Unexpected Hydrazinolysis Behaviour of 1-Chloro-4-methyl-5 H-pyridazino [ 4 , 5b ] indole and a Convenient Synthesis of New [ 1 , 2 , 4 ]-Triazolo [ 4 ’ , 3 ’ : 1 , 6 ] pyridazino [ 4 , 5b ] indoles

Reaction of the title compound with hydrazine in the presence of air gives the 1-unsubstituted parent system via oxidative dehydrazination of the 1-hydrazino intermediate. The latter can be obtained in high yield by carrying out the hydrazinolysis step under inert gas, and it is smoothly converted into [1,2,4]-triazolo[4’,3’:1,6]pyridazino[4,5b]indoles.


Introduction
Recently, we reported the synthesis of a series of new azacarbolines of the pyridazino [4,5-b]indole type, including 4-methyl-5H-pyridazino [4,5-b]indole, an aza isoster of the natural product, harman [1].Some of these tricyclic compounds had shown weak to moderate in-vitro cytostatic activity towards several human tumour cell lines.In the course of our ongoing search for new lead structures in the antitumour agent field, we became interested in further modifications of this type of compounds with the pyridazino [4,5-b]indole ring system as the core structure.In particular, annulation of a further ring onto the pyridazine unit, increasing the size of the heteroaromatic chromophore, was envisaged.For this purpose, 1-chloro-4-methyl-5H-pyridazino [4,5-b]indole (1), for which we had developed an efficient synthesis [1] via the corresponding pyridazinone [2], appeared to be a particularly useful key intermediate.The chloro function in 1 should be easily replaceable by a hydrazino group which, in turn, would permit the annulation of a further ring, e.g. a 1,2,4-triazole.Here, we describe the reaction behaviour of the tricyclic chloropyridazine 1 towards hydrazine as well as the convenient synthesis of a series of new [1,2,4]-triazolo [4',3':1,6]pyridazino [4,5-b]indole derivatives.

Results and Discussion
Initial attempts to transform the chloropyridazine 1 into the required 1-hydrazino compound by heating with excess hydrazine hydrate failed and after complete consumption of the starting material (48 hours), the 1-unsubstituted tricycle 2 was isolated in 50% yield as the sole reaction product.This compound had been prepared previously [1] by catalytic hydrogenation of 1 and it was unambiguously identified by its spectral data.The same result was obtained when the thione 3 [1] was employed as a substrate for hydrazinolysis: in this case 2 was obtained in 70% yield (Scheme 1).

