INVESTIGATIONS SYNTHESIS, CHARACTERIZATION AND EVALUATION OF THE ANTICACANCER ACTIVITY OF NOVEL 2-AMINOPHENYLTHIAZOLE DERIVED HETEROCYCLES

Aisha Y. Hassan1, Moshira A. El-Deeb2, Marwa T. Sarg2 and Maha A. Abou-Sharabia2. 1. Organic Chemistry Department, Faculty of Science (Girls), Al-Azhar University, Cairo. 2. Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Girls) Al-Azhar University, Cairo. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Novel substituted 2-aminophenylthiazole derivatives were synthesized via various synthetic pathways. Among which were compounds bearing different side chains attached to the thiazole backbone through occupying C 2 position 2-4, 6, 8-12,and 15-22. Also thiazole derivatives 23-25 bearing substituted pyrimidines were designed to be synthesized. Moreover, we have synthesized iminothiazolyl succinic acid derivatives 5 a and 5 b , fused thiazolothiadiazole analogue 7, as well as, the thiazolopyrimidine derivatives 13 and 14. The newly synthesized compounds were evaluated for their in vitro anticancer activity against human Liver cancer HepG2 and Breast cancer MCF7 cell lines compared to the reference drug Doxorubicin.

Materials and Methods:-
Melting points of all the synthesized compounds were determined in an open capillary method and were uncorrected. IR spectra were recorded using KBr disc technique on Nikolet IR 200 FT IR Spectrophotometer at Pharmaceutical Analytical Unit, Faculty of Pharmacy, Cairo University and values are represented in cm -1 .The 1 HNMR Spectra were recorded on Varian Gemini EM-300 MHZ, NMR Spectrometer at laboratories of the nuclear magnetic resonance, Chemical Warfare Department, Ministry of Defense. DMSO-d 6 was used as a solvent;

