Design and syntheses of 4-[3-(1-methoxycarbonyl-1,6-dihydropyridyl)]- and 4-[3-(1-methoxycarbonyl-4-substituted-1,4-dihydropyridyl)]- derivatives of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylates with calcium channel modulating activities

A group of 4-[3-(1-methoxycarbonyl-1,6-dihydropyridyl)]- derivatives of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylates 8a-e were synthesized by the regioselective reduction of the corresponding 4-(3-pyridyl)- analogs in the presence of methyl chloroformate using Li( t - BuO)3AlH. Alternatively, a related group of 4-[3-(1-methoxycarbonyl-4-substituted-1,4-dihydropyridyl)]- compounds 14-18 were prepared using a modified Hantzsch reaction involving the condensation of 1-methoxycarbonyl-4-substituted-1,4-dihydropyridyl-3-carboxaldehydes 11a-c with an alkyl 3-aminocrotonate 12 and nitroacetone 13 . In contrast to the 4-(3-pyridyl)- compound 2b , which


Introduction
5] Recently, we discovered a novel third generation class of isomeric isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(pyridyl)-5-pyridinecarboxylates 2a-c with different CC modulation activities. 6The 2-pyridyl isomer (±)-2a acted as a dual cardioselective calcium channel agonist (positive inotrope) / smooth muscle selective calcium channel antagonist (vascular relaxant).On the other hand, the 3-pyridyl (±)-2b and 4-pyridyl (±)-2c isomers acted as CC agonists on both cardiac and smooth muscle.The 2-pyridyl enantiomer (+)-2a exhibited agonist activity on both cardiac and smooth muscle, but the (-)-2-pyridyl enantiomer (-)-2a exhibited clinically desirable cardiac agonist and smooth muscle antagonist effects.1][12] The 1,4-versus 1,2regioselectivity of this reaction is dependent upon the activating N-substituent, the nucleophile, and the nature and position of pyridinium substituents. 10In the case of N-acylpyridinium salts, optimal 1,4-selectivity, using organometallic reagents, was obtained for organocopper reagents. 11e copper catalyzed reaction of a Grignard reagent with an activated pyridine was first reported by Loev et al. 13 A related reaction for the synthesis of 1,4-DHPs employing an organocuprate reagent was reported later by Piers et al. 14 Subsequently, Comins et al. 15 showed that reaction of an N-alkoxycarbonylpyridinium salt with a Grignard reagent in the presence of a catalytic amount of CuI resulted in nearly exclusive formation of the 1,4-DHP product.In this study, attempts to synthesize the novel class of unsymmetrical 1,4-DHP derivatives 14-18a-c by reaction of the pyridine derivatives 6 with an organocuprate reagent (n-BuMgBr + CuI), Grignard reagent (n-BuMgBr) or cadmium reagent (n-BuMgBr + CdCl 2 ) in the presence of MeOCOCl were not successful.This failure is attributed to an interaction between the 1,4-DHP nitro substituent of compounds 6 and the organometallic reagent since an earlier study showed that similar reactions employing dialkyl 1,4-dihydro-2,6-dimethyl-4-(3-pyridyl)-3,5pyridinedicarboxylates proceeded as expected to afford the corresponding 4-[3-(1methoxycarbonyl-1,4-dihydropyridyl)] products in good yield. 16 alternate synthetic strategy was therefore employed to by-pass the detrimental effect of the nitro group in compounds 8 starting from the 1-methoxycarbonyl-1,4-dihydropyridyl-3carboxaldehyde 11.In this regard, reaction 15 , 17 1].Although compounds 8 and 14-18 were less potent calcium channel agonist positive inotropes on heart than the reference drug Bay K 8644, both classes of compounds retained positive cardiac inotropic acitivity (see Table 1).The observation that compounds 14-18 exhibit positive inotropic effects over quite a large range (25-200% increase in cardiac contractile force at a 4.46 x 10 -5 M test compound concentration) indicates that the magnitude of the positive cardiac inotropic is dependent upon co-operative and/or interdependent contributions from the parent 1,4-DHP ring C-3, C-4 and C-5 substituents.
The results of this study show that a 4-[3-(1-methoxycarbonyl-1,6-dihydropyridyl)]-, or a 4-[3-(1-methoxycarbonyl-4-substituted-1,4-dihydropyridyl)]-, ring system is a suitable bioisostere for either the 4-(3-pyridyl)-substituent of 2b, or the 4-(2-trifluoromethylphenyl)-substituent of Bay K 8644, that are devoid of the smooth muscle constrictor effect exhibited by the latter compounds.This group of compounds 8, and 14-18, which could serve as valuable probes to study the structure-function relationships of calcium channels, constitute novel types of dihydropyridine cardioselective positive inotropes that provides a new drug design concept relevant to the treatment of congestive heart failure.Melting points were determined using a Thomas-Hoover capillary apparatus and are uncorrected. 1H NMR spectra were recorded using a Bruker AM-300 spectrometer, and the assignment of exchangeable protons (NH) was confirmed by the addition of D2O.Infrared spectra (IR) were acquired using a Nicolet 550-FT spectrometer.Microanalyses were performed by the Microanalytical Services Laboratory, Department of Chemistry, University of Alberta.
Preparative silica gel thin layer chromatography was performed with Macherey-Nagel silica gel.
Dihydropyridines 6a-e, 6 acetal 9 18 and nitroacetone 19 19 were prepared according to literature procedures.The methyl, ethyl and isopropyl 3-aminocrotonates 12 were purchased from the Aldrich Chemical Co.whereas, isobutyl and t-butyl 3-aminocrotonates 12 were prepared by passage of anhydrous ammonia through a solution of the alkyl acetoacetate in absolute EtOH according to the procedure of Joslyn et al. 20

