Electrophilic substitution of dimethyl 1-methylcarbazole-2,3-dicarboxylate: synthesis of new b-Fused carbazoles as potential antitumor agents

ARTICLE

Electrophilic substitution of dimethyl 1-methylcarbazole-2,3-dicarboxylate: synthesis of new b-Fused carbazoles as potential antitumor agents

Norbert Haider^{*, I}; Brigitte Marian^II; Thomas Nagel^I; Markus Tarnai^I; Katharina Tropper^I

^IDepartment of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

^IIInstitute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria

ABSTRACT

1-Methylcarbazole-2,3-dicarboxylic acid dimethyl ester, a key intermediate in the synthesis of b-fused carbazoles, was successfully transformed into its 6-bromo and 6-nitro derivative via electrophilic substitution in acetic acid solution, using N-bromosuccinimide or urea nitrate, respectively, as reagents. With N-chlorosuccinimide, a 6,8-dichloro congener was also obtained in substantial amounts. The 6-substituted carbazolediesters were used as building blocks for the preparation of several new pyridazine- or pyrrole-fused carbazoles with basic side chains, featuring the core structure of previously developed antitumor compounds.

Keywords: carbazoles, electrophilic substitution, bromination, nitration, antitumor activity

RESUMO

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 piridazino- ou 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.^13,14 Moreover, we had also employed a pyrimidine¹⁵ and a pyrrole subunit¹⁶ 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.⁵ A 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-methoxyindole¹³ 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.

Results and Discussion

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.²¹ This 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.²⁴ After 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 ¹H nuclear magnetic resonance (NMR), ¹³C 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 ºC (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-2,3-dicarboxylic 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.²⁵ The 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-2,3-dicarboxylic 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-d₆ (DMSO-d₆), tautomeric exchange processes can be assumed, as indicated by marked signal broadening of the C=O carbon resonances in the ¹³C 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.⁸ Thus, 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 ºC. 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 investigations¹⁶ 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 ºC). 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.²⁷ This 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-5-sulfophenyl)-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.

Experimental

Melting points were determined on a Kofler hot-stage microscope and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1605 FT-IR instrument. ¹H NMR (300 MHz) and ¹³C NMR spectra (75 MHz) were obtained on a Varian UnityPlus 300 spectrometer, using the residual signals of undeuterated solvent for calibration; chemical shift values (d) are reported in ppm, coupling constants (J) in Hz. Mass spectra (electron ionization, EI, 70 eV) were measured on a Shimadzu QP5050A DI50 instrument, high-resolution mass spectra were recorded on a Finnigan MAT 8230 at the Faculty of Chemistry, University of Vienna. Elemental analyses were performed at the Microanalytical Laboratory, Faculty of Chemistry, University of Vienna. For TLC, Merck aluminum sheets pre-coated with Kieselgel 60 F₂₅₄ were used; column chromatography was done on silica (Merck Kieselgel 60, 0.063-0.200 mm). Dimethyl 1-methyl-9H-carbazole-2,3-dicarboxylate (1) was prepared according to literature procedures.^22,23

Dimethyl 6-bromo-1-methyl-9H-carbazole-2,3-dicarboxylate (2)

To a suspension of 2.97 g (10 mmol) of the ester 1 in glacial acetic acid (100 mL) were added 1.87 g (10.5 mmol) of N-bromosuccinimide and the mixture was sonicated for 60 min at room temperature in an ultrasound bath. The solvent was distilled off under reduced pressure and the residue was taken up in CH₂Cl₂ (200 mL). This solution was washed consecutively with 5% aqueous Na₂SO₃ (50 mL) and water (2 × 50 mL), then it was dried (Na₂SO₄) and evaporated. The residue was recrystallized from methanol to afford 2.55 g (68%) of 2 as almost colorless needles, m.p. 220-221 ºC; IR (KBr) ν_max/cm^-1 3450, 3322, 2950, 1706, 1576, 1480, 1438, 1288, 1246, 1152, 1058, 786; ¹H NMR (300 MHz, CDCl₃) d 2.42 (s, 3H, 1-CH₃, shows positive NOE on irradiation at 8.58 ppm), 3.92 (s, 3H, OCH₃), 4.01 (s, 3H, OCH₃), 7.30 (d, 1H, J 8.7 Hz, 8-H, shows positive NOE on irradiation at 8.58 ppm), 7.51 (dd, 1H, J 8.7, 1.8 Hz, 7-H), 8.12 (s, 1H, 5-H, shows positive NOE on irradiation at 8.40 ppm), 8.40 (s, 1H, 4-H), 8.58 (s, 1H, NH); ¹³C NMR (75 MHz, CDCl₃) d 13.7, 52.3, 52.7, 112.7, 113.5, 117.6, 119.3, 121.3, 121.4, 123.5, 125.0, 129.6, 133.0, 138.8, 141.3, 166.6, 170.4; MS m/z (%) 377 (M⁺, 53), 375 (M⁺, 51), 346 (54), 345 (40), 344 (59), 343 (54), 331 (45), 330 (47), 329 (49), 328 (28), 301 (21), 300 (20), 287 (56), 285 (75), 259 (85), 258 (33), 257 (75), 213 (23), 179 (42), 178 (94), 177 (66), 172 (36), 166 (22), 164 (30), 162 (22), 152 (37), 151 (52), 150 (77), 149 (20), 139 (26), 89 (32), 83 (25), 77 (36), 76 (31), 75 (37), 75 (71), 74 (31), 73 (47), 71 (38), 69 (78), 67 (34), 63 (61), 62 (32), 59 (41), 57 (100), 55 (81), 51 (38); anal. calcd. for C₁₇H₁₄BrNO₄: C, 54.27%; H, 3.75%; N, 3.72%; found: C, 53.97%; H, 3.34%; N, 3.61%.

