key: cord-1007756-kwjp6doq authors: Verma, Vaijinath A.; Saundane, Anand R.; Meti, Rajkumar S.; Vennapu, Dushyanth R. title: Synthesis of novel indolo[3,2-c]isoquinoline derivatives bearing pyrimidine, piperazine rings and their biological evaluation and docking studies against COVID-19 virus main protease date: 2020-12-27 journal: J Mol Struct DOI: 10.1016/j.molstruc.2020.129829 sha: e5a35ab80caeb98cbfb70628d08097f71e4d52c2 doc_id: 1007756 cord_uid: kwjp6doq A series of hybrid indolo[3,2-c]isoquinoline (δ-carboline) analogs incorporating two pyrimidine and piperizine ring frameworks were synthesized. Intending biological activities and SAR we propose replacements of fluorine, methyl and methoxy of synthetic compounds for noteworthy antimicrobial, antioxidant, anticancer and anti-tuberculosis activities. Among these compounds 3a, 4a and 5e were progressively strong against E. coli and K. pneumonia. Whereas, compounds 4a, 5a and 6a with addition of various functional groups (OCH(3), CH(3)) were excellent against S. aureus and B. subtilis. Compound 5c exhibited strong RSA and dynamic ferrous ion (Fe(2+)) metal chelating impact with IC(50) of 7.88 ± 0.93 and 4.06±0.31µg/mL, respectively. Compound 5e was considerably cytotoxic against all cancer cells displaying activity better than the standard drug. Compounds 6b and 6e inhibited M. tuberculosis (MIC 1.0 mg/L) considerably. Molecular docking studies indicate that compounds 4d, 5a, 5b, 6b and 6f exhibited good interactions with 6LZE (COVID-19) and 6XFN (SARS-CoV-2) at active sites. The structure of the synthesized compounds were elementally analyzed using IR, (1)H, (13)C NMR and mass spectral information. Coronavirus disease 2019 (COVID-19), a fatal respiratory illness was reported in Dec 2019 from China. Initially the causal organism was named as 2019-nCoV. The earliest article in January 2020, related to 2019-nCoV revealed that 2019-nCoV belongs to the beta-coronavirus group, sharing ancestry with bat coronavirus HKU9-1, similar to SARS coronaviruses and that despite sequence diversity its spike protein interacts strongly with the human ACE2 receptor [1] . In February 2020, WHO renamed the virus as SARS-CoV-2 (severe acute respiratory syndrome corona virus 2). Several pre existing antiretroviral drugs showed potentiality against COVID-19 viz, oseltamivir [2] , lopinavir [3] , ritonavir [3] , remdesivir [4] , favipiravir [5] , ribavirin [5] , chloroquine and hydroxychloroquine. [6] alone or in combination. Out of these, ritonavir, remdesivir, chloroquine and hydroxychloroquine has shown efficacy at cellular level [7] . Nucleotide sequences of SARS-CoV-2 reveal it as a member of Betacoronaviruses, such as the SARS and MERS HCoVs [8] [9] [10] . HCoVs are positive-sense, long (30,000 bp), single-stranded RNA viruses SARS-CoV-2 genomic organization consists of a 50 -untranslated region (UTR), a replicase complex (orf1ab) encoding non-structural proteins (nsps), a spike protein (S) gene, envelope protein (E) gene, amembrane protein (M) gene, a nucleocapsid protein (N) gene, 30-UTR, and several unidentified non-structural open reading frames [11] . Cancer and Tuberculosis (TB) are two significant reasons for death, because still no widespread strong medications taking care of them have been synthesized. Concurring, World Health Organization (WHO) to factual information that eight million people attribute new infection with T.B. annually [12, 13] . Tuberculosis is a chronic bacterial contamination transmitted thru the air. This sickness is brought about by the bacteria Mycobacterium tuberculosis and primarily influences lungs (pulmonary TB). Mycobacterial cell membrane could initiate nitric oxide generation and reactive oxygen species (ROS) each involved in carcinogenesis [14] . National Cancer Institute review and reported that patients with pulmonary tuberculosis had more desirable danger of lung cancer and assessed a double rise in chance of lung cancer in males with tuberculosis [12, 15] . Furthermore, Cancer is lethal illness with strengthening cause for mortality and measurements with anticipated annual 20 million cases globally by 2020 every year [16] . It is conceivable that endogenous free radical reaction started by ionization radiation, can also end result in tumors arrangement. One of the most basic relationships among reactive oxygen species and cancer is the expanded demise rate from leukemia and malignant neoplasia of the breast, ovaries and rectum because of more prominent effect of lipid peroxidation [17] . The pathological classification of the disease has ample evidence of changes in free