Synthesis and evaluation of antitumor activity of some thiazolo [ 4 , 5b ] pyridines

Aim. To synthesize a series of novel 3H-thiazolo[4,5-b]pyridine-2-ones by structural modification of the core heterocycle in its N3and N6-positions and to evaluate their anticancer activity in vitro on several tumor cell lines. Methods. Organic synthesis, 1 H-NMR spectroscopy, trypan blue cell viability assay. Results. A new convenient synthetic approach was developed and optimized conditions were studied for the reaction of preparation of 3Hthiazolo[4,5-b]pyridin-2-one derivatives. 5,7-Dimethyl-3H-thiazolo[4,5-b]pyridin-2-one and 6-phenylazo-5,7dimethyl-3H-thiazolo[4,5-b]pyridin-2-one were obtained by [3 + 3]cyclocondensation of 4-iminothiazolidone2 with acetylacetone and -phenylazoacetylacetone in methanol medium in the presence of sodium methylate. They were used as starting compounds for further structural modification of the core thiazolo[4,5-b]pyridine heterocycle in its 3and 6-positions. On the basis of in vitro cytotoxicity studies of synthesized compounds several structure-functional relationships underlying anticancer potential of 5,7-dimethyl-3H-thiazolo[4,5-b]pyridin2-one derivatives were identified. Conclusions. 3H-thiazolo[4,5-b]pyridin-2-one can be considered as a promising molecular scaffold for rational design of potential anticancer drug candidates. Introduction of phenylazo substitute at C6-position of 3H-thiazolo[4,5-b]pyridin-2-one proved to be the most efficient, as it led to 3-fold increase of its anticancer potential.


Introduction. The development of diverse strategies
for the directed synthesis of potential drug candidates consists of several steps, starting from scaffold hopping towards the hit compounds identification and their further in silico rational design.Molecular scaffold-based design of 4-thiazolidine analogs is inspired by both the variety of their pharmacological actions and accessibility of chemical modification of their structure.A series of 4-thiazolidines are at different stages of clinical trials as anti-inflammatory, antitumor, antimicrobial, antiviral, cardiovascular and thrombolytic drug candidates [1].
The thiazolidine based heterocycles and their analogs fused w³th the pyridine ring serve as attractive tar-gets for developing efficient protocols to prepare thiazolo [4,5-b]pyridine scaffolds the biological activity of which still remains poorly studied.
There is little information available about the biological activity of thiazolo [4,5-b] pyridines.For example, some of their derivatives were described as useful antimicrobial agents effective against a variety of human and veterinary pathogens including Gram-positive and Gram-negative aerobic and anaerobic bacteria, as well as mycobacteria [2].Thiazolo [4,5-b]pyridines also show potent inhibitory activities for Ab42 fibrillization at a micromolar level for Alzheimer's disease treatment [3], some of them are H3 receptor antagonists [4], others were revealed to activate the GK enzyme in vitro and significantly reduce glucose levels [5].Some thiazolo [4,5-b]pyridine analogues are potent antistaphylococ- cal compounds with suboptimal drug-like properties [6].They act as inhibitors of the bacterial cell-division protein, FtsZ.Several compounds among these substances were found to possess fungicidal action [7] or act as antagonists of metabotropic glutamate receptors 5 (mGluR5) [8], as well as epidermal growth factor receptors [9] and a number of other enzymes [10,11].
However, the information on antitumor activity of the mentioned bicycle derivatives of 4-thiazolidones is still missing.Up to now just a few compounds of such chemical structure have been tested against some human tumor cell lines in vitro [1,12].Several studies point out that 4-thiazolidone based bicyclic analogues, in particular pyrazole and isoxazole derivatives, can act as inhibitors of HSP90, an ATP-dependent molecular chaperon [12].
In normal cells HSP90 is involved in protein folding, but in cancer cells it can stabilize oncoproteins v-Src, Bcr/Abl and mutant products of p53 gene, thus possessing strong anti-apoptotic activity [13].Several HSP90 inhibitors, such as geldanamycin, have already shown some promising effects in clinical trials [14], but search for other small molecule inhibitors of HSP90 still remains an active area of cancer research.
