Characterization of amide bond conformers for a novel heterocyclic template of N-acylhydrazone derivatives.

Herein we describe NMR experiments and structural modifications of 4-methyl-2-phenylpyrimidine-N-acylhydrazone compounds (aryl-NAH) in order to discover if duplication of some signals in their ¹H- and ¹³C-NMR spectra was related to a mixture of imine double bond stereoisomers (E/Z) or CO-NH bond conformers (syn and anti-periplanar). NMR data from NOEdiff, 2D-NOESY and ¹H-NMR spectra at different temperatures, and also the synthesis of isopropylidene hydrazone revealed the nature of duplicated signals of a 4-methyl-2-phenylpyrimidine-N-acylhydrazone derivative as a mixture of two conformers in solution. Further we investigated the stereoelectronic influence of substituents at the ortho position on the pyrimidine ring with respect to the carbonyl group, as well as the electronic effects of pyrimidine by changing it to phenyl. The conformer equilibrium was attributed to the decoplanarization of the aromatic ring and carbonyl group (generated by an ortho-alkyl group) and/or the electron withdrawing character of the pyrimidine ring. Both effects increased the rotational barrier of the C-N amide bond, as verified by the DG(≠) values calculated from dynamic NMR. As far as we know, it is the first description of aryl-NAH compounds presenting two CO-NH bond- related conformations.

1. Introduction

The bioactive class="Chemical">N-acylhydrazone (<class="Chemical">span class="Chemical">NAH) moiety has been identified in a great number of lead compounds that act on different types of molecular targets [1,2,3,4]. Because of the assemblage of amide and imine functions, NAH compounds may exist as C=N double bond stereoisomers (E/Z) (Scheme 1) and as syn/antiperiplanar conformers about the amide CO-NH bond (Scheme 1) [5].

Scheme 1

General structure and stereochemistry of NAH.

General structure and stereochemistry of <span class="Chemical">NAH.

A research program to develop a series of class="Chemical">2-phenyl-4-methylpyrimidine-N-acylhydrazone compounds with antinociceptive and anti-inflammatory activities led to the discovery of <class="Chemical">span class="Chemical">LASSBio-1083 (1, Figure 1), a promising lead compound that shows an ED50 of 27 µmol/Kg in the acetic acid-induced mouse writhing test [6]. Regarding full structural characterization, the 1H-NMR spectra revealed the duplication of some signals, indicating the presence of a mixture of stereoisomers or conformers.

Figure 1

Stereoelectronic modifications of the NAH derivative LASSBio-1083 (1).

Using class="Chemical">1H- and <class="Chemical">span class="Chemical">13C-NMR data, Palla and coworkers posited that NAH compounds derived from the condensation of hydrazides and aromatic aldehydes are present in solution as the E geometric isomer, which is less sterically hindered compared to the Z form [7]. However, starting from the pyridine-2-carboxaldehyde, the Z isomer can be detected in less polar solvents due to its stabilization with intramolecular H-bonds [5]. On the other hand, the duplication of NMR signals has been attributed to the presence of anti- and synperiplanar conformers in benzyl and alkyl NAHs [8,9,10], but there is no description of this phenomenon in aryl NAHs in the literature. Because the complete knowledge of structure, including stereochemistry, is essential for lead optimization in drug discovery, we herein describe our studies concerning the elucidation of the signal duplication in the 1H-NMR of 1 and the modifications of its structure leading to compounds 2a–d and 3a to better understand the stereoelectronic properties that promote this effect (Figure 1).

Stereoelectronic modifications of the <span class="Chemical">NAH derivative <class="Chemical">span class="Chemical">LASSBio-1083 (1).

2. Results and Discussion

2.1. Chemical Synthesis

2.1.1. Synthesis of Esters 4a–e

class="Chemical">Esters 4a and <class="Chemical">span class="Chemical">4b were chemoselectively obtained in 80% and 73% yield, respectively, through the condensation of benzamidine with (Z/E)-ethyl 2-((dimethylamino)methylene)-3-oxobutanoate (5) and (Z/E)-methyl 2-((dimethylamino)methylene)-3-oxopentanoate (6), respectively, in ethanol (for 4a) and methanol (for 4b) at room temperature (Scheme 2) [11,12]. The synthesis of the pyrimidine nucleus was confirmed by the presence of singlets at 9.17 and 9.12 ppm in the 1H-NMR spectra, which refer to the H-6 attached to the pyrimidine ring in esters 4a and 4b, respectively.

Scheme 2

Synthesis of 4-alkyl-2-phenylpyrimidinyl esters 4a and 4b.

class="Gene">Synthesis of <class="Chemical">span class="Chemical">4-alkyl-2-phenylpyrimidinyl esters 4a and 4b.

Reagents and conditions: (a) for 4a: class="Chemical">benzamidine hydrochloride, Na/<class="Chemical">span class="Chemical">EtOH, rt, 2 h, 80% and 4b: benzamidine hydrochloride, NaOH/MeOH, rt, 1 h, 73%.

class="Chemical">Ethyl 4-hydroxy-2-phenylpyrimidine-5-carboxylate (8) was obtained in 50% yield by condensation between <class="Chemical">span class="Chemical">benzamidine and diethyl 2-(ethoxymethylene)malonate (7) in ethanol at room temperature (Scheme 3) [12]. Two subsequent reactions from 8 furnished the ethyl ester 4c in 50% overall yield. The first step involved chlorination of 8 using POCl3 at 100 °C, and the second one was dehalogenation of 9 by a radical reaction using zinc and AcOH in THF (Scheme 3) [13,14]. The presence of a singlet at 9.22 ppm that integrated to 2H, confirmed the dehalogenation of C-4 and formation of the pyrimidinyl ester 4c.

Scheme 3

Synthesis of unsubstituted 2-phenylpyrimidinyl ester 4c.

<span class="Gene">Synthesis of unsubstituted <class="Chemical">span class="Chemical">2-phenylpyrimidinyl ester 4c.

Reagents and conditions: (a) class="Chemical">benzamidine hydrochloride, Na/<class="Chemical">span class="Chemical">EtOH, rt, 9 h, 50%; (b) POCl3, 100 °C, 1 h, 98%; (c) Zn, AcOH, THF, 60 °C, 23 h, 50%.

The class="Gene">synthesis of <class="Chemical">span class="Chemical">biphenyl ester 4d was achieved by Suzuki cross-coupling between methyl ester 10 and phenylboronic acid in the presence of potassium carbonate and PdCl2(PPh3)2 at 80 °C in 91% yield, while the ester 4e was obtained commercially (Scheme 4) [15].

Scheme 4

Synthesis of biphenyl esters 4d and 4e.

<span class="Gene">Synthesis of <class="Chemical">span class="Chemical">biphenyl esters 4d and 4e.

Reagents and conditions: (a) class="Chemical">phenylboronic acid, <class="Chemical">span class="Chemical">PdCl2(PPh3)2, K2CO3, toluene/MeOH, 60 °C, 3 h, 91%.

2.1.2. Synthesis of N-acylhydrazone Derivatives 1–3

The key precursors for the class="Gene">synthesis of <class="Chemical">span class="Chemical">NAH 1 and 2a–d, the phenylpyrimidine and biphenyl hydrazides 11a–e, were obtained in 79% to 95% yield by nucleophilic acyl substitution reaction of 4 with 100% hydrazine hydrate in ethanol at 55–80 °C (Scheme 5) [16]. The desired compounds 1 and 2a–d were synthesized using the classic acid-catalyzed condensation of hydrazides 11a–d with 4-dimethylaminobenzaldehyde in ethanol at room temperature in 85%–99% yield (Scheme 5 and Table 1). Analysis of the 1H-NMR in DMSO-d6 showed the formation of NAH 1 and 2a–d by the presence of signals attributed to the N=CH and CONH protons, but for some NAH (1, 2a–c), these signals appear to be duplicated (Table 1). Finally, the N-methyl-NAH derivatives 3a were obtained from a selective N-methylation of 1 using methyl iodide in a basic medium of K2CO3 at 43 °C in 77% yield (Scheme 5 and Table 1) [17]. The presence of signals referring only to the N-CH3 protons indicates the selective N-methylation of NAH. Additionally, no signal duplication was observed for these N-methyl-NAH derivatives.

