Synthesis and Cytotoxicity of Thieno[2,3-b]Pyridine Synthesis and Cytotoxicity of Thieno[2,3-b]Pyridine Derivatives Toward Sensitive and Multidrug-Resistant Leukemia Cells

A new series of substituted ethyl 7-cyclopropyl-2-(2-aryloxo)-3-nitro-4-oxo-4,7-dihydrothieno[2,3- b ]pyridine-5-car-boxylates 3a – e were prepared by utilizing ethyl 2-chloro-7-cyclopropyl-3-nitro-4-oxo-4,7-dihydrothieno[2,3- b ]pyri-dine-5-carboxylate ( 1 ) and replacing of the 2-chlorine with anions obtained from phenol ( 2a ), salicylaldehyde derivatives 2b – d or thiophenol ( 2e ), leading to the respective ethyl 7-cyclopropyl-2-(2-aryloxo)-3-nitro-4-oxo-4,7-dihydroth-ieno[2,3- b ]pyridine-5-carboxylates 3a – e . The new compounds were evaluated for their in vitro cytotoxicity towards sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. The screening revealed that compounds 3a , 3b , and 3e inhibited the growth of both cell lines. Compound 3b , with a phenol moiety, exhibited the highest growth inhibitory activity against CEM/ADR5000 and CCRF-CEM cells with IC 50 values 4.486 ± 0.286 and 2.580 ± 0.550 µM, respectively. Collectively, the presented results demonstrate that the synthesized thieno[2,3- b ]pyridines warrant further exploration for potential use as anti-cancer agents. Synthesis and Cytotoxicity of Thieno[2,3-b]Pyridine b ] resistance


Introduction
Thieno [2,3-b]pyridines were mentioned for the first time in 1913. 1 The chemistry of thieno [2,3-b]pyridines has been well documented during the past decades. 1 Various biological activities of this heterocyclic compounds class were described, 2 such as antimicrobial, [3][4][5][6] anti-inflammatory, 7-9 antioxidant, 6 antituberculosis, 4 and antimalarial activities. 10 Moreover, the incorporation of the N-cyclopropyl group with 4-oxothieno [2,3-b]pyridines showed a higher potency against Escherichia coli ATCC10536 than the N-ethyl and N-tert-butyl analogs. 11 It is important to mention that compounds containing thieno[2,3-b]-pyridines moieties attracted considerable interest regarding their potency as anti-cancer agents. [12][13][14] Despite severe undesired side effects, chemotherapeutics are considered effective treatments of primary and metastatic tumors (Figure 1). 15 One serious problem of cancer chemotherapy is the development of resistance towards multiple structurally and functionally unrelated anti-cancer drugs. [16][17][18] This phenomenon defined as multidrug resistance (MDR), where chemotherapy fails even at high drug, which leads to toxic side effects. 19 MDR is frequently caused by the overexpression of membrane efflux pumps of the ATPbinding cassette (ABC) transporter family. The best characterized ABC-transporter in this context is P-glycoprotein (Pgp), which causes increased transport of chemotherapeutic agents out of the cells. 20,21 Thieno [2,3-b]pyridines have been reported to exhibit chemopreventive effects suppressing carcinogenesis of numerous tumor types including breast, prostate, non--small cell lung, melanoma, leukemia, ovarian, liver, and colon cancer. 22,23 As a part of our continuing search for novel biological agents, [24][25][26][27] newly synthesized 4,7-dihydrothieno[2,3-b]pyridine derivatives were evaluated for their growth inhibitory activity towards multidrug--resistant CEM/ADR5000 cells in comparison to their pa- rental sensitive cell line, CCRF-CEM. This is the first report on the cytotoxicity of 4,7-dihydrothieno [2,3-b] pyridine against sensitive and multidrug resistance leukemia cells. Moreover, the structure-activity relationship of the synthesized set was also studied.
The new compounds 1 and 3a-e were characterized by IR, MS, and NMR spectral data. These data, given in the    Table 1. Thus, the mass spectra display the correct molecular ion peaks, for which the measured high-resolution mass spectra (HRMS) data were in good agreement with the calculated values. DEPT and 2D (COSY, HMQC, HMBC) experiments showed correlations that helped in the 1 H and 13 C signal assignments to the different carbons, and they are attached and/or neighboring hydrogens. H-6 proton resonating at 8.36 as a sharp singlet made a long-range correlation with C-3, C-4, C-1' and CO 2 Et. In addition, protons 2' , 3' show correlation with C-1' . On the other hand, the phenol ring correlated with aldehyde functionality (Table 1).

