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

Abstract

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 (CH₃); 0.5%).

Treatment of 3c with thionyl chloride in pyridine caused cyclization to furnish [1,2,6]thiadiazin-5-oxide 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%).

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-diphenylacetyl- tetrahydrocarbazole 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.

EXPERIMENTAL

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. ¹H-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.

1-Acetyl-1,2,3,4-tetrahydro-9H-carbazole (2)

Acetyl chloride (0.1 mol) was added to 1,2,3,4-tetrahydro-9H-carbazole (1) (0.1 mole) dissolved in glacial acetic acid (10 mL) and the mixture was refluxed for half an hour. The solution was evaporated and the residue was crystallized to give the title compound (

Table 1:. Physical and analytical data for synthesized compounds 

Compd No.	M.P.* °C	Yield %	Formula (M. Wt)	Elemental analyses Calculated/ Found, %
				C	H	N	Cl	S
2	122a	59	C14H15NO (213)	78.87 78.77	7.04 7.10	6.57 6.49		11.81 11.71
3a	190b	60	C15H17N3S (271.37)	66.38 66.21	6.31 6.11	15.48 15.32
3b	140b	65	C15H18N4O (270.31)	66.64 66.51	6.71 6.66	20.72 20.63
3c	160b	62	C15H18N4S (286.39)	62.90 62.81	6.33 6.25	19.56 19.46		11.19 11.09
3d	240c	66	C15H18N4 (254.32)	70.83 70.71	7.13 7.03	22.03 21.97
3e	170b	50	C16H18N2O2 (270.3)	70.03 71.00	6.66 6.57	10.36 10.42
5	135b	55	C17H17N3OS (311.38)	65.51 65.49	5.46 5.50	13.49 13.38		10.28 10.26
6	180b	57	C15H16N4OS2 (332.42)	54.19 54.01	4.85 4.69	16.85 16.72		19.29 19.15
7	220c	50	C16H16N4O2S2 (360.3)	53.33 53.29	4.47 4.39	15.55 15.44		17.76 17.66
8	190b	64	C18H19N3O4 (341.31)	63.33 63.11	5.61 5.51	12.31 12.08
9	100d	65	C20H18N2O (302.35)	79.44 79.31	6.00 5.95	9.26 9.16
10	155b	55	C20H18N4 (314)	76.43 76.26	5.73 5.71	17.83 17.88
11	160b	53	C16H15NO2 (253.26)	75.87 75.77	5.97 5.85	5.53 5.49
12	210b	63	C16H14NOCl (271.72)	70.71 70.63	5.19 5.00	5.15 5.03	13.04 13.00
13	140b	69	C18H20N2O (280.34)	77.11 77.00	7.19 7.13	9.99 9.82
14	180b	50	C28H25NO2 (407.48)	82.53 82.39	6.18 6.08	3.44 3.34
15a	100e	61	C25H21N3OS (411.42)	72.98 72.82	5.15 5.09	10.21 10.12
15b	160b	57	C30H27N3O (445.52)	80.87 80.79	6.11 6.01	9.43 9.31

* Recrystallization solvents: ^aMeOH, ^bEtOH, ^cDMF/H₂O, ^dbenzene, epetroleum. ether (b.p. 80-100°C).

).

General procedure for the preparation of 1-(2,3,4,9)-Tetrahydro-1H-carbazol-1-yl)ethylidene thiourea (3a), 1-(2,3,4,9)-tetrahydro-1H-carbazol-1-yl)-ethylidene semicarbazone (3b), 1- (2,3,4,9)-tetrahydro-1H-carbazol-yl)-ethylidene thiosemicarbazone (3c) and 1-(2,3,4,9- tetrahydro-1H-carbazol-1-yl)-ethylidene amino acetic acid (3e)

Compound 2 (0.01 mole) was dissolved in ethanol (20 mL), sodium hydroxide (10%; 10 mL) and thiourea, semicarbazide, thiosemicarbazide or glycine (0.01 mole) was added. The reaction mixture was refluxed for 4 hrs., cooled and dilute hydrochloric acid (10 mL) was added. The solid formed was filtered off and crystallized to furnish compounds 3a,b,c and e, respectively (Table 1).

1`-[1-(2,3,4,9-Tetrahydrocarbazol-1-ylidene)-N-(thiazolidin-4-one)ethanamine (5)

Chloroacetic acid (0.01 mole) was added to a solution of 3a (0.01 mole) in dry benzene (20 mL) containing a few drops of triethylamine. The reaction mixture was refluxed for 4 hr and left to cool. The precipitate formed was crystallized to give 5 (Table 1).

