Abstract
Background/Aim: Malignant diseases present a significant public health burden worldwide and their treatment is further complicated by the phenomenon of multidrug resistance. Derivatives of imidazopyridine exhibit several remarkable pharmacological activities and they could reverse the multidrug resistance of cancer cells due to overexpressing P-glycoprotein. Materials and Methods: A series of novel imidazo[4,5-b]pyridine derivatives were synthesized and their biological activities were evaluated in vitro using parental (PAR) and multidrug resistant (MDR; ABCB1-overexpressing) mouse T-lymphoma cells. The cytotoxic activity and selectivity of the tested compounds were assessed by the thiazolyl blue tetrazolium bromide (MTT) assay, the ABCB1 modulating activity was measured by rhodamine 123 accumulation assay using flow cytometry. Results: Six compounds (b, c, d, f, h and i) showed moderate-to-high cytotoxic activity on the tested cell lines, while derivative i presented with promising selectivity towards the MDR cell line. Derivatives a, d, f, g and i were proven to be effective modulators of the ABCB1 multidrug efflux pump, with two compounds showing efflux pump modulatory activity at 2 μM concentration. Conclusion: Based on our experimental results, compounds that showed potent activity are those with a short carbon side chain; a methoxy group on the benzene ring; a heterocyclic (triazole) side chain and the presence of an alkylated N-atom at position 4.
Malignant diseases present a significant public health burden worldwide, and even with the therapeutic armamentarium of surgical interventions, radiation therapy and chemotherapeutic treatment, these diseases still represent the second major cause of mortality in the developed world (1). The emergence of cancer cells that are resistant to structurally and functionally unrelated cytostatic drugs (this phenotype is termed multidrug resistance or MDR) further complicated the treatment of these diseases (2). A common mechanism for the reduction of chemotherapeutic efficacy is the overexpression of ATP-binding cassette (ABC) drug transporters (3).
Compounds containing the imidazo[4,5-b]pyridine moiety have been reported for various activities such as anticancer (4-6), antiviral (7-9) antimitotic (10), anti-inflammatory (11, 12) and tuberculostatic (13, 14). In addition, imidazo[4,5-b]pyridine may act as an antagonist of various biological receptors (15) including angiotensin-II, platelet activating factor (PAF) (16), subtype metabotropic glutamate V3 (17), AT1 receptor (18) and adenosine A2a (19). The imidazo[4,5-b]pyridine heterocyclic system is also a structural analogue of purine, whose derivatives readily interact with large biomolecules such as DNA, RNA and proteins in vivo. The structural homology with the adenine nucleus would allow imidazopyridine derivatives to be able to reverse multidrug resistance by inhibiting the activity of P-glycoprotein (ABCB1) (20).
In the present study, a series of newly-synthesized imidazo[4,5-b]pyridine derivatives (9 compounds) were investigated regarding their cytotoxicity and their ABCB1-modulating properties against parental and ABCB1-overexpressing MDR mouse T-lymphoma cells in vitro.
Materials and Methods
Compounds. The imidazo[4,5-b]pyridine derivatives tested were synthesized by alkylation (Figure 1A) and 1,3-dipolar cycloaddition (Figure 1B). Condensation of 5-bromo-2,3-diaminopyridine with 4-chlorobenzaldehyde or 4-methoxybenzaldehyde in water for 48 h at 100°C in the presence of I2 resulted in Derivative 1 (21). Derivatives 2, 2’, 3 were obtained by the reaction of Derivative 1 with mono-halogenated reagents under phase transfer catalysis conditions, using tetra-n-butylammonium bromide (BTBA) as a catalyst, K2CO3 as a base and N,N-dimethylformamide as solvent (22). Derivative 4 was obtained by the condensation of azides with acetylenic compounds by 1,3-dipolar cycloaddition. Substituents of the tested compounds, reaction yields and melting points are shown in Table I. Compounds were solved in DMSO to obtain stock solutions. Afterwards, working solutions were prepared by dilution in water, the concentration of DMSO was below 1% in all the experiments.
