Novel indolo [3,2-c]isoquinoline-5-one-6-yl [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazole analogues: Design, synthesis, anticancer activity, docking with SARS-CoV-2 Omicron protease and MESP/TD-DFT approaches

Indoloisoquinoline derivatives are associated with varieties of biological and pharmacological properties. Therefore, we herein reported the synthesis of novel series of indolo [3,2-c]isoquinoline incorporated with [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazole moieties. Spectroscopic methods were used to determine the chemical structures of these molecules. Whereas, the B3LYP functional with the def2-SVP basis set were used to improve TD-DFT geometries and solvent effects. Investigations, which are directly connected to the optical spectra (absorption and emission) of molecules. These findings reveals that the compound 3d-f with a strong electron acceptor NO2 exhibited UV−visible spectra peaks to near infrared (NIR) range in solvents. Compound 3e exhibited a lowest ∆E of 2.28 eV in MeCN. Further, among the newly synthesized compounds 3d and 3g exhibits highest activity against four cell lines with strongest potent cytotoxicity, as contrasted to the control drug (Doxorubicin). Docking experiments revealed that compounds in contrast to 3a and 3d had strong interactions with Asn322, Met323, Ala387,Ala386, Gln506 and Gly326 with a greater binding affinity which are important amino acid residues that play a key role in SARS-CoV-2 Omicron main protease (Mpro) through hydrophobic, hydrogen bonding, Pi-sigma, Pi-sulfur and van der Waals interactions.

Finally, newly synthesized compounds were compelled to theoretical calculations of TD-DFT. The goal of our was investigations to identify the characteristics of electronic transitions and their ramifications with electronic concerns resulting from electron donating and electron withdrawing substituents, for example CH 3 , H, NH 2, Cl and NO 2. Further, to investigate the electronic structure using UV spectroscopy and quantum chemistry approaches, which influences their reactivity and anti-cancer activity. Based on the spectroscopic (IR, 1 H NMR, 13 C NMR and mass) data, the information of elemental analysis structures of newly synthesized indolo [3,2c]isoquinoline compounds were confirmed.

Plausible mechanism
Protonation is the initial step of acid appeared by dehydration and attack at the same time of nitrogen lone pair to the electron deficient resonance-stabilized cation (acylium ion) to establish an intermediate. In the second stage, the intermediate is subjected to an adjacent group involvement with nucleophilic sulfur, this results in the formation of C-S bond by removing the water molecule ( Scheme 2 ). Ultimately, the title compounds are obtained as a result of deprotonation (3a-i).

Molecular electrostatic potential (MESP) analysis
Our initial endeavours were concentrated on molecular electrostatic potential (MESP), it was implemented to establish the electrostatic attraction among molecules. Similarly, the MESP assessment accounted for hydrogen bonding, dipole moment, electro-negativity, partial charges and chemical reactivity site of a molecule [15] . The density of electrons would be an essential component in determining the reactivity of electrophilic and nucleophilic regions, including hydrogen bonding interactions [ 16 , 17 ]. So, as to forecast the reactivity of electrophilic and nucleophilic areas attack with relation to compounds under investigation, we used the B3LYP level of the optimised geometry to MESP [18] . The color sequence red to yellow (negative regions) denotes low electrostatic potential and are associated with electrophilic reactivity. Whereas, blue to green (positive regions) denotes nucleophilic reactivity sites with strong electrostatic potential and the MESP represented ESP gradually increasing in intensity in the or-Scheme 2. Plausible mechanism of title compounds. der of red-yellow-green-blue (regions in between 0.03 to 0.94 a.u.). The high density of electrons on the surface of molecules is indicated by the red color which correlate to electrophilic attacks. While the blue color with high electrostatic potential is associated with nucleophilic reactivity sites and is accountable for atomic nuclei rejecting proton. The highest negative areas of the MESP maps are related to oxygen and chlorine atoms and were identified and as most favourable regions for electrophilic attack. Similarly, it is noteworthy that hydrogen atoms of indole NH are nucleophilic attack. MESP plots are depicted in Fig. 1 .

