Copper-catalyzed one-pot relay synthesis of anthraquinone based pyrimidine derivative as a probe for antioxidant and antidiabetic activity

Article abstract of DOI:10.1016/j.molstruc.2020.129668

Synthetic compounds have modernized the globe due to its vast applicable fields. Anthraquinones, as well as pyrimidine derivatives, are used as essential pharmacophores in the field of medicine. Maintenance of a green disease-free environment by using these derivatives is being acknowledged in developed as well as developing countries of the world. Considering the use of active catalysts in the synthesis of anthraquinone based derivatives are the era of concern for researchers due to their distinctive properties. Owing to the remarkable activities of anthraquinone and pyrimidine derivative, we synthesize compounds having both functionalities with the utilization of novel synergically active copper catalysts. This study explores the application of synthesized compounds using fast, ecofriendly and cost-effective approaches.¹H and ¹³C NMR, antioxidant, antidiabetic, molecular docking and QSAR studies were used for characterization and evaluation of newly synthesized anthraquinone based pyrimidine derivatives. The result of these techniques shows that our desired compounds were successfully synthesized and have potent applications. Among all synthesized compounds, G₂ and G₃ showed a remarkable antioxidant activity with IC₅₀ of 15.09 and 21.88 μg/ml respectively. While the compound G₂ and G₄ showed a strong inhibitory antidiabetic activity with the IC₅₀ value of 24.23 and 28.94 μg/ml respectively. Furthermore, molecular docking results for both of the proteins assist the experimental data and confirms the different interactions between binding domains and substituent moieties. SAR study also relates to the experimental facts by giving us positive results of synthesized compounds. According to the QSAR study, G₄ and G₂ emerged as the most stable and most reactive compound among other compounds respectively. While MEP shows moderate to good nucleophilic and electrophilic reactivity of all four compounds.

Full text of DOI:10.1016/j.molstruc.2020.129668

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Copper-catalyzed one-pot relay synthesis of anthraquinone based
pyrimidine derivative as a probe for antioxidant and antidiabetic
activity
Gul Zarren , Nusrat Shafiq , Uzma Arshad , Naila Rafiq ,
Shagufta Parveen , Zaheer Ahmad
PII:
S0022-2860(20)31981-5
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129668
MOLSTR 129668
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Journal of Molecular Structure
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Revised date:
Accepted date:
15 August 2020
1 November 2020
13 November 2020
Please cite this article as: Gul Zarren , Nusrat Shafiq , Uzma Arshad , Naila Rafiq ,
Shagufta Parveen , Zaheer Ahmad , Copper-catalyzed one-pot relay synthesis of anthraquinone
based pyrimidine derivative as a probe for antioxidant and antidiabetic activity, Journal of Molecular
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Highlights





One-pot relay synthesis of anthraquinone based-pyrimidine derivatives.
Novel synergistically active copper catalysts were used in the synthesis.
Study covers the biological aspects, Molecular docking and QSAR analysis.
Biologically and theoretically G2 and G4 is proposed as an effective compound.
All the comparative analysis of the advanced techniques are in good agreement.

Copper-catalyzed one-pot relay synthesis of anthraquinone based pyrimidine derivative as a probe for
antioxidant and antidiabetic activity
Gul Zarren1, Nusrat Shafiq1*, Uzma Arshad1, Naila Rafiq2, Shagufta Parveen1, Zaheer Ahmad3
1Department of chemistry, Government College Women University Faisalabad-38000
2Department of chemistry, Government College Women University Faisalabad-38000
3Department of chemistry, University of Wah, Wah Cantt.
Corresponding Author: Dr. Nusrat Shafiq, Assistant Professor, Department of chemistry, Government
College Women University Faisalabad-38000
Corresponding Address: gqumarin@gmail.com, dr.nusratshafiq@gcwuf.edu.pk

Abstract
Synthetic compounds have modernized the globe due to its vast applicable fields. Anthraquinones, as
well as pyrimidine derivatives, are used as essential pharmacophores in the field of medicine.
Maintenance of a green disease-free environment by using these derivatives is being acknowledged in
developed as well as developing countries of the world. Considering the use of active catalysts in the
synthesis of anthraquinone based derivatives are the era of concern for researchers due to their distinctive
properties. Owing to the remarkable activities of anthraquinone and pyrimidine derivative, we synthesize
compounds having both functionalities with the utilization of novel synergically active copper catalysts.
This study explores the application of synthesized compounds using fast, ecofriendly and cost-effective
approaches.1H and 13C NMR, antioxidant, antidiabetic, molecular docking and QSAR studies were used
for characterization and evaluation of newly synthesized anthraquinone based pyrimidine derivatives. The
result of these techniques shows that our desired compounds were successfully synthesized and have
potent applications. Among all synthesized compounds, G2 and G3 showed a remarkable antioxidant
activity with IC50 of 15.09 and 21.88 μg/ml respectively. While the compound G2 and G4 showed a strong
inhibitory antidiabetic activity with the IC50 value of 24.23 and 28.94 μg/ml respectively. Furthermore,
molecular docking results for both of the proteins assist the experimental data and confirms the different
interactions between binding domains and substituent moieties. SAR study also relates to the
experimental facts by giving us positive results of synthesized compounds. According to the QSAR study,
G4 and G2 emerged as the most stable and most reactive compound among other compounds
respectively. While MEP shows moderate to good nucleophilic and electrophilic reactivity of all four
compounds.
Keywords: Co-crystal approach; Copper catalysis; Antioxidant; Molecular docking; QSAR analysis
Highlights





One-pot relay synthesis of anthraquinone based-pyrimidine derivatives.
Novel synergistically active copper catalysts were used in the synthesis.
Study covers the biological aspects, Molecular docking and QSAR analysis.
Biologically and theoretically G2 and G4 is proposed as an effective compound.
All the comparative analysis of the advanced techniques are in good agreement.

