Abstract
A new series of 1H-pyrrolo[3,2-c]pyridine derivatives were designed and synthesised as colchicine-binding site inhibitors. Preliminary biological evaluations showed that most of the target compounds displayed moderate to excellent antitumor activities against three cancer cell lines (HeLa, SGC-7901, and MCF-7) in vitro. Among them, 10t exhibited the most potent activities against three cancer cell lines with IC50 values ranging from 0.12 to 0.21 μM. Tubulin polymerisation experiments indicated that 10t potently inhibited tubulin polymerisation at concentrations of 3 μM and 5 μM, and immunostaining assays revealed that 10t remarkably disrupted tubulin microtubule dynamics at a concentration of 0.12 μM. Furthermore, cell cycle studies and cell apoptosis analyses demonstrated that 10t at concentrations of 0.12 μM, 0.24 μM, and 0.36 μM significantly caused G2/M phase cell cycle arrest and apoptosis. The results of molecular modelling studies suggested that 10t interacts with tubulin by forming hydrogen bonds with colchicine sites Thrα179 and Asnβ349. In addition, the prediction of physicochemical properties disclosed that 10t conformed well to the Lipinski’s rule of five.
Introduction
Microtubules consist of α- and β-microtubulin heterodimers and microtubule-associated proteins, which exhibit dynamic properties of depolymerisation and polymerizationCitation1–3. As the critical elements and components of the cytoskeleton, microtubules play important and fundamental roles in a variety of biological processes, such as cell mitosis, cytokinesis, and signal transductionCitation4–6. Microtubule-targeting drugs (MTA) can disrupt the balance of microtubule dynamics by binding to specific binding sites of microtubule proteins, leading to mitotic arrest. Given their key role in cell growth, microtubules have emerged as a successful and attractive target for anticancer drug developmentCitation7,Citation8. To date, a total of seven MTA-binding sites have been confirmed in the literature. These include the colchicine-binding site, gatorbulin-binding site, laulimalide/peloruside-binding site, maytansine-binding site, pironetin binding-site, taxane binding-site, and vinblastine binding-siteCitation9–12. Actually, some MTA acting on paclitaxel- or vincristine-binding sites, such as paclitaxel and vincristine, have already been approved for tumour treatment by the FDA and have achieved significantly high therapeutic effects and salesCitation13–15. Notably, the colchicine-binding site is situated at the binding interface of the α/β tubulin dimer, colchicine-binding site inhibitors (CBSI) can inhibit microtubule dynamics, which in turn leads to mitotic arrest, thus exerting effective anticancer effects. In addition, CBSIs can overcome the limitations and drawbacks of paclitaxel- or vinblastine-binding site inhibitors. However, due to adverse effects such as neurotoxicity and haematotoxicity, no CBSI have been approved by the FDA for cancer therapy, but several CBSI are currently approved by the FDA for clinical trials, such as CA-4 (1, ), CA-4P (2, ), and OXI4503 (3, )Citation16–18. Therefore, the search for novel CBSI with different chemical structures and better anticancer effects is still encouraged. Many CBSIs (4–8, ) with different chemical structures and significant antitumour potency have been reported, some of which can overcome MDR and even show good bioavailabilityCitation19,Citation20.
Figure 1. Chemical structures of CBSI.
A potent anticancer agent, CA-4, a naturally cis-stilbene derivative derived from the African willow species Combretum caffrum, substantially reduced tubulin polymerisation by interacting with the colchicine-binding siteCitation21. Additionally, the (Z, E)-vinylogous CA-4 (9, ), produced by J. Kaffy and colleagues, also showed comparable efficacy to CA-4 in inhibiting tubulin polymerizationCitation22. However, the fact that the cis-olefin bonds of both CA-4 and 9 are susceptible to isomerisation in an acidic media led to a significant decrease in both antitubulin and antiproliferative activitiesCitation23,Citation24. One proven method for avoiding the stability issue with CA-4 and 9 is to substitute a suitable heterocyclic component for the cis-olefin bondCitation25–28. This drives us to validate whether the configuration-constrained strategy can be used for optimising the structures of CA-4 and 9.
Figure 2. The rational design of target compounds.
Due to their numerous pharmacological activities, particularly their anticancer potential, pyrrolo[3,2-c]pyridine scaffolds are highly superior in medicinal chemistry and have garnered a significant lot of interest from medicinal chemistsCitation29–31. Inspired by the fact that pyrrolo[3,2-c]pyridine scaffolds have attractive properties, we utilised the configuration-constrained strategy that replaced the cis-olefin bonds of both CA-4 and 9 with a rigid 1H-pyrrolo[3,2-c]pyridine scaffold to restrict bioactive configuration of CA-4 and 9 to design a series of ring-fused 1H-pyrrolo[3,2-c]pyridine derivatives (10a-t). We expect to discover new potent CBSI from these newly designed molecules.
