Synthesis of novel cytotoxic tetracyclic acridone derivatives and study of their molecular docking, ADMET, QSAR, bioactivity and protein binding properties

Acridone based synthetic and natural products with inherent anticancer activity advancing the research and generating a large number of structurally diversified compounds. In this sequence we have designed, synthesized a series of tetracyclic acridones with amide framework viz., 3-(alkyloyl/ aryloyl/ heteroaryloyl/ heteroaryl)-2,3-dihydropyrazino[3,2,1-de]acridin-7(1H)-ones and screened for their in vitro anti-cancer activity. The in vitro study revealed that compounds with cyclopropyl-acetyl, benzoyl, p-hydroxybenzoyl, p-(trifluoromethyl)benzoyl, p-fluorobenzoyl, m-fluorobenzoyl, picolinoyl, 6-methylpicolinoyl and 3-nicotinoyl groups are active against HT29, MDAMB231 and HEK293T cancer cell lines. The molecular docking studies performed for them against 4N5Y, HT29 and 2VWD revealed the potential ligand–protein binding interactions among the neutral aminoacid of the enzymes and carbonyl groups of the title compounds with a binding energy ranging from − 8.1394 to − 6.9915 kcal/mol. In addition, the BSA protein binding assay performed for them has confirmed their interaction with target proteins through strong binding to BSA macromolecule. The additional studies like ADMET, QSAR, bioactivity scores, drug properties and toxicity risks ascertained them as newer drug candidates. This study had added a new collection of piperazino fused acridone derivatives to the existing array of other nitrogen heterocyclic fused acridone derivatives as anticancer agents.

drugs, amongst such compounds acridone derivatives plays a key role as acridine based natural and synthetic compounds are a vital class of nitrogen heterocycles. These compounds are attentive as they exhibit an extensive array of pharmaceutical properties as it is being an integral part of natural products and important heterocycles in medicinal chemistry.
Similarly, 2-hydroxyacridone (X) and its substituted derivatives were reported as DNA topoisomerase II and protein kinase C inhibitors 26 (Fig. 1). It is also reported that some of the synthesized fluorinated acridone derivatives as potent anticancer agents against MCF7, A549 and HT29 cell lines 27 . Similarly, the versatile biological activity of acridone and piperazine hybrids/conjugates 28 like multifunctional cholinesterase inhibition acivity 29 , anticancer agents 30 , and antimicrobial activity 31 , hAChE and hBChE inhibitors 32 . This striking segment stimulated us to conjugate piperazine ring onto parent acridone ring to greatly induce its potentiality. This hypothesis has been realized by generating a piperazine fused acridone ring that is appended with an amide linker comprising of alicyclic/ aromatic/ hetero-aromatic rings by N-aroylation/ N-aroylation. In ultimate this study has realized the concept making the newer molecules with enhanced lipophilicity and binding interactions towards the interacting proteins. The merit of this accomplishment is that all existing nitrogen heterocycle fused acridone derivatives includes triazole, imidazole and pyrimidine rings in them, this is the first report on the synthesis and anticancer activity evaluation of the piperazine fused acridone derivatives. Moreover the tertiary amide linker (compared to the tertiary amines) impregnated on the piperazine ring has been proved as a prominent structural feature of the study.

Materials and methods
Chemistry. Reagent grade chemicals and analytical grade solvents were procured from Sigma-Aldrich and used for the synthesis, characterization and biological screening of title compounds. The analytical reagent [AR] grade solvents were purified by literature methods 33 . TLC monitoring was performed on alumina supported silica gel 60 F254 plates (Merck, Darmstadt, Germany) and visualized under UV light. All 1 H (400 MHz) and 13 C NMR (400 MHz) spectra were recorded on Bruker NMR spectrometer. Chemical shifts were reported in ppm (δ) with reference to TMS (internal standard) using CDCl 3 , DMSO-d 6 and TFA as solvents. Coupling constant (J) values were given in Hertz and the multiplicities were designated as br, broad; s, singlet; d, doublet; t, triplet; m, multiplet. Molecular weights of the synthesized compounds were checked by SHIMADZU LCMS-2020 series in ESI mode. Melting points were recorded in capillaries and on Buchi Melting Point B-540 and are uncorrected. IR data was recorded on Perkin Elmer Spectrum 100. Column chromatography was performed with 100-200 mesh silica.