1 Introduction
One of the key sources of potent agents for hundreds of years is the natural and synthetic products.
Presently, extraction and application of the natural products as bioactive secondary metabolites in the
discoveries of drugs and medicines are the cornerstone of the research field [1]. Various plants produce
potent secondary metabolites i.e. anthraquinone, alkaloids and its derivatives which is further used for a
wide range of applications in the textile industry, coloring agents and therapeutic agents for the treatment
of various diseases [2, 3]. Among secondary metabolites, anthraquinones are found naturally in plants,
lichens and fungi. They are widely studied for their structural evaluation and biological significance [4-6].
They are used as laxatives and to treat ulcers, skin disease, tumors, asthma, cancers, itching and eczema
[7, 8]. They are also used as antifungal, antimicrobial, insecticidal, antioxidant and as antidiabetic [9-12].
Synthetically, the synthesis of anthraquinone derivative has become the major concern to the chemist to
accomplish different chemical strategies. Therefore, various synthetic approaches and their
pharmaceutical applications have been examined. In literature, several methods are available for the
synthesis of simple to substituted anthraquinone through oxidation of anthracene [13], diels alder reaction
[14], and friedel craft reaction [15, 16].
Anthraquinones either synthesized or collected from natural sources are thought to interact with different
derivatives having -N group. The anthraquinone due to its planar ring structure permits interaction and
intercalation between 5´-pyrimidinepurine-3´GC sites of DNA base pairs in β conformation [17]. Here is
the review of some synthetic strategies which were opted in past to synthesize anthraquinone derivatives
having –N functionality. The anthraquinone derivatives named ametantrone were synthesized and the
final product of the synthesis having two different esterified amino acids L-serine and 6-amino hexanoic
acid effectively inhibits the proliferation of cancer cells by intercalating the DNA [18].
Also, anthraquinone based 2-aminopyridine and other anthraquinone derivatives which were coupled with
other rings by interaction through -N functionality have been synthesized that shows inactivity against
bacteria [19]. The series of dyes having anthraquinone basic nucleus interacted with other groups through
N-functionality have been studied that proved to be efficiently used in the medical application for
manufacturing the iris implants [20]. Recently, various anthraquinone derivatives with simple to high
molecular weight pyrrole and pyrazole rings were synthesized and investigated [3, 21, 22]. All of the
above mentioned anthraquinone derivatives interacted directly through the-N functionality either in a ring
system or in the aliphatic chain.
After reviewing the literature, we get the point that anthraquinone has a remarkable ability to effectively
interact with units having –N functionality. Pyrimidine also consists of a six-membered ring possessing
two nitrogen atoms at position 1 and 3 [23]. In addition to many pharmacophores with a variety of
potential biological activities, the structural skeleton of pyrimidine is a fundamental constituent of nucleic
bases and alkaloids [24]. Like anthraquinone, pyrimidine nucleus present in many classes of
chemotherapeutic agents and is in clinical use as an anticancer, antiviral, antifungal, antimalarial and
antibacterial agent [25]. Pyrimidine derivatives are cell cycle specific and eradicate only vigorously
growing cells [26]. They work by impeding the synthesis of nucleic acid (DNA and RNA) [27].
In recent times, even though there has been a great development in the identification and treatment of
diseases, still the treatment of infectious diseases attributes a challenging problem. So, there is a dare need
to develop novel and efficient chemotherapeutic agents for treating and controlling outspreading diseases

[28]. As we discussed, heterocyclic compounds having nitrogen moiety exhibit interesting biological
activities. The brief overview of remarked chemotherapeutic properties of anthraquinone, pyrimidine and
its characteristic interaction with –N based pyrimidine compounds gives us an idea to synthesize
compound having both functionalities.
In this study, we intend to synthesize novel anthraquinone based pyrimidine derivatives, which is not
discovered yet, through the simple, facile and efficient strategies. Most of the synthetic routes in the past
include multiple-step reactions and require expensive catalysts. Therefore, in this study, we follow the
facile one-pot protocol as an ideal strategy which not only avoids the formation of multiple bonds, reduce
waste production and save time but also give the desired compound having reasonable complexity.
Specifically, the coupling reactions catalyzed through the transition metals significantly construct C-C
and C-X (X= heteroatom) bond [29]. Hence, we supposed to synthesize our anthraquinone based
pyrimidine derivatives catalyzed through these transition metals. Based on this concept, we have used a
non-toxic copper catalyst to establish the direct coupling reaction between anthraquinone and pyrimidine
derivatives.
2. Materials and Methods
2.1 Chemicals
All the chemicals used in this study were of analytical grade, purchased from a commercial supplier and
used without further purification. The following chemicals are used in the synthesis of our first
anthraquinone precursor: Phthalic anhydride, Aniline, Phenol,4-chloro benzaldehyde, Alum and conc.
Hydro chloric acid (HCl). Benzaldehyde, 4-chloro-benzaldehyde, urea, ethylacetoacetate and cupric
chloride are used in the synthesis of our second pyrimidine precursor. Then Cupric oxide (CuO), Copper
chloride (CuCl2. 2H2O), Methanol, Distilled water and both precursors are used in the synthesis of our
targeted co-crystal. All the reactions were monitored by TLC and further checked under ultraviolet (254
nm) light.
2.2 General synthesis of precursors
The co-crystal of our desired product was synthesized by the following two steps. The first step is to
synthesize anthraquinone derivatives by following the procedure found in the literature with little
modification in the synthetic protocol of Madje et al. [15]. A combination of substituted benzene (2g),
phthalic anhydride (1g), water (5ml) and a catalytic amount of alum was collectively added in a 50ml one
neck round bottom flask. The reaction mixture was allowed to stir for 60-80 minutes at room temperature.
The reaction progress was monitored regularly by TLC. After that, the mixture was left until it turns
completely in a dry powder form. Then in the product, 3-5ml concentrated HCl was added, the liquid
frequently turns yellow. The filtrate was separated and dried to give the solid mass. The crude product
was washed with ethyl acetate, dried and checked for its solubility. The synthesized compound was
solubilized in methanol, filtered off and recrystallized. The progress of the reaction was again determined
with TLC. The schematic representation of the reaction is shown in Fig. . After the successful formation
of anthraquinone precursor, we synthesize pyrimidine derivative by following the procedure of Shafiq et
al. [30] replacing thiourea with urea. The schematic representation of the reaction is shown in Fig. .