Result and discussion
Chemistry
Scheme 1 shows the detailed synthesis method of designed 6-aryl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridines (10a–t). Briefly stated, commercially available 2-bromo-5-methylpyridine (11) was reacted with m-chloroperbenzoic acid to obtain 2-bromo-5-methylpyridine-1-oxide (12). Then 2-bromo-5-methylpyridine-1-oxide (12) was transformed to 2-bromo-5-methyl-4-nitropyridine 1-oxide (13) by using fuming nitric acid in sulphuric acid. Subsequently, 13 was reacted with N,N-dimethylformamide dimethyl acetal in N,N-dimethylformamide to afford the key intermediate (14). 6-bromo-1H-pyrrolo[3,2-c]pyridine (15) was synthesised from 14 in the presence of iron powder and acetic acid. 15 combined with 3,4,5-trimethoxyphenylboric acid, potassium carbonate, pyridine, and copper (II) acetate, produced the intermediate 6-bromo-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (16)Citation32. The Suzuki cross-coupling reaction between 16 and the corresponding arylboronic acids was used to generate the target compounds, 6-aryl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridines (10)Citation33.
Scheme 1. Reagents and conditions (a) m-chloroperbenzoic acid, N2 atmosphere, rt., overnight; (b) HNO3/H2SO4, 60–90 °C, 1 h; (c) N,N-dimethylformamide dimethyl acetal, N,N-dimethylformamide, 120 °C, 4 h; (d) iron powder, acetic acid, 100 °C, 5 h; (e) 3,4,5-trimethoxyphenylboric acid, K2CO3, pyridine, Cu(OAc)2, 1,4-dioxane, N2 atmosphere, 85 °C, microwave (M.W), 30 min; (f) Substituted phenylboronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O, N2 atmosphere,125 °C, M.W., 26 min.
Biological evaluation
In vitro antiproliferative activity
Using a standard MTT assay with CA-4 as the positive control, the in vitro antiproliferative activities against three human cancer cell lines-including the cervical cancer cell line HeLa, the gastric cancer cell line SGC-7901, and the breast cancer cell line MCF-7-were assessed. As shown in , most of these new compounds exhibited moderate to excellent antiproliferative activities, suggesting that locking the (Z, E)-butadiene of vinylated CA-4 using a rigid 1H-pyrrolo[3,2-c]pyridine scaffold is an effective strategy to maintain potent antiproliferative activity. Among them, 10t with indolyl as the B-ring showed the strongest antiproliferative activities against HeLa, SGC-7901, and MCF-7 cell lines with IC50 values of 0.12, 0.15, and 0.21 µM, respectively.
Table 1. Antiproliferative activity of all compounds.
The structure-activity relationship (SAR) of compounds in this series was summarised. With phenyl or aryl moieties as the B-ring, 10a–10o showed moderate to weak activities, and the introduction of electron-donating groups (EDGs), such as CH3 (10d) and OCH3 (10h), on the para-substitution of B-ring, led to an increase in antiproliferative activities. However, when electron-withdrawing groups (EWGs), such as F (10l), Cl (10 m), and NO2 (10n), were introduced on the para-substitution of B-ring, the antiproliferative activities were decreased, and the results indicated that EDGs located on the para-substitution of B-ring had better activities. Importantly, compounds with EDGs (e.g., OH and OCH3) on the meta-substitution of B-ring (10i and 10j) could improve the inhibitory activities. Furthermore, EDGs on the B-ring exhibited an order of potency being ortho- < meta- < para-substituted in generally (10b < 10c < 10d and 10f < 10 g < 10h). We next introduced different rigid aromatic groups such as naphthyl (8p), thienyl (10q), pyridyl (10r and 10s), and indolyl (10t) into the B-ring to explore the effect of different skeletons on antiproliferative activities against three different cell lines. Amongst these, 10t showed the best antiproliferative activity against HeLa cells (IC50 value of 0.12 µM), suggesting that the volume and electronegativity of B-ring may be the important factors affecting activity.
Effect on tubulin polymerisation
The tubulin polymerisation experiments were used to investigate the effect of target compounds on tubulin polymerisation in order to examine whether the inhibitory activity of compound 10t was induced by disrupting the tubulin system. 0.1% methyl sulfoxide (DMSO) was the blank control, CA-4 was the positive control, and paclitaxel was the negative control. Compared with the DMSO blank group, 5 μM of tubulin depolymerisation inhibitor paclitaxel accelerated the growth rate of fluorescence intensity, indicating an enhancement of tubulin polymerisation, while 3 μM of tubulin polymerisation inhibitor CA-4 decreased the growth rate of fluorescence intensity, suggesting a reduction of tubulin polymerisation. As with CA-4, 10t also reduced the growth rate of fluorescence intensity at concentrations of 3 μM and 5 μM, suggesting that 10t inhibited tubulin polymerisation in a manner similar to CA-4, and that 10t had the same strong inhibitory activity as CA-4 (. What these tubulin polymerisation tests show is that 10t and CA-4 inhibited polymerisation to approximately the same extent at equimolar concentrations. These results reveal that 10t is a new tubulin polymerisation inhibitor with the same mechanism of action as CA-4.
Figure 3. Effects of compound 10t on tubulin polymerisation.
