Supporting Information

Synthesis and bio evaluation of quaternary centered 3- hydroxy-3-(triflouromethyl)indolin-2-one derivatives for anticancer and antimicrobial activities ● R. Bikshapathi1 ● P. Sai Prathima*1 ● B. Yashwanth2 ● R. Pamanji2 ● G. Jagadeeshkumar1 ● R. Maheshwari2 ● J. VenkateswaraRao2 ● U.S.N Murty2 ● V. Jayathirtha Rao*1

 Sai Prathima Parvathaneni [email protected] Jayathirtha Rao, [email protected] 1 Crop Protection Chemicals Division, Organic II. 2 Biology Division, (IICT), Hyderabad. CSIR-Indian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka, Hyderabad –500607, India. Fax: +91-040-27193382, Tel: (+) 91 40 27193933.

CELL LINES AND CELL CULTURE The cell lines SKOV3 (Human ovary adenocarcinomacells), B16F10 (mouse melanoma),THP-1 (human acute monocyticleukemia) and PC3 (Human prostate Cancer)cell lines were obtained from the National Centre for Cellular Sciences (NCCS), Pune, India. Cells were cultured either in RPMI-1640 (THP-1) or DMEM (B16F10, SKOV3, PC3) media, supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1 mM NaHCO3, 2 mM -glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. All cell lines were maintained in culture at 37° C in an atmosphere of 5% CO2.

TEST CONCENTRATIONS:

Initially, stock solutions of each test substances were prepared in 100% Dimethyl Sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) with a final concentration of 8mg/ml. Exactly 25μl of stock was diluted to 1 ml in culture medium to obtain experimental stock concentration of 200μg/ml. This solution was further serially diluted with media to generate a dilution series of 1µg to 100µg/ml. Precisely, 100µl of each test concentration was added to 100µl of cell suspension (total assay volume of 200µl; efficacy of the derivatives were evaluated with three different set of experiments) and incubated for 24h at 37 °C in 5% CO2.

CYTOTOXICITY:

Cytotoxicty was measured using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, according to the method of Mossman (1983). Briefly, the cells (2 x 104) were seeded in each well containing 100µl of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO2, exactly 100µl of different test concentrations (1µg to 100µg/ml) were added to the cell suspension, which is equivalent to 0.2 to 20µg per 200µl of assay volume. The viability of cells was assessed after 24h, by adding 10μl of MTT (5 mg/ml) per well and incubated at 37°C for additional three hours. The medium was discarded and the formazan blue, which formed in the cells, was dissolved in 100μl of DMSO. The intensity of colour formation was measured at 570 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-5.4). The percent inhibition of cell viability was determined with reference to the control values (without test compound). The data were subjected to linear regression analysis and the regression lines were plotted for the best straight-line fit. The IC 50 (inhibition of cell viability) concentrations were calculated using the respective regression equation.1

Protein docking experimental procedure: To understand the drug receptor interaction of synthesized active compounds 11, 15 and 16 we carried out the molecular modeling studies by using Glide docking software2

Ligand preparation: Geometry optimized 2D structure of four active compounds 11, 15 and 16 were sketched and prepared for docking by using Ligprep, a versatile program of Glide, version 6.5, Schrödinger suite 2014.4 and minimized using OPLS-2005 force field. A total of ten conformations were generated.

Protein preparation: Crystal co-ordinates for nuclear xenobiotic receptor CAR (PDB ID: 1XLS), PIM1 kinase (PDB ID: 2O65) and CDK2 kinase (PDB ID: 3QHR) were taken from protein databank. Protein was prepared for docking studies using protein prep wizard of Glide, version 6.5, Schrödinger 2014.4. Bond orders and formal charges were added for hetero groups, and hydrogen’s were added to all atoms in the system. Water molecules with in 5 A0 distance were removed. For each structure, a brief relaxation was performed using an all-atom constrained minimization carried out with the Impact Refinement module (Impref) using the OPLS-2005 force field to alleviate steric clashes that may exist in the original PDB structure. The minimization was terminated when the energy converged or the RMSD reached a maximum cut off of 0.30 A0.

