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Intramolecular hydrogen bonds in 1,4-dihydropyridine derivatives

1,4-Dihydropyridine (1,4-DHP) derivatives have been synthesized and characterized by 1H, 13C, 15N nuclear magnetic resonance (NMR) spectroscopy, secondary proton/deuterium 13C isotope shifts, variable temperature 1H NMR experiments and quantum-chemical calculation. The intramolecular hydrogen bonds NH⋯O=C and CH⋯O=C in these compounds were established by NMR and quantum-chemical studies The downfield shift of the NH proton, accompanied by the upfield shift of the 15N nuclear magnetic resonance signals, the shift to the higher wavenumbers of the NH stretching vibration in the infrared spectra and the increase of the 1J(15N,1H) values may indicate the shortening of the N–H bond length upon intramolecular NH⋯O=C hydrogen bond formation.


Introduction
Since the original discovery of amlodipine, the 2-substituted 1,4dihydropyridines have attracted considerable attention owing to their various biological activities [1]. For the aimed synthesis of novel therapeutic agents, it is important to establish what structural factors influence their biological activity. To this end, the modification of 2,6-Me groups was performed. The original method was elaborated for obtaining both alkyl 4-substituted 2-acetoxymethyl-(3) and 2,6-bis-acetoxymethyl-1,4dihydropyridine-3,5-dicarboxylates (5) (scheme 1).
Non-covalent interactions have fundamental roles in supramolecular chemistry, drug design, protein folding, crystal engineering and other areas of molecular science. Hydrogen bond interactions are principal forces, which determine the molecular recognition and self-assembly processes as well as the structure of a great variety of chemical and biological systems [2]. The structures of the novel compounds 3 and 5 are interesting because there could be different types of intramolecular interactions. The investigated compounds 1, 3 and 5 can be divided into three groups according to their structure. In the first group (1) any intramolecular hydrogen NH· · · O bonds are absent, the second one (3)-could have one NH· · · O bond and the third group (5) may have two such bonds. The NH chemical shift (CS) in 1 H nuclear magnetic resonance (NMR) spectra in general reflects the strength of the intramolecular hydrogen bond [3]. Additionally, intramolecular hydrogen bonding can be characterized by means of secondary deuterium isotope effects NH(D) on 13 C NMR chemical shifts [4] as well as Fourier transform infrared (FTIR) spectroscopy [5]. Quantum-chemical calculations should give insight for energetic and geometrical parameters of the intramolecular hydrogen bond in the optimal conformations of the molecules.

General
All reagents were purchased from Acros, Aldrich, Alfa Aesar or Merck and used without further purification. Thin layer chromatography was performed on silica gel 60 F254 aluminium sheets 20 × 20 cm (Merck), as eluent using (20% EtOAc/hexane). The purities of compounds were determined by high performance liquid chromatography on a Waters Alliance 2695 system and Waters 2489 ultraviolet-vis detector equipped with Alltima C18 column (5 µm, 4.6 × 150 mm, Grace) using a gradient elution with acetonitrile/phosphoric acid (0.1%) in water, at a flow rate of 1 ml min −1 . Peak areas were determined electronically with a Waters Empower 2 chromatography data system.

Computational details
All calculations were performed in GAUSSIAN 09 [12] using the hybrid density functional theory (DFT) B3LYP functional with basis set 6-311++G(d,p) with empirical dispersion D3 version of Grimme with Becke-Johnson damping (GD3BJ) [13]. All geometries were optimized using the polarizable continuum model (PCM) solvation model for chloroform. For all optimized conformers, vibrational harmonic frequencies were calculated at the same level of theory, all the obtained harmonic frequencies were positive. For chemical shift calculation a gauge-independent atomic orbital (GIAO) method was used.

Results and discussion
The structures of 1, 3, 5 were analysed by multinuclear 1 H, 13 C, 15 N NMR spectroscopy (tables 1 and 2) and quantum-chemical methods.

