Topo II inhibition and DNA intercalation by new phthalazine-based derivatives as potent anticancer agents: design, synthesis, anti-proliferative, docking, and in vivo studies

Abstract This research presents the design and synthesis of a novel series of phthalazine derivatives as Topo II inhibitors, DNA intercalators, and cytotoxic agents. In vitro testing of the new compounds against HepG-2, MCF-7, and HCT-116 cell lines confirmed their potent cytotoxic activity with low IC50 values. Topo II inhibition and DNA intercalating activities were evaluated for the most cytotoxic members. IC50 values determination demonstrated Topo II inhibitory activities and DNA intercalating affinities of the tested compounds at a micromolar level. Amongst, compound 9d was the most potent member. It inhibited Topo II enzyme at IC50 value of 7.02 ± 0.54 µM with DNA intercalating IC50 of 26.19 ± 1.14 µM. Compound 9d was then subjected to an in vivo antitumor examination. It inhibited tumour proliferation reducing solid tumour volume and mass. Additionally, it restored liver enzymes, proteins, and CBC parameters near-normal, indicating a remarkable amelioration in their functions along with histopathological examinations.


Introduction
Cancer is characterised by uncontrolled cell growth and proliferation following genetic mutation. It represents one of the most important health issues worldwide and is the second leading cause of death 1,2 . Therefore, it represents one of the greatest challenges to medical researchers, especially with the continued failure of current therapies from one side and the development of drug resistance from the other side [3][4][5] .
The current search and discovery of new drug candidates with anticancer activities have become one of the most important issues for medicinal chemists nowadays [6][7][8][9][10][11] . Among the most important chemotherapeutic agents applied for cancer treatment are those that interact with DNA. Anticancer agents in the previously mentioned class belong to either alkylating agents, groove binders, or intercalating agents 12 . DNA intercalating agents got great attention from scientists due to their promising antitumoral activity [13][14][15][16][17][18] . They are classified into two major groups of compounds that intercalate between DNA base pairs (especially G and C, 70%) without covalent binding: 1) acridines and related compounds and 2) anthracyclines and related compounds 19 . These compounds produce local structural changes to the DNA molecule, including the lengthening of the DNA strand following the unwinding of its double helix. So, DNA intercalators are mutagenic due to their retardation or even inhibition of DNA transcription and replication 20 .
Doxorubicin is one of the two first isolated and introduced anthracyclines as antitumor agents. It works through two mechanisms of action; 1) intercalates into the DNA double helix without covalent binding, and 2) binds covalently to topoisomerase II (involved in DNA replication and transcription), poisons the cleavable complex of DNA and prevent its re-ligation, and finally results in an apoptotic action 21,22 .
Phthalazine moiety was recommended in the area of medicinal chemistry to have promising antitumor activity and primarily to act as DNA intercalator and topoisomerase II inhibitors as well 16,23,24 . On the other hand, many other organic moieties like triazoles, hydrazine amides, hydrazine thioacetamides, benzylidene hydrazones, sulphonamides, benzoic acid, and thioacetamides derivatives were identified and introduced as potential antitumor agents [25][26][27][28][29] . Some reported DNA intercalators and topoisomerase II inhibitors showing their common pharmacophoric features were depicted in Figure 1.

The rationale of molecular design
A ligand-based drug design approach 30 topoisomerase II inhibitors taking into consideration the basic pharmacophoric features of doxorubicin. It is worth mentioning that there are three crucial pharmacophoric features present in doxorubicin which guided our rationale. The first one is the planar polyaromatic system (chromophore) inserted in between the DNA base pairs. The second one is the presence of a groove binding side to occupy the minor groove of DNA. The third part is the cationic moiety, or a species having the ability to be protonated in the physiological PH to interact with the negatively charged phosphate group of DNA sugar moiety 32,33 . Molecular hybridisation of triazolo phthalazine moieties instead of the planar aromatic system of doxorubicin with different recommended anticancer moieties (hydrazine amides, hydrazine thioacetamides, benzylidene hydrazones, sulphonamides, benzoic acid, and thioacetamides derivatives) as the groove binding site with the presence of -NH-or -NH 2 group to act as a cationic site were designed and synthesised as depicted in Figure 2. 2. Results and discussion:

