Synthesis and Cytotoxicity of Novel Hexahydrothieno-cycloheptapyridazinone Derivatives

Designed as a new group of tricyclic molecules containing the thienocycloheptapyridazinone ring system, a number of 2N-substituted-hexahydrothieno-cycloheptapyridazinone derivatives were synthesized and their biological activity evaluated. Among the synthesized compounds, derivatives 7d and 7h were found to possess cytotoxic activity against non-small cell lung cancer and central nervous system cancer cell lines, respectively.


Introduction
Among diseases, cancer is not a single pathological state but a broad group of diseases characterized by a high proliferative index and the spread of aberrant cells from their site of origin [1]. Clinically, the therapeutic treatment of cancer is a combination of surgery and/or radiotherapy with chemotherapy [2,3].

OPEN ACCESS
Current chemotherapy consists of cytotoxic (cell-killing) agents and anti-hormonal drugs, which reduce the proliferation of the tumors [2,3]. The therapeutic use of anticancer drugs is complicated by systemic toxicity, usually observed in the bone narrow, the gastrointestinal (GI) tract and hair, and by development of resistance. Therefore, the search for novel chemical structures with broader therapeutic windows and acceptable resistance profiles is being actively pursued.
Preparation of the target compounds 7a-l was accomplished by treatment of the pyridazinones 15a,b with formaldehyde and appropriate amines. Scheme 1. Synthesis of novel pyridazinone derivatives 7a-l.
g h X and Y as reported in Table 1 .

Results and Discussion
A new series of twelve substituted pyridazinones 7a-l were synthesized and eight of them (7c-e,h-l) were evaluated at a single concentration of 10 -5 M (10 μM) for their antitumor activities. The evaluation established a primary screening where compounds were tested to determine their growth inhibitory properties against sixty different human tumor cell lines in vitro [18][19][20]. The compounds were added at a single concentration and the cell culture was incubated for 48 h. End point determinations were made using a protein binding dye, sulforhodamine B (SRB), which was used to estimate cell viability or growth [21]. The results for each compound are reported as percent growth of treated cells when compared to untreated control cells (Tables 2-3). Range of growth % shows the lowest and the highest growth % found among different cancer cell lines, where all tested compounds have demonstrated being scarcely active or completely inactive in the antitumor screening in vitro ( Table 2). 5-Fluorouracil (5-FU) was used as reference compound with the mean growth inhibitory effect (GI 50 ) of 2.45 × 10 -5 M which corresponds in logarithmic scale to 4.61 [22]. Nevertheless, compounds 7d and 7h displayed a higher anti-proliferative activity in the non-small cell lung cancer cell line EKVX and in the CNS cancer cell line SNB-75, which showed growth inhibitions of 27.94% and 27.86%, respectively (Table 3). Compounds 7k and 7l also showed cell growth inhibitory activity, even if weaker than the one expressed by 7d and 7h. In particular, compound 7l was found to be active as growth % inhibitor of the leukemia cell line RPMI-8226 with a value of 24.95%; the derivative 7k was active on leukemia cell line SR with a value of 24.83% and non-small cell lung cancer cell line EKVX with a value of 23.93%. Moreover, 7e was found to be active as growth % inhibitor of the non-small cell lung cancer cell line EKVX with a value of 21.09%. Finally, 7c and 7i showed a growth % inhibitor of non-small cell lung cancer cell line HOP-92 with values of 18.97, and 19.80%, respectively.

Conclusions
As part of our continuous search for potential biologically active compounds, a series of pyridazinone derivatives were synthesized and assessed for their anticancer activity. It was found that all new eight compounds tested showed weak or incomplete activity without significant differences between 9-substituted and unsubstituted derivatives. Specifically, two of them showed scant activity, while others showed no activity in the cell growth inhibition assay against sixty different human cancer cell lines panel in vitro. From these data, we may conclude compounds 7d and 7h were the most effective molecules for anti-proliferative activity, specifically in non-small cell lung cancer and CNS cancer respectively, so they might be useful as leads for designing new compounds with potential antitumoral activity. This structure was derived from pyridazinone with hydrogen or methyl group in the 9-position linked to the 4-methylpiperazine moiety by a methylene spacer. The obtained results prove the necessity for further investigations to clarify the molecular mechanisms involved in antitumor activities to acquire more information about the structural requirements for enhancing anticancer activities and minimizing neurotoxicities, the synthesis of more new derivatives with different substituents at other positions is needed.