Analysis of immunofluorescence staining
Using confocal immunofluorescent microscopy, we further examined the effect of compound 10t on tubulin polymerisation at the cellular level in vitro. Under fluorescence microscopy, the tubulin networks labelled with Alexa Fluor 488 were green, and the nuclei labelled with DAPI were blue. The microtubule networks of normal HeLa cells were evenly distributed in the cytoplasm and well assembled, as seen in . When HeLa cells were exposed to CA-4 (1 fold IC50) or 10t (1 fold IC50) for 24 h, respectively, the microscopic structure and shape underwent profound changes. Such changes were highlighted in two interrelated aspects: first, the cell morphology became rounded and the cytoskeletal microtubules were destroyed; second, the appearance of multinucleated cells. This phenomenon suggests that 10t may be a colchicine-site binder, similar to CA-4.
Figure 4. Effects of compound 10t (1 IC50) and CA-4 (1 IC50), on the cellular microtubule network and microtubule reassemble by immunofluorescence.
Cell cycle analysis
Typically, CBSIs arrest the cell cycle of tumour cells in the G2/M phase. To ascertain whether compound 10t could arrest HeLa cells in the G2/M phase, cell cycle analysis was performed on HeLa cells. For cell analysis by flow cytometry, the test groups were treated for 24 h with 10t at concentrations of 1 IC50, 2 IC50, and 3 IC50, whereas the control group received 0.1% DMSO as a treatment. According to , compared to the control group (3.8% in G2/M phase), there were 9.6%, 40.6%, and 67.3% more HeLa cells arrested in G2/M phase when 10t (1 IC50, 2 IC50, and 3 IC50) were present. These findings demonstrate that 10t resulted in cell accumulation in the G2/M phase in a dose-dependent manner.
Figure 5. Effects of CA-4 and compound 10t on cell cycle. HeLa cells were treated with compound 10t (1 IC50, 2 IC50, and 3 IC50) for 24 h.
Induction of cell apoptosis
Apoptosis may begin at mitotic arrest in cancer cells. Using a biparametric cytofluorimetric analysis, we quantitatively analysed the cellular distribution of early apoptotic (propidium iodide negative but Annexin V positive stained cells) and late apoptotic cells (propidium iodide and Annexin V-positive stained cells) after treatment with different concentrations of compound 10t for 48 h. shows that the proportion of apoptotic cells increased in the 10t groups compared to the total number of apoptotic cells in the control group (4.4%), reaching 16.8%, 24.5%, and 33.1% at concentrations of 1 IC50, 2 IC50, and 3 IC50, respectively. These all demonstrated that 10t might cause dose-dependent cell apoptosis in HeLa cells.
Figure 6. Analyses of apoptosis induction in Hela cells. Cells were harvested and stained with Annexin-V/PI for analysis after treatment with different concentrations of compound 10t (1 IC50, 2 IC50, and 3 IC50) and control for 48 h. The diverse cell stages were given as live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1).
Molecular docking analysis
In order to further investigate the binding interactions, molecular docking studies of CA-4, 7, and the most potent compound, 10t, with the tubulin crystal structure (PDB: 5LYJ) were also performed using the SchrödingerCitation34,Citation35. When CA-4 was first docked to the tubulin’s colchicine binding site, and its conformation was approximately duplicated with the co-crystal conformation, indicating that the method is suitable for docking analysis. As seen in , 10t had considerable overlap with CA-4 (the original co-crystal ligand) in the active site, with its 1H-pyrrolo[3,2-c]pyridine core overlapping with the (E, Z)-butadiene linker of 7 and forming an important hydrogen bond with Thrα179, and the nitrogen atom of the indole (B-ring) forming another key hydrogen bond with Asnβ349. It is worth noting that the docking score of 10t (Docking score: −12.4 kcal/mol) in the 5LYJ was lower than that of CA-4 (Docking score: −9.2 kcal/mol) and 7 (Docking score: −9.1 kcal/mol), which might be the reason why the antiproliferative activities of 10t were as potent as that of CA-4 and 7. The molecular docking study showed that 10t could be bound to tubulin at the colchicine-binding site.
Figure 7. Proposed binding modes for 10t (C) in comparison with CA-4 (A) and 7 (B) at the colchicine-site. Carbon atoms are shown in cyan for CA-4, in salmon for 7, and in pale purple for 10t. The residues from the α-tubulin chain are shown in pale yellow, whereas residues from β-tubulin are coloured in gray.
Physicochemical properties
To investigate the drug-like properties of 1H-pyrrolo[3,2-c]pyridine derivatives, traditional physicochemical properties of CA-4, 10i, and 10t were predicted via ChemBioDraw Ultra 14.0 software or a free online website (http://www.swissadme.ch/index.php) to fit Lipinski’s five rules. As shown in , CA-4, 10i, and 10t fit Lipinski’s five rules well.
Table 2. Prediction of physicochemical properties of CA-4, 9i, and 9pTable Footnotea.
