Promising anticancer agents should be necessarily developed as cancer remains as public health concern worldwide [1] along with the additional origin of a newer pandemic like . The drug repurposing strategy has been employed to reduce COVID-19 induced inflammation, coagulopathy and dysfunction by using anticancer agents like interleukin inhibitors, corticosteroids, kinase inhibitors, etc [2d] . The types, mutations [3] , metastasis [4] , resistance [5] , and after-effects of clinical treatment, make it a gruesome disease [6] without ideal treatment. Consequently, there lies a lacuna in the progression of novel agents to combat cancer. Inhibition of more than one target with a key role in cell replication and proliferation set forth magnificent directions in the generation of new chemical entities (NCEs) towards chemical research.
The discovery and comprehension of the structure and function of DNA are paramount of all the discoveries in the last century [7] . Hitherto, DNA is a major pharmacological target for small molecules which are effective in various ailments which originated from virus [8] , bacteria [9] and also in cancer [10] . Based on the interaction of the small molecules with DNA, they have been broadly divided into (i) Covalent inhibitors, which are irreversible binders that form covalent adducts, thereby cause serious damage to the DNA process and induce cell death. e.g., cisplatin (ii) Non-covalent inhibitors are reversible binders; further classified based on modes of binding with duplex DNA, (a) Surface binders (exhibits electrostatic interactions) (b) Intercalators (between DNA base pairs) (c) Groove binders (major and minor groove binding) [11] . Usually, the small molecules bind with DNA with more than one binding mode. Also, enzymes acting on DNA play a promising role in replication and repair. One such enzyme is DNA Topoisomerase II (Topo II), with a potential role in cell division and demarcation by causing nicking and relegation of DNA and is an imperative target in drug discovery [12] . There are two isoforms of Topo II with gene codes Topo IIα and Topo IIβ which are analogous in structure and are uniform in catalytic properties. However, topo IIα has positive coordination on the mitotic process of cells such as rate of cell proliferation, expression of mRNA, and also on different phases of the cell cycle nevertheless, cellular functions of topo IIβ are yet unclear [13] . Topo II, in general, modifies DNA topology and induces double-strand breaks through multiple steps that require ATP hydrolysis and Mg 2+ for activity [14a] and agents inhibiting it are categorized into interfacial poisons (IFPs) and catalytic inhibitory compounds (CICs).
Anticancer agents like doxorubicin and etoposide are renowned Topo II poisons that cause accretion of deplorable linear DNA by inhibition of the relegation of cleaved DNA strands through the stabilization of the Topo-DNA complex. Catalytic inhibitors such as novobiocin and merbarone, block binding and nicking of DNA respectively. On the contrary, agents such as suramin, a potent catalytic inhibitor block topo II's ATP binding site [14] . In addition to the above features, agents that induce apoptosis are considered as remarkable anticancer agents as they target an important hallmark of cancer [15] . Thus the discovery of new chemical entities targeting topo II through DNA intercalation is an important area of cancer research. Based on the literature, the rationale to exhibit intercalative topo II inhibition by NCE's is to possess a chromophore (head part) linked to a side chain (tail part) through a cationic species. The mechanistic details indicate that the chromophore is a polyaromatic ring system which intercalates between DNA base pairs. The linker and extending heterocycles impart stabilization by interacting with phosphate groups and by orienting into the groove of DNA respectively [16] .
The structural features of naturally occurring β-carboline alkaloids make it a potential chromophore to intercalate with DNA in between the base pairs. The tricyclic pyrido [3,4-b] ring system (norharmane A) possesses an affinity to stack between GC residues of DNA [17] . Furthermore, these Peganum harmala derived agents (harmine B) followed up by their chemical functionalization were reported as DNA damaging and photocleavage agents [18] .
In synergism, inhibitions of other enzymes like topoisomerases [12, 19] , kinases [20] make β-carboline a promising entity in cancer research along with indications in several diseases [21] .