Results and Discussion:-
2-Amino-4-phenylthiazole derivative1 a was prepared according to the reported procedure [13] which was further utilized as a building block to synthesize various phenylthiazole derivatives substituted either at the thiazole amino function or fused to the thiazole back bone.However, treatment of compound 1 a with phenylisothiocyanate furnished the corresponding thiourea derivative 2 that was further cyclized to its aminothiazoleanalogue 3. The 1 H NMR spectrum of compound 2 revealed a deuterium oxide exchangeable singlet signal at δ 8.76 ppm attributed to NH-C 6 H 5 while, the IR spectrum of compound 3 showed NH function absorption band at 3387 cm -1 . In addition to, absorption bands at 1240, 1090 cm -1 corresponding to C-S-C function. The 1 H NMR spectrum of compound 3 2366 revealed a deuterium oxide exchangeable singlet signal at δ 8.65 ppm attributed to NH. In addition,dihydrothiazole C 5 -H was observed as a singlet signal at δ 6.93 ppm.
We carried out the synthesis of the target N,N-dipropynylthiazole-2-amine derivative 4 by refluxing one equivalent of 2-amino-4-phenyl-thiazole 1 a with two equivalents of propargyl bromide.The 1 H NMR spectrum of compound 4 revealed a singlet signal at δ 4.15 ppm attributed to two CH 2 protons. In addition to a singlet signal at δ 8.5 ppm due to two acetylenic protons.
Previously iminosuccinic acid derivatives were obtained via treatment of different substituted aromatic amines with itaconic acid [14]. However, the reaction was postulated to proceed through addition of the ring nitrogen to the double bond of the methyl succinic acid (itaconic acid). The iminosuccinic acid derivatives 5 a,b were synthesized by stirring the appropriate 2-amino-4-phenylthiazole derivatives1 a,b with an equimolar amount of itaconic acid at room temperature. The IR spectra of compounds 5 a,b showed absorption bands at 3429-3383 cm -1 corresponding to OH function and absorption bands at 3267-3116 cm -1 due to NH function. In addition to, absorption bands at 1701 cm -1 corresponding to C=O function.
Furthermore, the thioformylbenzamide derivative 6 was prepared via the reaction of of2-amino-4-phenylthiazole 1 a with benzoyl isothiocyanatewhich was further cyclized to its correspondingthiadiazolederivative7 upon reflux with phosphorous oxychloride. The IR spectrum of compound 6 showed absorption band at 3369 cm -1 corresponding to OH tautomer. The 1 H NMR spectrum of compound 6 revealed two deuterium oxide exchangeable singlet signals at δ 5.45 and δ 7.68 ppm attributed to NH and OH tautomer; respectively. However, the 1 H NMR spectrum of compound 7 revealed two multiplet signals at δ 7.40-7.46 ppm and δ 7.59-7.61ppm attributed to C 3,5 and C 4 protons of CO-C 6  In our recent study, the benzamide derivative 8 was prepared via the reaction of equimolar amounts of 2formylbenzoic acid and 2-aminothiazole derivative 1 b in methanol under reflux conditions. The IR spectrum of compound 8 showed an absorption band at 3367 cm -1 corresponding to OH tautomer. In addition to, two absorption bands at 1755, 1710 cm -1 corresponding to two C=O functions. The 1 H NMR spectrum of compound 8 revealed two deuterium oxide exchangeable singlet signals at δ 7.41 and 9.08 ppm attributed to OH tautomer andthiazole NH; respectively.
It is well established in the literature that, thioglycolic acid was reported to be utilized in various cyclocondensation reactions with different Schiff's bases leading to their cyclized thiazolidinone analogues [8,15] . Therefore, schiff's base 9 was treated with thioglycolic acid in refluxing tetrahydrofuran containing zinc chloride as a catalyst to yield the target thiazolidinone derivative 10. The IR spectrum of compound 9 lacked absorption bands corresponding to NH 2 function while, the IR spectrum of compound 10 showed abroad absorption band at 3421 cm -1 corresponding to OH tautomer. In addition to, an absorption band at 1683 cm -1 corresponding to C=O function.
Chloroacetyl chloride has been widely reported to be involved in the preparation of various azetidinone derivatives through the reaction with different Schiff's bases [8,16]. Therefore, thiazolylazetidin-2-one derivative 11 was prepared via stirring equimolar amounts of Schiff's base derivative 9 and chloroacetyl chloride in dry benzene containing triethylamine. The 1 H NMR spectrum of compound 11 revealed a singlet signal at δ 2.30 ppm attributed to azetidinyl-C 4 -H proton. In addition to a multiplet signal at δ 4.05-4.18 ppm due to azetidinyl-C 3 -H proton.
Furthermore,2-aminothiazole derivative 1 b upon heating under reflux in ethanol containing piperidine with equivalent amount of diethyl oxalate yielded the corresponding carbamoyl formate derivative 12. The IR spectrum of compound 12 showed an absorption band at 3446 cm -1 corresponding to OH tautomer and two absorption bands at 1741, 1685 cm -1 corresponding to two C=O functions. The 1 H NMR spectrum of compound 12 revealed a triplet signal at δ 3.98 ppm attributed to CH 3 protons and a quartet signal at δ 4.32 ppm attributed to CH 2 protons. In addition to, two deuterium oxide exchangeable signals at δ 8.7 ppm and δ 13.10 ppm due to OH and NH of the tautomer respectively. In our recent study, 1 b upon treatment with excess of ethyl chloroformate yieldedthetarget N,N-diethylformate thiazole-2-amine 15.The IR spectrum of compound 15 lacked absorption bands corresponding to NH 2 function, besides to, two absorption bands at 1789, 1720 cm -1 due to the two ester carbonyl functions. In the present investigation, the target 1, 2, 4 thiazolidine-3, 5-dione derivative 16 was obtained by fusion of equimolar amounts of compound 15 and hydrazine hydrate. The 1 H NMR spectrum of compound 16 revealed two deuterium oxide exchangeable singlet signals at δ 7.68 ppm and δ 7.71 ppm attributed to two NH protons.(Scheme 3) Furthermore, 2-aminothiazole derivative 1 b was stirred with equimolar amounts of chloroacetyl chloride in dimethylformamide at room temperature to furnish the target chloroacetamide derivative 17.The IR spectrum of compound 17 showed an absorption band at 3446 cm -1 corresponding to tautomeric OH function and an absorption band at 3182 cm -1 due to NH function. In addition to, an absorption band at 1654 cm -1 due to C=O function. The 1 H NMR spectrum of compound 17 revealed two deuterium oxide exchangeable singlet signals at δ 7.14 ppm and δ 12.59 ppm attributed to OH and NH of the tautomers; respectively. In addition to, a singlet signal at δ 4.17 ppm due to CH 2 protons.
In addition, thecyclization of17 to the target thiazolylthiazolone derivative 18was achieved via heating equimolar mixture of 17 and ammonium thiocyanate in ethanol under reflux. The IR spectrum of compound 18 showed a broad absorption band at 3444 cm -1 corresponding to OH of the tautomer. The 1 H NMR spectrum of compound 18 revealed two deuterium oxide exchangeable singlet signals at δ 7.30 ppm and δ 7.46 ppm attributed to OH and NH protons.