Conclusion
Altogether, in order to limit the bioactive configuration of 1and 9, the rigid 1H-pyrrolo[3,2-c]pyridine scaffold was used to design a series of novel 6-aryl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridines as CBSI. With IC50 values in the micromolar to nanomolar range, the majority of target compounds demonstrated moderate to potent antiproliferative effects. The most effective activities against three cancer cell lines with IC50 values ranging from 0.12 to 0.21 µM were shown by 10t, which had an indolyl moiety as the B-ring and a 3,4,5-trimethoxyphenyl moiety as the A-ring. The SAR of these compounds was thoroughly examined. Tubulin polymerisation assay showed that 10t could notably inhibit tubulin polymerisation similarly to CA-4. Immunofluorescence analysis indicated that 10t could effectively disrupt the microtubule networks, and its inhibitory effect was similar to that of CA-4. Cell cycle analysis and induction of cell apoptosis studies suggested that 10t could significantly induce HeLa cells to arrest in G2/M phase and induce apoptosis. In addition, molecular docking studies revealed that 10t could occupy the colchicine-binding site on tubulin. Finally, the prediction of physicochemical properties indicated that 10t fits well with five Lipinski’s rule. All these results suggest that 10t is a promising inhibitor of tubulin polymerisation and is expected to be further investigated in anticancer drug development.
Experimental
Chemistry
Materials and methods
All of the reagents and solvents were purchased from chemical companies. TLC analysis was used for determining the extent of reactions under UV light (wavelength: 365 nm and 254 nm).1H NMR and 13C NMR spectra were tested in CDCl3 with TMS as the internal reference on a Bruker AVANCE (1H at 500 MHz and 13C at 126 MHz). High-resolution mass spectra (HRMS) were recorded by Agilent Accurate-Mass Q-TOF 6530 instrument in ESI mode. The microwave (M.W.) reactions were carried out in a single-mode cavity microwave synthesiser (CEM Corporation, NC, USA).
General synthetic procedure for 2-bromo-5-methylpyridine 1-oxide (12)
A mixture of 2-bromo-5-methylpyridine (1.7 mmol, 0.29 g) and m-chloroperbenzoic acid (2.6 mmol, 0.45 g) in dichloromethane (5 ml) was stirred overnight at room temperature under N2. The pH was adjusted to 8 with aqueous sodium bicarbonate and the reaction was extracted with dichloromethane (25 ml × 3). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to afford the title compound as a yellow solid.
General synthetic procedure for 2-bromo-5-methyl-4-nitropyridine-1-oxide (13)
To a reaction flask containing a solution of 2-bromo-5-methylpyridine-l-oxide (5.3 mmol, 1 g) in concentrated sulphuric acid (2 mL) was added a mixture of fuming nitric acid in sulphuric acid (3 ml, 1:1) dropwise with stirring at 60 °C. The resulting solution was stirred for 1 h at 90 °C, cooled to room temperature, and quenched by the addition of ice water. The reaction was filtered and the filtrate was extracted with ethyl acetate (30 ml × 3). The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo to afford the title compound as a yellow solid.
General synthetic procedure for (E)-2-bromo-5–(2-(dimethylamino)vinyl)-4-nitropyridine-1-oxide (14)
To a reaction vessel were added 2-bromo-5-methyl-4-nitropyridine-l-oxide (3.9 mmol, 0.91 g), N, N-dimethylformamide (5 ml), and N, N -dimethylformamide dimethyl acetal (5 mL). The resulting solution was stirred for 4 h at 120 °C. The reaction was cooled to 0 °C and the product was isolated by filtration and dried to afford the title compound as a black solid.
General synthetic procedure for 6-bromo-1H-pyrrolo[3,2-c]pyridine (15)
To a reaction vessel was added (E)-2-bromo-5–(2-(dimethylamino)vinyl)-4-nitropyridine-1-oxide (13.8 mmol, 3.97 g), iron powder (55.7 mmol, 3.11 g), and acetic acid (80 mL). The reaction mixture was stirred for 5 h at 100 °C, filtered, and concentrated in vacuo. The pH was adjusted to 8 with aqueous sodium carbonate and extracted with ethyl acetate. The organic portion was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by silica gel chromatography using a mixture of n-hexane/ethyl acetate (1:2) as an eluent to afford the title compound as a white solid.
General synthetic procedure for 6-bromo-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (16)
The starting material 6-bromo-1H-pyrrolo[3,2-c]pyridine (0.3 mmol, 0.06 g), 3,4,5-trimethoxyphenylboric acid (0.6 mmol, 0.13 g), K2CO3 (0.6 mmol, 0.083 g), pyridine (0.9 mmol, 0.071 g), and Cu(OAc)2 (0.6 mmol, 0.12 g) were dissolved in 1,4-dioxane (15 mL). Then the mixture was stirred at irradiated in a microwave reactor for 30 min at 85 °C. When the reaction was completed, the mixture was extracted with ethyl acetate (25 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and purified by silica gel column chromatography or recrystallized to afford 6-bromo-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (16). 1H NMR (500 MHz, CDCl3) δ 8.73 (s, 1H), 7.56 (s, 1H), 7.31 (d, J = 3.3 Hz, 1H), 6.74 (d, J = 3.3 Hz, 1H), 6.63 (s, 2H), 3.92 (s, 3H), 3.91 (s, 6H); HRMS calcd for C16H16BrN2O3 [M + H]+ 363.0344, found 363.0314.
