A Four-Component Domino Reaction: An Eco-Compatible and Highly Efficient Construction of 1,8-Naphthyridine Derivatives, Their In Silico Molecular Docking, Drug Likeness, ADME, and Toxicity Studies

A multicomponent domino reaction of enaminone, malononitrile, and o -phthalaldehyde has been established, providing direct access to novel highly functionalized pentacyclic cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine derivatives. The simplicity of execution, readily available substrates, high yields, excellent functional group tolerance, scalability, and good scores of en-vironmental parameters make this synthetic strategy more sustainable and worthy of further attention. This one-pot trans-formation, which involved multiple steps and did not require the use of a catalyst, constructed four new C-C bonds, two new C-N bonds, and three new rings, with eﬃcient use of all reactants. Furthermore, we performed in silico molecular docking analysis for prediction of anticancer (against human topoisomerase II β protein) and antimicrobial (against E.coli. DNA gyrase B protein) activities. Drug likeness and ADMETstudies were also predicted. Overall investigation indicates that compound 6i may serve as a candidate that could be developed as potential anticancer and antimicrobial agent among all.

Nevertheless, the true potential of this nitrogen containing heterocyclic framework has largely remained untapped owing to a lack of general diversity-oriented synthetic strategies. In the last two decades, growing concern about the environment has forced chemists to develop novel and sustainable chemical processes through the principles of "green chemistry" [28,29]. As a consequence, synthetic protocols that allow diversity and are operationally simple, mild, step-economical, high yielding, and compatible with green solvents are of prime importance [28,29]. To achieve these goals, multicomponent domino reactions (MDRs) come into play [30][31][32]. Significantly, MDRs enable assembly of three or more reactants in a single and an ordered event to render intricate and complex heterocyclic entities in an atom-and step-economical manner [33,34].
To date, the synthesis of 1,8-naphthyridine derivatives has been achieved via a variety of approaches including the condensation of 2-aminopyridine derivatives with carbonyl compounds under Conrad-Limpach, [35] Skraup, [36], and Friedlander [37] reaction conditions. e above methods suffer from the use of corrosive reagents, poor yields, multistep syntheses, and harsh reaction conditions. More importantly, all the reported methods need expensive and rare starting materials, the synthesis of which may demand significant scientific investment in itself. In the above light, a catalyst-free MDR-based route towards the assembly of novel pentacyclic cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine derivatives starting from cheap and abundantly available starting materials would be of great significance from both an economical and a chemical standpoint.
In continuation to our ongoing endeavors to prepare novel biologically active nitrogen containing heterocyclic entities, by using green and benign MCR protocols, [38,39] we herein report a domino four-component strategy for the efficient synthesis of highly functionalized pentacyclic   [1, 2, 3-de] [1,8] naphthyridine derivatives by utilizing cheap and commercially available reagents, that is, different enaminones, malononitrile, and o-phthalaldehyde under appropriate catalyst-free and mild microwave conditions (Scheme 1). Many chemical modifications have been carried out previously also at N-1, C-5, C-6, C-7, and C-3 positions, [21,[25][26][27] but this work represents a novel synthetic methodology as well as designing strategy to find out less toxic and potent anticancer agents. e pharmacokinetics (PK) and pharmacodynamics (PD) evaluations play a crucial role in drug development [40,41]. e evaluation of pharmacological parameters such as drug-likeness, ADME, and toxicity is paramount in identifying lead compounds [40,41]. Recently, in silico methods that include molecular docking analysis and online ADMET predictions (SwissADME and ProTox-II) facilitate the process of identifying the potential molecule [41][42][43][44][45]. In this present investigation, we report a new green synthetic methodology and characterization of novel 1,8-naphthyridine derivatives and their in silico anticancer and antimicrobial molecular docking analysis and pharmacological properties predictions such as drug-likeness, ADME, and toxicity.
e High-Resolution Mass Spectra (HR-MS) were performed on Bruker Daltonics micrOTOF-QII® spectrometer using ESI ionization. IR spectra were recorded on PerkinElmer Spectrum Two FT-IR spectrometer. Melting points were performed with OptiMelt automated melting point system. e reactions were performed in a G-10 Borosilicate glass vial sealed with Teflon septum in Anton Paar Monowave 300 reactor®, operating at a frequency of 2.455 GHz with continuous irradiation power of 0 to 300 W. Analysis of the reactions was done by thin layer chromatography (TLC) on Merck precoated silica gel TLC plates (Merck® 60F 254 ). Chemicals and reagents were purchased from Sigma-Aldrich and Alfa Aesar. All solvents, ethanol, petroleum ether, and ethyl acetate, were purchased from locally available commercial sources and used as received.