1 H, 15 N nuclear magnetic resonance and infrared spectra
The X-ray structures of 3,5-mono-and 3,5-dicarboxylates of 1,4-DHP as well as quantum-chemical calculations [6] show that the carbonyl groups adopt the s-cis, s-cis conformation relatively to C 3 =C 2 or C 6 =C 5 double bonds. This is unusual, because it is well known that for any dienes s-trans orientation is preferable, owing to the better conjugation. The reason for the peculiar s-cis conformation of the fragment -C=C-C=O is not clear. However, in the case of 1,4-DHP derivatives it was proposed that the s-cis orientation is favoured by weak intramolecular CH· · · O=C interaction [7]. Such interactions are possible in 1, 3, 5 too. The signals of the methylene groups of the alkoxy carbonyl chains in position 3 and 5 for compounds 3 and 5 show a rather complex splitting pattern, owing to the presence of a prochiral centre on C 4 leading to the diasteretopicity of the two hydrogens of the methylene group. The rest of the signals in 1 H NMR spectra are in agreement with the nature of aromatic or aliphatic hydrogen atoms (      The two methylene hydrogens of the substituents on C 2 and C 6 in 3 and 5 are also diastereotopic and they give an AB system at 5.24-5.44 ppm in the 1 H NMR spectra. The non-equivalence of these protons is characterized by the 1 H chemical shift difference (δ(H A ) − δ(H B )) which may be affected by such factors as the conformation of the 1,4-DHP ring, the anisotropic influence of the substituents, the hindered rotation of the substituents at C 2,6 and the intramolecular hydrogen bond of the type CH· · · O. The geminal constants 2 J(H A ,H B ) slightly increase from 14.8 Hz in 3 to 15.2 Hz in 5 (table 1). For the compounds 3 the difference in the chemical shifts of the AB methylene protons varies in the range of 0.043-0.130 ppm depending on the R substituents at C 4 .
1 H NOESY spectra of compounds 3 and 5, allow us to determine the relative position of the AB protons for the methylene groups-the NOE from NH to the more shielded protons H A is more intense than the NOE to H B . This means that the H A proton is located closer to the NH proton and cannot form a CH· · · O = C hydrogen bond. The 1 H NMR signals of these protons show unusual temperature dependence (figure 1) similarly to 4-aryl-2,6-bis-(bromo-methyl)-1,4-DHP [14]. The temperature dependence of the AB quartet excludes an ordinary exchange process, because the signals do not exhibit exchange line broadening prior to  coalescence and the chemical shifts of the both protons vary simultaneously. This is in contradiction with classical dynamic NMR theories [15].
At temperature variation, the CS of the H A proton in 3 and 5 changes linearly with the same coefficient as the CS of 2-CH 3 methyl protons in 1 (table 1). So, the H A proton should be available to the solvent (chloroform), to the same extent as in 1. At the same time the signal of H B changes nonlinearly and the curve is close to a negative parabolic (figure 2). The nonlinear dependence (δ(H B ) versus T) can point to the fact that H B is engaged in a weak intramolecular hydrogen bond C-H· · · O=C.
The unusual temperature influence on the diastereotopic methylene proton signals could be explained by changes in the conformer ratio owing to two conformational processes: the rotation of the substituent around the C 2,6 −CH 2 and around the C 3,5 −CO 2 bonds. These processes have relatively high barriers for transitions between the possible conformations (approximately 16-18 kcal mol −1 (66.94-75.31 kJ mol −1 ) and approximately 8-10 kcal mol −1 (33.47-41.84 kJ mol −1 ), respectively) and unusual temperature effects on the methylene proton signals are caused by changes in the conformer ratio. So, the observed AB proton resonances are the statistically averaged resonance signals of the various conformations, the populations of which are temperature dependent [14].  The measured coupling constants 1 J( 13 C, 1 H) for each of the methylene AB protons are different (table 1). The larger 1 J( 13 C, 1 H) value corresponds to the less shielded proton H B . This is consistent with the literature data, because if the CH proton is involved in a hydrogen bond the 1 J( 13 C, 1 H) value usually increases [16]. Indeed, in our previous work [9] the hydrogen bonded protons were characterized by the larger value of the 1 J( 13 C, 1 H). On the basis of the data obtained it can be concluded that the intramolecular CH· · · O=C hydrogen bond could be one of the reasons for the non-equivalence of the methylene protons in these systems and conformations with s-cis-s-cis carboxyl group orientation relative to the double bond in the dihydropyridine cycle.