General synthetic procedure for 6-aryl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridines (10a–t)
The intermediate 6-bromo-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (16, 0.1 mmol, 0.036 g), substituted phenylboronic acid (0.15 mmol), K2CO3 (0.5 mmol, 0.069), and Pd(PPh3)4 (0.006 mmol, 0.007 g) were dissolved in 1,4-dioxane (6 mL) and H2O (2 mL) and degassed with N2. Then the mixture was reacted in a microwave reactor for 26 min at 125 °C. When the reaction was completed, the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and purified by silica gel column chromatography to afford target compounds 6-aryl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridines (10a–t).
6-phenyl-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10a)
White solid; yield: 63%; 1H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.08 − 7.91 (m, 2H), 7.80 (s, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.41 − 7.34 (m, 2H), 6.80 (d, J = 3.2 Hz, 1H), 6.71 (s, 2H), 3.95 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.07, 154.07, 149.89, 143.18, 141.17, 137.58, 134.10, 130.28, 128.78, 128.78, 128.31, 127.88, 127.00, 127.00, 124.81, 102.92, 102.59, 102.45, 102.45, 61.06, 56.44, 56.44; HRMS calcd for C22H21N2O3 [M + H]+ 361.1552, found 361.1556.
6-(o-tolyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10b)
White solid; yield: 65%; 1H NMR (500 MHz, CDCl3) δ 9.08 (s, 1H), 7.50 (s, 1H), 7.43 (d, J = 6.7 Hz, 1H), 7.39 (d, J = 3.3 Hz, 1H), 7.28 (t, J = 3.8 Hz, 2H), 7.26 − 7.24 (m, 1H), 6.82 (d, J = 3.2 Hz, 1H), 6.69 (s, 2H), 3.91 (s, 3H), 3.89 (s, 6H), 2.40 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 157.84, 154.00, 154.00, 151.97, 142.93, 140.46, 137.45, 135.91, 134.21, 130.65, 129.99, 129.81, 127.88, 125.80, 124.28, 105.81, 102.76, 102.32, 102.32, 61.02, 56.38, 56.38, 20.49; HRMS calcd for C23H23N2O3 [M + H]+ 375.1709, found 375.1707.
6-(m-tolyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10c)
White solid; yield: 94%; 1H NMR (500 MHz, CDCl3) δ 9.09 (s, 1H), 7.96 (d, J = 11.1 Hz, 2H), 7.76 (s, 1H), 7.72 − 7.68 (m, 1H), 7.39 (d, J = 3.3 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 6.80 (d, J = 3.9 Hz, 1H), 6.70 (s, 2H), 3.95 (s, 3H), 3.91 (s, 6H), 2.42 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 159.03, 154.09, 154.09, 149.85, 142.82, 141.24, 138.54, 135.71, 134.01, 132.53, 131.76, 130.48, 129.24, 127.70, 124.73, 103.04, 102.77, 102.47, 102.47, 61.06, 56.44, 56.44, 21.53; HRMS calcd for C23H23N2O3 [M + H]+ 375.1709, found 375.1709.
6-(p-tolyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10d)
White solid; yield: 67%; 1H NMR (500 MHz, CDCl3) δ 9.07 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.77 (s, 1H), 7.36 (d, J = 3.3 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 3.2 Hz, 1H), 6.71 (s, 1H), 3.95 (s, 1H), 3.91 (s, 1H), 2.40 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 154.05, 154.05, 150.05, 143.17, 141.18, 138.19, 135.59, 134.19, 130.05, 129.49, 129.49, 128.71, 126.82, 126.82, 124.66, 102.87, 102.41, 102.41, 102.14, 61.05, 56.42, 56.42, 21.20; HRMS calcd for C23H23N2O3 [M + H]+ 375.1709, found 375.1708.
6–(3,4-dimethylphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10e)
White solid; yield: 49%; 1H NMR (500 MHz, CDCl3) δ 9.06 (s, 1H), 7.83 (s, 1H), 7.77 (s, 1H), 7.68 (d, J = 6.4 Hz, 1H), 7.34 (d, J = 3.3 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 6.78 (d, J = 3.2 Hz, 1H), 6.71 (s, 2H), 3.95 (s, 3H), 3.91 (s, 6H), 2.35 (s, 3H), 2.31 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 154.03, 154.03, 150.44, 143.40, 141.09, 137.86, 137.41, 136.94, 136.72, 134.33, 129.99, 129.75, 128.19, 124.66, 124.15, 102.75, 102.37, 102.37, 101.99, 61.06, 56.41, 56.41, 19.92, 19.54; HRMS calcd for C24H25N2O3 [M + H]+ 389.1865, found 389.1865.
6–(2-methoxyphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10f)
White solid; yield: 76%; 1H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 7.93 (s, 1H), 7.79 (dd, J = 7.6, 1.7 Hz, 1H), 7.38 (d, J = 3.3 Hz, 1H), 7.35 (ddd, J = 8.3, 7.5, 1.8 Hz, 1H), 7.08 (td, J = 7.5, 1.0 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 3.2 Hz, 1H), 6.73 (s, 2H), 3.92 (s, 3H), 3.91 (s, 6H), 3.83 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 158.41, 156.74, 153.94, 153.94, 147.82, 143.06, 140.29, 137.26, 134.39, 131.49, 129.79, 129.29, 124.56, 121.08, 111.53, 106.84, 102.84, 102.19, 102.19, 61.05, 56.36, 56.36, 55.83; HRMS calcd for C23H23N2O4 [M + H]+ 391.1658, found 391.1653.