UV-absorption and emission wavelength analysis
The quantum computation on electronic absorption spectra were done using TD-DFT/(B3LYP/def2-SVP) level to geometry optimized of the compounds and comprehended parameters for instance absorption wavelengths ( λ max), oscillator strengths ( ƒ), energy gap E (eV), ground state dipole moment (Debye, μ) and electronic transitions were determined in the gaseous phase to examine the solvatochromic effect of solvents (Hex, MeOH, and MeCN) for our compounds. The absorption and emission spectra, molecular extinction coefficient of all compounds ranges from 10,0 0 0 to 90,0 0 0 M −1 cm −1 were noticed. The spectra exhibited a consistent distribution with a most intense band at higher energies range between 340 and 758 nm, this shows the nature of ππ * transitions. It could be clear to observe that the maximum absorption ( λ abs ) and emission ( λ em ) wavelengths are influenced by the substituents. It denotes that the absorption transition has a distinctive intramolecular charge transfer (ICT) and as a consequence, the excited state is not the same as in the emission transition. In gas phase: the newly synthesized compounds have strong and wide absorption bands in order of absorption wavelengths which are clearly distinguishable: The maximum wavelength of absorption was by compound 3e ( λ abs = 494 nm) and 3 g the ( λ abs = 350 nm) minimum. When compared to other compounds, the absorption wavelength of 3e is red shift. The initial excited state was optimised using CPCM (conductor-like polarizable continuum model)-TDDFT, the status of excited peaks has been anticipated accurately by accredation to the transition from S 1 to S 0 . It's worth noting that the excited bands solvatochromism are considerably in the order: 3d ˃ 3f ˃ 3e ˃ 3c ˃ 3i ˃ 3a ˃ 3h ˃ 3b ˃ 3g . Compound 3d exhibited maximum ( λ abs max = 574 nm) in hexane and 3f ( λ abs max = 633 and 611 nm) in MeOH and MeCN, respectively.
As a consequence, the absorption wavelength increases as delocalization increases. All the compounds ( 3a-i ) emission maxima exhibited increasingly redshift and significantly increases reliance on the substituents included. Compound 3e noticed the highest red shift ( λ em max 564 nm) in the gas phase and λ em max 758 nm in the MeCN solvent. Because of the EDG-EWG [electron donating group (EDG) and strong electron-withdrawing group (EWG)], rings of indolo [3,2-c]isoquinoline with p-CH 3 substituted and 1,3,4-thiadiazolyl phenyl with p -NO 2 groups existence and excited state, ICT should be effective because of the conjugated molecule's ππ * electron transition. Alternatively, EWG-EDG, EDG-EWG and EWG-EWG substituents all showed gradual red shifts, maximum emission wavelength of 728 nm (MeCN) for 3d , 756 nm (MeOH) for 3e , and 754 nm (MeOH), 749 nm (MeCN) for 3f respectively. Some of the organic compounds with consistent donor-π -acceptor (D-π -A) like benzothiazole, benzo [e]indole and quinoline had significant emission maximum wavelengths in the ultraviolet-visible (UV-vis) to near infrared (NIR) range. The π linkage among the units that donate and accept electrons has improved ICT [22][23][24] .
In contrast, λ abs max to λ em max red shifted by 59 nm (3 h) in GP and 159 nm ( 3e ) in MeCN, both compounds connected with EDG-EWG (CH 3 , Cl and NO 2 ). Due to coulombic interaction between the dipolar solute and solvent molecules, [ 25 , 26 ] the excited state of 3e seems to be dramatically stabilized. The electron delocalization from the electron donor methyl moiety to the electron acceptor nitro group results in a red-shift (high stokes shift) λ em max in 3e . Due to the ππ * characteristic of the transition, the greater stability of the excited state in relation to the ground state results in a red-shift appearance in wavelengths of absorption and emission. Accordingly, Hammett's substituent constants are correlated [27] . The strong electron acceptor -NO 2 group at the p-position on phenyl ring has shown considerable electron delocalization within excited state, in contrast to the weak electron acceptor Cl group. These aspects might have an impact in a decrease of the energy band gap between donor and acceptor substitutions [28] . The substantial stokes shifts of title compounds 3e and 3f showed that the molecules alter structure when excited. In contrast to dipole moment for 3a-3c (2.16-6.17 debye) and 3 g-3i (4.27-7.76 debye), there is a significant shift in 3d −3f (7.22-9.52 debye). The dipole moment of the strong electron donor-acceptor-acceptor (NO 2 ) and the weak electron acceptor or donor groups (Cl, CH 3 , H and NH 2 ) differs. Oscillator strength ( ƒ) is the chance of electromagnetic radiation absorption or emission in transition between energy levels of atoms or molecules. The computed oscillator strength values ranged from 0.002 to 0.168. The structure and behavior of electrically excited states are revealed by quantum yield. The range of quantum yield( ) was shown to be between 1.206 to 1.372% and these findings demonstrated by compounds 3e and 3g . The absorp-tion spectrum was predicted by Avogadro software as represented in Table 1 and Fig. 2 .