Biological Evaluation. Cytotoxic Activities Against Lymphoblastic Leukemia Cells
As the first step, all compounds were tested at a fixed concentration of 10 µM ( Figure 2) in CCRF-CEM and CEM/ADR5000 cells. Compounds 3a, 3e, and 3b significantly inhibited cell viability (reduction to less than 10% growth).
From the structure-activity relationship (SAR) point of view, incorporating salicylaldehyde moiety to thieno[2,3-b]pyridine (3b) exhibited good activity against CCRF-CEM and CEM/ADR5000 cells with IC 50 values of 4.76 and 5.11 µM, respectively. However, incorporating the phenol moiety (3a) increased activity against CCRF--CEM and CEM/ADR5000 (Table 2). Changing the hydrogen on the para position of 3b with a halogen in 3d or with methyl in 3c led to reduced biological activity against both cell lines. While replacement of the phenol moiety with thiophenol in 3e retrieved biological activity against CCRF-CEM and CEM/ADR5000 cells with IC 50 values of 4.00 and 4.59 µM for 3e, respectively ( Table 2). This is a remarkable result since CEM/ADR cells are more than 1000-fold resistant to the established anti-cancer drug doxorubicin. 28 Hence, these compounds inhibited multidrug-resistant cells with similar efficacy as sensitive cells, possibly qualifying them as candidates for further development as treatment of unresponsive cancers.
The resulting mixture was refluxed at 85 °C for 2 h, then cooled to room temperature, and washed with water (3 × 2 mL). The organic layer was separated, dried over MgSO 4 and the solvent was then evaporated to dryness to obtain the desired product 6. A stirred solution of 6 in dichloromethane (50 mL) was treated dropwise with cyclopropyl amine (2.85 g, 50 mmol) at 2-4 °C. The resulting mixture was then stirred at 25 °C for 24 h. The solvent was evaporated and the residue was soaked with hexane to obtain a yellow precipitate product 7. Sodium hydride (0.8 g, 17.5 mmol, 55%) in dry THF (60 mL) was added to the pure 7. The reaction mixture was stirred at room temperature for 30 min. Then, the temperature was increased to 60 °C for 3 h. The solvent was evaporated, and the residual white precipitate 8 was washed with water and dried. The product crystallized from the CHCl 3 /ethanol mixture (1:2). 11 Finally, the nitration of 8 was achieved by dissolving 8 in concentrated sulfuric acid (6 mL). The latter solution was slowly and dropwise added for 30 min to -5 °C stirred solution of fuming nitric acid (2 mL) and concentrated sulfuric acid (5 mL). The mixture was allowed to warm to 5 °C and poured into the ice bath (50 mL). The solid product 1 was filtered and crystallized from DMF/ethanol (1:9) [mp 180-182 °C dec., total yield 30%]. 1

4 Resazurin Reduction Assay
The cytotoxic effects of compounds on drug-sensitive leukemia CCRF-CEM and multidrug-resistant P-glycoprotein-overexpressing CEM/ADR5000 cells were evaluated by the resazurin assay as previously described. [29][30][31][32][33] All compounds were first tested at a single concentration of 10 µM (Figure 2) against CCRF-CEM and CEM/ADR5000 cells. Compounds 3a, 3e, and 3b, which significantly inhibited cell viability (reduction to less than 10% growth) were further tested in a concentration range from 0.001 to 10 µM to determine the 50% inhibitory concentrations (for IC 50 ) in both, CCRF-CEM and CEM/ ADR5000 cell lines. The fluorescence was measured using an Infinite M2000 Pro TM plate reader (Tecan, Crailsheim, Germany) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. All experiments were performed three times with every six parallel measurements. The viability was evaluated based on a comparison with untreated cells. The IC 50 values represent the concentrations of the compounds required to inhibit 50% of cell viability and were determined from a calibration curve by linear regression using Microsoft Excel.

Conclusion
A series of novel thieno [2,3-b]pyridine derivatives have been synthesized and screened for their in vitro cytotoxicity towards sensitive CCRF-CEM and multidrug resistance CEM/ADR5000 leukemia cells. Compounds 3a, 3b, and 3e inhibited the growth of both cell lines incorporating phenol without substitution at para position, which can be considered as lead structures for further drug development.