6–(3-methoxyphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10 g)
White solid; yield: 61%; 1H NMR (500 MHz, CDCl3) δ 9.08 (s, 1H), 7.76 (s, 1H), 7.56 (s, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 3.3 Hz, 1H), 7.35 (s, 1H), 6.93 (dd, J = 8.5, 2.9 Hz, 1H), 6.81 (d, J = 2.7 Hz, 1H), 6.70 (s, 2H), 3.95 (s, 3H), 3.91 (s, 6H), 3.88 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 160.07, 159.05, 154.10, 154.10, 142.64, 141.24, 137.69, 133.90, 132.03, 130.68, 129.81, 124.86, 119.39, 114.42, 112.44, 103.09, 102.97, 102.48, 102.48, 61.06, 56.44, 56.44, 55.43; HRMS calcd for C23H23N2O4 [M + H]+ 391.1658, found 391.1656.
6–(4-methoxyphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10h)
White solid; yield: 51%; 1H NMR (500 MHz, CDCl3) δ 9.05 (s, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.73 (s, 1H), 7.35 (d, J = 3.3 Hz, 1H), 7.00 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 3.2 Hz, 1H), 6.71 (s, 2H), 3.94 (s, 3H), 3.91 (s, 6H), 3.86 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 159.94, 154.05, 154.05, 149.76, 143.11, 141.27, 137.49, 134.21, 129.94, 128.15, 128.15, 124.41, 114.16, 114.16, 113.49, 102.85, 102.42, 102.42, 101.61, 61.05, 56.43, 56.43, 55.36; HRMS calcd for C23H23N2O4 [M + H]+ 391.1658, found 391.1654.
2-methoxy-5–(1-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridin-6-yl)phenol (10i)
White solid; yield: 54%; 1H NMR (500 MHz, CDCl3) δ 9.04 (s, 1H), 7.71 (s, 1H), 7.68 − 7.66 (m, 1H), 7.59 (dd, J = 8.4, 2.2 Hz, 1H), 7.35 (d, J = 3.3 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 3.3 Hz, 1H), 6.70 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.05, 154.05, 147.06, 145.82, 142.89, 141.30, 137.55, 134.12, 132.95, 131.89, 130.10, 124.47, 118.99, 113.10, 110.86, 102.82, 102.47, 102.47, 101.87, 61.05, 56.43, 56.43, 56.02; HRMS calcd for C23H23N2O5 [M + H]+ 407.1607, found 407.1607.
6–(3,4-dimethoxyphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10j)
White solid; yield: 55%; 1H NMR (500 MHz, CDCl3) δ 9.05 (s, 1H), 7.74 (s, 1H), 7.64 (d, J = 1.3 Hz, 1H), 7.55 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 3.3 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 3.2 Hz, 1H), 6.71 (s, H), 4.00 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 160.23, 154.08, 154.08, 149.54, 142.76, 141.29, 137.55, 134.09, 132.67, 129.71, 124.52, 119.14, 117.27, 111.23, 110.42, 102.98, 102.40, 102.40, 101.94, 61.06, 56.43, 56.43, 56.11, 55.97; HRMS calcd for C24H25N2O3 [M + H]+ 421.1763, found 421.1760.
6–(4-ethoxyphenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10k)
White solid; yield: 57%; 1H NMR (500 MHz, CDCl3) δ 9.05 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.71 (s, 1H), 7.35 (d, J = 3.2 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 3.2 Hz, 1H), 6.70 (s, 2H), 4.07 (q, J = 6.9 Hz, 2H), 3.94 (s, 3H), 3.91 (s, 6H), 1.43 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 159.43, 154.06, 154.06, 142.74, 141.38, 137.56, 137.42, 134.10, 130.20, 128.19, 128.19, 124.34, 114.77, 114.77, 113.95, 102.96, 102.96, 102.46, 101.75, 63.53, 61.05, 56.44, 56.44, 14.81; HRMS calcd for C24H25N2O4 [M + H]+ 405.1814, found 405.1815.
6–(4-fluorophenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10l)
White solid; yield: 85%; 1H NMR (500 MHz, CDCl3) δ 9.04 (s, 1H), 8.00 − 7.92 (m, 2H), 7.73 (s, 1H), 7.36 (d, J = 3.3 Hz, 1H), 7.14 (t, J = 8.7 Hz, 2H), 6.78 (d, J = 3.9 Hz, 1H), 6.70 (s, 2H), 3.94 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 162.98, 154.06, 154.06, 149.39, 143.63, 141.04, 137.55, 136.54, 134.19, 129.99, 128.61, 128.61, 124.80, 115.55, 115.55, 102.72, 102.48, 102.48, 102.13, 61.05, 56.44, 56.44; HRMS calcd for C22H20FN2O3 [M + H]+ 379.1458, found 379.1456.