Scheme 5

Synthesis of functionalized N-acylhydrazone derivatives 1, 2a–d and 3a.

Table 1

Synthesis of N-acylhydrazone and N-methyl-N-acylhydrazone derivatives 1–3.

Compound	X	R1	R3	Yield (%) a,b	Ratio a:b
1	N	CH3	H	66	2:1
2a	N	H	H	21	4:1
2b	N	CH2CH3	H	49	1.7:1
2c	C	CH3	H	81	5:1
2d	C	H	H	84	c
3a	N	CH3	CH3	51	c

a Global yield; b The analytical results for C, H and N were within 0.4% of the calculated values; b Ratio obtained by signal integration of the duplicate N=CH peaks in the 1H-NMR spectra; c Only one signal is present.

<span class="Gene">Synthesis of functionalized <class="Chemical">span class="Chemical">N-acylhydrazone derivatives 1, 2a–d and 3a.

Reagents and conditions: (a) class="Chemical">NH2NH2.<class="Chemical">span class="Chemical">H2O, 55–80 °C, 2–8 h, 79%–95%; (b) 4-(dimethylamino)benzaldehyde, catalytic HCl, EtOH, rt, 85%–99%; (c) CH3I, K2CO3, acetone, 43 °C, 24 h, 77%.

class="Gene">Synthesis of <class="Chemical">span class="Chemical">N-acylhydrazone and N-methyl-N-acylhydrazone derivatives 1–3.

a Global yield; b The analytical results for C, H and N were within 0.4% of the calculated values; b Ratio obtained by signal integration of the duplicate N=CH peaks in the <span class="Chemical">1H-NMR class="Chemical">spectra; c Only one signal is present.

2.2. NAH Stereochemistry Elucidation

2.2.1. Determination of the Relative Configuration of the Imine Double Bond

The class="Chemical">1H-NMR class="Chemical">spectrum of the analgesic lead compound <class="Chemical">span class="Chemical">LASSBio-1083 (1) is presented in Figure 2A and shows the duplication of the hydrogen signals. Assuming that these duplications could be attributed to the presence of the two possible isomers (E/Z) of the imine double bond, we decided to proceed with differential Nuclear Overhauser Effect (differential NOE) experiments to assess the spatial proximity of 1H-1H. The hydrogen atom selected for irradiation was the NH of the amide. Due to the presence of two singlets related to the NH hydrogen, we chose the one that shifted less and that was more prevalent at 11.67 ppm (Figure 2B).

Figure 2

(A) 1H-NMR and (B) NOEdiff spectra for irradiation of NH (the major peak) of NAH derivative 1.

Although only one class="Chemical">amide signal at 11.67 ppm was selected for irradiation in the NOE experiments, the <class="Chemical">span class="Chemical">amide hydrogen at 11.79 ppm presented the same irradiated signal phase. Furthermore, both signals for H6 and N=CH showed increased intensities, presenting a positive NOE of 11% and 15%, respectively (Figure 2B).

Because of the angle and distance, a positive NOE on N=CH was not expected from NH irradiation of the Z isomer. Calculations using Mspin 1.03 software [18] were performed to simulate the NOE effect at a radius of 5 Å around the irradiated <span class="Chemical">hydrogen. For this simulation, more stable E (A/B) and Z (C) stereoisomers, obtained through a process of geometric optimization by molecular mechanics followed by conformational analysis using the semi-empirical method <class="Chemical">span class="Species">AM1 in the PC Spartan Pro 1.0.5 software [19], were selected (Table 2). Although the B conformer was not as stable as A, the choice was based on observations from other NAH compounds in X-ray diffraction studies (Table 2) [17].

Table 2

Theoretical relative NOE obtained in MSpin software for the E and Z geometric isomers of LASSBio-1083 (1).

Form	N H–N=CH	N H–H6	N H–H2′′ a	N H–H3′′	N H–H6′′ a	N H–CH3
A	0.063	0.650	0.033	-	-	0.015
B	0.746	0.497	0.006	-	0.014	0.018
C	0.064	0.586	0.811	0.023	0.022	0.017

a The pair H2” and H6” is chemically identical in a dynamic model, but the software only considers a static form and gives different values for pairs of equal hydrogens.

(A) <span class="Chemical">1H-NMR and (B) NOEdiff class="Chemical">spectra for irradiation of NH (the major peak) of <class="Chemical">span class="Chemical">NAH derivative 1.

The theoretical results for <span class="Chemical">amide NH irradiation showed that this effect should be observed mainly in H6 and N=CH for both E (A and B) conformers and be more pronounced at N=CH in the B conformer (Table 2). The same analysis for the Z stereoisomer showed that the greatest positive NOE should be observed at H2” followed by H6, but no significant positive NOE would be expected for N=CH (Table 2).

To clarify the reason for the negative phase of the minor NH signal and exclude the possibility of a technical artifact due to the proximity of signals, we decided to irradiate the signal of the less prevalent H2”/H6” at 7.20 ppm. This location was relatively distant from the multiplet and doublet neighbor signals, whereas the signal corresponding to H2′′/H6′′ in greater proportion was located inside a multiplet at 7.54 <span class="Species">to 7.57 ppm (Figure 3). The irradiation led again to two negative signals correclass="Chemical">sponding to both signals of H2′′/H6′′ (7.20 and 7.55 ppm) and a positive one for both signals of N=CH and H3′′/H5′′, presenting a positive NOE of 9% and 8%, reclass="Chemical">spectively (Figure 3). Curiously, only the H2′′/H6′′ signal shifted at 7.55 ppm inside the multiplet, and no other <class="Chemical">span class="Chemical">hydrogen presented a negative signal , showing that the inversion of phase was not an artifact of the technique due to the proximity of signals (Figure 3).

Figure 3

(A) Expanded 1H-NMR and (B) NOEdiff spectra for irradiation of H2”/H6” (in minor proportion) of NAH derivative 1.

Additionally, we obtained a 2D-NOESY spectrum and verified that each expected diagonal signal from a conformer presented a cross-correlation in-phase with the correspondent signal of the other conformer (supplementary material). This effect was dependent on the experimental mixing time (D8 of 40, 80 and 300 ms), with longer times resulting in more intense cross-correlations.

Theoretical relative NOE obtained in MSpin software for the E and Z geometric isomers of <span class="Chemical">LASSBio-1083 (1).

a The pair H2” and H6” is chemically identical in a dynamic model, but the software only considers a static form and gives different values for pairs of equal <span class="Chemical">hydrogens.

(A) Expanded <span class="Chemical">1H-NMR and (B) NOEdiff class="Chemical">spectra for irradiation of H2”/H6” (in minor proportion) of <class="Chemical">span class="Chemical">NAH derivative 1.

Normally this type of cross-correlation can be attributed to an artifact because of a chemical exchange with solvent. However, this is not the case, as we observed a cross-correlation for all protons including those that were not exchangeable. The cross-correlation is most plausibly due to an exchange among multiple conformers in which a <span class="Chemical">1H has a distinct chemical shift in each conformer. The time required for one conformer to become another explains the increase in intensity with longer mixing times.

