NMR structural study of the prototropic equilibrium in solution of schiff bases as model compounds

Article abstract of DOI:10.3390/molecules19010459

An NMR titration method has been used to simultaneously measure the acid dissociation constant (pK_a) and the intramolecular NHO prototropic constant δKNHO on a set of Schiff bases. The model compounds were synthesized from benzylamine and substituted ortho-hydroxyaldehydes, appropriately substituted with electron-donating and electron-withdrawing groups to modulate the acidity of the intramolecular NHO hydrogen bond. The structure in solution was established by ¹H-, ¹³C- and ¹⁵N-NMR spectroscopy. The physicochemical parameters of the intramolecular NHO hydrogen bond (pK_a, δKNHO and δδG°) were obtained from ¹H-NMR titration data and pH measurements. The Henderson-Hasselbalch data analysis indicated that the systems are weakly acidic, and the predominant NHO equilibrium was established using Polster-Lachmann d-diagram analysis and Perrin model data linearization.

Full text of DOI:10.3390/molecules19010459

Molecules 2014, 19, 459-481; doi:10.3390/molecules19010459
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
NMR Structural Study of the Prototropic Equilibrium in
Solution of Schiff Bases as Model Compounds
David Ortegón-Reyna 1, Cesar Garcías-Morales 2, Itzia Padilla-Martínez 3, Efren García-Báez 3,
Armando Aríza-Castolo 2, Ana Peraza-Campos 1 and Francisco Martínez-Martínez 1,*
1
Laboratorio de Posgrado, Facultad de Ciencias Químicas, Universidad de Colima, Km 9 Carretera
Colima-Coquimatlán, Colima 28400, Mexico; E-Mails: dortegon@ucol.mx (D.O.-R.);
peraza@ucol.mx (A.P.-C.)
2
Departamento de Química, Centro de Investigación y Estudios Avanzados del Instituto Politécnico
Nacional, Av. IPN 2508, San Pedro Zacatenco 07360, México D. F., Mexico;
E-Mails: cgarciam@cinvestav.mx (C.G.-M.); aariza@cinvestav.mx (A.A.-C.)
3
Departamento de Ciencias Básicas, Unidad Profesional Interdisciplinaria de Biotecnología del
Instituto Politécnico Nacional, Av. Acueducto s/n Barrio la Laguna Ticomán, México D. F. 07340,
Mexico; E-Mails: ipadillamar@ipn.mx (I.P.-M.); efren1003@yahoo.com.mx (E.G.-B.)
* Author to whom correspondence should be addressed; E-Mail: fjmartin@ucol.mx;
Tel./Fax: +52-312-316-1163.
Received: 28 October 2013; in revised form: 12 December 2013 / Accepted: 13 December 2013 /
Published: 31 December 2013
Abstract: An NMR titration method has been used to simultaneously measure the acid
dissociation constant (pKa) and the intramolecular NHO prototropic constant ΔKNHO on a
set of Schiff bases. The model compounds were synthesized from benzylamine and
substituted ortho-hydroxyaldehydes, appropriately substituted with electron-donating and
electron-withdrawing groups to modulate the acidity of the intramolecular NHO hydrogen
1
13
15
bond. The structure in solution was established by H-, C- and N-NMR spectroscopy.
The physicochemical parameters of the intramolecular NHO hydrogen bond (pKa, ΔKNHO
1
and ΔΔG°) were obtained from H-NMR titration data and pH measurements. The
Henderson–Hasselbalch data analysis indicated that the systems are weakly acidic, and the
predominant NHO equilibrium was established using Polster–Lachmann δ-diagram
analysis and Perrin model data linearization.
Keywords: Schiff bases; NHO prototropic tautomerism; NMR titration; δ-diagram