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The thiazolyl hydrazine derivative 19 was prepared by treatment of2-aminothiazole derivative 1 b with an equimolar amount of hydrazine hydrate in ethylene glycol. The 1 H NMR spectrum of compound 19 revealed two deuterium oxide exchangeable singlet signals at δ 3.72 and 4.38 ppm attributed to NH and NH 2 .
However, the thiazolyl hydrazine derivative 19 furnished the correspondingthiazolylamino-N-iminomethylphenol derivative 20upon heating with oneequivalent of5-bromosalicylaldehyde in methanol under reflux. The IR spectrum of compound 20 showed a broad absorption band at 3406 cm -1 corresponding to OH function.
Our aim was extended to study the effect of cyclization of the hydrazone derivative 20 tothiazolylindazole derivative 21 in refluxing glacial acetic acid/ methanol mixture on the anticancer activity study. The IR spectrum of compound 21 lacked the absorption band due to OH and NH functions of its precursor. (Scheme 4).
It is well established in the literature that, various guanidine derivatives could be achieved via treatment different of amines with guanidinylation reagents [17,18]. Therefore, we utilized cyanamide in the reaction with 2aminothiazole derivative 1 b to obtain the thiazolylguanidine intermediate 22. The 1 H NMR spectrum of compound 22 revealed three deuterium oxide exchangeable singlet signals at δ 6.99 ppm, δ 8.30 ppm and δ 12.20 ppm attributed to NH 2 , NH and C=NH protons; respectively. Our efforts was directed to study the anticancer activity of various substituted pyrimidine heterocycles attached to the thiazole backbone through its amino function, a step that was achieved via cyclization of the side chain of the intermediate 22 by the aid of variousactive methylene bearing carbonyl compounds.
The thiazolylguanidine22 upon treatment with either with acetyl acetone or benzoyl acetone furnished the target thiazolylmethylpyrimidine derivatives 23 and 24; respectively. The 1 H NMR spectra of compounds 23 and 24 revealed singlet signals at δ 3.00 ppm due to two CH 3 protons of 23 and a singlet signals at δ 2.44 ppm attributed to CH 3 protons of 24 besides to, the NH proton deuterium oxide exchangeable singlet signal in each at δ 3.47 ppm and δ 10.53 ppm for compounds 23 and24; respectively. In addition, the 1 H NMR spectra of both 23 and 24 revealed the pyrimidine C 5 -H singlet signal at δ 8.15 ppm and δ 8.27 ppm; respectively.
Finally, utilizing diethylmalonate in the cyclization of the guanidine derivative 22yielded the target thiazolylaminopyrimidindione derivative 25. The IR spectrum of compound 25 showed abroad absorption band at 3431 cm -1 corresponding to OH function. In addition to, absorption band at 1685 cm -1 corresponding to C=O function. However,the 1 H NMR spectrum of compound 25displayed three deuterium oxide exchangeable singlet signals at δ 7.71, 12.21 and 12.44 ppm attributed to NH, pyrimidine N-H and OH tautomeric protons; respectively.

Scheme 5 Biological Activity:-
Anticancer screening studies:-Twenty fourof the synthesized compounds (2-25) were screened for their in vitro cytotoxic activity against human hepatocellular liver carcinoma (HepG2) and human breast cancer (MCF7) cell lines in the regional center for mycology and biotechnology, at Al-Azhar University. Doxorubicinwas used as the reference drug in this study. It is 2372 well documented that doxorubicin induces its antitumor activity through several mechanisms including inhibition of topoisomerase II, DNA intercalation, generation of reactive oxygen species and DNA single and double strand breaks.
Cytotoxicity evaluation using viability assay:-For cytotoxicity assay, the cells were grown as monolayers in growth RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50μg/ml gentamycin. The monolayers of 10,000 cells adhered at the bottom of the wells in a 96-well micro titer plate incubated for 24h at 37 o C in a humidified incubator with 5% CO 2 . The monolayers were then washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 μl from different dilutions of tested sample in fresh maintenance medium and incubated at 37 o C. A control of untreated cells was made in the absence of tested sample. A positive control containing Doxorubicin drug was also tested as reference drug for comparison. Six wells were used for each concentration of the test sample. Every 24h the observation under the inverted microscope was made. The number of the surviving cells was determined by staining the cells with crystal violet followed by cell lysing using 33% glacial acetic acid and read the absorbance at 590nm using ELISA reader (SunRise, TECAN, Inc, USA) after well mixing. The absorbance values from untreated cells were considered as 100% proliferation.The number of viable cells was determined using ELISA reader as previously mentioned before and the percentage of viability was calculated as [1-(ODt/ODc)]x100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots [19,20].    Sample concentration µg/mL) % Growth Inhibition