Dimethyl 6-chloro-1-methyl-9H-carbazole-2,3-dicarboxylate (3)

To a solution of 0.297 g (1 mmol) of the ester 1 in glacial acetic acid (20 mL) were added 0.147 g (1.1 mmol) of N-chlorosuccinimide. The mixture was sonicated for 10 min at room temperature in an ultrasound bath, and then it was refluxed with stirring for 2 h. The solvent was distilled off under reduced pressure and the residue was taken up in CH₂Cl₂ (40 mL). This solution was washed consecutively with 5% aqueous Na₂SO₃ (20 mL) and water (2 × 20 mL), then it was dried (Na₂SO₄) and evaporated. The residue was subjected to column chromatography, eluting with light petroleum/ethyl acetate (2:1, v/v). Evaporation of the first fraction gave 0.080 g (22%) of the dichloro compound 4 (for characterization details, see below), evaporation of the second fraction yielded 0.196 g (59%) of 3 as colorless crystals, m.p. 183-185 ºC (methanol); IR (KBr) ν_max/cm^-1 3320, 2950, 1732, 1694, 1606, 1440, 1340, 1292, 1264, 1150, 1060, 780, 598; ¹H NMR (300 MHz, CDCl₃) δ 2.42 (s, 3H, 1-CH₃, shows positive NOE on irradiation at 8.62 ppm), 3.92 (s, 3H, OCH₃), 4.01 (s, 3H, OCH₃), 7.34 (d, 1H, J 8.4 Hz, 8-H, shows positive NOE on irradiation at 8.62 ppm), 7.38 (dd, 1H, J 8.7, 1.8 Hz, 7-H), 7.96 (d, 1H, J 1.5 Hz, 5-H, shows positive NOE on irradiation at 8.40 ppm ), 8.40 (s, 1H, 4-H), 8.62 (s, 1H, NH); ¹³C NMR (75 MHz, CDCl₃) d 13.7, 52.3, 52.7, 112.3, 117.6, 119.2, 120.5, 121.3, 121.5, 124.4, 126.2, 127.0, 132.9, 138.5, 141.5, 166.7, 170.5; MS m/z (%) 333 (M⁺, 26), 331 (M⁺, 60), 302 (23), 301 (38), 300 (68), 299 (68), 286 (21), 285 (69), 284 (64), 243 (29), 242 (27), 241 (90), 215 (31), 214 (46), 213 (100), 212 (30), 179 (22), 178 (46), 177 (35), 152 (26), 151 (39), 150 (91), 83 (22), 77 827), 76 (28), 75 (67), 74 (23), 73 (39), 71 (41), 69 (67), 67 (25), 63 (24), 59 (27), 57 (83), 56 (20), 55 (59), 51 (38); anal. calcd. for C₁₇H₁₄ClNO₄^•0.4H₂O: C, 60.25%; H, 4.40%; N, 4.14%; found: C, 60.21%; H, 4.01%; N, 4.03%.