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1. Introduction
Schiff bases are a great topic of basic research, that to date have an important place in organic
chemistry and they have a great versatility in different fields of study. They have different biologic
applications as antitumor agents [1–5], in the strengthening of immune response for cancer, in
leukemia, in HIV, as anticonvulsant, antibacterials, antifungal, antiinflammatory, as prodrugs [6–15] and
as study models in the intramolecular hydrogen bond from cofactor pyridoxal-5-phosphate [16–20].
They are also of interest because of their solvatochromic, thermochromic and photochromic properties
with applications in optical recording technology, molecular electronics and photonics [21–30].
The Schiff bases derived from ortho-hydroxyaromatic aldehydes that are pentaconjugated
non-symmetric systems [31] in which proton transfer from the oxygen hydroxyl to the nitrogen of imine,
through the NHO hydrogen bond is observed (Scheme 1) have been extensively studied in recent
years [32–46].
Scheme 1. Prototropic 1,5 rearrangement in Schiff bases.
R1
+
H
R1
H
-
O
N
N
O
KNHO
R2
R2
In these investigations several analytical methods for determining the prototropic equilibrium have
been applied, such as FT-IR spectroscopy and X-ray diffraction in the solid state [45–48], as well as
solution 1H-, 13C- and 15N-NMR [33,34,49–51]. This 1,5 tautomeric equilibrium is directly affected by
the substituents [52–56] attached to both the phenyl group and the imine nitrogen which exert a strong
influence on the acidity of the OH group, the basicity of the nitrogen atom and thus the NHO bond
strength. Substituents also greatly increase the stability of the compounds by the effect of hydrogen
bonding assisted by resonance (RAHB) [57–59]; preferences have been found in the position of the
hydrogen either linked to oxygen (N···H-O) or nitrogen (N-H···O) [32,33,46,60] atoms and even being
in the middle of both (O−···H···N+) [60,61].
For a prototropic acid-base-system HA in equilibrium, its equilibrium constant Ka is expressed by
Equation (1), which after logarithms becomes the Henderson-Hasselbalch Equation (2):
[H ][A ]
(1)
Ka 
[HA]
[A ]
(2)
pH  pKa  log
[HA]
Equation (2) is directly related to the chemical shifts of active nuclei in NMR, which are dependent
on pH changes, this leads to Equation (3):
max  obs
obs  min
pH  pKa  log
(3)

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The pKa is experimentally obtained, using the tabulation of log[(δmax − δobs)/(δobs − δmin)] against
pH, where δmin and δmax are the chemical shifts in the inflection points in the titration curve, while δobs
is the observed chemical shift during the course of the titration, so the equilibrium point is at point
zero, which corresponds to pH = pKa [44,62–67]. This method has been extensively used because of
its simplicity, however is limited by variability in pH readings and accuracy in measuring the volumes
of the titrant.
Polster and Lachmann postulated the Gibbs triangle method, which later emerged as the absorbance
diagram (A-diagram) or chemical shift diagram (δ-diagram), depending on the spectrometry used for
the analysis of data from a titration, for the study of acid-base systems [62,68]. This method allows the
evaluation of the quotient of acidity constants (ΔKa) of two or more compounds, mainly in diprotic and
polyprotic acid-base systems [68], on the bases of a ratio of distances from the Gibbs triangle which is
independent of pH readings [68].
Later, Perrin et al. [69–72] also developed a mathematical model for the determination of ΔKa for
mixtures of isomers in equilibrium with independency from the pH readings by drawing δ-diagrams
also, so this model can be applied to the analysis of acid-base equilibrium mainly in monoprotic
systems. Then for two acids HA and HB, the quotient of their acidity constants ΔKa, can be measured
by the variation in chemical shifts due to changes in the acidity of the systems:
KaHA
[A ][HB]
K 

(4)
KaHB [HA][B ]
Equation (4), written in terms of chemical shifts when ΔKa ≠ 1, allows the evaluation of ΔKa as the
slope of a straight line, Equation (5):

b B


AH a

 K
a A BH b
 
(5)
where δA°, δB° are the chemical shifts from species at the start of the titration, δa, δb are the chemical shifts
observed during the titration, andδHA, δHB are the chemical shifts from species at the end of the titration.
In this contribution, both the Perrin and Polster-Lachmann models are applied to the study of
intramolecular hydrogen bonds that involve prototropic equilibrium with the aim to find with
accuracy and selectivity the position of the proton on the oxygen or nitrogen atoms. The model
compounds were a set of Schiff base derivatives of 5-nitrosalicylaldehyde, 5-chlorosalicylaldehyde,
5-bromo-salicylaldehyde, salicylaldehyde, 5-methoxysalicyladehyde and 5-hydroxysalicyladehyde
with benzylamine (compounds 1–6, Figure 1). The substituents were selected in order to cover a broad
range of both electrodonating (ED) and electrowithdrawing (EW) groups whose electronic effects could
modulate the NHO hydrogen bonding scheme. 1H-NMR spectrometry was used as the titration method.
Figure 1. Schiff base derivatives of 1–6.
7
3
15
9
4
2
R
14
13
N
10
11
8
5
1
OH
6
12
1 R = NO2 4 R = H
2 R = Cl 5 R = OCH3
3 R = Br 6 R = OH

