Synthesis and evaluation of biological activity of 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones

Aim. To accomplish the synthesis and screening of anticancer and antimicrobial activities of 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones 2-10. Methods. The in vitro anticancer activity of compounds 4, 6, 8-10 has been established by DTP(Developmental Therapeutics Program) of the National Cancer Institute. The antibacterial and antifungal activities of synthesized thiazole-based derivatives were evaluated in vitro with the agar diffusion and broth microdilution methods to wards Gram-positive, Gram-negative bacteria and yeasts. For the synthesized compounds, the in silico drug-likeness screening using SwissADME online server is reported. Results. The novel 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones were synthesized from 1-[2-amino-4-methylthiazol-5-yl]ethanones and various aromatic aldehydes in the Claisen–Schmidt condensation. The synthesized compound 9 was moderately active against the leukemia CCRF-CEM and HL-60(TB), renal cancer UO-31 and breast cancer MCF7 cell lines. The antimicrobial screening led to identification of the active compound 10 against Staphylococcus aureus , Pseudomonas aeruginosa , and Candida albicans . Conclusions. The results obtained herein provide a platform for structure-based optimization of these newly identified thiazole-based compounds for the anticancer and antibacterial drug design.


Introduction
Nitrogen-and sulfur-containing heterocycles underlie a large number of innovative drugs and biologically active compounds. The significant pharmacological potential of the mentioned compounds is related to their high affinity for biotargets and the structural similarity of various metabolites [1]. In the context of searching for potential drug-like molecules, a prominent interest among heterocyclic systems is attracted to the thiazole derivatives, which constitute a large number of antibiotics, sulfonamides, NSAIDs, antiulcer drugs and vitamins [2][3][4]. Additionally, several lead compounds with insecticidal [5], antitumor [6], anti-inflammatory [7], antioxidant [8], antimicrobial [9], analgesic [10] properties have been identified among the thiazole derivatives, which undergo various stages of clinical and preclinical studies. Besides, substituted thia-zole derivatives are well known as COX-2 [11,12], serine protease urokinase (uPa) inhibitors [13], adenosine A1 receptor [14] and metabotropic glutamate receptor 5 (mGluR5) antagonists [15]. Noteworthy, the thiazole cycle's uniqueness in the construction of new druglike molecules is based on the wide possibility of their functionalization providing different classes of condensed and non-condensed thiazoles [16][17][18][19][20]. Moreover, the structural modification of this heterocycle allows obtaining compounds with a new pharmacological profile, increased activity or reduced toxicity.
Among the thiazole derivatives, a significant interest is attracted to aminothiazoles as active binucleophiles, which are important reagents in modern heterocyclic chemistry, allowing the reaction with electrophiles to obtain different classes of heterocyclic com-  pounds [21,22]. The most investigated area of aminothiazole chemistry is the condensation reactions (Claisen-Schmidt condensation, Mannich reaction etc.) and various reactions involving heterocyclic ring formations with a,b-unsaturated ketones or a-ketoacids yielding fused azoloazines [23,24]. Thus, the purpose of our work was the design and synthesis of 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones, and evaluation of their anticancer and antimicrobial activities.

Chemistry
All materials were purchased from Sigma-Aldrich and used without purification. Melting points were measured in open capillary tubes and were uncorrected. The elemental analyses were performed using the Thermo Scientific FlashSmart Elemental Analyzer. The 1 H NMR spectra were recorded on Varian Gemini ( 1 H NMR at 400 MHz) instrument in DMSO-d 6 . Chemical shifts (δ) are given in ppm units relative to tetramethylsilane as refe rence (0.00). LC-MS spectra were obtained on a Finnigan MAT INCOS-50. The purity of all obtained compounds was checked by TLC on Silufol-254 plates (Eluent Benzene: EtOAc 1:1). 2-The starting 2-amino-4-methyl-5-acetylthiazole 1 was prepared according to a reported method [25].

Cytotoxic activity against malignant human tumor cells
Anticancer in vitro assay was performed on the human tumor cell lines panel derived from nine neoplastic diseases, following the Drug Evaluation Branch protocol, National Cancer Institute, Bethesda, MD, USA [26][27][28]. Tested compounds were added to the culture at a single concentration (10 -5 M), and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, Kiton Red 620. Results for each tested compound were reported as the GP% of the trea ted cells compared with the untreated control cells. GP% was evaluated spectrophotometrically vs controls not treated with test agents.

