Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3′,4′,5′-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3′,4′,5′-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3′,4′,5′-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 μM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.


Introduction
Cancer, which is the result of deviation of cell growth control mechanisms, has become a major worldwide disease and the second leading cause of mortality in developed countries, with almost 20 million deaths annually [1,2]. Many of the current treatments, involving an integrated treatment of surgery, radiation, and chemotherapy, have problems with toxicity and drug resistance [3,4]. Therefore, there is a strong demand for the discovery and development of effective chemotherapeutic agents with high selectivity and low toxicity [5][6][7].
The central role of microtubules in cell division and mitosis makes them an important target for the development of potential new anticancer agents [8][9][10]. A large number of small molecules displaying wide structural diversity and derived from natural sources or obtained by chemical synthesis have been identified within the last several decades [11][12][13] and have been shown to interfere with microtubules, polymeric structures composed of αand β-tubulin heterodimers [14][15][16].
Prominent examples of tubulin-binding compounds used clinically for cancer treatment include microtubule-stabilizers that inhibit microtubule function by promoting abnormally high levels of tubulin polymerization. Examples of such drugs include paclitaxel (Taxol) and its semisynthetic analogue docetaxel (Taxotere) [17,18]. In contrast, the vinca alkaloids vincristine, vinorelbine, and vinblastine destabilize microtubules and act as microtubule depolymerizers by inhibiting tubulin polymerization [19]. Both taxoids and vinca alkaloids interact with β-tubulin, but at different sites, named the taxol and vinca sites, respectively [20]. The colchicine-site binders that inhibit tubulin assembly [21] are another class of compounds in discovery and development, including combretastatin A-4 phosphate (CA-4P) [22], that bind to the colchicine site located between the αand β-tubulin subunits in the heterodimer, distinct from the vinca site located between adjacent αand β-tubulin heterodimers [23].
In an earlier published study, a series of molecules with general structure 1 (Figure 1), based on the 2-amino-3-(3 ,4 ,5 -trimethoxybenzoyl)-4,5,6,7-tetrahydrothieno [2,3-c]pyridine skeleton, yielded the promising derivatives 1a and 1b. These compounds inhibit cancer cell growth with IC 50 values ranging from 25 to 440 nM against a panel of four cancer cell lines and exert strong inhibition of tubulin polymerization by binding to the colchicine site of tubulin [45]. For this series of molecules, the presence of a N-methoxy/ethoxycarbonyl moiety at the 6-position of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine system was needed for potent antiproliferative activity.
In order to investigate the selective antiproliferative activities of derivatives 3a and 3b in normal human cells, both these molecules were tested in vitro against human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. After treatment of both resting and phytohemagglutin (PHA)-stimulated PBMC for 72 h, the IC 50 values were over 20 µM, indicating that 3a and 3b did not substantially affect the viability of these cells, suggesting that these two compounds have cancer cell selective killing properties.

Inhibition of Tubulin Polymerization and Colchicine Binding
To investigate whether the antiproliferative activities of these compounds were related to an interaction with the microtubule system, the two most active compounds of the series (3a and 3b) as antiproliferative agents were evaluated for their inhibitory effects on tubulin polymerization and on the binding of [ 3 H]colchicine to tubulin (in the latter assay, tubulin was examined at a concentration of 1 µM, while compounds and colchicine were at 5 µM). For comparison, CA-4 was examined in contemporaneous experiments as a reference compound (Table 2). Derivatives 3a and 3b inhibited the assembly reaction with IC 50 values of 3.6 and 3.8 µM, respectively, considerably higher than the value obtained with CA-4 (IC 50 : 0.54 µM), which is consistent with the reduced antiproliferative activity of 3a and 3b with respect to CA-4. These results indicated that tubulin may be the intracellular target of compounds 3a and 3b. In competition experiments, both these compounds inhibited the binding of [ 3 H]colchicine to its binding site on tubulin, with 31% and 29% of inhibition, respectively. These derivatives were less potent than CA-4, which in these experiments inhibited colchicine binding by 98%. Table 2. Inhibition of tubulin polymerization and colchicine binding by compounds 3a, 3b, and CA-4.
The results are consistent with the conclusion that the antiproliferative activity of these compounds derives from an interaction with the colchicine site of tubulin and interference with microtubule assembly. In conclusion, manipulation of the scaffold of compounds with general structures 1 and 2 led to the successful identification of a new series of inhibitors of tubulin assembly, characterized by a 3,4,5-trimethoxy aniline and a cyano function at the 2-and 3-positions, respectively, of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine nucleus.

