Synthesis of tetra-and pentacyclic carbazole-fused imides as potential antitumor agents

A series of tetra-and pentacyclic imides with a carbazole skeleton and a basic side chain at the imide nitrogen was synthesized by cyclization of carbazole-2,3-dicarboxylic acid esters with an appropriate amine or via an N -aminoimide as a reactive intermediate. The target compounds 6 and 9 were tested in vitro for tumor cell-growth inhibition


Introduction
Based on the planar, aromatic tetracyclic skeleton of the antitumor alkaloids, ellipticine and olivacine, 1 a number of drug candidates with enhanced antineoplastic properties has been developed in the past decades. 2Examples include compounds like retelliptine, 3 pazelliptine, 4 datelliptium, 5 or S16020-2. 6It has been shown that the presence of a basic side chain of the N,Ndialkylaminoalkyl type enhances the drugs' DNA affinity. 3The latter is essential for the biochemical mode of action, through stabilisation of the complex formed between DNA and the enzyme, topoisomerase II. 7Among the numerous drugs targeting topoisomerase II, also a group of compounds featuring a naphthalene (or higher annulated) ring system fused to a sixmembered cyclic imide structure with a basic N-substituent has received considerable attention.The drug molecules, amonafide 8 and azonafide 9 can be regarded as prototypes for this type of agent.Remarkably, amonafide as well as the pyrido [4,3-b]carbazole derivative, S16020-2 have been found to escape the P-glycoprotein-mediated multi-drug resistance, 10,11 the latter being a common problem in tumor chemotherapy.As it has been claimed that certain carbazoles fused to a N-substituted cyclic imide also exhibit pronounced antitumor properties, 12,13 we became interested in the combination of some structural features of ellipticine-type and amonafide-type agents, in continuation of our previous studies aimed at the synthesis and antitumor activity of heterocycle-annulated carbazoles. 14,15Here, we wish to report on the preparation of a series of new tetra-and pentacyclic compounds with a carbazole-2,3-dicarboximide core structure and their in-vitro tumor cell-growth inhibitory activity.

Results and Discussion
The easily accessible diester 1a 17 (Scheme 2) was chosen as a synthon featuring the 1,4dimethylcarbazole motif of ellipticine as well as the required functional groups which should be suitable for the construction of an imide unit.Initial experiments showed that 1a can be transformed into a N-substituted carbazole-2,3-dicarboximide derivative simply by heating with an excess of a high-boiling amine such as benzylamine (affording compound 2), but reacts much more sluggishly with low-boiling amines.In the latter case, e.g. for the preparation of the Nbutyl substituted imide 4, employment of a reactive intermediate is necessary. 18For this purpose, the N-aminoimide 3 19 which is easily formed on treatment of 1a with hydrazine hydrate, can be conveniently used as an "anhydride equivalent", thus giving 4 in good yield upon refluxing in excess n-butylamine.The formation of the five-membered cyclic imide structure is clearly evidenced by the characteristic IR absorption bands of the products at 1740-1750 cm -1 and 1680-1700 cm -1 , apart from their mass spectra and microanalytical data.For the preparation of the N-unsubstituted parent compound 5, heating of 1a in formamide/formic acid was found to be the method of choice, affording 5 in 88% yield.Introduction of the desired basic side chain was accomplished by prolonged heating of the diester 1a with excess N,N-diethyl-1,3-propanediamine in DMSO solution under argon atmosphere.This method, which is similar to that described for the preparation of carbazole-1,2-dicarboximides from the corresponding diesters, 13 gave the diethylaminopropyl-substituted target compound 6a in high yield.In an analogous fashion, the mono-methyl-substituted carbazole-2,3-diester 1b 20 (lacking the methyl group at position 4) was smoothly cyclized into the imide 6b.

Scheme 2
Enlarging the tetracyclic skeleton into a pentacyclic one, introduction of a three-carbon bridge between the indole nitrogen and the adjacent ring C was envisaged, thus adding a structural feature of the cytotoxic canthine alkaloids. 21The requisite carbazolediesters of type 7 (Scheme 3) had been made available by us previously, 15 making use of an intramolecular inverse-electron-demand Diels-Alder reaction of appropriately substituted 3-[3-(indol-1yl)propyl]pyridazine-4,5-dicarboxylates.A similar cycloaddition approach had been used by Snyder's group for the construction of bridged β-carbolines. 16Interestingly, the tetracyclic esters 7a,b did not undergo direct cyclization into the target imides by heating with excess primary amine under various conditions, despite their steric and electronic similarity to the ester 1a.However, 7a,b could be easily transformed into the pentacyclic N-aminoimides 8a,b.Like in the case of compund 3, these structures were found to be sufficiently reactive in order to undergo an exchange reaction with excess amine, thus furnishing the desired N-substituted imides 9a,b in good yields. 15-76%

Scheme 3
The target compounds 6a,b and 9a,b as well as the N-unsubstituted imide 5 which represents the core structure were tested in vitro for tumor cell-growth inhibition, using the XTT assay. 22The results are summarized in Table 1, they show that 6a is superior as compared to 6b, the latter lacking the second methyl group at the aromatic scaffold.Likewise, when 9a is compared to its methoxy derivative 9b, the latter structural modification is clearly beneficial.Expectedly, the reference compound 5 with an unsubstituted imide nitrogen shows only very weak activity, which demonstrates the importance of the basic side chain in this type of agent.At concentrations lower than 3.16 µg/mL, also compounds 6a,b and 9a,b exhibited only weak to moderate effect.In conclusion, a series of tetra-and pentacyclic imides with a carbazole skeleton and a basic side chain attached to the imide nitrogen has been made conveniently available either by direct cyclization of a carbazole-2,3-diester with an appropriate primary amine or by a two-step sequence involving an N-aminoimide as a more reactive intermediate.The target compounds show significant tumor cell-growth inhibition in vitro at a concentration of about 10 µmol/L.

Experimental Section
General Procedures.Melting points were determined on a Kofler hot-stage microscope.IR spectra (KBr pellets) were recorded on a Perkin-Elmer 1605 FT-IR instrument. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded on a Varian Unityplus 300 spectrometer (δ values in ppm).Mass spectra were obtained on a Shimadzu QP 5050A DI 50 instrument, highresolution mass spectra (HRMS) were recorded on a Finnigan MAT 8230 spectrometer at the Institute of Organic Chemistry, University of Vienna.For column chromatography, Merck Kieselgel 60 (0.063-0.200 mm) was used.Microanalyses 23 were performed at the Microanalytical Laboratory, Faculty of Chemistry, University of Vienna.
The almost colorless, crystalline material was collected by filtration and the filtrate was subjected to column chromatography (AcOEt/light petroleum/Et