Dimethyl 6,8-dichloro-1-methyl-9H-carbazole-2,3-dicarboxylate (4)

To a solution of 0.297 g (1 mmol) of the ester 1 in glacial acetic acid (10 mL) were added 0.268 g (2 mmol) of N-chlorosuccinimide. The mixture was sonicated for 10 min at room temperature in an ultrasound bath, and then it was refluxed with stirring for 40 h. The solvent was distilled off under reduced pressure and the residue was taken up in CH₂Cl₂ (40 mL). This solution was washed consecutively with 5% aqueous Na₂SO₃ (20 mL) and water (2 × 20 mL), then it was dried (Na₂SO₄) and evaporated. The residue was purified by column chromatography, eluting with CH₂Cl₂/ethyl acetate (19:1, v/v), followed by recrystallization from methanol to afford 0.270 g (72%) of 4 as colorless crystals, m.p. 170-171 ºC; IR (KBr) ν_max/cm^-1 3294, 2948, 1716, 1568, 1486, 1438, 1326, 1288, 1260, 1152, 1060, 786; ¹H NMR (300 MHz, CDCl₃) d 2.52 (s, 3H, 1-CH₃), 3.95 (s, 3H, OCH₃), 4.02 (s, 3H, OCH₃), 7.47 (d, 1H, J 1.8 Hz, 7-H), 7.92 (d, 1H, J 1.8 Hz, 5-H), 8.37 (s, 1H, NH), 8.49 (s, 1H, 4-H); ¹³C NMR (75 MHz, CDCl₃) d 13.9, 52.4, 52.8, 117.0, 118.3, 119.2, 120.2, 121.7, 121.9, 125.3, 126.1, 126.4, 133.7, 135.9, 141.1, 166.3, 169.9; MS m/z (%) 369 (M⁺, 22), 367 (M⁺, 45), 365 (M⁺, 44), 336 (31), 335 (39), 334 (72), 333 (53), 321 (28), 320 (33), 319 (31), 318 (51), 277 (59), 275 (100), 250 (20), 249 (62), 248 (53), 247 (100), 246 (34), 213 (27), 211 (31), 184 (21), 178 (21), 177 (51), 176 (35), 175 (39), 168 (54), 164 (23), 150 (29), 124 (25), 123 (21), 98 (20), 88 (25), 83 (22), 75 (38), 74 (38), 71 (26), 69 (539, 67 (24), 63 (22), 59 (369, 57 (56), 55 (50); anal. calcd. for C₁₇H₁₃Cl₂NO₄^•0.5H₂O: C, 54.42%; H, 3.76%; N, 3.73%; found: C, 54.27%; H, 3.25%; N, 3.69%.

Dimethyl 1-methyl-6-nitro-9H-carbazole-2,3-dicarboxylate (5) and dimethyl 1-methyl-8-nitro-9H-carbazole-2,3-dicarboxylate (6)

