Synthesis of 1-Substituted Carbazolyl-1,2,3,4-tetrahydro- and Carbazolyl-3,4-dihydro-β-carboline Analogs as Potential Antitumor Agents

A series of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-β-carboline analogs have been synthesized and evaluated for antitumor activity against human tumor cells including KB, DLD, NCI-H661, Hepa, and HepG2/A2 cell lines. Among these, compounds 2, 6, 7, and 9 exhibited the most potent and selective activity against the tested tumor cells. As for inhibition of topoisomerase II, compounds 1–14 and 18 showed better activity than etoposide. Among them, compounds 3, 4, 7, 9, and 10 exhibited potent activity. The structure and activity relationship (SAR) study revealed correlation between carbon numbers of the side chain and biological activities. The molecular complex with DNA for compound 2 was proposed.


Introduction
Marine invertebrates are rich in -carboline alkaloids [1][2][3]. These natural -carboline metabolites have been found to possess interesting antitumor and antiviral activities [4][5][6]. Eudistomins [7,8] and manzamines [9,10], which were isolated from tunicates and sponges, respectively, are of particular interest. The antiviral eudistomines C and E were active against HSV-2, Vaccinia virus and RNA viruses [11]. In addition, the novel manzamines exhibited potent antitumor, antibacterial, antifungal and anti-HIV activities [12][13][14]. The major compound, manzamine A, showed most potent activity against murine P-388 cells [15]. Our previous paper reported that manzamines A-D and H showed significant cytotoxicities against human KB-16, A-549 and HT-29 tumor cells [16]. The -carboline and 3,4-dihydro--carboline moieties in manzamines appear to be essential for the biological activity. In order to investigate the structure and activity relationship (SAR) and bioactive center of manzamine A, the synthesis of 1-substituted carbazolyl-1,2,3,4-tetrahydro-and carbazolyl-3,4-dihydro--carboline derivatives were initiated [17]. The limited source of manzamine A make this current investigation more urgent and important. The purpose of this study does not include all SAR studies of manzamines and -carboline analogs. However, a combination of -carboline and various carbazole analogs is addressed. An attempt to realize the possible active center of manzamine A and the importance of the alkyl substituents on the nitrogen atom of carbazole ring was conducted. Thus, a facile synthetic method by the application of N-alkylation, Duff reaction [18], Pictet-Spengler reaction [19,20] and DDQ oxidation [21] succeeded in the production of compounds 1-18. In this communication, we wish to report the preparation, structural elucidation and bioactivities of 1-substituted carbazolyl-1,2,3,4-tetrahydro-and carbazolyl-3,4-dihydro--carboline analogs.

Analog Design and Chemistry
The basic strategy for the synthesis of the target substances involved molecular modeling and SAR studies of manzamine analogs. To improve our knowledge of the main structural requirements needed for high antitumor activity, we synthesized two new series of 1-substituted carbazolyl-1,2,3,4-tetrahydro--carboline and carbazolyl-3,4-dihydro--carboline derivatives based on the analog design of manzamine A. These new compounds bear an N-alkyl carbazole conjugated with a -carboline-like nucleus. The main part of manzamine A illustrated in Figure 1 is similar in both shape and size to our target compounds. The distances between N atom on carbazole and N atoms on carboline-nucleus are almost the same as those in the main part. With the aim of studying the SAR, we focused on the lengh of the N-alkyl side chain on the carbazole ring. Compounds 19-26 were prepared by N-alkylation of carbazole with the appropriate alkyl bromide as depicted in Scheme 1. Subsequent synthesis of compounds 27-34 was achieved by Duff reaction, which required hexamethylenetetramine/trifloroacetic acid and the appropriate N-alkyl carbazole. Compounds 1, 3, 5, 7, 9, 11, 13, 15, and 17 (A series) were furnished from tryptamine and series of N-substituted 3-carbazolyl carboxyaldehydes (27-34, and N-ethyl-3-carboxyaldehyde, which was purchased from Sigma-Aldrich Co.) by application of Pictet-Spengler cyclization. Subsequent oxidation of 1,2,3,4-tetrahydro--carbolines (A) by DDQ yielded 3,4-dihydro--carboline derivatives (B series: 2, 4, 6, 8, 10, 12, 14, 16, 18). The preparation and spectral data for 1-34 are described in the Experimental Section.

