Cancer is a disorder that results from genetic or epigenetic alterations in the somatic cells and has abnormal cell growth which may be spread to other body parts. They form a subset of neoplasm. The unregulated growth of cells in a group is called neoplasm or tumor and they form a lump or mass and may be distributed diffusely [1] [2] . Cancer is a leading cause of death globally (about 9.6 million deaths) and approximately 15 million new cancer cases will be diagnosed as the world population reached 7.5 billion by 2020 [3] , and about 420 million new cases of cancer by 2025 [2] . Cancer cells utilize multiple strategies such as high glycolytic flux, redox signalling and modulation of autophagy to avoid cell death and overcome nutritional deficiency [4] .

Topoisomerase (Topo) is an established target for anticancer drugs and is known to be responsible for regulating the topological constraints in DNA. Topo II inhibitors are classically divided into catalytic inhibitors and Topo II poisons, according to their mechanism of action. According to Okoro and Fatoki [5] , topo II catalytic inhibitors destroy cancer cells through the inhibitions of Topo II enzymatic activities, thus preventing the formation of topo II-DNA complex without increasing DNA cleavage, via the mechanisms of action that include interfering with DNA binding, inhibiting cleavage of the DNA molecule, ATP hydrolysis, and binding to the ATP binding site, whereas Topo II poisons destroy cancer cells by increasing the amount of covalent Topo II-DNA complexes and preventing the religation of the cleaved DNA strands, thus forming unwanted double strand breaks that are toxic to the cells, and, subsequently, leading to apoptosis.

Most of the first-line agents for treating cancer are Topo II poisons, such as etoposide (non-intercalator), doxorubicin, and m-amsacrine (intercalator), but due to side effects, such as risk of cardiotoxicity and secondary malignancies, that are often encountered during the use of DNA poisonous drugs, research is now shifting towards the discovery of Topo II catalytic inhibitors, which have good pharmacokinetics profiles [5] .

In silico approaches that involve virtual high-throughput screening (VHTS), three-dimensional quantitative structure activity and relationship (3D-QSAR), molecular docking, and ADME/Tox prediction have been applied to study potential inhibitors of Topo IIα [6] [7] . The present study builds on the previous report from our lab on some acridone derivatives [8] . The aim of this study was to synthesize and computationally evaluate pharmacological potential of some derivatives of 9-amino-3-phenylacridone, as topoisomerase II inhibitors.

The molecular weight of the ten derivatives of 3-phenyl-9-aminoacridone that were synthesized in this work have molecular weights ranging from 306.33 g/mol (58c) to 382.84 g/mol (58e); with melting points ranging between 250 C (58a) to 301 (58g), as shown in Table 1. The structure of the ten derivatives of 3-phenyl-9-aminoacridone together with standard drugs, amsacrine and etoposide, are shown in Figure 2.

All the 10 derived compounds have moderate solubility, high gastrointestinal absorption and inhibitory effect on cytochromes which are similar to that of amsacrine. Two derivatives (58e and 58j) were predicted to be non-permeant of

blood-brain barrier, and this property was found similar to that of amsacrine and etoposide, as indicated in Table 2. Furthermore, ADMET results in Table 3 indicate that the intestinal absorption of compounds 58e was predicted to be slightly higher than that of amsacrine and etoposide, and that all the synthetic compounds have intestinal absorption that are much higher than that of etoposide. Also, all the synthetic compounds as well as amsacrine and etoposide were predicted to be inhibitors of p-glycoprotein I and II. The results indicate that compounds 58d-i have cytochrome P450 inhibitory profiles that are similar to that of amsacrine. The toxicity results showed that only compound 58e has no AMES toxicity potential, which is similar to that of etoposide, and that only etoposide was not a potential inhibitor of hERG II with no potential hepatotoxicity.

Note: Physicochemical properties: Molecular weight (MW), Molar Refractivity (MR), Total polar surface area (TPSA). Lipophilicity: Consensus Log P. Water Solubility: ESOL Log S, ESOL Class. Pharmacokinetics: Gastrointestinal absorption (GIA), Blood-brain barrier (BBB), P-glycoprotein (P-gp) substrate, Inhibition of Cytochrome P450 (CYPs) type CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, Skin permeation (Log Kp). Druglikeness: Bioavailability Score (BS), Medicinal Chemistry: Synthetic accessibility (SA).

Also, compound 58f-h was predicted to have highest T. Pyriformis toxicity.

