Novel indole-bearing combretastatin analogues as tubulin polymerization inhibitors

Background The combretastatins are a class of natural stilbenoids. These molecules generally share three common structural features: a trimethoxy "A"-ring, a "B"-ring containing substituent often at C3′ and C4′, and an ethene bridge between the two rings, which provides necessary structural rigidity. Members of the combretastatin family possess varying ability to cause vascular disruption in tumors. Combretastatin binds to the colchicine binding site of β-subunit of tubulin. Despite having a similar name, combretastatin is unrelated to statins, a family of cholesterol-lowering drugs. Results New combretastatin 2-(1-acetyl-1H-indole-3-yl)-3-(phenyl) propenoic analogues (2a to 2y), bearing indole moiety at the place of ring A of combretastatin (CA4), were synthesized and evaluated for anticancer activity against various cancer cell lines such as THP-1 (leukemia), A-549 (lung), IGROV-1 (ovary), HEP-2 (liver), MCF-7 (breast), and DU-145 (prostate). Compound 2d showed anti-cancer activity against THP-1 and MCF-7 with IC50 0.80 and 0.37 μM, respectively, and 2y showed against MCF-7 with IC50 3.60 μM comparable to paclitaxel. Conclusions The target compounds bind to the colchicine binding site which is situated at α and β interface of tubulin and prevent polymerization as it was confirmed by immunofluorescence technique. The molecular docking further confirmed the binding of the potent compound 2d to the colchicine binding site at α and β interface of tubulin.


Background
Tubulin is a useful biochemical target for various clinically used anticancer drugs like paclitaxel, vincristine, and vinblastine ( Figure 1) [1]. It plays an important role in the formation of the mitotic spindle which provides the structural framework for the physical segregation of the chromosomes during the mitosis [2]. Microtubules, made from tubulin are highly dynamic cytoskeleton elements in eukaryotic cells and play a critical role in various processes like mitosis, cell shape, intracellular organelle, transport and cell-cell interactions, and signal transduction [3,4]. They have highly polar structure and favor growth at the plus (+) end and shrinkage at the minus (−) end. This dynamic equilibrium can be affected by treatment with some agents like phenstatin, chalcone, lignan, colchicine, and podophyllotoxin ultimately prevent mitosis, thus used for cancer treatment [5,6]. Drugs which target tubulin interact with mitotic spindle and act by binding to the following binding sites: (1) colchicine binding site, (2) vinca alkaloid binding site, (3) rhizoxin/maytansine binding site, (4) tubulin sulfhydryl binding site, and (5) unknown binding sites [1]. Taxol binds to the β-tubulin of colchicine binding site and stabilizes microtubule against depolymerization. Colchicine binds to the α-tubulin of colchicine binding site and blocks the polymerization of microtubule [7]. Combretastatins binds to β-tubulin of colchicine binding site, strongly inhibits tubulin polymerization [8][9][10], and disrupts the normal mitotic spindle function [11]. It was first isolated from the bark of African willow tree Combretum caffrum [12]. Various analogues have been synthesized and showed very good anticancer activity by inhibiting tubulin polymerization [13]. Earlier synthesized compounds were having two rings in cis-configuration, but Pettit et al. have synthesized resveratrol type of compounds, β-nitrostyrenes, in which two rings are in transconfiguration and have marked antitumor activity. They also have synthesized E-indole nitrostyrene which showed marked anticancer activity [14]. Anticancer agents vincristine and vinblastine derived from Catheranthus roseus have been useful in cancer treatment, and this effect is due to the interaction of these drugs with tubulin [8,15]. It was thought worthwhile to incorporate another ring system having indole nucleus to rationally design combretastatinlike compounds replacing one ring with indole moiety because vincristine, vinblastine, elliptinium [16], and bromoindirubin contain indole moiety and have good tubulin polymerization inhibitory property [17]. Resistance remains a significant problem in the treatment of cancer with taxol like many chemotherapeutic agents, and it is caused by alterations in microtubule dynamics [18]. Our aim was to synthesize tubulin inhibitors having indole ring as basic moiety.
Materials and methods of immunofluorescence confocal microscopy THP1 cells (8 × 10 [4] cells/well) were seeded onto 18-mm square coverslips in six well plates. Cells were allowed to adhere for 24 h before dosing with required concentrations of 2d. Paclitaxel 1 μM was used as a positive control. After the treatment period, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized using 0.5% Triton-X (Sigma-Aldrich, MO, USA) in PBS for 5 min. The cells were blocked with 10% goat serum for 20 min at room temperature. Microtubules were detected with a monoclonal α-tubulin antibody (Sigma Corporation, Cream Ridge, USA) diluted 1:100 in 0.1% Triton X-100 in PBS for 1 hr 37°C and Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, USA) diluted 1:1,000 in PBS for 1 hr at room temperature. Cells were then washed three times in PBS and stained with DAPI diluted 1:1,000 in PBS [5]. The coverslips were mounted over glass slides, and the cells were imaged by confocal microscopy using an Olympus Fluoview FV1000 laser scanning microscope (Olympus Inc., Center Valley, USA).

