A hydrogen bond was observed
In the current study, the focus was on the molecular docking of between the electronegative oxygen atom of (4) and SER606 with a
1-(bromomethyl)-3,5-dimethoxybenzene (4) within the active site bond length of 2.99 Å. Additional interactions include hydrophobic
of both RNR and tyrosinase enzymes, which could potentially lead associations with SER217, ARG293, ALA605, THR604, THR607,
to the discovery of a novel drug candidate with dual activity against ALA201, MET602, and SER202. The best conformation yielded a
these two biotargets. docking score of −20 kJ mol-1. The putative interactions are illustrated
in Figure 6.
Interpretation of molecular interactions The binding orientation of compound 4 was also investigated
Hydrogen bonds and hydrophobic contacts were prominent inside active pocket of tyrosinase enzyme. Tyrosinase plays important

Figure 6. The presumed 2D and 3D binding orientation of 1-(bromomethyl)-3,5-dimethoxybenzene with in active pocket of RNR protein. The grey colored
compound in 3D orientation is representing 1-(bromomethyl)-3,5-dimethoxybenzene (4)
Vol. 47, No. 5 Efficient synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene 7

Figure 7. The presumed 2D and 3D binding orientation of 1-(bromomethyl)-3,5-dimethoxybenzene with in active pocket of tyrosinase enzyme. The grey colored
compound in 3D orientation is representing 1-(bromomethyl)-3,5-dimethoxybenzene (4)

role in melanogenesis and ultimately lead to skin disorders. The

current study delved into the binding mechanism of (4) within the
active pocket of the tyrosinase enzyme. The docked posture of
(4) revealed significant molecular interactions with the enzyme,
encompassing both hydrophilic and hydrophobic associations. The
stability of the complex was reinforced by a hydrogen bond between
the oxygen atom of (4) and SER394 of the enzyme. This bond,
with a length of 2.69 Å, indicates its strong nature. Hydrophobic
interactions involved residues such as PHE400, HIS381, THR391,
LEU382, GLY389, ASN378, HIS215, TYR362, GLU360, HIS377,
and HIS192. The docking score for the complex stood at −24 kJ mol-1.
Figure 7 displays these anticipated interactions. Figure 8. HOMO and LUMO orbitals and optimized structure of compound

Density functional theory amount of energy to add a molecule to the system, while keeping
the temperature and volume constant. The electrophilicity index of
Density functional theory (DFT) is an extensively used technique compound 4 is 4.084, suggesting that it has a tendency to accept
of quantum mechanics for determination of electronic densities of electrons. The properties of compound 4 could make it useful in
the compounds. It is used to analyze the electronic configuration, various applications, depending on the specific context and desired
reactivity, efficiency and energy surface simulation of a compound. outcomes. The detailed reactivity descriptors are provided in Table 5.
All DFT calculations in this research were conducted using
Gaussian-09, utilizing the RHF calculation method and the STO‑3G Molecular dynamics simulation
basis set. The primary analysis involved determining energies and
the energy gaps between the highest occupied molecular orbital Molecular dynamics modeling is employed to predict the
(HOMO) and the lowest unoccupied molecular orbital (LUMO). thermodynamic properties of living systems under physiological
Compound 4’s structural geometry was designed to have the steepest settings. The Desmond MD simulation software was utilized to
energy gradient, and no phantom frequencies were detected that analyze the best-docked conformation. These 100 ns simulations
would have pointed to the precise local minima of the optimized provided pivotal insights into the stability of the protein-ligand
geometry. The compound’s polarizability and dipole moment were complex. From the MD trajectories, multiple analytical measures such
64.83 and 1.06 debye, respectively. Frontier molecular orbitals as RMSD, contact profile, RMSF, and ligand interaction profile were
(FMOs) investigation also demonstrated the compound’s chemical derived. Figure 9 depicts the RMSD plot for the protein’s C-alpha,
reactivity. The low energy gap between HOMO/LUMO orbitals the protein-ligand complex, and the ligand atoms. Protein C-alpha
were predictive of high chemical reactivity of the compound. The atoms’ RMSD patterns remained noticeably constant. The structural
HOMO/LUMO orbitals with their energies along with optimized changes were barely noticeable at the start of the trajectory but
structure are shown in Figure 8. showed structural reorganizations by the end of the MD simulations.
Compound 4 is a highly reactive compound with a low chemical These changes, which had an overall range of 1.1 to 2.9 Å, were kept
hardness of 0.008, indicating it can easily undergo chemical changes, within acceptable bounds. Protein C-alpha atoms have an average
and a high chemical softness of 66.66, indicating it is very easy for RMSD of 2.5 Å.
electrons to flow into or out of the compound. The compound has
a relatively low electronegativity of 0.248, which suggests it does Interpretation of protein-ligand stability
not strongly attract electrons towards itself in a chemical bond. The protein-ligand complex’s RMSD pattern showed a steady
The dipole moment of the compound is 5.88 debye, indicating a pattern. At first, the reference frame was used to align all 1000 frames,
significant separation of electrical charges within the molecule. The and an RMSD pattern was generated. The protein-ligand complex’s
chemical potential is −2.48, indicating that it requires a relatively low average RMSD was determined to be 2.8 Å. The compound first