General Procedure for the Synthesis of Synthons (Enaminones) (3a-i).
A mixture of cyclopentane-1, 3-dione 1 (1.0 mmol), and the suitable amine 2 (2.0 mmol) was introduced in a G-10 glass vial capped with a Teflon septum and was subjected to microwave irradiation with the initial ramp time of 1 minute at 60°C and then the temperature was raised to 160°C with a holding time of 10 minutes in neat. e reaction was monitored by TLC. Synthons 3a-i were obtained for further use in the synthesis of final products 6ai. All synthons 3a-i were characterized by 1 H NMR and 13 C NMR spectroscopies.

General Procedure for the Synthesis of Products (6a-i).
A mixture of enaminone 3 (1.0 mmol), malononitrile 4 (2.2 mmol, 2.2 equiv), and o-phthalaldehyde 5 (1.0 mmol) was introduced in a G-10 glass vial capped with a Teflon septum and was subjected to microwave irradiation with the initial ramp time of 1 minute at 60°C and then the temperature was raised to 110°C with a holding time of 20 minutes with DMF (2 mL) as a solvent. e reaction was monitored by TLC. After the completion, the reaction mixture was then cooled to room temperature and diluted with cold water (40 mL) and then filtered; the precipitate was collected and purified by recrystallization from 95% EtOH except 6h which was purified by silica gel column chromatography (eluent 15-20% ethyl acetate/pet. ether). e analytical data for representing compounds are shown below.

Preparation of Ligands.
e 2D structures (.mol) of all synthesized compounds (6a-i) were drawn and each individual structure was analyzed by using ChemDraw 16.0. All the compounds (6a-i) were converted to 3D structure (.pdb) using Chem3D 16.0. e 3D coordinates (.pdb) of each molecule were loaded onto Chem3D for energy minimization.

Preparation of Macromolecule.
e crystal structures of receptor molecule E. coli gyrase B (PDB ID: 6F86) and human topoisomerase IIβ with DNA (PDB ID 3QX3) were downloaded from protein data bank. e protein preparation was done using the reported standard protocol by removing the cocrystallized ligand, selected water molecules, and cofactors, and the target protein file was prepared by leaving the associated residue with protein by using autopreparation of target protein file AutoDock 4.2 (MGLTools 1.5.7).

AutoDock Vina Analysis.
e graphical user interface program AutoDock 4.2 was used to set the grid box for docking simulations. e grid was set so that it surrounds the region of interest in the macromolecule. e docking algorithm provided with AutoDock Vina was used to search for the best docked conformation between ligand and protein. During the docking process, a maximum of nine conformers were considered for each ligand. e conformations with the most favorable (least) free binding energy were selected for analyzing the interactions between the target receptor and ligands by Discovery Studio Visualizer and PyMOL.
AutoDock Vina with standard protocol was used to dock the proteins (PDB ID: 6F86 and 3QX3) and synthesized novel 1,8-naphthyridine ligands (6a-i) into the active site of proteins. e molecular docking studies were carried out using AutoDockTools (ADT) [44], which is a free graphic user interface (GUI) for the AutoDock Vina program. e grid box was constructed using 15 × 15 × 15, pointing in x, y, and z directions, respectively, with a grid point spacing of 0.375Å. e center grid box is of 35.58 × 95.63 × 52.38Å and 62 × 30 × 62Å for 3QX3 and 6F86, respectively. Nine different conformations were generated for each ligand scored using AutoDock Vina functions and were ranked according to their binding energies. e ligands are represented in different color, and H-bonds and the interacting residues are represented in stick model representation.