radicals and ROS with inflammation, malignant tumors, myocardial infarction, rheumatism and neurodegenerative problems [18] [19] [20] [21] [22] [23] . ROS can also promote lipid peroxidation, integrated restriction of mitochondrial respiratory complexes and inactivation of GAP-DH(glyceraldehydre-3-phosphate dehydrogenase). Inhibition of Na + /K + -ATPase activity, sodium channel membrane inactivation and other oxidative modification in proteins. Totally these impacts are probably to play an imperative part in stock pathophysiology, inflammation and ischemia/reperfusion (I/R) injury [24] . The literature is flooded with reports variety of biological activities of α-, β-γ-and δcarboline (pyrido-indole or indolo [3,2-c] isoquinoline) analogues [25] [26] [27] [28] [29] . Among them, δcarbolines are the least studied class of compounds as compared to their α-, β-and γcarboline analogues. Effective drugs have not been found yet among these class of heterocycles. Nevertheless, a rather broad spectrum of biological activity has been reported for several compounds of δ-carbolines such as antibacterial [30] , antiplasmodial [30] , anti-malarial [31] and potent cancer therapeuticals [32]. In addition, tetracyclic isoquinolinone units fused with indole are established in the central structure of various effective bioactive compounds displaying antitumor [33] , cytotoxic [34] , anticancer [35] , and DNA cleavage [36] . An insight into literature survey showed that many hybridized α-, δ-carboline (indolo [2,3c] isoquinoline, indolo [3,2-c] isoquinoline) analogs demonstrate diverse biological activities like bactericidal, fungicidal [37] , anticancer [38] , antineoplastic [39] , antitumor [40] , antihistaminic [41] , oxytocic [42, 43] , analgesic and anti-inflammatory activities [44] . In recent years, applications of indolo [3,2-c] isoquinoline include development of antimicrobial, antioxidant, anticancer and anti-TB [45] [46] [47] properties from our laboratory. Based on this speculation with a broader outlook in medicine citations, this area was considered. these results encouraged to continue the research work in the synthesis of biologically significant heterocyclic compounds, herein we have focused mainly synthesis of indolo [3,2-c] isoquinoline (δ-carboline) base hybrid analogs. All these compounds were screened for their in vitro antimicrobial, antioxidant and anticancer activities. Synthesis of marked compounds were accomplished as delineated in Scheme 1. The starting material ethyl 8-fluoro-5-oxo-5H-indolo[3,2-c]isoquinoline-6(11H)-carboxylate 1a were synthesized as per the procedure [42] . The compound 1a was subjected to N-methylation with methyl iodide in dimethylformamide (DMF) using potassium carbonate (K 2 CO 3 ) as base and as phase transfer catalyst tetra-n-butyl ammonium bromide (n-TBAB) was used to acquire 2a which has indicated a strong ingestion at 2954 cm-1 relating to N-CH 3 . Assimilation at 1715 and 1657 cm -1 relates to carbonyl stretching. 1 H NMR spectrum of 2a has shown multiplets between δ 6.9 and δ 8.2 (m, 7H, Ar-H) relating to fifteen aromatic protons within the molecule. Peaks at δ 3.7 and δ 4.0 ppm are allotted for the methyl protons respectively. 13 [3,2- c]isoquinoline N-CH 3 . Assimilation at 1676 and 1638 cm -1 relates to C=O stretching respectively. The 1 H NMR spectrum of 3a which has displayed a singlet at δ 10.6 was assigned to hydroxyl group and multiplets between δ 7.1 and δ 8.3 ppm corresponds to seven aromatic protons present in the molecule. Methyl protons assigned peaks at δ 3.9 ppm. 13 C NMR spectrum of 3a manifested signals at δ 164.7 and δ 160.5 integrated for two symmetric carbonyls by the indolo[3,2-c]isoquinolinone ring and δ 36.6 signals for methyl carbon. The mass spectrum of compound 3a has shown molecular ion peak at m/z 311 [M+ H] + . An attempt to chloroethyl ester, 4a obtained from 3a by cyclization with dichloroethane and anhydrous K 2 CO 3 in acetone, the reaction was refluxed for 6 h. Within the IR spectrum of 4a absorption at 2962 cm -1 relates to (N-CH3) an absorption at 1715 and 1657 cm -1 which corresponds to carbonyl stretch. The 1 H NMR spectrum of 4a has multiplets in the range δ 7.0 and 8.3 ppm corresponds to seven aromatic protons present in the molecule. Peaks at δ 4.9 and 4.6 were attributed to the protons of oxy-ethane and chloro-ethane, respectively. 