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

Article abstract of DOI:10.3390/molecules181011683

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 ΔG^≠ 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.

Full text of DOI:10.3390/molecules181011683

Molecules 2013, 18, 11683-11704; doi:10.3390/molecules181011683
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Characterization of Amide Bond Conformers for a Novel
Heterocyclic Template of N-acylhydrazone Derivatives
Alexandra Basilio Lopes 1,2, Eduardo Miguez 3, Arthur Eugen Kümmerle 4,
Victor Marcos Rumjanek 4, Carlos Alberto Manssour Fraga 1,2,5,* and Eliezer J. Barreiro 1,2,5,
*
1
Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio),
Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, P.O. Box 68.023,
Rio de Janeiro 21941-902, RJ, Brazil; E-Mail: alexandralopesb@yahoo.com.br
Programa de Pós-Graduação em Química, Instituto de Química,
2
3
Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949-900, RJ, Brazil
Instituto de Macromoléculas Professora Eloisa Mano,
Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão,
Rio de Janeiro CEP 21941-598, RJ, Brazil; E-Mail: emiguez@ima.ufrj.br
4
5
Departamento de Química, Instituto de Ciências Exatas,
Universidade Federal Rural do Rio de Janeiro, Seropédica 23.890-000, RJ, Brazil;
E-Mails: akummerle@hotmail.com (A.E.K.); victor.rumjanek@gmail.com (V.M.R.)
Programa de Pesquisa em Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, PO Box 68023, Rio de Janeiro 21941-902, RJ, Brazil
* Authors to whom correspondence should be addressed;
E-Mails: cmfraga@ccsdecania.ufrj.br (C.A.M.F.); ejbarreiro@ccsdecania.ufrj.br (E.J.B.);
Tel.: +55-21-2562-6644 (E.J.B.); Fax: +55-21-2562-6478 (C.A.M.F.).
Received: 4 August 2013; in revised form: 10 September 2013 / Accepted: 13 September 2013 /
Published: 25 September 2013
Abstract: 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

Molecules 2013, 18
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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 ΔG≠
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.
Keywords: N-acylhydrazones; pyrimidines; conformers; stereochemistry; NMR
1. Introduction
The bioactive N-acylhydrazone (NAH) moiety has been identified in a great number of lead
compounds that act on different types of molecular targets [1–4]. 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.
A research program to develop a series of 2-phenyl-4-methylpyrimidine-N-acylhydrazone
compounds with antinociceptive and anti-inflammatory activities led to the discovery of LASSBio-1083
(1, Figure 1), a promising lead compound that shows an ED50 of 27 µmol/Kg in the acetic acid-induced
1
mouse writhing test [6]. Regarding full structural characterization, the H-NMR spectra revealed the
duplication of some signals, indicating the presence of a mixture of stereoisomers or conformers.
1
Using H- and 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 [8–10], but
there is no description of this phenomenon in aryl NAHs in the literature. Because the complete

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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).
Figure 1. Stereoelectronic modifications of the NAH derivative LASSBio-1083 (1).
2. Results and Discussion
2.1. Chemical Synthesis
2.1.1. Synthesis of Esters 4a–e
Esters 4a and 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.
Reagents and conditions: (a) for 4a: benzamidine hydrochloride, Na/EtOH, rt,
2 h, 80% and 4b: benzamidine hydrochloride, NaOH/MeOH, rt, 1 h, 73%.

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Ethyl 4-hydroxy-2-phenylpyrimidine-5-carboxylate (8) was obtained in 50% yield by condensation
between 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.
Reagents and conditions: (a) benzamidine hydrochloride, Na/EtOH, rt, 9 h, 50%; (b) POCl3,
100 °C, 1 h, 98%; (c) Zn, AcOH, THF, 60 °C, 23 h, 50%.
The synthesis of 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.
Reagents and conditions: (a) phenylboronic acid, 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 synthesis of 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
1
Table 1). Analysis of the H-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