2.3 General synthesis of targeted co-crystal (G1-G4)
Anthraquinone derivative (0.2g), pyrimidine derivative (0.2g), methanol (10ml), a catalytic amount of
copper chloride and cupric oxide was collectively added in a 100ml one neck round bottom flask. The
reaction mixture was allowed to stir for 20-30 minutes at ambient temperature. The reaction progress was
monitored regularly by TLC. After the completion of the reaction, the mixture was left till it turns
completely in a dry powder form. The formulated compound (Fig. , G1-G4) was washed with cold water
and dried. The newly synthesized compound was solubilized in hot methanol, filtered off and slowly
recrystallized to afford the pure compound. The general schematic representation of reactions is shown in
Fig. .
2.4 Characterization
Synthesized compounds obtained in solid form by following the above-mentioned methods were
characterized by proton and carbon NMR to validate the successful synthesis of compounds.
2.4.1 N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-6-methyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (G1, C26H19N3O4):
Dark brown solid, % yield: 55,1H NMR (300 MHz, DMSO): δ (ppm): 9.14 (s, 1H, NH), 7.69 (m, 2H),
7.69 (m, 2H), 7.57 (m, 2H), 7.52 (m, 1H), 7.27 (m, 2H), 7.20 (m, 2H), 5.09 (d, J = 9.5, 0.8 Hz, 2H, NH),
2.50 (d, J = 1.0 Hz, 3H, CH3).13C-NMR (300 MHz, DMSO): δ (ppm): 182.35 (C=O), 180.83(C=O), 165.7
(C=O), 148.80 (C=O), 145.27 (2C), 131.15 (2C), 128.90 (2C), 127.78 (3C), 126.69 (5C), 99.67 (C), 54.38
(C), 18.28 (CH3).
2.4.2 Ethyl 4-(4-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino)phenyl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (G2, C28H23N3O5):
Dark brown solid, % yield: 62,1H NMR (300 MHz, DMSO): δ (ppm): 9.24 (s, 1H, NH), 7.97 (s, 1H), 7.97
– 7.90 (m, 1H), 7.77 (m, 1H), 7.53-7.51 (dd, J = 7.4, 4.8, 1.8 Hz, 3H), 7.46 (m, 1H), 7.43-7.40 (m, 1H),
7.37 (d, J = 9.7 Hz, 1H), 7.25 – 7.25 (m, 2H), 7.22 (m, 2H), 5.14 – 5.13 (m, 3H), 4.01 (1H, NH), 3.98 (m,
2H, CH2), 2.24 (d, J = 0.9 Hz, 3H, CH3), 1.11 (t, J = 8.0 Hz, 3H, CH3). 13C-NMR (300 MHz, DMSO): δ
(ppm): 183.14 (C=O), 182.30 (C=O), 165.69 (C=O), 152.41 (C=O), 149.19 (C), 144.21 (C), 132.26 (6C),
129.35 (3C), 128.88 (4C), 128.66 (2C), 99.29 (C), 59.77 (CH2), 53.85 (CH), 18.25 (CH3), 14.51 (CH3).
2.4.3 Ethyl 4-(4-(9,10-dioxo-9,10-dihydroanthracen-1-yloxy)phenyl) -6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (G3, C28H22N2O6):
Yellow-brown solid, % yield: 60,1H NMR (300 MHz, DMSO): δ (ppm); 10.12 (s, 1H, NH), 9.16 (s, 1H,
NH), 8.38 – 8.12 (m, 2H), 7.71 (m, 2H), 7.62 (m, 1H), 7.52 (m, 1H), 7.33 (dd, J = 7.5, 1.5 Hz, 1H), 7.27
(d, J = 9.7 Hz, 2H), 7.20 (m, 2H), 5.09 (d, J = 9.7, 0.9 Hz, 1H), 3.95 – 3.90 (m, 2H, CH2), 2.50 - 2.20 (d, J
= 1.0 Hz, 3H, CH3), 1.06 (t, J = 8.0 Hz, 3H, CH3). 13C NMR (300 MHz, DMSO): δ (ppm): 192.85 (C=O),
165.75 (C=O), 148.80 (4C), 140.50 (2C), 131.67 (3C), 131.12 (2C), 128.84 (C), 127.71 (2C), 126.67 (C),
124.76 (4C), 99.66 (C), 59.65 (CH2), 54.38 (CH), 18.23 (CH3), 14.39 (CH3).
2.4.4 Ethyl 1-(4-formyl-9,10-dioxo-9,10-dihydroanthracen-1-yl)-6-methyl-2-oxo-4-phenyl-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (G4, C29H22N2O6):
Dark Yellow solid, % yield: 53, 1H NMR (300 MHz, DMSO): δ (ppm): 9.12 (s, 1H, NH), 7.69 (d, J = 6.8,
2.1 Hz, 3H), 7.61 (m, 2H), 7.55 (m, 2H), 7.30 (m, 2H), 7.27 – 7.25 (m, 3H), 5.11 (s, 1H, CH), 4.72 (m,
2H, CH2), 2.20 (s, 3H, CH3), 1.06 (t, J = 8.0 Hz, 3H, CH3). 13C NMR (300 MHz, DMSO): δ (ppm):182.79

