Effects of 2-Alkylamino-Substituted Dihydropyridines on Rabbit Jejunum

1. Introduction

      Structurally diverse groups of compounds are known to be effective as calcium antagonists. The most potent known class of  antagonists are 1,4-dihydropyridines, which include the widely used nifedipine . This class of compounds has been the subject of many structure-activity relationship studies [1]. In this study, we also synthesized several 2-alkylamino-substituted dihydropyridines, and investigated their calcium channel blocking activities. Starting from dialkyl 1,4-dihydro-2,6-dimethyl-4-(1-benzyl-2- alkylthio-5-imidazolyl)-3,5-pyridinedicarboxylates (5), whose synthesis and effects as calcium channel antagonist on guinea-pig ileum has been reported, previously [2],

      dialkyl 1,4-dihydro-2-[2-(dimethylamino) ethyl]-6-methyl-4-(1-benzyl-2-alkylthio-5 imidazolyl)-3,5-pyrdinedicarboxylates (6) were synthesized as illusterated in scheme 1.

Scheme 1: 1-benzyl-5-hydroxymethylimidazole (2) was synthesized from benzylamine hydrochloride (1). Reaction of 2 with alkyl halides gave corresponding substituted alkylthioimidazoles (3). Oxidation of 3 with manganese dioxide in chloroform afforded 2-alkylthio-5-formyl-1-benzylimidazoles (4). Symmrtrical dihydropyridines (5a-f) were synthesized by classical Hantzch condensation. Then dihydropyridines (5a-f) were reacted with paraformaldehyde and dimethylamine hydrochloride to give the title dialkyl 1,4-dihydro-2-[2-(dimethylamino)ethyl]-6-methyl-4-(1-benzyl-2-alkylthio-5-imidazolyl)-3,5- pyrdinedicarboxylates (6a-f).

      Rabbit jejunum was used to determine the relaxant or antagonistic activity of the synthsized  compounds  (6c-e).  This  test model, particularly allows to examine the relaxant, spasmolytic, or antagonistic activi- ties of unknown compounds, directly with- out the use of an agonist [3]. We report here that  the  synthesized  compounds  inhibited the  spontaneous  contractile  activity  dose- dependently and completely, while high-K+ contracted tissues were relaxed partially.

2. Materials and methods

2.1. Chemistry

     Melting points  were  determined using the  capillary  apparatus  with  a  system  of Gallenkamp. 1H-NMR spectra were run on a Bruker AC-80 spectrometer. Infrared spectra were recorded on a FT-IR Perkin-Elmer Paragon 1000 spectrophotometer. Compounds 2 to 5 were synthesized as described previously [2]. The developed procedure [4] is exemplified with the obtaining of 1,4-dihydro-2-methyl-6-[2-(dimethylamino) ethyl]-4-(1-benzyl-2-ethylthio-5-imidazolyl)-3,5-pyridinedicarboxylate dimethyl ester (6b).

2.2. Synthesis of 1,4-dihydro-2-methyl-6-[2-(dimethylamino)ethyl]-4-(1-benzyl-2-ethylthio-5-imidazolyl)-3,5-pyridinedicarboxylate dimethyl ester (6b) 

      A solution of 5b (1.2g, 2.72 mmoles), dimethylamine hydrochloride (0.33 g; 4mmol), paraformaldehyde (0.12 g, 4 mmol)and 0.05 ml of concentrated hydrochloric acid in ethanol (5 ml), while protected from light, was heated at reflux for 10 h. The solvent was then evaporated and the residue was partitioned between hydrochloric acid (2 M; 30 ml) and ethyl acetate (15 ml). The aqueous phase was separated, basified with aqueous ammonia, and extracted into diethyl ether (3x30 ml). The extract was dried and the residue was chromatographed to give 0.4 g   (30%)  of     1,4-dihydro-2-methyl-6-[2- (dimethylamino)ethyl]-4-(1-benzyl-2-ethyl- thio-5-imidazolyl)-3,5-pyridinedicarboxy- late  dimethyl ester (6b) as a brown oil. IR (KBr):  1704,  1690cm-1  (C=O);  1H-NMR (CDCl3): 7.65-6.92 (m, 7H, arom, H-C4 imi- dazole, NH), 5.59(s, 2H, CH2N), 5.3 (s, 1H, H-C4 dihydropyridine), 3.6 (s, 6H, CH3O), 3.15-2.68 (m, 6H,  CH2), 2.5 (s, 6H, NCH3), 2.34 (s, 3H, CH3), 1.34 (t, 3H, CH3).