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2. Results and Discussion
2.1. NMR Spectra
1
1
Synthesized compounds were identified by H-, 13C- and 15N-NMR. The H-NMR spectra of
compounds 1–6 in DMSO-d6 solution showed remarkable changes in the chemical shift of the acidic
proton NHO in the range of 12.53–14.34 ppm, in response to the electronic character of the substituent
R. Since a larger value in the chemical shift indicates a greater acidity of the proton, compound 1 has
the largest acidity and compound 6 has the lowest acidity. Simultaneously the chemical shift of protons
H3, H5, H6 and H7 were affected too.
13
The C spectra of all compounds showed clear shielding and deshielding effects, according to the
substituent, mainly from C1 to C7. The chemical shifts of compound 1 were more affected than those
of compounds 2–6, especially the carbon atoms C1 and C4. Compound 1, the NO2 derivative, showed
a chemical shift of 175.8 and 136.9 ppm for C1 and C4, respectively, where C1 is in the range of
carbonyl chemical shifts (170 to 200 ppm) while C4 is in the range of nitro Schiff base compounds
(130 to 150 ppm). The 15N chemical shift of compound 1 was −162.1 ppm, indicating an average
between imine-enamine forms, therefore in this last compound the zwitterionic structure (Scheme 2a)
is favored and the hydrogen H8 is localized with the nitrogen atom (+N–H···O).
Scheme 2. Possible resonance and equilibrium structures for compounds 1–6.
O
N
O
N
(a)
O
N
H
Ph
O
N
H
Ph
O
O
Zwitterionic
Keto
(b)
R
R
N
H
Ph
N
Ph
H
O
O
Zwitterionic
Neutral
R
R
N
H
Ph
N
Ph
H
O
O
o-Quinonoid
Keto
In the case of compounds 2–6 the chemical shifts of C1 appear at lower frequencies from 160.4 to
153.5 ppm, a region characteristic of OH structures (150–160 ppm) and the chemical shifts of the
15
imine C7=N appear from 165.8 to 167.4 ppm, a less significant variation. The N chemical shifts for
compounds 2–6 were in the range of −79.7 to −81.8 ppm (−50 to −90 ppm for imine), in agreement
with a neutral N···H–O tautomeric form with the hydrogen H8 is localized with the oxygen atom
(Scheme 2b). The NMR chemical shifts of compounds 1, 3, 4 and 5 have already been reported [73]

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and are in agreement with the above mentioned results, except for the nitro derivative 1 for which the
authors conclude that the N–H tautomer is present in solution instead of the zwitterion form
proposed herein.
2.2. NMR Titration
All compounds were titrated in CD3OD solution with NaOD, and only compound 2 was further
1
titrated with DCl. H-NMR spectra were recorded after each aliquot of titrant and simultaneously the
pH was measured following each recorded spectrum. The resonances of H6 and H9 were used to plot
pH vs. δ1H, because these protons were most affected by deprotonation of the labile hydrogen.
Compound 2 was initially titrated with DCl, however hydrolysis occured with the acid titrant and
1
only five H spectra and their corresponding pH readings could be recorded, so subsequently all
compounds were titrated with NaOD (Figure 2A).
Figure 2. (A) Titration curve of compounds 2–4; compound 2 was titrated with DCl; only
the titration region with NaOD was used to calculate the pKa values with the Henderson-
Hasselbalch equation; (B) δ-Diagram of δ1 vs. δ1 of compounds 2–4; the initial data
H6
H9
obtained was not linearized but after using the Perrin model the data became for linearized
compounds 2 (Cl), 3 (Br) and 4 (H) to obtain the slope ΔKNHO as shown in Scheme 3.
The Henderson-Hasselbalch equation was used to measure pKa values of the compounds by a
graphical method with plots of pH against log[(δH9 − δ obs)/(δ
− δ min)] (Figure 3) from the
H9
max
obs
H9
H9
titration curve, while ΔKNHO was obtained from the δ-diagram (Figure 2B) using the Perrin model
e
e
linearization [(δH9 − δH9°)(δH6 − δH6) vs. (δH6 − δH6°)(δH9 − δH9)] for compounds 2, 3, 4 and by Polster
and Lachmann analysis for compounds 1, 5 and 6. Table 1 summarizes the physicochemical
parameters obtained by the graphical methods mentioned above.