Tyrosine Kinases (TK) are other possible anticancer targets for 4-thiazolidone derivatives.TK play a crucial role in the action of epidermal growth factor receptor (EGFR), and mutation of the ATP-binding site of EGFR is closely associated with TK activity, being one of the main reasons of cell malignization by disrupting the formation of tumorigenic signals.That's why the development of TK inhibitors is suggested as one of the most perspective strategies for cancer treatment, and some of them, such as trastuzumab (Herceptin ® ), gefitinib, imatinib have been already widely used in cancer treatment.Several five-membered heterocycle derivatives and fused heterocycle analogs were also found to possess the TK enzymes inhibitory activity and now are at different phases of clinical trials [15].
Based on these data, the main aim of current work was the development of novel innovative approaches for organic synthesis of thiazolo [4,5-b]pyridine derivatives with an eye towards the discovery of isosteric fused ring compounds with the nitrogen heteroatom as a potential atom interacting with the anticancer targets mentioned above.

Materials and methods.
Synthetic routine for the novel compounds preparation was carried out according to common approaches used for the potential drug candidates rational design with proper «Merck» (Germany) chemicals implementation.
The structures of all synthesized compounds were established on the basis of quantitative elemental analysis and spectral 1 H-NMR data («Varian Mercury VX-400», DMSO-d 6 was used as a solvent, tetramethylsilane as a standard).
Murine leukemia cells of L1210 line were obtained from the cell culture collection of R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, human breast adenocarcinoma cells of MDA-MD-231 lines were obtained from the cell culture collection of Ludwig Institute for Cancer Research (Sweden), and human melanoma cells of SK-MEL-28 line were obtained from the cell culture collection of Nencki Institute of Experimental Biology PAN (Poland).Cells were cultured in DMEM medium supplemented with 10 % fetal calf serum («Sigma Chemical Co.», USA), 50 µg/ml streptomycin («Sigma Chemi cal Co.»), 50 units/ml penicillin («Sigma Chemical Co.») in 5 % CO 2 -containing humidified atmosphere at 37 o C.
Experiments were performed in triplicate and repeated 3 times.Significance of the difference in a typical experiment was assessed by Student's t-test.The level of significance was set at 0.05.
Further structural modification of thiazolo [4,5-b]pyridine scaffold in its N3 and C6 positions was investigated resulting in the preparation of its novel derivatives.
The acidic character of the proton in the core heterocycle N3-position of compounds 1 and 2 promotes tendency of these compounds to transform into corresponding potassium salts under their treatment with potassium hydroxide.The obtained salts possess nucleophilic properties and can be involved into reactions with electrophilic reagents in mild conditions.Thus the potassium salt of 5,7-dimethyl-3H-thiazolo [4,5-b]pyridin-2-one and the same salt of 6-phenylazo-5,7-dimethyl-3H-thiazolo [4,5-b]pyridin-2-one (6) were alkylated with monochloracetic acid ethyl ester.The next step of our strategy was the utilization of the products in the hydrazinolysis reaction with the respective hydrazides preparation like 5,7-dimethyl-3Hthiazolo [4,5-b]pyridin-2-one hydrazide (7).In the same way the compound 2 hydrazide is easily available while its further utilization in condensation with heterocyclic aldehyde reaction led us to the compound 8 formation.Using 3-bromopropene-1 as an alkylation agent for the compound 7 treatment allows us to get compound 9 (Fig. 2).
The reduction decoupling reaction of 5,7-dimethyl-6-phenylazo-3H-thiazolo [4,5-b]pyridine-2-one (2) was studied in order to obtain 6-amine-5,7-dimethyl-3H-thiazolo [4,5-b]pyridine-2-one as a reagent for thiazolo [4,5-b] pyridines C6-position transformations.A high yield of the targeted compound 10 was found to occur in acetic acid-pyridine medium, zinc powder being used as a reduction agent.The amine group presence in its C6-position allows transforming it into appropriate 6-acetylaminoderivative (11).Dioxane was found to be the most suitable medium for the reaction of compound 10 with acetic acids chloroanhydride.The product of the compound 10 acylation with chloroacetylchloride made it possible to design 6-sulfanylacetamidederivative of the core bicycle (12) by treatment it with the appropriate thiol (Fig. 3).