3-(Pyridin-3-yl)-2,3-dihydropyrazino[3,2,1-de]acridin-7(1H)-one (7s
ADMET properties 39 . ADMET properties of 7a-s have calculated on preADMET online server 40 , which assisted to understand their ADMET potentialities In continuance, the prediction of tumarogenic, mutagenic, irritant and reproductive effects have assisted to ascertain the toxicity properties. All the ADMET properties were identified with in the potential limits of safe drugs as presented in Table 8. QSAR studies. As ADMET properties are foremost requisites for drug candidates to reach clinical stage, in addition the oral bio-availability comprehends them by precise poise among partitioning and solubility as evolved from QSAR studies. Similarly, obeying Lipinski's rule of five 41 is a notable tool for screening potentiality of newer molecules and was predicted by Molinspiration 42 software. The computation of Veber Rule and other parameters like partition coefficient (octanol to water) and percentage of absorption in addition fulfils the QSAR studies as presented the results in Table 9.
Bioactivity and toxicity risk studies. The QSAR descriptors of 7a-s have been predicted on molinspiration online server 42 as properties were explored with molinspiration engine v2018.10 and bioactivity scores were explored with molinspiration engine v2018.03 and the results proved them as safer drugs. Similarly, the Osiris online property explorer toolkit 43 has provided the toxicity risks and drug properties as presented in Table 10. This study helped in understanding the physico-chemical interactions of the synthesized compounds against their targets and eventually facilitated in determining their drug properties.
BSA protein binding assay. The protein binding assay of title compounds was performed with Bovine serum albumin (BSA), a standard protein, to correlate their evaluated anticancer activity through mutual interactions, where such interactions made these anticancer agents as transportable in blood. The UV-Visible absorption spectroscopy has adapted to track the changes in the absorption bands induced by conformational change showing the formation of the protein bound compound. The binding constant K b predicted from the BSA protein binding assay was given in Table 11.
In assay, prepared standard BSA protein solution [2.5 mg in 10.0 mL of Tris-HCl buffer (5 mM Tris-HCl + 10 mM NaCl @ pH = 7.4) was preserved at refrigeration conditions. Title compound solutions were incubated at room temperature for nearly 30 min before the process. Then the UV-Visible absorption spectra of title compounds of a conserved concentration of 25 μM in combination with prepared BSA solutions ranging www.nature.com/scientificreports/ from 5 to 500 μM were analyzed in the wavelength ranging from 200 to 400 nm. The UV-visible spectral studies were performed in a mixed solvent system (1:9 DMSO and Tris-HCl buffer) and absorption spectra were recorded by using 1-cm-path quartz cuvettes at room temperature. Then the Binding constant K b is calculated from Benesi-Hildebrand equation shown below. Where, A o and ε f are the absorbance and molar extinction coefficients of title compounds in free form, A and ε b are absorbance and molar extinction coefficients of respective title compounds bound with BSA protein.