(C=O), 182.36 (C=O), 165.81 (C=O), 148.76 (C=O), 145.14 (2C), 131.29 (7C), 128.85 (2C), 128.75
(3C), 127.78 (3C), 126.66 (3C), 99.78 (C), 59.72 (CH2), 54.34 (CH), 18.18 (CH3), 14.48 (CH3).
2.5 Biological assay
2.5.1 Antioxidant assay
All the synthesized compounds were assessed for their radical scavenging activity by diphenyl
picrylhydrazyl (DPPH) free radical. To perform the antioxidant activity, the stock solution of compounds
G1-G4 having a concentration 22mg/ml has been prepared. The solutions were then diluted to 250μg,
200μg, 150μg, 100μg and 50μg concentration. DPPH solution of 4mM was prepared in pure methanol.
Ascorbic acid was used as a standard. DPPH with methanol was used as a control. In test-tube 1ml of
sample/standard solution with 2ml of DPPH solution was added and kept in dark for 30 minutes. Then
absorbance of the mixture was measured by using Hitachi U-2900 spectrophotometer at 517nm and
expressed as their percentage inhibition, calculated by using the formula mentioned by Marinova and
Batchvarov[31].
For comparing results, IC50 values of each compound (G1-G4) were calculated by plotting the graph and
performing non-linear regression analysis on GraphPad Prism 8.0.2.
2.5.2 Antidiabetic assay
Antidiabetic activity of all the compounds (G1-G4) was screened by following the method reported by
Eseyin et al. [32] with little modification described here. The mixture containing 0.5ml of sample
compound from each diluted solution (250μg, 200μg, 150μg, 100μg and 50μg) along with 0.5ml buffered
solution of the enzyme was incubated at room temperature for 10min. After this 0.5ml of the starch
solution was added and again incubated for 10min. Then 1ml of DNSA was added to the mixture and kept
in boiling water bath for 5 minutes, cooled down and diluted with 20ml of distilled water. The control
sample was prepared without any sample mixture. Acarbose was used as a standard. The absorbance of
the mixture was taken at 540nm using Hitachi U-2900 spectrophotometer. The inhibitory percentage was
calculated using the same formula used for antioxidant activity. For comparing results, IC50 values of each
compound (G1-G4) were calculated from the plots by performing non-linear regression analysis on
GraphPad Prism 8.0.2.
2.6 Molecular Docking study
Basically, molecular docking is performed to screen compounds for their biological activity/ potential on
the basis of possible orientations and conformations for binding site of ligand. To scrren molecules for
their activity, first of all, reference molecules (arcabose used standard for anti-diabetic potential and
ascorbic acid as standard for anti-oxidant) of respective protein were docked standardize the protocol. The
molecular mechanism of synthesized compounds G1-G4 was explored by conducting molecular docking
study by using Molegro Virtual Docker version. Crystal structures of Proteins used for docking analysis
were obtained from RSCB protein data bank online at RCSB, http://www.rcsb.org and saved as pdb. PDB
ID for anti-diabetic activity is human pancreatic amylase (PDB:1HNY) with resolution of 1.8 Å was used
as target receptor site whereas for anti-oxidant activity Drosophila melanogaster carboxypeptidase
having PDB ID (3MN8) was used as target crystal structure. All these structures were downloaded as

PDB formate (gz) [33-35]. Synthesized compounds G1-G4 were drawn in BIOCHEM draw 2D-structures,
moved to 3Dbio-Chemdraw by copy-paste and then saved as Mol2 file. Open the docking software and
import the protein molecule as pdb. Prepared template and all compounds G1-G4 were run one by one.
Before docking, co-factors and water molecules of each protein were removed. Literature study revealed
that MVD Molegro Virtual Docker (MVD 2013 6.0.1, Molegro ApS) showed best results for binding
affinity in the form of Mol Dock score in comparison to other docking softwares e.g., autodock, Moe,
Glide etc. Binding interaction of receptor protein molecule with top rank pose of ligand was observed
via hydrogen bonding interactions [33, 35-37]. All the data related to study as parameters like MolDock
score, bond length, residues interacting along with groups of ligand interacting were tabulated in tables 3
and 4. All the results of docking for each compound were summarized and discussed in results and
discussion (Fig. 6a, 6b, 7a, 7b, 8).
2.7 Quantitative Structure Activity Relationship (QSAR) studies using DFT
Computational studies of synthesized compounds provide us with optimized molecular structures and
predict the robustness of molecular reactivity [35]. Quantum mechanically computed descriptors
optimized by density functional theory provide the quantitative structure activity relationship for
synthesized organic compounds [38, 39]. DFT/B3LYP has been preferred for QSAR studies, optical and
electronic properties of compounds because this method is systematic and provides an efficient balance
between chemical precision, biological activity and computational tariff [40-42].
2.7.1 Calculation of Micromolecular Descriptors
All synthesized compounds have been computed with Gaussian 09 program favored by the Gauss View
5.1 interface for an imaginal presentation of optimized structures and graphics 39. The molecule is
geometrically optimized by using hybrid type B3LYP functional with 321-G basis set in the configuration
of DFT, which provides HOMO-LUMO geometries, energy gap, net charge, dipole moment and other
molecular descriptors [38, 39]. Quantum chemical parameters based upon DFT computations for the
purpose of SAR studies at DFT/B3LYP/3-21G(d) were calculated.
3 Results and Discussion
A novel sequential synthesis of anthraquinone based pyrimidine derivatives has been done through a one-
pot relay process. Firstly, anthraquinone and pyrimidine derivatives have been synthesized according to
the methods found in the literature with little modification mentioned above in the synthesis protocol.
Then by combining both of the precursors in the presence of a suitable catalyst, our novel targeted co-
crystal have been synthesized. To assess the synthetic process for the preparation of anthraquinone based
pyrimidine derivatives, we explored the conditions required to simply combine our model substrate that is
anthraquinone derivative 3 and pyrimidine derivative 5. It has already been recognized that among the
transition metal catalyst, copper (Cu) catalyst proved to be potently used in several organic syntheses.
Copper contributes to effectively catalyze several reactions like C-C and C-N cross-coupling, heterocyclic
synthesis, amide linkage formation and conjugate addition [39, 41]. According to one school of thought,