1,3,4-Thiadiazole is another privileged heterocyclic scaffold with excellent therapeutic potential against diverse health problems. The potential of this bioactive scaffold was far recognized as an antiepileptic (acetazolamide C), antibacterial, antiprotozoal (megazol D), and to treat glaucoma [22] . Farooqi et. al., revealed the affinity of 1,3,4-thiadiazole towards DNA interaction by hybridization of 1,3,4-thiadiazole to the clinical drugs (Ibuprofen and Ciprofloxacin) [23] . Plech et. al., proved the affinity of 1, 3, 4-thiadiazole towards topo II by disubstitution on the heterocycles [24] . Indeed, this entity reveals wide pharmacological activities such as antimicrobial, antitubercular, anti-inflammatory, antiviral, anticonvulsant, and activity against central nervous system (CNS) disorders, cardiovascular system (CVS) diseases and cancer [22] .
Indole is a versatile and privileged scaffold with an extensive range of biological actions principally in cancer by targeting diverse enzymes (Topoisomerases, Tubulin polymerases, Aromatase) pathways (NF-kB/mTOR/PI3K/AKT), and mechanisms like DNA binding and apoptosis [25] . Naturally occurring antitumor antibiotic like duocarmycin has a strong affinity for DNA interaction, nonetheless unable to reach the clinic. However, synthetic analogs like adozelesin with substitution on 2 nd position (E) reached clinical trials in the treatment of cancer. Highly effective D-64131(F) and D 68144 (G) were recognized as potent anticancer agents even in paclitaxel-resistant tumors [26] (Figure 1 ).
<Insert Figure 1 here>
In maintaining our persistence to achieve the goal of reaching successful clinical candidates towards cancer [27] , by considering the advantage of structural features of the chemically diverse scaffolds like β-carboline, 1,3,4-thiadiazole, indole and exploiting pharmacophore hybridization to carboxamide formation, we have methodologically designed a new series (12a-k) of DNA intercalative topo II inhibitors with classical apoptosis-inducing features. The rationale for this design has been established based on literature and structural functionalities as illustrated in Figure 2 . Foremost requirements for intercalative topoisomerase II inhibition are the presence of a chromophore (intercalation into DNA), adjoining cationic center (electrostatic interaction with base pairs), and a groove extending side-chain/heterocycles (stabilization). DNA intercalative topoisomerase II inhibitors such as doxorubicin and PD-115934 also possess this kind of arrangement (chromophore + cationic center + groove extending side chain) [16] and support the rationale to this design. The chemical synthesis of β-carboline acid from amino acid Ltryptophan satisfies the utmost feature as a chromophore for dual activity. Further, the cationic center of 1,3,4-thiadiazole on the 2 nd position of indole (extending heterocycles) has been constructed by utilizing chemical functionalities of indole-2-acid and thiosemicarbazide followed by cyclization resulting in the free amino moiety. The chromophore has been adjoined with the rest of the scaffold by pharmacophore hybridization [28] by carboxamide formation through acid amine coupling.
<Insert Figure 2 here> 
The desired hybrids 1,3,4-thiadiazolo carboxamide bridged β-carboline-indole hybrids 12a-k were synthesized via amide formation between β-carboline acid and indol-2-yl-1,3,4thiadiazol-2-amine as depicted in Scheme 2. 1-Aryl-9H-pyrido [3,4-b] indole-3-carboxylic acids 6a-f were synthesized by earlier reported procedures, [12] from commercially available L-tryptophan (Spectrochem; CAS NO: 73-22-3) 1 which was esterified 2 and subjected to Pictet-Spengler condensation using acid catalyst trifluoroacetic acid with substituted aromatic benzaldehydes 3a-f to obtain tetrahydro-β-carboline esters 4a-f. The βcarboline esters 5a-f were obtained by further aromatization of 4a-f using KMnO 4 and were subjected to basic hydrolysis to yield the respective β-carboline acids 6a-f (Scheme 1).
The amine components 11a-c were attained by esterification of indole-2-acid 7, which (8) was further alkylated using different alkyl halides to yield alkylated indole-2-esters 9ac. Thus obtained esters, on basic hydrolysis result in respective alkylated indole-2-acids 10a-c. This acid component undergoes cyclization with thiosemicarbazide in POCl 3 to succumb amine counterpart. Further, the title compounds 12a-k was achieved by acidamine coupling between 6a-f and 11a-c in good yields (Scheme 2). All these compounds were purified by using column chromatography.