In Silico Pharmacokinetics and Toxicity.
Drug-likeness is a prediction that determines whether a particular pharmacological agent has properties consistent with being an orally active drug. e chemical structure of the compounds (6a-i) was converted to their canonical simplified molecular input line entry (SMILE) system and submitted to SwissADME tool to estimate in silico pharmacokinetic parameters. SwissADME predictor provides information on the numbers of hydrogen donors, hydrogen acceptors, and rotatable bonds, as well as total polar surface area of a compound. e ligands were also subjected to Lipinski et al., screened using SwissADME and PreADMET predictors. e organ toxicities and toxicological endpoints of the ligands and their LD 50 were predicted using ProTox-II and OSIRIS Property Explorer [42,43]. e analyses of the compounds were compared with that of Vosaroxin, a clinical-trial phase III drug.

Results and Discussion
3.1. Chemistry. A series of novel 1,8-naphthyridine derivatives are synthesized via a four-component domino reaction with no more than 20 minutes of microwave irradiation.
is one-pot transformation, which involved multiple steps and did not require the use of a catalyst, constructed four new C-C bonds, two new C-N bonds, and three new rings with efficient use of all reactants, which are readily available and cheap. Moreover, the proposed strategy involves neither tedious workup nor column purification steps. Furthermore, the methodology has excellent green credentials, scoring well in a number of green metrics, hence showing this approach to be an ideal green and sustainable process even at gram scale.
Furthermore, it is noteworthy that we synthesized the starting material enaminone (3a-i) under microwave irradiation, starting with cyclopentane-1, 3-dione (1), and various amines (2a-i) in neat (catalyst-and solvent-free conditions) at 160°C for 10 min. e reaction and products are summarized in Scheme 2. is work represents the first example of synthesizing different enaminones through this simple and green methodology. Hence, synthesis of compounds (3a-i) was accomplished with readily available ammonium acetate (2a) aromatic or benzylic amines (2b-i) and the commercially available cyclopentane-1,3-dione.
After synthesis of synthons 3a-i, we further used a 1:2:1 mixture of 3-aminocyclopent-2-enone (3a), malononitrile (4), and o-phthalaldehyde (5) in ethanol [46] at 100°C for 20 minutes under microwave irradiation, without using catalyst. Only 38% yield of the desired compound 6-amino- [1,8] naphthyridine-4a1, 7-dicarbonitrile (6a) could be afforded. is synthesized 6a derivative was characterized using IR, 1 H NMR, 13 C NMR, and HR-MS spectroscopies (see supplementary data (Figures S1-S18)). Optimization data are shown in Table 1. e addition of catalysts such as L-proline [47], piperidine [48], p-toluenesulfonic acid, [49] and silica sulfuric acid [50] did not improve the yields (Table 1, entries 2-5). DMF [51] was chosen as the solvent for all further reactions due to positive results. When the reaction was carried out at 90°C, 100°C, 110°C, and 120°C, 6a was obtained in yields of 70%, 75%, 89%, and 80% (Table 1, entries 6 and 11-13), respectively [52]. We checked the reaction with DMF at 110°C for 15 min., but it further reduced the yield and multiple spots Journal of Chemistry appeared at TLC (Table 1, entry 14). ese experiments showed that conditions of 110°C in DMF, without a catalyst, under microwave irradiation for 20 minutes provided the highest yield with column-free protocol. Using the optimal conditions, we investigated the substrate scope of the transformation. e results are summarized in Table 2. As shown in Table 2, phenyl groups bearing either electronwithdrawing or electron-donating groups on the enaminone ring as well as the unsubstituted enaminone as in the case of 6a were well tolerated under the reaction conditions, leading to the final products in satisfactory yields (82 ± 10%).
To conclude our analysis of this MDR, we have calculated green metrics [53,54] comprising E-factor, atom economy (AE), process mass intensity (PMI), reaction mass efficiency (RME), and carbon efficiency (CE) for the process.
A catalyst-free eco-friendly domino methodology towards the assembly of hexahydro-4a1H-cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine-4a1,7-dicarbonitrile derivatives has been developed from commercially available cyclopentane-1, 3-dione, aromatic amine (2b-i) (or shown by the fact that up to six new bonds (four C-C bonds and two C-N bonds) and three new rings (a tricyclic 5-6-6 skeleton consisting of cyclopentene and two pyridines) were readily formed in domino fashion. is work represents the construction of these special types of polyhydronaphthyridine skeletons in one pot.