Results and discussion
Chemistry. The acridone fused piperazino-carboxamide derivatives (7a-s) synthesized in the present study are novel as the piperazine ring has been constructed on the bridged carbon (linking ring A and B) rather simply fusing on ring A or C of basic acridone skeleton. This kind of construction has evolved some triazolo-, imidazolo-and pyrimidino-fused acridones so far, this piperazine fused acridone frame linking alkyl/ aryl moieties with amide linker is a novel accomplishment to the existing array of acridones. As Pd(OAc) 2 -Xantphos is used as a potential catalyst-ligand system for C-N coupling 44 , here we have extended the use of Cs 2 CO 3 -Pd(OAc) 2 -Xantphos system in producing the acridone ring by forming a C-N linkage through N-arylation process. The chemical structures of the synthesized compounds (7a-s) of the study were confirmed by IR, 1 H, 13 C and mass spectrometry analyses and all the spectral responses were observed in their expected standard range, and cor-  www.nature.com/scientificreports/ Table 8. ADMET properties predicted for compounds 7a-s. a BBB (Blood-Brain Barrier) penetration = [Brain]/ [Blood]; b Caco-2 cells derived from human colon adenocarcinoma, possessing multiple drug transport pathways through intestinal epithelium; c HIA (Human intestinal absorption), the sum of absorption and bioavailability evaluated from ratio of excretion in urine, bile and feces etc.; d MDCK cell system is used as tool for rapid permeability screening; e % of drug that binds to plasma protein; f in vitro Ames test by Metabolic and Non-metabolic activated TA100 and TA1535 strains collected from rat liver homogenate.

Entry
In vivo blood-brain barrier penetration (C. brain/C. blood) a    45 of percentage of inhibition. The potential compounds 7k, 7r, 7l, 7o, 7a, 7d, 7b, 7e, 7i and 7q having the substitutions like p-hydroxybenzoyl, p-fluorobenzoyl, benzoyl, p-(trifluoromethyl)benzoyl, picolinoyl, cyclopropyl-acetyl, 3-nicotinoyl, 6-methylpicolinoyl, trifluoromethylpicolinyl and 3-fluorobenzoyl groups on acridone fused piperazine moiety. The significant inhibition of title compounds is due to the greater electron releasing capacity, better intermolecular hydrogen bonding interactions, higher electronegativity, molecular volume and steric hindrance, which are making them to get interact with targeted cell lines and have boosted the pharmacokinetic, physicochemical, liphophilic, properties of the title compounds that led to the metabolic destruction of cells 45 . Significantly the tested profiles on HEK293T normal cell lines revealed them as safer compounds which are more potent against HT29 and MDA-MB-231 cell lines as shown in Tables 1, 2  ADMET properties. The investigation of ADMET properties for a group of analytes under study assists to comprehend the physico-chemical interactions of those analytes and helps us to evaluate their drug-likeness properties. This type of high-throughput screening helps in distinguishing a lead compound of a large group in the fascinated domain of a target 47 . This critical study assists in identifying the pharmacokinetic properties of 7a-s and to unveil their drug-like interactions. The Human intestinal absorption describes carrying of the effective composites to the target cell tissues via blood stream and made them to interact mutually. In oral administration of a drug-like compound its degree of absorption has been considered, where it again depends on its inherent bioavailability properties. There the absorbed quantity of compound be itself distributed into the muscles and there to other organs by the circulation via extracellular sites. Then the compound's distribution lowers its plasma concentration independently and then metabolizes, from there those metabolites will be distributed by the enzymatic redox reactions. In pharmacological aspects, potential metabolites distributed will work proficiently on cellular systems, inactive metabolites deactivates the administered compound and diminishes its effect in vivo, and the inert metabolites will be automatically excreted from kidneys. The analysis of the obtained ADMET properties (Table 8) of 7a-s informed that the in vivo BBB penetration potentiality ratio is effective with a range of 0.1523-3.7909 and confirms their high CNS significance and approves their greater permeability permeability for the self-distribution in vivo. It is strengthened on the basis of their in vitro Caco-2 cell permeability