These NOE experiment results led to the hypothesis that each signal could be related to the presence of two conformers. Moreover, we could exclude the Z stereoisomer because all NOE results are in agreement with the more stable E stereoisomer [20], as theoretically predicted. Thus, the <span class="Chemical">hydrogen of NH could be <class="Chemical">span class="Gene">synperiplanar (sp) or antiperiplanar (ap) in relation to the carbonyl oxygen atom of the E isomer (Figure 3). This hypothesis was corroborated by HPLC studies in which only one compound was detected as well as by the change in the ratio between the pair of duplicated signals when CDCl3 was used in place of DMSO-d6 for the 1H-NMR analysis, as previously observed by Palla and coworkers for alkyl-NAH [7].

2.2.2. NAH Derivative 1 Conformers Determination

Based on the work of Quattropani and colleagues [10], who described the presence of conformers for class="Chemical">alkyl NAH compounds (Figure 4), we carried out a <class="Chemical">span class="Chemical">1H-NMR experiment in DMSO-d6 at 80 °C to determine whether the coalescence of duplicated signals would be observed (Figure 5). A complete coalescence was observed for the NH, H6, H3′′/H5′′, N(CH3)2 and CH3 signals, while partial coalescence was observed for N=CH and H2′′/H6′′. The reversibility of these changes was verified when the experimental temperature was returned to 20 °C. This result corroborated the hypothesis of the conformers: the energy required to faster overcome the rotational barrier is reached upon an increase in temperature, leading to a rapid conversion between the conformers. Traditionally, the amide group is represented by the canonical forms 1 and 3.

Figure 4

Representation of ap/sp conformers of the amide bond of NAH derivative 1 and its canonical forms.

Figure 5

1H-NMR spectra of the NAH derivative 1 at 20 °C (A) and 80 °C (B).

Representation of ap/sp conformers of the <span class="Chemical">amide bond of <class="Chemical">span class="Chemical">NAH derivative 1 and its canonical forms.

<span class="Chemical">1H-NMR class="Chemical">spectra of the <class="Chemical">span class="Chemical">NAH derivative 1 at 20 °C (A) and 80 °C (B).

The resonance effect between the lone pair on the class="Chemical">nitrogen class="Chemical">sp2 atom and the <class="Chemical">span class="Chemical">carbonyl π bond contributes to the double bond character of the amides in the planar form. In this context, the resonance structure 3 is considered more significant for the double bond character and rotational barrier [21].

Converting a conformer into another requires rotation around the C-N bond, which consequently changes the <span class="Chemical">nitrogen class="Chemical">sp2 hybridization to a <class="Chemical">span class="Disease">pyramidal arrangement. In the pyramidal geometry, the lone pair of nitrogen is placed in an orbital with high s character for stabilization. The disruption of favorable interactions between N and C raises the rotational barrier. In the case of formamide, the barrier is 18 kcal mol−1 [22,23,24,25].

Wiberg et al. conducted theoretical studies concerning the class="Chemical">amide bond rotation. Their calculations showed that the C-N bond lengthens by 0.08 Ǻ upon rotation from the planar form to the transition state, whereas the C-O bond shortens slightly (by 0.01 Ǻ). Moreover, an insignificant difference in charge density at the <class="Chemical">span class="Chemical">oxygen was observed in the planar form and the transition state. Hence, the oxygen is assumed to simply polarize the C-O bond, resulting in a large δ+ on the carbon [22].

As the earlier model with two canonical structures did not explain these results, one that incorporated a <span class="Chemical">carbonyl dipolar canonical form was suggested (2). Therefore, the rotational barrier is attributed mainly to the resonance structures 2/3 and 5/6 [22,23].

N-class="Chemical">Methylation of <class="Chemical">span class="Chemical">NAH 1, which results in N-methyl-NAH 3a, causes conformational restriction of the amide bond, thus preventing its rotation, as previously described by our group [17,26]. In fact, this modification abolished the duplication of the signals in the NMR spectra. Another approach used to evaluate the presence of two conformers was the synthesis of isopropylidene hydrazones. The latter was based on the works of Wyrzykiewicz and Prukala [27] and Palla [5], to eliminate the possibility of forming E/Z stereoisomers. The compound (isopropylidene)-2-phenyl-4-methyl-pyrimidine-5-hydrazide (12) (Figure 6) was synthesized by condensation of 2-phenyl-4-methyl-pyrimidine-5-hydrazide (11a) with acetone. Its 1H-NMR spectrum presented two singlets for each NH, H6 and CH3a and another three for CH3b and CH3c. Regarding CH3a, the signal present to a lesser extent showed the same chemical shift of the solvent DMSO-d6 (2.48 ppm). Likewise, the signal for the smaller proportion of CH3c and that for the greater proportion of CH3b also coincided (both were at 1.9 ppm). This result confirmed the presence of conformers.

Figure 6

1H-NMR signal duplications and its proportions for the methyl groups in NAH derivative 12.

class="Chemical">1H-NMR signal duplications and its proportions for the <class="Chemical">span class="Chemical">methyl groups in NAH derivative 12.

Considering the proportion of the conformers, we believe that the more deshielded signal from N=CH matches the conformer ap, based on the work by Palla in which the more deshielded signal was attributed to <span class="Chemical">imine hydrogen of conformer ap in <class="Chemical">span class="Chemical">DMSO-d6 and in CDCl3 [7]. Furthermore, in the ap conformation, the two dipolar moments (Cδ+=Oδ− and Nδ−-Hδ+) are aligned, which does not occur in the sp conformer [21].

According to literature reports, the presence of conformers for the class="Chemical">NAH compounds is typically observed in those with a class="Chemical">spacer between the <class="Chemical">span class="Chemical">carbonyl group and the aromatic ring or in alkyl-N-acylhydrazones. However, NAH compound 1 does not structurally match the compounds described in previous reports. Perhaps an ortho steric effect, exerted by the methyl group on the carbonyl, and/or an electronic effect induced by the pyrimidine ring could be related to the observed phenomenon [28]. Thus, we decided to study the stereoelectronic requirements for the existence of conformers.

2.2.3. Stereoelectronic Effects on NAH Derivative (1) ap-sp Amide Bond Rotamers

To evaluate the ortho steric effect, the class="Chemical">methyl group was replaced by an H atom (2a) and an ethyl group (2b). The first modification represents the absence of any steric effect while the second one increases the steric effect. Both compounds showed duplicated signals in the <class="Chemical">span class="Chemical">1H-NMR spectra, as shown in Table 3. Additionally, the pyrimidine ring was replaced with a phenyl ring to verify the influence of the electronic effect on the amide bond, generating compounds 2c and 2d. Thus, we could study the influence of the heterocyclic ring both in the presence (2c) and absence (2d) of an ortho steric effect. Only 2d did not show duplicated signals. Therefore, we found that the steric effect and/or the pyrimidine ring induced the formation of conformers in this class of NAH.

Table 3

Chemical shifts (ppm) and relative integration (between parentheses) of the main hydrogen signals for the N-acylhydrazone derivatives 1 and 2a–d.

cpd a	CO-N H		H6		N=C H		H2′′/H6′′		H3′′/H5′′		N(CH3)2		R1
1 b	11.79 (34)	11.67 (66)	8.91 (66)	8.81 (34)	8.17 (66)	7.98 (34)	7.55 (66)	7.24 (34)	6.76 (66)	6.64 (34)	2.97 (66)	2.88 (34)	2.66 (66)	2.53(34)
2a c	11.91 (20)	11.87 (80)	9.31 e	9.29 e	8.31 (80)	8.02 (20)	7.60–7.58 d (80) g	7.4 (20)	6.78 e	6.73 e	2.99 (80)	2.95 (20)	-	-
2b b	11.86 (37)	11.74 (63)	8.90 (63)	8.79 (37)	8.15 (63)	7.9 (37)	7.56–7.53 d (63) g	7.21 (37)	6.75 (63)	6.62 (37)	2.97 (63) g	2.87 (37)	-	-
2c c	11.54 (17)	11.51 (83)	7.75–7.70 multiplet		8.17 (83)	7.95 (17)	7.61–7.39 d (83) g	7.23 (17)	6.77 (83)	6.64 (17)	2.99 (83)	2.90 (17)	2.46 (83)	2.34 (17)
2d b	11.60		8.00 f		8.34		7.54–7.38 d		6.77		2.98		8.00 f

a cpd = compound; b 40 °C; c 20 °C; d inside a multiplet; e signals are not totally separated; f represents 2H because R1 = H; g calculated by the difference from the other conformer.