<Insert Scheme 2 here>
The representative compound 12a of synthesized derivatives was thus characterized by 1 H NMR spectroscopy. The appearance of singlet at δ = 12.14 corresponds to the amidic N-H proton and confirms the formation of titled compounds. The two singlets at δ = 12.58 and 12.19 ppm account for the NH proton of indole and 9H-indolic proton of β-carboline ring respectively. The proton at the C4 position of β-carboline appeared as a singlet at δ = 9.13 ppm. All the other 14 aromatic protons of both β-carboline and indole are accounted for in the range of δ = 8.55 to 7.13 ppm. Further analysis by 13 C NMR spectroscopy disclosed that amidic carbonyl carbon resonated at δ = 164.1 ppm. The signals at δ = 158.2 and 158.0 ppm account for amide attached C2 carbon and indole attached C5 carbon of 1,3,4-thiadiazole respectively. All the remaining carbons of 12a appeared in the region of δ = 142. 1-104.5 ppm. Next, IR spectra show the band at 1673 cm -1 which indicates the C=O stretching of the amide of the compound 12a. The HRMS exhibited specific [M + H] + peaks that matched with their molecular formula. Similarly, spectral data ( 1 H, 13 C, FT-IR) of all the remaining compounds 12b-k matched accurately with their respective structures.
Thus, the synthesized compounds 12a-k were evaluated for their cytotoxic potential against a panel of human cancer cell lines and compared with the standard harmine and doxorubicin, VP-16 (etoposide) and represented in Table 1 . The MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [29a] was performed for all the derivatives on different cancer cell lines such as (i) adherent cell lines viz lung cancer (A549), breast cancer (MDA-MB-231, BT-474), colon cancer (HCT-116), and (ii) suspension cell lines viz leukemia (HL-60) and acute monocytic leukemia (THP-1) ( Figure   S1 and S2 of ESI) and compared to normal human cell line . Notably, all the tested compounds exhibited potent cytotoxicity against all the tested cell lines, particularly 12c with the highest cytotoxic potential of IC 50 of 2.82 ± 0.10 µM in the A549 cell line.
Further, it has leading cytotoxicity in all the cell lines tested with an IC 50 of 3.18 ± 0.49 µM and 3.83 ± 0.94 µM in THP-1 and HL-60 respectively. Also, in both breast cancer cell lines, 12c expressed an IC 50 of 3.28 ± 0.43 µM and 4.09 ± 0.59 µM in MDA-MB-231 (triplenegative) and BT-474. Also, the compound 12c has exhibited in vitro cytotoxicity in HCT-116 with an IC 50 value of 4.78 ± 1.29 µM and the order of cytotoxicity for compound 12c in different cell lines is A549 > THP-1 > MDA-MB-231 > HL-60 > BT-474 > HCT-116.
<Insert Table 1 here> Encouragingly, the compound 12c displayed the lowest in vitro cytotoxicity with an IC 50 value of 71.25 ± 7.95 µM in normal human lung epithelial cells (BEAS-2B).
Remarkably, the compounds 12a, 12f, 12h, and 12j exhibited notable cytotoxicity potential in all the cell lines tested with overall IC 50 ≤ 10 µM. Moreover, compounds 12b, 12d, 12i, and 12k also expressed a reasonable in vitro cytotoxicity on A549 and MDA-MB-231 cell lines with an IC 50 value < 10 µM. From the detailed analysis of the in vitro cytotoxicity table, it was comprehended that 12c is a potent compound and possesses the least inhibition in BEAS-2B and the cytospecificity profile on all the human cancer cell lines along with normal human cell line was represented in graphical form in Figure 3a .
<Insert Figure 3a Based on the established in vitro cytotoxicity data against human cancer cell lines, the structure-activity relationship (SAR) was constructed to provide insights regarding the pattern of efficiency of the synthesized NCEs as illustrated in Figure 4 . Two-point diverse modifications have been performed on the C1-phenyl of the β-carboline (R 1 ) and nitrogen of indole (R 2 ). Noticeably, the compounds 12a, 12c, 12h, and 12j without substitution on indole (R 2 = H) were found to be potent than substituted derivatives 12b, 12d-g, 12i, and 12k. Additionally, increase in length of alky chain (R 2 = H > Me > Et) on indole resulted in derivatives with lower cytotoxicity potential (12c > 12d > 12e & 12a > 12b). The better cytotoxicity was observed when R 1 is substituted by electron-donating group (EDG) (12c-g, 12k: R 1 = 4-Me, 4-OMe & 3,4,5-triOMe) than no substitution (12a, b: R 1 = H) and followed by electron-withdrawing group (EWG) (12h-j: R 1 = 4-F & 4-Cl). The in vitro cytotoxic potential was compared with EDG and EWG in the following order: (a) EDG: (i) In derivatives where R 2 = methyl; and R 1 is 3,4,5-trimethoxy substituted, such derivative exhibited superior inhibition than 4-methoxy containing corresponding derivative (12f > 12d); (ii) In compounds with R 2 = ethyl; the compound with 4-methyl substitution on R 1 displayed better inhibition than derivative with 3,4,5-trimethoxy followed by 4-methoxy substituted compound (12k > 12g > 12e); (b) EWG: In compounds with R 2 = H; the derivative with 4-chloro substitution at R 1 position exhibited higher inhibition than 4-fluoro substituted derivative (12j > 12h). Interestingly, a similar pattern of inhibition was observed for all the compounds in the tested cell lines.