13 C NMR spectrum for 4a showed signals in a falling field at δ 164.7 and less deshielded peak at δ 160.8 integrated for the C = O, indolo [3,2- c]isoquinolinone ring and δ 39.5 integrated for N-methyl carbon. The mass spectrum of compound 4a showed a peak of the molecular ion at m/z 420 [M + +2]. The spectral data presented confirm the formation of compound 4a. Spectral data of the compounds 4c-4d confirm their structures. The development and unfolding of antimicrobial resistance has become foremost serious concern across the globally with reference to public health. Antimicrobial resistance is flexibility to inactivate, exclude or block the inhibitory or lethal mechanism of the antimicrobial agents by microbes [48] . Electron-rich heteroatoms such as nitrogen, sulfur, halogen heterocycles have a essential role in diverse medicinal chemistry [49] . The synthesized compounds were investigated Table- 1. Based on the initial microbiological concept, it was found that the antibacterial activity of all compounds against pathogenic bacteria was moderately good, except for compounds with fluoro, hydrogen substitutes into in the indolo [3,2-c] isoquinoline. For pyrimidine rings of compounds 3a, 4a and 5e good activity was observed against E. coli and K. pneumonia. Increased free radicals in the body have been involved in harming human life [53, 54] . Antioxidant agent properties are significant in checking the injurious impacts on nourishments and biological systems by free radicals [55] . values of μg/mL usually indicated best antioxidant properties. Antioxidants scavenge free radicals by donating DPPH electrons [56] . DPPH, a static free radical, has an odd electron and therefore has a tough retention at 517 nm. As electron gets combined off to saturation, the consumption diminishes stoichiometrically in terms of the number of absorbing electrons or hydrogen particles. This reaction is significantly embraced to examine the ability of molecules as free radical scavengers. We evaluated the scavenging powers of synthetic compounds on DPPH radical. The effects were in contrast with the standards 2-terbutyl-4-methoxy phenol (butylated hydroxyl anisole, BHA). All the examined compounds showed a decrease in the activity of the absorption of free radicals, when, in contrast to BHT (IC 50 Ferrous ion is one of the types of metal ions. Deficiency of ferrous ions protects the oxygenrelated reaction and from lipid generation. Ferrous ion chelators evacuate ferrous ion (Fe 2+ ) that will take part in generation of hydroxyl radical. The chelating impact of ferrous ion on synthesized compounds was resolved and results were contrasted with BHA as standard as mentioned in the method of Dinis et al [57] . In this strategy, ferrozine can quantitatively form the ferrous ion complex. The chelating agents disrupt the complex system leading to a decrease in redness of the complex. Therefore, Table 3 . Structure activity relationship (SAR) has shown noteworthy RSA and metal chelating activity of the analogs with replacement or substitution on the aromatic rings of indoloisoquinoline, pyrimidine and piperizine with the CH 3 group. Compounds 5c, 5b, 5g and 6h with electron-donating group methyl group at the para-position were better antioxidants than electron retreating fluorine moiety due to enhanced stability of the radical and metal chelating activities. The cytotoxic activities of newly synthesized compounds 3-4(a-d) and 5,6(a-h) were assessed by an in vitro assay carried out against four human tumor cell lines, e.g. MCF-7 (breast), A549 (lung), HeLa (cervical) and Panc-1 (pancreas) using 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) [58] with doxorubicin as positive reference. The results are presented in Table- The in vitro anti-tuberculosis activity (anti-TB) was decided by the broth microtiter dilution method [59] Table 5 . For preliminary confirmation of the physio-chemical properties, ADMET and drug likeness for molecules is crucial in their underlying distinctive proof as a synthetic lead and establishes a benchmark, against which incorporated compounds were measured amid lead advancement. Stages of Absorption, Distribution, Metabolism, Excretion of the ligand molecules were screened using SwissADME software [60] . This investigation aims mainly to approximate the pharmacokinetics profile of the examined compounds intrigued. The drug likeness profiles of newly synthesized compounds were predicted and ADMET properties are illustrated in Table 6 3. Molecular docking tool aids in recognizing novel drug-like compounds that display high binding affinity with specific targets and rational ADMET characters. Docking is the most widespread program for specification of protein-ligand interactions. Docking results bought for every ligand with the receptors had been scrutinized aside from docking energy and