Novel Xanthene-1,8-dione Derivatives Containing the Benzylic Ether Tail as Potent Cytotoxic Agents: Design, Synthesis, In Vitro, and In Silico Studies

. Seventeen new xanthene-1,8-dione derivatives were synthesized and evaluated as cytotoxic agents against the lung carcinoma cell line (A549). Compound 9-(4-(benzyloxy)phenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione ( 4a ) showed good cytotoxic efects (34.59 μ M) in comparison to cis-platin. Docking results showed 4a could interact with DNA as intercalation. Calculated ligand efciency of compound 4a was more than daunomycin. Based on the results, it can be concluded that compound 4a is a suitable DNA binding agent.


Introduction
Today, cancer has become a big problem for the treatment systems of countries due to its widespread prevalence and the difculty of treatment.Comprehensive studies have been conducted to determine the causes, prevention, and treatment of this disease [1].DNA chain is known as the cellular target of many anticancer compounds.Te interaction of a drug with the DNA chain has a special role in the pharmacological efects as well as the mechanism of action of that drug.Understanding how the complex afects both the mechanical and structural properties of DNA is an important step toward elucidating the functional mechanism of binding agents and may also provide information for more selective drug design [2].
Drug binding to the DNA chain can be classifed into two general categories: covalent interactions and noncovalent interactions.Te most important examples of covalent interactions are DNA-alkylating drugs.Te main advantage of alkylating compounds is their high binding power to the nucleotide bases of the DNA chain.As a result of alkylation, the DNA chain undergoes structural changes that afect both transcription and replication processes [3].Temozolomide (I), carmustine (II), and cyclophosphamide (III) are three famous examples of DNA alkylating drugs (Figure 1) [4].
Noncovalent interactions are divided into two categories.DNA grooves Interacting agents and intercalating agents.Some small chemical compounds are attached to the minor groove of DNA by forming van der Waals and hydrogen bonds.Tese molecules deform the DNA by binding to the minor groove and disrupting DNA function [5].Berenil (IV) is an example of these compounds [6].Due to the formation of π-π interactions with DNA nucleotide bases, intercalators are placed perpendicular to the DNA chain and between nucleotide bases.Intercalator drugs cause a change in the twist of DNA and cause a change in function, leading to the inhibition of transcription, replication, and repair processes in DNA [7].Daunomycin (V) is an intercalating agent [8].
Xanthenes are a family of oxygen-containing heterocycles.Te pyran ring is the central building block of them.
Te tricyclic ring system of xanthene-1,8-diones resembles the A-B-C ring system in anthracyclines such as daunomycin (VI in Figure 1).As Figure 1 shows, if there is a suitable basic substitution in the benzyl or phenoxy moieties, it has the same orientation as the amino group in the aminoglycoside of Daunomycin.Te similarity between the ring systems of these two classes of anticancer agents prompted us to investigate the interaction of xanthene-1,8dione derivatives with DNA chain.

Materials and Instrumental.
Chemical substances were purchased from Merck and Sigma.Uncorrected melting points were measured by a Stuart SMP3 apparatus.An alpha-BRUKER IR device recorded the IR spectra of products on KBr disks.A Bruker 500-NMR spectrometer recorded the 1 H NMR spectra.An Agilent technology (HP 5975C MSD) mass spectrometer operating at an ionization potential of 70 eV, recorded the mass spectra of the products.Te A549 cancer cell line was purchased from the Pasteur Institute of Iran (IPI).PBS, FBS, RPMI, trypsin-EDTA, and Pen-strep were prepared from Kiazist Company.

General Procedure for the Synthesis of Xanthene-1,8-dione
Derivatives Containing the Benzylic Ether Tail.4-(Benzyloxy)benzaldehyde derivatives were synthesized as per the previous reported method [17].In a 25 mL roundbottom fask, 4-(benzyloxy)benzaldehyde derivatives 3 (1 mmol) and 2 mmol 1,3-cyclohexanedione were added to 3 mL ethylene glycol.Ten the reaction mixture was stirred at 80 °C for 24 hours.After the reaction was complete, a water/ice mixture was added to the reaction mixture to precipitate the products.Te precipitate was washed with the boiling CH 3 OH.For further purifcation, it was recrystallized with methanol.1.18 Å) was downloaded from the RCSB database [18].Te water molecules were removed, and the Daunomycin and apo form of DNA saved in pdb format separately.Te molecular docking procedure and ADME prediction were performed according to our previously reported paper [17].
Te synthetic routes for the preparation of xanthene-1,8dione derivatives containing the benzylic ether tail (4a-q) were illustrated in Scheme 1.For this purpose, the reaction between (benzyloxy)benzaldehydes (3) and 1,3-cyclohexanedione was carried out in ethylene glycol at 80 °C to obtain the desired products 4a-q in moderate to good yields.Te structures of new xanthene-1,8-dione derivatives were confrmed by IR, 1 H NMR, and mass spectroscopy.
Te mechanism of xanthine ring formation in the products is shown in Scheme 2. First, intermediate VII is formed by the condensation of one equivalent of 1,3cyclohexanedione and aldehyde.Ten the second equivalent of 1,3-cyclohexanedione reacts with intermediate VII, and after removing H 2 O, the xanthene ring is formed.