Antimicrobial activity
The 16 reference and clinical strains of microorganisms were used as test objects, including the methicillin-sensitive strain of S.aureus (MSSA), strain "Iv", the methicillin-resistant strain of S.aureus (MRSA), strain ICA-5, (isolated from a patient with retroperitoneal abscess), clinical strain "Fedk" of E. coli (from a patient with cystitis) and Bacillus subtilis (wild isolate), as well as with a strain of the yeast Candida albicans ST-1 (from patients with protein stomatitis). Isolates were identified using biochemical microtests "STAPHYtest 16", "ENTEROtest 24" (Lachema, Czech Republic), as well as taking into account the complexes of morphological and cultural properties by the recommendations of the 9th edition of "Bergi bacteria Determinant." The C. albicans culture was identified based on 40 biochemical tests using VITEK 2 systems with the VITEK 2 YST identification card (biomerieux, France). Few strains were multidrug resistant (MDR) to antibiotics.
The antimicrobial activity of the synthesized compounds was studied by the agar diffusion method. Test culture suspensions standardized according to the optical turbidity standard (1*10 7 CFU/ml) were evenly inoculated on the nutrient agar surface in Petri dishes. The agar wells with a diameter of 4.0 ± 0.1 mm were made, and 20 μl of the tested compound (concentration of 1000 μg/ml) were added. The solvent was a solution of alcohol/ DMSO/water 2:1:1. After cultivation for 24-48 hours, the diameter of the growth inhibition zone was measured. A pure solvent was added to the control wells. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using serial dilution technique [29,30].

Chemistry
The starting thiourea has been used as S,Nbinucleophile with 2-chloroacetylacetone as the equivalent of dielectrophilic synton [C2] 2+ in the [2+3]-cyclocondensation reaction providing 2-amino-4-methyl-5-acetylthiazole with good yield. The reaction was performed in glacial acetic acid and anhydrous sodium acetate as catalyst according to the method described previously [25].

In vitro evaluation of the anticancer activity
The synthesized compounds contain the enone fragment in the structure, which makes them potentially Michael acceptors, the object of intensive study in modern medicinal chemistry [31]. Moreover, Michael acceptors are now considered a "new old tool" for creating new drug-like molecules, especially anticancer agents (Michael acceptors are one of the most effective activators of Nrf2, effective covalent inhibitors of several cancer biotargets [32]), and became the reason for the synthesis of target 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones. However, in some reports, it was found that the conjugation of a double bond with the carbonyl group in mentioned compounds gives the properties of "promiscuous inhibitors" and causes probable lack of selectivity in the interaction with potential biotargets [33].
The synthesized 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones 4, 6, 8-10 were submitted and evaluated at a 10 -5 M single concentration towards a panel of approximately sixty cancer cell lines. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the US NCI protocol

Antimicrobial activity
Compounds 7 and 10 were selected for screening the antimicrobial activity. The research objects were chosen to specify the effect of molecular fragments of target compounds on the pharmacological effect. Moreover, an essential aspect of the study was the experimental establishment of the dependence of the antimicrobial activity of the synthesized compounds on the structural environment of the thiazole moiety. In particular, the nature of the aryl substituent of the thiazole nucleus, the establishment of the role of the «Michael acceptors» properties in the realization of a particular pharmacological effect.
Thus, according to the screening results, compounds 7 and 10 showed the moderate antimicrobial activity of different levels with  Note: *MDR strain ADME prediction ADME prediction of all the synthesized molecules was determined using the SwisAdme online server of the Swiss Institute of Bioinformatics [34]. All the molecules showed acceptable physicochemical parameters with low Lipinski violation of potential drug candidates (Table 4).

Conclusion
New thiazole-based derivatives 2-10 have been synthesized with high yields via Claisen-Schmidt reaction using 1-(2-amino-4-methylthiazol-5-yl)-ethanone and aromatic aldehydes as starting compounds. The synthesized compound 9 displayed moderate antitumor activity against the leukemia, renal, and breast cancers cell lines. The preliminary results on antimicrobial activity allowed us to identify the active compound 10, which has shown the best activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The drug-likeness of 1-[2-amino-4-methylthiazol-5-yl]-3-arylpropenones was calcu- lated through SwissADME online server. The synthesized derivatives fulfilled all the conditions specified by Lipinski without any violation and could be considered as the lead compounds for the anticancer and antibacterial drug design.