The protein targets of the ten derivatives of 3-phenyl-9-aminoacridone that were synthesized in this work includes acetylcholinesterase (ACHE), butyrylcholinesterase (BCHE), carboxylesterase 4A/5A/1 (CES4A/CES5A/CES1), carboxyl ester lipase (CEL), and neuroligin 1 (NLGN1). These targets did not match any of targets of amsacrine and etoposide (Table 4).

The results of molecular docking of the ten derivatives of 3-phenyl-9-aminoacridone that were synthesized in this work, showed that the synthetic compounds (58a-j) and the standard drugs have overall best binding affinities for human acetylcholine esterase than butyrylcholinesterase, and overall best binding affinities for human topo IIα than human topo IIβ as shown in Table 5. The docking pose of interaction of some of the compounds with the molecular targets are presented in Figure 3 and Figure 4, which indicated the involvement of hydrogen bonding and pi-stacking in some of the ligand-protein complexes.

Ten derivatives of 3-phenyl-9-aminoacridone were synthesized in this work. The results of the ADMET in this study indicate that compounds 58a-j have profiles that are nearly identical to that of amsacrine. The results of molecular target prediction pointed the compounds 58a-j towards acetylcholinesterase (AChE), butyrylcholinesterase, carboxyl ester lipase, and neuroligin 1 proteins.

Neuroligin 1 (NLGN1) encodes a trans-synaptic protein that acts as a postsynaptic adhesion molecule involved in the regulation of glutamatergic transmission. A study has shown that increased mRNA and protein levels of NLGN1 expression were associated with worse overall survival or recurrence-free survival in colorectal cancer patients [17] . Moreover, it was found that Neuroligin 1

Note: A: Acetylcholinesterase (ACHE). B: Butyrylcholinesterase (BCHE). C: Carboxylesterase 4A/5A/1 (CES4A/CES5A/CES1). D: Carboxyl ester lipase (CEL) E: Neuroligin 1 (NLGN1) F: Topoisomerase (DNA) II beta (TOP2B). G: Topoisomerase (DNA) II alpha (TOP2A). H: Tumor protein p53 (TP53). I: Potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). J: matrix metallopeptidase 2 (MMP2). K: Topoisomerase (DNA) I (TOP1). L: B-cell CLL/lymphoma 2 (BCL2). M: Caspase 2, apoptosis-related cysteine peptidase (CASP2). N: Werner syndrome, RecQ helicase-like (WRN). O: UDP-galactose-4-epimerase (GALE).

promotes colorectal cancer progression by modulating the tumor suppressor adenomatous polyposis coli (APC), thus impacting WNT/β-catenin pathway [18] . Not all cancer types exhibit high AChE activities, and some of the examples of cancers which possess high AChE activity than normal tissues are: non-small cell lung cancer (NSCLC) such as lung adenocarcinoma, squamous cell lung carcinoma, large cell carcinoma; human leukemias; breast cancer; thyroid cancer, pancreatic cancer, as well as high grade glioma, medulloblastoma and oligodendroglioma [19] .

The synthetic compounds investigated in this study have tacrine (9-amino-1,2,3,4-tetrahydroacridine) scaffold in their structure. Tacrine has been found that to be an effective inhibitor of acetylcholinesterase and butyrylcholinesterase, as well serves as a relatively weak catalytic inhibitor of Topo II when compared with 9-aminoacridine [20] [21] [22] . However, tacrine was withdrawn from Alzheimer’s disease therapy due to its hepatotoxicity and other detrimental side effects in Alzheimer’s disease patients [23] . Tacrine is being currently used as a versatile scaffold in medicinal chemistry for designing novel hybrid compounds with improved pharmacological and toxicological profiles affecting several pathological mechanisms.

Recent studies have explored the anti-cancer activity of tacrine and tacrine-derivatives in human cancer. Roldan-Pena et al. [24] , synthesized tacrine dimers, sulfide tacrine dimers and selenotacrine dimers and tested their growth-inhibitory activity in a panel of six human cancer cell lines, and the results showed that these tacrine dimers were approximately 10-fold more potent in inhibiting the enzyme activity of AChE than tacrine itself, and in all cell lines the IC₅₀ values of the tacrine dimers were approximately 100-fold lower than tacrine and 20-fold lower than standard chemotherapeutic drugs like 5-fluorouracil and cisplatin [24] .