<Insert 
Intercalation of the ligand into DNA doesn't always cause alterations in hydrogen bonds between base pairs, but the ligand-DNA complex is stabilized by van der Waals, charge transfer, π-π stacking with the planar scaffold, hydrophobic and hydrogen-bonding interactions [31] . Intercalation stiffens, lengthens, reduces helical twist, and unwinds the DNA double helix [32] , thereby resulting in inhibition of cell proliferation and triggers cell death. [33] Among various classes of DNA binding agents, intercalating agents were widely studied by considering different biophysical assays. By relating absorbance and viscosity measurements using CT-DNA with a concentration of the tested compound, these assays suggest the mode of binding of the novel synthesized compounds.
To understand the binding mode of newly synthesized series, a relative viscosity experiment has been performed for potent compounds 12c and 12a using CT-DNA as per the reported protocol [34] . The different modes of binding of ligand result in alterations in the viscosity of DNA. The increase in viscosity is a result of an increase in the axial length of DNA to accommodate the ligand in between the base pairs, which indicates the intercalative mode of binding. However, no or minimal alteration in viscosity is observed with compounds possessing minor groove binding affinity. Conversely, covalent binders such as cisplatin result in a decrease of viscosity due to absolute functional inhibition of DNA through irreversible binding [35] . Ethidium bromide (EtBr) and Hoechst33258 generally cause an increase and comparative alteration in relative viscosity of DNA indicating their mode of binding as intercalator and minor groove binder respectively [34] .
In the current study, different concentrations of compound 12c and 12a along with harmine, Hoechst 33258, and EtBr are incubated with CT-DNA, and changes in viscosity were recorded. The controls EtBr and harmine displayed an increase in viscosity whereas;
Hoechst33248 exhibited a minimal change in viscosity with the increase in concentration.
The tested compounds exhibited an increase in viscosity in a concentration-dependent manner indicating intercalation as their mode of DNA binding. Therefore, a graph is plotted with the concentration of compound [compound/CT-DNA] on X-axis and viscosity [η/η 0 ] 1/3 on Y-axis and is represented in Figure 6 .
<Insert Figure 6 here>
Intercalation of ligands with DNA results in a slight bathochromic and significant hypochromic shift in absorbance at the specific wavelength [36] . This principle has been applied to find the efficacy of compounds 12c and 12a using CT-DNA.
Nanodrop is a kind of UV-spectrophotometer which uses a small quantity (nano quantity) to measure the quality of the nucleic acids [37] . The specific absorbance was measured at 260/230 nm wavelength with the different concentrations of compound 12c and 12a along with control CT-DNA. Harmine, Dox, Hoechst-33258, and EtBr were used as standards. It is evident from Figures 7A and 7B , that compound 12c and 12a caused hypochromic shift at specific wavelength respectively, and are potent intercalators.
<Insert Figure 7 here>
In addition to the nanodrop method, the absorbance alterations induced by compounds 12a and 12c on CT-DNA were evaluated at a specific concentration along with potent intercalator EtBr as a positive control. The plain CT-DNA without the addition of any ligand was used as a control, and from Figure 8 , it is evident that the order of absorption is EtBr > 12c > 12a. The results of the absorbance spectroscopic methods are consistent with relative viscosity measurements and establish the intercalative mode of binding of compounds tested with CT-DNA. [38] <Insert Figure 8 here>
Programmed cell death is evaded by cancer cells and prevails in the different organs; however, new cancer agents that induce apoptosis are always the prime choice of cancer research [39] . To understand the ability of compound 12c in apoptosis induction, various qualitative (morphological) and quantitative assays were performed on the A549 lung cancer cell line.