Biological Activity
Cytotoxicity of new products 1-18 was tested against KB (human mouth epidermoid carcinoma); DLD (human colon adenocarcinoma), NCI-H661 (human lung large cell carcinoma) and Hepa (human hepatoma), HepG2/A2 (human hepatoma) tumor cells in vitro. The IC 50 values of these compounds are summarized in Table 1. Table 1 shows that compounds 1-12 exhibited significant and/or selective cytotoxic activities. Among them, compounds 2 and 6 are most potent against KB tumor cells selectively. Compound 7 is more potent than 2 against NCI-H661 tumor cells although compound 2 shows most promising activity against all tumor cells. On the other hand, compounds 14-18 are inactive toward four tumor cells while compounds 11-13 exhibit weak, marginal or no activity. Table 1 also shows that these compounds have selective cytotoxicity against HepG2/A2 tumor cells. Compounds 2-4 and 8 are active while others are inactive. In this assay, compound 2 exhibits most potent activity against the HepG2/A2 system. The SAR study revealed that there was not a linear relationship between the carbon number of the side chain at N atom in carbazole and cytotoxicity. Nevertheless, we observed that, in general, elongation of the alkyl chain resulted in a decrease in activity. In fact, compounds 11-18 showed very weak or no activity toward HepG2/A2, KB and NCI-H661 cells even though compound 11 was active in DLD assay.
Inhibition activity of compounds 1-18 was evaluated against human DNA topoisomerases I and II. Table 2 indicates that compounds 1-14 and 18 are more potent inhibitors of human DNA topoisomerase II than etoposide. Among the tested compounds, 3, 4, 7, 9 and 10 were most potent. On the other hand, all compounds were inactive or showed mild activity against DNA topoisomerase I. Compounds 3, 4, 9 and 10 were 10-to 15-fold more potent against topoisomerase II (compared to etoposide) and superior to compounds 1 and 2. On the other hand, compound 18 showed great activity while compounds 15-17 did not.
Compound 7 showed much better activity than that of 2 suggesting that the different degrees of activity might be explained by the differences in binding affinity or bioavailability such as drug uptake and fate of metabolism.

Molecular Modeling
To rationalize the biological activity results obtained for this novel series of carbazolyl-3,4-dihydro--carbolines, we carried out a molecular modeling study of compound 2. The molecular modeling was performed by making use of two double strand DNA fragments (CGCTAGGG) 2 and (CGCGAATTCGCGG) 2 . Figure 2 shows the result of d(CGCATGGG) 2 -compound 2 complex. It was found that the binding conformation of this complex was formed by intercalation. Unlike the usual planar intercalators, compound 2 inserts into base pairs of DNA in a scissor-like conformation. We observed that the carbazole moiety of compound 2 was parallel with the thymidine base. However, the conformation of d(CGCGAATTGCG) 2 -compound 2 complex, as illustrated in Figure 3, was formed by minor groove binding. In this case, compound 2 interacts with DNA by putting the carbazole moiety in the minor groove leaving the -carboline chromophore outside. The estimated binding energy values for these two interaction forms are −16 and −186 kcal/mole, respectively. In these experiments, the occurrence of hydrogen bonding was observed in both conformations and resulted in the formation of a stable drug-DNA complex. The hydrogen bonding between the N-9 of -carboline and the oxygen atom of C-2 in thymidine had a bonding length 1.86 and 2.02 Ǻ, respectively [22].  The three-dimensional structures of DNA duplexes was obtained from the Brookhaven Protein Databank (PDB code: 108d). The 3D structure of Mana-Hox was constructed as a protonated form and was assigned Gasteiger-Huckel partial charges using Sybyl 6.5. The AutoTors module was used to specify two rotatable bonds in Mana-Hox for AutoDock. The grid maps were calculated with AutoGrid using a grid-point spacing of 0.3 Å, and 100 x 76 x 76 grid points for 108d. 200 independent docking runs were carried out using the Lamarckian genetic algorithm (LGA) with a maximum number of 500,000 energy evaluations and a population of 50 randomly initiated individuals. The lowest-energy docked conformations proposed the energetically favorable binding modes of Mana-Hox to DNA. a, Line plot; b, space-filling plot. The following coloring scheme is used: Mana-Hox, red; DNA duplex, blue; Drug-DNA hydrogen bond, green.

General Experimental Procedures
All of melting points were taken on a Buchi mp B-540 apparatus and were uncorrected. UV and IR spectra were recorded on Hitachi U-3210 and JASCO A-100 IR spectrophotometers, respectively. EIMS spectra were obtained on a MAT 112S-JMS D300 spectrometer, using direct inlet systems. HRMS data were taken on a JMX 110 mass spectrometer. 1 H-and 13 C-NMR spectra were recorded on a Bruker FT-300 spectrometer. Analytical thin-layer chromatography (TLC) was carried out on Kiesel gel GF 254 plates and detection was made under UV light. EM Kieselgel 60 (230-400 mesh ASTM) was used for column chromatography.