It was reported that tacrine-coumarin conjugates containing seven, eight and nine methylene groups in the spacer moiety decreased the viability of human colorectal cancer, breast cancer and mouse mammary carcinoma cells [25] . Small molecule synthetic AChE-inhibitors have many pleiotropic biological effects apart from suppressing AChE activity. Recently, studies show that tacrine and its analogs are not just strong AChE inhibitors they also potently block carbonic anhydrase activity [26] , and DNA topoisomerase I and II [27] [28] .

The binding of amsacrine to topo IIα is similar to that of etoposide based on the interacting amino acid residues, thus only compounds 58a, 58c, 58f, and 58i showed similar binding, which could be used to infer their anticancer properties. Similar results had been reported for the docking interaction of human topoisomerase IIα (PDB: 1ZXM) with naphthalimide–benzothiazole conjugates and etoposide, which indicated amino acid residues Val57, Gln59, Gln60, Met61, Trp62, Tyr72, Phe77, Pro79, Tyr82, Lys83, Lys306, Gln309, Ile311, Phe313, Ala318, Ser320, Lys321 and Glu379 [29] .

The binding of amsacrine to topo IIβ is different from that of etoposide based on the interacting amino acid residues, thus only compounds 58a, 58c, 58f and 58g showed binding similar to that of amsacrine, while compounds 58b, and 58e showed binding similar to that of etoposide. A study has reported that amino acid residues Gly488, Gly506, Ser763, Ser800, Ala801, Ser802, and Pro803, as well as ASP463, Arg487, and Met766 are involve in the Topo II binding interactions near DNA region [30] [31] [32] , and it is evident that most of the first-line agents for treating cancer are Topo II poisons, such as etoposide (non-intercalator), and m-amsacrine (intercalator) [5] . A study on novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives has reported that Cl, F, and Br substituted at C7 acted as covalent, rather than interfacial, topoisomerase II poisons and that an amino group at C9 was critical for activity [33] . Thus, compound 58b and 58e could be DNA non-intercalator of topo IIβ while others will be non-Topo II poisons but catalytic inhibitors of topo IIα and topo IIβ.

The binding of amsacrine to acetylcholinesterase is slightly the same with that of etoposide based on the interacting amino acid residues, thus only compounds 58a showed binding property that is different from the standard drugs. Also, binding of amsacrine to butyrylcholinesterase is markedly different from that of etoposide based on the interacting amino acid residues, thus only compounds 58a, 58b, 58d, 58h and 58j showed binding similar to that of amsacrine, while compounds 58c, 58e, 58f, 58g, and 58i showed binding similar to that of etoposide.

A study has observed that there was an increase in AChE expression in the apoptotic cells induced by the DNA topoisomerase inhibitors etoposide or excisanin A, in colon cancer cell line SW620 [34] . Moreover, the implication of 3-phenyl-9-aminoacridone derivatives as anticancer properties will be by inhibition of Topo IIα/β through AChE and BChE pathway, while anti-neurological properties will be by inhibition of Topo IIα/β through neuroligin pathway, although physiological functions of topo IIβ are yet to be fully understood [5] [35] [36] [37] .

In this study, 10 derivatives of 3-phenyl-9-aminoacridone were synthesized and characterized. The potential pharmacological indications of these compounds were computationally predicted. Overall, the results of this study suggest that the synthetic compounds 58a, 58c, 58f, 58g, and 58i could probably inhibit topo IIα by catalytic inhibition as seen with amsacrine, but only 58b and 58e possessed DNA non-intercalation properties as seen with etoposide, serving as topo II poison. Further work will be done to validate the reported properties of these synthetic compounds on various cancer cell lines; especially those are characterized with high AChE than normal cell.

The authors acknowledge the institutional support from the Department of Chemistry, Tennessee State University (USA), Department of Biochemistry, Vanderbilt University School of Medicine, Grant Number 1R01 GM 126363 (NO), USA, Department of Biochemistry, Federal University Oye-Ekiti (Nigeria).

The authors declare no conflicts of interest regarding the publication of this paper.

Oyedele, A.S., Fatoki, T.H., Dalvie, E., Osheroff, N. and Okoro, C.O. (2023) Synthesis, SAR, and in Silico ADME Screening Studies of Some 9-Amino-3-Phenylacridone Derivatives as Topoisomerase II Inhibitors. Open Journal of Medicinal Chemistry, 13, 15-34. https://doi.org/10.4236/ojmc.2023.132002