Synthesis of 6-alkylsulfanyl-1,4-dihydropyridines as potential multidrug resistance modulators

Introduction

Multidrug resistance (MDR) against various anticancer drugs remains an important problem in anticancer drug therapies over the last decades. ABCB-1 transporters [1] are the most studied target for reverting MDR. From all the numerous efforts to overcome MDR, like transcription control of ABCB-1 expression, the most promising approach has been the development of MDR modulators, which are able to increase the intracellular drug levels in co-application with MDR substrates by efflux pump inhibition. Substances of different classes have been used as transport protein inhibitors [1, 2]. The calcium channel blocker verapamil is the most investigated and often used as a reference compound but, unfortunately, cardiotoxicity is observed in combination with actual anticancer drugs [3].

Rational approach to drug design – structural analogy with known active agents – has been used in our research to develop effective MDR modulators on the basis of thieno[2,3-b]pyridines [4]. A pharmacophore model has been created assuming one part of verapamil as the linker and methoxyphenyl groups as essential features for the pharmacophore (Figure 1).

Figure 1

Pharmacophore approach with a modified linker.

We have shown previously that substitution of 3-aminothieno[2,3-b]pyridine-5-carboxylate or pyridine-5-carboxylate scaffolds with methoxyphenyl groups (hydrophobic aryl groups and methoxy groups as hydrogen bond acceptors) in position 2 and 4 leads to potent P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP-1) and breast cancer resistance protein (BCRP-1) inhibitors. Such derivatives of 3-aminothieno[2,3-b]pyridine-5-carboxylates significantly exceed the activity of verapamil, MK-571 and reversan [4], but the activity of pyridine derivatives is comparable to that of verapamil [5].

Results and discussion

In continuation of our research we used the pharmacophore approach mentioned above to modifying the linker from thieno[2,3-b]pyridine and pyridine to 1,4-dihydropyridine (DHP). There are many publications in the last years in which DHPs Ca²⁺ channel blockers are investigated as promising MDR reversal agents [6, 7]. It is known that modification of substituents on the DHP ring can lead to the loss of calcium antagonistic properties [6] which in this case would be a positive result. DHPs 5a–d were prepared in 73–89% yields by one-pot reaction of ethyl 2-arylmethylidenacetoacetate 1 with 2-cyanothioacetamide (2) in the presence of equimolar amount of piperidine (3) as base in ethanol followed by subsequent alkylation of the resultant thiolate with substituted 2-bromoacetophenone 4 (Scheme 1, pathway A). In turn, DHPs 5e,f were prepared in 73–89% yields by treatment of the thiolate 6 (prepared according to the procedure described in [8] and used as crude product) with substituted 2-bromoacetophenone 4 (pathway B). Alternative one-pot preparation was the treatment of ethyl acetoacetate 7 with 2-cyano-3-(3,4-dimethoxyphenyl)thioacrylamide 8 in the presence of equimolar amount of piperidine (3) in ethanol followed by alkylation of the intermediate thiolate with 2-bromoacetophenone 4 or 2-iodoacetamide 9, which gave DHP 5c and 5g in 75% and 64% yields (pathway C). As can be seen, the pathway A to 5c and 5g is more efficient.

Scheme 1

SAR data for the thieno[2,3-b]pyridine and pyridine series indicate the necessity of both hydrogen bond donors and hydrogen bond acceptors to reach optimum activity [4]. Pyridines [5] and DHPs 5 are intermediates to thieno[2,3-b]pyridines. It was expected that similarly to the thieno[2,3-b]pyridine scaffold, the DHP pharmacophore model (Figure 1) the methoxyphenyl groups would play an essential role in activity.

As shown in the Table 1, DHP 5a at 20 μm concentration displays high P-gp inhibition activity, the activity of DHPs 5c is comparable, DHPs 5b,d are slightly less active than verapamil. The aryl group with substituent R¹ in position 4 and benzoylmethylsulfanyl group with substituent R² in position 2 of the pyridine ring are essential for MDR modulating activity. The active compound 5a contains a 3,4,5-(OMe)₃C₆H₂ group in position 4 of DHP, while its substitution with 4-OMeC₆H₄ [5] or 4-ClC₆H₄ group leads to a reduced activity from 12.0±1.6 (compound 5a) to 1.8, 2.4 and 1.2 (compounds 5e, 5f and 5g). The highest MDR modulating activity is observed in the case where substituent R² in SCH₂COAr group is 4-OMe, but substituents 2,4-(OMe)₂, 4-Cl or 4-F lead to a significantly reduced activity.

As shown in Table 1, DHPs 5a–d at 20 μm concentration are slightly more potent MRP-1 inhibitors than verapamil. Compounds 5e–g without methoxyphenyl groups appear inactive or display weak potency.

Compounds 5a–d reveal a weak influence on Ca²⁺ antagonist effect and are less toxic (LD₅₀=1777–2528 mg/kg) in comparison to verapamil (LD₅₀=962 mg/kg). Application of the above mentioned pharmacophore approach with a modified linker to search for new MDR modulators demonstrates that compounds with the 1,4-dihydropyridine core as linker are less active in comparison with the thieno[2,3-b]pyridine [4] and pyridine [5] derivatives.

Experimental

Melting points were determined on OptiMelt MPA100 apparatus and are uncorrected. ¹H NMR spectra were recorded in CDCl₃ on a Varian Mercury BB 400 MHz spectrometer. The IR spectra have been recorded on Shimadzu IR Prestige-21 spectrometer in nujol. The progress of the reactions was monitored using silica gel 60 F₂₅₄ plates (Merck) eluting with chloroform/hexane/acetone (2:2:1).