The low field shift of the N 1 H proton signal in the sequence 1, 3, 5, may reflect the formation of an intramolecular N-H· · · O hydrogen bond in compounds 3 and 5 between the N 1 H proton and the oxygen atoms of the OCOMe substituents at C 2 and C 6 carbons [17,18]. On the other side the low-field shift of N 1 H resonance could correspond partly to the decrease of the electron density on N 1 H atom as calculated by the B3LYP-NBO method in the sequence 1, 3, 5 (figure 3).
The temperature coefficient ( δ/ T) of the N 1 H signal is usually used to detect the presence of intramolecular H-bonds [18,19]. The breaking of hydrogen bonds, induced by increasing the temperature, moves the resonance signal of N 1 H upfield. The temperature coefficients with the values more positive than −4.5 ppb K −1 are strongly indicative of intramolecular H-bonds.
The temperature coefficients of the NH proton resonance in 1, 3, 5 have been measured recording the 1 H NMR spectra in CDCl 3 in the temperature range 223-323 K (table 1). The obtained values fall between −2.5 and −10.4 ppb K −1 . The NH protons of 3 have the δ/ T values more positive than −4.5 ppb K −1 (approx. −3 ppb K −1 ) which indicates the involvement of these NH protons in the intramolecular Hbonding. In compounds 5, that have two acetoxymethyl substituents and, consequently, may have two hydrogen bonds, the δ/ T values are lower than in 3 (approx. −3.6 ppb K −1 ). This could be the consequence of the weaker NH· · · O=C hydrogen bond in case of 5 when compared to 3.
The absolute values of the measured coupling constants 1 J( 15 N, 1 H) 92-96 Hz slightly increase in the sequence 1, 3, 5 (figure 4) and suggest that the hydrogen is stably bonded to the nitrogen atom and there is little intermolecular exchange, this is in contradiction, because increased positive charge on the NH proton (deshielding) should decrease the absolute value of the 1 J( 15 N, 1 H) [20,21]. However, when the nitrogen of the proton donor is sp 2 hybridized the changes in 1 J( 15 N, 1 H) are quadratically related to the length of the corresponding NH distances of nitrogen bases: the shorter d(NH) corresponds to the larger 1 J( 15 N, 1 H) value [22]. This could point to the slight decrease of the NH distance in the sequence 1-3-5.
According to [23] if NH participate in the N-H· · · X hydrogen bond, the 1 J( 15 N, 1 H) coupling is affected by mainly two factors, the electrostatic effect and the n(X) → (N-H)* charge transfer interaction. The first one is known to cause an increase in the absolute value of 1 J( 15 N, 1 H) and the second leads to a decrease in the absolute value of 1 J( 15 N, 1 H) coupling. Their influence on the IR stretching frequency of the N-H bond are also opposite to each other, so the charge transfer interactions correspond to a red shift while the electrostatic effect corresponds to a blue shift. Usually, hydrogen bonds lead to an elongation of the X-H covalent bond and its stretching vibration ν XH shifts to the lower wavenumbers (red shift) [5,18].    shortening of the NH distance (so-called blue shift). Such a kind of effect has been registered for the C-H and N-H bonds previously [24]. The IR spectral data of 1a-c with no hydrogen bonds are characterized by the stretching ν(NH) bands in the range 3344-3336 cm −1 . The NH groups engaged in one hydrogen bond in compounds 3a-c display the NH-stretching vibration in the interval 3347-3348 cm −1 and the NH groups engaged in two hydrogen bonds in compounds 5a-c display the ν(NH) bands in the range 3363-3437 cm −1 (figure 5). The ν(NH) band moves up to 8 cm −1 on going from 1b to 3b, and up to 64 cm −1 on going from 1b to 5b. The IR stretching ν(NH) band frequencies and the 1 J( 15 N, 1 H) values in compounds 1-5 indicate that both of these parameters (ν(NH), 1 J( 15 N, 1 H)) could be partly caused by the same reason-the difference in the length of the NH bond [25].
The 15 N HMQC spectra with the value of the direct NH coupling constants ( 1 J( 15 N, 1 H) = 95 Hz) were used to measure the chemical shift for the nitrogen nucleus [26,27]. The 15     accompanied by the shielding of the adjacent nitrogen atom [28]. Commonly the NH bond elongates on hydrogen bond formation [29]. According to [30] the observed 15  In the data obtained by the natural bond orbital (NBO) method, the average π-orbital character of the N atom in NH-bond decreases in the sequence 1(74.2%)-3(73.0%)-5(72.2%) (figure 7). That should lead to a diamagnetic shift of the 15   A very popular explanation of diamagnetic shift of heavy nuclei is sterical perturbation of the electronic cloud nearby. In our case this may be owing to the spatial affinity between the acetoxy substituent and 15 N nucleus. According to the literature data [31] such effect could shift 15 N resonance signal up to 10 ppm to higher fields.