N H
Obviously, the initially formed hydrazinopyridazine 4 is very susceptible towards oxidation by air oxygen, and thus undergoes oxidative dehydrazination under the conditions required for nucleophilic displacement of the leaving group at the 1 position.The oxidative removal of a hydrazino group from arylhydrazines is a well-established method [3] and has also found many applications in pyridazine chemistry [4].However, in most cases such transformations require the use of oxidants like copper(II) salts or mercury(II) oxide, although there are also examples in which molecular oxygen acts as the oxidant, typically in strongly alkaline media [5][6][7][8][9][10][11].As a mechanism of the observed transformation, we can propose a dehydrogenation of the N-N bond of the hydrazino function into a diazene structure, followed by spontaneous loss of molecular nitrogen, (Scheme 2).
Consequently, strict exclusion of oxygen during the nucleophilic substitution step should permit the preparation of 4 from its precursor.This was found to be the case: when the chloro compound 1 was refluxed in hydrazine hydrate under argon, a nearly quantitative yield of the hydrazino product was obtained.However, the latter proved to be very unstable on attempted purification or storage and thus was immediately used for further transformations.Treatment of 4 with benzoyl chloride in refluxing dioxane afforded the benzhydrazide 5 as a stable derivative (Scheme 3).The open-chain structure of 5, which was isolated as the hydrochloride, clearly follows from its mass spectrum (M + peak at m/z = 317) and a strong IR absorption band at 1668 cm -1 (C=O stretching).Compound 5 can be smoothly dehydrated by heating in phosphorus oxychloride, affording the fused triazole 6a in high yield.When phenylpropionyl chloride is employed in the reaction with 4, the inititally formed hydrazide cyclizes spontaneously into the phenethyl-substituted triazole 6b.In a similar fashion, heating of 4 in excess acetic anhydride gives 6c.For the synthesis of the 3-unsubstituted and the 3-ethyl congeners 6d and 6e, heating of the hydrazine 4 in the appropriate ortho ester (triethyl orthoformate or triethyl orthopropionate, respectively), was found to be a suitable method.Also with high-boiling carboxylic acid esters, analogous cyclocondensations can be effected, as exemplified by the one-step preparation of the esters 6f and 6g from 4, using diethyl oxalate or diethyl malonate, respectively.The lower yields mainly result from losses during purification.
Another new type of [1,2,4]-triazolo[4',3':1,6]pyridazino [4,5-b]indoles was made available by reacting the hydrazine 4 with carbon-dioxide-type building blocks (Scheme 4).Heating of 4 with 1,1'-carbonyldiimidazole (CDI) in dry dioxane smoothly gave the fused triazolone 7, whereas the corresponding tetracyclic thione 8 was obtained in satisfactory yield on treatment of 4 with carbon disulfide in ethanolic potassium hydroxide.Expectedly, alkylation of the latter compound was found to take place preferentially at the sulfur atom [12].Thus, the two alkylsulfanyl derivatives 9a,b, featuring a basic side chain at position 3 of the condensed system were prepared from 8 by reaction with the respective alkyl chloride in ethanolic solution in the presence of sodium acetate.The location of the newly introduced alkyl residue in 9a,b is supported by the absence of a thiocarbonyl band in the IR spectrum.Moreover, the 1 H-NMR spectra clearly show for both compounds that the only exchangeable hydrogen is attached to the indole nitrogen (7-N), as proven by a NOE which is observed on irradiation of the 8-H resonance.In an in-vitro screening of the new compounds, only 5, 6b, 6e and 9a showed weak to moderate antitumour activity.Cell-growth inhibitory activities generally did not exceed 50% at a fixed sample concentration of 3.16 µg/mL, with the exception of the (diethylamino)ethylsulfanyl-substituted compound 9a (67% growth inhibition for NCI-H460 non-small-cell lung cancer cells, 72% for RKOp27 colon adenocarcinoma cells).

Conclusions
We could demonstrate that hydrazinolysis of the indole-fused chloropyridazine 1 can afford either the 1-unsubstituted 3-azaharman 2 or the 1-hydrazino compound 4, both with preparatively useful yields, and entirely dependent on the presence or absence of oxygen in the atmosphere.The hydrazine derivative 4 was found to be a versatile key intermediate for the synthesis of a series of new [1,2,4]triazolo[4',3':1,6]pyridazino [4,5-b]indoles.

General
Melting points (uncorrected) were determined on a Kofler hot-stage microscope (Reichert). 1 H-NMR spectra were recorded on a Bruker Avance DPX 200 (200 MHz) or on a Varian UnityPlus 300 (300 MHz) spectrometer.IR spectra were taken on a Perkin-Elmer 1605 FT-IR instrument.Mass spectra were obtained on a Shimadzu QP5050A DI 50 instrument, high-resolution mass spectra were recorded on a Finnigan MAT 8230 spectrometer at the Department of Organic Chemistry, University of Vienna.Column chromatography was carried out on Merck Kieselgel 60, 0.063-0.200mm, thin layer chromatography was done on Merck aluminium sheets pre-coated with Kieselgel F 254 .Microanalyses [13] were performed at the Institute of Physical Chemistry (Microanalytical Laboratory), University of Vienna.
Method B: A mixture of the thione 3 [1] (215 mg, 1 mmol) and hydrazine hydrate (2 mL, 0.04 mol) in EtOH (10 mL) was refluxed for 48 h.The volatile components were removed under reduced pressure and the residue was triturated with water (10 mL).The product was collected by filtration and recrystallized from EtOH to give 2 (129 mg, 70%) as colourless crystals (see above).