copper catalyst along with some metal oxides synergistically enhance the selective conversion. Copper
together with metal oxide provide a basic metal site to simultaneously accelerate the retro aldol
condensations and isomerization reaction [43]. Recently, studies have shown that copper oxide
synergistically coactive copper chloride and facilitate the electrophilic reactions [44]. Therefore, after
exploring the conditions required for the subsequent reaction, copper chloride along with cupric oxide as
a catalyst and methanol as a solvent was used to make our coupling reaction feasible. The overall one-pot
relay procedure involves intermolecular coupling reaction between anthraquinone 3 and pyrimidine 5
derivatives catalyzed through synergistically active copper catalyst to form four novel anthraquinone
based pyrimidine derivatives (G1-G4) as shown in Fig. . After the successful synthesis, all the synthesized
compounds were confirmed trough the interpretation of their spectral data.
The 1H-NMR and 13C-NMR spectra of compound G1-G4 showed characteristic peaks which enable us to
identify and characterize the corresponding compounds. The 1H-NMR spectra of G1 displayed
characteristic protonic singlet peak at δ 9.14 which confirms the presence of amide group present at the
junction of the anthraquinone and pyrimidine ring. While the characteristic carbon peak in the 13C-NMR
of G1 at δ 165.7 is due to carbonyl functionality directly linked with –NH group. These distinguishing
peaks of G1 confirm the successful formation of our targeted product.
All other peaks of anthraquinone and pyrimidine rings in the spectra of compound G1 are in consonance
with the literature [45].
1
The H-NMR spectra of G2 showed a characteristic protonic singlet peak at δ 4.01 is due to –NH group
which joins the anthraquinone and pyrimidine ring together. While the little upfield chemical shift value
at 144.21 and 132.26 ppm in the 13C-NMR of G2 is due to the effect of directly linked -NH group. These
distinguishing peaks of G2 confirm the successful linkage between anthraquinone and pyrimidine ring.
All other peaks of anthraquinone and pyrimidine rings in the spectra of compound G2 are similar to G1
except the additional peaks at δ3.98 and 1.11 in 1H-NMR and 59.7 and 14.5 in 13C-NMRwhich originates
1
due to the presence of –OC2H5 group. In the H-NMR spectra of G3 the missing singlet peak at δ 5.00,
which should be shown in case of the presence of unreacted hydroxyanthraquinone, confirms that there
must be a linkage. While in the 13C-NMR spectra of the same compound, the characteristic upfield
chemical shift values of carbon at 148.80 ppm shows that these carbons must be attached directly in C-O-
C linkage. These two characteristic peaks confirm the successful synthesis of our compound G3. All other
peaks of anthraquinone and pyrimidine rings in the spectra of compound G3 are similar to G2.
In the 1H-NMR spectra of G4 again one missing singlet peak of –NH at δ 9.00-10.00, which should there
if pyrimidine derivative left unreacted, confirms that there must be some sort of linkage. While the 13C-
NMR spectra of the same compound showed the distinctive up field chemical shift value of carbon at
148.80 ppm. This distinguishing peak shows that this carbon must be directly attached to the –NR group.

