The first Q-Tube based high-pressure synthesis of anti-cancer active thiazolo[4,5-c]pyridazines via the [4 + 2] cyclocondensation of 3-oxo-2-arylhydrazonopropanals with 4-thiazolidinones

A novel and efficient protocol for the synthesis of thiazolo[4,5-c]pyridazine derivatives was developed. The approach utilizes a high pressure Q-Tube reactor to promote cyclocondensation reactions between 3-oxo-2-arylhydrazonopropanals and 4-thiazolidinones. The process has a significantly high atom economy and a broad substrate scope, as well as being applicable to gram scale syntheses. The in vitro cytotoxic activities of the synthesized thiazolo[4,5-c]pyridazine derivatives were examined utilizing a MTT colorimetric assay with doxorubicin as a reference anti-cancer drug and three human cancer cell lines including HCT-116 (colon), MCF-7 (breast) and A549 (lung). The results show that thiazolopyridazines 7c, h, k and p have high cytotoxic activity against the MCF-7 cell line with respective IC50 values of 14.34, 10.39, 15.43 and 13.60 μM. Moreover, the thiazolopyridazine derivative 7s also show promising cytotoxic activity against the HCT-116 cell line with IC50 = 6.90 μM . Observations made in this effort serve as a basis for further investigations into the design and preparation of new anti-cancer drugs.


Results and Discussion
The initial phase of the current investigation, which was aimed at developing a new method for synthesis of thiazolo [4,5-c]pyridazine derivatives utilizing cyclocondensation reactions between 4-thiazolidinones and 3-oxo-2-arylhydrazonopropanals, concentrated on the preparation of 4-thiazolidinones. We observed that by using a slight modification of a two-step procedure described previously 29 , aromatic or heteroaromatic amines  Table 1. Optimization of reaction of 4-thiazolidinone 4a and arylhydrazonal 5a. Reaction conditions: a 4-thiazolidinone 4a (5 mmol), arylhydrazonal 5a (5 mmol) and additive (10 mmol) in solvent (10 mL), b 4-thiazolidinone 4a (2 mmol), arylhydrazonal 5a (2 mmol) and additive (4 mmol) in solvent (5 mL).  www.nature.com/scientificreports www.nature.com/scientificreports/ ammonium acetate (4 mmol) in acetic acid (4 mL) in a 10 mL MW tube which was irradiated at 125 °C (250 watt, 25 min). This process produces 7a in 65% yield (Table 1, entry 6). On the other hand, a larger scale reaction of [4a (5 mmol), 5a (5 mmol) and ammonium acetate (10 mmol) in acetic acid (10 mL)] conducted in a 35 mL borosilicate glass tube in a Q-tube reactor at 150 °C for 30 min forms 7a in a much higher 81% yield (Table 1, entry 7). The noticeable enhancement in the yield of this reaction when a Q-tube reactor is employed may in part be a consequence of the fact that a temperature higher than the boiling points of the solvent and reagents is used and this leads to increase the reaction rate 30,31 . In addition, the increased pressure achievable using a Q-tube reactor reduces the volume of the reaction, thus increasing the effective concentrations of the reactants and their collision frequency, so the competing degradations of the reactants is minimized, allowing more clean reaction pattern. After clearly demonstrating the effect of using the Q-tube reactor on the efficiency of the thiazolo [4,5-c] pyridazine forming reaction (Table 1, entry 7), we assessed the effects of additives and temperature on the process. The results showed that the use of anhydrous sodium acetate rather than ammonium acetate and acetic acid as solvent is optimal (Table 1, entry 9). Also, we observed that temperature plays a role in determining the efficiency of this reaction. Accordingly, reactions performed at 150, 160 and 170 °C lead to formation of 7a in 86, 92 and 98% respective yields, (Table 1, entries 9-11).
