Synthesis and anticancer activity evaluation of some new 1,2,3,5-tetrazine derivatives attached to benzothiazole moiety

A series of novel tetrazine derivatives, containing benzothiazole framework, were prepared during the coupling reactions of some diazotized 2-aminobenzo[d]thiazole derivatives with p-acetaminophen. Their structures were elucidated based on NMR and MS spectrometry. The anticancer activity and the safety of the synthesized compounds along with the entire precursors were assessed against three human cancer cell lines and a normal cell line. All the synthesized compounds showed selective cytotoxic activity against the cancer cell lines used in comparison to the normal Vero cell line. Their IC50 values varied from 2.02 to 171.67 µM.


Introduction
Cancer is one of the most important problems in public health and is the second leading cause of death in the world. 1 Currently, cancer chemotherapy is one of the most effective methods to treat cancer, which represents a constant, global, and interdisciplinary research effort and can heavily promote and extend the quality of life. 2 New antitumor therapies point to novel compounds with potently selective effects and must therefore exhibit a cytotoxic effect on malignant cells, without damaging normal ones.7][8] Additionally, synthetic tetrazine derivatives have been used in molecular imaging.Examples included targeted labelling, delivery of radionuclide, and visualization of tumors in lung cells and mice; fluorescent label proteins on the surface and inside mammalian cells, [9][10][11][12] and covalent bonding of tetrazine molecules to the surface of cells to study cell-to-cell interactions 13 .5][16][17][18] The coupling of tetrazine to tetrazine and dienophile has been used for surface structuring and in the fabrication of microarrays. 19,20][23] There is, however, still a continuous research interest in tetrazines due to the reactivity of their unsaturated heterocyclic ring which can serve as electron donor or acceptor depending on the reaction conditions.Using tetrazine as a reactant in the search for new biologically active compounds is therefore of great importance.Thus, this work aimed to synthesize tetrazine type compounds via coupling reactions of diazonium ions of benzothiazole substrates followed by the determination of their anticancer properties in cultured cells.The process led to the synthesis of three unusual tetrazine derivatives and this might open a line of thought on the synthesis of these compounds.

Results and Discussion
Chemistry Two 2-aminobenzothiazole derivatives were diazotized with nitrosylsulfuric acid at low temperature (0 -5 °C).Coupling reaction was carried out by adding the diazonium solution of 1 to a solution of p-acetaminophen 3 in DMSO (Scheme 1).The chemical structures of 4 were confirmed by the available spectroscopic and elemental analysis data.

Scheme 1. Reactions' sequences to compounds 4.
The reaction of the diazonium salt solution 2a with 3 gave the two coupling products 4a and 4a' (Scheme 1).Compound 4a was obtained as a red powder with a sharp melting point at 119 -121 °C.The elemental analysis and the High Resolution Electron Impact Mass Spectroscopy-experiments were used to establish the gross formula as C17H22N4O5S showing that the coupling product crystallized with three molecules of H2O.The HRMS showed the molecular ion peak at m/z 395 corresponding to (M +. + H).Characteristic ion fragments were rationalized from the spectrum at m/z = 338 (M +. + H -N2 -2CH3), 332 (M +. + H -H2O -3CH3), 310 (M + -3H2O -2CH3), 284 (M +. + H -NCOCH3-3H2O).These results were confirmed by the IR-experiment which exhibited a large band in the higher frequency region around 3248 cm -1 due to the combined stretching frequencies of the phenolic-OH and the hydroxyl groups of H2O.The stretching frequencies of the aromatic C-H appeared at 2922 cm -1 , whereas the combined stretching frequencies of the carbonyl groups could be observed as an intense band around 1659 cm -1 .The characteristic stretching frequencies of the diazo bridges were exhibited between 1450 and 1483 cm -1 .On the 1 H-NMR spectrum, the benzothiazole protons H-4 and H-7 resonated at δH = 7.89 and 7.84 ppm as a singlet, while the singlets observed at δH = 10.63 and 9.94 ppm were respectively assigned to the OH-group and NH proton of the acetaminophen.Dye 4a showed a singlet peak at 2.36 ppm for methyl groups fixed on benzothiazole ring and a peak at 2.01 ppm for COCH3 protons on the acetaminophen ring.Besides, one poorly resolved "dd"-signals [centered at 7.60 ppm (H-6', J = 2.00 and 8.00 Hz) and a set of two couples of poorly resolved "d"-signals [centered at 8.10 ppm (H-2', J = 2.00 Hz) and 7.06 ppm (H-5', J = 8.00 Hz) overlapping in the range 7.06 -8.10 ppm, were attributed to the ABX proton systems in the acetaminophen unit of the coupling product.
The 13 C-NMR-spectrum exhibited 17 relevant signals, out of which eight could be assigned to the methyl carbon and the aromatic tertiary C-H carbons, and nine to the quaternary carbon atoms.The assignment of 1 H-and 13 C ( 1 H)-NMR data for compound 4a was done by comparison with simulated values (table 1).
In the coupling reaction of 2a with 3, it was observed that the anticipated compound 4a is first formed and a part of it subsequently undergoes in situ nucleophilic addition with another part of unreacted 2a to yield tetrazine 4a' (Scheme 2).A plausible mechanism for the reaction may consist in the first step of the nucleophilic attack of the diazo function of diazonium salt 2a by the heteroaromatic nitrogen atom of the azobenzothiazole 4a as shown in Scheme 2. In the second step, the heteroaromatic N atom of the newly introduced benzothiazole fragment operates an intramolecular nucleophilic attack on the highly electron deficient sp 2 C of the other thiazole ring to form the unstable fused five ring intermediate system incorporating the tetrazine unit.The latter subsequently rearranges with proton abstraction from the hydrogen sulfate ion (HSO4 -), resulting into the opening of the thiazole ring of the first benzothiazole moiety with the formation of the thiol function.This hypothesis is in agreement with recent reports 24,25 mentioning the high reactivity of the heteroaromatic N atom of benzothiazole under similar reactions conditions.Scheme 2. The plausible mechanism for the synthesis of 4a'.
The structure of compound 4a' has been elucidated by various spectroscopic techniques such as 1 H-NMR and 13 C-NMR spectra and elemental analysis.