To a solution of 0.297 g (1 mmol) of the ester 1 in glacial acetic acid (5 mL) were added 0.462 g (3 mmol) of urea nitrate and the mixture was refluxed with stirring for 20 h. It was then cooled, diluted with water (30 mL) and extracted with CH₂Cl₂ (3 × 15 mL). The extract was washed with water (2 × 10 mL), then it was dried (Na₂SO₄) and evaporated. The residue was subjected to column chromatography, eluting with CH₂Cl₂/ethyl acetate (19:1, v/v). Evaporation of the first fraction gave 0.093 g (27%) of the 8-nitro compound (6) as yellow crystals, m.p. 213-214 ºC (methanol); IR (KBr) ν_max/cm^-1 3434, 3412, 2948, 1726, 1712, 1610, 1526, 1484, 1432, 1340, 1254, 1060, 740; ¹H NMR (300 MHz, CDCl₃) d 2.57 (s, 3H, 1-CH₃, shows positive NOE on irradiation at 10.00 ppm), 3.95 (s, 3H, OCH₃), 4.04 (s, 3H, OCH₃), 7.39 (t, 1H, J 8.1 Hz, 6-H), 8.35 (d, 1H, J 8.4 Hz, 5-H, shows positive NOE on irradiation at 8.55 ppm), 8.38 (d, 1H, J 8.7 Hz, 7-H), 8.55 (s, 1H, 4-H), 10.00 (s, 1H, NH); ¹³C NMR (75 MHz, CDCl₃) δ 13.9, 52.5, 52.8, 112.3, 117.6, 118.8, 120.1, 121.3, 122.9, 124.4, 126.2, 128.0, 132.9, 138.5, 141.2, 166.2, 169.6; MS m/z (%) 342 (M⁺, 34), 311 (57), 310 (75), 306 (20), 295 (32), 265 (30), 252 (100), 250 (47), 224 (68), 206 (20), 179 (25), 178 (70), 177 (37), 151 (29), 150 (31), 75 (31); anal. calcd. for C₁₇H₁₄N₂O₆: C, 59.65%; H, 4.12%; N, 8.18%; found: C, 59.43%; H, 4.06%; N, 7.98%. Evaporation of the second fraction gave 0.240 g (70%) of the 6-nitro compound (5) as yellow crystals, m.p. 168-169 ºC (methanol); IR (KBr) ν_max/cm^-1 3632, 3352, 2954, 1718, 1612, 1580, 1516, 1484, 1436, 1332, 1304, 1270, 1238, 1132, 1060, 1014; ¹H NMR (300 MHz, DMSO-d₆) δ 2.46 (s, 3H, 1-CH₃), 3.85 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 7.63 (d, 1H, J 9.0 Hz, 8-H), 8.30 (dd, 1H, J 9.0, 2.4 Hz, 7-H), 8.81 (s, 1H, 4-H, shows positive NOE on irradiation at 9.24 ppm), 9.24 (s, 1H, 5-H), 12.4 (br s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 13.8, 52.2, 52.3, 111.8, 118.2, 118.9, 119.5, 121.7, 121.8, 122.1, 122.1, 133.0, 142.5, 144.2, 166.0, 168.8; MS m/z (%) 342 (M⁺, 42), 311 (68), 310 (98), 295 (32), 266 (29), 265 (51), 253 (21), 252 (100), 250 (26), 249 (23), 224 (54), 206 (22), 179 (34), 178 (70), 177 (33), 164 (22), 152 (20), 151 (30), 150 (38), 140 (26), 139 (26), 76 (21), 75 (34), 69 (21), 63 (25), 59 (23), 57 (30), 55 (21); anal. calcd. for C₁₇H₁₄N₂O₆^•0.5H₂O: C, 58.12%; H, 4.30%; N, 7.97%; found: C, 58.10%; H, 4.27%; N, 7.75%.

General procedure for the preparation of 9-substituted 5-methyl-2,3-dihydro-1H-pyridazino[4,5-b]carbazole-1,4(6H)-diones (7-9)

A solution of the corresponding precursor, 6-substituted dimethyl 1-methyl-9H-carbazole-2,3-dicarboxylate (2, 3, or 5) (1 mmol) in 100% hydrazine hydrate (6 mL) was heated to reflux for 2 h. Excess reagent was distilled off under reduced pressure, the residue was taken up in ethanol (10 mL) and again evaporated under reduced pressure. The last step was repeated three times in order to remove residual hydrazine hydrate. The solid residue was recrystallized from ethanol to afford the pure product.

9-Bromo-5-methyl-2,3-dihydro-1H-pyridazino[4,5-b]carbazole-1,4(6H)-dione (7)

Yield: 0.333 g (97%) as yellow crystals; m.p. > 350 ºC; IR (KBr) ν_max/cm^-1 3422, 3162, 3010, 1646, 1466, 1394, 1282, 1240, 1122, 1050, 802; ¹H NMR (300 MHz, DMSO-d₆) δ 3.09 (s, 3H, CH₃), 7.54 (d, 1H, J 8.7 Hz, 7-H), 7.64 (dd, 1H, J 8.7, 1.8 Hz, 8-H), 8.61 (d, 1H, J 1.8 Hz, 10-H, shows positive NOE on irradiation at 8.78 ppm), 8.78 (s, 1H, 11-H), 11.21 (s, 2H, NH), 11.88 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 15.2, 111.8, 113.5, 115.8, 119.6, 121.2, 122.9, 124.2, 124.4, 124.5, 130.3, 140.5, 142.3, 154.0, 158.6; MS m/z (%) 345 (M⁺, 10), 343 (M⁺, 10), 71 (30), 69 (45), 67 (20), 59 (100), 57 (71), 55 (50); anal. calcd. for C₁₅H₁₀BrN₃O₂^•0.5H₂O: C, 51.01%; H, 3.14%; N, 11.90%; found: C, 51.19%; H, 3.06%; N, 11.64%.

