Synthesis and Biological Activity of New Thiopyrano[2,3-d]thiazoles Containing a Naphthoquinone Moiety

Novel 11-substituted 3,11-dihydro-2H-benzo[6,7]thiochromeno[2,3-d][1,3]-thiazole-2,5,10-triones 4a–i were synthesized in 75–90% yields via the hetero-Diels-Alder reaction of 5-arylidene-4-thioxo-2-thiazolidinones with 1,4-naphthoquinone. The synthesized compounds were evaluated for their antineoplastic and antimycobacterial activities. A moderate selectivity against melanoma cancer cells (GI50 (UACC-257-melanoma) = 0.22 μM) was demonstrated for 4i, whereas derivatives 4a, 4c, 4g, and 4h showed promising antimycobacterial activity at a low toxicity level.


Introduction
Drug resistance remains an important problem in the pharmacotherapy of cancer [1] with many medicinal chemists being involved in the search for new effective antitumor agents. The anticancer activity was shown in our earlier studies [2,3] for norbornane-containing fused thiopyrano [2,3-d]thiazoles. Subsequently, we have decided to modify the structure of the latter compounds towards their planarization using the naphthoquinone scaffold (Sch. 1). The naphthoquinone fragment can be found both in the well-known anticancer drugs, such as doxorubicin, daunorubicin, mitoxantrone, and mitomycine C [4][5][6], and in the new promising antimycobacterial agents [7]. This article presents our findings on the anticancer and antimycobacterial activities of the synthesized compounds. Optimization of anti-cancer activity Sch. 1.
The synthesized novel thiopyrano [2,3-d]thiazoles 4a-i were characterized by 1 Н and 13 С NMR, LC-MS spectra, and elemental analyses (see Experimental section). Spontaneous in situ dehydrogenation was confirmed by the 1 H NMR spectra containing a singlet peak of the 11H-proton. The latter was highly displaced in the weak magnetic field (5.40-5.75 ppm) because of the neighboring carbonyl group. This signal shift can be increased even more with an ortho-OH-substituted aryl substituent (compound 4a), and is affected most probably by the intra-molecular hydrogen bonding. The signals of naphthoquinone moiety protons and aryl substituents in position 11 were within 6.63-8.09 ppm.
Compounds 4f and 4i were tested initially at a single concentration of 10 -5 M against a full panel of 60 cancer cell lines derived from leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer (Tab. 1).
Compounds 4f and 4i showed a considerable level of activity in the primary test and were chosen for advanced assays against the full panel (approx. 60 cell lines) at five 10-fold dilutions (100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM). Compound 4e was tested in the latter assays without primary pre-screening.
The full panel of individual GI 50 values (µM) for each cell line is presented in Table 2 and  the results of five concentrations' screenings are summarized in Table 3. Selectivity analysis highlighted melanoma cell lines as the most sensitive targets for compounds 4f and 4i (GI 50 (µM) = 1.26; 0.22 respectively). Compound 4e possessed a considerable activity level, however, the distinctive selectivity of cytotoxicity towards cancer cell lines was not observed. The most potent compound 4i showed high cytotoxic activity (GI 50 < 1µM) against the following cancer cell lines:   [16].
The COMPARE analysis evaluates the similarity of the compounds' cytotoxicity patterns with those of known anticancer standard agents and NCI synthetic compounds present in public databases [17][18][19]. The COMPARE analysis revealed moderate correlations at the GI 50 level of the 4f pattern with panсratistatin (Pearson correlation coefficient, PCC = 0.603), didemnin B (PCC = 0.525), S-trityl-L-cysteine (PCC = 0.473), and the compound 4i pattern with trimethyltrimethylolmelamine (PCC = 0.473). The highest obtained correlation indicated certain similarity of 4f with the pro-apoptotic product, panсratistatin, which selectively influenced cancer cells. This substance is a natural compound initially extracted from Spider Lily. According to the literature data [20], the anticancer activity of pancratistatin is realized via FAS (fatty acid synthase) receptor inhibition, which launches caspase-3-mediated apoptosis. Based on the COMPARE analysis data, one could suggest that 4f could have a similar mechanism of anticancer activity to that of pancratistatin. Additionally, it was observed that 4-OH group alkylation of 11-aryl fragment leads to the decrease in antineoplastic activity. This point can be explained by the formation of a hydrogen bond (HB) between the 4-OH group of compound 4i and some hydrogen acceptor of the biotarget. This HB could cause a 10-fold increase in GI 50 in comparison with 4e and 4f.