Scheme 3. HRMS fragmentation patterns of compound 4a'.
These results were also confirmed by the IR-experiment which exhibited a large band in the higher frequency region around 3887-3284 cm -1 due to the combined stretching frequencies of the phenolic-OH and the hydroxyl groups of H2O.The stretching frequencies of the aromatic C-H appeared at 2920 cm -1 while those of S-H appeared at 2324 cm -1 .Two additional bands due to the tetrazine group 26 appear at 1370 and 889 cm -1 .
The structure of synthetic tetrazine was determined based on the 1 H and 13 C NMR chemical shifts and on the proton-proton coupling constants.On the 1 H-NMR spectrum, the protons of the methyl on benzothiazole ring resonated at δH = 2.50, 2.39, 2.37, and 1.23 ppm respectively as singlets, while the singlet observed at δH = 2.04 ppm was assigned to the methylketone protons. 27In the phenolic moiety, we have an ABX Proton system at 8.13 (d, 1H, J = 2.8 Hz, H-6'), 7.63 (dd, 1H, J = 8.8 and 2.8 Hz, H-4') and 7.09 (d, 1H, J = 8.8 Hz, H-3') confirming the proposed regio-orientation of the electrophilic substitution of the benzothiazole diazonium ion at the ortho-position to the OH-group in the acetaminophen reagent.
The 13 C-NMR spectrum of the tetrazine shows 24 signals attributable to 26 carbon atoms.In addition, the characteristic signals of five methyl groups (CH3) appearing at δ = 23.82ppm, 23.78 ppm, 19.86 ppm, 19.67 ppm and 19.61 ppm, are in agreement with the presence of two benzothiazoles units in the assigned structure of 4a'.A remarkable feature in the 13 C-NMR spectrum of this compound is the intense downfield shifts (Δδ = 30.5 ppm) of the δC of the carbon 4 of the tetrazine rings add to diazo function respectively from 168.14 ppm in the benzothiazole substrate 27 4a to 198.6 ppm in the tetrazine product.
The UV spectrum of the tetrazine exhibits absorptions between 270 and 640 nm, in the visible range.Table 2 below recapitulates some characteristics wavelengths maxima and the corresponding molar extinction coefficients.A comparison of the UV spectrum of the tetrazine with that of the starting azo dye allows us to observe a weak bathochromic effect and the appearance of new bands on the spectrum of the tetrazine, in agreement with the extension of the chromophore system resulting from the condensation reaction.The above assumption that the involvement of the heteroaromatic N of the benzothiazole in the interand intramolecular nucleophilic attack respectively in the first and second step of the reaction's mechanism of scheme 2 is due to the lack of suitable electrophilic positions on the aromatic ring of the benzothiazole used as coupler, prompted us to investigate the behavior of the diazonium salt 2b in the presence of 1b (Scheme 4).As anticipated, tetrazine 4c and the coupling product of two equivalents of 2b molecules 4c' formed as previously described 27 were isolated from this reaction.The formation of compound 4c certainly results from the subsequent nucleophilic addition of the heteroaromatic nitrogen atom of the benzothiazole ring (Scheme 5).

