Synthesis, Characterization of Poly Heterocyclic Compounds, and Effect on Cancer Cell (Hep-2) In vitro

A synthesis series of new heterocyclic derivatives (A2-A7) (pyrrole, pyridazine, oxazine and imidazol) derived from 4-acetyl-2,5-dichloro-1-(3,5-dinitrophenyl)-1H-pyrrole-3-carboxylate(A1) have been synthesised. Synthesis of compound (A2) by the reaction of starting material (A1) with hydroxyl amine hydrochloride in the presence of pyridine. Compound (A2) was reacted with hydrazine hydrate in dry benzene to give (A3) derivative. The compound )A3( deals with sodium nitrite to give diazonium salt, and the reaction diazonium salt with ethyl acetoacetate to produce compound (A4). To a mixture of compound (A4) and hydroxyl amine with sttired to yield (A5).Compound (A6) was prepared by reaction compound (A4) with thiosemicarbazide in presence of drops of acetic acid. Synthesis of 1compound (A7) by reaction compound (A6) with ethyl chloro acetate. The reactions have been monitored by TLC and the synthesized compounds were characterized using spectrophotometric methods FT-IR, 1H NMR.The biological effects of the prepared compounds on the cancer cells were studied in vitro. The results indicated that these Synthesized compounds (A1–A7) inhibited1 the cancer1 cells1 efficiently, the compound (A6) was activity inhibited on the cancer cells.

They are known to inhibit1 reverse transcriptase [human immunodeciency virus1 type (HIV-1)] and cellular DNA polymerases protein kinases.
Preparation of pyrrole is through a number of known interactions such as: Hantzsch synthesis, Knorr synthesis, Barto-Zord synthesis, Piloty-Rabinson synthesis, cycloaddition based routes and other methods (11)(12)(13)(14)(15)(16)(17)(18). Pyridazine is a sixmembered heterocyclic with low adjacent (N) atoms, Its used as an intermediate compound for the preparation of compounds containing pyrazole in structural (19,20). It is also found as a structural unit in the preparation of natural products. Pyridazines skeleton can be considered as one of the most important molecules which grant potential biological and pharmaceutical activity. Pyridazine derivatives constitutes framework of the molecule used in herbicides such as credazine, pyridafol and pyridate. It is also an important pharmacophore of top selling harmaceutical drugs such as, Azelastine, Ameziniummetalilsulfate, Emorfazone, Cadralazine, Hydralazine, Minaprine and Sulfamethoxypyridazine. Pyridazine molecule and its derivatives are also known to possess a wide range of biological activities, such as anticancer, antiviral, antituberculosis, antidepressant, analgesic, antimicrobial and in platelet aggregation (21)(22)(23)(24)(25). The aim of the reasearch is the synthesis and characterization of organic compounds and study the effect of compounds on cancer cell line (Hep-2).

Material and Methods:
Melting points were determined on galenkamp (MFB-600), m.p apparatus and are uncorrected. FT-IR spectra of compounds were recorded on (FT-IR 8400S-spectrometer) as KBr disk). ¹H -NMR spectra (solvent DMSO-d6) were recorded on Bruker DMX-500 spectrophotometer -300MHz with in solution with TMS as internal standard. Measurements were made at the Chemistry Department, AL-Albait1 University, Jordan.
1) In a 50ml Erlenmeyer flask ( 0.001 , 0.399 gm) of compound (A 3 ) was dissolved in (15ml) of water containing( 2ml ) of 6M HCl. solution is cooled to (0-5)°C in an ice bath, and a solution of (0.24gm) of sodium nitrite in 2ml of water was slowly added. Cool the Erlenmeyer flask in an ice water bath for (l0 min.). The diazonium salt precipitates, keep the suspension in the ice bath so we need it in the next step of synthesis compound (A 3 ).
2) ethyl acetoacetate (0.05 mol ,6.5 gm) was added at room temperature to a stirred suspension of 1diazonium salt of compound A 3 (0.01 mol) in water (20 mL) and stirring was continued for 10 min. The reaction temperature was then gradually raised to 50 o C, and after 10 minutes a clear faintly yellow solution was obtained. The solution was then treated with KOH (0.1 mol) in methanol (20 mL) and this mixture was heated to reflux and then left to crystallize. Crystals were collected by filtration, washed with acetone (yield: 72%, m.p. 203-205 °C).