Chemistry
The new triazolo phthalazine members were synthesised following the reactions outlined in Schemes 1 and 2. 2,3-Dihydrophthalazine-1,4-dione 2 was prepared by reaction of phthalic anhydride 1 with hydrazine hydrate in absolute ethanol 34 . Compound 2 was then chlorinated with phosphorus oxychloride to afford 1,4-dichlorophthalazine 3 35 , which was then heated with hydrazine hydrate in boiling ethanol 36 to furnish 1-chloro-4-hydrazinylphthalazine 4. A solvent-free reaction was performed to  cyclize compound 4; thus, compound 4 was heated with butyric anhydride to give the cyclized member 5 37 . Reflux of compound 5 with hydrazine hydrate in boiling ethanol afforded the target hydrazinyl triazolo derivative 6. Compound 6, however, was allowed to react with different isocyanates and/or isothiocyanates to afford the corresponding semicarbazides 7a,b, and/or Scheme 1. General procedure for synthesis of target compounds 7a,b, 8a,b and 9a-d; Reagents and conditions: (i) NH 2  thiosemicarbazides 8a,b, respectively. Furthermore, treating the hydrazinyl compound 6 with appropriate substituted benzaldehyde derivatives with a catalytic amount of glacial acetic acid afforded the corresponding imines (Schiff's bases) 9a-d. IR charts of the later compounds revealed the loss of NH 2 absorption band of compound 6 and the presence of NH absorption bands in the range of 3180 to 3242 cm À1 . In contrast, 1 H NMR spectra of members 9a-d displayed characteristic singlet signals in the range of d 8.12 À 8.72 ppm representing the new benzylidene protons. The 13 C NMR spectra of compounds 9a-d, however, showed a characteristic downfield peak around d 141 ppm corresponding to the new benzyledine carbon (Scheme 1).
Upon cyclisation of hydrazinyl triazolo derivative 6 with carbon disulphide in alcoholic potassium hydroxide, the corresponding mercaptotriazole derivative 12 was afforded. 16 1 H NMR spectrum of 12 displayed a singlet D 2 O exchangeable signal at d 14.24 ppm corresponding to the SH proton. The potassium salt 13 was then obtained upon heating compound 12 with potassium hydroxide in absolute ethanol 29 . The potassium salt 13 was heated with the appropriate N-aryl-2-chloroacetamide derivatives 11a-c and/or 2chloroacetamide in the presence of a catalytic amount of potassium iodide in DMF following the reported procedure to afford the corresponding thioacetamide derivatives, 14a-c and 15, respectively (Scheme 3).

In vitro anti-proliferative activities
Anti-proliferative activities of the target compounds were assessed via the standard MTT method [38][39][40] against three cancer cell lines, namely, hepatocellular carcinoma (HepG-2), colorectal carcinoma (HCT-116), and human breast adenocarcinoma (MCF-7). Doxorubicin was used in this test as a positive control.
As illustrated in Table 1, the obtained results revealed that most synthesised compounds showed remarkable anti-proliferative activities against the tested cell lines.
In general, compounds 9d and 14a were found to be more active than the reference drug, doxorubicin, against the three tested cell lines. In particular, compound 9d was the most potent counterpart with IC 50 values of 5.08, 4.74, and 4.95 mM as it was 1.63, 2.03, and 1.34 times more active than doxorubicin (IC 50 ¼ 8. 28, 9.62, and 7.67 mM) against HepG2, HCT-116, and MCF-7 cell lines, respectively. While, compound 14a was about 1. 46