9-Chloro-5-methyl-2,3-dihydro-1H-pyridazino[4,5-b]carbazole-1,4(6H)-dione (8)

Yield: 0.259 g (86%) as pale yellow crystals, m.p. > 350 ºC; IR (KBr) ν_max/cm^-1 3450, 3152, 2950, 1646, 1470, 1282, 1122, 1060, 866, 818, 732, 700; ¹H NMR (300 MHz, DMSO-d₆) δ 3.09 (s, 3H, CH₃), 7.52 (dd, 1H, J 8.7, 1.8 Hz, 8-H), 7.60 (d, 1H, J 8.7 Hz, 7-H), 8.46 (d, not resolved, 1H, 10-H, shows positive NOE on irradiation at 8.78 ppm), 8.78 (s, 1H, 11-H), 11.19 (s, 2H, NH), 11.86 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 15.1, 113.0, 115.8, 119.5, 121.1, 121.3, 122.8, 123.5, 124.0, 124.5, 127.6, 140.1, 142.4, 154.0, 158.4; MS m/z (%) 301 (M⁺, 11), 299 (M⁺, 36), 83 (20), 71 (42), 69 (46), 67 (28), 57 (100), 56 (24), 55 (78); anal. calcd. for C₁₅H₁₀ClN₃O₂^•0.85H₂O: C, 57.19%; H, 3.74%; N, 13.34%; found: C, 57.25%; H, 3.51%; N, 12.95%.

5-Methyl-9-nitro-2,3-dihydro-1H-pyridazino[4,5-b]carbazole-1,4(6H)-dione (9)

Yield: 0.230 g (74%) as yellow crystals, m.p. > 350 ºC; IR (KBr) ν_max/cm^-1 3414, 2892, 1626, 1518, 1472, 1324, 1242, 1128, 822; ¹H NMR (300 MHz, DMSO-d₆) δ 3.04 (s, 3H, CH₃), 7.60 (d, 1H, J 9.0 Hz, 7-H), 8.29 (d, J 8.7 Hz, 1H, 8-H), 8.80 (s, 1H, 11-H), 9.20 (s, 1H, 10-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 15.1, 111.5, 116.2, 118.7, 120.5, 121.8, 121.9, 122.9, 123.5, 125.2, 140.4, 142.9, 145.0, 153.8, 158.4; MS m/z (%) 311 ([M+1]⁺, 22), 310 (M⁺, 98), 207 (48), 179 (22), 178 (48), 177 (24), 151 (20), 150 (32), 83 (22), 77 (22), 76 (25), 75 (34), 74 (29), 73 (24), 71 (27), 70 (21), 69 (62), 63 (34), 57 (67), 56 (40), 55 (70), 53 (20), 51 (27); anal. calcd. for C₁₅H₁₀N₄O₄^•0.6H₂O: C, 56.11%; H, 3.52%; N, 17.45%; found: C, 55.96%; H, 3.35%; N, 17.68%.

9-Bromo-1,4-dichloro-5-methyl-6H-pyridazino[4,5-b]carbazole (10)

A mixture of 1.72 g (5 mmol) of compound 7, N,N-diethylaniline (6.5 mL) and phosphorus oxychloride (75 mL) was refluxed for 2 h under exclusion of moisture. During the reaction, a clear solution was obtained which later became turbid and a yellowish precipitate was formed. The excess reagent was distilled off under reduced pressure and the solid residue was neutralized with dilute ammonia under ice cooling. The material was collected by filtration and washed several times with cold water. After air-drying, the filter cake was washed several times with diethyl ether and dried in vacuo to afford 1.85 g (97%) of 10 as yellow crystals, m.p. > 350 ºC (decomp.); ¹H NMR (300 MHz, DMSO-d₆) d 3.12 (s, 3H, CH₃), 7.55 (d, 1H, J 8.7 Hz, 7-H), 7.73 (dd, 1H, J 8.7, 1.8 Hz, 8-H), 8.78 (d, 1H, J 1.8 Hz, 10-H), 9.09 (s, 1H, 11-H), 12.19 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 16.9, 112.4, 113.5, 116.0, 117.0, 120.6, 122.7, 123.3, 125.3, 127.4, 131.9, 141.4, 143.8, 152.9, 155.2; MS m/z (%) 385 (M⁺, 6), 383 (M⁺, 36), 381 (M⁺, 100), 379 (M⁺, 51), 363 (23), 361 (21), 311 (20), 309 (23), 230 (20), 202 (18), 176 (18), 101 (23), 87 (24), 75 (26), 69 (28), 57 (41); anal. calcd. for C₁₅H₈BrCl₂N₃^•0.3H₂O: C, 46.62%; H, 2.24%; N, 10.87%; found: C, 46.71%; H, 2.61%; N, 10.80%.

