Electrophilic Substitution of Dimethyl 1-Methylcarbazole-2 , 3-dicarboxylate : Synthesis of New b-Fused Carbazoles as Potential Antitumor Agents

O ácido dimetiléster 1-metilcarbazol-2,3-dicarboxílico, um intermediário chave na síntese de carbazóis b-fusionados, foi transformado com êxito nos seus 6-bromo e 6-nitro derivados via uma substituição eletrofílica numa solução de ácido acético, usando como reagentes N-bromosuccinimida ou nitrato de uréia, respectivamente. Com a N-clorosuccinimida foi também obtido o derivado 6,8-dicloro congênere, em quantidade apreciável. Os carbazodiésteres 6-substituídos foram usados como matéria-prima para a preparação de vários novos derivados piridazinoou pirrolocarbazóis fusionados com cadeias laterais básicas, levando à estrutura básica de compostos antitumorais previamente desenvolvidos.


Introduction
Polycyclic heteroaromatic compounds featuring a b-fused carbazole skeleton have been known for some time as anticancer agents.As prototypical lead structures can be regarded the pyrido [4,3-b]carbazole alkaloids, ellipticine and olivacine 1-5 (Figure 1), which are targeting the DNA-topoisemerase-II complex.Their scaffolds (sharing a methyl group at position 5) have been used as the core motif for a larger number of structural variations, leading to compounds with significantly enhanced activity, such as the drugs (or drug candidates, respectively) elliptinium, 6  datelliptium, 7 retelliptine, 8 pazelliptine, 9 or S16020-2. 10 Among several other research groups, 11 we had previously reported such structural modifications, 12  focusing mainly on the bioisosteric replacement of the pyridine unit (ring D) of the pyrido [4,3-b]carbazole system with a pyridazine ring. 13,14Moreover, we had also employed a pyrimidine 15 and a pyrrole subunit 16 as ring D of such tetracyclic compounds, and various side chains have been introduced into this region and evaluated for their effect.In the context of our department's ongoing research program in the field of antitumor agents, 12,17-20  we became interested in the systematic enlargement of our compound library by also modifying the substitution pattern of ring A. This region of the scaffold is known to be relevant in terms of binding affinity, and some examples among the drug molecules mentioned above demonstrate that, e.g., an oxygen substituent at ring A is advantageous. 5A comparable effect had been observed previously by ourselves in the pyridazino [4,5-b]carbazole series, 13 but the limited synthetic accessibility of the corresponding building blocks had prevented us from more extensive investigations.In particular, a 6-methoxy-1-methylcarbazole-2,3-dicarboxylic ester which served as the key intermediate had to be prepared in a multistep synthesis from 5-methoxyindole 13 and such an approach was found to be unattractive for broader variation of the target structures.Here, we wish to report on the facile introduction of heteroatom substituents into position 6 of the 1-methylcarbazole-2,3-dicarboxylate intermediate by electrophilic substitution and on the exploitation of these new synthons for the preparation of several hitherto inaccessible b-fused carbazoles as potential antitumor agents.

