Synthesis and Anticancer Activities of Novel 1,4-disubstituted Phthalazines

A series of novel 1-anilino-4-(arylsulfanylmethyl)phthalazines were designed and synthesized. The structures of all the compounds were confirmed by IR, 1 H-NMR, elemental analysis and MS. The analogues


Introduction
Phthalazine derivatives, like the other members of the isomeric benzodiazine series, have been widely applied as therapeutic agents due to their anticonvulsant, cardiotonic, vasorelaxant and antiinflammatory properties [1,2].To our knowledge, however, there have been no reports on the anticancer activities of 1-anilino-4-arylsulfanylmethylphthalazines.We describe here the synthesis of some novel 1-anilino-4-arylsulfanylphthalazine derivatives 9-19, several of which exhibited higher activity than the cisplatin control.
Phthalazines were previously synthesized from 2-aryl-3-hydroxyinden-1-ones or β-diketones by condensation with hydrazine hydrate [2][3][4][5].However, these routes do not allow for the desired incorporation of thiophenylmethyl groups into the phthalazine 4-position.This report describes a convenient access to 1,4-disubstituted phthalazines with such substituents.The compounds obtained in were characterized by IR, 1 H-NMR, MS and elemental analysis and their anticancer activities were evaluated in vitro.

Chemistry
Our syntheses of the requisite phthalazines are illustrated in Schemes 1 and 2. Refluxing phthalic anhydride and malonic acid in pyridine gave 2-acetylbenzoic acid (1) [6], which was then esterified with dimethyl sulfate.In the next step, the acetyl group was brominated with phenyltrimethylammonium tribromide (PTT), a selective brominating reagent for ketones or ketals [7], to give intermediate 3, which facilitated the introduction of various thiophenol substituents.It should be noted that side-products could be formed if bromine was used as the halogen source.Compound 3 was then treated with thiophenol or 3,4-difluorothiophenol using K 2 CO 3 as base.Cyclization of 5a/5b with hydrazine hydrate led to the generation in 90-96% yields of the phthalazines 6a/6b, which were treated with POCl 3 to give 1-chloro-4-substituted-phthalazines 7a and 7b.Treatment of 7a/7b with substituted anilines provided the target compounds 9-15.The target compounds 16-18 could be obtained by oxidization of the corresponding compounds 13-15 with H 2 O 2 .However, the sulfanyl substituted phthalazine derivatives could not be easily converted to the corresponding sulfonyl ones.MCPBA has been used as the oxidizing reagent in some cases [8], but in our synthetic route this could easily lead to an undesired N-oxidized side-product, due to the presence of the amino groups.We have found, however, that H 2 O 2 /Na 2 WO 4 was very effective for oxidizing sulfanyl to sulfonyl groups in good yield and high purity.

Anticancer activities
The anticancer activities of compounds 9-19 were evaluated in vitro by the MTT method and the results are summarized in Table 1.Compounds 12 and 13 showed more in vitro activity than cisplatin against the two cancer cell lines tested.The phthalazine derivative 11 showed activity comparable to cisplatin.The remaining compounds exhibited slight to moderate activities.

Conclusions
A series of novel 1,4-disubstituted phthalazines have been prepared.From the biological test results the following conclusions can be reached about their structure-activity relationships: (a) incorporation of a substituted thiophenol group into position 4 of the phthalazine ring appears to increase anticancer activity, compared to that of an unsubstituted thiophenol; (b) replacement of a sulfanyl with sulfinyl or sulfonyl groups decreases the anticancer activity.Further investigations are in process.

General
Melting points were determined by the capillary tube method, and the thermometer was uncorrected.Mass spectra were obtained on an Agilent 1100 HPLC-MS instrument. 1 H-NMR spectra were run in DMSO-d 6 , with TMS at the internal standard, on a Bruker ARX-300 instrument operating at 300 MHz.IR spectra (KBr disks) were recorded on a Bruker IFS 55 instrument.Elemental analysis was performed with a Carlo-Erba 1106 Elemental analysis instrument.

Methyl 2-(bromocarbonyl)benzoate (3)
To a solution of 2 (100.0 g, 0.56 mol) in anhydrous tetrahydrofuran (200 mL), a solution of PTT (210.5 g, 0.56 mol) in anhydrous tetrahydrofuran (80 mL) was added dropwise.During this addition a white precipitate was formed and the solution became yellow.After stirring at room temperature for 15 h, the resulting mixture was filtered.The filtrate was stirred into a mixture of petroleum ether/water (200 mL, 1:1 v/v), then separated and concentrated to give 116. 4  To a mixture of K 2 CO 3 (8.3g, 0.06 mol) and 4a (11 g, 0.1 mol) in methanol (150 mL), a solution of 3 (25.7 g, 0.1 mol) in acetone (200 mL) was added dropwise while the temperature was kept below 0 o C. The reaction mixture was stirred for an additional 1.5 h at this temperature.After filtration and concentration, the residue was dissolved in dichloromethane (200 mL).The organic phase was washed with saturated sodium carbonate solution (100 mL×3) and dried with MgSO 4 .Concentration gave 5a as an oil, 23.5 g (82%, GC purity: 96.9%), which could be used in next step without purification.

Pharmacology
The anticancer activities of compounds 9-19 were evaluated in vitro on Bel-7402 (Human Liver Cancer cell lines) and HT-1080 (Human Fibro Sarcoma cell lines) by measuring cell viability by the MTT method, with cisplatin as the positive control.The cells were seeded in RPM I 1640 medium (100 µL) in a 96-well plate at a concentration of 4000 cells per well.After culturing for 12 h at 37 o C and 5% CO 2 , cells were incubated with various concentrations of the samples for 24 h.MTT was added at a terminal concentration of 5µg/mL and incubated with the cells for 4 h.The formazan crystals were dissolved in DMSO (100 µL) in each well and the optical density was measured at 492 nm (for the absorbance of MTT formazan) and 630 nm (for the reference wavelength).The IC 50 was calculated using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.