Chemical shifts (ppm) and relative integration (between parentheses) of the main <span class="Chemical">hydrogen signals for the <class="Chemical">span class="Chemical">N-acylhydrazone derivatives 1 and 2a–d.

a cpd = compound; b 40 °C; c 20 °C; d inside a multiplet; e signals are not totally separated; f represents <span class="Chemical">2H because R1 = H; g calculated by the difference from the other conformer.

Thus, the decoplanarization between the class="Chemical">carbonyl and the aromatic ring (induced by the ortho-alkyl group) as well as the electron withdrawing effect exerted by the <class="Chemical">span class="Chemical">pyrimidine ring on the carbonyl group increase polarization. Both effects are disadvantageous in regard to the transition state between one conformer and the other. The transition state between amide rotamers is when the C-N bond is turned 90° and the nitrogen is not conjugated with the carbonyl (resulting in a pyramidalized N atom). As stated by Olsen and colleagues for nicotinamide and picolinamide, in the transition state, the π electrons from the aromatic ring can stabilize the resonance forms B, lowering the transition state energy and facilitating amide bond rotation (Figure 7). As shown, 2d and 2a are capable of coplanarizing their aromatic π electrons in the B canonical form with C+-O−. However, while this feature stabilizes the canonical form B1 in compound 2d, the recognized electron withdrawing effect of pyrimidine reduces stabilization (form B2 in compound 2a), leading to increases in the transition state energy and rotational barrier (Figure 7). This type of effect is supported by the observed 5.4 kcal/mol difference in the amide bond rotational barrier between nicotinamide and picolinamide [25]. Additionally, due to the more difficult coplanarization generated by ortho-alkyl groups, compounds 1 and 2c present a more energetic transition state B3 and thus a higher rotational barrier.

Figure 7

Resonance structure stabilization in the transition state of the amide bond rotamers.

The class="Chemical">13C-NMR class="Chemical">spectra support these assumptions, as the <class="Chemical">span class="Chemical">carbon from the carbonyl bond is deshielded by approximately 2 and 3 ppm after the introduction of an ortho-alkyl group or the replacement of the pyrimidine ring with a phenyl, respectively (Table 4).

Table 4

13C-NMR chemical shifts (ppm) of the carbonyl groups of NAH derivatives 1 and 2a–d.

cpd	Ring	Ortho group	δ C=O (ppm)
1	pyrimidine	Me	167.42
2a	pyrimidine	H	165.47
2b	pyrimidine	Et	169.75
2c	phenyl	Me	164.83
2d	phenyl	H	162.91

Resonance structure stabilization in the transition state of the <span class="Chemical">amide bond rotamers.

class="Chemical">13C-NMR chemical shifts (ppm) of the <class="Chemical">span class="Chemical">carbonyl groups of NAH derivatives 1 and 2a–d.

The higher class="Chemical">sp2 amide <class="Chemical">span class="Chemical">nitrogen character attributed to compounds 1 and 2a–d was evaluated using HMBC, which allowed us to distinguish between the aromatic carbons directly attached to a carbonyl that is cis and those attached to a carbonyl that is trans to the amide N-1H three bonds away. Due to the local bond constraints and planarity, two vicinal bond angles (0° and 180°) are present in the stable conformers. The HMBC correlations show that for compounds 1 and 2a–c, the signals of the amide N-1H of each conformer are coupled differently to the aromatic 13C (13C-CO-N-1H). Only the minor conformer, which has a trans correlation between these atoms, shows a 3JCH coupling (Table 5). These results agree with the literature showing a higher coupling for the trans coupling compared to cis [29,30]. On the other hand, compound 2d did not show a 3JCH coupling in the same experiments, possibly due to the minor sp2 amide nitrogen character and the longer length of the C-N bond, which results in a decrease in the rotational barrier compared to the other compounds (1, 2a–c). Because of this small barrier, 2d does not possess the fixed trans conformation necessary for 3JCH coupling in this series. In addition, 3JCH coupling of the amide N-1H and imine 13C=N was present in all compounds, indicating the similar chemical environments with no great differences in conformation for this moiety of NAH compounds.

Table 5

HMBC for NAH derivatives 1, 2a, 2c and 2d.

cpd	Long-range correlations for the amide N-H
	2 JCH (maj)	2 JCH (min)	3 JCH (maj)	3 JCH (min)
1	C=O	C=O	C=N	C=N, C-5
2a	C=O	C=O	C=N	C=N, C-5
2c	C=O	C=O	C=N	C=N, C-4
2d	C=O	a	C=N	a

a compound 2d presents only one signal.

<span class="Chemical">HMBC for <class="Chemical">span class="Chemical">NAH derivatives 1, 2a, 2c and 2d.

The Gibbs free energy of activation (∆G≠) related to the transition state among one conformer and another can be calculated using Equation 1. Through a dynamic NMR process, we could estimate the coalescence temperature (Tc), which provides, in association with the maximum peak separation (Δv in Hz) at low-temperature (i.e., slow exchange between conformers), the energy of the rotational barrier (∆G≠) [31]: ∆

The ∆G≠ values showed that the steric effect exerted by the <span class="Chemical">methyl group on the ring decoplanarization and the electron withdrawing effect of <class="Chemical">span class="Chemical">pyrimidine have similar effects on the rotational barrier because compounds 2a and 2c have almost the same ∆G≠ (Table 6).

Table 6

Results for restricted rotation about the CO-NH bond of NAH derivatives 1 and 2a–c.

cpd	Signal	Tc (K)	vc (Hz)	G≠ (kcal mol−1)
1	N H	353.15	52.0	17.4
2a	N H	313.15	16.52	16.1
2b	N H	356.15	64.3	17.4
2c	N H	313.15	12.12	16.3

Results for restricted rotation about the CO-NH bond of <span class="Chemical">NAH derivatives 1 and 2a–c.

A small additional increase in the barrier was observed when both effects are present, as observed for 1 and 2b, while no change was found when replacing the <span class="Chemical">methyl group with an ethyl group.

3. Experimental

3.1. General Procedures

NMR spectra were recorded on a 200/50 MHz Bruker DPX-200, 250/62.5 MHz Bruker DPX-250, 400/100 MHz Bruker DPX-400, 500/125 MHz Bruker Avance-500, 400/100 MHz Varian 400-Mr, 300/75 MHz Varian Unity-300 spectrometer. The spectra were performed in class="Chemical">DMSO-d and <class="Chemical">span class="Chemical">CDCl3 with TMS as internal standard. Chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz. Melting points were determined with a Quimis Q340.23 apparatus and are uncorrected. Elemental analyses were carried out on a Thermo Scientific Flash EA 1112 Series CHN-Analyzer. Thin layer chromatography was performed on Merck Kieselgel 60 HF 254 plates and detection took place using UV (254 and 365 nm). All reagents and solvents were purchased from commercial suppliers and used without further purification.

