Synthesis of polysubstituted pyridines as potential multidrug resistance modulators

Introduction

Chemotherapy has found clinical application in the treatment of almost every type of cancer. Multidrug resistance (MDR) appears as a major obstacle in cancer chemotherapy. ABCB1 transporters [1] are the most studied targets for reverting MDR. From all the numerous efforts to overcome MDR, such as transcription control of ABCB1 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 ABCB1 (P-glycoprotein) inhibitors [1, 2], with Ca²⁺ channel blocker verapamil being the most investigated and often used as the reference compound. Unfortunately, cardiotoxicity is observed in combination with verapamil in actual anticancer drugs [3].

A rational approach of drug design (structural analogy with known medicines) was used by our research group to develop effective MDR modulators on the basis of thieno[2,3-b]pyridines [4]. Pharmacophore model was created assuming one part of verapamil as a linker and methoxyphenyl groups as essential moieties for the pharmacophore (Figure 1).

Figure 1:

The pharmacophore model with a modified linker.

We have shown that the substitution of thieno[2,3-b]pyridine scaffold with hydrophobic aryl groups in positions 2 and 4, an ester group in position 5, an amino group in position 3 (hydrogen bond donor), and methoxyphenyl groups (bearing appropriate amount of hydrogen bond acceptors) leads to potent MDR modulators [4].

Results and discussion

In continuation of our research, we used the above-mentioned pharmacophore model, modifying a linker from thieno[2,3-b]pyridine to pyridine. To the best of our knowledge, there are no studies in which polysubstituted alkylsulfanylpyridines are tested for revealing MDR-modulating properties. Contrary to pyridines, 1,4-dihydropyridines as Ca²⁺ channel blockers are of great interest, and in the last years, promising results in these series have resulted in the development of MDR reversal agents [5, 6].

It is known that the oxidation of 1,4-dihydropyridines to pyridines is accompanied by loss of calcium antagonistic properties [7]. Thus, in the case of pyridines, less side effects could be expected. The synthesis of 1,6-dihydro-6-thioxopyridines 1 is described in the literature [4]. On the treatment of thiones 1 with substituted bromoacetophenones 3 in the presence of piperidine (2) in ethanol, the alkylation of 1 takes place, giving rise to 2-aroylmethylsulphanyl-3-cyanopyridines 4a,b in 91% to 92% yield (Scheme 1). Piperidinium bromide 5 is formed as a waste product. It is worth to mention that the yield of products 4a,b is increased in the presence of piperidine in comparison with the reaction conducted in the presence of NaOH [4]. Pyridine 4c was prepared in 67% yield in an alternative route by the oxidation of the corresponding 1,4-dihydropyridine 6 with manganese acetate in acetic acid media (Scheme 1). The starting 1,4-dihydropyridine 6 is easily accessible in 83% yield by the treatment of thione 7 [8] with bromoacetophenone 3a in the presence of piperidine in ethanol. The comparison of the two-stage path A (synthesis of 1 in 42%–50% yield [4] followed by alkylation in 91%–92% yield) and the three-stage path B (synthesis of 7 in 42% yield [8] followed by alkylation in 67% yield and then oxidation in 83% yield) shows that path A is simpler and more efficient.

Scheme 1

SAR data in thieno[2,3-b]pyridine series indicate the necessity of the presence of both hydrogen bond donors and hydrogen bond acceptors in the molecules to reach optimum activity [4].

The measurement of P-glycoprotein activity was conducted according to the procedure described by Krauze et al. [4], and the results are shown in Table 1. As a Ca²⁺ channel blocker, verapamil in combination with actual anticancer drugs reveals cardiotoxicity [3], and the influence of some obtained pyridines 4 on cardiovascular system and their toxicity were also tested [4].

As shown in Table 1, pyridine 4b displays P-gp inhibition activity comparable with that of verapamil. The aryl group in position 4 and the aroylmethylsulfanyl group in position 2 of the pyridine are essential for MDR-modulating activity. The most active compound 4b bears a 3,4,5-trimethoxyphenyl functionality in position 4 of pyridine. Activity decreases with the substitution of this group with 3,4-dimethoxyphenyl and 4-methoxyphenyl groups. Concerning the substituents R³, it was found that only one OMe group in the para position of the phenyl ring is desired. The analogous 1,4-dihydropyridine 6 is not active as P-gp inhibitor.

As shown in Table 1, only pyridine 4a reveals a weak influence on Ca²⁺ antagonist effect. By contrast, pyridines 4a–c are less toxic (LD₅₀=1621 or >2000 mg/kg) in comparison with verapamil (LD₅₀=962 mg/kg).

Conclusion

Polysubstituted pyridines 4 have been prepared by the alkylation of 6-thioxo-1,6-dihydropyridine 1 or by the oxidation of 1,4-dihydropyridine 6 with manganese triacetate. The comparison of both paths showed that the first one is preferable. The MDR-modulating (P-glycoprotein inhibition) activity of pyridine derivatives 4 is comparable with that of the reference compound verapamil.

Experimental

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

The measurement of P-glycoprotein activity was conducted according to the procedure described in reference [4]. The use of Ca²⁺ channel blocker verapamil in combination with actual anticancer drugs reveals cardiotoxicity [3]. The effect of pyridines 4 on cardiovascular system as well as their toxicity were tested according to the procedure described previously [4].