General procedure for the syntheses of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-[3-(1methoxycarbonyl-1,6-dihydropyridyl)]-5-pyridincarboxyates 8a-e
A solution of Li(t-BuO) 3 AlH in THF (1 mL of 1M) was added to a solution of the respective pyridyl compound 6a-e (1 mmol) in dry THF (15 mL) at -78 °C with stirring.After 10 min, a solution of MeOCOCl (94.5 mg, 1 mmol) in THF (10 mL) was added drop wise via a syringe during 10 min, and the reaction was allowed to proceed at -78 °C for 5 h with stirring.The reaction mixture was allowed to warm to 0 °C, a saturated solution of aqueous NH 4 Cl (10 mL) was added to quench the reaction, and the mixture was poured onto water (50 mL).Extraction with CH2Cl2 (5 x 25 mL), drying the CH 2 Cl 2 extract (Na 2 SO 4 ), and removal of the solvent in vacuo afforded a yellow residue.Purification by silica gel column chromatography using EtOAchexane (3:7, v/v) as eluant yielded the respective product 8a-e.The physical, spectral and microanalytical data for 8a-e are listed below.In the 1

General procedure for the syntheses of 1-methoxycarbonyl-4-substituted-1,4dihydropyridine-3-carboxaldehydes 11a-c
Me 2 S (0.846 g, 13.6 mmol) and CuI (10 mg, 0.05 mmol) were added to a solution of the acetal 9 (0.151 g, 1 mmol) in dry THF (20 mL) under a nitrogen atmosphere, and the mixture was stirred at 25 °C for 15 min.After cooling to -23 °C, a solution of MeOCOCl (94.5 mg, 1 mmol) in dry THF (10 mL) was added, followed by addition of a solution of the respective Grignard reagent (R 1 MgBr, R 1 = Me, n-Bu, Ph) in THF (1.1 mmol of a 1.4M THF solution) during 10 min, and the reaction was allowed to proceed at -23 °C for 4 h with stirring.The reaction mixture was allowed to warm to 25 °C, a solution of 20% aqueous NH 4 Cl (20 mL) was added, and the mixture was extracted with ether (3 x 20 mL).The combined organic extracts were washed with 28% aqueous NH 4 OH-20% aqueous NH 4 Cl (1:1, v/v) until the aqueous layer was colorless.The organic fraction was washed consecutively with water (2 x 15 mL), 5% HCl (20 mL) and then water (10 mL).The organic fraction was dried (Na 2 SO 4 ) and the solvent was removed in vacuo at 25 °C to give a yellow oil that was purified by preparative silica gel thin layer chromatography using hexane-EtOAc (5:1, v/v) as the development solvent to afford the respective aldehyde 11ac.Products 11a-c were used immediately for the subsequent preparation of compounds 14-18a-c.
The physical, spectral and microanalytical data for 11a-c are listed below.

In vitro calcium channel antagonist and agonist assays
Calcium channel antagonist activity was determined as the molar concentration of the test compound required to produce 50% inhibition of the muscarinic receptor-mediated (carbachol, 1.67 x 10 -7 M) Ca 2+ -dependent contraction (tonic response) of guinea pig ileum longitudinal smooth muscle (GPILSM) using the procedure previously reported. 9The IC 50 value (± SEM), n = 3) was determined graphically from the dose-response curve.
Calcium channel agonist activity (positive inotropic effect on heart) was determined as the percentage (%) increase in contractile force of isolated guinea pig left atrium (GPLA) induced by a 4.46 x 10 -5 M (maximum concentration used) test compound concentration relative to its basal contractile force in the absence of test compound. 6

a 6 ISSN
The molar concentration of the test compound causing a 50% decrease in the slow component, or tonic contractile response (IC50 ± SEM, n = 3), in guinea pig ileum smooth longitudinal smooth muscle (GPILSM) by the muscarinic agonist carbachol (1.67 x 10 -7 M) was determined graphically from the dose-response curves .b The cardiac calcium channel agonist effect was calculated as the (+)-percentage increase (positive inotropic effect) in contractile force of isolated guinea pig left atrium (GPLA) relative to its basal contractile force in the absence of test compound (n = 3 unless otherwise stated).c Data for the racemate 2b is taken from a previous study.