Frontier molecular orbital energies (FMOs)
The FMOs (highest occupied molecular orbital HOMO and the lowest unoccupied molecular orbital LUMO) play a significant role in influencing molecular features such as UV-visible absorption, optical and electronic characteristics [29] . The energies of HOMO and LUMO diagrams shows the electronic structural and excitation properties quantitatively. Compound 3e exhibit a lowest HOMO-LUMO energy gap of 2.28 eV in MeCN solvent and 2.53 eV, 2.44 eV and 2.3 eV, respectively for gas phase, hexane, MeOH, respectively. Consequently, resulting in a decrease in the HOMO-LUMO energy gap on altering the polarity of the solvents from n-hexane to acetonitrile. Further, HOMO-1, −2 to LUMO + 1, + 2, transitions for electron donating groups (CH 3 , NH 2 and H), largest E gap ranges between 3.87-4.34 eV compounds of 3b and 3c , while electron accepting groups (Cl and NO 2 ), transitions E gap ranges between 3.53-4.53 eV. Interestingly, HOMO-1, −2 to LUMO + 1, + 2, transition energy gap are increased when electron-donating replaced by EWGs. However, when p-CH 3 , p-NO 2 substitutions are introduced at indolo [3,2-c]isoquinoline and 1,3,4-thiadiazolyl phenyl rings, the energy gap decrease in all solvents, while incorporation Table 1 TDDFT(B3LYP/def2-SVP) UV spectra of absorption ( λ abs max , nm) and emission ( λem max , nm) for compounds 3a-i in gas phase and various solvents, Stockes shift cm −1 (SS), ground state dipole moment (Debye) ( μ), oscillator strength ( ƒ), a emission quantum yield( ), energy gap E (eV), and transition electronic and Assignment. π π * of p-NH 2 , p-Cl and p-H groups in other compounds ranges from 2.56 to 2.53 eV in gas phase Table 1 and graphical presentation in Fig. 3 (see figs. S1-S3 and table S1 supplementary data) show the frontier orbital energies from HOMO-1, −2 to LUMO + 1, + 2, as well as the HOMO-LUMO energy gaps. The various global reactivity variables are derived from the following equations [30] , for example chemical potential (energy which received or discharged) μ = -( I + A )/2; Global hardness (the resistance to deformation is described to as hardness) η = (I-A)/2; Electronegativity (attractiveness of electrons) χ = ( I + A )/2; Global softness (indicate the ease with which a molecule may polarised) S = 1/2 η; Global electrophilicity index (ability to accept electrons) ω = μ 2 /2 η. The qualities of global hardness ( η) and softness (S) are used to evaluate their significance in terms of charge transfer reduction, stability and reactivity of molecules. The hardness value of compound 3d was high compared to 3c (6.80 eV and 2.75 eV, respectively) and values of S (6.80 eV and 5.51 eV, respectively) which were low. On the other hand, the electrophilicity index is a measure of a chemical species' ability to take any number of electrons owing to a maximal flow of electrons from a donor environment. Compound 3b exhibited the highest electrophilicity index (0.277 eV), while compound 3e showed the lowest (0. 0.120 eV), in current investigation. Furthermore, electronegativity, which is defined as the negative of the chemical potential in DFT, is a measure of the inclination to fascinate electrons in a chemical bond [31] . Consequently, 3b has the highest 1.75 eV, while 3e has the lowest 1.26 eV as shown in Table 2 .

Biological evaluation
Aqueous solubility (log S) and Lipophilicity (log P) are determined before considering the molecule's anticancer activity. Swis-sADME is used to evaluate log P and log S. [32] Log P is directly connected to drug transport and contact to receptors, whereas log S is strongly linked to bioavailability. Compound 3f shows Log P 2.96, indicating that the chemical can diffuse through cell membranes, as a result of which it can be used in drug delivery applications. The compound 3c value log S ( −5.85) confirms the permeability of molecule through cell membranes. The PASS online program is used to estimate various antineoplastic activities of the title compounds. Cancers of the brain, lung, cervical, ovarian, gastric, pancreatic, and bladder are examples. The PASS software's estimated findings have an average accuracy of roughly 85%. Only antineoplastic activities were listed in this investigation [33] as presented in Pa > 70% in Fig. 4 (Table S2. see supplementary information).

ADME and drug-likeness analyses
A molecule's ADME (absorption, distribution, metabolism and excretion) are important procedures to examine during the phases of drug development. [33] All of the synthesized compounds 3a-3i were initially evaluated using ADME-Profiling and the results are highlighted in Table 4 . The drug-likeness features of the chosen compounds were analysed using the Lipinski rule of five (L 5 ).