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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.
Reagents and conditions: (a) NH2NH2.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%.
Table 1. Synthesis of N-acylhydrazone and N-methyl-N-acylhydrazone derivatives 1–3.
Compound
X
N
N
N
C
C
N
R1
CH3
H
R3
H
Yield (%) a,b
Ratio a:b
2:1
1
66
21
49
81
84
51
2a
2b
2c
2d
H
4:1
CH2CH3
CH3
H
H
1.7:1
H
5:1
c
H
c
3a
CH3
CH3
a
b
b
Global yield; The analytical results for C, H and N were within 0.4% of the calculated values; Ratio
obtained by signal integration of the duplicate N=CH peaks in the 1H-NMR 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 1H-NMR spectrum of the analgesic lead compound 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).
Although only one amide signal at 11.67 ppm was selected for irradiation in the NOE experiments,
the 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

Molecules 2013, 18
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effect at a radius of 5 Å around the irradiated 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 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].
Figure 2. (A) 1H-NMR and (B) NOEdiff spectra for irradiation of NH (the major peak) of
NAH derivative 1.
(A)
(B)
The theoretical results for 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 to 7.57 ppm (Figure 3). The irradiation led again to two negative signals
corresponding 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%, respectively (Figure 3). Curiously, only

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the H2′′/H6′′ signal shifted at 7.55 ppm inside the multiplet, and no other 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).
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.
Table 2. Theoretical relative NOE obtained in MSpin software for the E and Z geometric
isomers of LASSBio-1083 (1).
Form
A
NH–N=CH
0.063
NH–H6
0.650
0.497
0.586
NH–H2′′ a
0.033
NH–H3′′
NH–H6′′ a
-
NH–CH3
0.015
-
-
B
0.746
0.006
0.014
0.022
0.018
C
0.064
0.811
0.023
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.
1
Figure 3. (A) Expanded H-NMR and (B) NOEdiff spectra for irradiation of H2”/H6”
(in minor proportion) of NAH derivative 1.
(A)

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Figure 3. Cont.
(B)
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
1
among multiple conformers in which a H 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 hydrogen of
NH could be 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
1
was used in place of DMSO-d6 for the H-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
1
for alkyl NAH compounds (Figure 4), we carried out a H-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.

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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).
A
B

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The resonance effect between the lone pair on the nitrogen sp2 atom and the 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 nitrogen sp2 hybridization to a 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 [22–25].
Wiberg et al. conducted theoretical studies concerning the 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 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 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-Methylation of 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)
1
with acetone. Its H-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.
1
Figure 6. H-NMR signal duplications and its proportions for the 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

Molecules 2013, 18
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attributed to imine hydrogen of conformer ap in 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 NAH compounds is
typically observed in those with a spacer between the 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 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
1
increases the steric effect. Both compounds showed duplicated signals in the H-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
1 b
CO-NH
H6
N=CH
H2′′/H6′′
7.55
H3′′/H5′′
N(CH3)2
R1
11.79 11.67 8.91
8.81 8.17 7.98
7.24 6.76
6.64
(34)
2.97 2.88 2.66 2.53
(66) (34) (66) (34)
2.99 2.95
(34)
11.91 11.87
(20) (80)
11.86 11.74 8.90
(37) (63) (63)
11.54 11.51 7.75–7.70
(17) (83)
11.60
(66)
(66)
(34)
(66) (34)
8.31 8.02 7.60–7.58 d
(66)
(34) (66)
7.4
2a c
2b b
9.31 e 9.29 e
6.78 e 6.73 e
-
-
(80) (20)
(80) g
7.56–7.53 d 7.21 6.75
(63) g
(37) (63)
8.17 7.95 7.61–7.39 d 7.23 6.77
(20)
(80) (20)
8.79 8.15 7.9
6.62
2.97 2.87
-
-
(37)
(63) (37)
(37) (63) g (37)
6.64
(17)
2.99 2.90 2.46 2.34
(83) (17) (83) (17)
2c c
multiplet
8.00 f
(83) (17)
(83) g
7.54–7.38 d
(17) (83)
2d b
8.34
6.77
2.98
8.00 f
a
b
c
d
e
f
cpd = compound; 40 °C; 20 °C; inside a multiplet; signals are not totally separated; represents 2H
because R1 = H; g calculated by the difference from the other conformer.