General procedure for the preparation of 3-methyl-2,4,5,6-tetrahydro[1,2,6]thiadiazino[4,3,2-jk]- carbazol-2-yl-2-thiourea-1-oxide (6) and 7-chloro-1,2,3,3a-tetrahydro-[3,2,1-jk]carbazol-4-one (12)

Thionyl chloride (0.05 mole) was added portionwise to a solution of 3c or 11 (0.01 mole) in pyridine (20 mL) respectively. The reaction mixture was refluxed on a water bath for 30 minutes, excess solvent was evaporated and the resulting solid was washed with petroleum.ether (b.p. 60-80°C) and crystallized to furnish compounds 6 or 12 (Table 1).

3-Methyl-2,4,5,6-tetrahydro[1,2,6]thiadiazino[4,3,2jk]carbazol-2-yl amino-(thioxo)methylamino- methanone-1-oxide (7)

Ethyl formate (0.01 mol) was added to a solution of 6 (0.01 mol) in ethanol (20 mL) and hydrochloroic acid (10%, 10 mL). The reaction mixture was refluxed for 4hr, left to cool and the solid  was crystallized to give compound 7 (Table 1).

3-Oxo-3-[3-(1-2,3,4,9-pentahydrocarbazole-1-ylidene)-ethyl]ureidopropionic acid (8)

A mixture of compound 3d (0.01 mol) and ethyl cyanoacetate (0.01 mol) in ethanol (25 mL) with 10 mL (10%) sodium hydroxide was refluxed for 4 hrs then cooled and neutralized with dilute hydrochloric acid. The solid thus formed was filtered off and crystallized to give 8 (Table 1).

3-Pyridin-3-yl-1-(2,3,4,9-tetrahydro-1-carbazol-1-yl)prop-2-en-1-one (9)

A mixture of 2 (0.01 mol) and pyridine 3-carbaldehyde (0.01 mol) in ethanol (20 mL) containing a few drops of piperidine was refluxed for 4 hrs. The product precipitated after cooling, was filtered off and crystallized to give compound 9 (Table 1).

1-(5-Pyridin-3-yl-1H-pyrazol-3-yl)2,3,4,9-tetrahydro-1H-carbazole (10)

A mixture of 9 (0.001 mol) and hydrazine hydrate (0.003 mol) in ethanol (20 mL) was refluxed for 3 hr, then left to cool. The solid was filtered off and crystallized to give 10 (Table 1).

7-Ethylamino-1,2,3,3a-tetrahydro[3,3,1-jk]carbazole-4-one (13)

Compound 12 (0.01 mol) was fused with ethylamine (0.012 mol) at 150°C for 30 min. The solid product that obtained after trituration with ethanol was filtered and crystallized to afford 13 (Table 1).

1,2,3,3a-Tetrahydroazepino[3,2,1-jk]carbazole-4,7-dione (11) and 1,9-diphenylacetyl-1,2,3,4- tetrahydrocarbazole (14)

Sodium hydride (0.04 mole) was added to compound 1 (0.01 mol) dissolved in dry dimethylformamide (75 mL) and the mixture was heated at 90°C for 1 hr., left to cool and a solution of succinoyl chloride or phenyl acetyl chloride (0.01 mole) in dry dimethyl formamide (20 mL) was added slowly to the mixture, which was then heated at 90°C for 4 hrs., left to cool and treated with ice. The precipitate formed was filtered off, washed with water, dried and crystallized to afford 11 or 14, respectively (Table 1).

2(2,3,4,9-Tetrahydrocarbazol-1-yl)-1-cyano-1(5-hydro-4-oxo-1,3-thiazol-2yl)-3-phenyl-1-propene (15a) and [1-(2,3,4,9-tetrahydrocarbazol-1-yl)-2-phenyl-1-[(3-methyl-1-phenyl)pyrazol-5-one-1- ylidine]ethane (15b)

A solution of 14 (0.001 mol) in ethanol (20 mL) was treated with 2-cyanomethyl thiazol-4-one or 3- methyl-1-phenylpyrazol-5-one (0.01 mol) in presence of anhydrous potassium carbonate (2 g). The reaction mixture was refluxed for 4 hr, left to cool and filtered. The precipitate so formed was crystallized to furnish compounds 15a,b (Table 1).

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°C in a humidified atmosphere of 5% CO₂. The cell concentration was determined by counting BT₂₀ cell in a hemocytometer. Results were expressed as the dose that inhibited 50% control growth after incubation period (ED₅₀) the value were estimated from a semilog plot of the drug concentration (μg/mL) VS the percent of viable cells (%),

Table (5):. Cytoxic activity against breast cancer B₂₀ 

Compound No.	ED50
2	30
3a	42
3c	38
6	37
7	35
14	29
15a	26

.

The ranking of anticancer activity (Table 5) was found to be 15a, 14, 2, 7, 6, 3c and 3a in descending order.