SYNTHESIS OF NOVEL SUBSTITUTED-BENZO [ d ] THIAZOLE-2 , 4-DICARBOXAMIDES HAVING KINASE INHIBITION AND ANTIPROLIFERATIVE ACTIVITY

A series of novel derivatives containing N4-(4-fluorophenyl)-N2-substitured-benzo[d]thiazole-2,4-dicarboxamides were synthesized via an efficient, mild and convenient multistep reaction protocol with excellent yields. The structure of the synthesize compounds were confirmed by IR, 1H NMR, 13C NMR, 19F NMR, mass spectra, elemental analysis and purity was checked by HPLC. All synthesized compounds were screened for anticancer activity against A-549 and Du-145 cancer cell lines by MTT assay. The preliminary bioassay suggests that most of the compounds show anti-proliferation with different degrees. The synthesized compound shows IC50 values in the range of 1.52-17.18 μM in both cell lines. The compounds having electron donating groups had higher anticancer activity compared compounds with electron withdrawing substitutions.


INTRODUCTION
The kinases plays important role in cell functioning.There are over 500 kinases comprising in the human kinome, and all are associated with the functioning of cells. 1 Different types of kinases are responsible for different functioning of cells, some kinases are target of rapamycin (TOR) signaling for cell growth. 2,3 ome are protein tyrosine kinase inhibitors.By considering the importance of kinases we need to develop new kinase inhibitors with diversified activity.
In present work we have chosen substituted thiazol nuclei and its derivatives for cell line and kinases study.Substituted benzothiazole are known for diversified biological activities like anti-tubercular, 4 MAP kinase inhibitors. 5Kinases plays key role in cancer initiation and progression. 6,7Thiazoyl-sulfonamides act as carbonic anhydrase inhibitors 8 and anticancer. 91][12] Neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) activating small molecule-drug conjugates enzymes inhibitors, 13 Raf kinase inhibitor protein (RKIP), 14 poly-ADP-ribose polymerases (PARP) and topoisomerase (TOPO) inhibitors. 157] By considering the diversified biological activity of benzo [d]thiazol and continuation of our research work, [18][19][20] we have synthesized a series of substitutedbenzo [d]thiazole derivatives and all the synthesized compounds were tested for their biological activity in cell line and enzymatic study.

RESULTS AND DISCUSSION
We have synthesized a series N 4 2) has already been prepared in DMF and N-methyl piperidione at high temperature. 21,22We have used readily available 2-amino-3chlorobenzonitrile (1) as a staring material.We have optimized step a by heating the compound 1 with Nmethylpiperidinone at 150 °C for 1 h and adding the potassium ethylxanthate in DMF.The N-methylpiperidinone acts as base as well as high boiling solvent as higher temperature is required for the reaction to proceed.We heated that reaction mixture at 180 °C for 12 h to get >96 % yield.We have used mixture of solvents for solubility of compound and it helps in clean isolation of pure compound.The compound 2 was characterized as it shows clear S-C and S-H bonds in the IR spectrum at 730 cm -1 and 2560 cm -1 , respectively and in 1 H NMR the NH2 protons were vanished.
In step b the compound 2 was chlorinated by using POCl3 in 2 h at 100 °C to obtain crude compound with quantitative recovery, and the crude compound 3 was hydrolysed.The presence of compound 3 was confirmed with IR spectroscopy, the bands for C-S and C-H bonds were disappeared.
Compound 3 was hydrolyzed by using 20 % aqueous NaOH for 12 h at room temperature and later a routine acidbase treatment was used to isolate compound 4 with 90% isolated yield. 23It has been used further without purification.Compound 4 was characterized by TLC and 1 H NMR as in TLC it shows trailing spot at base and in NMR it shows an acidic peaks at δ 12.2 ppm.
In step d compound 4 reacted with 4-fluoroaniline (5) by using peptide coupling condition to obtain compound 6 with 90 % yield.Formation of Compound 6 was confirmed with TLC, mass and 1 H NMR spectra, as its aromatic region shows A2-B2 pattern of peaks.
In step e we have done displacement reaction on compound 6 by using sodium cyanide in methanol to obtain compound 7 with 85 % yield.The presence of compound 7 conformed from IR spectrum as it shows distinct band at 2200 cm -1 for nitrile group.
The compound 7 was subjected to hydrolysis to get compound 8. Using basic hydrolysis conditions led to quantitative yield of compound 8.The compound 8 is a key intermediate for the synthesis of desired compounds 10a-10j.
The compound 8 was converted to its acid chloride and lateer reacted with different amines 9a-9j without base to obtain all the final compounds 10a-10j with >80 % yields for all the derivatives.For the compounds 2 to 8 we have purified all the compounds by washing with different solvent combinations and avoided column purifications.For final compounds 10a-10j we have used column purification to get required compounds with >95 % purity.The detailed experimental procedure and characterization of final compounds and key intermediates were given in experimental section.