perceived with 21.6738-44.8966 nm/s range, which institutes their persistent permeability to bind with plasma proteins and to penetrate in to BBB system. The in vitro PPB affinity in 89.0836-98.4091% range authorizes the robust binding capability of the compounds to plasma proteins. The in vitro MDCK cell permeability in 0.0454-215.3520 nm/s range reveals them as good permeable. Similarly the %HIA in the range of 96.2654-98.7226 ratio assures their interactions with the proper species in the anticipating target of domains. The negative sign of the toxicity predictions indicate that compounds 7a-s are non-toxic and safer drugs. Inclusively this ADMET analysis has been revealed the potential physico-chemical interactions of 7a-s and their drug-likeness properties. QSAR studies. QSAR results (Table 9) indicate that analogues 7a-s under study with molecular weights ranging from 409.37 to 313.36 (less than 500 daltons) demonstrated log P in the range of 3.69 to 1.51 (less than 5) suggesting their better permeability through cell membranes. Similarly number of hydrogen bond acceptor and donors are in the line of Lipinski's rule as < 10 and < 5 respectively. The molecular refractivity from 90.33 to 103.57 cm 3 /mol as in the standard range i.e., 40-130 cm 3 /mol ascertains as all analogues are obeying the Lipinski rule of five and all they are considerably orally active drugs with good drug likeness properties. Moreover the total polar surface area contributed by the sum of polar atoms such as oxygen, nitrogen and attached hydrogens 48 , which is ranging from 38.13 to 62.54 is also obeying the Veber rule as it is less than 140 Å 2 . Hence, these molecules are estimated to be easily diffused, absorbed and transported. Here the total polar surface area is very much correlated with the hydrogen bonding of a molecule and is associated with the transport properties of drug across the membranes, prediction in the BBB and intestinal crossing. Molecules with total polar surface area in the range ≤ 160 Å 2 have good intestinal absorption and ≤ 60 Å 2 has BBB penetration 49  www.nature.com/scientificreports/ able bonds in all the compounds are limited to 1-2 which is as per the Veber rule (i.e., less than 7) and hence in total they also obeying the Veber's rule and again ascertains them as orally administrable drugs. Furthermore, the percentage of absorption ranging from 87.42 to 95.85, density in the range of 1.37 to 1.53 gm/cc, solubility ranging from − 5.52 to − 7.10, Van der Waals volume in the range 269.89 to 333.61 Å 3 and ClogP in the range of 3.43 to 5.28 ascertains all the compounds as significantly safer drug-like compounds. These calculations are in understanding the physico-chemical interactions of synthesized analogues with their targets and eventually helped in determining their drug properties by associating with the bioactivity and toxicity risks studies.
Bioactivity and toxicity risk studies. The prediction of bioactivity and toxicity risk studies of the synthesized analogues 7a-s (Table 10) revealed their bioactivity properties like GPCR ligand property, ion channel modulator, kinase inhibitor, nuclear receptor ligand interactions, protease inhibitor and enzyme inhibitor interactions, and drug properties like drug-likeness and drug scores have measured and ascertained as potential non-toxic molecules. This Molinspiration prediction extensively helps to investigate the cheminformatics of the compounds under study by correlating with the database of in vitro and in vivo studies of established drugs based on mutual functional group similarity. The toxicity risk results clearly indicate that 7a-s are safer as showing low or no risks of mutagenicity, tumorigenicity, irritant and low or no effect on reproductive system and conformed drug like behavior. The positive value of drug likeness states that the molecule contains predominantly fragments which are frequently present in commercial drugs whereas majority of compounds accounts for negative values 38 . Solubility is an important factor which aids in the movement of a compound from the site of administration into the blood stream and poor solubility leads to poor absorption 50 . Alike drug score is a harmonizing parameter of druglikeness, ClogP, logS, molecular weight and toxicity risks and used to judge the compound's overall potential to qualify for a drug. Ultimately it is predicted that all the synthesized analogues 7a-s exhibited higher scores than the standard drug.