13 C nuclear magnetic resonance spectra
The 13 C NMR spectra of compounds 1, 3, 5 display signals in the carboxyl, aromatic and aliphatic regions (table 2). The 13 C resonances in the 1,4-DHP ring (table 2) are relatively insensitive to the nature of the substituents on C 3 , C 4 and C 5 . The 13 C NMR spectra of compounds 1, 3, 5 show the signals for C 3 and C 5 atoms at the lower values (ca 102-104 ppm), than those expected for the typical olefinic carbons atoms, while the C 2 and C 6 signals appear at higher values (approx. 141-145 ppm). These findings have been accounted for by the strong push-pull effect of the groups linked to the olefinic double bonds like ones previously observed in other related systems. The carbon signal of C 4 is in agreement with the previous data [32,33] and appears at 28.5 ppm for the aliphatic R and in the interval 34.2-39.8 ppm when R is aromatics.

Proton/deuterium isotope effects on 13 C chemical shifts
Measurement of the secondary n 13 C(proton/deuterium (H/D)) deuterium isotope effects (IE) is known to be a very good tool in studying intramolecular hydrogen bonds [4]. In compounds 1, 3, 5 the replacement of H by D at the nitrogen atom produces only intrinsic isotope effects, because the values of 1 J ( 15 N, 1 H)   effects are of vibrational origin and are owing to the anharmonicity of the potential curve of the N-H· · · X bond. Hydrogen bonding increases this anharmonicity. The changes in δ( 13 C) are the result of the shortening of the NH/D bond upon deuteration [34]. The values of IE ( n 13 C(H/D) = δ( 13 C(NH)) − δ( 13 C(ND))) have been measured from the 13 C spectra of partly deuterated samples where the signals of the two isotopomers could be easily identified on the basis of their relative intensity. All measured isotope effects in 1, 3, 5 were positive (table 3).
The substituent effects on IE values are mainly described by two factors: the hydrogen bond and the transmission of the electronic effects of substituents [35].
The N(H/D) isotope effects on the acetoxymethyl carbonyl 13 C atoms have been registered (table 3). For compounds 3 where the NH atom is involved in one hydrogen bond, the 5  If we consider the influence of the N(H/D) replacement on the 13 C resonances inside the 1,4-DHP cycle (table 3) there is little change in the sequence 1-3-5. This corresponds to the small change of the 13 C chemical shifts (table 2). Interestingly, that the N(H/D) IE on the C 2,6 -13 CH 3 atom of the methyl groups separated from the N 1 H proton by three bonds, is similar to the effect on the 13 C 2,6 carbons separated by only two bonds. At the same time, the N(H/D) IE values for the 13 C 3,5 atoms are much smaller (table 3). The 13 C isotope shifts of the methyl groups at C 2 and C 6 in 1 and 3 are considerably larger than the corresponding values of the methylene groups at the same atoms in compounds 3 and 5 (table 3).
No 13 C(NH/D) isotope effects are observed on the C 4 carbon atom in the 1,4-DHP cycle, however in 4-Me substituted compounds 1a, 3a and 5a the positive long-range isotope effects 5 C 4−Me (H/D) approximately 26-28 ppb are observed. The observation of such a long-range isotope effect at the methyl group C 4 -CH 3 is rather unusual. Since there is no general theory about the long-range IE we have problems in interpretation of the long ranges 13 C IE observed in 1, 3, 5 compounds.