Molecular docking study
The COVID-19 panic pandemic is a significant threat to mankind and it has been spreading rapidly over the last two years. Newly identified SARS-CoV-2 Omicron virus variant B.1.1.529 was first reported by WHO on 24 November 2021 [35] . Hence, in present investigation, we looked into a molecular docking study to identify feasible binding affinity among newly synthesized compounds ( 3a-i ) with SARS-CoV-2 Omicron PDB ID:7T9L (Cryo-EM structure of SARS-CoV-2 Omicron spike protein complex with human ACE2, EMD-25,761) obtained from https://www.rcsb.org/structure/7T9L . Docking study was performed with online server ( https://mcule. com/apps/1-click-docking ) and for visualisations Discovery Studio visualizer-2021 was employed [36] . The investigation of docking, appears that all the indolo [2,3-c]isoquinoline analogues interact with the SARS-CoV-2 Omicron protease. As a result, the evaluated compounds may be successful in accomplishing binding interactions into the omicron PDB binding pocket and endeavours of structure-binding showed that hydrophobic and H-bonds are interplayers of key bindings. The results of the molecular docking computations revealed that the best docking scores were −8.4 and −8.2 kcal/mol shown for synthesised molecules 3a and 3d , respectively. Energetically, the NH and carbonyl groups in compound 3a revealed four hydrogen bonds with Asn 322 , Met 323 , Ala 386 and Ala 387 at bond distance 2.95, 2.33, 2.98 and 2.42 Å , respectively. The p-NH 2 attached to 1,3,4-thiadiazolyl phenyl has a Pi-sigma hydrophobic interaction with Thr 354 (3.86 Å ) and Phe 356 (bond distance 5.82 Å ) forms a Pi-sulfur with 1,3,4-thiadiazole moiety of the compound 3a with binding sites of SARS-CoV-2 Omicron main protease (M pro ). The NO 2 group of compound 3d exhibited two hydrogen interactions with Gln 506 , Gly 326 and NH of 1,2,4-triazole ring system to Asn 322 (2. 26 (Table S3, Supplementary materials).

Conclusion
In this investigation, numerous indolo [3,2-c]isoquinolinyl-[1,2,4]triazolo [3,4-b] [1,3,4]thiadiazole analogous were synthesized. The time-dependent density functional (TD-DFT) in conjunction with B3LYP (Becke, 3-parameter, Lee-Yang-Parr) function and def2-SVP basis set were accustomed to the predict spectrum of absorption and emission of newly synthesized compounds. The outcomes indicate that compound 3f exhibits maximum absorption spectra at 633 and 611 nm in MeOH and MeCN, respectively. In MeCN, compound 3e exhibited maximum emission spectra at 758 nm and 2.28 eV is the lowest E of HOMO-LUMO. Further, compounds 3d and 3g highlighted the most effective activity against human cancer cell lines. The molecular docking findings revealed that compounds 3a and 3d demonstrated appropriate affinity for the amino acids at the active site of SARS-CoV-2 Omicron main protease. Ultimately, the findings indicated that both molecules might be investigated further in the search for a novel antiviral drug to SARS-CoV-2 Omicron. Nevertheless, further wet laboratory validation is required. Consequently, DFT is often used to investigate electronic properties and HOMO-LUMO energy gap, which could be the key reasons for its indicated innate biolog-ical properties. The global chemical reactivity features suggest that this molecule has a proclivity for chemical reactions. The MESP is in inverse relation to the electrical density and an important distinction among electrophilic and nucleophilic attack sites, contacts, including hydrogen bonding interactions [37]. The MESP surface of the title compounds are suited for the pharmacological action which contains electrophilic sites located around the indolo [3,2c]isoquinoline and [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazole ring systems. That has also been recognized to residue sites besides its biological values, molecular docking strategy was accustomed to screen. It is indeed worth noting that the inclusion of chloro and oxygen atoms provide the maximum strengths on the description compounds which possess the greatest possible electron density and would preferentially converse with microorganisms and amplify the anticancer potential.

Anticancer activity
Four different human cancer cell lines such as MCF-7 (breast), A549 (lung), HeLa (cervical) and Panc-1 (pancreas) were used to investigate the anticancer activities of synthesized compounds. The synthesized compounds were diluted in Dimethyl Sulfoxide (DMSO) to different concentrations (10, 5, 2.5, and 1.25 g ML-1) and evaluated using 3-(4, 5-Dimethyl-2-yl-2, 5-diphenyl tetrazolium bromide (MTT assay). Cells were treated with various concentrations of the described compounds and their anticancer activity was tested. The control was maintained, untreated cells (negative control) and Doxorubicin (positive control). The independent t -test in the SPSS 12 software was used to examine the statistical significance of the sample and negative control. Non-linear regression analysis was used to calculate the compounds concentrations necessary to kill half of the cell population (IC 50 ). The average IC 50 of three separate studies was used to calculate cytotoxic activity.

Declaration of Competing Interest
The authors confirm that there is no conflict of interest in the content of this article.