Fused Thiopyrano[2,3-d]thiazole Derivatives as Potential Anticancer Agents

rel-(5aR,11bR)-3,5a,6,11b-tetrahydro-2H,5H-chromeno[4′,3′:4,5]thiopyrano[2,3-d][1,3]thiazol-2-ones formed by the stereoselective Knoevenagel-hetero-Diels-Alder reaction were functionalized at the nitrogen in position 3 via reactions of alkylation, cyanoethylation, and acylation. The synthesized compounds were evaluated for their anticancer activity in NCI60 cell lines. Among the tested compounds, 3f was found to be the most active candidate with the greatest influence on leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, prostate cancer, and breast cancer subpanel cell lines with GI50 values over a range of 0.37–0.67 μM.


Introduction
Thiopyrano [2,3-d]thiazole derivatives as well as their synthetic precursors, namely 5-ylidene-4-thiazolidinones [1,2], exhibit various pharmacological activities and may be considered as a possible source of innovative drug-candidates. The earliest reports of thiopyrano [2,3-d]thiazole biological activity is related to the antifungal and antituberculosis activities. It is important to note that these derivatives were highly active towards pathogenic isolates of Trichoderma harzianum, Penicillium simplex, Candida albicans, Mucor hiemalis, Aspergillus oryzae, Actynomices sp., A. fumigatus at the level of fluconazole [3][4][5].
Modern research allowed the identification of antitumor potential of different 4-thiazolidinone derivatives and abovementioned heterocycles [6]. Moreover, some directions of highly active compounds design and optimization have been proposed (figure 1). Approach to the design of target compounds was based on the number of generated hypotheses and facts. The presence of substitutes at the C5 position of basic heterocycle is critical for 4-thiazolidinone derivatives antitumor effect realization and value [1,2,[7][8][9]. Fixation of highly active 5-arylidene-4-thiazolidinone in thiopyranothiazole system usually saves the activity vector and opens up new possibilities of obtained derivatives optimization [10][11][12][13]. Moreover, the introduction of different substitutes in the N3 position is one of the effective methods of target compounds optimization. This approach allowed substantive increasing in the level and/or selectivity of the investigated compounds antitumor effect in comparison with N-unsubstituted analogues [6,8,14].  [10,13] [12] [13] direction of modification
Starting 1a-c contain NH-acidic centers at position N3 of basic heterocycles that from chemical point of view was the rationale for the synthesis of various 3-substituted derivatives. Such synthetic approach is reasonable because of anticancer activity intensification of structurally simpler N-3-substituted 4-azolidinones containing carboxylic acids moieties in 3-d position. On the other hand SAR analysis showed that simultaneous presence of substitutes in the positions C5 and N3 of basic heterocycle is perspective direction of new antitumor agents rational design based on 4-azolidinones and related heterocycles [1,6,8].
Alkylation reactions of 1a-c were carried out through the stage of potassium salts formation that later were used in the reactions with bromoacetophenones, ethylchloroacetate and different acetamides (Scheme 2).
To expand the number of N-substituted chromeno [4',3':4,5]thiopyrano [2,3-d]thiazole-2-one derivatives and obtain new pharmacologically attractive structures 3-(2-carboxyethyl)chromeno [4',3':4,5]thiopyrano [2,3-d]thiazole-2-one 5 was synthesized (scheme 3). We worked out two alternative methods of its synthesis. The first one is based on the alkylation reaction of starting compound with sodium 3-chloropropionate via intermediate potassium salt. The above method has a significant disadvantage. The target product contains impurities of starting substance and requires multistage purification. In technological term the best is the second method we have proposed, based on the cyanoethylation reaction. So, via interaction of the compound 1a with acrylonitrile in the pyridine medium propionitrile 4 was obtained with high yield. Hydrolysis of the latter allowed obtainment of target acid 5 qualitative characteristics of which far outweigh the product obtained in the alkylation reaction. For carboxylic group functionalization corresponding acid chloride was synthesized and used in the reaction of aromatic amines acylation. Sch. 3. Two alternative methods for the synthesis of 5 and its further modification.

Biological activity
The main focus of biological activity studies was the search for compounds with antitumor activity. The newly synthesized compounds were selected by the National Cancer Institute (NCI) within the Developmental Therapeutic Program (www.dtp.nci.nih.gov) for in vitro cell line screening. Anticancer assays were performed according to US NCI protocol, which was described elsewhere [15][16][17][18]. The compounds were first evaluated at one dose primary anticancer assay towards approximately 60 cell lines (concentration 10 −5 M Other compounds tested in one-dose primary assay didn't show any impressive anticancer activity and therefore can't be considered as prospective anticancer agents.
Whereas the GI 50 may be viewed as a growth-inhibitory level of effect, the TGI signifies a "total growth inhibition" or cytostatic level of effect. The LC 50 is the lethal concentration, "net cell killing" or cytotoxity parameter. If tested parameters (logGI 50 , logTGI and logLC 50 ) specified in negative log units are less then < −4.00 these compounds are assigned as active [19][20][21]. Results estimation of the compound 5 showed that the most sensitive tumor cell lines to this compound are leukemia.  Taking into consideration high antitumor potential of the compounds 3a, 3f and 3q and prospects for their enhanced pharmacological studies we have calculated the quantitative indicators of selectivity effects on different types of cancer based on the experimental results of in-depth screening. The selectivity ratios (SR) were calculated by dividing the full panel MID GI 50 and MID TGI (the average sensitivity of all cell lines towards the test agent) by their individual subpanel MID (the average sensitivity of all cell lines of a particular subpanel towards the test agent). Selectivity ratios of 3-6 refer to moderate selectivity; ratios greater than 6 indicate high selectivity towards the corresponding cell line, while compounds not meeting either of these criteria are rated non-selective.
Compound 3a was found to be non-selective (SR were between 0.66-2.06 and 0.84-1.66 at the GI 50 and TGI levels respectively). Two other "hit-compounds" 3f and 3q showed moderate selectivity toward the prostate cancer subpanel with selectivity ratios SR = 3.24 at GI 50 level for 3f and SR = 4.41 at TGI level for 3q.