binding modes, interaction of every ligand with the functional residues of PDBs. showed the highest conventional hydrogen bonding interactions with 6XFN. with various amino acids present in the binding pockets of receptor is expressed in Fig. 1 suggests that compounds 4d, 5a, 5b, 6b and 6f display a positive range of drug likeness. In summary, the procedure of molecular hybridization has been effectively connected to the All reagents were commercially purchased and used without further purification. Melting points were determined using open capillaries and were not corrected The purity of the compounds was verified by TLC using aluminum plates coated with silica gel G (Merck), hexane:ethyl acetate isoquinoline-6(11H)-carboxylates(1a-1d) was by following literature procedure [42] . To a combination of 1a-1d (0.1 mol), potassium carbonate (0.2 mol) in dimethylformamide (DMF) (40 mL), Tetra-n-butylammonium bromide(n-TBAB) as catalyst was added at 0 °C in mixture. Then, the mixture was stirred at room temperature for 30 min following which methyl iodide (0.3 mol) was added dropwise and continuously stirring for 12h. The reaction progress was checked by using TLC. Then the reaction mixture was poured into ice-cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO 4 and evaporated under vacuum. The crude residue was purified by column chromatography (petroleum ether: ethyl acetate (4:1)) to obtain the pure compounds 2a-2d. To a solution of compounds 2a-d(0.01 mol) in ethanol (30 mL), 4N NaOH (30 mL) was added and heated under reflux for 3h. The reaction mixture was cooled and acidified with 2N HCl. The completion of reaction was monitored by TLC using silica gel coated plates using solvents (ethyl acetate: petroleum ether 1:1) as eluent and observed in UV light. The solid residue was filtered off and washed with cold water, dried and purified from ethanol to get 3a-3d. White To a solution of 8-substituted-11-methyl-5-oxo-5H-indolo[3,2-c]isoquinoline-6(11H)carboxylic acids (3a-3d) (1 mol), dichloroethane (2 mol) and anhydrous K 2 CO 3 (2 mol) in acetone (40 mL) was added and resulting solution was refluxed for 6 h. The resulting residue was washed with petroleum ether and distilled water progressively and separated under reduced pressure. Then the residue was dried and purified by silica gel chromatography (petroleum ether: ethyl acetate (2:1)) as eluent to afford compounds 4a-4d. Orange crystals, yield: 72%, m. 134.3, 134.0, 132.2, 132.0, 128.9, 128.1, 126.9, 126.4, 125.3,124.3, 124.2 Green 134.3, 134.2, 132.5, 132.2, 131.2, 129.4, 126.7, 125.6, 124.7,124.2, 123.3, 120.2 Compounds 4a-d (3 mol), substituted pyrimidine-2-thiol (3 mol) and dry K 2 CO 3 (3 mol) were introduced to dry DMF (20 mL). The reaction mixture was stirred at room temperature for 8-10 h. When the reaction was completed (by means of TLC), the reaction mixture was poured into ice water (100 mL) and acidified with acetic acid. The obtained residue was filtered, washed with water, dried and purified by silica gel (petroleum ether: ethyl acetate (3:1)) to afford the compounds 5a-5h. White Pale Compounds (3a-3d) (0.001 mol), hydroxylbenzotriazole (0.012 mol) in dry Tetrahydrofuran (THF) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCH) (0.012 mol) were mixed and stirred for 30 min. To the reaction mixture, substituted phenylpiperazine (0.001 mol) was included under ice cold temperature and the reaction mixture was further stirred at room temperature for 6 h. After finish of reaction as checked by TLC, Reaction mixture was separated with ethyl acetate, collected organic layer was dried over Na 2 SO 4 and concentrated under vacuum to give compounds 6a-6h. Hexanine as solvent for TLC. White 6. Biological procedure The synthesized compounds were tested against four bacteria and four fungi. The bacterial strains incubated at 106 microbe/mL concentration by the broth dilution method (concentrations 100, 75, 50, 25, 12.5, 6.5 3.12 and 1.5 (µg/mL). The optical density was measured at 655 nm wavelength for each sample and compared with standard drugs streptomycin and fluconazole for antibacterial and antifungal activities, respectively. The lowest concentration of the test compound was considered as precise MIC values. At this concentration, growth of bacteria and fungi were completely inhibited. The RSA of the test compounds was carried out in methanolic solution at concentrations 25, 50, 75 and 100 µg/mL containing freshly prepared DPPH solution (0.004 % w/v) and compared to standards using reported method [57] . All the analysis were performed on averaged of triplicates. The results are