6–(4-chlorophenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10m)
White solid; yield: 32%; 1H NMR (500 MHz, CDCl3) δ 9.06 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.76 (s, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 3.3 Hz, 1H), 6.80 (d, J = 3.0 Hz, 1H), 6.70 (s, 2H), 3.95 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.08, 154.08, 148.89, 143.56, 141.02, 138.61, 137.62, 134.19, 134.09, 130.24, 128.86, 128.86, 128.15, 128.15, 124.98, 102.82, 102.50, 102.50, 102.27, 61.06, 56.44, 56.44; HRMS calcd for C22H20ClN2O3 [M + H]+ 395.1162, found 395.1164.
6–(4-nitrophenyl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10n)
Yellow solid; yield: 51%; 1H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.19 (d, J = 8.7 Hz, 2H), 7.87 (s, 1H), 7.47 (d, J = 3.1 Hz, 1H), 6.88 (d, J = 3.0 Hz, 1H), 6.70 (s, 2H), 3.96 (s, 3H), 3.92 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 162.10, 154.21, 154.21, 147.70, 143.23, 141.03, 138.05, 133.54, 131.61, 127.68, 126.13, 125.56, 124.13, 124.13, 115.66, 103.93, 103.29, 102.69, 102.69, 61.08, 56.50, 56.50; HRMS calcd for C22H20N3O5 [M + H]+ 406.1403, found 406.1403.
4–(1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridin-6-yl)phenol (10o)
White solid; yield: 50%; 1H NMR (500 MHz, CDCl3) δ 9.03 (s, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.67 (s, 1H), 7.36 (d, J = 3.3 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 3.2 Hz, 1H), 6.70 (s, 2H), 3.94 (s, 3H), 3.90 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 157.87, 154.06, 154.06, 149.89, 142.30, 141.51, 137.59, 134.03, 132.70, 131.87, 128.37, 128.37, 124.19, 116.12, 116.12, 103.02, 102.45, 102.45, 101.99, 61.05, 56.44, 56.44; HRMS calcd for C22H21N2O4 [M + H]+ 377.1501, found 377.1498.
6-(Naphthalen-2-yl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10p)
White solid; yield: 52%; 1H NMR (500 MHz, CDCl3) δ 9.13 (s, 1H), 8.51 (s, 1H), 8.14 (dd, J = 8.6, 1.7 Hz, 1H), 7.94 (dd, J = 10.1, 5.6 Hz, 3H), 7.89 − 7.83 (m, 1H), 7.52 − 7.45 (m, 2H), 7.38 (d, J = 3.3 Hz, 1H), 6.81 (d, J = 3.2 Hz, 1H), 6.75 (s, 2H), 3.96 (s, 3H), 3.93 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.08, 154.08, 150.17, 143.81, 141.08, 137.68, 137.51, 134.30, 133.66, 133.20, 129.96, 128.56, 128.32, 127.60, 126.19, 126.09, 125.94, 124.95, 124.86, 102.78, 102.64, 102.47, 102.47, 61.07, 56.44, 56.44; HRMS calcd for C26H23N2O3 [M + H]+ 411.1709, found 411.1710.
6-(Thiophen-3-yl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10q)
White solid; yield: 59%; 1H NMR (500 MHz, CDCl3) δ 8.97 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.69 (s, 1H), 7.61 (dd, J = 5.1, 1.1 Hz, 1H), 7.40 − 7.39 (m, 1H), 6.80 (d, J = 3.1 Hz, 1H), 6.69 (s, 2H), 3.95 (s, 3H), 3.92 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.15, 154.15, 141.87, 141.41, 137.87, 133.62, 131.24, 126.77, 126.00, 125.71, 125.17, 124.42, 123.72, 103.55, 102.57, 102.51, 102.51, 61.07, 56.48, 56.48; HRMS calcd for C20H19N2O3S [M + H]+ 367.1116, found 367.1115.
6-(Pyridin-3-yl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10r)
White solid; yield: 55%; 1H NMR (500 MHz, CDCl3) δ 9.18 (s, 1H), 9.10 (s, 1H), 8.62 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 7.83 (s, 1H), 7.41 (d, J = 3.3 Hz, 2H), 6.82 (d, J = 3.2 Hz, 1H), 6.71 (s, 2H), 3.95 (s, 3H), 3.92 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.13, 154.13, 148.95, 147.94, 147.06, 143.89, 140.88, 137.72, 136.67, 134.64, 133.92, 130.54, 125.29, 123.74, 102.92, 102.81, 102.45, 102.45, 61.06, 56.45, 56.45; HRMS calcd for C21H20N3O3 [M + H]+ 362.1505, found 362.1502.
6-(Pyridin-4-yl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10s)
White solid; yield: 32%; 1H NMR (500 MHz, CDCl3) δ 9.11 (s, 1H), 8.74 (s, 2H), 7.99 (s, 2H), 7.90 (s, 1H), 7.43 (d, J = 3.3 Hz, 1H), 6.83 (d, J = 3.8 Hz, 1H), 6.70 (s, 2H), 3.95 (s, 3H), 3.92 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.15, 154.15, 149.61, 147.93, 146.76, 144.13, 144.07, 141.08, 140.68, 137.81, 133.86, 130.91, 130.91, 126.05, 103.25, 102.96, 102.58, 102.58, 61.06, 56.47, 56.47; HRMS calcd for C21H20N3O3 [M + H]+ 362.1505, found 362.1503.