The morphological assays reveal the formation of characteristic apoptotic features in the cells with the aid of different nuclear affinity dyes. Henceforth, these are the elemental line of apoptosis identification in diverse cancer cells.
The changes in the morphological features of A549 cells were observed after the treatment with compound 12c at different concentrations along with the untreated control cells. Further, images reported in Figure 9A were captured using phase-contrast microscopy after 72 h, reveal the characteristic apoptotic features like changes in morphology (shape, shrinkage) of the cell, reduction in the number of live cells.
Acridine orange is selective toward nucleic acid and penetrates live cells and stains the nuclei green [40] . In the present study, A549 cells after treatment with compound 12c for 72 h exhibited the formation of apoptotic features such as the appearance of membrane blebs and inverse proportion in the number of cells with a concentration of compound tested. In addition, the cationic dye has the potential to bind lysosomes and thereby also exhibiting the vacuoles as represented in Figure 9B .
The initial discovery of 4,6-diamidino-2-phenylindole (DAPI) in the treatment of protozoal diseases retarded due to a strong binding affinity towards DNA base pairs. Later, this ability of passage through cell membranes made it an efficient stain to detect nuclear changes in the apoptosis cells [41] . The DAPI staining was executed according to the reported procedure [40] to detect apoptosis induction. Compound 12c treated A549 cells after 72 h, on staining with DAPI visualized the chromatin condensation, pyknotic (inset of 1.25 μM), and condensed (bright colored: inset of 2.5 μM) nuclei formation as depicted in 
Cancer cells detach from the site of origin, migrate, and develop the disease at the secondary site is known as metastasis, which makes the disease perilous and abating the survival rate of the patient [4, 44] .
To understand the capability of 12c on cell migration, wound healing assay has been performed on A549 cells. This assay is based on the ability of the healing of an artificial wound called "scratch" created using a sterile pipette tip (200 μL).
[45] The cells were treated with a range of concentrations varying from 1.25-5 μM of 12c, along with control, and images were captured at 0 and 72 h and compared. It is inferred from Figure 13 , that the wound in control was practically healed, whereas, the cell migration is inhibited in 12c treated cells after the period of treatment.
<Insert <Insert Figure 14 here>
The analysis of docking interactions indicated that all the structural fragments (β-carboline, thiadiazole, and indole) of ligand 12c established key interactions with residues in the active pocket such as the formation of two hydrogen bonds between amide attached thiadiazole's nitrogen and indolic nitrogen with dC8 (d = 3.65 Å) and dT9 (d = 3.51 Å) residues respectively as represented in B of Figure 14 . Further, π-cation interaction was exhibited by tricyclic pyridine NH with dA12 residue and π-π interaction was observed with phenyl ring of β-carboline with dA12 and dG13 residues. Notably, several hydrophobic interactions were observed with different amino acid residues such as Leu486, Met762, Met766, Ile769, Ala801, and Pro803. Additionally, compound 12a exhibited another crucial interaction on the β-carboline viz hydrogen bond between indolic N-H and Arg487 (d = 2.87 Å), along with other interactions as displayed in D of Figure 14 . The docking results reveal the topo II inhibition is a collective contribution by all the structural fragments ( Figure S3 , ESI) of the compounds and are compatible with the in vitro biological evaluation and encourage the structural diversification on the ligands in the near future.
Binding energies for the molecules were calculated to identify the affinity and complex The drug likeliness of the potent molecules and standards (Harmine, Etoposide) was analyzed by the evaluation of the Lipinski rule of five and is incorporated in Table 2 .
Although the compounds other than 12a violate the Lipinski rule in terms of molecular weight, the optimal range [49] obtained by evaluating the 95% of known drugs recommends the acceptable range up to 725 Daltons reveals the compounds tested satisfies the entire parameters and possess good drug-like properties [50, 51] .