N'-(9-Bromo-4-chloro-5-methyl-6H-pyridazino[4,5-b]carbazol-1-yl)-N,N-diethylpropane-1,3-diamine (11a) and N'-(9-bromo-1-chloro-5-methyl-6H-pyridazino[4,5-b]carbazol-4-yl)-N,N-diethylpropane-1,3-diamine (11b)

A mixture of 0.381 g (1 mmol) of the dichloro compound (10) and N,N-diethylpropane-1,3-diamine (4.0 mL, 25.3 mmol) in DMSO (20 mL) was stirred and heated to 130 ºC for 3 h. The volatile components were removed by Kugelrohr distillation (10^-1 mbar, 80 ºC) and the residue was dissolved in CH₂Cl₂ (50 mL) and washed with dilute ammonia (3 × 10 mL). The solution was dried (Na₂SO₄) and evaporated under reduced pressure. The residue was subjected to column chromatography, eluting with CH₂Cl₂/methanol/triethylamine in v/v ratios of 97:3:2 (elution of apolar side products), followed by 95:5:2 (elution of 11a) and finally 90:10:2 (elution of 11b). Evaporation of the corresponding fraction gave 0.170 g (36%) of 11a as yellow crystals, m.p. 261-263 ºC (CH₂Cl₂/diethyl ether); ¹H NMR (300 MHz, DMSO-d₆) d 1.17 (t, 6H, J 6.9 Hz, NCH₂CH₃), 2.10 (quint, 2H, J 7.5 Hz, NHCH₂CH₂CH₂NR₂), 3.13 (s, 3H, 5-CH₃), 3.12-3.33 (m, 6H, NHCH₂CH₂CH₂NR₂, NCH₂CH₃), 3.66 (q, 2H, J 9.6 Hz, NHCH₂CH₂CH₂NR₂), 7.59 (d, 1H, J 8.4 Hz, 7-H), 7.72 (dd, 1H, J 8.7, 1.8 Hz, 8-H), 7.80 (m, 1H, 1-NHR, shows positive NOE on irradiation at 9.27 ppm), 8.37 (d, 1H, J 1.5 Hz, 10-H, shows positive NOE on irradiation at 9.27 ppm), 9.27 (s, 1H, 11-H), 12.01 (s, 1H, carbazole-NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 8.4, 17.1, 23.0, 46.0, 48.9, 111.6, 113.6, 114.0, 114.5, 115.0, 121.8, 123.7, 123.8, 125.1, 130.8, 141.0, 142.7, 143.2, 154.6; MS m/z (%) 446 (2), 444 (2), 440 (2), 439 (2), 438 (2), 403 (13), 402 (18), 401 (20), 400 (14), 375 (25), 373 (19), 367 (12), 312 (21), 311 (13), 310 (23), 231 (10), 230 (12), 203 (11), 202 (10), 201 (11), 161 (9), 155 (9), 113 (21), 112 (53), 100 (22), 98 (23), 86 (100), 84 (33), 72 (30), 71 (16), 58 (79), 57 (27), 56 (64); HRMS m/z calcd. for C₂₂H₂₆BrClN₅ ([M+H]⁺): 474.1060; found: 474.1063. Evaporation of the last fraction gave 0.081 g (17%) of 11b as yellow crystals, m.p. 231-235 ºC (ethanol/diethyl ether); ¹H NMR (300 MHz, DMSO-d₆) d 0.96 (t, 6H, J 6.9 Hz, NCH₂CH₃), 1.85 (quint, 2H, J 6.6 Hz, NHCH₂CH₂CH₂NR₂), 2.47-2.58 (m, 6H, NHCH₂CH₂CH₂NR₂, NCH₂CH₃), 3.10 (s, 3H, 5-CH₃, shows positive NOE on irradiation at 6.68 ppm), 3.54 (q, 2H, J 5.7 Hz, NHCH₂CH₂CH₂NR₂), 6.72-6.64 (m, 1H, 4-NHR), 7.56 (d, 1H, J 8.7 Hz, 7-H), 7.69 (dd, 1H, J 8.7, 1.8 Hz, 8-H), 8.74 (d, not resolved, 1H, 10-H), 8.87 (s, 1H, 11-H), 11.93 (s, 1H, carbazole–NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 11.5, 16.4, 25.6, 46.5, 50.8, 111.7, 113.3, 115.2, 115.4, 118.3, 119.9, 123.8, 124.7, 125.3, 130.8, 141.0, 142.6, 145.8, 155.1; MS m/z (%) 475 (M⁺, 1), 473 (M⁺, 1), 440 (4), 438 (4), 403 (5), 401 (4), 389 (5), 387 (4), 376 (4), 374 (4), 369 (9), 367 (10), 363 (4), 361 (7), 359 (4), 312 (8), 310 (8), 231 (4), 230 (6), 203 (5), 202 (6), 113 (15), 112 (100), 100 (8), 98 (10), 86 (64), 84 (17), 72 (13), 70 (7), 58 (31), 56 (27); anal. calcd. for C₂₂H₂₅BrClN₅^•0.2H₂O: C, 55.23%; H, 5.35%; N, 14.64%; found: C, 55.37%; H, 5.13%; N, 14.25%.