General
Melting points were determined using a Reichert-Köfler hot-stage apparatus and are uncorrected. Infrared spectra were recorded with a Perkin-Elmer Paragon 500 FT IR spectrophotometer (KBr pellets, in Nujol mulls, as well as in film). 1 H-NMR spectra were recorded on a Varian XL 200 FT NMR spectrometer using CDCl 3 as solvent, unless otherwise specified. Chemical shifts are reported in δ or ppm and coupling constants (J) in Hertz (Hz), downfield from tetramethylsilane (TMS).
Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Reactions were monitored by analytical thin-layer chromatography (TLC) using SiO 2 Polygram SIL and ALOX N/UV 254 precoated plastic sheets and with visualization by irradiation with a UV lamp and/or iodine vapor for detection. Flash chromatography was performed using Merck silica gel type 60 (230-400 mesh ASTM). Electron ionization mass spectra (70 eV) were recorded on a Hewlett-Packard 5790-5970 MSD gas chromatograph/mass spectrometer. Atmospheric Pressure Ionization Electrospray (APIES) mass spectra, when reported, were obtained on a Agilent 1100 series LC/MSD spectrometer. All moisture sensitive reactions were performed under nitrogen atmosphere, using oven-dried glassware. Anhydrous DCM, THF and DMF was obtained from Aldrich, Lancaster or Merck. All starting materials and reagents were commercially available from Aldrich, Lancaster and Avocado. Evaporation was performed in vacuo (rotary evaporator). Anhydrous sodium or magnesium sulfate was always used as the drying agent. Elemental analyses were performed in a Perkin-Elmer 240C elemental analyzer, and the results were within ± 0.4% of the theoretical values, unless otherwise noted.

General procedure for the synthesis of 5-oxopentanoic acids 9a,b
To a suspension of anhydrous AlCl 3 (17.54 mmol) in dry CH 2 Cl 2 (20 mL) cooled with an ice bath, a solution of glutaric anhydride (19 mmol) in dry CH 2 Cl 2 (20 mL) was added dropwise under a N 2 atmosphere, and the whole mixture was stirred at RT for 0.5 h. Then a solution of thiophene 8a,b (17 mmol) in dry CH 2 Cl 2 (15 mL) was added dropwise and the reaction mixture stirred at the same temperature for an additional 0.5 h. The mixture was poured into crushed ice and conc. HCl was slowly added followed by warming until the suspended materials dissolved. The aqueous phase was separated and extracted with CH 2 Cl 2 . The combined organic phase was washed with H 2 O and then extracted with 2N NaOH aqueous solution (5 × 7 mL): the solid separated upon acidification of the alkali layer, was filtered off and air dried to yield the desired product.

General procedure for the synthesis of pentanoic acids 10a,b
A mixture of 5-oxopentanoic acid 9a,b (2.00 g, 9.3 mmol), diethylene glycol (DEG, 24 mL), potassium hydroxide (0.035 mol) and hydrazine hydrate (0.045 mol) was refluxed with a Dean-Stark apparatus for 3 h. The solution, after cooling to RT, was poured into cold water (50 mL), washed with ether, acidified with 6 N HCl and then extracted with ether (4 × 5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo to give the title compounds.

General procedure for the synthesis of ketones 11a,b
To a solution of pentanoic acid 10a,b (13 mmol) in toluene (35 mL), were added Celite ® (4.52 g) and phosphorus pentoxide (23 mmol). The mixture was refluxed for 2 h, then cooled and filtered. The filtrate was washed with 5% aqueous NaHCO 3 solution (2 × 5 mL), dried (Na 2 SO 4 ), filtered and concentrated in vacuo, to give the ketones as oils.

General procedure for the synthesis of Mannich bases 12a,b
Acetic anhydride (39 mmol) was added dropwise to a solution of dimethylamine hydrochloride (10 mmol) and 37% formaldehyde (29 mmol) at 85-90 °C and the mixture was stirred for 0.5 h. Then tetrahydrocyclohepta[b]thiophen-4-one 11a,b (7 mmol) was added to the mixture and the whole stirred at 75 °C for 3h. After cooling, the mixture was evaporated under reduced pressure and the resulting crude residue was crystallized from acetone (12a) or triturated with diisopropyl ether (12b) to afford the desired product.

General procedure for the synthesis of nitriles 13a,b
To a solution of the Mannich base 12a,b (4 mmol) in methanol (8 ml), an aqueous solution of NaCN (22 mmol, 10 ml) was dropwise added, at RT, and the mixture was stirred at 55 °C for 4 h, then poured onto cold H 2 O and afterwards extracted with CH 2 Cl 2 (3 × 5 mL). The resulting organic layer was washed with H 2 O, brine, dried (Na 2 SO 4 ), filtered and evaporated in vacuo.

General procedure for the synthesis of acids 14a,b
To a solution of nitrile 13a,b (3.5 mmol) in AcOH (3.6 ml), HCl conc. (2.5 ml) was dropwise added at RT, then the reaction mixture was refluxed for 3 h (TLC). After cooling to RT, the mixture was diluted with cold H 2 O and afterwards extracted with CH 2 Cl 2 (4 × 5 mL). The resulting organic layer was washed with H 2 O, brine, dried (Na 2 SO 4 ), filtered and evaporated in vacuo. 6,7,