Here both the characteristic peak values confirm the successful formation of compound G4. Furthermore,
all other peaks of anthraquinone and pyrimidine rings in the spectra of compound G4 are similar to G2
except the additional protonic peak at δ 9.12 which arises due to the presence of the aldehyde group.
3.1 Biological evaluation
3.1.1 Antioxidant activity
Unstable free radicals have shown a main role in developing a number of diseases and antioxidants are
primarily the source of eliminating these radicals by inhibiting the oxidation rate and protecting the body
from damage [46]. Oxidative stress caused by these free radicals in atherosclerosis, stroke, diabetes and a
number of other diseases are treated with antioxidants nowadays [47, 48]. In order to evaluate in vivo and
in vitro antioxidant activity, several procedures have been developed recently [49-51]. In our study, the
antioxidant ability of our compound was evaluated by DPPH radical scavenging activity. DPPH is known
to be one of the stable free radical that can accept an electron or hydrogen radical from the corresponding
donor to form a diamagnetic molecule. When the DPPH accepts a hydrogen from donor, its solution
readily covert from dark purple to yellow [52-54]. In vitro antioxidant activity of all the synthesized
compounds (G1-G4) have shown the results summarized in Table 1. Standard ascorbic acid show 51.25
percent inhibition. At various concentration, compound G1-G4 were screened for their percent inhibition.
In comparison to standard ascorbic acid, compound G3 with a concentration of 50μg/ml have shown the
excellent antioxidant activity with 49.75 percent inhibition. While the other compounds G1, G2 and G4
have shown the maximum 30.78, 45.76 and 36.94 percent inhibition respectively with moderate to good
antioxidant activity as compared to the standard ascorbic acid.
To compare the overall results, we have calculated the IC50 value for each sample compound through
GraphPad Prism analysis and the results are shown in Fig. . The lower IC50 values indicate the greater
antioxidant activity of compounds. Certainly, the compound G2 and G3 showed the excellent DPPH
radical scavenging activity with IC50 of 15.09 and 21.88 μg/ml respectively. This remarked activity may
be attributed to the combined effect of anthraquinone and pyrimidine precursor. While the other two
compounds i.e. G1 and G4 have shown a weak inhibitory activity with the IC50 value of 64.31 and
97.51μg/ml respectively due to the structural differences of compounds.
3.1.2 Antidiabetic activity
The disease of high blood glucose levels is characterized as diabetes, which becomes a serious problem
nowadays. So, the main goal of the scientist is to develop treatment or medication that can effectively
treat diabetes by controlling the levels of blood sugar. In this modern age, there is a great development in
the field of medication or treatment which can treat diabetes with hyperglycemia and hypoglycemia
agents [55, 56]. Despite these developments, all the treatment modes and medications are still related to
some side effects which opens up the ways for further investigation. In this study, we have checked the
antidiabetic activity of newly synthesized compounds (G1-G4) by in vitro alpha-amylase analysis. In vitro
antidiabetic activity of all the synthesized compounds have shown the results summarized in Table 2.
Acarbose was used as a standard and exhibit 61.70 percent inhibition. At various concentration,
compound G1-G4 were screened for their percent inhibition. In comparison to standard acarbose,
compound G2 exhibits good antidiabetic activity with 57.80 percent inhibition. While the other

compounds G1, G4 and G3 in comparison to standard acarbose have shown moderate antidiabetic activity
with a maximum 40.96, 39.36 and 37.94 percent inhibition respectively.
Furthermore, the inhibitory effects of each synthesized compound were evaluated with the calculation of
IC50 which interprets the concentration of the inhibitor that is required to inhibit 50% of its targeted
enzyme. The lower IC50 values indicate the greater antidiabetic activity of compounds. In comparison to
the overall result, compound G2 and G4 (Fig.) showed a strong inhibitory activity with an IC50 value of
24.23 and 28.94 μg/ml. respectively. Whereas, the other compounds i.e. G1 and G3 have shown a weak
inhibitory activity with the IC50 value of 89.37 and 78.82μg/ml respectively. The overall difference
showed in the results may be attributed to the structural differences of a compound.
3.2 Molecular Docking study analysis
Docking studies were performed to explore the action mechanism of targeted compounds G1-G4 as
antidiabetic and antioxidant agents. G1-G4 showed multiple interactions with active sites of binding
proteins receptors (Error! Reference source not found.a, 6b, 7a, 7b and 8) explored by Docking results.
The more feasible binding modes of molecules G1-G4 for anti-diabetic protein (human pancreatic alpha
amylase) revealed their docking score range from -119.48 to -131.536 Kcal/mol while of standard drug
acarbose used was -111.57 kcal/mol respectively. Best possess of ligand that fit into receptor sites were
observed in MVD. G1 and G3 compounds showed zero interactions (Fig. 6a-b and table 3). G2 showed two
interactions as oxygen of 30-O with Thr 163 and oxygen of aldehyde group at 11´ with His 305 having
bond lenths of 2.602 and 2.44 A◦ respectively (Fig.6a-b). Compound G4 interact with Ile 235 by its
oxygen of aldehyde group at 11´-position having bond length of 2.89 A◦ (Fig. 6a-b and Table 3). Capacity
of all compounds G1-G4 for binding affinity and anti-diabetic activity were observed in a good co-
relation. Experimental results of antidiabetic activity of compound G2 showed best %age inhibition
among all compounds (Tale 2) which was in accordance with MolDock scores and additionally quantity
of hydrogen bond interactions with receptor also confirm the G2 as a good inhibitor of target enzyme [56,
57].
Synthesized compounds were further screened for their antioxidant potential by using ascorbic acid as
standard. Their molecular docking results showed that best fit modes of compounds G1-G4 have Mol
dock score in range of -131.96 to -184.273 kcal/mol while standard ascorbic acid has mol dock score of -
80.346 kcal/ mol (Table 4). From molDock score it has cleared that all the compounds G1-G4 have good
anti-oxidant activity as compared to standard (Fig. 7a-b, Tbale 4)[55]. 3D interface of best fit poses as
ligand into receptor site are shown in MVD. In vitro anti-oxidant activity of synthesized compounds is
greatly correlated with molecular docking results. Compound G1 showed three interactions with
receptor as 15-O with Lys 370, 16-O with Gln 79 and 31-O with Ser 86 having bond lengths of 2.72, 2.61
and 3.27 A˚ respectively. G2 showed only two interactions of 15-O with Lys 208 and oxygen of aldehyde
at 11-position with Arg 367 having bond lenths of 3.091 and 3.093 A˚ respectively whereas compound
G4 also have three hydrogen bond interactions as; oxygen of aldehyde group at 11ʹ-position with Lys 370
at distance of 3.108 A, 15-O and 28-O with Asn 203 at bond lenth of 3.17 and 3.01 A respectively (Fig.
7a-b and table 4). Among all compounds, G3 showed interactions as 15-O linked with Rag 68 and 16-O
linked with Asn 203 having hydrogen bond lenths of 3.58 and 3.41 A˚ respectively. 25-NH and oxygen of
aldehyde present at 26-position interact with same receptor Phe 65 at different bond lenths 2.60 and