Molecules 2013, 18
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Thus, the decoplanarization between the carbonyl and the aromatic ring (induced by the ortho-alkyl
group) as well as the electron withdrawing effect exerted by the 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.
The 13C-NMR spectra support these assumptions, as the 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).
Figure 7. Resonance structure stabilization in the transition state of the amide bond rotamers.
Table 4. 13C-NMR chemical shifts (ppm) of the carbonyl groups of NAH derivatives 1 and 2a–d.
cpd
1
Ring
Ortho group δ C=O (ppm)
pyrimidine
pyrimidine
pyrimidine
phenyl
Me
H
167.42
165.47
169.75
164.83
162.91
2a
2b
2c
2d
Et
Me
H
phenyl

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The higher sp2 amide 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
13
N-1H of each conformer are coupled differently to the aromatic C (13C-CO-N-1H). Only the minor
3
conformer, which has a trans correlation between these atoms, shows a JCH coupling (Table 5).
These results agree with the literature showing a higher coupling for the trans coupling compared to
3
cis [29,30]. On the other hand, compound 2d did not show a JCH 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
3
13
series. In addition, JCH coupling of the amide N-1H and imine C=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.
Long-range correlations for the amide N-H
cpd
2JCH (maj) 2JCH (min) 3JCH (maj)
3JCH (min)
C=N, C-5
C=N, C-5
1
C=O
C=O
C=O
C=O
C=O
C=O
C=N
C=N
C=N
C=N
2a
2c
2d
C=O
a
C=N, C-4
a
a compound 2d presents only one signal.
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]:
∆G≠ = 4.57.10−3T (9.972 + log Tc/Δv)
(1)
The ∆G≠ values showed that the steric effect exerted by the methyl group on the ring decoplanarization
and the electron withdrawing effect of 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
1
Signal
NH
Tc (K)
353.15
313.15
356.15
313.15
νc (Hz)
52.0
G≠ (kcal mol−1)
17.4
16.1
17.4
16.3
2a
2b
2c
NH
16.52
64.3
NH
NH
12.12

Molecules 2013, 18
11696
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 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 DMSO-d6 and 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 (4a)
In a round bottomed flask under argon atmosphere were successively added anhydrous 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-d6) 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-d6) 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 (4b)
To a solution of benzamidine hydrochloride (0.085 g, 0.540 mmol) and 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,
1
affording the title compound as a white amorphous solid in 73% yield. H-NMR (400 MHz,
DMSO-d6) 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-d6) 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).

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3.4. Ethyl 2-phenylpyrimidine-5-carboxylate (4c)
In a round bottomed flask under argon atmosphere were successively added anhydrous 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%)
1
afforded the title compound as a white crystalline solid in 50% yield. Compound 8: H-NMR (200
MHz, DMSO-d6/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-d6) 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-d6/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-d6/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 (4d)
In a round bottomed flask into ice-bath, were added methanol (15 mL), 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
1
white crystalline solid in 91% yield, after evaporation under vacuum. Compound 10: H-NMR

Molecules 2013, 18
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(200 MHz, DMSO-d6/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-d6/TMS) δ 167.18; 142.31; 134.65; 132.51; 129.54;
1
129.16; 126.33; 52.58; 21.18. Compound 4d: H-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);
13
2.60 (s, 3H, CH3). C-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 (11a)
To a solution of ethyl 4-methyl-2-phenylpyrimidine-5-carboxylate (4a, 0.710 g, 2.92 mmol) in
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
1
compound was obtained as a white crystalline solid in 83% yield. H-NMR (200 MHz, DMSO-d6) 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-d6) 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 (11b)
To a solution of 4b (0.191 g, 0.788 mmol) in 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
1
compound as a white amorphous solid in 79% yield. H-NMR (400 MHz, DMSO-d6) 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-d6) δ
170.12 (C=O); 165.40 (C4het); 163.66 (C2het); 156.09 (C6het); 137.23 (C1′Ph); 131.71 (C4′Ph); 129.31
(C3′Ph, C5′Ph); 128.44 (C2′Ph, C6′Ph); 126.68 (C5het); 28.62 (CH2); 13.14 (CH3).
3.8. 2-Phenylpyrimidine-5-carbohydrazide (11c)
To a solution of ethyl 2-phenylpyrimidine-5-carboxylate (4c) (0.080 g, 0.350 mmol) in 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-d6/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-d6/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).