9-Bromo-N,N'-bis[3-(diethylamino)propyl]-5-methyl-6H-pyridazino[4,5-b]carbazole-1,4-diamine (12)

A mixture of 0.533 g (1.4 mmol) of the dichloro compound (10) and N,N-diethylpropane-1,3-diamine (12 mL, 76 mmol) was stirred and heated to 160 ºC for 168 h under an argon atmosphere. The volatile components were removed by Kugelrohr distillation (10^-1 mbar, 80 ºC) and the residue was subjected to column chromatography, eluting with ethyl acetate/ethanol/triethylamine (25:25:2, v/v) to afford 0.246 g (62%) of 12 as yellow crystals, m.p. > 350 ºC (ethyl acetate/ethanol); ¹H NMR (300 MHz, DMSO-d₆) d 0.92-1.09 (m, 12H, NCH₂CH₃), 1.92 (quint, not resolved, 4H, NHCH₂CH₂CH₂NR₂), 2.62-2.79 (m, 12H, NHCH₂CH₂CH₂NR₂, NCH₂CH₃), 3.09 (s, 3H, 5-CH₃), 3.40-3.50 (m, 4H, NHCH₂CH₂CH₂NR₂), 4.04-4.12 (m, 2H, 1-NHR and 4-NHR), 7.56 (d, 1H, J 8.7 Hz, 7-H), 7.66 (dd, not resolved, 1H, 8-H), 8.30 (d, not resolved, 1H, 10-H), 9.10 (s, 1H, 11-H), 12.03 (s, 1H, carbazole–NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 10.3, 10.5, 16.8, 25.2, 25.5, 45.7, 46.0, 46.1, 46.3, 49.9, 50.3, 111.4, 113.5, 114.5, 116.3, 118.7, 123.4, 123.9, 130.2, 140.7, 142.3, 150.2, 150.5; MS m/z (%) 567 (M+, 0.2), 538 (1), 496 (29), 494 (30), 467 (4), 465 (3), 422 (3), 410 (7), 408 (10), 396 (8), 394 (9), 382 (9), 380 (6), 368 (9), 367 (9), 354 (4), 340 (3), 327 (2), 310 (5), 247 (4), 230 (6), 203 (6), 177 (3), 157 (3), 143 (2), 112 (19), 98 (10), 86 (100), 84 (12), 73 (14), 72 (27), 58 (70), 56 (16); HRMS m/z calcd. for C₂₉H₄₃BrN₇ ([M+H]⁺): 568.2758; found: 568.2765.

8-Bromo-2-[3-(diethylamino)propyl]-4-methylpyrrolo[3,4-b]carbazole-1,3(2H,5H)-dione (13)

A solution of 0.376 g (1 mmol) of the diester 2 and 1.30 g (10 mmol) of N,N-diethylpropane-1,3-diamine in DMSO (5 mL) was stirred and heated to 130 ºC for 24 h under an argon atmosphere. The volatile components were removed by Kugelrohr distillation (10^-1 mbar, 80 ºC) and the residue was purified by column chromatography, eluting with CH₂Cl₂/ethyl acetate/triethylamine (95:95:10, v/v), followed by recrystallization from ethanol to afford 0.258 g (58%) of 13 as yellow needles, m.p. 223-226 ºC; ¹H NMR (300 MHz, DMSO-d₆) δ 0.90 (t, 6H, J 7.2 Hz, NCH₂CH₃), 1.71 (quint, 2H, J 6.9 Hz, NCH₂CH₂CH₂NEt₂), 2.41-2.50 (m, 6H, NCH₂CH₂CH₂NEt₂, NCH₂CH₃), 2.85 (s, 3H, 4-CH₃), 3.58 (t, 2H, J 7.5 Hz, NCH₂CH₂CH₂NEt₂), 7.54 (d, 1H, J 8.7 Hz, 6-H), 7.61 (dd, 1H, J 8.7, 2.1 Hz, 7-H), 8.56 (s, 1H, 10-H), 8.58 (d, 1H, J 2.1 Hz, 9-H), 12.22 (s, 1H, carbazole–NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 11.4, 12.0, 25.5, 35.7, 46.1, 49.9, 112.3, 113.8, 114.7, 121.4, 122.5, 123.8, 124.0, 124.5, 125.3, 129.6, 139.5, 142.6, 168.0, 168.9; MS m/z (%) 443 (M⁺, 1.2), 441 (M⁺, 1.3), 428 (2), 426 (2), 414 (4), 412 (4), 371 (2), 369 (2), 343 (3), 341 (3), 262 (2), 259 (2), 257 (2), 214 (2), 203 (3), 178 (4), 177 (4), 151 (3), 150 (3), 112 (5), 87 (8), 86 (100), 84 (6), 72 (20), 58 (14), 56 (6); anal. calcd. for C₂₂H₂₄BrN₃O₂^•0.1H₂O: C, 59.49%; H, 5.49%; N, 9.46%; found: C, 59.48%; H, 5.45%; N, 9.06%.