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Figure 3. The experimental pKa were found using the plot of the Henderson-Hasselbalch
equation, when log[(δH9max − δH9obs)/(δH9obs − δH9min)] = 0 then the pH intercept is the pKa value.
Table 1. Physicochemical parameters of compounds 1–6 at 296.15 ± 1 K in CD3OD solution.
Compound Ka/10−9 pKa
ΔKNHO
ΔpKNHO ΔΔG° [a]
−0.017 −0.097
1
2
3
4
5
6
15.1
2.13
1.44
3.33
1.23
0.17
7.8
1.04(±0.05)
8.6 1.031(±0.002) −0.0133 −0.075
8.8 0.986(±0.002)
0.006
0.036
0.426
8.4 0.841(±0.005) 0.0754
8.9 1.021(±0.014) −0.01
−0.052
−0.004
9.7
1.02(±0.02)
−0.001
[a] ΔΔG° = −RTlnΔKNHO (kJ mol−1 K−1).
The pKa values obtained by the Henderson-Hasselbalch equation for all compounds were greater
than 7 and less than 11, showing that these compounds are weak acids, the pKa value increases in the
order NO2 < H < Cl < Br < OMe < OH. Only compound 6 showed two pKa values, the value of 9.7
belongs to NHO and the second value of 9.8 to the phenolic hydroxyl group C4-OH. On the other hand
the ΔΔG° values, associated with the prototropic NHO equilibrium, are favored in the order
NO2 > Cl > OMe  OH > Br > H.
Figure 2 shows the titration curve with full pH scale (A) and the δ-diagram (B) of compounds 2 to 4;
only the data region titrated with NaOD was taken for the pKa value calculation. All compounds
should show the same shape of δ-diagram as they were titrated with NaOD. However, the titration
curves of compounds 2, 3 and 4 showed an almost linear behavior, whereas those of compounds 1 and 5
showed one inflection point and those of compound 6 two inflection points (see Supporting
Information). These results indicate that the initial structure of compounds 1–6, at the beginning of the
titration, was not the same in agreement with the NMR data discussed above.
On the other hand, the prototropic 1,5-rearrangement (Scheme 3) can be envisaged as composed by
two equilibria as depicted by in Figure 2. The quotient of the equilibrium constants KHN and KHO is
defined as ΔKNHO, corresponding to the equilibrium constant of the prototropic 1,5-tautomerism. The
chemical shifts of H6 and H9 are the most sensitive to changes in the equilibrium positions, thus they
were used as probes for KHO and KHN measurements, respectively.

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Scheme 3. Equilibria variation in the prototropic 1,5-tautomeric equilibrium. The quotient
of the equilibrium constants KHN and KHO is ΔKNHO
.

N

O
H
H
N
O
6
6
KHO
R
R
O
9
9

N

O
H
+
H
-
N
KHN
R
R
Thus, the ΔKNHO value allows one to establish the position of the NHO equilibrium. Therefore, if
the ΔKNHO value is equal to 1 then the system is in equilibrium Nδ+···H···Oδ− and both ΔpKNHO and
ΔΔG° are equal to zero; if the value of ΔKNHO is higher than 1, both ΔpKNHO and ΔΔG° are less than
zero and labile hydrogen is predominantly positioned on the N atom, +N–H···O; and finally if ΔKNHO is
less than 1 then ΔpKNHO and ΔΔG° are greater than zero and therefore the labile hydrogen is
predominantly positioned on the O atom, N···H–O.
Figure 4. Linearization of 1H chemical shifts from the δ-diagram for compounds 2, 3 and 4
e
e
by Perrin model. (δH9 − δH9°)(δH6 − δH6) = ΔKNHO (δH − δH6°)(δH9 − δH9), ΔKNHO is the
slope of the straight line. Compounds 2 (Cl) y = 1.031(±0.002)x + 8*10−5, R = 0.996;
3 (Br) y = 0.986(±0.002)x − 2*10−5, R = 0.994; 4 (H) y = 0.841(±0.005)x − 9*10−5, R = 0.983.
Compounds 2, 3 and 4 were treated with the Perrin model (Figure 4) for the nonlinear behavior in
the δ-diagram. In the case of compounds 1, 5 and 6 the Polster-Lachmann analysis seems to be more
appropriate, because of the shape of the Gibbs triangle taken in the δ-diagram (Figure 5).