Chemistry
Electrophilic aromatic substitution was regarded as the method of choice for the synthesis of 6-halo and 6-nitro derivatives of the 1-methylcarbazole-2,3-dicarboxylic ester (1), because one of the two benzene rings in this substrate is strongly deactivated by the electron-withdrawing ester groups, whereas the other benzene unit should be sufficiently reactive towards electrophilic reagents.The directing effect of the (unprotected) NH function should ensure preferential attack of electrophiles at positions 6 and 8, with the latter site being somewhat less favored due to steric reasons. 21This assumption could be verified when we subjected the ester 1 (which is an easily accessible synthon) 22,23 to bromination with a slight excess of N-bromosuccinimide (NBS) in acetic acid solution at room temperature under sonication (Scheme 1), as described recently for pyrrolo [3,2-b]quinolone derivatives. 24After 1 hour, the starting material was completely consumed and a single product was formed.This compound was isolated in 68% yield and it was found to be the 6-bromo derivative 2, based on its 1 H nuclear magnetic resonance (NMR), 13 C NMR, infrared (IR), mass spectrometry (MS) and microanalytical data.In particular, the pronounced nuclear Overhauser enhancement (NOE) which can be observed between 4-H (singlet at 8.40 ppm) and 5-H (singlet at 8.12 ppm) and another NOE between the NH proton (broad singlet at 8.58 ppm) and 8-H (doublet at 7.30 ppm, J 8.7 Hz) are indicative for the substitution pattern in 2.
In an attempt to also prepare the analogous 6-chloro compound, the ester 1 was treated with a slight excess of N-chlorosuccinimide (NCS) under identical conditions as described above.However, at room temperature there was almost no conversion, obviously because of the lower reactivity of NCS compared to NBS.Thus, the reaction temperature was increased to 117 °C (reflux temperature of acetic acid), which effected complete consumption of the substrate within 2 hours, albeit at the expense of regioselectivity (Scheme 1).In contrast to the smooth and selective transformation of 1 into the 6-bromo compound 2, two products were formed in the chlorination reaction.After chromatographic separation, the main product (isolated in 59% yield) was identified as the desired 6-chloro compound 3, whereas the minor product (22% yield) was found to be the 6,8-dichloro derivative 4. Performing the chlorination with a smaller amount of reagent gave a mixture of 3, 4 and unreacted 1, whereas the yield of the dichloro ester 4 could be raised to 72% by employing two equivalents of NCS.As we were also interested in 1-methylcarbazole-2,3dicarboxylic esters with a nitro substituent at position 6, the ester 1 was subjected to nitration both under classical conditions (concentrated nitric acid) as well as with the solid reagent, urea nitrate, 25 in acetic acid solution (Scheme 2).Similar results were obtained with both methods, but the slightly better yields and easier reagent handling made the variant with urea nitrate the method of choice, although a larger excess of reagent (3 equivalents) had to be employed in order to achieve complete conversion, as opposed to just one equivalent which had been described for use with non-deactivated carbazoles as substrates. 25The resulting mixture was separated by column chromatography to afford the 6-nitro compound 5 as the main product (70% yield) and the 8-nitro isomer 6 as the minor component (27% yield).No disubstitution was observed in this case, which is obviously due to the strongly deactivating effect of the first nitro group that is introduced.
The three 6-substituted 1-methylcarbazole-2,3dicarboxylic esters 2, 3 and 5 are well suited for the construction of pyridazine-fused carbazole derivatives (featuring a 3-aza-ellipticine/olivacine skeleton), as demonstrated by their smooth cyclization into the tetracycles 7, 8 and 9 upon heating in neat hydrazine hydrate (Scheme 3), in analogy to previously reported cyclocondensation reactions of similar building blocks. 16,26 These pyridazinediones can exist in different tautomeric forms, of which the dihydroxy form can be safely excluded at least for the solid state: the IR spectra of all compounds show a strong C=O stretching band at 1630-1650 cm -1 .In a solution of dimethyl sulfoxide-d 6 (DMSO-d 6 ), tautomeric exchange processes can be assumed, as indicated by marked signal broadening of the C=O carbon resonances in the 13 C NMR spectra, in particular for the halogen compounds.
The bromo derivative 7, being most conveniently accessible from the precursor 1 as described above, was chosen for further structural modification with the aim of increasing the molecule's solubility as well as its DNA affinity.For this purpose, the introduction of basic side chains is known to be a successful strategy. 8Thus, the pyridazinedione 7 was first transformed into a 1,4-dichloro derivative (10) by heating in phosphorus oxychloride in the presence of N,N-diethylaniline, which afforded 10 in almost quantitative yield.This reactive intermediate was then treated with excess N,N-diethylpropane-1,3-diamine in DMSO solution at 130 °C.After 3 hours, the dichloro compound was completely consumed and the formation of two products was detected by thin layer chromatography (TLC).The mixture could be separated by column chromatography, giving the two isomeric monosubstitution products in yields of 36% (11a) and 17% (11b), respectively (Scheme 4).The position of the newly introduced alkylamino residues clearly follows from NOE experiments which show the spatial proximity of the alkylamino NH and the adjacent 11-H (for compound 11a) or the 5-methyl group (for compound 11b).Interestingly, no disubstituted product was found in the reaction mixture despite the excess of nucleophile and high reaction temperature.This is obviously caused by the strongly deactivating effect of the first alkylamino group that is introduced.Replacement of both chloro functions with N,N-diethylaminopropylamino side chains could be finally accomplished under even more drastic conditions, by refluxing 10 in the neat amine for 7 days under argon atmosphere.After work-up, the 1,4-disubstituted compound 12 was obtained in 62% yield.Like the two monosubstitution products 11a and 11b, compound 12 was selected for evaluation of its in vitro cytotoxicity.
As a further structural modification, we envisaged ring closure of the ortho-diester function with an appropriate primary amine to afford carbazole-fused cyclic imides, again with a basic side chain attached to ring D of the tetracyclic skeleton thus formed.Previous investigations 16 had shown that the corresponding ring-A-unsubstituted pyrrolo [3,4-b] carbazole-1,3(2H,5H)-diones (bearing one or two methyl groups at ring C) can significantly inhibit the growth of various tumor cells in vitro.Accordingly, our new diesters 2 and 5 were subjected to this type of cyclocondensation with N,N-diethylpropane-1,3-diamine under the conditions we had previously used (prolonged heating with 10 equivalents of amine in DMSO solution at 130 °C).By this method, the 8-bromo compound 13 and the 8-nitro congener 14 could be obtained in acceptable yields (58 and 57%, respectively, Scheme 5).Also in this case, the spectral data of the new compounds are in excellent agreement with those of their ring-A-unsubstituted counterparts. 16