<Insert Table 2 
The prediction of pharmacokinetic parameters provides an idea regarding the acceptable range for human use which was obtained by analyzing 95% of known drugs [51, 52] . The Physico-chemical parameters of the five best performers in the cytotoxicity table and standards (Harmine, Etoposide) were predicted by using the QikProp module [53] of Schrödinger and some key pharmacokinetic descriptors along with the recommended range are tabulated. The evaluation of computed parameters such as QPlogKhsa predicts the binding of a ligand to serum albumin which determines the availability of free drug and assessment of QPPCaco, where Caco cells act as a model for gut-blood barrier and QPPMDCK, where MDCK (Madin-Darby Canine Kidney) cells are a mimic for blood-brain barrier assist in the estimation of bioavailability. From the in silico data delineated in Table   3 , all the newly synthesized compounds assessed through QikProp fall in the optimal range and exhibit pharmaceutical significance.
<Insert Table 3 here>
In order to understand the toxicity profile of the synthesized compounds and harmine, TOPKAT (TOxicity Prediction by Komputer Assisted Technology) tool from Discovery Studio v19.1.0.18287 was utilized. TOPKAT employs robust and cross-validated Quantitative Structure Toxicity Relationship (QSTR) models to predict toxicological endpoints of given molecules chemical structure. Toxicity model development involves the use of 2D molecular, spatial and electronic descriptors. The optimal Predictive Space validation method of TOPKAT gives assurance to the prediction [54, 55] .
In silico toxicity profiling results for some of the synthesized compounds and harmine was depicted in Table 4 . The toxicity profile assessment has been predicted using different animal models. The toxicity of synthesized compounds was assessed and compared with harmine in different animal models. Broadly, the probability values of the synthesized compounds showed a lower toxicity profile than harmine. All the analyzed compounds including harmine have shown higher toxicity probabilities values for Ames mutagenicity. <Insert Table 4 here>
In summary, new molecules 12a-k were rationally designed by taking into consideration of structural fragments and pharmacophoric hybridization approach to build intercalative topoisomerase IIα inhibitors targeting cancer. Based on the earlier presented pharmacophoric profile of these heterocycles (β-carboline, indolo-thiadiazole), the synthesis [3,4-b] indole -3-carboxamide (12a-k) To a mixture of 1-aryl-9H-pyrido [3,4-b] indole-3-carboxylic acid (6a-f, 1.1 equiv.), EDC (1.5 equiv.), HOBt (1.5 equiv.) under nitrogen conditions add dichloromethane and allowed to stir at room temperature for 15 min. Later, the amino component (5-(1-alkylindol-2-yl)-1,3,4-thiadiazol-2-amine 11a-c, 1 equiv.) dissolved in dichloromethane, was added and the reaction was allowed to stir at room temperature till complete consumption of the starting materials as determined by TLC. The reaction mixture was extracted with dichloromethane and water (3 × 50 mL) and purified by column chromatography to yield N- [3,4-b] indole-3carboxamide 12a-k in moderate to good yields (72-85%).
10. 3, 1, 158.2, 156.0, 142.1, 142.1, 137.9, 137.5, 137.3, 135.5, 130.3, 129.7, 129.5, 129.4, 129.2, 128.4, 128.2, 123.9, 122.6, 121.6, 121.4, 121.1, 120.4, 115.6, 113.3, 112.4, 104.5 [M+H] + .
pyrido [3,4-b] 
pyrido [3,4-b] 
9H-pyrido [3,4-b] 
9H-pyrido [3,4-b] [3,4b] 1, 141.1, 137.9, 137.3, 135.4, 133.9, 131.9 (d, J = 8.4 Hz), 130.4, 129.4, 128.4, 128.2, 123.9, 122.6, 121.6, 121.4, 121.1, 120.4, 116.2, 116.1, 115.9, 115.6, 113.3, 112.4, 104.5 
pyrido [3,4-b] 3, 158.3, 156.3, 142.1, 141.1, 139.4, 137.2, 135.4, 134.0, 131.9, 131.9, 130.4, 129.4, 129.0, 127.3, 124.1, 121.6, 121.5, 121.1, 120.8, 116.1, 116.0, 115.7, 113.3, 111.0, 106.6, 32. [3,4b] 3, 156.0, 142.2, 140.7, 137.9, 137.4, 136.7, 136.3, 135.5, 134.5, 131.5, 131.0, 130.5, 129.5, 129.3, 129.1, 128.4, 128.2, 123.9, 122.7, 121.6, 121.4, 121.1, 120.4, 113.3, 112.4, 104.5 [3,4b] 139.3, 138.4, 137.1, 135.5, 134.7, 130.2, 129.8, 129.4, 128.1, 127.5, 124.1, 122.6, 121.7, 121.0, 120.8, 115.4, 113.3, 110.9, 107.0, 21.4, 15.8 
Normal 
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a dye that 
The 
DNA intercalation is determined by UV-Visible spectroscopic analysis. In our experiment, we incubated the 50 μM of calf thymus DNA (Sigma-Aldrich, USA) with compound 12c and 12a, and Ethidium bromide (EtBr), for 10 min at 1 μM concentration and compared with control (i.e. CT-DNA alone). Later, the TAE buffer used as blank and absorbance and concentrations were determined by UV-visible Spectrophotometer (Jasco;
V-650).