Table 5. Reactivity descriptors of selected compound

Compound Chemical hardness Chemical softness Electronegativity Dipole moment Chemical potential Electrophilicity index
(η) (S) (X) (debye) (µ) (ω)
4 0.008 66.66 0.248 5.88 −2.48 4.084
8 Saeed et al. Quim. Nova

oscillated at 2.5 Å and was firmly anchored to the active pocket’s to 1.2 Å. The overall protein displayed an average RMSF of 1.07 Å,
amino acid residues. Inside the binding region, there were a few which is considered within acceptable limits. Figure 10 depicts the
minor structural changes, although these variations were within RMSF progression, with green lines signifying interactions between
acceptable bounds. A ligand molecule was in interaction with the the ligand and specific amino acid residues.
amino acid residues SER202, PRO294, ALA605, and TYR625 in
both hydrophilic and hydrophobic ways. 3.3 Å separated the atoms of
SER202 and the ligand molecule. The interatomic distances between
the other residues specified varied from 2.6 to 3.7 Å as well. The
overall RMSD pattern of the trajectory was stable, and the ligand
made adequate contact with the targeted protein. When the ligand
displayed minor rearrangements and the RMSD pattern increased to
3.5 Å after 70 ns, a little strange behavior was seen. The trajectory’s
equilibration, which stayed stable and equilibrated for the remainder
of the trajectory, was encouraging.

Figure 10. Evolution of RMSF for amino acid residues of targeted protein

The connections between a ligand and protein are heavily

influenced by their contact interactions. The nature and duration
of these interactions directly impact the stability of their combined
structure. In the studied complex, a variety of interactions, including
hydrogen bonds, water bridges, and hydrophobic connections,
were identified. Key hydrogen bonds were notably formed with
SER202 and SER606 from the target protein. Hydrophobic contacts,
particularly with PRO294, TYR625, ALA605, LEU428, and PRO603,
were sustained for over 10% of the simulation period, enhancing the
complex’s stability. Figure 11 displays the interaction profile of the
simulated ensemble.
Figure 9. RMSD trajectory analysis for protein (C‑alpha), protein ligand
complex (blue trajectory) and ligand atoms (orange colored trajectory) Interpretation of protein-ligand stability
Through MD simulations, the stability of tyrosinase in complex
The root mean square fluctuations (RMSF) analysis is vital for with 1-(bromomethyl)-3,5-dimethoxybenzene was also assessed. A
pinpointing local structural alterations in proteins. In this research, further confirmation of putative molecular interactions in the initial
a residue-specific RMSF was computed for every residue. While docked conformation of the ligand with the tyrosinase came from
most residues showed optimal fluctuations, certain residues, namely MD simulations. Following a 100 ns simulation of the complex, it
HIS108, ARG12, GLY264, THR265, ASN266, and ASN109, was discovered that the C-alpha atoms of the protein and tyrosinase-
displayed significant structural changes, varying between 3 and 4.3 Å. ligand combination had an important stability pattern. The stability
Notably, these residues are part of the C and N terminals, which are pattern of the evolution of protein RMSD was examined, and it was
typically less dense than the central backbone and alpha strand regions found that the protein remained stable and equilibrated with little
of the protein. It was observed that amino acid residues including variations. The protein’s average RMSD was found to be 1.8 Å, which
ASN144, TYR145, PHE146, THR150, ARG153, TYR155, ALA201, is completely acceptable.
SER202, PRO203, SER217, SER244, ALA245, LYS292, ARG293, The protein-ligand complex displayed a stable and equilibrated
PRO294, PHE329, MET602, PRO603, THR604, ALA605, SER606, trajectory, with fluctuations of about 0.8 Å. The minute fluctuations
THR607, ALA608, GLN609, LEU611, GLY612, ASN613, GLU615, showed that the ligand was still adequately bound to the amino acid
SER622, TYR625 and ARG740 were in contact with ligand molecule. residues in the active site. Significant contact was made by the ligand
All these residues exhibited very few fluctuations ranges from 0.6 molecule with HIS215, TYR362, HIS377, ASN378, LEU382, and