Dna intercalation assay (DNA/methyl green colorimetric assay)
DNA/methyl green assay was carried out for the synthesised derivatives using doxorubicin as a positive control following the reported procedure described by Burre et al. 41 , to give extra quantitative data about the binding affinity of the target compounds towards the DNA molecules. DNA-binding affinities of the target compounds were represented as IC 50 values and are summarised in Table 2. Compounds 8a, 9d, and 14a exhibited excellent DNA binding affinities more than the reference drug with IC 50 values of 29.63 ± 1.41, 26.19 ± 1.10, and 28.74 ± 1.71 mM, respectively. In addition, compounds 7a, 7b, 8b, and 14b showed remarkable activities but slightly less than the reference drug with IC 50 values of 37.14 ± 2.0, 36.57 ± 1. 8

Topoisomerase II inhibitory activity
Seven compounds that exhibited significant DNA binding affinities (8a, 8b, 9a, 9c, 9d, 14a, and 14b) were further estimated to determine their inhibitory activities towards topoisomerase II. The activity of topoisomerase II was determined according to the reported procedure described by Patra et al. 42 . Doxorubicin was utilised as a positive control in this test. The results were reported as IC 50 values and summarised in Table 2. Compounds 8b, 9d, and 14a was found to be the most potent derivatives with IC 50 values of 8.91 ± 0.77, 7.02 ± 0.54, and 7.64 ± 0.66 mM, which were more active than the reference drug, doxorubicin (IC 50 ¼ 9.65 ± 0.77 mM). The other tested compounds, 8a, 9a, 9c, and 14 b, exhibited moderate to weak activities with high IC 50 values ranging from 13.66 ± 1.02 to 13.66 ± 1.02 mM.

In vivo antitumor activity
To examine the in vivo anticancer activity of compound 9d, adult female Swiss albino mice (30 mice) inoculated with I.P. injection of Solid Ehrlich Carcinoma (SEC) tumour cell lines in a volume of 0.2 ml physiological saline contains 1 Â 10 6 viable cells for 24 h.
These mice were randomly divided into four groups (7 mice/ group). The 1 st group (normal saline-control group) was used as a negative control, the 2 nd group (the SEC-control group) was injected with the SEC, the 3 rd group (compound-treated group) was injected with SEC then with compound 9d, and the 4 th group was injected with the SEC then with a standard anticancer drug, doxorubicin (DOX), as described in the experimental section. Bodyweight and survival were recorded daily until the 24 th day in both treated and control groups. At the end of the experiment, the blood of each group was collected under light anaesthesia for the estimation of hematological and biochemical assays. The anaesthetised animals were then sacrificed to evaluate of the antitumor activity and to conduct hematological, biochemical, and histopathological assays, Figure 3.

Hematological and biochemical assays (Blood parameters assay).
At the end of the experiment, animals from different groups were sacrificed, and blood samples were collected for hematological parameters, including Hb, RBC's, and WBC's levels, and serum for determination of liver enzymes ALT, AST levels, and proteins.
Liver enzymes ALT and AST were significantly increased to 63.4, 64.67 (U/L), respectively, following tumour inoculation as shown in Table 3, compared with normal mice at 45.14 and 53.67 (U/L) because of hepatocellular damage. While liver protein and albumin were decreased to 6.13 and 2.97 (g/dL). Treatment with compound 9d substantially reduced liver enzymes to 42.9, 55.6 U/ L, respectively, and increased liver protein and albumin to 8.04 and 6.25 (g/dL), indicating a remarkable amelioration in the hepatocellular functions.
In terms of hematological parameters in SEC-bearing mice, all CBC parameters were changed in the SEC control, with Hb content and RBCs significantly decreased to 5.36 (g/dL) and 3.33 (10 6 /mL), respectively. When compared to normal control levels, the WBC count was significantly increased to 6.21 (10 3 /mL). Tumour propagation is routinely associated with decreased haemoglobin, RBC, and WBC counts 43,44 . After treatment with compound 9d, CBC levels were nearly restored to normal, where it elevated the Hb (8.2 g/dL), RBC's (5.37 10 6 /mL) and reduced the WBC's (3.72 10 3 /ml) levels.
Interestingly our results following previous studies 45,46 , illustrated the anticancer activity by improving hematological and biochemical parameters after treatment with the tested compound. Taken together, treatment of SEC mice with compound 9d improved hematological and biochemical parameters, as well as tumour weight and volume.