expressed as the ratio of absorption decrease in the presence of test compounds and absorption of DPPH solution in the absence of test compounds at 517 nm on ELICO SL171 mini spect spectrometer in percentage. The percentage scavenging activity of the DPPH free radical was determined using the following equation: DPPH radical scavenging (%) = [(Ac-As/Ac)] X 100 Where, Ac is the absorbance of the control reaction and As is the absorbance of the sample or standards. The metal chelating activity of the ferrous ions (Fe 2+) towards the synthetic compounds was determined by the Denis method by the ferrous ions and BHA as standards. In this strategy, samples of ethanol solution (0.4 ml) in a solution of ferrous chloride (0.05 ml, 2 ml) were included in 25, 50, 75, and 100 μg / mL at concentrations. The reaction was initiated by the addition of ferrozine (0.02 mL, 5 mM) and the total volume was made up to 4 mL in ethanol. The mixture was shaken vigorously and incubated at room temperature for 10 min and absorbance at 517 nm were estimated using a UV-Visible spectrophotometer. The percentage of inhibition of the ferrozine complex formation was determined using the accompanying equation. Absorbance of Control The anticancer activity of synthesized compounds were tested against three human cancer cell lines including MCF-7 (breast carcinoma), A-549 (lung carcinoma) and HeLa (Cervical carcinoma). The synthesized compounds were diluted to various concentrations (10, 5, 2.5 and 1.25μg ML -1 ) in Dimethyl Sulfoxide (DMSO) and assessed using 3-(4, 5-dimethyl-2-yl-2, 5diphenyl tetrazolium bromide (MTT assay). Anticancer activity was determined for cells treated with various concentrations of the titled compounds, the untreated cells (negative control) and Doxorubicin (positive control). A statistical significance was tested between sample and negative control using independent t-test by SPSS 12 program. The concentration of compounds required to kill half of the cell population (IC 50 ) were determined by non-linear regression analysis. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments. The anti-TB activity of the synthesized compounds were determined as per reported method. The DMSO dissolved test compounds were further diluted in cation adjusted Mueller-Hinton broth (MHB) to a concentration of 256 mg/L. To each well of 125 μL of MHB, 125 μl of each test solution were added to obtain 128 mg/L starting concentration. A twofold serial dilution of test solutions of range 128-0.25 mg/L was prepared. Columbia blood agar supplemented with 7% defibrinated horse blood was the used media. Bacterial suspension with a turbidity of 0.5 on the MacFarland scale was made in 0.9% NaCl solution and diluted to inoculum density of 5 x 10 5 cfu/mL. Each well was inoculated with 125µL of inocula, except the blank. The plates were incubated at 37 0 C for 72 h. The lowest concentration of test compounds that completely fail to produce macroscopic growth (MIC) were detected by incubation at 35 0 C for 20 min with 20µL methanolic solution of tetrazolium redox dye (MTT, 3.5 mg/mL). Tests were done in triplicates and ethambutol, isoniazid and rifampicin were used as positive controls. All the synthesized ligands were subsequently prepared using Chemdraw ultra 12 in CDX format. The ligands were converted into three-dimensional structure by Minimize Energy to Minimum RMS Gradient of 0.100 in Chem3D ultra and saved as MDL, MolFiles. The molfile of ligands were then converted to smiles format using Open Babel. The molecular docking studies were performed for PDBs: 6LZE and 6XFN the crystal structure of COVID-19 and SARS-CoV-2 (COVID-19) main proteases downloaded from the Protein Data Bank (https://www.rcsb.org). Protein was prepared using BIOVIA Discovery Studio Visualizer 2020 to define and edit binding site after the preparation of protein and saved as PDB format. Further the ligands' smiles format and prepared protein (PDB) have been imported into mcule (mcule.com: online drug discovery platform) for molecular docking and the docked proteinligands interactions poses were downloaded for docking studies. 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M. Bohra Commerce Degree College, Shorapur-585 224, Yadgir, Karnataka, India for provide laboratory facilities. Authors are grateful to the Directors, IIT Madras, Chennai, India provide spectral data, National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL) and National Centre for Cell Science (NCCS), Pune, India to providing test materials. The authors confirm that there is no conflict of interest in the content of this article.