Grid generation:

Glide energy grid was generated using binding pocket of reported co-crystallized inhibitors of nuclear xenobiotic receptor CAR (PDB ID: 1XLS),3 PIM1 kinase (PDB ID: 2O65)4 and CDK2 kinase (PDB ID: 3QHR).5

Glide docking : GLIDE module of Schrödinger suite was used to get the compounds 11, 15 and 16 docked into binding pockets of co-crystallized inhibitor of nuclear xenobiotic receptor CAR (PDB ID: 1XLS), PIM1 kinase (PDB ID: 2O65) and CDK2 kinase (PDB ID: 3QHR). Standard extra precision docking was performed. A total of ten ligand conformations were allowed and finally top score conformation was selected as an active conformation for each of the compounds.

Fig 1: 2D and 3D receptor (PDB ID: 1XLS) ligand interactions and docked pose of compound 11, 15 and 16 are depicted with thick rods green colour and the binding pocket of protein is shown with thinner rods. Crystal structure of nuclear xenobiotic receptor CAR (PDB ID: 1XLS) was selected. Ligands were prepared using ligprep, optimized by OPLS-2005 force field and docked into the active site of the protein using Glide docking software. Docking studies were performed on the mosts active molecules 11, 15 and 16. Docking results shows that Compound 11, 15 and 16 shows same receptor ligand interactions which are hydrophobic interactions with Val 265, 342, 349, Ile 268, 310, 324, 345, Ala272, Trp 305, Leu 309, 433, 436, Phe 313, 346, 439, Cys 432, polar interactions with Asn 306, Hid 435. Fig 2: 2D and 3D receptor (PDB ID: 3QHR) ligand interactions and docked pose of compound 11, 15 and 16 are depicted with thick rods green colour and the binding pocket of of protein is shown with thick rods (LYS67 and LEU44) and remaining with thinner rods. Crystal structure of CDK2 kinase (PDB ID: 3QHR) was selected5. Ligands were prepared using ligprep, optimized by OPLS-2005 force field and docked into the active site of the protein using Glide docking software 2. Docking studies were performed on the mosts active molecules 11, 15 and 16. Docking results found that Compound 11, hydroxyl group oxygen shows hydrogen bonding back bone interaction with Gln 131, hydrophobic interactions with Ile 10, Val 18, 64, Ala 31, 144, Phe 80, 82, Leu 83, 134, charged positive interactions with Lys 33, 89, charged negative interactions with Asp86, 145, polar interactions with Hid 84, Gln 85, 131, Asn 132; compound 15 shows hydrophobic interactions with Ile 10, Val 18, 64, Ala 31, Phe 80, 82, Leu 83, 134, charged positive interactions with Lys 20, 89, charged negative interactions with Glu 8, 12, 81, Asp 86, polar interactions with Hid 84, Gln 85, 131, Asn 132; glycine interactions with Gly 11; compound 16 shows hydrophobic interactions with Ile 10, Val 18, 64, Ala 31, 144, Phe 80, 82, Leu 55, 83, 134, charged positive interactions with Lys 33, 89, charged negative interactions with Glu 51, Asp 86, 145, polar interactions with Hid 84, Gln 85, 131, Asn 132; glycine interactions with Gly 11. ANTIMICROBIAL ACTIVITY: Invitro Antibacterial activity and MIC Determination: The minimum inhibitory concentrations (MIC) of various synthetic compounds were tested against three representative Gram-positive organisms viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis( MTCC 435) and Gram- negative organisms viz Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 741), and Klebsiella pneumoniae (MTCC 618) by Microdilution method recommended by CLSI Standard Protocol (1) in liquid medium (Nutrient agar) distributed in 96-well plates, serial dilutions of the tested compounds were performed (concentrations from 150 μg/mL to 0.97 μg/mL) in a 200 μL culture medium final volume, afterwards each well was seeded with a 50 μL microbial suspension of 0.5 MacFarland density. In each test a microbial culture control and a sterility control (negative) were performed. The plates were incubated for 24 hours at 37°C. The lowest concentration which inhibited the visible microbial growth was considered the MIC (μg/mL) value for the tested compound. Penicillin and Streptomycin were used as standard drugs. The minimum inhibitory concentration (MIC) values are presented in the table.6 Anti Fungal Activity: In vitro antifungal activity of the newly synthesized compounds was studied against the fungal strains, Candida albicans (MTCC 227) and Saccharomyces cervisiae (MTCC 36) of yeasts and Aspergillus flavus (MTCC 277), and Aspergillus niger (MTCC 282), by Agar Well Diffusion Method (2). The Potato Dextrose Agar (PDA) medium was suspended in distilled water (39 g in 1000 ml) and heated to boiling until it dissolved completely, the medium and Petri dishes were autoclaved at pressure of 15 lb/inc2 for 20 min. Agar well bioassay was employed for testing antifungal activity. The medium was poured into sterile Petri dishes under aseptic conditions in a laminar air flow chamber. When the medium in the plates solidified, 0.5 ml of (week old) culture of test organism was inoculated and uniformly spread over the agar surface with a sterile L- shaped rod. Solutions were prepared by dissolving the compound in DMSO and Chloroform and different concentrations were made. After inoculation, wells were scooped out with 6 mm sterile cork borer and the lids of the dishes were replaced. To each well different concentrations of test solutions were added. Controls were maintained. The treated and the controls were kept at 270 C for 48 h. Inhibition zones were measured, the diameter calculated in millimeter and the corresponding results were tabulated.7 References:

1. Mosmann T. J Immunol Methods. 1983, 65, 55.

2. Glide, version 6.5, Schrödinger, LLC, New York, NY, 2014.

3. Suino, K. L.; Peng, R.; Reynolds, Y.; Li, J. Y.; Cha, J. R.; Joyce, A. K.; Steven H. E.; Xu, Molecular Cell, 2004, 16, 893. 4. Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer, R.; Neidigh J. W. ; Lilly, M. B. Mol Cancer Ther 2007, 6, 163. 5. Bao, Z. Q.; Jacobsen, D. M.; Young, M. A. Structure 2011, 19, 675. 6. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Tests; Eighteen informational supplements M100-S18 (2008). 7. Linday, M.E., 1962. Practical Introduction to Microbiology. E and F.N. Spon Ltd., United Kingdom, p. 177.

8. Schenck HA, Lenkowski PW, Mukherjee IC, Ko SH, Stables J P, Patel MK, Brown ML.

(2004) Bioorg. Med. Chem. 12:979.

Table1.

Structure of Oxindole analogs 1 to 16 Table 2. In vitro cytotoxicity of oxindoles derivatives against different cancer cell lines by

MTT assay

*IC50 (µg/ml) S.N Compound

o Codes SkoV3 B16F10 PC3 THP1 1. CF-1 61.60±2.04 95.34±1.98 67.64±2.06 64.05±1.28 2. CF-2 >100 >100 >100 >100 3. CF-3 97.25±1.27 >100 >100 >100 4. CF-4 >100 >100 >100 >100 5. CF-5 59.08±1.78 >100 70.43±1.26 65.21±1.18 6. CF-6 99.98±2.13 >100 86.14±1.17 64.24±1.90 7. CF-7 >100 >100 >100 >100 8. CF-8 58.54±1.83 96.14±2.08 64.39±1.38 60.55±1.45 9. CF-9 >100 >100 >100 >100 10. CF-10 >100 >100 >100 >100 11. CF-11 27.33±1.18 19.78±1.66 13.20±1.12 6.14±1.48 12. CF-12 >100 >100 >100 >100 13. CF-13 >100 >100 >100 >100 14. CF-14 >100 >100 >100 >100 15. CF-15 28.49±1.46 25.70±1.96 19.75±1.61 47.09±1.74 16. CF-16 15.09 ±1.63 17.13±1.40 27.54±1.21 43.62±1.07 17. Etoposide 4.37±1.31 4.88±1.11 8.04±1.34 4.02±1.04

Exponentially growing cells were treated with different concentrations of oxindolederivatives for 24h and cell growth inhibition was analyzed through MTT assay. *IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control cultures in the absence of an inhibitor and were calculated using the respective regression analysis. The values represent the mean ± SE of three individual observations. Table 3. MIC determination of the synthetic compounds and antibiotics (Penicillin and

Streptomycin 30 µg/ml) against various Gram positive and Gram negative bacteria by microdilution method

MIC

(µg/ml ) Compou B.Subtili S.epider P.aerogi K.pneum

nd code s S.aureus midis E.coli nosa oniae CF-1 75 75 37.5 75 75 75 CF-2 75 >150 37.5 37.5 >150 37.5 CF-3 >150 75 >150 37.5 75 75 CF-4 75 75 >150 18.75 >150 75 CF-5 75 37.5 9.375 75 >150 75 CF-6 75 18.75 75 75 >150 75 CF-7 75 37.5 37.5 37.5 >150 >150 CF-8 75 9.375 18.75 18.75 >150 75 CF-9 75 9.375 18.75 75 >150 75 CF-10 75 75 75 75 >150 75 CF-11 75 18.75 9.375 37.5 >150 75 CF-12 75 75 >150 >150 >150 >150 CF-13 75 75 >150 >150 >150 75 CF-14 75 75 150 >150 >150 75 CF-15 75 4.68 37.5 37.5 37.5 75 CF-16 75 9.375 18.75 37.5 37.5 37.5 Penicillin 1.562 1.562 3.125 12.5 12.5 6.25 Streptom