The substrate scope of the novel cyclocondensation reaction, conducted using the conditions described in entry 11 of Table 1, was explored next. An assortment of 4-thiazolidinones 4a-c, prepared by using the same sequence illustrated in Scheme 2, and several 3-oxo-2-arylhydrazonopropanals 5, containing electron-withdrawing or -donating groups on both aryl substituents were used for this purpose. The results depicted in Table 2 show that reactions between 4a−c and 5 produce the corresponding thiazolo [4,5-c]pyridazine derivatives 7a−u in high yields and, consequently, that the existence of either electron-donating or electron-withdrawing substituents on both aryl moieties in the arylhydrazonal substrate has no effect on the efficiency of the new cyclocondensation process. The thiazolo [4,5-c]pyridazine 7 m (Fig. 1), generated in this exploratory effort was used to carry out X-ray crystallographic analysis to prove the assigned structures to the products and show that only the (Z)-isomer of 6-(benzothiazol-2-ylimino)-1,6-dihydrothiazolo [4,5-c]pyridazine 7 is formed in the reactions.
In vitro anti-cancer screening. Owing to the general propensity of thiazolopyridazine derivatives to display interesting properties, we have embrked on a program to assess potential biological activities of the substances synthesized using the process described above. In the preliminary phase of this effort, we evaluated the cytotoxic activities of 7a-u towards three human cancer cell lines including HCT-116 (colon cancer), MF-7 (breast cancer) and A549 (lung cancer), in addition to the normal human cell line MCF10A (breast cell line). This assessment was performed using the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay 32,33 , and utilizes doxorubicin as a reference anti-cancer agent [34][35][36][37][38] . For this purpose, three independent determinations were made using treatments with three concentrations (12.5, 25 and 50 μM) of 7a-u for a 48 h incubation period. Analysis of the results of the MTT assays gave thew IC 50 values for 7a-u that are listed in Table 3 and Fig. 2. Inspection of the data shows that in comparison with doxorubicin the thiazolopyridazine derivatives have good to excellent cytotoxic activities against the tested cancer cell lines with IC 50 in the range of 6.90-51.46 μM (vs. 11.26-23.47 μM for doxorubicin). While all of the tested substances display IC 50 values in the ten micromolar range, several trends in their activities are worthy of brief mention. Firstly with respect to the MCF-7 cell line, 7c, 7 h, 7k and 7p show the highest cytotoxic activities with IC 50 values 14.34, 10.39, 15.43 and 13.60 μM, respectively (vs. doxorubicin 19.35 μM), while compounds 7b, 7e, 7j, 7 l and 7n exhibits comparable IC 50 to the reference drug. In addition, the results show that 7a-p, which contain benzothiazole moieties, have higher cytotoxicities than do 7q-s and 7t,u, which contain benzenesulfonamide and [5-(1-methyl-1H-indole-3-carbonyl)-2-phenyl-2H- [1,2,3] triazol-4-ylimino] moieties, respectively. Substituents on the 1-N-aryl group play a significant rule in governing      www.nature.com/scientificreports www.nature.com/scientificreports/ cytotoxic activity, as highlighted by the fact that the presence of electron-withdrawing substituents enhance activity (F > NO 2 > Cl) while electron-donating substituents on the N-aryl moiety lower activity (H > Me > OMe). Moreover, 7s which possesses a benzenesulfonamide moiety and NO 2 (electron-withdrawing) substituent on the 1-N-aryl group displays higher cytotoxic activity (IC 50 = 6.90 μM) than doxorubicin (IC 50 = 11.26 μM), against the HCT-116 cell line. Also, 7q, 7r and 7u are potent against the HCT-116 cell line. Furthermore, the respective IC 50 values of 7f-h against the A549 lung cancer cell line are 25.73, 24.13 and 22.96 μM respectively, which reflects the combined importance of the benzothiazole moiety and two electron-withdrawing substituents on the N-aryl moiety, along with the two fluorine substituents which show better cytotoxicity. In order to decide whether the cytotoxic characteristics of the synthesized compounds in this investigation, has no fatal cytotoxic effect on the normal human cells, and mostly selective to the cancer cells, the compounds showed the highest cytotoxic profile against the used cancer cell line like 7c, 7 h, 7k, 7p and 7s, are screened and evaluated utilizing the same procedure against the MCF-10A cell line (normal breast cell line). The obtained IC 50 values for this non-malicious cell (MCF-10A), gave promising data since their values [7c (32.98 ± 1.11 μM), 7h (38.69 ± 1.85 μM), 7k (32.15 ± 1.47 μM), 7p (35.64 ± 0.87 μM), 7s (26.88 ± 1.59 μM)] were comparable or higher than IC 50 (28.74 ± 1.38) of doxorubicin when it was applied to the same normal cell line.