Structure activity relationship studies. The comparative structural analysis infers that amide linker is beneficial as it highly elevated the activity of all the compounds. In over-all increase in number of hetero atoms in 7h (thiophene) and 7j (thiazole) increased the activity among the tested array of cells. Similarly among the positional isomers 7a (2-pyridyl), 7b (3-pyridyl) and 7c (4-pyridyl), nitrogen atom at second position in 7a is diminishing the activity and its lower commotion is due to lone pair-lone pair repulsions present on the oxygen and nitrogen (Fig. 3). Ultimately, nitrogen at fourth and third positions is more favourable for stimulating the activity. Similarly, isomer series of 7m (o-CF 3 ), 7n (m-CF 3 ) and 7o (p-CF 3 ) and 7p (o-F), 7q (m-F) and 7r (p-F), have followed the activity trending in the order of meta > para > ortho by following the negative inductive effect and demonstrated the higher activity for meta position over other two. In the series of 7e-g, the increasing order of activity is observed as 7g (3-methylpicolinoyl) < 7e (6-methylpicolinoyl) < 7f (5-methylpicolinoyl) as di functional substitutions diminishing the activity based on steric factors. In brief, the para substituted compounds with respect to the amide bond are acting as electron releasing groups and improving the activity (Fig. 3). www.nature.com/scientificreports/ BSA protein binding assay. The study of BSA protein binding interactions of a drug substance infers about its possible transportability with BSA, as it is a standard protein carrier for almost all drugs and metabolites. Here, all the title compounds were identified as potentially bound to the BSA protein, compounds 7i, 7g, 7e and 7f having 2-trifluoromethyl, 2-methyl, 5-methyl and 4-methyl substitutions on picolinoyl groups were exhibited the highest binding constant (K b ) values (Table 11) and absorption maxima at 280 nm and showed the hyperchromic effect. The values for these ranging from 1.5415 × 10 4 M −1 to 1.4033 × 10 4 M −1 are better in comparison to the Doxorubicin drug standard (0.7865 × 10 4 M −1 ) of the study. Hence, the BSA protein binding assay also ascertained the drug properties of the title compounds along the cytotoxic activity screened for them.

Conclusion
An array of novel tetracyclic acridone derivatives (7a-s) have been synthesized in good yields from quinoxaline, where Cs 2 CO 3 -Pd(OAc) 2 -Xantphos used as a potential catalytic system in generating the acridone ring by forming a C-N linkage. Similarly, the anticancer activity evaluation had revealed that 7k, 7r, 7l, 7o, 7a, 7d, 7b, 7e, 7i and 7q were identified as effective anticancer agents as assessed by MTT assay against HT29, MDAMB231 and HEK293T cancer cell lines. In addition, the molecular docking studies, QSAR, ADMET, bioactivity and toxicity risk properties predicted for them have strengthened their drug-likeness and were well correlated with in vitro anticancer activity and BSA protein binding assay results. The BSA binding studies confirms the stronger bond of title compounds with BSA as evidenced from the binding constants ranging from 1.5415 × 10 4 M −1 to 1.4033 × 10 4 M −1 than the Doxoruicin drug reference. The molecular docking studies have inferred that compounds 7a-s have potentially bound to Glycine and Lysine (neutral aminoacids) present on enzymatic proteins 4N5Y, 1IGT and 2VWD with a binding energy ranging from − 9.0887 to − 6.9915 kcal/mol. These potential inter molecular hydrogen bonding interactions between hydrogen atoms of amides of enzymatic proteins and carbonyl groups of acridinone and 1-piperazinoyl fragments are responsible for cell growth inhibition by 7a-s. Ultimately, compounds 7a-s were identified as potential excitatory protein donor antagonists to reduce and block the cell functionality by neuronal damages and death of the cells. Therefore the idea of fusing acridone core with piperazine ring and linking various alkyl/ aryl/ heteroaryl to them via piperzinoyl carbon has been ascertained as an admiring task in designing these potential anticancer agents. Prospectively this study is serving as a trustworthy tool in accomplishing more potential acridone derivatives by novel structural modifications in 7a-s, which are under study and are exercising for higher activity. These results demonstrate strong evidence for innovative investigations involving in designing more analogous compounds and methodologies to explore mechanistic aspects of their anticancer activity.