Other chemicals used in the study as reagents were: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St Louis, MO, USA), sodium dodecyl sulphate (SDS; Sigma-Aldrich, St Louis, MO, USA), rhodamine 123 (R123; Sigma, St. Louis, MO, USA), verapamil (EGIS Hungarian Pharmaceutical Company, Budapest, Hungary) and dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA). Stock solutions of R123 were prepared in phosphate buffered saline and verapamil was dissolved in water. All solutions were prepared on the day of the assay.
Cell lines. L5178Y mouse T-cell lymphoma cells (PAR) (ECACC Cat. No. 87111908, obtained from FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described by Cornwell et al. (23). The ABCB1-expressing cell line L5178Y (MDR) was selected by culturing the infected cells with colchicine. The L5178Y human ABCB1-transfected subline was cultured in McCoy's 5A medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich), 200 mM L-glutamine (Sigma-Aldrich) and a penicillin-streptomycin (Sigma-Aldrich) mixture in concentrations of 100 U/l and 10 mg/l, respectively. The cell lines were incubated at 37°C, in a 5% CO2, 95% air atmosphere.
Assay for cytotoxic effect. The effects of increasing concentrations of the tested imidazo[4,5-b]pyridine derivatives on cell growth were evaluated in 96-well microtiter plates. The parental (PAR) and multidrug resistant (MDR) mouse T-lymphoma cells were cultured using McCoy's 5A medium supplemented with 10% heat-inactivated horse serum. The density of the cells was adjusted to 1x104 cells per well (in 100 μl of medium per well) and then added to the 96-well flat-bottomed microtiter plates containing the dilutions of the tested compounds. The culture plates were incubated at 37°C, in a 5% CO2, 95% air atmosphere.
The culture plates were incubated at 37°C for 24 h; at the end of the incubation period, 20 μl of MTT (Sigma) solution (from a stock solution of 5 mg/ml) were added to each well. After incubation at 37°C for 4 h, 100 μl of sodium dodecyl sulfate (SDS) (Sigma) solution (10% in 0.01 M HCI) were added to each well and the plates were further incubated at 37°C overnight. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA) (24). Inhibition of the cell growth was determined according to the formula below:
Rhodamine 123 accumulation assay. The following method is based on a fluorescence-based detection system which uses verapamil as reference inhibitor of the ABCB1 efflux pump (25). The parental and multidrug resistant (MDR) subline of mouse T-lymphoma cells were adjusted to a density of 2×106 cells/ml and re-suspended in serum-free McCoy's 5A medium and distributed in 500 μl aliquots. The tested compounds (1 and 10 μl from a stock solution of 1 mM, respectively) were added at different concentrations (final concentrations of 2 μM and 20 μM, respectively), and the samples were incubated for 10 min at room temperature. Verapamil was used as positive control at 20 μM (from a 5 mg/ml stock solution) and DMSO was used as solvent control (at 2 V/V%). After the first incubation period, 10 μl (5.2 μM final concentration) of rhodamine 123 were added to the samples and the cells were further incubated for 20 min at 37°C, washed twice with phosphate buffered saline (PBS) and re-suspended in 1 ml PBS for analysis. The fluorescence intensity of the gated cell population was measured with a Partec CyFlow flow cytometer (Partec, Munster, Germany). The mean fluorescence intensity was calculated for the treated MDR and parental mouse T-lymphoma cells lines as compared to the untreated cells (26, 27). The fluorescence activity ratio (FAR) was calculated based on the following equation which relates the measured fluorescence values:
Results
The IC50 values (thus, the cytotoxic activity) of the tested compounds on the parental subline of the mouse T-lymphoma cells can be divided into three groups: compounds with IC50 values above 100 μM (a and e), compounds with IC50 values between 20-70 μM (in decreasing order: c, g, f, b, d and h) and derivative i, which presented with an IC50 value of 1.73 μM. The IC50 values of the tested compounds on the MDR mouse T-lymphoma cells can be divided in a comparable way (compounds with IC50 values above 100 μM: a, e and g; compounds with IC50 values between 10-75 μM: c, b, f, d and h; and derivative i, which presented with an IC50 value of 0.20 μM) (Table II). While the majority of the compounds had no selective cytotoxicity against the efflux pump overexpressing subline of mouse lymphoma cells (with IC50MDR values higher than IC50PAR; SI values ranging between 0.55 and 1.49), derivative i, which was proven to be the most cytotoxic among the tested imidazo[4,5-b]pyridine derivatives (IC50PAR=1.73 μM, IC50MDR=0.20 μM) showed potent selectivity (SI=8.66) towards the MDR cells (Table II).