Molecules 2013, 18
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3.9. 3-Methyl-biphenyl-4-carbohydrazide (11d)
To a solution of methyl ester (4d) (0.60 g, 2.65 mmol) in 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-d6/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-d6/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 (11e)
To a solution of methyl biphenyl-4-carboxylate (4e, 0.50 g, 2.36 mmol) in 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-d6/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-d6/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 1, 2a–d
To a solution of an aromatic cabohydrazide (1.10 mmol) in 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-(Dimethylamino)benzylidene)-4-methyl-2-phenylpyrimidine-5-carbohydrazide
(1):
The
condensation of 4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) and 4-dimethylamino-
1
benzaldehyde, afforded the title compound as a yellow solid (mp. 206 °C) in 99% yield. H-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(CH3)2); 2.66,
13
2.53 (2s, 3H, CH3). C-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-(Dimethylamino)benzylidene)-2-phenylpyrimidine-5-carbohydrazide (2a): The condensation of
2-phenylpyrimidine-5-carbohydrazide (11c) and 4-dimethylaminobenzaldehyde, afforded the
1
title compound as a yellow amorphous solid (mp. 269 °C) in 99% yield. H-NMR (300 MHz,

Molecules 2013, 18
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DMSO-d6/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-d6/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-(Dimethylamino)benzylidene)-4-ethyl-2-phenylpyrimidine-5-carbohydrazide
(2b):
The
condensation of 4-ethyl-2-phenylpyrimidine-5-carbohydrazide (11b) and 4-dimethylamino-
1
benzaldehyde, afforded the title compound as a yellow solid (mp. 240 °C) in 85% yield. H-NMR
(200 MHz, DMSO-d6) 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-d6) 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-(Dimethylamino)benzylidene)-3-methylbiphenyl-4-carbohydrazide (2c): The condensation of
3-methyl-biphenyl-4-carbohydrazide (11d) and 4-dimethylaminobenzaldehyde, afforded the title
1
compound as a yellow amorphous solid (mp. 238 °C) in 94% yield. H-NMR (300 MHz,
DMSO-d6/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,
13
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). C-NMR
(75 MHz, DMSO-d6/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-(Dimethylamino)benzylidene)-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-d6/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);
13
2.96 (s, 6H, N(CH3)2). C-NMR (75 MHz, DMSO-d6/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.

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3.12. (E)-N′-(4-(Dimethylamino)Benzylidene)-N,4-Dimethyl-2-Phenylpyrimidine-5-Carbohydrazide (3a)
To a solution of N-acylhydrazone 1 (0.14 mmol) and 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
1
solid (mp. 188–189 °C) in 77% yield. H-NMR (300 MHz, DMSO-d6/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-d6/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 (12)
A mixture of 4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) 0.07 g, 0.31 mmol) in acetone
(7 mL) was stirred for 15 h at 45 °C. After, the solvent was evaporated to give a white solid in 85%
1
yield (mp. 186–188 °C). H-NMR (400 MHz, DMSO-d6/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-d6/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 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 carbonyl group 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.

Molecules 2013, 18
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4. Conclusions
In this work, we revealed that duplicated signals observed in NMR spectra of 4-methyl-2-
phenylpyrimidine-N-acylhydrazone correspond to the presence of two CO-NH bond- related 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
1
H2’’/H6’’ irradiations, 2D-NOESY and dynamic H 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.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/10/11683/s1.
Acknowledgments
The authors thank CAPES (BR), CNPq (BR), FAPERJ (BR) and INCT-INOFAR
(BR, #573.564/2008-6) for financial support and fellowships.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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