ycin 6.25 6.25 3.125 6.25 1.562 3.125

The series of the synthetic compound were evaluated against different bacteria in 96-well plate for 24 hours at 37OC. Gram-positive organisms viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis and Gram-negative organisms viz Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 741), and Klebsiella pneumoniae (MTCC 618) were used for MIC determination. Penicillin and Streptomycin is used as positive control. DMSO was used as negative control.

Table 4: Invitro Antifungal activity of synthetic compound (100 and 150 µg/ml) and fungicide (30 µg/ml) against fungal species tested by Agar well diffusion method. Zone of Inhibiton (mm) Compo Candid Sacharr Aspergi Aspergillus flavus und a omyces llus code albican cerevisi niger s ae 100µg 150µg 100µg 150µg 100µg 150µg 100µg 150µg CF-1 11 13 10 11 11 12 11 12 CF-2 10 11 10 11 11 12 10 11 CF-3 10 12 10 13 11 13 10 11 CF-4 10 12 11 12 10 12 10 11 CF-5 10 12 11 12 10 12 10 13 CF-6 10 11 10 12 10 12 10 12 CF-7 10 11 10 12 10 13 10 11 CF-8 10 12 10 11 10 11 10 11 CF-9 10 11 10 12 10 11 10 11 CF-10 10 11 10 12 10 12 10 11 CF-11 10 12 10 13 10 12 10 11 CF-12 10 12 10 11 10 12 10 12 CF-13 10 11 10 12 10 12 10 11 CF-14 10 12 10 11 10 12 10 11 CF-15 10 11 10 12 10 11 10 12 CF-16 10 11 10 12 10 11 10 11 Ampoth 23.5 22 25 25 ericin-B In vitro antifungal activity of the newly synthesized compounds was studied against the fungal strains, Candida albicans (MTCC 227), and Saccharomyces cervisiae (MTCC 36) of yeasts and Aspergillus flavus (MTCC 277), and Aspergillus niger (MTCC 282), by Agar Well Diffusion Method. Ampothericin-B is used as positive control.DMSO was used as negative control. NMR Spectra of 1-benzyl-3-hydroxy-3-(trifluoromethyl)indolin-2-one (1) NMR spectra of 1-ethyl-3-hydroxy-3-(trifluoromethyl)indolin-2-one (2) NMR spectra of 3-hydroxy-1-methyl-3-(trifluoromethyl)indolin-2-one (3) NMR spectra of 5-chloro-3-hydroxy-1-methyl-3-(trifluoromethyl)indolin-2-one (4) NMR spectra of 1-allyl-5-bromo-3-hydroxy-3-(trifluoromethyl)indolin-2-one (5) NMR spectra of 1-allyl-5-chloro-3-hydroxy-3-(trifluoromethyl)indolin-2-one (6) NMR spectra of 5-chloro-1-ethyl-3-hydroxy-3-(trifluoromethyl)indolin-2-one (7) NMR spectra of 1-ethyl-3-hydroxy-5-iodo-3-(trifluoromethyl)indolin-2-one (8) NMR spectra of 1-allyl-3-hydroxy-3-(trifluoromethyl)indolin-2-one (9) NMR spectra of 3-hydroxy-1-(prop-2-ynyl)-3-(trifluoromethyl)indolin-2-one (10) NMR spectra of 1-benzyl-5-chloro-3-hydroxy-3-(trifluoromethyl)indolin-2-one (11) NMR spectra of 5-bromo-3-hydroxy-1-propyl-3-(trifluoromethyl)indolin-2-one (12) NMR spectra of 3-hydroxy-1-phenyl-3-(trifluoromethyl)indolin-2-one (13) NMR spectra of 5-chloro -3-hydroxy-1-(prop-2-yn-1-yl)indolin-2-one (14) NMR spectra of 1-benzyl-5-bromo -3-hydroxy-3-(trifluoromethyl)indolin-2-one (15) NMR spectra of 1-benzyl-3-hydroxy-5-iodo-3-(trifluoromethyl)indolin-2-one (16)