3.1 A˚. Likewise, 30-O and 31-O iteract with Ser 207 at distance of 3.10 and 2.60 A˚ respectively [33, 56,
58]. In case of ascorbic acid, it’s all hydroxyl group that showed hydrogen bonding interactions with Lys
370, Ser 207, Arg 87, Ser 369 and oxygen of ring showed interaction with Lys 370 respectively. Length of
all the hydrogen bonds involved were found within the range 2.83 -3.28 Å (Fig.8, Table 4) [57, 59].
By observing tabulated data (
Table 3 and

Table 4), it has been confirmed that the differences of interactions were due to structural
differences among target molecule G1-G4 and this theoretical data was found very close to experimental
data evaluated for their biological action mechanism. Docking results (
Table 3) explored the maximum interactions for G2 in PDB:1NHY and pose which reflect
maximum interaction was shown in Error! Reference source not found.a-b. Docking results for
PDB:3MN8 (

Table 4) were displayed that the maximum interactions were found for G3 (Error! Reference
source not found.a-b) and this confirmed the G3 as an excellent inhibitory agent. By comparing these
results, it was suggested that inhibitory potential of compounds G1-G4 is due to their interactions with
their binding domains and dependent upon substituent moieties.
3.3 Quantitative Structure Activity Relationship (QSAR) analysis
Quantitative Structure Activity Relationship analysis of our compounds (G1-G4) leads us to fundamental
results which are discussed below.
3.3.1 Global Reactivity Descriptors
Electronegativity (χ), chemical potential (µ), global hardness (η), global softness (S), electrophilicity index
(ω) and Electronic energy (E) are the molecular reactivity parameters.

Table 5 showed the calculated values for each synthesized compound. Dipole moment (µ) is the
measure of the bond properties and charge densities in the molecule. Fig. showed the vector of the
dipole moment of all the synthesized compounds. Compound G3 has a large value of dipole moment
which showed that it has the best charge distribution and increasing distance of bonds. This means that
compound G3 showed the best conductivity improved through oxidation [60]. Highest electrophilicity
value (ω = 0.086 eV) and electronegativity values (χ = 0.1720 eV) indicate that compound G4 is the best
electron acceptor or good electrophile [61]. The large negative value of electronic energy (E) indicates
the stability of compound G4 as compared to other compounds that referred to the Coulombic forces,
dipole-dipole interactions and hydrogen bonds contributed towards the higher binding affinity, more
solute-solvent interactions and lowIC50 value [62]. Hence, the low antidiabetic IC50 value of compound G4
(Table 2) is proved to be in accordance with this theoretical evaluation.
3.3.2 Molecular Electrostatic Potential (MEP)
MEP help to predict the hydrogen bonding interaction, molecular recognition process and interpretation
of electrophilic and nucleophilic reactions. This model is the check of responsiveness of molecule
towards a binding substrate and behavior of molecule with other compounds. Also, this modal is a visual
scheme for checking the relative polarity of the molecule. Fig. showed the molecular electrostatic
potential map based on SCF energy. The positive (blue) region of molecular electrostatic potential
showed nucleophilic reactivity while the negative (red and yellow) regions showed the electrophilic
reactivity [36]. The blue color is an indication of the site of the nucleophilic attack, in all the compounds,
hydrogen atoms are capable to react with a nucleophile. While red or yellow color is an indication of an
electrophilic attack, so all the oxygen atoms are capable to react with an electrophile. While benzene
rings remain neutral as green color indicates the neutral reactivity [63].
3.3.3 Frontier Molecular Orbital Analysis (FMO)
According to this theory, HOMO and LUMO are vital factors as these orbitals are modeled for molecular
reactivity and pharmacological properties. In which HOMO acts as an electron donor and LUMO acts as
an electron acceptor. Fig. shows the energy level and dispersion of LUMO and HOMO orbitals calculated
at the B3LYP/3-21G(d) [64].
Values of HOMO and LUMO predicted that HOMO is more stable than the LUMO and energy gap having
small difference showed that molecule is soft, tend to be more polarized and biologically active in
inhibiting the enzymatic activity. This energy gap showed that the molecule is chemically more reactive
and kinetically less stable [63]. Homo is related to ionization potential and LUMO is linked to electron
affinity [40, 44].
Compound G1 has the highest value of HOMO energy (EHOMO = -0.06878eV). In the same way, the
compound G4 has the lowest value of LUMO energy (ELUMO = -0.11641 eV). HOMO-LUMO diagram of four
compounds (Fig. ) showed that the best electron donor moieties in the molecules are amide group and
the oxygen atoms of the quinoline molecule. In the same way, the best electron moiety is the
anthraquinone moiety of the G4 compound due to the presence of LUMO orbitals on this moiety. The
compound G2 has the lowest energy gap (ΔEgap = 0.10122 eV) which tends to make it softer and more

reactive as compared to another molecule. Chemical hardness and softness of G2 (η = 0.05061 eV, S =
9.8794 eV) are lesser among all molecules so the molecule is more reactive of all other compounds [61].
Conclusively, the compounds are optimized by using density functional theory calculations using B3LYP
as a basis set with 3-21G as a functional level using Gaussian 09 package. FMO studies showed that
compound G2 has a lower energy gap, therefore, considered to be more reactive as compared to all
other compounds. Hence, compound G2 showed the best antioxidant and antidiabetic activities
described in biological evaluation, which is in accordance with its theoretical results. Molecular
electrostatic potential studies showed that all the compounds have positive region around hydrogen
atoms and the negative region around the oxygen atom of carbonyl groups and anthraquinone group
[33]. Other reactivity parameters calculations shown in