Cell transplant and implantion conditions
In this study, Hep -2 cells type have been utilized. These cells have been grown into a singlelayer form of spindle shape. They were implanted in a RPMI 1640 medium with a 10% complementary fetal calf serum,50 mg /ml streptomycin and 1000 units/ml Penicillin (atnibiotic drug against microbes). Cells line had grown into a form of one single layer under humid atomospher, temperature at 37 • C and 5% CO 2 . Matianing pH number of the medium at a fixed value.Those experiments have been conducted on living cells at specific logarithm growth.

Cytotoxicity study
This method, previously mentioned by Vrshena (2000), has been followed. Cells suspention was prepared by adding (2) ml of Trypsin solution into (25) cm 3 flask of implanted cells .During suspention a cell is shown in ( 20ml ) of growth medium and then completed with 10% of fetal calf serum. The strength of the cells was calculator by the use of blue dye(Alatriepan) and should be more than 95%. After cell suspention mixed well, then was followed by transfering (200) m to 95 different deep flat plates, with pipette containing (1 × 105 cells / plate). The plate was incubated at 37 • C untill 60-70 % after a adding (0.001 M) of prepared compounds. Plates were incubated at 37 • C in an incubator1,with the addition of CO 2 as a complementary for 72 hour,when incubation period has completed,50 ml/plate of neutral red stain was added. Then plate's content was removed by washing the cells three times with phosphate buffer solution. To remove the exess stain from vital cells, 100 ml of elution buffer (phosphate buffer solution and ethanol) was added .Data was analyzed on 492 nm wavelength. Inhibition rate for each compound has been determind1 according to the following equation:

Figure 2 . FT-IR spectrum for compound (A 2 )
Reacting A 2 with hydrazine afforded compound A 3 . The structure of this compound was confirmed by IR spectrum (Fig. 3) which display appearance of absorption bands at (3265-3137) cm-1 due to NH2, and in the same time carbonyl group absorption band at 1685 cm -1 disappeared.

Figure 3 . FT-IR spectrum for compound ( A 3 )
Compound A 4 was prepared by reacting A 3 with ethyl acetoacetate and (NaNO 2 /HCl). The reaction mixture was cooled and stirred for the required time, and the band at (3165-3137) cm -1 ,which assigned to NH2 functional group in compound (3) disappeared. The absence of this band indicates the convertion of the NH2 group to azo group.
Compound A 5 was formed by ring closure. Using hydroxyl amine hydrochloride in amixture1 of ethanol and pyridine. This reaction resulted in a formation of compound A 5 . The compound was identifed by IR spectra (Fig. 4) . It showed one peak at 1727 cm -1 which attributed for (C=O) group, associted with ring formation. Compound A 6 was prepared by Schiff's reaction.
Compound A 5 reacted with thiosemicarbazide in ethanol, with few drops of acetic acid, which leads to the formation of the targted compound. The IR spectrum of compound A 6 ( Fig. 5) shows band absorption disappearance at 1720 cm -1 . However, new absorption bands appeared at 3300-3250 cm -1 , which was signed for (NH 2 ) stretching, and 3245 cm -1 for (NH).

Figure 5 . FT-IR spectrum for compound (A 6 )
Compound A 7 was prepared by the reaction of compound A 6 with concentrated sulphuric acid.The structure of this synthesized compound was assigned on the basis of IR, 1HNM spectral data .The IR spectra for this compound ( Fig. 6) showed the disappearnace of two bands at 3300-3250 cm -1 ( NH 2 stretching absorption bands), and the appearance of one absorption band at 2230cm -1 , which attributed for (SH) stretching. Unexpectedly, the IR spectra of this compound shows two bands associated with (NH,SH), which was attributed to the compound having two different tautumirasim forms.

The effect of compounds (A 1 -A 7 ) on cancer cell line (Hep-2)
One1 type of cancer cell line has been used to1investigate how the compounds1 under study. Affect the cell 1growth in vitro. The cell line used was Hep1-2 type, In this method, the 1number of 1cells is calculated 1under the optimal1condition for cell growth. Then the prepared compounds were added to investigate their 1effects on cell growth (Hep-2).
At the end1 of the test of the prepared 1compounds, it was found that the value of T(cytotoxic to MT-4Cells at CC 50 ) was 2.873 at the degree of freedom 53, and the value of the P-value was 10.023, which is less than 0.05, indicating a statistically significant1 difference between the compounds (Tables 1.2)

Conclusion:
The present work reports a new investigation of heterocyclic systems (pyrrole, pyridazine, oxazine and imidazol) derived from 4-acetyl-2,5-dichloro-1-(3,5-dinitrophenyl)-1H-pyrrole-3-carboxylate And the systems were prepared with the objective of developing better antibacterial molecules. Note that when the number of heterocyclic increase in the compound its inhibitory capacity increases. The seven compounds have the highest inhibition capacity.