Scheme 5. The plausible mechanism for the formation of compound 4c.
Based on the structure of 1,2,3,5-tetrazine, this molecule not only has π* orbitals, but also nonbonding n orbitals.So, these orbitals contribute to two kinds of transition: n→π* transition and π→π* transition.The absorption spectra of tetrazine 4c, shows two main absorption bands: one around 260 nm corresponding to the π→π* transition analogue to benzene and a second is around 450 nm due to the n→π* transition.According to Mason, the broad absorption band appearing around 352 nm in solutions is due to an additional n→π* transition. 28Similar observations were reported earlier. 29ompound 4c was obtained as an orange powder with a melting point in the range of 245 -247 °C.The elemental analysis and the electrospray mass spectra-experiments were used to establish the gross formula as C14H18N6O14S3 showing that the coupling product crystallized with one sulfate ion, and six molecules of H2O.The electrospray mass spectrum showed the molecular ion peak at m/z 590 corresponding to [M] +. .Surface ligand exchange behaviours of compounds 4c by primary alcohols using electrospray ionization mass spectrometry (ESI-MS) were clearly established. 27Methanol was used as a model primary alcohol for the analysis.As clearly proven by peaks at m/z = 568, the surface of tetrazine 4c is partly exchanged by methanol within 1 min to give peaks (M +. -3H2O + MeOH).The ion-fragments at m/z = 513 (M +. -3H2O + MeOH -2H2O), 450 (M +. -3H2O + MeOH -H2 -H2SO4), 378 (M +. -3H2O + MeOH -C6), 294 (M +. -3H2O + MeOH -C4H2 -2HO -) and 247 (M +. -3H2O + MeOH -NO2), were assigned as in scheme 6, confirming the above hypothesis (Scheme 6).

Scheme 6. HRMS fragmentation patterns of compound 4c highlighting surface ligand exchange behaviours by MeOH.
The FTIR spectrum of compound 4c shows typical absorption bands due to aromatic C-H-stretching vibrations at 3097 cm -1 , aromatic C=C-stretching and ring deformation vibrations at 1514 and 718 cm -1 respectively.Two additional bands due to the tetrazine moiety appear at 1336 and 908 cm -1 . 29he  4 Hz, H-7'), attributable to two ABX systems of nitrobenzothiazole, confirming the proposed regio-orientation of the nucleophilic addition of the heteroaromatic N of the benzothiazole 1b with diazonium ion 2b instead of electrophilic substitution to yields compounds 4C and/or 4C' (figure 1) as anticipated.The 13 C-NMR spectrum of compound 4c exhibited 13 carbon signals instead of 14 as required by the molecular formula.This could be explained by the overlapping of the signals of carbon C-5 and C-5' at δC 122.5 ppm due to their magnetic and chemical equivalence.The analysis made on the HSQC spectrum of compound 4c shows six correlation spots which confirm the presence of six methines (=C-H) in its structure.This spectrum also allows us to clearly establish that, although carbons C-5 and C-5' have the same chemical shift, they correlate with two distinct protons at δH 8.25 and δH 8.11 ppm respectively, proving that the signal at δC 122.5 ppm corresponds to two carbons.
The structure of compounds 4b and 4c' were assigned based on their analytical and spectral data following similar reasonings as above.

Anticancer activity
The anticancer activity and the safety of the synthesized compounds along with the entire precursors were assessed against three human cancer cell lines (A549, Hela and MCF-7) and a normal cell line (Vero cells).Doxorubicin was used as a reference control drug.Their median inhibitory concentrations (IC50) are presented in Table 3. Benzothiazole possesses wide spectrum biological activities including anti-inflammatory, antibacterial, antioxidant, antiviral, anti-tumour, anticancer and anti-proliferative, among others. 31In addition, tetrazine and benzothiazole derivatives have been an attractive subject due to their potential anticancer activity in medicinal chemistry. 32Therefore, all the synthesized compounds were tested for their cancer cell proliferation inhibiting activity.Results showed selective cytotoxicity against the cancer cell lines tested compared to the normal Vero cell line.Their IC50 values varied from 2.02 µM to 171.67 µM.Compared to their precursors, all the synthesized compounds had increased inhibition of the three human cancer cells.Interestingly, none of the tested compounds had any significant cytotoxicity against non-cancerous Vero cells.Among the tested compounds, compound 4b, a tetrazine derivative, had the most potent inhibitory activity on the three cancer cell lines (A549, Hela and MCF-7) with IC50 values of 2.02 µM, 2.059 µM, and 13.65 µM, respectively.The activity of compound 4b against A549 and Hela cells was close to that of the standard cytotoxic anticancer drug doxorubicin (IC50 values of 0.88 µM and 0.33 µM respectively).According to recommended in vitro cytotoxic activity cutoff values a compound is considered to be cytotoxic if the IC50 < 10 μM. 33Based on this recommended value, the cytotoxicity of compounds 4a, 4a' and 4b can be considered significant.This finding is in agreement with relevant literature since several compounds containing the 1,2,4,5-tetrazine skeleton have been synthesized and evaluated for their antitumor activities and these compounds have been shown to exhibit potent antiproliferative activities against a panel of cancer cells including MCF-7. 34,35Although the two 2-aminobenzothiazole compounds 1a and 1b used as starting materials in this study showed moderate anticancer activity, several reports have documented benzothiazole derivatives as anticancer agents. 31pecific killing of cancer cells without affecting healthy cells is a key safety feature of any cancer chemotherapy; therefore, the selectivity index for cancer cell lines A549, Hela and MCF-7 was calculated from the ratio of their respective IC50 values and that of Vero normal cells (Table 3).The SI values of the tested compounds ranged from 0.89 to 7.15.As the SI value highlights the differential activity of a compound, the greater the SI value the more selective is the compound.The SI significance of compounds has been classified as follow: SI ≤ 1 = no selectivity, 1 < SI < 5 = moderate selectivity, SI ≥ 5 = strong selectivity. 30Considering these criteria, it can be deduced that compounds with good anticancer activity also displayed strong selectivity index values in terms of activity against Vero cells.SI values less than 1 indicate the general toxicity of the pure compound.