ADME Prediction.
All new derivatives (4a-q) of xanthene-1,8-dione were investigated to evaluate their druglikeness via Lipinski's rule of 5. Data in Table 2 show that all compounds passed Lipinski's rule and met the criteria for drug-likeness.
Daunomycin had a larger MW than the xanthene-1,8dione derivatives that violated the RO5.But the high polarity of Daunomycin (ClogP � 0.92) could compensate for the violation of the MW criterion.Te main diferences between the drug-likeness profles of Daunomycin and xanthene-1,8dione derivatives were the number of HBDs and polarity.Te xanthene-1,8-dione derivatives were lipophilic compounds (in comparison to Daunomycin) that did not have any HBD groups.
According to the obtained results, we concluded that the synthesized xanthene-1,8-dione derivatives were more lipophilic than approved anticancer drugs.Tis physicochemical property might help the entrances of molecules into the cells but decreases the selectivity profle of the proposed scafold.Directional hydrogen bonds (donor or acceptor) orient drugs to form specifc interactions in the binding site that lead to more selective drugs.

Biological Assay.
Our results on the evaluation of the antitumor activity of the compounds 4a-q against the lung cancer cell line (A549) are presented in Table 3. Te results show that only compound 4a has signifcant activity against A549 with an IC 50 value 34.59 µMin comparison to Cisplatin as a reference antitumor agent [20] with an IC 50 value 20.86 µM.It surprised us that the other derivatives did not have good activity against this cell line.

Docking Studies.
Binding energies of Daunomycin and 4a with DNA strand D (CPGPTPAPCPG) calculated by the docking process are presented in Table 4. Self-docking of crystallographic ligand via the mentioned procedure led to an RMSD equal to 0.95 Å. Te comparison of the predicted and crystallographic pose of Daunomycin showed minor deviation.Accordingly, we found that the molecular docking method was valid.Also, the validation was repeated for 3 times with diferent initial conformers of Daunomycin.Except for the population of the high-rank cluster (9.4% deviation), all the results were the same.Considering the obtained cytotoxic results, just compound 4a was evaluated in a molecular docking study.
Te comparison of the estimated free energy of binding after 106 bootstrapping cycles and the 95% confdence interval showed a signifcant diference in binding energy between Daunomycin and 4a.Te standard deviation of RMSD between conformers in the top cluster of Daunomycin and 4a was ±2.7 × 10 −2 and ±5.8 × 10 −3 , respectively.Te larger conformational space of 4a in the top cluster of the docking result was related to the structural variation of the benzyloxy tail in the minor groove of DNA.Te Journal of Chemistry diference in conformational variation afected the range of the confdence interval.Due to diference in the core structure of Daunomycin and 4a, ligand efciency was calculated to compare the participation of each atom in binding to DNA.Both ligands had the same LE (0.32 kcalmol −1 /heavy atoms).Although the designed xanthene-1,8-dione possessed fewer atoms and consequently more binding interactions than Daunomycin, the efciency of the atoms of 4a was higher than that of Daunomycin in the binding process.Te docking study of 4a showed that the xanthene-1,8dione ring intercalated between nucleobases (Figure 3).In this orientation, the phenoxybenzyl tail occupied the minor groove.Considering the predicted binding pose, the active

Figure 2 :
Figure 2: Chemical structures of compounds VI and VII.

Figure 3 :
Figure 3: Orientation of Daunomycin and 4a in complex with D(CPGPTPAPCPG) DNA strand.Ligands are shown by orange sticks.Te proper substitutions at meta position of phenoxy or ortho position of benzyl can occupy the same binding region of aminoglycoside.

Table 4 :
Binding energy of Daunomycin and 4a with DNA strand D (CPGPTPAPCPG).Energy is reported in Kcal mol −1 .Te confdence interval was calculated by applying 106 cycles of bootstrapping.
that adding a basic functional group at the metaposition of phenoxy or orthoposition of benzyl moieties could improve binding afnity and the physicochemical properties of the designed xanthene-1,8-dione derivatives.