Material and methods for molecular docking
The coordinates of tubulin were obtained from protein data bank [PDB:1SA0] [19,20]. The structure of 2d was drawn in ChemDraw [21] and subjected to energy minimization in the MOPAC module, using the AM1 procedure and implemented in the CS Chem3D Ultra (Cambridge Soft Corporation, Cambridge, USA). The 2d was docked in to the colchicine binding site of tubulin using the GOLD 5.0.1 software [22]. GOLD performs genetic algorithm-based ligand docking to optimize the conformation of the ligand at the receptor binding site. It utilizes Gold score fitness function to evaluate the various conformations of the ligand at the binding site and comprises four components: protein-ligand hydrogen bond energy, protein-ligand van der Waals (vdw) energy, ligand internal vdw energy, and ligand torsional strain energy. The compound was docked ten times, and each pose was ranked according to its Gold score fitness function. The conformation with the highest score was selected for discussion.

Results and discussion
Chemistry Twenty-five combretastatin analogues (2a to 2y) were synthesized by condensation of indole-3-acetic acid (1) with different substituted aldehydes (2) using triethyl amine and acetic anhydride as reported previously (shown in Scheme 1) [23]. The reaction was monitored by TLC. The compounds were purified (yield 48% to 63%) by column chromatography using silica gel mesh size of 60 to 120. All the synthesized compounds ( Figure 2) were characterized by spectroscopic techniques like infrared spectrometry (IR), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR) (1H NMR, 13C NMR, heteronuclear multiple bond correlation (HMBC) NMR ( Figure 3 and Table 1), and heteronuclear single quantum coherence (HSQC). 1H NMR indicates that H-7′ is highly deshielded as it appears to lie in the deshielding cone of N-acetyl group. H-3 is deshielded because of its nearby carbonyl of N-acetyl. H-3 is also deshielded because of its β position to carbonyl, but it is less deshielded than H-7′ because it does not come under the influence of magnetic anisotropy of carbonyl. No change in the integration of the H-3 was observed when H-2′ was doubly irradiated. These results indicated that H-2′ and H-3 are not in the close proximity. This is possible only when two rings are trans to each other. If two rings would have been in cis configuration, double irradiation of H-2′ would have resulted in signal enhancement of H-3 due to nuclear Overhauser effect (nOe). It was further confirmed that when a compound with hydroxyl located at 2″ was synthesized, the free OH (at 2″) is used in the formation of lactone because the geometry is such where OH and C = O are close in space and prefer to form lactone. H-3 gets more deshielded than H-7′ because it comes in the deshielding cone in the plane of benzene ring as well as the deshielding cone of carbonyl. IR value of carbonyl shifted to 1,763 from 1,710 because of lactone formation. Further Pschorr reaction [24] is fully supported in the condensation of phenyl acetic acid with aldehyde, resulting into combretastatin with cis configuration. When 2-hdroxybenzaldehyde is used for the condensation with phenyl acetic acid, no lactone formation occurred ( Figure 4). From the above observations, it was thus concluded that synthesized compounds are having two rings in trans-configuration.
The italicized values indicate significant activity. Synthesized compounds, in spite of having basic indole moiety, bind to colchicine binding site instead of vinblastine binding site because vincristine and vinblastine molecules are very large molecules, and thus, their binding sites have enlarged cavity as the binding site, while the present synthesized compounds, being much smaller in overall dimensions, appear to roll around in the vinblastine site. Further, this was confirmed by immunofluorescence confocal microscopy.