Histopathological examinations.
Histopathological examinations of liver tissues of the SEC-bearing mice in different treatments were illustrated in Figure 5. According to compound 9d ability to improve liver enzymes and proteins, its treatment was able to keep liver structure close to normal.   7). Signs of Ã and # are values with significant differences in tumour weight and tumour volume, respectively compared to SEC control using an unpaired t-test (P 0.05) using GraphPad prism. Values are expressed as Mean ± SEM (n ¼ 7). # Significant difference between treated groups and SEC control using unpaired t-test (P 0.05) using the GraphPad prism7. DNA binding site of Topo II (PDB ID: 3qx3). Docking investigation was carried out using Discovery Studio 2.5 software. An X-ray crystallographic structure of Topo II with its co-crystallised ligand, etoposide, was downloaded from the Protein Data Bank (PDB). Redocking of the co-crystalized ligand was initially performed aiming to validate the used docking protocol. The simulation of the   re-docked ligand successfully regenerated the same binding mode of the co-crystalized one inside the DNA binding site of Topo II with RMSD of 0.81 Å, which indicates the validity of the docking process, Figure 6.
The predicted binding pattern of the co-crystallised ligand, etoposide, revealed an affinity value of À30.13 kcal/mol with the formation of six H-bonds. The planar aromatic system occupied the hydrophobic pocket formed by Glu477, Gly478, Asp479, Leu502,  Arg503, Gln778, Met782, and Pro819. It was also stacked between different DNA nucleotides, namely, Cytosine (DC-8 and DC-14), Guanine (DG-7, DG-10, and DG-13), Adenine (DA-12), and Thymine (DT-9). The sugar moiety of etoposide was directed towards the DNA minor groove and stabilised by the formation of two H-bond interactions with Gln778 and DG-13. Similarly, its phenolic OH group formed two H-bond interactions with Asp479. Two H-bonds were also formed between the etoposide oxygen atoms and the DNA nucleotides DG-13 and DA-12 Figure 7.
The proposed binding mode of doxorubicin, with an affinity value of À33.50 kcal/mol, revealed that the doxorubicin planar aromatic chromophore formed aromatic stacking interactions with the different key residues Glu477, Gly478, Asp479, Leu502, Arg503, Gln778, Met782 in addition to the DNA nucleotides DT-9, DC-8, DC-11, DG-13, and DA-12. The sugar moiety of doxorubicin was oriented into the minor groove of DNA and stabilised by two H-bonds with Asp479. The rest of the compound was involved in several H-bond interactions with Arg503, DG-13, and DA-12, Figure 8.  A general investigation of docking results revealed that the designed compounds displayed a binding pattern comparable to that of the native ligand with predicted binding energy scores ranging from À18.49 to À29.91 kcal/mol.
The predicted binding mode of compound 9d as illustrated in Figure 9. Its triazolo phthalazine planner moiety was inserted between the DNA nucleotides with the formation of many hydrophobic interactions with DT-9, DC-8, DG-13, and DA-12 as well as Gly776, Gln778, and Ala779 amino acids. In addition, the 4-nitrophenyl part was oriented in the minor groove of DNA, forming hydrophobic interactions with DA-12 and Arg503. The nitro group of 9d, however, interacted with Arg503 via an H-bond interaction.
Compound 14a showed an affinity value of À27.36 kcal/mol. The planar aromatic system occupied the hydrophobic pocket formed by DT-9, DC-8, DG-13, and DA-12 nucleotides in addition to Arg503, Gly504, Gly776, Gln778, and Ala779 residues forming several pi-pi interactions. The benzoic acid moiety was directed towards the DNA minor groove with the formation of H-bond interaction with Arg503 residue Figure 10.