Analysis of Effects on the Cell Cycle
Since molecules exhibiting effects on tubulin assembly should cause alteration of cell cycle parameters, leading to a preferential G2/M blockade, the effects of compounds 3a and 3b on cell cycle distribution were investigated on K562 cells using flow cytometry. The cells were cultured for 72 h in the presence of two different concentrations (IC 50 and IC 75 ) for each tested derivative and the obtained results were compared with non-treated K562 cells as control. Both compounds caused a significant (p < 0.01) and dose-dependent accumulation of the cells in the G2/M phase of the cell cycle, with a concomitant decrease of cells in the other phases of the cell cycle (especially the G0/G1 phase). As shown in Figure 2 (panels I and L), treatment of K562 cells at different concentrations of 3b (IC 50 : 0.70 µM and IC 75 : 0.90 µM) increased the percentage of G2/M-phase cells from 22.9% ± 0.7% (control group) to 30.3% ± 1.3% and 33.5% ± 0.6% respectively, indicating that compound 3b impact the cell cycle distribution in a dose-dependent manner. Similarly, for derivative 3a (IC 50 : 0.75 µM and IC 75 : 1.0 µM), an increase of the percentage of G2/M-phase cells from 22.2% ± 0.7% (control group) to 33.6% ± 1.1%, and 34.9% ± 1.3% was observed at the values, respectively. These data confirm that compounds 3a and 3b act selectively on the G2-M phase of the cell cycle, as expected for inhibitors of tubulin assembly.

Effects on Apoptosis
In order to characterize the mode of cell death induced by compounds 3a and 3b, a biparametric flow cytometry analysis was performed using propidium iodide (PI), which stains DNA and is permeable only to dead cells, and fluorescent immunolabeling of the protein annexin-V, which binds to the phospholipid phosphatidylserine (PS) in a highly selective manner. This phospholipid flips from the inner to the outer leaflet of the plasma membrane during apoptosis. Positive staining with annexin-V correlates with the loss of plasma membrane polarity, but this staining precedes the complete loss of membrane integrity that accompanies the later stages of cell death, resulting from either apoptosis or necrosis. In contrast, PI can only enter cells after complete loss of membrane integrity. Thus, dual staining for annexin-V and with PI permits discrimination between unaffected cells (annexin-V − /PI − ), early apoptotic cells (annexin-V + /PI − ), late apoptotic cells (annexin-V + /PI + ), and necrotic cells (annexin-V − /PI + ). The results obtained are shown in Figure 3.

Molecular Modeling Studies
The potential interaction between compounds 3a and 3b and the colchicine site was investigated through molecular docking studies, using Glide. [54] The colchicine-tubulin complex (PDB ID: 4O2B) crystal structure was selected as the protein for the docking simulation. [55] Both compounds seem to occupy the binding site partially overlapping the co-crystallized colchicine, with the trimethoxyphenyl ring orientated towards the nearby α-tubulin subunit, with interactions αSer178 and αThr179 ( Figure 4A,B). The N-methoxy/ethoxycarbonyl moiety at the 6-position of the tetrahydrothieno[2,3-c]pyridine ring is placed in proximity and interacts with either the polypeptide backbone of β-tubulin or with the sidechain of βCys241, a key interaction point for tubulin polymerization inhibition. Interestingly, the two molecules adopt a different orientation than the canonical binding previously reported for several tubulin inhibitors, including combretastatin A-4, in which the trimethoxyphenyl ring is placed in the β-tubulin subunit in proximity to βCys241 [56,57]. This occupation of the colchicine site seems to be fundamental for strong inhibition of tubulin polymerization, and therefore, the different orientation found for compounds 3a and 3b could explain their apparently weaker binding to tubulin and causing their reduced inhibitory effects on tubulin assembly/colchicine binding described above.