2.3. Evaluation of pharmacological activity

      Male adult, healthy rabbits, ranging from 1500150 grams, were purchased from the animal  house  of  Mashhad  University  of Medical Sciences. Animals were fasted for 12 h but had free access to water before the experiments.  After  cervical  dislocation, jejunum was isolated, the adjacent tissues were removed , and 2-3 cm long pieces were cut. Each piece was  hanged diagonally in a 10 ml organ bath filled with the Tyrode’s solution (composition in mM: KCl 2.7, NaCl 136.9, MgCl2 1.1, NaHCO3 0.4, CaCl2 108, Glucose 5.6) and was aerated with carbogen (95% O2 and 5% CO2 ) at 37 °C. The contractions  were  recorded  with  a  model  7Grass  polygraph. A preload of  1.0  g  was applied to each tissue. After stabilizing the tissue with 3x10-7 M norepinephrine, the test compounds  were  added  in  a  cumulative dose-fashion.   All of the compounds were dissolved in 10% DMSO and their effect on the  spontaneous  activity  of   the  rabbit jejunum  was  determined. The  experiment was repeated five times for each compound.

     In a second set of experiments, calcium channel blocking activity of the compounds was studied. Eigthy mM KCl was used to induce a sustained contraction. At plateau, the compounds were added in the cumula- tive  dose-fashion.  The  experiments  were repeated at least three times for each com- pounds. The Prism pad was used to present the data in graphical form; the same software also calculated I/C 50 values. Student t-test was employed and the level of significance was taken at p 0.05.

3. Results and discussion

      The effects of compounds 6c, 6d, and 6e in rabbit jejunum are presented in Fig1. The compound 6d and 6e showed a dose-depend- ent inhibition of jejunum movement, while compound 6c sharply inhibited the move- ments after the third dose, and then followed a dose-dependent pattern. The results pre- sented in Table 1 shows the  IC50 values of the     test     compounds,     comparatively. Compound 1 is more potent in this tissue.

     The  dose-dependent  inhibition  of  spontaneous  contractile  activity  by  a  test  com- pound is a characteristic of a calcium channel antagonist [5]. To determine the calcium channel antagonistic activity of the synthesized  compounds,  high-K+     was  used  to induce sustained contraction in the tissues. A high-K+ is known to cause the contraction in the smooth muscles due to entry of Ca++ into the cells through the voltage-dependent cal- cium channels (VDCs) [6, 7]. The cytoplas- mic calcium [Ca++]i is responsible for acti- vating the contractile element in the smooth muscle preparations [8]. Compounds which inhibit  a  high-K+   induced  contraction  is thought to be a calcium channel antagonist[9].

     As shown in Figure 1, compounds 6c and 6d did not relaxed the pre-contracted tissue significantly, while compound 6e significantly relaxed the jejunum which suggest that it has partial calcium channel blocking activity.

Figure 1. Effects of compounds 6c,d ,e on spontaneous rabbit jejunum movements.

Figure 2. Effects of compounds 6c,d ,e on K+-contracted rabbit jejunum

Table 1- IC50 values of compounds 6c,d, e on spontaneous rabbit jejunum.

Acknowledgement

The work was partially supported by a grant from the Research Council of Medical Sciences, Mashhad  University of  Medical Sciences.