In silico ADMET analysis
ADMET studies were carried out for the synthesised compounds using doxorubicin as a reference compound. The predicted ADMET parameters were listed in Table 4.
The results revealed that compounds 7 a , 9 d , 14 a , 14 b , and 14 c had very low Blood Brain Barrier penetration power. Accordingly, such compounds were expected to be safe to CNS. Aqueous solubility of the synthesised compounds ranged from low to very low. Compounds 7a, 7 b , 8 a , 8 b , 9 a , 9 b , 9 c , and 15 showed good absorption level. Except compounds 9a and 9b, all members were predicted as non-inhibitors of CYP2D6. Except compounds 9 c , 14 a , 14 b , and 15, all compounds were expected to bind plasma protein more than 90% (Figure 11).

Toxicity studies
Toxicity prediction was carried out based on the validated and constructed models in Discovery studio software 47,48 . As shown in Table 5, most compounds showed in silico low adverse effects and toxicity against the tested models. Regarding developmental toxicity potential (DTP), all the tested compounds were predicted to be non-toxic. For Carcinogenic Potency TD 50 (Rat), all compounds showed higher values (from 0.873 to 34.570 mg/kg body weight/day) than that of doxorubicin (0.861 mg/kg body weight/ day) except compound 7a (0.651 mg/kg body weight/day). For rat maximum tolerated dose model, compounds 7 b, 8a, 8 b, 9a, 9 b, and 9c showed higher levels than doxorubicin. The tested compounds showed high oral LD 50 values ranging from 0. 0.229to 12.326 mg/kg body weight/day which were higher than that of Figure 11. The expected ADMET study of the target compounds. doxorubicin (0.227 mg/kg body weight/day) Moreover, except compounds 9a and 9d, all compounds were predicted to be mild and non-irritant against ocular irritancy and skin irritancy models, respectively.

Conclusion
A new series of phthalazine derivatives was designed hoping to discover novel Topo II inhibitor and DNA intercalator agents as well. Twelve compounds were synthesised and tested in vitro for their anti-proliferative activities against three human cancer cell lines, HepG-2, MCF-7, and HCT-116. The tested members exhibited a promising cytotoxic effect with IC 50 values ranging from 4.35 ± 0.19 to 78.49 ± 2.04 mM. All compounds were further estimated for their in vitro DNA intercalating effects. Amongst, seven compounds were further examined for their in vitro inhibitory activity against Topo II enzyme. Three compounds, 8b, 9d, and 14a, out of the seven exhibited potent Topo II inhibitory activities with IC 50 values of 8.91 ± 0.77, 7.02 ± 0.54, and 7.64 ± 0.66 mM, respectively. Finally, in vivo antitumor studies were carried out for compound 9d. In vivo study exhibited that treatment with compound 9d substantially inhibited tumour proliferation reducing solid tumour volume and mass. Additionally, it restored liver enzymes, proteins, and CBC parameters near-normal, indicating a remarkable amelioration in their functions along with histopathological examinations. Hence, compound 9d was investigated as a novel anti-cancer agent through Topo II inhibition and DNA-binding affinity. To conclude, compounds presented in the current study were proved to be potent Topo II inhibitors with DNA intercalating efficacy that can be further adopted for hit optimisation and/or lead discovery.

Chemistry
Starting materials and reagents were purchased from Sigma-Aldrich and used without purification. Melting points measurement was carried out by a Gallen lamp melting point apparatus and are uncorrected. Reactions progress was monitored by TLC (Merck, Germany), the spots were detected by exposure to UV lamp at k 254 nm. IR spectra were recorded by pye Unicam SP 1000 IR spectrophotometer using KBr discs and expressed in wavenumber (cm À1 ). 1 H and 13 C NMR spectra were recorded with Bruker Advance 400 spectrophotometer operating at 400 MHz and 100 MHz, respectively and the chemical shifts were given in d as parts per million (ppm) downfield from tetramethylsilane (TMS) as internal standard. The mass spectra were recorded on Varian MAT 311-A (70 e.v.).