Binding Mode of Analysis of Synthesized 1,8-Naphthyridine Derivatives Docked against Human Topoisomerase II Beta (PDB ID 3QX3).
It has been reported that topoisomerase IIβ is one of the elucidated targets for antitumor agents such as 1,8-naphthyridine derivatives (e.g., Vosaroxin) [58,59]. erefore, in this study, the molecular docking analysis of the synthesized compounds (6a-i) was carried out to investigate their binding interaction within the binding sites of human topoisomerase IIβ and the results were compared with standard anticancer agent in class (Vosaroxin). e synthesized compounds (6a-i) were found to have minimum binding energy ranging from -6.2 to -6.7 kcal/mol (Table 4). Compared with Vosaroxin (-5.6 kcal/mol), the synthesized compounds (6a-i) have shown high binding affinity and similar residual and DNA interaction profile with amino acid residues Asp-479, Ser-480, Glu-477, Ala-481, Arg-503, Gly-478, and Gly-504 and nucleic acid residues DT-8, DT-9, DG-10, DA-12, and DG-13. Compounds 6c and 6g have no hydrogen bonding interaction with amino acid residues within the binding pocket. e compounds 6a (-6.6 kcal/mol), 6b (-6.7 kcal/ mol), and 6i (-6.5 kcal/mol) have shown better binding affinity and similar residual amino acid and DNA binding within the binding pockets of 3QX3 as compared to Vosaroxin (Figure 2). e in silico interaction results have shown that all the synthesized compounds (6a-i) have better binding affinity compared to Vosaroxin; among them compounds 6b (-6.7 kcal/mol) and 6f (-6.7 kcal/mol) revealed better binding affinity. e inhibition constant K i values showed much better results for synthesized compounds 6a-i (0.726 to 3.493 nm) as compared to Vosaroxin (22.993 nm). Among them, 6b and 6f showed minimum value (0.726 nm), 6a was slightly higher (0.994 nm), and compound 6i showed more than 10-fold decrement (1.361 nm) to Vosaroxin (22.993 nm). Based on the molecular docking analysis results, all the synthesized compounds have shown comparable residual interactions and better docking scores than Vosaroxin (see supplementary materials ( Figures S19-S28)). Hence, these compounds might prove to be better anticancer agents than Vosaroxin. e binding affinity, inhibition constant, H-bond, and residual interaction of all the synthesized compounds are summarized in Table 4.