Materials and Methods
1 H experiments were recorded on either a Bruker AC 200 or a Varian 400 Mercury Plus spectrometer, while 13 C-NMR spectra were recorded on a Varian 400 Mercury Plus spectrometer. Chemical shifts (δ) are given in ppm upfield, and the spectra were recorded in appropriate deuterated solvents, as indicated. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing Finnigan MAT 95 instrument with BE geometry. Melting points (mp) were determined on a Büchi-Tottoli apparatus and are uncorrected. All products reported showed 1 H and 13 C-NMR spectra in agreement with the assigned structures. The purity of tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at least 95% as compared with the theoretical values. All reactions were carried out under an inert atmosphere of dry nitrogen, unless otherwise indicated. Standard syringe techniques were used for transferring dry solvents. Reaction courses and product mixtures were routinely monitored by Thin Layer Chromatography (TLC) on silica gel (precoated F254 Merck plates), and compounds were visualized with aqueous KMnO4. Flash chromatography was performed using 230-400 mesh silica gel and the indicated solvent system. Organic solutions were dried over anhydrous Na 2 SO 4 .
Following general procedure B, the crude residue was purified by flash chromatography, using ethyl acetate:petroleum ether 0.5:9.5 (v:v) as eluent, to furnish 5p as a colorless oil. Yield: 68%.

Cell Growth Conditions and Antiproliferative Assay
Murine leukemia L1210, human T-lymphocyte leukemia CEM, human cervix carcinoma (HeLa), and human chronic myelogenous leukemia K562 cells (3-5 × 10 4 cells) and a serial (5-fold) dilution of the test compounds were added to a 96-well microtiter plate. The cells were incubated for 72 h at 37 • C in a humidified 5% CO 2 atmosphere. At the end of the incubation period, the cells were counted in a Coulter Counter (Coulter Electronics Ltd., Harpenden Herts, United Kingdom). The IC 50 (50% inhibitory concentration) was defined as the concentration of compound that inhibited cell proliferation by 50%. The IC 50 values represent the average (± standard deviation) of three independent experiments. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by centrifugation with Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala).

Effects on Tubulin Polymerization
Bovine brain tubulin was purified as described previously [58]. To evaluate the effect of the compounds on tubulin assembly in vitro [59], varying concentrations were preincubated with 10 µM tubulin in 0.8 M glutamate buffer at 30 • C and then cooled to 0 • C. After addition of GTP, the mixtures were transferred to 0 • C cuvettes in a recording spectrophotometer and warmed to 30 • C, and the assembly of tubulin was observed turbidimetrically. The IC 50 was defined as the compound concentration that inhibited the extent of assembly by 50% after a 20 min incubation. The ability of the test compounds to inhibit colchicine binding to tubulin was measured as described earlier [60], except that the reaction mixtures contained 1 µM tubulin, 5 µM of [ 3 H]colchicine, and test compounds 3a or 3b.

Effects on the Cell Cycle
Human leukemia K562 cells were treated with compounds 3a or 3b, and analysis of the distribution of the cells through the cell cycle was performed with the Muse Cell Analyzer instrument (Millipore Corporation, Billerica, MA, USA). Assay and cell counting were performed according to the instructions supplied by the manufacturer. Samples were collected after the treatment and washed in PBS. For analysis, 5 × 10 5 cells were resuspended in 200 µL of Muse Cell Cycle Reagent, incubated for 30 min at room temperature, and the cell suspension was transferred to a 1.5 mL microcentrifuge tube prior to analysis. Cells were analyzed by fluorescence-activated cell sorting analysis using a Muse Cell Analyzer.