Dna intercalation assay (DNA/methyl green colorimetric assay)
The DNA/methyl green assay was estimated in vitro for all the target derivatives using doxorubicin as a reference drug, adopting the protocol described by Burres et al. 41 as shown in the Supplementary data.

Measurement of topoisomerase II activity
Compounds (8a, 8b, 9a, 9c, 9d, 12a, and 12b) that showed the better results in anti-proliferative and DNA/methyl green assay were further evaluated for their in vitro inhibitory activities against Topoisomerase II using doxorubicin as a reference drug following to reported procedure described by Patra et al. 42  with free access to standard laboratory mice food and water. All procedures related to care and maintenance of the animals were performed according to the international guiding principles for animal research and approved by the Faculty of Science, Suez Canal University bioethics and animal ethics committee (Approval number REC-07-2021).
Solid Ehrlich carcinoma (SEC) was purchased from the National Cancer Institute (Cairo University, Egypt). The tumour cell line was proliferated in mice through serial intraperitoneal (I.P.) transplantation of a volume of 0.2 ml physiological saline contains 1 Â 10 6 viable cells for 24 h. SEC cells were collected 7 days after I.P. implantation. The harvested cells were diluted with saline to obtain a concentration of 5 Â 10 6 viable SEC cells/mL. A volume of 0.2 ml saline contains 1 Â 10 6 SEC cells that were I.P. implanted into each normal mouse. SEC cells (1 Â 10 6 tumour cells/mouse) were implanted subcutaneously into the right thigh of the hind limb.
The experimental animals were randomly divided into four groups. Group 1 served as the normal saline control (5 ml/kg B.Wt., I.P.). Group 2 served as the SEC control (1 Â 10 6 cells/ mouse). Group 3 served as the compound-treated group (5 mg/kg B.Wt., I.P.). Group 4 received the standard anticancer drug doxorubicin (5 mg/kg BW, I.P.) and is considered as a reference control. Bodyweight and survival were recorded daily until the 24 th day in both treated and control groups. At the end of the experiment, the blood of each group was collected under light anaesthesia to the estimate of hematological and biochemical assays. The anaesthetised animals were then sacrificed for evaluation of the antitumor activity and histopathological examination.

Antitumor potentiality.
It includes tumour volume, weight, and tumour inhibition ratio (TIR%). Time interval measurements of tumour volume using digital Vernier calliper (Tricle Brand, Shanghai, China). Measure tumour length and width using a clipper and then calculate tumour volume using formulations V ¼ (L Â W Â W)/2, where V is tumour volume, W is tumour width, L is tumour length. While TIR% was calculated according to the following equation At the end of the experiment, animals from different groups were sacrificed, and blood samples were collected for hematological parameters including, Hb, RBC's, and WBC's levels, and serum for determination of liver enzymes ALT, AST levels, and proteins. Complete blood count (CBC) was investigated using the Abbott CELL-DYNV R 1800 automated haematology analyser (USA) using ready-made kits (Abbott Laboratories, Abbott Park, IL, USA). Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated using commercial kits (ELITech clinical systems, France). Serum albumin level was determined by kit purchased from STANBIO Company (USA). Protein content was determined by colorimetric method using ready-made kits produced by Instrumentation Laboratory SpA, Inova diagnostics, Milano, Italy.

Histopathological study.
Specimens of liver-sacrificed mice were fixed in 10% saline formalin. The fixed liver specimens were dehydrated in ascending series of ethyl alcohol and embedded in paraffin. Sections at 5 mm thicknesses were stained with haematoxylin and eosin and examined under the light microscope.

4.3.
In silico studies 4.3.1. Docking study Discovery Studio 2.5 software was used to perform docking and visualisation according to the described protocol. 16

In silico ADMET analysis
ADMET studies were performed according to the reported procedure as adescribed in Supplementary data 51-53 .

Toxicity studies
Toxicity studies were performed according to the reported procedure as adescribed in Supplementary data [54][55][56] Disclosure statement No potential conflict of interest was reported by the author(s).