Biological evaluation
Compounds 11a, 11b, 12, 13 and 14 were tested in vitro for their cytotoxic activity against the human colon carcinoma cell line, SW480, at a fixed concentration of 10 µM, using the well-established XTT assay. 27This is a colorimetric assay which measures the activity of the mitochondrial dehydrogenase of living cells.The water soluble, yellow-colored 2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) is taken up by living cells and it is reduced to an intensely colored formazan dye by a mitochondrial dehydrogenase.As the amount of product is proportional to the number of viable cells in the culture, this assay is suitable to assess the number of viable cells.The tested compounds showed moderate inhibitory activity of 15% (for 13), 83% (for 11b), 83% (for 11a), 63% (for 12), and 84% (for 14).In addition, compounds 11b and 13 were tested against a small panel of different cell lines consisting of A549 (lung carcinoma), Hep3b (hepatocarcinoma), U373 (glioblastoma) and HTB65 (melanoma) at two fixed concentrations of 10 and 5 µM.The results are summarized in Table 1: only at the higher concentration, significant inhibition of tumor cell growth was observed.Interestingly, the two compounds show (at least partially) marked differences in cell-type specificity, with 13 being most active against HTB65 and 11b against the SW480 line.One must conclude, however, that in comparison with our previous lead structures, 13,16 the introduction of electron-withdrawing substituents at ring A does not improve cytotoxic activity.

Conclusions
We have demonstrated that the diester 1, a key intermediate for the preparation of b-fused carbazoles, can be efficiently and selectively brominated at position 6 with NBS in acetic acid under sonication at room temperature.In contrast, chlorination of 1 with NCS lacks this selectivity and gives also disubstitution at positions 6 and 8. Nitration with urea nitrate in acetic acid affords the 6-nitro compound in satisfactory yields along with minor amounts of the 8-nitro isomer.Expectedly, the functionalized diesters 2 and 5 could be smoothly cyclized into the tetracyclic imides 13 and 14, and compound 2 was transformed into the mono-or dialkylamino-substitited fused pyridazines 11a,b and 12 via dichloropyridazine 10.The target compounds showed only weak to moderate activity in an in vitro antitumor assay.On the other hand, our synthetic route offers some potential for further structural modifications, especially in view of the versatility of the bromo and the nitro functionality.

Table 1 .
In vitro evaluation of 11b and 13 for cytotoxic activity against human tumor cells: percentage inhibition of tumor cell growth room temperature in an ultrasound bath.The solvent was distilled off under reduced pressure and the residue was taken up in CH 2 Cl 2 (200 mL).This solution was washed consecutively with 5% aqueous Na 2 SO 3 (50 mL) and water (2 × 50 mL), then it was dried (Na 2 SO 4 ) and evaporated.