However, the hydrolysis of compound T-3 side chain weakened its anticancer potential -we observed 18 % inhibition of L1210 cells, while for carcinoma cells no statistically significant differences between T-3 and T-4 were found -60 % inhibition of MDA-MD-231 cells (compared to 64 % in case of T-3) and 56 % inhibition of SK-MEL-28 cells (compared to 66 % in case of T-3).Thus, the terminal cyano group at N3-position of 5,7-dimethyl-3H-thiazolo [4,5-b]pyridin-2-one derivatives seems to play some role in their antileukemic activities.

% of SK-MEL-28 cells compared to 72 % by T-2).
These results give an additional proof that no substitutes should be placed at N3-position for preservation of the highest anticancer activity of T-2 scaffold .
The next part of the work was dedicated to studying the influence of different substitutes in C6-position of T-1 core scaffold.At the first step phenylazo fragment was reduced to amino group, and the resulting T-10 compound completely preserved its anti-melanoma cytotoxic effect (72,5 % growth inhibition of SK-MEL-28 cells compared to 72 % by T-2), while its anticarcinoma effect (52 % of growth inhibition of MDA-MD-231 cells compared to 82 % by T-2) and antileukemia activity (29 % of growth inhibition compared to 65 % by T-2) were significantly weaker than those of parental compound.Thus, the exchange of phenylazo group at C6-position for amino group somehow decreases the affinity of 5,7-dimethyl-3H-thiazolo [4,5-b] pyridin-2-one scaffold to leukemia cells.A similar phenomenon was observed when cyano terminal motif in T-3 was hydrolyzed to acid in T-4, as mentioned before.
The acylation of amino group at C6-position of T-10 scaffold led to further decrease in its cytotoxic activity -the resulting compound T-11 was weaker than its predecessor in carcinoma and melanoma cells (14 % growth inhibition of MDA-MD-231 cells compared to 52 % by T-10 and only 37 % inhibition of SK-MEL-28 melanoma cells compared to 72.5 % by T-10), while sensitivity of L1210 leukemia cells remained the same (30 % growth inhibition of L1210 leukemia cells compared to 29 % in case of T-10).
Under the interaction of a product of acylation of aminogroup of T-10 by chloroacetylchloride with one of thiols, the T-12 compound was prepared.Interestingly, we observed a slight increase in anti-carcinoma effect of T-12 on MDA-MD-231 cells (66 % growth inhibition compared to 52 % effect of T-10), while its anti-leukemia action remained the same as of the parental compound (34 % growth inhibition of L1210 cells compared to 29 % effect of T-10).In contrast to these data, SK-MEL-28 melanoma cells demonstrated decreased sensitivity to T-12 (34 % growth inhibition compared to 72.5 % by T-10).Thus, further steric modifications decrease the anticancer potential of the aminogroup at C6-position of the core scaffold and thus are not effective.
According to these results, several principal suggestions could be derived.Despite the introduction of various substitutes into the 5,7-dimethyl-3H-thiazolo [4, 5-b]pyridin-2-one core molecule, we did not succeed in significant enhancement of their anticancer activity.The average cytotoxic activity of the most potent compound with anticancer activity T-2 (6-phenylazo-5,7dimethyl-3H-thiazolo [4,5-b]pyridin-2-one) towards three cell lines was only 50 µM, which is still 10 times more than that of some clinically used anticancer drugs.Thus, further modifications in the structure of 6N-derivatives of 5,7-dimethyl-3H-thiazolo [4,5-b]pyridin-2one should be done in order to enhance its anticancer potential, as well as to perform in-depth studies of its potential targets in tumour cells.Previously we reported that conjugation of various side groups to 4-thiazolidone core could lead to the induction of different cell death pathways by such compounds [18].However, in that case the chemical structure of substitutes had a little effect on the selectivity of action of these 4-thiazolidones toward leukemia and carcinoma cells.Here, one can see completely different situation, when exchange of a single atom can lead to complete loss of the compound activity towards specific cell line (e.g., leukemia, carcinoma, melanoma).Such data indicate a distinctive mechanism of action of thiazolo [4,5-b]pyridine derivatives compared to the previously studied 4thiazolidones.