Table 5 confirm that these electronic properties help us to synthesize the leading compound for future
research.
4 Conclusion
The summary of our whole work is as follows. Four new biologically active compounds G1, G2, G3 and
G4 were synthesized in a one-step procedure by the incorporation of substituted anthraquinone and
pyrimidine derivatives. The incorporation of these derivatives was done by the one-pot relay process
using a synergically active copper catalyst. Among the four novel compounds, the compound ethyl 4-(4-
(9,10-dioxo-9,10-dihydroanthracen-1-ylamino) phenyl) -6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (G2) showed remarkable biological applications and proved to be a potent antioxidant and
antidiabetic drug. Other synthesized compounds showed good to moderate biological applications.
Molecular docking results further assist the experimental results and confirms that among all the
synthesized compounds (G1-G4), ethyl-4-(4-(9,10-dioxo-9,10-dihydroanthracen-1-yloxy) phenyl)-6-
methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (G3) showed maximum interactions with both
evaluated proteins. QSAR analysis further relates to the experimental study and shows superior stability
of compound G4 with the large negative value of electronic energy and low IC50 value of this compound.
Also, MEP predicts the hydrogen bonding interaction and shows the reactivity of all four compounds
having moderate to good nucleophilic and electrophilic reactivity. Furthermore, FMO analysis gives us
confidence about the synthesis and successful application of our novel compounds and showed the
lower energy gaps and higher reactivity of compound G2 compared to all other compounds, which is in
accordance with the experimental results of the biological analysis. Hence, overall the compounds
synthesized here proved to be the potential compounds that may cause evolutionary success in the field
of medicine and synthesis. Conclusively, the synthetic strategy opted here can be of prime interest for the
research community and the novel compounds synthesized here can be used in the future as an effective
organic drug due to their safety, inexpensive, sustainability, enhanced activity and eco-friendly nature.
Credit Author Statement
Gul Zarren1: Methodology, Experimental and writing related portion
Nusrat Shafiq1*: Conceptualization, Designing Reactions and research theme, Molecular Docking study,
Writing- reviewing
Uzma Arshad1: DFT calculations, visualization, Software Validation and writing related portion
Naila Rafiq2: Bioactivity Portion writing
Shagufta Parveen1: Reviewing and Editing
Zaheer Ahmad3: Supervision

Declaration of Competing Interest
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.
There is no conflict of interest
Acknowledgement
Authors are thankful to Higher Education Commission of Pakistan to provide facilities to conduct this
research under HEC/SRGP.1142 research Grant.
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List of Figures
Fig. 1. General synthesis of anthraquinone precursor.
Fig. 2. General synthesis of pyrimidine precursor.
Fig. 3. Schematic synthesis of targeted anthraquinone based pyrimidine derivatives.
Fig. 4. Graphical representation of antioxidant activity and estimation of IC50 value by non-
linear regression analysis in GraphPad Prism.
Fig. 5. Graphical representation of anti-diabetic activity and estimation of IC50 value by
non-linear regression analysis in GraphPad Prism.
Fig. 6a. Leading pose of compound G1, G2, G3, G4 into active pocket of 1HNY along with standard drug
ACARBOSE; (A1) coupling of compound G1 with residues of 1HNY; (A2) coupling of compounds G2 with
residues of 1HNY; (A3) coupling of compound G3 with residues of 1HNY; (A4) coupling of compound G4
with residues of 1HNY; (A5) coupling of compound A5 (acarbose) with residues of 1HNY
Fig. 6b. 2D visualization of compound G1, G2, G3, G4 by Discovery Studio Visualizer;
(A1) 2D model of compound G1; (A2) 2D model of compound G2; (A3) 2D model of compound G3; (A4)
2D model of compound G4
Fig. 7a. Leading pose of compound G1, G2, G3, G4 into active pocket of 3MN8 along with standard drug
QUERCETIN: (B1) coupling of compound G1 with residues of 3MN8; (B2) coupling of compound G2 with
residues of 3MN8; (B3) coupling of compound G3 with residues of 3MN8; (B4) coupling of compound G4
with residues of 3MN8; (B5) coupling of QUERCETIN with residues of 3MN8
Fig. 7b. 2D visualization of compound G1, G2, G3, G4 by Discovery Studio Visualizer: (B1) 2D model of
compound G1; (B2) 2D model of compound G2; (B3) 2D model of compound G3; (B4) 2D model of
compound G4
Fig. 8. 2D schematic illustration by Discovery Studio Visualizer of reference compounds A5 (Acarbose)
and Quercetin
Fig. 9. The optimized geometry, numbering system, and the vector of the dipole moment of
compound (G1-G4) using B3LYP/3-21G(d).
Fig. 10. Molecular Electrostatic Potential (MEP) of compounds (G1-G4) based on DFT studies.
Fig. 11. Frontier Molecular Orbitals of Compounds (G1-G4).

List of Figures
O
O
O
R1
R2
R1
R2
1. Alum, H2O, rt
2. conc. HCl
+
O
O
3
1
2
R1 = OH, NH2, CI
R2 = H, CHO
Fig. 1.
R3
O
R3
H2N
OH
NH2
O
O
C2H5O
NH
H3C
OC2H5
CHO
H3C
N
H
O
CuCl2.2H2O
4
5
R3 = H, Cl
Fig. 2.

Fig. 3.

120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50 = 64.31 μg/ml
IC50 = 15.09 μg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
log [G1]
log [G2]
120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50 = 97.51 μg/ml
IC50 = 21.88 μg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
log [G3]
log [G4]
Fig. 4.

120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50 = 24.23 μg/ml
IC50 = 89.37 μg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
log [G1]
log [G2]
120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50 = 78.82 μg/ml
IC50 = 28.94 μg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
log [G3]
log [G4]
Fig. 5.

Fig. 6A

Fig. 6B