Synthesis of Novel 1-Substituted and 1,9-Disubstituted-1,2,3,4tetrahydro-9H-Carbazole Derivatives as Potential Anticancer Agents

Condensation of 1-acetyl-1,2,3,4-tetrahydro-9H-carbazole (2) with some amino compounds furnished the corresponding imino derivatives 3a-e. Compound 3a reacted with chloroacetic acid and underwent cyclization to give the thiazolidine derivative 5. Also, treatment of 3c with thionyl chloride caused cyclization to yield the [1,2,6]thiadiazino derivative 6, which gave the corresponding N-formyl derivative 7 upon heating with ethyl formate. In addition, interaction of 3d with ethyl cyanoacetate yielded the monoamide of malonic acid derivative 8. Acylation of carbazole 1 with succinoyl chloride or phenylacetyl choride produced the corresponding azepine (11) and 1,9-diphenyl acetyl derivatives (14), respectively. Compounds 11, 14 were further reacted to give the carbazole derivatives 12, 13 and 15a,b. The cytotoxic activity for some of the prepared compounds against breast cancer B20 is discussed.


Introduction
The clinical need for therapeutic agents which restore or enhance an immune response in immunocompromised patients such as that which occurs in viral infections, cancer, autoimmune diseases and acquired immune deficiency syndrome (AIDS) has led to the search for novel immunostimulants [1].Interferon-γ (IFN-γ) is a potent activator of the immune system and has been used in the treatment of infections in human.A fused pyrrolo [2,3-c]carbazol-6-one (Figure 1) reportedly potentiates the INF-γ induction of MHC-class π molecules [2].
Other carbazole derivatives like ellipticin, and the alkaloids vincristine and vinblastine have a well established role in the treatment of cancer [3][4][5][6][7][8].The present work is a part of our program aimed at developing new approaches for synthesis of fused heterocyclic systems containing the carbazole moiety and evaluating their anticancer activity.

Results and Discussion
Acetylation of 1,2,3,4-tetrahydrocarbazole (1) using acetyl chloride and glacial acetic acid afforded 1-acetyl tetrahydrocarbazole (2).Compound 2 was used as a starting material in the synthesis of many heterocyclic compounds containing a carbazole moiety.Thus, condensation of 2 with thiourea, thiosemicarbazide, guanidine hydrochloride and glycine produced the corresponding imino derivatives 3a-e.
Interaction of 3a with chloroacetic acid can procede via the formation of an intermediate mercaptoacetic acid derivative 4 which cyclizes through elimination of water to give the thiazolidine derivative 5, as depicted in Scheme 1.Its mass spectrum showed the expected m/z 296 (M-15 (CH 3 ); 0.5%).
Treatment of 3c with thionyl chloride in pyridine caused cyclization to furnish [1,2,6]thiadiazin-5oxide derivative 6 [9] via elimination of two moles of HCl.The elemental analysis and spectral data are in good agreement with the proposed structure.Formylation of 6 was done by treatment with ethyl formate to yield the N-formyl derivative 7 (Scheme 1).The mass spectrum of compound 7 exhibited a molecular ion peak at m/z 360 (M+, 1.0%), 344 (M-16 (O), 0.7%).

Scheme 1
Furthermore, interaction of the imino derivative 3d with ethyl cyanoacetate gave a product with analytical data indicating that one mole of ethanol was eliminated, followed by hydrolysis of the cyano and imino groups.This product was formulated as the monoamide of malonic acid derivative 8.The structure of compound 8 was demonstrated on the basis of its elemental analysis and spectral data.
Condensation of acetyltetrahydrocarbazole 2 with pyridine-3-carboxaldehyde yielded the arylidine derivative 9, which upon treatment with hydrazine hydrate in ethanol gave the pyrazole derivative 10.The IR spectrum of 10 showed the complete disappearance of the CO band present in the parent compound.
Acylation of 1,2,3,4-tetrahydrocarbazole 1 with succinoyl chloride in presence of sodium hydride caused cyclization via elimination of two molecules of HCl to give the azepine derivative 11.
Chlorination of 11 with thionyl chloride afforded the corresponding chloroderivative 12, which upon treatment with ethylamine furnished the corresponding ethylamino derivative 13.
In contrast to the behaviour of 1 towards acetyl chloride and succinoyl chloride, the reaction of 1 with phenylacetyl chloride consumed two moles of the reagent to produce the 1,9-diphenylacetyltetrahydrocarbazole derivative 14.
Compound 14 was reacted with some active methylene compounds such as 2-cyano-methylthiazolone [10] and a pyrazolone derivative.Reaction took place via condensation of the methylene group with the carbonyl group and hydrolysis of the 9-amide to give the corresponding thiazolo and pyrazolo derivatives 15a,b (Scheme 2).The spectral data was all in agreement with the proposed structures.(12)

General
All melting points are uncorrected.Microanalytical data were obtained at the Microanalytical Data Unit at Cairo University.IR spectra were recorded in KBr disks on a Unicam SP 200 spectrophotometer. 1 H-NMR spectra were measured on a Jeol DFF 100 (270 Mhz) with TMS as an internal reference.The mass spectra were recorded at Cairo University on a Shimadzu-GC-MS-QP 100 EX using the direct inlet system.

Pharmacological Study: Cytotoxic assay
The cytotoxicity assay performed using a microculture tetrazolium (MYY) sigma method [11].The cultured cells at a long phase of their growth were treated in triplicate with various concentrations of chemical ranging from 0.5-100 µg/ mL.Synthesized compounds 2,3a, 3c, 6, 7, 14 and 15a were dissolved in DMSO by adding 10 µL DMSO to each tested compound followed by gentle shaking.The cultured cells were then incubated for 18 hrs at 37 o C in a humidified atmosphere of 5% CO 2 .The cell concentration was determined by counting BT 20 cell in a hemocytometer.Results were expressed as the dose that inhibited 50% control growth after incubation period (ED 50 ) the value were estimated from a semilog plot of the drug concentration (µg/mL) VS the percent of viable cells (%), Table 5.