Figure 11. Contact profile of the protein-ligand complex. Green peaks indicate hydrogen bonds, while purple highlights hydrophobic interactions
Vol. 47, No. 5 Efficient synthesis of 1-(bromomethyl)-3,5-dimethoxybenzene 9

HIS381. Initial ligand rearrangements were visible in the RMSD

pattern, but these rearrangements became stable as the RMSD pattern
shrank to 2.8 Å. The average RMSD of the protein-ligand combination
was discovered to be 3.2 Å. The results of the molecular dynamics
simulation of the complex showed that the HIS215, GLU216,
ARG321, GLU360, TYR362, HIS377, ASN378, HIS381, LEU382,
GLY388, GLY389, GLN390, and HIS404 were all in contact with
the ligand molecule. Thankfully, all of these residues belonged to
the active site and showed minimal variation. The typical separation
between the ligand and these residues was between 2.5 and 3.3 Å.
These patterns represent the complexes with the fewest structural
variations and sufficient stability under physiological conditions.
Figure 12 is illustrating the evolution of RMSD pattern for protein Figure 13. Illustration of RMSF for tyrosinase enzyme
and protein ligand complex.
50% of simulation duration. Additionally, for 10% of the simulation
time, hydrophobic interaction was also seen with LEU382.

CONCLUSIONS

Conclusively, based on the desire of the potential candidate

for the treatment of skin cancer in this study, a title compound
1-(bromomethyl)-3,5-dimethoxybenzene (4), an intermediate towards
a range of natural products, was successfully synthesized and its
structure was confirmed through different spectroscopic methods.
The synthesized crystal was subjected to a thorough analysis,
including Hirshfeld analysis and comprehensive in-silico studies, in
order to determine its properties, reactivity and potential of the title
compound against the tyrosinase and RNR. The results of molecular
Figure 12. Evolution of RMSD pattern for tyrosinase and protein-ligand docking, density functional theory (DFT), and molecular dynamics
complex (MD) simulation studies revealed that the compound has significant
inhibitory potential and chemical reactivity, particularly as a dual
Variability in the tyrosinase enzyme by individual residues was inhibitor of the RNR and tyrosinase enzymes. The further research
examined. Apart from a few amino acids in the C and N terminals, the is needed to fully understand the mechanism of action and potential
C-alpha atoms of the selected protein maintained a prominent stability efficacy of dual inhibition of RNR and tyrosinase for the treatment of
pattern. Noteworthy fluctuations exceeding 3 Å were identified in skin cancer. Based on the in-silico findings, the title compound was
residues like HIS81, GLY202, VAL203, GLN205, GLU206, and found as a promising candidate for the synthesis of more potential
ASP82. In contrast, most residues showed minor structural variations candidate which can be explored at molecular level in addition to the
and consistently remained stable throughout the simulation. Ligand- computational investigations.
bound residues such as HIS215, GLU216, ARG321, GLU360,
TYR362, HIS377, ASN378, HIS381, LEU382, GLY388, and SUPPLEMENTARY MATERIAL
GLY389 registered slight fluctuations, ranging between 0.4 and
1.5 Å. The entire protein’s average RMSF was calculated as 1.20 Å. Complementary material for this work is available at
Figure 13 visualizes the RMSF values for the tyrosinase enzyme. http://quimicanova.sbq.org.br/, as a PDF file, with free access.
Figure 14 shows the contact profile of the chosen ligand.
Significant hydrophobic and hydrogen bonding interactions were ACKNOWLEDGEMENTS
found to be stabilizing the protein ligand complex. In particular,
hydrophobic contact with TYR362 persisted for 40% of simulation All the authors want to extend their appreciation to researchers
time while hydrophobic bonding with ASN378 persisted for more than supporting project (project number RSP2024R357), King Saud

Figure 14. Contact profile of ligand with amino acid residues of tyrosinase enzyme
10 Saeed et al. Quim. Nova

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