3.2. Ethyl 4-methyl-2-phenylpyrimidine-5-carboxylate (

In a round bottomed flask under class="Chemical">argon atmoclass="Chemical">sphere were successively added anhydrous <class="Chemical">span class="Chemical">ethanol (20 mL), sodium metal (0.745 g, 32.4 mmol), benzamidine hydrochloride (5.07 g, 32.4 mmol) and (E/Z)-ethyl 2-((dimethylamino)methylene)-3-oxobutanoate (6.00 g, 32.4 mmol). The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under vacuum and the resulting residue was filtered and washed with cold water. The title compound was obtained as a white crystalline solid in 80% yield. 1H-NMR (200 MHz, DMSO-d) δ 9.17 (s, 1H, H6het); 8.46–8.44 (m, 2H, H2′Ph, H6′Ph); 7.58–7.55 (m, 3H, H3′Ph, H4′Ph, H5′Ph); 4.36 (q, 2H, J = 8.0 Hz, OCH2); 2.80 (s, 3H, CH3het); 1.35 (t, 3H, J = 8.0 Hz, CH3Et). 13C-NMR (50 MHz, DMSO-d) δ 168.13 (C=O); 164.50 (C2het); 164.31 (C4het); 158.90 (C6het); 136.11 (C1′Ph); 131.76 (C4′Ph); 128.84 (C3′Ph, C5′Ph); 128.37 (C2′Ph, C6′Ph); 121.30 (C5het); 61.30 (OCH2); 24.28 (CH3het); 14.01 (CH3Et).

3.3. Methyl 4-Ethyl-2-Phenylpyrimidine-5-Carboxylate (

To a solution of class="Chemical">benzamidine hydrochloride (0.085 g, 0.540 mmol) and <class="Chemical">span class="Chemical">sodium hydroxide (0.022 g, 0.550 mmol) in methanol (2 mL), was added (E/Z)-methyl 2-((dimethylamino)methylene)-3-oxopentanoate (0.10 g, 0.540 mmol). The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The precipitate was filtered and washed with cold water, affording the title compound as a white amorphous solid in 73% yield. 1H-NMR (400 MHz, DMSO-d) δ 9.12 (s, 1H, H6het); 9.48–9.46 (m, 2H, H2′Ph, H6′Ph); 7.46–7.41 (m, 3H, H3′Ph, H4′Ph, H5′Ph); 3.89 (s, 3H, CH3het); 3.18 (q, 2H, J = 7.5 Hz, OCH2); 1.32 (t, 3H, J = 7.5 Hz, CH3Et). 13C-NMR (100 MHz, DMSO-d) δ 172.98 (C=O); 165.42 (C4het); 159.19 (C6het); 136.88 (C1′Ph); 132.50 (C4′Ph); 128.88 (C3′Ph, C5′Ph); 128.50 (C2′Ph, C6′Ph); 120.16 (C5het); 52.14 (OCH3); 29.84 (CH2); 12.36 (CH3).

3.4. Ethyl 2-phenylpyrimidine-5-carboxylate (

In a round bottomed flask under class="Chemical">argon atmoclass="Chemical">sphere were successively added anhydrous <class="Chemical">span class="Chemical">ethanol (5 mL), sodium metal (0.426 g, 18.5 mmol), benzamidine hydrochloride (2.90 g, 18.5 mmol) and diethyl 2-(ethoxymethylene)malonate (4.0 g, 18.5 mmol). The reaction mixture was stirred at room temperature for 9 h, and then poured into ice. The precipitate was filtered, washed with coldwater and recrystallized in EtOH-MeOH-CHCl3 (1:1:1) (45 mL), giving the ethyl 4-hydroxy-2-phenylpyrimidine-5-carboxylate (8) as a white crystalline solid in 50% yield. The derivative 8 (1.2 g, 4.91 mmol) and POCl3 (9.8 g, 63.9 mmol) were refluxed at 100oC for 1 h. The excess of POCl3 was removed under vacuum, ice was added on the resulting solid into the reaction flask, followed by filtration and washing with cold water. The chloride compound 9, obtained as a white amorphous solid in 98% yield, was successively dehalogenated with zinc powder (0.176 g, 2.69 mmol, 4 equiv) in anhydrous THF (2 mL). The reaction mixture was stirred at 60 °C for 1 h, and then 5 drops of acetic acid were added to the reaction vessel. After stirring at 60 °C for 23 h, the reaction mixture was cooled to room temperature, followed by addition of CH2Cl2 (3 mL), filtration and evaporation of solvent. Purification by silica gel column chromatography (n-hexane-EtOAc 0.6%) afforded the title compound as a white crystalline solid in 50% yield. Compound 8: 1H-NMR (200 MHz, DMSO-d/TMS) δ 12.22 (s, 1H); 8.64 (s, 1H); 8.16 (d, 2H, J = 6.0 Hz); 7.68–7.51 (m, 3H); 4.25 (q, 2H, J = 6.0 Hz); 1.28 (t, 3H, J = 6.0 Hz). 13C-NMR (62.5 MHz, DMSO-d) δ 163.9; 161.3; 160.1; 159.0; 133.0; 131.9; 129.1; 128.8; 115.0; 60.8; 14.5. Compound 9: 1H-NMR (200 MHz, CDCl3/TMS) δ 9.19 (s, 1H); 8.50 (dd, 2H, J = 2.2 Hz, 8.0 Hz); 7.61–7.46 (m, 3H); 4.46 (q, 2H, J = 7.0 Hz); 1.45 (t, 3H, J = 7.0 Hz). 13C-NMR (50 MHz, CDCl3/TMS) δ 166.57; 162.94; 160.61; 160.52; 135.32; 132.48; 129.29; 128.85; 121.61; 62.28; 14.23. Compound 4c: 1H-NMR (250 MHz, DMSO-d/TMS) δ 9.27 (s, 2H, H4het, H6het); 8.45–8.41 (m, 2H, H2′Ph, H6′Ph); 7.59–7.52 (m, 3H, H3′Ph, H4′Ph, H5′Ph); 4.36 (q, 2H, J = 7.5 Hz, OCH2); 1.33 (t, 3H, J = 7.5 Hz, CH3). 13C-NMR (63 MHz, DMSO-d/TMS) δ 166.30 (C=O); 163.84 (C2het); 158.67 (C4het, C6het); 136.49 (C1′Ph); 132.45 (C4′Ph); 129.37 (C3′Ph, C5′Ph); 128.90 (C2′Ph, C6′Ph); 122.28 (C5het); 61.87 (OCH2); 14.47 (CH3).

3.5. Methyl 3-methyl-biphenyl-4-carboxylate (

In a round bottomed flask into ice-bath, were added class="Chemical">methanol (15 mL), <class="Chemical">span class="Chemical">acetyl chloride (8.72 g, 111 mmol) and 4-bromo-2-methylbenzoic acid (1.0 g, 4.65 mmol). The reaction mixture was stirred at room temperature for 24 h, and then concentrated under reduced pressure. The resulting residue was extracted with CH2Cl2. The organic layer was washed with 10% sodium carbonate solution, dried over anhydrous Na2SO4, filtered and evaporated, giving the methyl 4-bromo-2-methylbenzoate 10 as a colorless oil in 86%, which was directly used in cross coupling reaction. The compound 10 (0.30 g, 1.31 mmol) was transferred to a round bottomed flask with a mixture of toluene-methanol (9:1, 4.5–0.5 mL), followed by addition of phenylboronic acid (0.192 g, 1.57 mmol), PdCl2(PPh3)2 (0.037 g, 4 mol %) and potassium carbonate (0.543 g, 3.93 mmol). The reaction mixture was stirred at 60 °C for 3 h, and after cooling to room temperature, was filtered over Celite. The resulting oil was purified by silica gel column chromatography (n-hexane-EtOAc 0%–1%), affording the title compound as a white crystalline solid in 91% yield, after evaporation under vacuum. Compound 10: 1H-NMR (200 MHz, DMSO-d/TMS) δ 7.73 (d, 1H, J = 8.0 Hz); 7.58 (s, 1H); 7.51 (d, 1H, J = 8.0 Hz); 3.82 (s, 3H); 2.49 (s, 3H). 13C-NMR (50 MHz, DMSO-d/TMS) δ 167.18; 142.31; 134.65; 132.51; 129.54; 129.16; 126.33; 52.58; 21.18. Compound 4d: 1H-NMR (200 MHz, CDCl3/TMS) δ 7.92 (d, 1H, J = 8.0 Hz, H5Ph); 7.55–7.51 (m, 2H, H2Ph, H6Ph); 7.40–7.25 (m, 5H, H2′Ph-H6′Ph); 3.83 (s, 3H, OCH3); 2.60 (s, 3H, CH3). 13C-NMR (50 MHz, CDCl3/TMS) δ 167.97 (C=O); 144.75 (C1Ph); 140.91 (C1′Ph); 140.14 (C3Ph); 131.36 (C5Ph); 130.49 (C2Ph); 128.95 (C3′Ph, C5′Ph); 128.30 (C4Ph); 128.09 C4′Ph); 127.33 (C2′Ph, C6′Ph); 124.47 (C6Ph); 51.89 (OCH3); 22.08 (CH3).