Out of the 9 tested imidazo[4,5-b]pyridine derivatives, 5 compounds (a, d, f, g and i) presented with potent ABCB1-modulating activity (the intracellular concentration of rhodamine 123 was increased) at the same concentration as verapamil (20 μM), surpassing the activity of the positive control by a considerable margin (with FAR values ranging between 19.65 and 57.55 vs. FARverapamil=11.83). Based on the results presented in Table III, the potency of the active compounds is 1.66-4.86-fold higher than verapamil's. In some cases, the efflux pump modulating activity of the compounds approaches (i, FAR quotient: 89.50%) or surpasses (a, FAR quotient: 143.35%) the activity of the positive control at concentrations that are ten times smaller (Table III). The case of compound a is especially compelling, because despite the fact that this compound was not the most potent inhibitor of the ABCB1 transporter among the tested derivatives (FAR20L>μM=28.09), it exerted its activity without any toxicity (IC50 >100 μM) on the tested cell lines.
Synthesis of imidazo[4,5-b]pyridines derivatives synthesized by alkylation (A) and 1,3-dipolar cycloaddition (B).
Substituents of novel imidazo[4,5-b]pyridine derivatives.
Discussion
Compounds containing the imidazo[4,5-b]pyridine moiety represent a special group of compounds with numerous pharmacological activities (28, 29, 30). In the present study, the anticancer and efflux pump modulating activity of nine novel imidazo[4,5-b]pyridine derivatives were evaluated. Five compounds (c, b, f, d and h) showed moderate cytotoxicity on the MDR cell line, while derivative i presented with potent selectivity and a very low IC50 value.
Cytotoxicity of tested compounds against parental (PAR) and multidrug-resistant (MDR) mouse lymphoma cells and selectivity indices (SI).
Five of these compounds exhibited significantly better MDR-reversing activity than the reference (verapamil) with a fluorescence ratio of 19.65 to 57.55 compared with 11.83 for verapamil. It should be noted that compounds that lack the potent efflux pump modulatory activity of their counterparts are those with a long carbon chain in position 3 or 4 (n>4C), which suggests that this moiety is not suitable for pronounced activity. Both compounds h and i are alkylated at the same position with a propargyl group, however, i has a methoxy and h has chlorine group. By comparing their activity of ABCB1 pump inhibition, it can be deduced that the methoxy group is the most favorable for activity. However, the activity of compound f is greater than that of product g. This amelioration of activity for the compound containing a chlorine group can be attributed to the position of the carbon chain (f at position 4, g at position 3). This suggests that alkylation at the 4-position would be better for the MDR-reversing activity. In addition, compounds a and e both have a long side chain, but for product a, the chain is carried by a triazole group. Therefore, the presence of heteroatoms on the side chain, in particular nitrogen, improves the MDR-reversing activity of the imidazo[4,5-b]pyridine derivatives. It can be concluded that the novel imidazo[4,5-b]pyridine derivatives presented with potent MDR-reversing activities together with anticancer properties to a varying degree. For additional understanding of the structure-activity relationships (SAR) of these compounds, the synthesis of additional derivatives and further biological assays are warranted.
Rhodamine 123 accumulation assay in the presence of imidazo[4,5-b]pyridine derivatives against multidrug resistant (MDR) mouse T-lymphoma cells.
Acknowledgements
This study was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’. Gabriella Spengler was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Márió Gajdács and Gabriella Spengler received funding from the Márton Áron Research Programme (2017/18) financed by the Hungarian Ministry of Foreign Affairs and Trade. Márió Gajdács was supported by the ÚNKP-17-3 New National Excellence Program of the Ministry of Human Capacities. The study was supported by the Szeged Foundation for Cancer Research.
- Received April 24, 2018.
- Revision received May 24, 2018.
- Accepted May 29, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