A549 cells were plated in 12 well culture plates with a cell density of 1 × 10 6 cells/mL and allowed to adhere overnight. The cells were incubated with different (1.25, 2.5, and 5 µM) concentrations of the compound 12c for 72 h treatment. Later cells were checked for the morphological changes, and images were taken using a phase-contrast microscope (Nikon, Inc. Japan).
A549 cells were plated at a concentration of 1 × 10 6 cells/mL and treated with various concentrations of compound 12c and the plates were incubated for 72 h. 10 µL of fluorescent dye, Acridine Orange (AO) was added into each well in equal volumes (10 µg/ml) respectively then the cells were visualized immediately under a fluorescence microscope (Nikon, Inc. Japan) with excitation (488 nm) and emission (550 nm) at 200X magnification.
DAPI staining aids in the observation of morphological changes in the nucleus. After treatment with compound 12c for 72 h, lung cancer A549 cells were washed with PBS and permeabilized with 0.1% Triton X for 10 min followed by staining with 1 µM DAPI.
Control and treated cells were observed with a fluorescence microscope with excitation at 359 nm and emission at 461 nm using DAPI filter at 200X magnification.
This assay was performed by the method 
In general, the novel compounds exert their cytotoxic or growth inhibitory effect by arresting the specific checkpoint in the cell cycle. Flow cytometric analysis (FACS) was performed to calculate the distribution of the cell population in various cell cycle phases.
Here A549 cancer cells were incubated with compound 12c at various concentrations from 
The DNA crystal structure has been retrieved from Protein Data Bank (PDB ID:
5GWK) [46] . The protein preparation tool was used for the preparation of the human topoisomerase II alpha with DNA. The missing atoms will be added up and peripheral water molecules will be removed with a distance of more than 5 Å from the pocket through the protein preparation wizard. The grid is generated by picking the active site where the cocrystal is located and grid box of 10 × 10 × 10 Å (Schrödinger 2017-1) [47] . The potent hybrids were sketched (12c & a) by using 2D sketcher and energy minimized, and prepared using Ligprep for the generation of different conformers (Schrödinger 2017-1). The different conformers thus obtained were subjected to molecular docking with SP Glide (Schrödinger 2017-1). The poses generated were evaluated and the best one was described.
Five potent compounds and standards were subjected to ligand preparation and the diverse conformers thus generated were envisaged for their Physico-chemical parameters using
QikProp [52] . The different descriptors were thus reported.
prediction of carcinogenicity and mutagenicity of chemicals by the TOPKAT program. Mutation Research, 1994, 305, 47- Table 3 . Physico-chemical parameters of some of the potent thiadiazolo-carboxamide bridged β-carboline-indole hybrids and standards predicted by QikProp [53] [3,4-b] indole-3carboxylic acids) N H CH 2 Cl 2 , rt, 12 h, 72-85% 11a-c 12a: R 1 = phenyl, R 2 = H 12b: R 1 = phenyl, R 2 = Me 12c: R 1 = 4-methoxyphenyl, R 2 = H 12d: R 1 = 4-methoxyphenyl, R 2 = Me 12e: R 1 = 4-methoxyphenyl, R 2 = Et 12f: R 1 = 3, 4, 5-trimethoxyphenyl, R 2 = Me 12g: R 1 = 3, 4, 5-trimethoxyphenyl, R 2 = Et 12h: R 1 = 4-fluorophenyl, R 2 = H 12i: R 1 = 4-fluorophenyl, R 2 = Me 12j: R 1 = 4-chlorophenyl, R 2 = H 12k: R 1 = 4-methylphenyl, R 2 = Et Scheme 2. Synthesis of thiadiazolo-carboxamide bridged β-carboline-indole hybrids 12a-k.