EXPERIMENTAL SECTION
All chemicals, unless otherwise specified, were purchased from commercial sources and were used without further purification.The major chemicals were purchased from Sigma Aldrich and Avra labs.The development of reactions was monitored by thin layer chromatography (TLC) analysis on Merck pre-coated silica gel 60 F254 aluminum sheets, visualized by UV light.Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected.The 1 H NMR spectra were recorded on a 400 MHz Varian NMR spectrometer.The 13 C were recorded on a 100 MHz Varian NMR spectrometer.The chemical shifts are reported as NMR spectra δ (ppm) units and standard is tetramethylsilane (TMS).The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br s).LC-MS mass spectra were taken with Micromass-QUATTRO-II of WATER mass spectrometer.In the reaction mixture of compound 1 (1.52 g, 0.01 mol) in N-methylpiperidinone (15 mL) was heated for 1 h at 150 °C.Potassium ethylxanthate (3.2 g, 0.02 mol) dissolved in DMF (7 mL) was added dropwise and the reaction mixture was stirred at 180° C for 12 h.Progress of reaction was monitored by TLC and LCMS.After completion the reaction, the reaction mixture was cooled to room temperature and poured into cold H2O (15 mL).The reaction mixture was acidified by using 4 M aq.HCl solution up to pH 4, when a solid was precipitated.The formed precipite was filtered in vacuo, washed with cold water (15 mL) and diethyl ether (10 mL) to afford 2-mercaptobenzo[d]thiazole-4-carbonitrile (2, 1.85 g, crude) as a brown solid.The crude obtained was used further for the next reaction without purification.

Step-b: Synthesis of 2-chlorobenzo[d]thiazole-4-carbonitrile (3):
To the reaction mixture of compound 2 (1.92 g, 0.01 mol) in a round bottom flask, POCl3 (1.9 mL) was added slowly at room temperature.The reaction mixture was stirred at 100 °C for 2 h.Progress of reaction was monitored by TLC and LCMS.The reaction mixture was cooled to room temperature and evaporated under reduced pressure to obtain 2-chlorobenzo[d]thiazole-4-carbonitrile (3, 2.5 g, crude) as gray semisolid compound.The crude obtained was used further for the next reaction without purification.

Step-c: Synthesis of 2-chlorobenzo[d]thiazole-4-carboxylic acid (4):
To a stirred solution of compound 3 (2.50 g) in a round bottom flask.20 % aq NaOH solution (25 mL) was added at room temperature.The reaction mixture was stirred at room temperature for 12 h.Progress of reaction was monitored by TLC and LCMS.
The reaction mixture was poured into cold H2O (10 mL) and extracted with DCM (15 mL).The aqueous layer was collected and acidified by using 4 M aq.HCl solution up to pH 3, when a solid was precipitated out.The formed precipitate was filtered off in vacuo, washed with water (25 mL), brine (20 mL) and diethyl ether (25 mL) to afford 2chlorobenzo[d]thiazole-4-carboxylic acid (4, 2.52g, crude) as a white solid.The crude obtained was used further for the next reaction without purification.