3.6. 4-Methyl-2-phenylpyrimidine-5-carbohydrazide (

To a solution of class="Chemical">ethyl 4-methyl-2-phenylpyrimidine-5-carboxylate (4a, 0.710 g, 2.92 mmol) in <class="Chemical">span class="Chemical">ethanol (5 mL) was added hydrazine monohydrate 100% (2.93 g, 2.84 mL, 58.4 mmol). The reaction mixture was stirred at 55 °C for 4 h. After cooling the reaction to room temperature, the round bottomed flask was placed in an ice-bath and the solid, filtered and washed with cold water. The title compound was obtained as a white crystalline solid in 83% yield. 1H-NMR (200 MHz, DMSO-d) δ 9.80 (s, 1H, NH); 8.76 (s, 1H, H6het); 8.44–8.39 (m, 2H, H2′Ph, H6′Ph); 7.56–7.52 (m, 3H, H3′Ph-H5′Ph); 4.62 (s, 2H, NH2); 2.63 (s, 3H, CH3). 13C-NMR (50 MHz, DMSO-d) δ 165.43 (C=O); 164.80 (C2het); 163.02 (C4het); 155.56 (C6het); 136.54 (C1′Ph); 131.16 (C4′Ph); 128.72 (C3′Ph, C5′Ph); 127.92 (C2′Ph, C6′Ph); 126.32 (C5het); 22.63 (CH3).

3.7. 4-Ethyl-2-phenylpyrimidine-5-carbohydrazide (

To a solution of class="Chemical">4b (0.191 g, 0.788 mmol) in <class="Chemical">span class="Chemical">ethanol (3 mL) was added hydrazine monohydrate 100% (0.789 g, 15.8 mmol). The reaction mixture was stirred at 50 °C for 5 h, cooled to room temperature and poured into ice. The solid was filtered and washed with cold water, giving the title compound as a white amorphous solid in 79% yield. 1H-NMR (400 MHz, DMSO-d) δ 9.77 (s, 1H, NH); 8.72 (s, 1H, H6het); 8.42–8.40 (m, 2H, H2′Ph, H6′Ph); 7.54–7.51 (m, 3H, H3′Ph, H5′Ph); 4.59 (s, 2H, NH2); 2.90 (q, 2H, J = 8.0 Hz, CH2); 1.27 (t, 3H, J = 8.0 Hz, CH3). 13C-NMR (125 MHz, DMSO-d) δ 170.12 (C=O); 165.40 (C4het); 163.66 (C2het); 156.09 (C6het); 137.23 (C1Ph); 131.71 (C4Ph); 129.31 (C3Ph, C5Ph); 128.44 (C2′Ph, C6′Ph); 126.68 (C5het); 28.62 (CH2); 13.14 (CH3).

3.8. 2-Phenylpyrimidine-5-carbohydrazide (

To a solution of class="Chemical">ethyl 2-phenylpyrimidine-5-carboxylate (4c) (0.080 g, 0.350 mmol) in <class="Chemical">span class="Chemical">ethanol (2 mL) was added hydrazine monohydrate 100% (0.351 g, 7.01 mmol). The reaction mixture was stirred at room temperature for 2 h. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a white amorphous solid in 85% yield. 1H-NMR (250 MHz, DMSO-d/TMS) δ 10.12 (s, 1H, NH); 9.20 (s, 2H, H4het, H6het); 8.43–8.39 (m, 2H, H2′Ph, H6′Ph); 7.55–7.52 (m, 3H, H3′Ph-H5′Ph); 4.64 (s, 2H, NH2). 13C-NMR (63 MHz, DMSO-d/TMS) δ 165.04 (C=O); 162.88 (C2het); 156.71 (C4het, C6het); 136.78 (C1′Ph); 131.99 (C4′Ph); 129.31 (C3′Ph, C5′Ph); 128.56 (C2′Ph, C6′Ph); 125.03 (C5het).

3.9. 3-Methyl-biphenyl-4-carbohydrazide (

To a solution of class="Chemical">methyl ester (4d) (0.60 g, 2.65 mmol) in <class="Chemical">span class="Chemical">ethanol (5 mL) was added hydrazine monohydrate 100% (3.98 g, 79.5 mmol). The reaction mixture was stirred at 50 °C for 15 h, and then concentrated under reduced pressure. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a white amorphous solid in 95% yield. 1H-NMR (200 MHz, DMSO-d/TMS) δ 9.46 (s, 1H, NH); 7.69–7.66 (m, 2H, H2Ph, H5Ph); 7.54–7.37 (m, 6H, H6Ph, H2′Ph-H6′Ph); 4.49 (s, 2H, NH2); 2.42 (s, 3H, CH3). 13C-NMR (50 MHz, DMSO-d/TMS) δ 168.85 (C=O); 141.68 (C1Ph); 140.04 (C1′Ph); 136.98 (C3Ph); 135.06 (C4Ph); 129.50 (C3′Ph, C5′Ph); 129.28 (C5Ph); 128.54 (C2Ph); 128.28 (C4′Ph); 127.26 (C2′Ph, C6′Ph); 124.25 (C6Ph); 20.08 (CH3).

3.10. Biphenyl-4-carbohydrazide (

To a solution of class="Chemical">methyl biphenyl-4-carboxylate (4e, 0.50 g, 2.36 mmol) in <class="Chemical">span class="Chemical">ethanol (5 mL) was added hydrazine monohydrate 100% (2.36 g, 47.1 mmol). The reaction mixture was stirred at 80 °C for 8 h, and then concentrated under reduced pressure. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a pale amorphous solid in 85% yield. 1H-NMR (200 MHz, DMSO-d/TMS) δ 9.85 (s, 1H, NH); 7.93 (d, 2H, J = 8.0 Hz, H3Ph, H5Ph); 7.77–7.69 (m, 4H, H2Ph, H6Ph, H2′Ph, H6′Ph); 7.52–7.36 (m, 3H, H3′Ph-H5′Ph); 4.55 (s, 2H, NH2). 13C-NMR (50 MHz, DMSO-d/TMS) δ 166.13 (C=O); 143.17 (C1Ph); 139.74 (C1′Ph); 132.66 (C4Ph); 129.57 (C3Ph, C5Ph); 128.55 (C4′Ph); 128.17 (C3′Ph, C5′Ph); 127.37 (C2Ph, C6Ph); 127.08 (C2′Ph, C6′Ph).