Conclusions
Herein, a straightforward synthetic approach for the preparation of 1,2,3,5-tetrazine with benzothiazole moieties as pendant groups was introduced.The new 1,2,3,5-tetrazines containing benzothiazole backbones were successfully synthesized at a temperature of 0-5 °C and fully characterized by available elemental and spectroscopic data.Compound 4b exhibited potent activity against A459 and Hela cells, with strong selectivity calculated in terms of activity against non-cancerous Vero cells.These findings could be helpful for the design and synthesis of lead compounds with the structural scaffold of 1,2,3,5-tetrazine for the development of new anticancer therapeutic agents.Future studies will involve the determination of the mechanism of anticancer activity of these compounds.

Experimental Section
Chemistry General.Melting points were determined on a Buchii melting point apparatus and are uncorrected.The Thin Layer Chromatography (T.L.Cs) was carried out on Eastman Chromatogram Silica Gel Sheets (13181; 6060) with fluorescent indicators.A mixture of hexane and ethyl acetate (4:6) was used as the eluent and iodine was used for the visualization of the chromatograms.The IR spectra were measured with a Fourier Transform Infrared spectrometer JASCO FT/IR-4100 and a Perkin Elmer FT-IR 2000 spectrometer.The UV spectra were recorded with a Beckman U-640 Spectrophotometer, using samples' solutions of concentration 5×10 −5 mol.L −1 .Combustion analyses were carried out with a Euro EA CHNSO analyser from Hekatech company, and the results were found to be in good agreement (±0.3%) with the calculated values.Positive ion electrospray mass spectra were recorded on a Waters Xevo TQD tandem quadrupole mass spectrometry system running in MS scan mode, 1 minute of acquired spectra were combined and centroided. 1 H-NMR spectra were recorded in DMSO-d6 with a 400 MHz spectrometer NMR Bruker Advance 400. 13C-NMR spectra were recorded in DMSO-d6 with a 100 MHz spectrometer NMR Bruker Advance 400.Tetramethylsilane (TMS) was used as the internal reference.Simulated 1 H-and 13 C( 1 H)-NMR-spectra were performed using http://www.nmrdb.org/spectral simulation software.
Preparation of the reagents and starting materials.All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without further purification.Preparation of diazonium salt solution.In a similar manner as earlier described 27 , dried sodium nitrite (0.69 g, 10 mmol) was slowly added over a period of 30 minutes to concentrated sulphuric acid (10 mL) with occasional stirring.The solution was cooled to 0-5 °C.Compound 1 was dissolved in DMSO (10 mL) and cooled to 0-5 °C.The nitrosyl sulphuric acid solution was added to the solution of 1 and the temperature was maintained between 0-5 °C.The clear diazonium salt solution thus obtained consisting of the in situ-formed intermediate 2, was used immediately in the coupling reactions.General procedure for the preparation of the coupling products 4. Acetaminophen (3) (1.51 g, 10 mmol) or 2amino-6-nitrobenzothiazole (1b) (1.952 g, 10 mmol) was dissolved in DMSO (10 mL) and then cooled in an icebath at 0-5 °C.The previously prepared diazonium solution of 2 was added drop wise over 1 hour, and then 15 mL of sodium acetate solution (10%) was added to the mixture.The pH of the mixtures was in the range 9-11.The solid precipitate was collected on a filter and crystallised from methanol to give the title compound.

Table 1 .
Comparison of 1 H-and 13 C ( 1 H)-NMR data of 4a with the simulated values

Table 3 .
Cytotoxicity of synthesized compounds and reference anticancer drugs