Synthesis and In-vitro Antitumor Activity of 1-[3-(Indol-1-yl)prop-1-yn-1-yl]phthalazines and Related Compounds

A series of novel 3-(indol-1-yl)prop-1-yn-1-yl-substituted phthalazines and related azines was prepared via a concise pathway by palladium-catalyzed cross-coupling of appropriate halo-azines and N-propargylindoles. Some of the compounds exhibited significant antitumor activity in an in-vitro assay.


Introduction
During the course of a research program at our department, focusing on the synthesis and antitumor activity of polycyclic hetarenes, especially condensed carbazoles of the ellipticine/olivacine type [1][2][3][4][5] (cf. Figure 1) and polycyclic quinones [6][7][8], we recently described the preparation of novel pentacyclic ellipticine analogs via a route featuring an intramolecular inverse-electron-demand Diels-Alder reaction of indolylpropyl-substituted 1,2-diazines as the key step (Scheme 1) [9].In a routine invitro screening for cytotoxic activity, not only the target compounds, but also one of the intermediates, namely 1-[3-(indol-1-yl)prop-1-yn-1-yl]phthalazine, showed significant tumor cell-growth inhibition.Therefore, this compound with a 1,3-disubstituted propyne unit as the central element was selected as a new lead structure for further exploratory investigations.Here, we report on the synthesis and the results of preliminary in-vitro antitumor tests of a focused compound library featuring the same propyne motif with one electron-rich and one electron-deficient hetarene attached at the terminal carbon atoms.Scheme 1. Synthesis of bridged ellipticine/olivacine analogs [9].

Syntheses
Following the first step of the pathway above (Scheme 1), the target compounds were prepared essentially by a Sonogashira cross-coupling reaction of an appropriate propargyl-substituted indole or indoline synthon with an iodohetarene or bromohetarene, respectively.Two series of compounds were synthesized, keeping always one of the two heterocyclic subunits constant (either the indole or the azine) and varying the other one.The propynyl-substituted educts were obtained in good yields by treatment of the N-unsubstituted precursors with propargyl bromide in toluene/50% sodium hydroxide, using tetrabutylammonium bromide as a phase-transfer catalyst [10].

An improved synthesis of 1-iodophthalazine
During the preparation of the requisite starting materials, it turned out that the transformation of 1-chlorophthalazine into 1-iodophthalazine by treatment of the former with potassium iodide and hydroiodic acid in acetone, as described by Hirsch and Orphanos [11], gives very unreliable results if the original work-up procedure is applied (dissolving the initially formed hydroiodide salt of 1-iodophthalazine in water, followed by neutralization and filtration of the free base).In several runs, the compound underwent complete decomposition within a very short time.We found that this crude material is highly acid-sensitive and is prone to an autocatalytic decomposition process if exposed to traces of acid.Therefore, it is essential to keep the pH after liberation of the free base strictly alkaline.Instead of collecting the product by filtration, it is extracted quickly into dichloromethane and the organic extract is immediately basified by addition of triethylamine (see Experimental section).Evaporation of this solution gives a pure product which can be stored under refrigeration for several weeks.

Biological Activity
All compounds were subjected to a preliminary screening for antitumor activity at a fixed sample concentration of 3.16 µg/mL, using the XTT in-vitro assay [19].As can be concluded from the results (see Table 1), replacement of the phthalazine unit by a monocyclic 1,2-diazine (3i) or by an isoquinoline (3c, 3d) leads to a marked drop in activity, whereas substitution of the phthalazine at position 4 [methyl (3a) or phenyl (3b)] is well tolerated.In the indole part of the molecule, the 5methoxy substituent appears to be most favorable among the variations studied.Replacement of an indole by an indoline structure (3e) results in lower activity.

Conclusions
Sonogashira reaction of 1-iodophthalazines and related haloazines with the terminal acetylene unit of N-propargylindoles provides a convenient access to the title compounds, which are of pharmaceutical interest due to their activity in an in-vitro antitumor screen.Further investigations will be required for a more detailed evaluation of these agents.

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 Institute 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 were performed at the Microanalytical Laboratory, Faculty of Chemistry, University of Vienna.

Synthesis of Compounds 3 by Pd-Catalyzed Cross-Coupling
Reaction.General Procedure.To a solution of the aryl halide 2a, 2b, 2c, 2d, or 2e (2.6 mmol), respectively, and the appropriate alkyne 1a, 1b, 1c, or 1d (3.25 mmol), respectively, in dry THF (6 mL) were added triethylamine (1.0 mL, 7.2 mmol), CuI (0.015 g, 3 mol%) and Pd(PPh 3 ) 2 Cl 2 (0.055 g, 3 mol%), and the mixture was flushed with argon.It was then stirred under an argon atmosphere under the conditions (room temperature or reflux) and for the time listed in Table 1.The insoluble material was filtered off and washed carefully with THF.The combined filtrates were evaporated under reduced pressure and the residue was purified by column chromatography (eluent: ethyl acetate or ethyl acetate/light petroleum).

Table 1 .
Structures, reaction conditions, yields, and biological activities for the title compounds.