Binding Mode of Analysis of Synthesized 1,8-Naphthyridine Derivatives
Docked against E. coli DNA Gyrase B (PDB ID 6F86). DNA gyrase B, an enzyme belonging to a member of bacterial topoisomerase, controls the topology of DNA during transcription, replication, and recombination by introducing transient breaks to both DNA strands [60,61]. Hence, the bacterial DNA gyrase is paramount for bacterial survival and therefore necessary to disrupt as an antibacterial drug target [62]. erefore, in this study, the molecular docking analysis of the synthesized compounds was carried out to investigate their binding pattern with DNA gyrase and the results were compared with standard antibacterial agent (Ciprofloxacin). e synthesized compounds (6a-i) were found to have minimum binding energy ranging from -6.5 to -9.4 kcal/mol (Table 5), with the best result achieved using compound 6i (-9.4 kcal/mol). Compared to Ciprofloxacin, the synthesized compounds (6a-i) have shown high binding affinity (except 6d) and similar residual interaction profile with amino acid residues Glu-50, Gly-77, Ile-94, Ile-78, Pro-79, Ala-47, r-165, and H-bond with Asp-73, Arg-76, and Asn-46. All the synthesized ligands have revealed the crucial interaction between the ligand, Asp-73, and the water molecule with E. coli DNA gyrase B (6F86). It has been previously reported that binding modes between ligand, Asp-73, and water molecule are crucial for the inhibition for the DNA gyrase B [63]. Compounds 6a (Asp-49), 6b (Gly-77 and r-165), 6f (Glu-50), and 6i (Ala-47) have additional hydrogen bonding interaction with amino acid residues. e compounds 6c, 6d, 6e, 6g, and 6h have shown similar hydrogen bond (Gly-77) and residual amino acid binding within the binding pockets of 6F86 (Ile-78, Glu-50, Ile-94, and Pro-79). e in silico interaction results of the synthesized compounds (6a-i) showed better binding affinity in comparison with Ciprofloxacin; among them, compounds 6b (-8.1 kcal/mol) and 6i (-9.4 kcal/mol) revealed the best binding affinity results. Compound 6d has shown smaller docking affinity (-6.5 kcal/mol) and partially matching amino acid residues interactions in comparison with Ciprofloxacin ( Figure 3). e inhibition constant K i values showed better results for synthesized compounds 6a-i (0.00015 to 1.36144 nm) as compared to Ciprofloxacin (0.08061 nm). Among them, 6i showed minimum value (0.00015 nm) and 6b was slightly higher (0.00895 nm), while 6h (0.02295 nm), 6a (0.03142 nm), and 6c (0.05888 nm) are comparable to Ciprofloxacin (0.08061 nm). Based on the molecular docking analysis results, all the synthesized compounds (except 6d) have shown comparable residual interactions and better docking scores than Ciprofloxacin (see supplementary materials ( Figures S29-S38)). erefore, these compounds might have potential to be promising antibacterial agents. e binding affinity, inhibition constant,  calculation up to the crude product.

Journal of Chemistry 9
H-bond, and residual interaction of all the synthesized compounds are summarized in Table 5.