Density functional theory calculations
In compounds 3 and 5 there could be four possible types of the intramolecular H-bonds (NH· · · O=, NH· · · -O-, CH· · · O = and CH· · · -O-) defining the optimal configuration of the molecules.
To establish which of the available types of bonds is dominant in stabilizing the optimal geometry of compounds 3 and 5, the quantum-chemical calculations of the possible conformations of compounds 3a and 5a were performed.
The most stable conformations of 3a and 5a and the calculated geometric parameters of the intramolecular hydrogen bonds CH· · · O and NH· · · O are presented in figures 9 and 10 and table 4.    The optimal conformations of the side chain 3,5-carbomethoxy groups were shown before to be s-cis, s-cis [9,26]. The obtained results reveal that in 3a' the energy minima correspond to the conformations with s-cis-s-cis carboxyl group orientation (A, D) relative to the double bond in the dihydropyridine cycle and there is a possibility to form the C 2,6 H···O bonds with the oxygens of the carboxyl groups at C 3 and C 5 . These additional H-bonds stabilize the molecule, as the structures with s-cis, s-trans (B, E) and s-trans, s-trans (C, F) carboxyl groups orientation have the higher energy.   According to the calculations, the distances d(CH· · · O) increase, and the angles α(C-H· · · O) decrease on going from A-C to D-F, indicating weakening of the CH· · · O hydrogen bonds between the methylene protons and the oxygens of the carboxyl groups. At the same time in conformations A-C only one methylene proton participates in the CH· · · O hydrogen bond, and in D-E conformations both methylene protons form CH· · · O bonds. Although the first one is stronger because it is characterized by the shorter distance d CH···O and the larger angle α CH···O , two weaker CH· · · O bonds in D-E might have the same stabilizing effect for the molecule optimal conformations. This tendency is true for any orientations of the carbomethoxy groups at C 3 , C 5 positions.
In 5a the optimal conformation A is characterized by s-cis, s-cis O=C 3,5-carboxyl group orientation relative to the double bond in the dihydropyridine cycle and the NH· · · O hydrogen bond with two O=C oxygens (A).
The structures with s-cis-s-trans (B) and s-trans-s-trans (C) orientation of the 3,5-carboxyl groups in 5a similarly to 3a have the higher energy.
The calculations of 3,5-side chain conformations of 5a show that, the distances d(CH· · · O) increase, and the angles (C-H· · · O) decrease on going from --C to D-F, indicating a weakening of the CH· · · O hydrogen bonds between the methylene protons and the oxygens of the carboxyl groups. Similarly to 3a' in conformations A-C of 5a only one methylene proton participates in the CH· · · O = hydrogen bond, and in D-E conformations of 5a both methylene protons form approximately equal CH· · · -O-bonds. Although the first hydrogen bond is stronger because it is characterized by the shorter distance d CH···O and the larger angle α CH···O , two weaker CH· · · O bonds in D-E of 5a might have the same stabilizing effect on the optimal conformations of the molecule. This tendency is true for any orientations of the carbomethoxy groups at C 3 , C 5 positions.
In conformations A-C of the compounds 3a and 5a, the methylene protons at C 2 , C 6 are nonequivalent, because only one participates in the CH· · · O hydrogen bond. In conformations D-F of 3a and 5a, both methylene protons are equivalent because they form two equal CH· · · O hydrogen bonds (table 4). Owing to the energy advantage of the conformation A over D of compounds 5 and energetic equivalence of A and D conformations in 3, the difference of the CS of the methylene protons (δ(H A )-δ(H B )) in 5 is twice as high than in 3.
The calculations performed demonstrate that in case of compounds 3, the NH proton could preferably form a 5-membered H-chelate cycle with the alkoxy oxygen. In compounds 5 two 7-membered H-chelate cycles with the C=O oxygens in 2 and 6 substituents are formed. Different types of the NH· · · O bonds in 3 and 5 can be the reason for the increase of the values of the secondary n 13 C(NH/D) isotope shifts in 13 C NMR spectra on the carboxyl group carbon in substituents at C 2,6 on going from 3 to 5. The greater isotope effect of the NH/D substitution through 6 bonds to the 13 C CS of the carboxylic carbons in substituents at positions 2 and 6 in compounds 5 ( 6 13 C(NH/D)), than through 4 bonds ( 4 13 C(NH/D)) in 3 proves that the NH···O hydrogen bond in the seven-membered H-chelate cycle formed by the C=O group is stronger than the one in the five-membered H-chelate formed by the alkoxy group.
In monosubstituted compounds 3, the NH proton preferably forms a five-membered H-chelate cycle with the alkoxy oxygen, while in disubstituted compounds 5, two 7-membered H-chelate cycles with the carbonyl oxygens of the carboxyl groups in 2-and 6-substituents are preferred.
The downfield shift of the 1 H NMR signal of the NH proton, accompanied by the upfield shift of the 15 N NMR signals and the increase of the 1 J( 15 N, 1 H) values in the sequence from dimethyl 4-substituted 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1) to 3 and 5, may indicate the shortening of the N-H bond length upon hydrogen bond formation. This is supported by the shift of NH stretching band to the higher wavenumbers in the FTIR spectra of 3 and 5 The optimal conformers of 3 and 5 with the lowest energy have the 3,5-carbonyl groups in s-cis, s-cis position relative to the double bonds of the DHP cycle (C 3 =C 2 and C 6 =C 5 ). They are stabilized by weak intramolecular hydrogen bonds of the CH· · · O type. The diastereotopic protons of the methylene groups at positions C 2 and C 6 show unusual temperature dependence due to the changes in the populations of the optimal conformations. Data accessibility. Supporting data are available at Dryad (http://dx.doi.org/10.5061/dryad.j3m73) [36].