Conclusion
In summary, the study described above led to the development of an efficient Q-tube reactor based high-pressure protocol for synthesizing unprecedented series of thiazolo [4,5-c]pyridazines via [4 + 2] cyclocondensation reactions between 3-oxo-2-arylhydrazonopropanals and 4-thiazolidinones. The process has a high functional group tolerance and atom economy, and it is performed using simple, safe and environmentally compatible conditions. The synthesized thiazolopyridazines were shown to possess a potent cytotoxicities against MCF-7 (breast), HCT-116 (colon), and A549 (lung) cancer cell lines. The next target of this study in the future, after obtaining these promising primary anticancer activity results, is to conduct more comprehensive studies to determine how the newly prepared thiazolopyridazine derivatives work to promote cell death (the mode of action) and to optimize biological activities.

Experimental
General. Melting points were recorded on a Griffin melting point apparatus and are uncorrected. IR spectra were recorded using KBr disks and a Jasco FT-IR-6300 spectrophotometer. 1 H NMR (400 MHz) or (600 MHz) and 13 C{ 1 H} NMR (100 MHz) or (150 MHz) spectra were recorded at 25 °C using DMSO-d 6 or (TFA-d) as solvents with TMS as an internal standard on a Bruker DPX 400 or 600 super-conducting NMR spectrometer. Chemical shifts (δ) are reported in ppm. Low-resolution electron impact mass spectra [MS (EI)] and high-resolution electron impact mass spectra [HRMS (EI)] were performed using a high resolution GC-MS (DFS) thermo spectrometer at 70.1 eV and a magnetic sector mass analyzer. Following the courses of reactions and checking homogeneity of products were performed using thin layer chromatography (TLC). The Q-Tube assisted reactions were performed in a Q-tube pressure monitor safe reactor from Q Labtech (distributed by Sigma-Aldrich), equipped with stainless steel adapter attached with pressure gauge (300 psi), high pressure adapter (180 psi), a needle adapter, a borosilicate glass pressure tube (35 mL), Teflon sleeve, a PTFE faced silicon septa and a catch bottle. The X-ray crystallographic data were collected by using a Bruker X8 Prospector at room temperature by using Cu-Kα radiation. The structures were solved by using direct methods and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. The structures were solved and refined using the Bruker SHELXTL Software Package (Structure solution program-SHELXS-97 and Refinement program-SHELXL-97) 39 . Data were corrected for the absorption effects using the multi-scan method (SADABS). The crystal image was created by the software Mercury (version 4.3.1) 40 . Compounds 3a-c and 4a-c were prepared according to literature procedures with slight modification in case of 4a-c 29 . The three human cancer cell lines including HCT-116 (colon cancer), MCF-7 (breast cancer) and A549 (lung cancer) were obtained from the American Type Culture Collection (ATCC).

General procedure for the preparation of 2-Chloro-N-(heteroaryl)acetamides 3a-c. Solutions
of amines 1a-c (10 mmol) and chloroacetyl chloride 2 (1.12 g, 10 mmol) in chloroform (for 3a,b) or dioxane (for 3c) (50 mL) containing K 2 CO 3 (15 mmol) were stirred at reflux for 10 h. The solutions were concentrated in vacuo giving residues that were diluted with water (100 mL) and filtered. The solid product is then washed with 5% NaHCO 3 and subsequently with water, dried and crystallized from appropriate solvent to furnish pure 3a-c. 29  www.nature.com/scientificreports www.nature.com/scientificreports/ 2-Chloro-N- [5-(1-methyl-1H-indole-3-carbonyl)-2-phenyl-2H- [1,2,3] General Procedure for the Synthesis of 2-(Arylimino)thiazolidin-4-ones 4a-c. Borosilicate glass pressure tubes (35 mL) of the Labtech Q-tube were charged with 2-chloro-N-(heteroaryl or aryl)acetamides 3a-c (10 mmol) and ammonium thiocyanate (15 mmol) in absolute ethanol (15 mL) (for 4a,b) or dioxane (15 mL) (for 4c). A PTFE faced silicon septa was placed on the top of the tubes and the appropriate cap andpressure adapter were used. The mixtures were heated in an oil bath at 130 °C for 30 min. The course of each reaction was monitored by using TLC and GC/MS. The reaction mixtures were cooled to room temperature, and the formed solids were separated by filtration, washed with water and then recrystallized from the appropriate solvent.

General procedure for the preparation of thiazolo[4,5-c]pyridazine derivatives 7a-u. Borosilicate
glass pressure tubes (35 mL) of the Labtech Q-tube were charged with 4-thiazolidinone 4a-c (5 mmol), arylhydrazonal 5 (5 mmol), anhydrous sodium acetate (10 mmol) and glacial acetic acid (10 mL). A PTFE faced silicon septa was placed on the top of each tube and the appropriate cap and pressure adapter were used. The mixtures were heated in an oil bath at 170 °C for 25 min. The progress of each reaction was monitored by using TLC and GC/MS. The mixtures were cooled to room temperature, and the formed solids were separated by filtration, washed with ethanol and recrystallized from the appropriate solvent (see below) to give the thiazolo[4,5-c]pyridazine derivatives 7a-u as pure products.   [4,5-c] OCH 3 ), 113.7, 118.3, 118.8, 122.6, 125.3,  129.5, 130.7, 130.8, 130.9, 131.7, 133.0, 136.4, 137.4, 138.00, 138.2, 139.0, 162.2, 170.4, 171.7, 174 www.nature.com/scientificreports www.nature.com/scientificreports/ MTT in vitro cytotoxic assay. The in vitro cytotoxic activities of 7a-u were determined toward three human cancer cell lines including HCT-116 (colon cancer), MCF-7 (breast cancer) and A549 (lung cancer), and one normal cell MCF10A (breast cell), by employing the MTT colorimetric assay. The cell lines were grown in a MEM (Minimum Essential Media) medium boosted with 10% fetal bovine serum (FBS), penicillin, and streptomycin at 37 °C in 5% CO 2 humidified atmosphere. The tested compounds were dissolved in DMSO and the resulting solutions were diluted by adding culture medium to the required concentrations. Each cell line was distributed into 96 well sterilized plates in a medium boosted with 10% FBS then treated with the three concentrations of the tested compounds and doxorubicin. In all cases, the incubation time was 48 h in 5% CO 2 incubator at 37 °C. Then the medium was removed and a solution of MTT was added and the mixture was incubated again for 4 h. After incubation, the MTT solution was removed and the obtained purple formazan dye was dissolved in DMSO. Finally the absorbance at 490 nm was measured utilizing a microplate spectrophotometer and triplicate experiments were carried out. The IC 50 values (Table 3) were calculated using the GraphPad Prism Scientific graphing explore software version 7.0.