Immunofluorescence confocal microscopy
The immunofluorescence technique was used to evaluate the effect of 2d on microtubules. The effect on microtubule structure was determined using confocal microscopy. Figure

Molecular docking of 2d
In order to investigate the recognition process of these inhibitors at the colchicine-binding site of tubulin, a flexible docking study was performed on the most potent compound 2d with the help of GOLD software [22]. The colchicine binding site encompasses the residues of both α and β subunits of tubulin ( Figure 6). To validate the docking procedure for the prediction of the correct binding mode of inhibitor at the colchicine-binding site, the colchicine was extracted from the original X-ray structure [PDB:1SA0] [19,20] and re-docked using GOLD.21. The highest scoring conformation was selected and compared with X-ray structure conformation. The docked conformation of colchicine using GOLD was found to be similar with the original X-ray structure ( Figure 7A). The root mean square deviation between the best scored conformers from docking and X-ray structure was found to be 0.72 Å. The compound 2d was docked at the colchicine binding site of tubulin, and the best fit conformation was selected on the basis of Gold score and visual inspection. The Figure 7B shows the binding conformation of 2d at the colchicine binding site. The compound 2d gets stabilized at the colchicine-binding site of tubulin by hydrophobic and hydrogen bond interactions. The aromatic part of indole ring gets positioned in a hydrophobic cavity formed by Leu255β, Ala250β, Ala316β, Val318β, and Ala354β. The carbonyl oxygen (carbonyl group at indole) forms a hydrogen bond with sulfhydryl group of Cys241β. This is similar to the interaction of colchicines with the tubulin [25]. Another hydrogen bond was formed between the hydroxyl group of the carboxylic acid part of 2d and nitrogen of Leu255β. These two hydrogen bonds play crucial role in stabilizing the conformation of 2d at the colchicine-binding site. The unsaturated intermediate chain between rings A and B of 2d is placed in close vicinity of the side-chain of Leu248β ( Figure 7B). The ring B of 2d is extended towards the α-chain of the tubulin. The docking study was helpful to find the binding conformation of 2d at the colchicine binding site of tubulin.

Experimental
The reagents were purchased from Sigma-Aldrich (MO, USA), LobaChemie (Mumbia, India), and Central Drug House (New Delhi, India) and used without further purification. All yields refer to isolated products after purification. Products were characterized by spectroscopic data (IR, [1] H NMR, and [13] C NMR spectra). The spectra were measured in DMSO-d 6 relative to TMS (0.00 ppm). IR (KBr pallets) spectra were recorded on a Fourier transform infrared Thermo spectrophotometer (ThermoFisher Scientific Inc., Waltham, USA). Melting points were determined in open capillaries and were found to be uncorrected.
Typical experimental procedure for the synthesis combretastatin 2-(1-acetyl-1H-indole-3-yl)-3-(phenyl) propenoic analogues (2a to 2y) A mixture of IAA (0.0057 mmol), substituted benzaldehyde (0.0057 mmol) and triethylamine (2 ml) in Ac 2 O (4 ml), was heated until the reaction was complete. After cooling, the reaction mixture was acidified with 35% aqueous HCl (6 ml) and kept at room temperature overnight when precipitates appeared as such or on dilution with   water. The products were purified by column chromatography using silica gel of mesh size 60 to120.     Figure 6 Colchicine binding site at the interface between α and β subunits of tubulin. The inhibitor 2d is shown in silver color.         Figure S2, Additional file 3: Figure S3, Additional file 4: Figure S4, Additional file 5: Figure S5 and Additional file 6: Figure S6.

Biological evaluation
In vitro cytotoxicity against five human cancer cell lines was determined using 96-well tissue culture plate [26]. The cells were allowed to grow in carbon dioxide incubator (37°C) for 24 h. Test materials in complete growth medium (100 μl) were added after 24 h of incubation to the wells containing cell suspension. The plates were further incubated for 48 h in a carbon dioxide incubator. The cell growth was stopped by gentle layering trichloroacetic acid (50%, 50 μl) on top of the medium in all the wells. The plates were incubated at 4°C for 1 h to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove trichloroacetic acid, growth medium low-molecular weight metabolites, serum proteins, etc. and air-dried. The plates were stained with sulforhodamine B dye (0.4% in 1% acetic acid, 100 μl) for 30 min. The plates were washed five times with 1% acetic acid and then air-dried [27]. The adsorbed dye was dissolved in Tris-HCl buffer (100 μL, 0.01 M, pH 10.4), and the plates were gently stirred for 10 min on a mechanical stirrer. The optical density (OD) was recorded on ELISA reader at 540 nm. The cell growth was determined by subtracting the mean OD value of respective blank from the mean OD value of the experimental set. Percent growth in presence of test material was calculated considering the growth in the absence of any test material as 100%, and in turn, percent growth inhibition in presence of test material was calculated.