Effects on Apoptosis
Apoptosis assays on K562 cells were performed with the Muse Cell Analyzer instrument, and assays were performed according to the instructions supplied by the manufacturer. The Muse Annexin V & Dead Cell Kit employs annexin V to detect phosphatidyl serine (PS) on the external membrane of apoptotic cells. A fluorescent DNA intercalator (7-ADD: 7-aminoactinomycin D) is also used as an indicator of cell membrane integrity. From live, healthy cells, as well as early apoptotic cells, 7-ADD is excluded while it is able to bind DNA in cells in late apoptosis and in dead cells. Cells were washed with sterile PBS 1X, suspended, and diluted (1:2) with the Muse Annexin V & Dead Cell reagent. Samples were gently mixed and incubated at room temperature, protected from the light for 15 min. Samples were analyzed using a Muse Cell Analyzer, and data from prepared samples were acquired and recorded utilizing the Annexin V and Dead Cell Software Module (Millipore) [61].

Molecular Modeling Methods
All molecular docking studies were performed on a Viglen Genie Intel ® Core TM i7-3770 vPro CPU@ 3.40 GHz x 8 running Ubuntu 18.04. Molecular Operating Environment (MOE) 2019.10 [62] and Maestro (Schrödinger Release 2019-3) [54] were used as molecular modeling software. The tubulin structure was downloaded from the PDB data bank (http://www.rcsb.org/; PDB code 4O2B). The protein was preprocessed using the Schrödinger Protein Preparation Wizard by assigning bond orders, adding hydrogens and performing a restrained energy minimization of the added hydrogens using the OPLS_2005 force field. Ligand structures were built with MOE and then prepared using the Maestro LigPrep tool by energy minimizing the structures (OPLS_2005 force field), generating possible ionization states at pH 7 ± 2, generating tautomers and low-energy ring conformers. After isolating a tubulin dimer structure, a 12 Å docking grid (inner-box 10 Å and outer-box 22 Å) was prepared using as centroid the co-crystallized colchicine. Molecular docking studies were performed using Glide SP precision, keeping the default parameters and setting 5 as the number of output poses per input ligand to include in the solution. The output database was saved as a mol2 file. The docking results were visually inspected for their ability to bind the active site.

Conclusions
In conclusion, we have described the synthesis and biological evaluation of two classes of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiop hene skeletons, both characterized by the presence of a common 3 ,4 ,5 -trimethoxyanilino moiety at the 2-position and a cyano or different alkoxycarbonyl groups at the 3-position. In the series of 2-(3 ,4 , 5 -trimethoxyanilino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivatives, the results indicated that inhibition of cell growth was strongly dependent on the substituent at the 3-position, with the greatest activity occurring with the cyano group, the least with alkoxycarbonyl moieties such as methoxy/ethoxy/tert-butoxycarbonyl. We also demonstrated that replacement of the 4,5,6,7-tetrahydro thieno [2,3-c]pyridine moiety with a 4,5,6,7-tetrahydrobenzo[b]thiophene system was detrimental for antiproliferative activity. The 2-(3 ,4 ,5 -trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahy drothieno [2,3-c]pyridine derivative 3a and the corresponding 6-ethoxycarbonyl homologue 3b were the most promising compounds in this series, which inhibited cancer cell growth at low micromolar concentrations and interacted with tubulin by binding to the colchicine site. Comparing the two 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile derivatives 3a and 3b, the latter was about 1.5-fold more active than 3a against L1210 and HeLa cells, while both these derivatives were equipotent against CEM cells. We also showed by flow cytometry that derivatives 3a and 3b had cellular effects typical of agents that bind to tubulin, causing accumulation of cells in the G2-M phase of the cell cycle. Apoptotic cells also increased in a concentration-dependent manner, with an increase in the percentage of apoptotic K562 cells observed at the IC 50 values examined for 3a and 3b (0.75 and 0.70 µM, respectively). Finally, the same compounds showed high sensitivity towards cancer over normal cells as they had no significant antiproliferative activity toward both quiescent and phytohemoagglutinin-stimulated cultures of PBMC (IC 50 > 20 µM), suggesting that derivatives 3a and 3b may have selectivity against cancer cells.