Step-f: Synthesis of 4-(4-fluorophenylcarbamoyl)benzo[d]thiazole-2-carboxylic acid (8):
To a stirred solution of compound 7 (2.97 g) in a round bottom flask.20 % aq.NaOH solution (25 mL) was added at room temperature.The reaction mixture was heated at 100 °C for 2 h.Progress of reaction was monitored by TLC and LCMS.The reaction mixture was poured in cold H2O (10 mL) and extracted with DCM (15 mL), the aqueous layer was collected and acidified by using 2 M aq.HCl solution up to pH 3. A solid was precipitated out .The formed precipitate was filtered off in vacuo, washed with water (25 mL), brine (20 mL) and diethyl ether (25 mL) to afford 4-(4-fluorophenylcarbamoyl)benzo[d]thiazole-2-carboxylic acid (8, 2.85 g, crude) as a white solid.The crude product obtained was used further for the next reaction without purification.

Biological evaluation
All the synthesized compounds were tested for their in vitro anticancer activity against various cancer cell lines.
The anticancer activity test is performed according to the procedure developed by the National Cancer Institute (NCI, USA) in the 'In vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye Sulforhodamine B (SRB) to assess cell growth. 24,25  the newly synthesized compounds 10a-10j are evaluated for their anti-proliferative activities against human lung cancer cell line (A-549) and human prostate cell line (DU-145).The results are summarized in Table 1.These values represent the concentration required to inhibit 50 % cell population compared with the control cells treated with DMSO and positive control Doxorubicin under similar conditions.
For lung cancer cell line (A549) the compounds 10a, 10d, 10g, 10h, 10i, and 10j are the most active with IC50 value in the range of 1.52 µM and 2.86 µM.The remaining compounds are moderately to less active with IC50 values in the range of 3.18 to 12.17 µM.For Du-145 cell line the compounds 10e, 10g, 10h, 10i and 10j are the most active with IC50 value in the range of 1.68 to 2.58 µM.The remaining compounds are moderate to less active with IC50 value is in the range of 3.17 to 17.18 µM.
Compounds 10g is the most active for A-549 and Du-145 cell line, 10g is 1.125 and 1.083 times more effective than the Doxyrubicin standard.The compounds having strongly electron donating groups like t-butyl (10f) or strongly electron withdrawing group (10l) seem to be very less active.
The SAR can be drawn like compound having methyl, ethyl, propyl etc. substituents are moderate to less active as they all are having electron donating tendency.For the remaining groups as the electron donating groups are present along with some electron deficient nitrogen atom shows promising activity.The compounds only having electron withdrawing groups are also less active to inactive.The SAR can be drawn like that for better activity electron donating nature of substituent should be there along with some electron deficient atom to enhance the activity.The compounds 10g, 10h and 10i were found to be the most active in cell line studies, so further we have tested them for their activity against a panel of eight human kinase at 10 µM concentration.
The inhibition results are summarized in Table 2. Protein kinase plays a key role in cell proliferation, differentiation of cell, migration of cell, survival of cell and angiogenesis of cells.The compound 10g, 10h and 10i show inhibition in the range of 28 % to 61 % for Aurora-A and Aurora-B kinases, but the activity are not the same for both kind of Aurora kinases.
The compounds show inhibitions in the rage of 19 to 51 %. for CDK/cyclin A, CDK/cyclin E, CDK5/P25 kinases belong to CMGC kinase family.For MAP kinase family the all compounds show inhibition in the range of 49 to 66%.For EGFR and JNK kinases all three compounds shows inhibitions >58 %.

CONCLUSION
We have synthesized N4-(4-fluorophenyl)-N2-substituredbenzo[d]thiazole-2,4-dicarboxamide derivatives (10a-10j) from aniline through a series of reactions including benzthiazol synthesis, chlorination, hydrolysis, cynation and amide coupling.We have reported simple reaction condition, easy workup, short reaction time and good to high yields.The synthesized compounds were screened for anticancer activity against A-549 and Du-145 cancer cell lines.Most of the compounds were active for tested cell lines with IC50 value in the range of 1.52 to 17.18 µM.The compounds 10g, 10h and 10i are most active with IC50 values in the range of 1.52-1.98µM.The compounds 10g, 10h and 10i were shows promising inhibitions (> 58%) for EGFR and JNK human kinases.The compounds having electron donating substituents along with electron deficient atom shows promising activity and greater inhibitions for protein kinases.

Table 2 .
Inhibitory activity of selected compounds against panel of eight human kinases.