8-Nitro-2-[3-(diethylamino)propyl]-4-methylpyrrolo[3,4-b]carbazole-1,3(2H,5H)-dione (14)

A solution of 0.547 g (1.6 mmol) of the diester 5 and 2.08 g (16 mmol) of N,N-diethylpropane-1,3-diamine in DMSO (8 mL) was stirred and heated to 130 ºC for 24 h under an argon atmosphere. The volatile components were removed by Kugelrohr distillation (10^-1 mbar, 80 ºC) and the residue was purified by column chromatography, eluting with ethyl acetate/triethylamine (19:1, v/v), followed by recrystallization from ethyl acetate/diethyl ether to afford 0.372 g (57%) of 14 as yellow crystals, m.p. 248-251 ºC; ¹H NMR (300 MHz, DMSO-d₆) d 0.90 (t, 6H, J 7.2 Hz, NCH₂CH₃), 1.70 (quint, 2H, J 7.2 Hz, NCH₂CH₂CH₂NEt₂), 2.39-2.46 (m, 6H, NCH₂CH₂CH₂NEt₂, NCH₂CH₃), 2.79 (s, 3H, 4-CH₃), 3.56 (t, 2H, J 6.9 Hz, NCH₂CH₂CH₂NEt₂), 7.60 (d, 1H, J 9.0 Hz, 6-H), 8.25 (dd, 1H, J 9.0, 2.1 Hz, 7-H), 8.56 (s, 1H, 10-H), 9.22 (d, 1H, J 2.4 Hz, 9-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 11.5, 11.9, 25.6, 35.8, 46.1, 49.9, 112.0, 115.0, 118.6, 121.8, 122.1, 122.2, 123.5, 125.0, 126.0, 140.8, 143.7, 144.0, 167.7, 168.5; MS m/z (%) 408 (M⁺, 1), 393 (1), 379 (2), 336 (1), 308 (3), 265 (1), 262 (2), 249 (3), 203 (14), 178 (3), 177 (3), 112 (4), 105 (11), 91 (4), 87 (6), 86 (100), 84 (3), 77 (4), 72 (15), 58 (21), 57 (7), 56 (10), 55 (5); HRMS m/z calcd. for C₂₂H₂₅N₄O₄ ([M+H]⁺): 409.1876; found: 409.1874.

Biological evaluation

Antiproliferative activity of compounds was assessed using an XTT²⁷ assay (EZ4U®) on SW480 (colon carcinoma), A549 (lung carcinoma), Hep3B (liver carcinoma), U373 (glioblastoma) and HTB65 (melanoma) cell lines. Cells were seeded at a density of 10000 cells (SW480, A549, HEP3B) or 6000 cells (U373, HTB65) per well into 96-well plates and after treatment with active compounds at fixed concentrations of 10 µM and 5 µM for 24 h, the supernatant was replaced with 100 µL of freshly prepared EZ4U® solution (EZ4U®, Biomedica, Vienna, Austria). The cells were incubated at 37 ºC for 3 h and the absorbance was measured at 450 and 620 nm in a Tecan Infinite 200 Pro microplate reader (Tecan, Männedorf, Switzerland).

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

Submitted: June 6, 2014

Published online: August 1, 2014

Supplementary Information

The supplementary material is available in pdf: [Supplementary material]

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Publication Dates

• Publication in this collection
  13 Nov 2014
• Date of issue
  Nov 2014

History

• Received
  06 June 2014
• Accepted
  01 Aug 2014