Synthesis of 3-azaharman and other new azacarbolines of the pyridazino[4,5-b ]indole type

Starting from the indole-fused pyridazinone 3 , a series of new pyridazino[4,5-b ]indoles, functionalized at positions 1, 2, or 5 was prepared, including the two tetracyclic compounds 12 and 13 , which represent new ring systems. Reductive dehalogenation of the chloro compound 8 gave a 3-aza isoster of the natural product, harman.


Introduction
The pyridazino [4,5-b]indole ring system (A) has been known for several decades and so far a variety of biological activitiy has been reported for a large number of its derivatives, such as antihypertensive, 1 antiarrhythmic, 2 positive inotropic, 3 thromboxane A 2 synthetase inhibitory, 4 MAO inhibitory, 5 serotonine antagonistic, 6 antihistaminic, 7 anxiolytic, 8 or HIV-1 reverse transcriptase inhibitory 9 activities.Moreover, the pyridazino [4,5-b]indole ring system can be regarded as an aza analog of β-carboline as well as γ-carboline which both, in turn, are the parent systems of many other bio-active natural and synthetic compounds.In this context, the 1-methylβ-carboline, harman (B), 10 and its cytotoxic congeners as well as the antitumor γ-carbolines of type C (Figure 1) should be particularly mentioned.The latter compounds 11 had been designed as tricyclic analogs of tetracyclic anticancer agents of the ellipticine type, and they had been found to exhibit significant cytotoxic activity despite "shrinking" the pyrido [4,3-b]carbazole into a pyrido [4,3-b]indole scaffold.As we had previously demonstrated that also pyridazino [4,5b]carbazoles (3-azaellipticines) bearing appropriate substituents show comparable activity in antitumor assays, 12 we became interested in the synthesis of 4-methylpyridazino [4,5-b]indoles as tricyclic analogs of these 3-azaellipticines, representing another new type of potential antitumor agents.

Results and Discussion
The key intermediate 3 (Scheme 1) had been prepared previously by Zhungietu et al. 13 by condensation of 2-acetylindole-3-carboxylic acid 1 with hydrazine hydrate at elevated temperature.This reaction, however, suffers from poorly reproducible yields which reflect the high decarboxylation tendency 14 of the starting material, causing a significant side reaction (formation of 2-acetylindole or its hydrazone, respectively).We now could substantially improve this step by first transforming the keto acid 1 into a suitable activated derivative under mild conditions and subsequent treatment of this intermediate with hydrazine hydrate.For this purpose, the imidazolide 2 was found to be a good choice, as it can be easily prepared in an inert solvent at room temperature by treatment of 1 with 1,1'-carbonyldiimidazole (CDI), and without isolation this compound smoothly undergoes hydrazinolysis to afford the pyridazinone 3. Also monosubstituted hydrazines can be employed in the cyclization reaction with 2 to afford the 2substituted products 4a and 5, although yields are somewhat lower and, in the case of methylhydrazine, formation of small amounts of an isomeric side product 4b was observed.

Scheme 1
On the other hand, it was found that alkylation of the condensed pyridazinone 3 preferentially takes place at the indole nitrogen.Thus, reaction of 3 with one equivalent of diethylaminoethyl chloride in the presence of potassium carbonate in dimethylformamide solution gives the 5substituted product 6 in moderate yield, whereas employment of two equivalents of the alkylating agent affords the 2,5-disubstituted compound 7 (Scheme 2).

Scheme 2
As an intermediate for functionalization at the 1-position, the chloropyridazine 8 was prepared in excellent yield by heating 3 in phosphorus oxychloride (Scheme 3).This compound turned out to be remarkably inert towards nucleophilic attack.For instance, 8 can be easily recrystallized from boiling ethanol/acetonitrile (2:1) without noticeable solvolysis.Obviously, this lack of reactivity is mainly caused by considerable steric shielding of the chloro function by the 9-H atom at the benzene ring, in addition to electronic factors (annulation of an electron-rich indole system onto the chloropyridazine moiety).Nucleophilic substitution of the chloro function in 8 with amines requires relatively harsh conditions, e.g.heating in a high-boiling amine in the absence of a solvent.By this method, the benzylamino compound 9 could be obtained in good yield.Likewise, the potential anticancer agent 10, bearing a 3-(diethylamino)propylamino side chain as well as the hydroxyethylamino derivative 11 were prepared, albeit in lower yields owing to work-up losses and some decomposition during the substitution reaction.The alcohol 11, when heated in thionyl chloride, is transformed into the corresponding chloro derivative which spontaneously cyclizes into the imidazo[2',1':6,1]pyridazino [4,5-b]indole 12 (obtained as the hydrochloride), which represents a new ring system.Another representative of a hitherto unknown ring system, the tetrazolo[5',1':6,1]pyridazino [4,5-b]

Scheme 3
Attempts to convert the pyridazinone 3 into the corresponding thione by refluxing with phosphorus pentasulfide in pyridine gave only a very low yield of the desired compound, whereas employment of Lawesson's reagent met with a complete failure.However, reaction of the chloropyridazine 8 with thiourea in ethanol, followed by alkaline hydrolysis of an intermediate isothiourea derivative (14) was found to afford the pyridazinethione 15 in satisfactory yield (Scheme 4).Expectedly, reaction of this compound with alkylating agents takes place at the sulfur atom exclusively, as demonstrated by the transformation of 15 into the alkylsulfanyl compounds 16-18, which are obtained by treatment of the thione with methyl iodide, diethylaminoethyl chloride, or ethyl bromoacetate, respectively, in ethanolic solution in the presence of a weak base (sodium acetate).The position of the newly introduced substituent clearly follows from NOE difference spectra which confirm the proximity of the S-alkyl residue and the 9-H proton.
In contrast to the sluggish nucleophilic displacement reactions with the chloropyridazine 8, reductive dehalogenation takes place very smoothly when 8 is subjected to catalytic transfer hydrogenation in refluxing methanol, employing ammonium formate as the hydrogen source and palladium on carbon as the catalyst.Thus, the 1-unsubstituted tricycle 19 which represents an aza isoster of the natural product, harman, is obtained in 64% yield.In a preliminary in-vitro screening of the new azacarbolines, only compounds 6, 7, 9, 10, 11, and 13 showed weak to moderate antitumor activity, with cell-growth inhibitory activities generally not exceeding 50% at a fixed sample concentration of 3.16 µg/mL.Further investigations aiming at the synthesis of new functionalized and/or annulated derivatives of the pyridazino [4,5-b]indole system with potential biological activity are in progress.

indole 13, was prepared from 8 in
a single step by refluxing with excess sodium azide in dimethylformamide solution.