In Silico Pharmacokinetics (Drug-Likeness) and
Toxicity Analysis. e drug-likeness of the synthesized 1,8naphthyridine derivatives was characterized according to "Lipinski's rule of five." As per Lipinski's rule, the potential molecules should have the following physicochemical properties [64], such as (i) less than 5 hydrogen bond donors (HBDs), (ii) less than 10 hydrogen bond acceptors (HBAs), (iii) a molecular mass less than 500 Da, (iv) log P not greater than 5, and (v) total polar surface area (TPSA) which should not be >140Å. e SwissADME computed results showed that all the synthesized compounds (6a-i) in the present study are satisfying Lipinski's rule of five with zero violations (Table 6) [65]. Hence, all the synthesized compounds might be candidates for anticancer and antimicrobial studies.
Evaluation of ADMET properties of newly synthesized compounds is pivotal in drug discovery [45]. e skin absorption of molecules is indicated by skin permeability value (Kp) in cm/s. e more negative the value of log K p , the less the skin absorption [41,66]. e skin permeability, K p , values of all molecules ( 7.12 to −8.11 cm/s) are within the range of Vosaroxin ( 8.98 cm/s) inferring low skin permeability. In addition to that, absorption and distribution of drug molecules are measured by gastrointestinal (GI) and blood brain barrier (BBB) permeation. e SwissADME prediction parameters have shown that all the compounds 6a-h and 6i (except 6g) have high gastrointestinal (GI) absorption and none of the compounds have blood brain barrier (BBB) permeation. Drug metabolism has been mainly governed by a range of cytochromes (CYP's). Among them, CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 are paramount for biotransformation of drug molecules [41]. SwissADME prediction has shown that all the compounds including Vosaroxin (except 6h) are substrates of permeability glycoprotein (P-gp). e prediction result exhibits that all the compounds are found to be noninhibitor for CYP2C19 and CYP2D6. e compounds 6b, 6d, 6e, 6h, and Vosaroxin are potential inhibitors for CYP1A2, and compounds 6a, 6c, 6f, 6g, and 6i are noninhibitors. For CYP2C9 and CYP3A4, the compounds 6a and Vosaroxin are noninhibitors, and the other compounds 6b-i are potential inhibitors. e in silico computed results of absorption, distribution, metabolism, and excretion (ADME) for synthesized compounds 6a-i and Vosaroxin are given in Table 7.     Acute toxicity predictions result such as LD 50 values and toxicity class classification [1 (toxic) to 6 (non-toxic)] reveals that none of the ligands have shown acute toxicity and were found to be similar to Vosaroxin. e synthesized compounds 6a-i have shown toxicity class classification 4 (harmful if swallowed). e toxicological prediction gives results of endpoints such as hepatotoxicity, carcinogenicity, mutagenicity, immunogenicity, and cytotoxicity. Compounds 6b, 6e, and 6i were predicted to be nonhepatotoxic, nonimmunotoxic, nonirritant, and noncytotoxic. However, the compounds 6b and 6e have shown carcinogenicity and mutagenicity. Among all the compounds, 6i was found to be completely nontoxic. ProTox-II and OSIRIS property explorer prediction analyses are shown in Table 8. Hence,

Conclusions
In this investigation, we have developed a green synthetic procedure for the facile synthesis of various potentially biologically active hexahydro-4a1H-cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine derivatives, based on a novel fourcomponent domino reaction. Using this method, a diverse collection of 1,8-naphthyridine derivatives were rapidly constructed with excellent yields in short reaction times by simply heating a mixture of different enaminones, malononitrile, and o-phthalaldehyde in DMF, without a catalyst, under microwave irradiation. Furthermore, in silico molecular docking, drug-likeness, and ADMET studies while comparing with clinically proven drugs Vosaroxin and Ciprofloxacin revealed the possible potency of synthesized compounds towards anticancer and antimicrobial activities. Based on the molecular docking analysis results, all the synthesized compounds have shown better docking scores than Vosaroxin and Ciprofloxacin. Hence, these compounds might prove to be better anticancer agents than Vosaroxin and might have potential to be very good antibacterial agents.
e SwissADME prediction results showed that all the synthesized compounds (6a-i) in the present study satisfy Lipinski's rule of five with zero violations. Better performing compounds 6b, 6e, and 6i were predicted to be nonhepatotoxic, nonimmunotoxic, nonirritant, and noncytotoxic. However, the compounds 6b and 6e have shown carcinogenicity and mutagenicity. Compound 6i was found to be completely nontoxic. e result of the present investigation suggested that 6-amino-1-oxo-4-(4-trifluoromethyl)phenyl)-1, 2, 3, 4, 7a, 11b-hexahydro-4a1H-cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine-4a1,7-dicarbonitrile (6i) may serve as a candidate that could be developed into potent human topoisomerase IIβ and E. coli. DNA gyrase B inhibitor. So, it is worthy to carry out further in vitro, in vivo, and preclinical studies of the most active compound to develop potent anticancer and antimicrobial agent. Based on the preliminary prediction results of this investigation, we are working on a new series of 1,8-naphthyridine derivatives to explore better structure activity relationship and to find out more potent, less toxic, and better biologically active scaffolds.

Data Availability
e Excel data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.