Unexpected Diastereotopic Behaviour in the 1 H NMR Spectrum of 1 , 4-Dihydropyridine Derivatives Triggered by Chiral and Prochiral Centres

Derivados de 1,4-dihidropiridina constituem um importante grupo farmacológico para o tratamento de doenças cardiovasculares. Sintetizamos neste trabalho uma série de 4-(5'-nitro-2'-furil)-1,4dihidropiridina derivados, os quais foram caracterizados por H RMN. Observamos que grupos carboxílicos em C-3 e C-5 no anel 1,4-dihidropiridine mostram um sinal muito mais complexo no espectro de H RMN, tanto quando C-4 é um centro quiral ou pseudo-quiral.


Introduction
1,4-dihydropyridine derivatives (DHPs) of the nifedipine compound type, are potential antihypertensive drugs based on their Ca +2 -channel antagonistic activity.The precise mode of interaction is believed to involve the insertion into a binding site of the alpha 1 subunit of the L-type voltage gated channels present in skeletal and cardiac muscle. 1 The presence of ester groups, at the 3 and 5 positions on the 1,4-dihydropyridine ring, is of crucial importance for the pharmacological effects.It has been suggested that these groups produce hydrogen bonding with the receptor site. 2 Attempts to find improved bioavailability, longer duration of therapeutic action and other pharmacological applications, have encourage the synthesis of many structural analogues of nifedipine.Furthermore, in a previous work we have reported that some 1,4-DHPs inhibit the oxygen consumption by T. cruzi epimastigotes, Tulahuén strain. 3n this scope, recently, we have synthesized some 4-(2'-furyl-5'-nitro)-1,4-DHP derivatives, compounds 1-2.
5][6][7] The spectrum shows a more complex splitting pattern.[10] Herein, we communicate the undescribed observation of non-equivalence for the two hydrogens of the carboethoxy methylene group in compounds of the dihydropyridine's type (1-4 Figure 1).In addition, the full 1 H NMR data of these compounds is reported.

Results and Discussion
The labelling system used for the hydrogens of 1-5 is shown in the structural formula (Figure 1).Chemical shifts and coupling constants for 1-5 are summarized in Table 1.
The 1 H NMR spectrum corresponding to the methylene group on the carboethoxy substituent shows a rather more complex splitting pattern than a simple quartet (Figure 2).This behavior is due to a non-equivalence of the two hydrogens of the methylene group.A geminal coupling is observed (J gem 10.91 Hz) equally split by the neighbor hydrogens (J vic 7.10 Hz) for the compound 1.Thus, the non-equivalent hydrogens of the CH 2 (H-9A and H-9B) are split into a doublet, and each peak of the doublet is split into a quartet.The methyl hydrogens are split into a doublet, and each peak of the doublet into another doublet with equal or very similar coupling constants (H-10).The spectrum belongs to the three-spin ABX 3 type.This observation has been confirmed by the simulation of the system 11 (Figure 3).
Continuing with our efforts to find similar splitting patterns in other compounds of the series, we synthesized the compounds 3 and 4, and a similar effect has been observed.This behavior arises because C-4 is a prochiral center in compounds 1, 3 and 4 (although, actually it should be described as a "pseudo-prochiral" center because the R 1 and R 2 ligands, which would make it a prochiral center, are part of the ring and cannot be separately removed as required in the test for prochirality) and an asymmetric center in compound 2. The methylene hydrogens of the carboethoxy group in these compounds are diasterotopic and they are five bonds from the pseudoprochiral or chiral center. 12,13Prochiral groups (including hydrogens) are intrinsically non-equivalent by the chemical shift criterion (i.e., diasterotopic) when there is not a symmetry plane, s, bisecting the R 1 -C-R 2 angle.If geminal hydrogens (CH 2 ) in a molecule cannot be interchanged through a symmetry element, those hydrogens are diastereotopic to one another; then each has a different chemical shift, except for coincidental overlap.The occurrence of diastereotopic methylene hydrogens has been observed in some achiral compounds, such as: citric acid, glycerol, diethyl acetal, 3-hidroxiglutaric acid.In these molecules the chemical shift nonequivalence effect has been measured through two and three bonds between the chiral center and the methylene hydrogens.Although, the methylene groups in the 1,4-dihydropyridine derivatives are not chemical shift equivalent even the hydrogens are five bonds removed from the chiral or prochiral center. 14,15To confirm the influence of the chirality on the splitting pattern in the studied compounds, we synthesized the non substituted compound 5, where the C-4 is a C2 center, therefore, these hydrogens are enantiotopic and, consequently, the corresponding spectrum of this compound showed the methylene group signal as a simple quartet.
Also, we have found that commercially available DHPs, nitrendipine, nisoldipine, isradipine exhibit a complex splitting pattern in their 1 H NMR spectra.In the first case, due to a non-equivalence of the two hydrogens of the carboethoxy methylene group and due to a nonequivalence of the two methyl groups of the isopropoxy moiety in the other two cases.
In conclusion, we have found that the pseudo-prochiral C-4 on the 1,4-dihydropyridine ring, (compound 1, 3 and 4) and the chiral center (compound 2), are responsible on the splitting pattern.The two methylene hydrogens are diastereotopic and they give an ABX 3 spin system.

Experimental
The compounds 1-5 were synthesised by a modified method from the Hantzsch synthesis. 16Nitrendipine, nisoldipine, isradipine were obtained from Sanitas Laboratories, Chile Laboratories and Sandoz Laboratories, respectively.
Hydrogen NMR spectra were acquired using a Bruker AVANCE DRX 300 spectrometer operating at a hydrogen frequency of 300.13 MHz.Acquisition and data treatment were carried out with XWIN-NMR 3.0 Bruker program.All measurements were performed at a probe temperature of 300ºK, using solutions of compounds 1-5 in CDCl 3 containing tetramethylsilane (TMS) as an internal standard.
1 H spectra were obtained with a spectral width of 4500 Hz, a 90º flip angle (11 ms) and 1s relaxation delay in 16 scans.An exponential function with Lb = 0.3 Hz was applied before Fourier transformation to enhance the spectral resolution.The number of data points employed was 16384 and the digital resolution was 0.261281 Hz.
The hydrogen spectrum was simulated using Win-Daisy version 4.05 Bruker Program.The system was simulated as an ABX 3 group.To perform the spectrum simulation the following main parameters were considered: number of spins or groups with magnetically equivalent nuclei: 3; number of spins to use: 5 (all hydrogens that give rise to signals in the spectrum).The C 3 methyl-group was treated with the Composite Particle theory as one particle, so no point group symmetry was present.Three resonance frequency values were considered, two non-equivalent methylene hydrogens and the methyl group.The line width used was 1.4 Hz.Finally, the values for geminal and vicinal coupling constants introduced to simulate the spin system were 10.91 and 7.10 Hz, respectively.