Synthesis of Compounds 23-26
To a stirred solution of carbazole (1.0 g, 0.6 mmol) and KOH (0.5 g) in EtOH (20 mL), 1-bromo-3-methylbuane, 2-bromopentane, 1-bromohexadecane, 1-bromoeicosane were slowly added, respectively (each 1 mL or 1 g). The reaction mixture was stirred at 50 °C for 24 h. After filtration and evaporation of the solvent under vacuum, the residue was chromatographed on a silica gel column (30 g) and eluted with n-hexane to afford compounds 23-26 with a yield which varied in a range of 50-75%.

Cytotoxicity Assay
The cytotoxic activities of compounds against KB (human mouth epidermoid carcinoma), DLD (human colon adenocarcinoma), NCI-H661 (human lung large cell carcinoma), Hepa (human hepatoma), and HepG2/A2 (human hepatoblastoma) cells were assayed by the MTT{3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide} colorimetric assay as previously described [23]. The cells for assay were cultured in RPMI-1640 medium supplemented with 5% CO 2 in an incubator at 37 °C. The cytotoxicity assay depends on the binding of methylene blue to fixed monolayers of cells at pH 8.5, washing the monolayer, and releasing the dye by lowering the pH value. Samples and control standard drugs were prepared at a concentration of 1, 10, 40, and 100 μg/mL. After seeding 2880 cells/well in a 96-well microplate for 3 h, 20 μL of sample or standard agent was placed in each well and incubated at 37 °C for 3 days. After removing the medium from the microplates, the cells were fixed with 10% formaldehyde in 0.9% saline for 30 min, then dyed with 1% (w/v) methylene blue in 0.01 M borate-buffer (100 μL/well) for 30 min. The 96-well plate was dipped into a 0.01 M borate-buffer solution four times in order to remove the dye. Then, 100 μL/well of EtOH-0.1 M HCl (1:1) was added as a dye eluting solvent, and the absorbance was measured on a microtiter plate reader (Dynatech, MR 7000) at a wavelength of 650 nm. The ED 50 value was defined by a comparison with the untreated cells as the concentration of test sample resulting in 50% reduction of absorbance. Doxorubicin was used as a standard compound.

Relaxation Assay of Topoisomerases I and II
Topoisomerases I and II (topo I and II) assays were measured by assessing relaxation of supercoiled pBR322 plasmid DNA according to [24]. Using camptothecin (CPT) as topo I and etoposide (VP-16) as topo II positive controls, test samples were dissolved in 5% (v/v) DMSO and then diluted to appropriate concentrations. In summary, topo I (TopoGen) was mixed with the test sample and 10 volume of assay buffer (100 mM Tris-HCl, 10 mM EDTA, 1.5 M NACl, 1.0% BSA, 1 M spermidine, and 50% glycerol), and then supercoiled DNA (pBR322) was added. In topo II assay, the mixture contained test sample and buffer including 50 mM Tris-HCl, 120 mM KCl, 10 mM MgCl 2 , 0.5 mM ATP, 0.5 mM dithiothreitol, 2 g BSA, pBR322 plasmid DNA (0.25 g), and 3U of topo II (TopoGen) in final volume of 20 L. After incubation of topo I or topo II mixture for 30 min at 37 °C, 2 λ 10% SDS and 2.5 λ proteinase K were added for 1 h. The reaction mixtures were electrophoresed on a 2% agarose gel (50 V, 20 min; 100 V, 30 min; 110 V, 30 min) and stained with ethidium bromide. Finally, by a densitometer of ImageMaster ® (Fujifilm thermal imaging system, FTI-500), the gels were directly scanned and the area representing supercoiled DNA was calculated. Concentrations for 50% inhibition (IC 50 ) were determined by interpolation from plots of topoisomerases I or II activity versus inhibitor concentration. Etoposide was used as a standard.

Conclusions
In summary, a series of 1-substituted carbazolyl-1,2,3,4-tetrahydro-and carbazolyl-3,4-dihydro-carboline analogs have been synthesized and evaluated as potential antitumor agents. Among them, compound 7 and 6 showed the most potent and selective activity against NCI-H661 and KB tumor cells, respectively. Compound 9 possessed most potent activity against DLD tumor cells. Compound 2 exhibited most promising activity against KB, NCI-H661 and Hepa (or HepG 2 /A 2 ) tumor cell lines. Inhibition of human DNA topoisomerase II revealed that compounds 3 and 4 are quite promising for further development of enzyme inhibitors. The SAR revealed that there was a lack of complete correlation between carbon numbers of the side chain and biological activities. However, the negative correlation was present between the alkyl side chain length in cytotoxicity tests. The optimal chain length is between 2 and 5 carbons. The optimal chain length for anti-topoisomerase II may be 3 and 5 carbons. The DNA binding capacity, which favored minor groove binding, was thus influenced by the alkyl substitution on carbazole chromophore and proton donor on -carboline chromophore. On the basis of the SAR study, synthesis of analogs of lead compounds 2, 3, 6, 7 and 9, in order to search for more potent activity, is currently in progress.