6-(1H-indol-6-yl)-1–(3,4,5-trimethoxyphenyl)-1H-pyrrolo[3,2-c]pyridine (10t)
White solid; yield: 51%; 1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H), 8.69 (s, 1H), 7.94 (s, 1H), 7.71 − 7.64 (m, 2H), 7.55 (d, J = 2.6 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.25 (t, J = 2.8 Hz, 1H), 6.96 (s, 1H), 6.82 (d, J = 3.3 Hz, 1H), 6.74 (s, 2H), 3.92 (s, 3H), 3.91 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 154.01, 154.01, 151.27, 143.30, 140.82, 137.28, 136.64, 134.40, 132.92, 129.75, 126.04, 124.84, 124.54, 122.10, 120.04, 111.15, 104.84, 102.88, 102.27, 102.16, 102.16, 61.04, 56.37, 56.37; HRMS calcd for C24H22N3O3 [M + H]+ 400.1661, found 400.1659.
Biological evaluation
Cell culture
HeLa, SGC-7901, and MCF-7 cells were purchased from Shanghai Institute of Cell Resources Centre of Life Science (Shanghai, China). All cells were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), penicillin, and streptomycin at 37 °C in humidified atmosphere with 5% CO2.
In vitro antiproliferative activity
Target compounds 10a–t and CA-4 were tested for their in vitro antiproliferative activities using the established MTT assay, as previously describedCitation36. In 96-microwell plates, the cell lines were cultured for 24 h at 37 °C in a humidified 5% CO2 incubator. Following that, test compounds were applied to triplicate wells at different concentrations for an additional 72 h. Medium was removed and 20 ml of MTT (5 mg/mL in PBS) was added to incubate for another 4 h, the medium with MTT was removed and 150 μL of DMSO was added to each well. Using a microplate reader, the optical density was detected at 490 nm. The dose-dependent curves were used to calculate the IC50 values.
Effect on tubulin polymerisation
Using the BK011P-tubulin polymerisation assay kit, the tubulin polymerisation experiment was carried outCitation33. In a nutshell, free tubulin (2 mg/mL) was used in a buffer that also included 15% glycerol and 1 mM GTP. Following that, the test compounds (CA-4, paclitaxel, and compound 10t) were added to the tubulin solution, and changes in the fluorescence intensity were determined by kinetic reading at 37 °C using a fluorescent plate reader (ex = 370 nm, em = 445 nm).
Analysis of immunofluorescence staining
Immunofluorescence studies were performed following a reported methodCitation37. After being seeded into 24-well plates, HeLa cells were treated for 24 h with vehicle, CA-4, or 10t. Cells were permeabilized with 0.2% (v/v) Triton X-100, fixed with 4% formaldehyde, and blocked with 5% bovine serum albumin (BSA) for 30 min. With 2% BSA in PBS, the primary α-tubulin antibody was diluted (1:100) and incubated for an overnight period at 4 °C. Following a PBS wash to remove any remaining primary antibody, the cells were treated for 3 h at 37 °C with FITC-conjugated anti-mouse secondary antibody that had been diluted 1:100 with 2% BSA in PBS. The nuclei were stained with DAPI, the cells were washed with PBS to remove binding secondary antibody, and a confocal microscope was used to detect immunofluorescence.
Cell cycle analysis
Cell cycle analysis was carried out using the method previously mentionedCitation36. HeLa cells from 10t and 0.05% DMSO (control) treated cultures were collected at the indicated time, trypsinized, washed in PBS, fixed by the addition of ice-cold 70% ethanol, and left for 2 h at −20 °C. The cells were then washed twice in PBS, stained with 50 μg/mL of propidium iodide in PBS, treated with 50 µg/mL of PI/RNase staining solution at room temperature to avoid light for 30–60 min, and analysed by flow cytometry using FACSVerse device. The results were examined with FlowJo. V10.
Induction of cell apoptosis
After being cultured with different doses of 10t for 48 h, HeLa cells were washed twice in PBS, centrifuged, and then resuspended in 500 μL binding bufferCitation33. The cells were stained for 15 min in complete darkness with 5 μL of PI and 5 μL of Annexin V-FITC. FACScan flow cytometry was then used to examine the samples.
Molecular docking analysis
The ligands in .sdf format applied in molecular docking were created with ChemBioDraw Ultra and prepared by LigPrep in Schrödinger package (version 2018). For protein preparation, the crystal structure of Tubulin (PDB code: 5LYJ) in complex with CA-4, 9, and 10t was downloaded from RCSB PDB Bank (http://www.rcsb.org/) and prepared by Protein Preparation Wizard in Schrödinger package (version 2018). The ligands were prepared by LigPrep Wizard in Schrödinger package (version 2018). All hydrogen atoms were added to residues, and all bond orders were assigned. Whereafter, the OPLS3 force field was applied to minimise the protein energy and eliminate steric hindrance. During the docking, a 15 Å × 15 Å × 15 Å grid box was generated around the active site of protein. The docking was performed by Ligand Docking in Schrödinger package (version 2018). The docking results were analysed using PyMOL (https://pymol.org/2/). Docking protocol which used was validated with RMSD.
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References
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