3.11. General Procedure to Synthesize the N-Acylhydrazones

To a solution of an aromatic class="Chemical">cabohydrazide (1.10 mmol) in <class="Chemical">span class="Chemical">ethanol (5 mL) were added HCl catalytic (2 drops) and the appropriate aromatic aldehyde. The reaction mixture was stirring at room temperature for ca 30 min, and then poured into ice. The precipitate was filtered, washed with cold water, petroleum ether and recrystallized in EtOH.

N′-(4-(Diclass="Chemical">methylamino)benzylidene)-<class="Chemical">span class="Chemical">4-methyl-2-phenylpyrimidine-5-carbohydrazide (1): The condensation of 4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) and 4-dimethylamino- benzaldehyde, afforded the title compound as a yellow solid (mp. 206 °C) in 99% yield. 1H-NMR (200 MHz, DMSO-d6) δ 11.79, 11.67 (2s, 1H, NH); 8.91, 8.81 (2s, 1H, H6het); 8.45–8.41 (m, 2H, H2′Ph, H6′Ph); 8.17, 7.98 (2s, 1H, N=CH); 7.56–7.54 (m, 5H, H3′Ph–H5′Ph, H2′′ap, H6′′ap); 7.24 (d, 2H, H2′′sp, H6′′sp, J = 8.7 Hz); 6.76, 6.64 (2d, 2H, H3′′, H5′′, J = 8.7 Hz, 9.0 Hz); 2.97, 2.88 (2s, 6H, N(CH)2); 2.66, 2.53 (2s, 3H, CH). 13C-NMR (50 MHz, DMSO-d6) δ 167.42, 165.96 (C=O); 164.37, 163.48 (C2het); 162.95, 161.59 (C4het); 156.37, 156.10 (C6het); 151.97, 151.73 (C4′′); 149.60, 145.96 (N=CH); 136.97, 136.84 (C1′Ph); 131.47, 131.34 (C4′Ph); 128.99 (C3′Ph, C5′Ph); 128.26 (C2′Ph, C6′Ph, C2′′, C6′′); 127.64, 126.72 (C5het); 121.09, 121.03 (C1′′); 111.73 (C3′′, C5′′); 39.87 (N(CH3)2); 23.02, 22.89 (CH3). Anal. Calcd for C21H21N5O: C: 70.18; H: 5.89; N: 19.48. Found: C: 70.17; H: 5.89; N: 19.62.

N′-(4-(Diclass="Chemical">methylamino)benzylidene)-<class="Chemical">span class="Chemical">2-phenylpyrimidine-5-carbohydrazide (2a): The condensation of 2-phenylpyrimidine-5-carbohydrazide (11c) and 4-dimethylaminobenzaldehyde, afforded the title compound as a yellow amorphous solid (mp. 269 °C) in 99% yield. 1H-NMR (300 MHz, DMSO-d/TMS) δ 11.79 (broad s, 1H, NH); 9.28 (s, 2H, H4het, H6het); 8.47–8.44 (m, 2H, H2′Ph, H6′Ph); 8.30, 8.01 (2s, 1H, N=CH); 7.57–7.55 (m, 5H, H3′Ph, H4′Ph, H5′Ph, H2′′, H6′′); 7.39 (d, 2H, H2′′, H6′′, J = 4.0 Hz); 6.77–6.69 (m, 2H, H3′′, H5′′); 2.97, 2.92 (2s, 6H, N(CH3)2). 13C-NMR (75 MHz, DMSO-d/TMS) δ 165.47, 165.33 (C=O); 164.66, 160.02 (C2het); 158.90, 157.43 (C4, C6); 152.45, 152.22 (C4′′); 150.42, 146.48 (N=CH); 137.11 (C1′Ph); 132.26 (C4′Ph); 129.54 (C3′Ph, C5′Ph); 129.35 (C2′′, C6′′); 128.90 (C2′Ph, C6′Ph); 126.70, 125.96 (C5het); 121.88 (C1′′); 112.50 (C3′′, C5′′); 40.56 (N(CH3)2). Anal. Calcd for C20H19N5O: C: 69.55; H: 5.54; N: 20.28. Found: C: 69.20; H: 5.54; N: 20.02.

N′-(4-(Diclass="Chemical">methylamino)benzylidene)-<class="Chemical">span class="Chemical">4-ethyl-2-phenylpyrimidine-5-carbohydrazide (2b): The condensation of 4-ethyl-2-phenylpyrimidine-5-carbohydrazide (11b) and 4-dimethylamino- benzaldehyde, afforded the title compound as a yellow solid (mp. 240 °C) in 85% yield. 1H-NMR (200 MHz, DMSO-d) δ 11.86, 11.74 (2s, 1H, NH); 8.90, 8.79 (2s, 1H, H6het); 8.47–8.43 (m, 2H, H2′Ph, H6’Ph); 8.15, 7.96 (2s, 1H, N=CH); 7.56–7.53 (m, 5H, H3′Ph–H5′Ph, H2′′, H6′′); 7.21 (d, 2H, H2′′, H6′′, J = 6.6 Hz); 6.75 (d, 2H, H3′′, H5′′, J = 6.6 Hz); 6.62 (d, 2H, H3′′, H5′′, J = 6.6 Hz); 2.99–2.94 (m, 8H, CH2, N(CH3)2); 2.87 (s, 6H, N(CH3)2); 2.81 (q, 2H, CH2, J = 7.5 Hz); 1.32–1.25 (m, 3H, CH3). 13C-NMR (50 MHz, DMSO-d) δ 169.75, 167.93 (C=O); 167.22, 163.24 (C4het); 162.65, 161.25 (C2het); 155.93, 155.80 (C6het); 151.61, 151.36 (C4′′); 149.18, 145.50 (N=CH); 136.81, 136.67 (C1′Ph); 131.15, 131.00 (C4′Ph); 128.70, 128.56 (C3′Ph, C5′Ph); 127.92, 127.08 (C2′′, C6′′); 126.19 (C2′Ph, C6′Ph); 121.03 (C1′′), 120.95 (C5het); 111.69 (C3′′, C5′′); 39.64, 39.46 (N(CH3)2); 28.30, 28.08 (CH2); 12.49, 12.03 (CH3). Anal. Calcd for C22H23N5O: C: 70.76; H: 6.21; N: 18.75. Found: C: 70.62; H: 6.19; N: 18.80.

(E)-N′-(4-(Diclass="Chemical">methylamino)benzylidene)-3-<class="Chemical">span class="Chemical">methylbiphenyl-4-carbohydrazide (2c): The condensation of 3-methyl-biphenyl-4-carbohydrazide (11d) and 4-dimethylaminobenzaldehyde, afforded the title compound as a yellow amorphous solid (mp. 238 °C) in 94% yield. 1H-NMR (300 MHz, DMSO-d/TMS) δ 11.41 (s, 1H, NH); 8.17, 7.94 (2s, 1H, N=CH); 7.72–7.67 (m, 2H, H2Ph, H5Ph); 7.58–7.35 (m, 8H, H6Ph, H2′Ph-H6′Ph, H2′′, H6′′); 7.22 (d, 2H, H2′′, H6′′, J = 8.4 Hz); 6.74, 6.62 (2d, 2H, H3′′, H5′′, J = 8.9 Hz, J = 8.4 Hz); 2.96, 2.87 (2s, 6H, N(CH3)2); 2.45, 2.33 (2s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d/TMS) δ 164.83 (C=O); 151.74, 151.48 (C4′′); 148.44, 144.70 (N=CH); 141.66, 140.62 (C1Ph); 139.69 (C1′); 136.84, 135.66 (C3Ph); 134.82 (C4Ph); 129.17, 129.06 (C3′Ph, C5′Ph, C2′′, C6′′); 128.63, 128.32 (C2′Ph, C6′Ph); 127.85 (C4′Ph); 126.97 (C2Ph, C5Ph); 124.03, 123.46 (C6Ph); 121.84 (C1′′); 112.03, 111.29 (C3′′, C5′′); 39.95 (N(CH3)2); 19.69 (CH3). Anal. Calcd for C23H23N3O: C: 77.28; H: 6.49; N: 11.76. Found: C: 76.92; H: 6.47; N: 11.59.

