A New Type of Synthesis of 1 , 2 , 3-Thiadiazole and 1 , 2 , 3-Diazaphosphole Derivatives Via-Hurd-Mori Cyclization

We present a short and efficient synthesis of the title compounds starting with cheap and readily available camphor and derivatives of acetophenone. The optimized sequence allows the large-scale preparation of this new type of synthesis in a few steps. New 1, 2,3-thiadiazole and 1,2,3diazaphosphole derivatives 11-20, were prepared from the ketones 1-5 via the corresponding semicarbazones 6-10. The Hurd-Mori and Lalezari methods were used, respectively, for the preparation of these 1,2,3-thiadiazole and 1,2,3-diazaphosphole derivatives. These derivatives exhibit anticancer effect due to their high potential biological activity.


Experimental Section
Melting points were taken on Gallen Kamp melting apparatus and were uncorrected.Thin layer chromatography was performed with fluorescent silica gel plates HF 254 (Merck), and plates were viewed under UV 254 and 265 light.Infrared spectra (-cm -1 ) were recorded on Bruker Vector Germany and on Mattson FT-IR 1000, using KBr disks.Mass spectra are measured on GCQ Finnigan MAT. 1 H-NMR spectra were recorded on Gemini-200 MHZ NMR spectrometer in DMSO-d 6 spectra were internally referenced to TMS.Peaks are reported in ppm.Downfield of TMS.The antibacterial activity were determined in microanalytical center Cairo University and anticancer activity was done in National Cancer Institute, Cancer Biology Department, Pharmacology, Cairo University.D-(+)-camphor, di-methoxy acetophenone p-bromo acetophenone, p-nitro acetophenone p-amino acetophenone were obtained from Fluka or Aldrich.
General Procedure for the Preparation of 1,2,3-thiadiazole (11,12,13,14,15)  An excess amount of thionyl chloride was stirred at room temperature and the thiosemicarbazones 6 or 7 or 8 or 9 or 10 were added in several portions.The mixtures were stirred at r.t.overnight until no more hydrogen chloride was produced, the product was washed with diethyl ether to give a good yields of the corresponding 1, 2, 3thiodiazoles as fine powders.A recrystallization from ethyl alcohol was carried out.
General procedure for the preparation of 1,2,3-diazaphosphole compounds (16,17,18,19,20)  Each of thiosemicarbazones 6 or 7 or 8 or 9 or 10 (1 mol) and triphenyl phosphine (1 mol) were dissolved in tetrahydrofuran(10 ml) and the mixture was stirred at room temperature for 2 days.The product was washed with diethyl ether to give a good yields of the corresponding 1,2,3-diazaphospholes as five powders.A recrystallization from ethyl alcohol was carried out.

Results and Discussion
[32] Scheme (1): Ketones used in the preparation of new 1,2,3-thiadiazole and 1,2,3diazaphosphole compounds The general equations for the preparation of 1,2,3-thiadiazole and 1,2,3diazaphosphole derivatives are shown in scheme 2.   Compound ( 14) was of particular interest as it represents the unexpected compound in contrast the previous structures.Scheme 3 shows the proposed mechanism for the formation of compound (14).Its structure was confirmed by I.R, 1 H-NMR, mass spectrometry.Characterization data of newly compounds hydrazones (6-10) and 1,2,3thiadiazole (11-15) and 1,2,3-diazaphosphole (16-20).The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH.Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method.This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values.

R CH
Blank paper disks (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated 10 µ of tested concentration of the stock solutions.
When a filter paper disc impregnated with a tested chemical is placed on agar the chemical will diffuse from the disc into the agar.This diffusion will place the chemical in the agar only around the disc.The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc.If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical.This area of no growth around the disc is known as a "Zone of inhibition" or "clear zone".

Antitumor Screening
Chemotherapy is a major therapeutic approach for the treatment of both localized and metastasized cancers.In the present work newly compounds 1,2,3-thiadiazole and 1,2,3diazaphosphole were evaluated as inhibitors of the growth of breast cancer cell line in comparison to the known anticancer drug: Doxorubicin as a trial to get more effective and less toxic agent.
Preliminary experiments were done using the human tumor cell line to identify the potential toxicity of four chosen selected newly synthesized compounds (11,12,16,17) (Fig. 1) in comparison to the known anticancer drug: doxorubicin by SRB using the method Skehan et al. 38  Cells were plated in 96-multiwell plate (10 4 cells/well) for 24 hrs before treatment with compounds to allow attachment of cell to the wall of the plate.
 Different concentration of the compound under test (0, 1, 2.5, 5 and 10g/ml) were added to the cell monolayer triplicate wells prepared for each individual dose.
 Monolayer cells were incubated with the compounds for 48 hrs at 37 o C and atmosphere of 5% CO 2 .
 After 48 hrs, cells were fixed, washed and stained with sulfo-rhodamine - stain.
 Excess stain was washed with acetic acid and attached stain was recovered with tris EDTA buffer.
 Color intensity was measured in an ELISA reader.
 The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound.

Conclusion
In connection with our work on derivatives of acetophenone herein we describe a new type procedure for the synthesis of 1,2,3-thiadiazole and 1,2,3-diazaphosphole compounds via-Hurd Mori cyclization.The Hurd-Mori reaction is by far the most widely used method in the research on 1,2,3-thiadiazoles, and some reactions are carried out on an industrial scale.In view of these facts, the aim of the present study was to obtain 1,2,3-thiadiazole and 1,2,3-diazaphosphole derivatives where synthesized as antitumor agents.The antitumor activity results indicated that all the four derivatives showed antitumor activity against the tested breast cancer cell line but with varying intensities in comparison to the known anticancer drug: doxorubicin.

Table ( 1
) shows the structures of the newly prepared compounds, melting point ranges and the percentage yields of these compounds.

Table ( 3
): Effect of some selected newly synthesized compounds on breast cancer cell line.Dose of the compound which reduces survival to 50%.