Tab. 3.
Selectivity ratios for the "hit-compounds" 3a, 3f and 3q COMPARE analysis was performed for compounds 3a, 3b and 3f to investigate the similarity of their cytotoxicity pattern (mean graph fingerprints) with those of known anticancer standard agents, NCI active synthetic compounds and natural extracts, which are present in public available databases (http://dtp.nci.nih.gov/docs/compare/… …compare.html) [22][23][24][25]. Such analysis is based on comparing the patterns of differential growth inhibition for cultured cell lines and can potentially gain insight into the mechanism of the cytotoxic action. If the data pattern correlates well with that of compounds belonging to a standard agent database (Pearson's correlation coefficient (PCC) >0.6), the compound of interest may have the same mechanism of action. On the other hand, if the activity pattern does not correlate with any standard agent, it is possible that the compound has a novel mechanism of action. Standard COMPARE analysis was performed at the GI 50 level.

Tab. 4.
Results of the COMPARE analysis at the GI 50 level. It was established that tested compounds do not have any high correlation levels with the NCI tested drugs or other biological active substances. This may be an argument in favor of the compounds unique mechanism of action that differs from the traditional links of influence on oncogenesis of known anticancer agents. However in our opinion some COMPARE analysis results deserve attention. Thus, there is correlation of the "hitcompounds" anticancer profile with macbecin II (NSC:S330500 Endpt:GI50 ExpId:AVGDATA hiConc: −6.0) which is DNA antimetabolite, which mechanism of action is in part due to heat shock protein Hsp90 protein inhibition [26]. Also one should mention that when comparing the GI 50 values on each cancer cell line for 3f there is some correlation with rhizoxin as inhibitor of tubulin (NSC:S332598 Endpt:GI50 ExpId:AVGDATA hiConc: −5.3) р=0.686. It is interesting that among the possible ways of "hit-compounds" antitumor effect realization COMPARE analysis does not deny the possible induction of apoptosis, which is an argument for the experimental confirmation of this hypothesis considering the anticancer mechanism of action of structurally related 5-(4-methylbenzylidene)-2-phenylamino-1,3-thiazolidin-4-one (MMPT) [27].

Preparation of for potassium salts (2a-c)
To a stirred suspension of 1a-c (0.01 mol) in 30 ml of ethanol potassium hydroxide (0.011 mol) in 15 ml of ethanol was added. Reaction mixture was stirred at room temperature for 1 h. The formed potassium salt was filtered, washed with ethanol, diethyl ether and used in the following transformations without additional purification.

Preparation of compounds 3c,d
To a suspension of 2a (0.01mol) in the ethanol 0.011 mol of p-fluoro-or p-chlorobromoacetophenone was added. The reaction mixtures were refluxed for 3-9 h, the progress of the reactions was monitored by TLC; formed precipitates were collected by filtration and crystallized from appropriate solvents.

Preparation of 3a,b and 3e-t
The mixture of 0.01 mol 2a-c, 0,015 mol of ethylchloroacetate or appropriate chloroacetamide, 5 ml of DMF, 15 ml of ethanol and catalytic amounts of potassium iodide and potassium carbonate was refluxed for 3 h. The product of the reaction was collected by filtration, washed with water and diethyl ether. Esters 3a,b were recrystallized from ethanol. Amides 3e-t were recrystallized from a mixture of DMF-ethanol (1:2)

Preparation of compound 5
The solution of 4 (0.01 mol) in 30 ml of acetic acid and 15 ml of hydrochloric acid was refluxed for 3 h. After cooling the reaction mixture was precipitated with water. After 24 h, the white solid was collected by filtration and treated with toluene, whereupon it was crystallized from ethanol. [4',3':4,5]thiopyrano [2,3-d]- [1,3]

Preparation of Compounds 6a-c
To the solution of 5 (0.01 mol) in anhydrous dioxane thionyl chloride (0.057 mol) was added. Reaction mixture was refluxed for 30 min, after cooling it was precipitated with hexane. Obtained acid chloride was used for further transformations without additional purification. To the solution of acid chloride (0.01 mol) in 10 ml of dioxane a mixture of appropriate aniline (0.01 mol) and triethylamine in 10 ml of dioxane was added. Reaction mixture was heated for 10 min at 100°С and poured into water. Filtered precipitate was crystallized from acetic acid. [4',3':4,5]thiopyrano [2,3-d] [1,3]

Cytotoxic activity against malignant human tumor cells
Primary anticancer in vitro assay was performed at human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [15][16][17][18]. Tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents.
The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10 −4 to 10 −8 M. A 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth. Using the seven absorbance measurements [time zero, (Tz), control growth in the absence of drug, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as: [(Ti − Tz) / (C − Tz)] x 100 for concentrations for which Ti ≥ Tz, [(Ti − Tz) / Tz] x 100 for concentrations for which Ti < Tz. Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(Ti − Tz)/(C − Tz)] x 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC 50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz) / Tz] x 100 = -50. Values were calculated for each of these three parameters if the level of activity is reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. The logGI 50 , logTGI, logLC 50 were then determined, defined as the mean of the logs of the individual GI 50 , TGI, LC 50 values. The lowest values are obtained with the most sensitive cell lines.