Tab. 3.
Summary of the dose-dependent assay on 60 cancer cell lines

Evaluation of antimycobacterial activity
Synthesized compounds 4a-i were evaluated in vitro for antimycobacterial activity in collaboration with the Tuberculosis Antimicrobial Acquisition and Coordinating Facility [21] using the BACTEC 460 radiometric system at a concentration 6.25 µg/mL [22][23][24]. The assays [25] were performed on the Mycobacterium tuberculosis H 37 R v strain (АТСС 27294) with the determination of inhibition percentage and evaluation of MICs (Tab. 4).

Tab. 4.
Pre-screening of the antimycobacterial activity and acute in vivo toxicity in mice. According to the data, compounds 4a, 4g, and 4h showed up with low MIC 90 values between 0.6 and 2.7 µg/mL. However, the low ratio between their MIC 90 and cytotoxicity (IC 50 ) indicates a possible association of antimycobacterial activity with the toxicity on the mammal cells. That is why further evaluation of the acute toxicity of in vivo investigated compounds was essential to clarify their toxicological profiles.

Evaluation of acute toxicity in vivo
The synthesized compounds were evaluated for their approximate LD 50 in male mice [27,28]. The mice were kept under a constant temperature and humidity in sterile cages with water and food. The stock solutions of the compounds used in this study were prepared immediately before usage and injected intraperitoneally (ip). The LD 50 values (Tab. 3) were calculated using the method described by Litchfield and Wilcoxon [27]. The results (Tab. 3) indicated that most of the tested compounds were non-toxic and welltolerated by the experimental animals as demonstrated by their LD 50 values (>500 mg/kg).

Biological screening
In Human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For the typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 µL of media. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population at the time of drug addition. Experimental drugs were solubilized in dimethyl sulfoxide at a 400-fold final test concentration and stored frozen. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to two-fold of the desired test concentration with complete medium supplemented with 50 µg/ml gentamicin. Additional four, 10-fold, or ½ log serial dilutions were made to provide a total of five drug concentrations plus the control. Three dose-response parameters were calculated for each experimental agent. GI 50 represents the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. TGI represents the drug concentration resulting in total growth inhibition. The LC 50, the concentration of the drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning, indicates a net loss of cells following treatment.

Antimycobacterial screening
In vitro evaluation of antimycobacterial activity against Mycobacterium tuberculosis H37Rv.
The primary screen was conducted at 6.25 µg/mL (or molar equivalent of the highest molecular weight compound in a series of congeners) against M. tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA) [21]. Compounds exhibiting fluorescence were tested in the BACTEC 460-radiometric system [23]. Compounds effecting <90% inhibition in the primary screen (MIC > 6.25 µg/mL) were not evaluated further.
BACTEC radiometric method of suspectibility testing.
The inocula for suspectibility testing were either from a positive BACTEC isolation vial with a growth index (GI) of 500 and more or from the suspension of organisms isolated earlier on the conventional medium. The culture was well-mixed and 0.1 mL positive BACTEC culture was added to each of the vials containing the test drugs. The drug vials were supplemented by rifampicin (0.25 µg/mL). A control vial was inoculated with a 1:100 microdilution of the culture. A suspension equivalent to the McFarland no.1 standard was prepared in the same manner as a BACTEC positive vial when growth from a solid medium was used. Each vial was tested immediately with BACTEC to provide CO 2 in the headspace. The vials were incubated at 37 ºC and tested daily with a BACTEC instrument. When the GI in the control read was at least 30, the increase in GI (ΔGI) from the previous day in the control was compared with that in the drug vial. The following formula was used to interpret the results: ΔGI control > ΔGI drug = susceptible ΔGI control < ΔGI drug = resistant If a clear suspectibility pattern (the difference in ΔGI of the control and the drug) was not seen at the time the control ΔGI was 30, the vials were read for one or two additional days to establish a definite pattern of ΔGI differences.

Median lethal dose (LD 50 ) evaluation.
The median lethal dose (LD 50 ), the dose of the selected compounds that causes 50% mortality in mice, was determined from dose-response curves with at least four doses by the method of Litchfield and Wilcoxon [27].