(E)-N′-(4-(Diclass="Chemical">methylamino)benzylidene)-<class="Chemical">span class="Chemical">biphenyl-4-carbohydrazide (2d): The condensation of biphenyl-4-carbohydrazide (11e) and 4-dimethylaminobenzaldehyde, following the general procedure for the synthesis of N-acylhydrazones, afforded the title compound as a yellow amorphous solid (mp. 289 °C) in 99% yield. 1H-NMR (300 MHz, DMSO-d/TMS) δ 11.58 (s, 1H, NH); 8.32 (s, 1H, N=CH); 7.98 (d, 2H, H3Ph, H5Ph, J = 8.0 Hz); 7.8 (d, 2H, H2Ph, H6Ph, J = 8.0 Hz); 7.74–7.72 (m, 2H, H2′Ph, H6′Ph); 7.55–7.47 (m, 4H, H2′′, H6′′, H3′Ph, H5′Ph); 7.42–7.38 (m, 1H, H4′Ph); 6.75 (d, 2H, H3′′, H5′′, J = 8.0 Hz); 2.96 (s, 6H, N(CH3)2). 13C-NMR (75 MHz, DMSO-d/TMS) δ 162.27 (C=O); 151.52 (C4′′); 148.64 (N=CH); 142.97 (C1Ph); 139.12 (C1′Ph); 132.53 (C4Ph); 129.02 (C3Ph, C5Ph); 128.42 (C3′Ph, C5′Ph); 128.14 (C2′′, C6′′); 126.85 (C2Ph, C6Ph, C4′Ph); 126.58 (C2′Ph, C6′Ph); 121.62 (C1′′); 111.81 (C3′′, C5′′). Anal. Calcd for C22H21N3O: C: 76.94; H: 6.16; N: 12.24. Found: C: 76.65; H: 6.15; N: 11.96.

3.12. (E)-N′-(4-(Dimethylamino)Benzylidene)-N,4-Dimethyl-2-Phenylpyrimidine-5-Carbohydrazide (

To a solution of class="Chemical">N-acylhydrazone 1 (0.14 mmol) and <class="Chemical">span class="Chemical">potassium carbonate (0.42 mmol) in acetone (5 mL) in an ice-bath was added methyl iodide (0.42 mmol). After, the reaction mixture was stirred at 43 °C for 24 h. The addition of 0.42 mmol of methyl iodide was needed, and then the reaction mixture was stirred at the same temperature for more 20 h. The reaction mixture was concentrated under vacuum, ethanol (1 mL) was added and the resulting mixture poured into ice. After filtering, the solid was heated into petroleum ether, and then refiltered to give the title compound as a yellow amorphous solid (mp. 188–189 °C) in 77% yield. 1H-NMR (300 MHz, DMSO-d/TMS) δ 8.73 (s, 1H, H6het); 8.46–8.42 (m, 2H, H2′Ph, H6′Ph); 7.94 (s, 1H, N=CH); 7.54–7.52 (m, 3H, H3′Ph-H5′Ph); 7.22 (d, 2H, J = 9.0 Hz, H2′′, H6′′); 6.61 (d, 2H, J = 9.0 Hz, H3′′, H5′′); 3.50 (s, 3H, N-CH3); 2.85 (s, 6H, N(CH3)2); 2.43 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d/TMS) δ 167.81 (C=O); 164.18 (C2het); 163.12 (C4het); 156.09 (C6het); 152.13 (C4′′); 143.46 (N=CH); 137.53 (C1′Ph); 131.65 (C4′Ph); 129.57 (C3′Ph, C5′Ph); 129.36 (C2′′, C6′′); 128.76 (C2′Ph, C6′Ph); 128.62 (C5het); 122.34 (C1′′); 112.50 (C3′′, C5′′); 40.32 (N(CH3)2; 28.92 (N-CH3); 23.29 (CH3). Anal. Calcd for C22H23N5O: C: 70.76; H: 6.21; N: 18.75. Found: C: 70.41; H: 6.05; N: 18.43.

3.13. 4-Methyl-2-Phenyl-N’-(Propan-2-Ylidene)Pyrimidine-5-Carbohydrazide (

A mixture of class="Chemical">4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) 0.07 g, 0.31 mmol) in <class="Chemical">span class="Chemical">acetone (7 mL) was stirred for 15 h at 45 °C. After, the solvent was evaporated to give a white solid in 85% yield (mp. 186–188 °C). 1H-NMR (400 MHz, DMSO-d/TMS) δ 10.9, 10.6 (2s, 1H, NH); 8.83, 8.70 (2s, 1H, H6het); 8.42–8.40 (m, 2H, H2′Ph, H6′Ph); 7.54–7.53 (m, 3H, H3′Ph-H5′Ph); 2.60, 2.48 (2s, 3H, CH3); 1.99, 1.91, 1.77 (3s, 6H, N=C-(CH3)2). 13C-NMR (125 MHz, DMSO-d/TMS) δ 168.57, 165.73 (C=O); 164.32, 163.47 (N=C); 162.97, 162.39 (C2het); 159.30, 153.61 (C4het); 156.47, 156.30 (C6het); 137.15, 137.12 (C1′Ph); 131.72, 131.60 (C4′Ph); 129.31, 129.25 (C3′Ph, C5′Ph); 128.43, 128.38 (C2′Ph, C6′Ph); 127.43 (C5het); 25.62, 25.56 (N=C-CH3); 23.14, 23.09 (CH3het); 18.55, 17.98 (N=C-CH3). Anal. Calcd for C15H16N4O: C: 67.15; H: 6.01; N: 20.88. Found: C: 66.93; H: 6.05; N: 20.52.

3.14. Molecular Modeling

The sketching, geometry optimization and conformational search of compounds were performed in PC Spartan Pro 1.0.5 software. They were subjected to structural minimization by the use of molecular mechanics, using the base MMFF. Subsequently, the conformational analysis was performed, using the semi-empirical <span class="Species">AM1.

The NOE prediction study was performed by selecting the minimum conformation of each stereoisomer (E/Z), and additionally the election of a second conformer (B) for the E isomer which ΔHf = 114.68 kcal/mol. Conformers that have the <span class="Chemical">carbonyl group <class="Chemical">span class="Gene">synperiplanar related to NH hydrogen were obtained by a dihedral angle search in 12 steps of 30° on the amide bond CO-NH, leaving the other angles free. Then we selected the lowest energy conformer, generated by the conformational analysis with the semi-empirical AM1 method, which presented the carbonyl synperiplanar related to the NH. The NOE calculations were carried out in the MSpin 1.03 software from the selected structures from the conformational analysis.

4. Conclusions

In this work, we revealed that duplicated signals observed in NMR spectra of <span class="Chemical">4-methyl-2-phenylpyrimidine-N-acylhydrazone correclass="Chemical">spond to the presence of two CO-NH bond- related <class="Chemical">span class="Gene">syn and antiperiplanar conformers. To our knowledge, this was the first description of conformers in aryl-NAH compounds. Our assumptions were based on NMR data from NOEdiff spectra of NH and phenyl H2’’/H6’’ irradiations, 2D-NOESY and dynamic 1H NMR and also by synthesis of isopropylidene hydrazone. The possibility of conformer’s observation rises from the increase of rotational barrier (∆G≠) among them, which results from both the decoplanarization of the aromatic ring and carbonyl group (induced by an ortho-alkyl group) and the electron withdrawing nature of the pyrimidine ring.