Molecules 2014, 19
Figure 5. Polster-Lachmann analysis of the δ-diagram from compound 5. Point “A” shows
466
the initial state, point “B” indicates a change on the compound (neutralization point), point
“C” the final state and point “BC” is the experimental ΔKNHO of the system. The dotted line
A to C shows the shape of a triangle; the dotted line from vertex A to point BC shows the
intercept in an ΔKNHO equilibrium point.
The Polster-Lachmann analysis is based on a ratio of distances established by the Gibbs triangle and
the ΔKNHO values are obtained from the chemical shifts of the titration data, hence from points A, B, C
and BC in the δ-diagram, in agreement with Equation (6):
K HN (BC)

B


KNHO


(6)
K HO
(BC) C

From δ-diagrams, the mechanism occurring in the course of the titration with NaOD, can be
proposed (Scheme 4).
Scheme 4. Mechanism proposed during the titration with NaOD; the scheme is according
to points in the δ-diagram of Figure 5.
R
R
R
R
N
H
Ph
N
H
Ph
N
H
Ph
N
Ph
NaOD
p.p.
NaOD
-H+
Na
O
O
O
O
Point "A"
Initial state
Point "C"
Conjugate base
Point "B"
Neutral enol form
Compounds 1, 2, 5 and 6 begin at an initial state as acidic species (Point “A”), with a small increase
+
of pH, the intramolecular hydrogen bond equilibrium is shifted from N-H···O− to N···H-O, reaching
the neutralization point of the solution (Point “B”); then, as long as the pH is increased compounds are
deprotonated to become into the conjugated bases that precipitate as a salt (Point “C”). In the case of

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compounds 3 and 4, the initial state is at point “B” with the intramolecular hydrogen bond in the N···H-O
form, the addition of NaOD aliquots only shift the equilibrium to point “C” the conjugate base.
The δ-diagrams show the initial state in all compounds and indicate the most stable species in a
methanol solution, so the stability of the NHO intramolecular hydrogen bond is affected by the
electronic nature of the substituent as well as solvation of methanol; therefore, the structure of
compounds 1, 5 and 6 with NO2, OMe and OH substituents, respectively, stabilizes and direct the
NHO equilibrium position by both mesomeric and inductive effects, although they have different
ΔKNHO values, whereas the halogen substituent in compounds 2 and 3 exert both electronegative and
inductive effects; none of such effects are present in compound 4. Thus from the obtained ΔKNHO
values, the predominant NHO equilibrium in compounds 3 and 4 are the neutral N···H–O form, while
+
for the rest of the compounds the zwitterionic N–H···O− form is present (Scheme 2). In the
1
13
particular case of compound 1, it is as an imine-enamine tautomeric form in agreement with H, C
and 15N pfg-HMQC spectroscopy mentioned above.
Finally, the obtained ΔKNHO values (Table 1) are very close to the equilibrium point Nδ−···H+···Oδ−
(ΔKNHO = 1), which indicate a fast interchange of intramolecular hydrogen bond and the effect produced
by both the substituent and the solvent that stabilize the systems in a preferred tautomeric form.
3. Experimental
3.1. General Remarks
Schiff bases 1 to 6 were obtained by condensation of the appropriate aromatic ortho-hydroxyaldehyde
with benzylamine in toluene at 25 °C (Scheme 5). Solids products were filtered and dried under a
1
vacuum. Compound 4 was a liquid and the excess of toluene was eliminated under vacuum. H and
13C-NMR spectra were recorded in DMSO-d6 on a JEOL ECA-500 spectrometer (1H, 500.1 MHz;
15
15
1
13C, 125.7 MHz; N, 50.7 MHz) and the N chemical shifts were obtained by correlation of H,15N
pfg-HMQC (see Supporting Information).
Scheme 5. Synthesis of Schiff bases.
O
7
3
6
15
12
9
4
2
Toluene
Stir R. T.
R
R
14
13
N
H
NH2
10
11
8
+
5
1
OH
OH
1 R = NO2 4 R = H
2 R = Cl
3 R = Br
5 R = OCH3
6 R = OH
(E)-2-((Benzylimino)methyl)-4-nitrophenol (1). Compound 1 was prepared as reported [73] by
condensation of 5-nitrosalicylaldehyde (0.5 g, 2.99 mmol) with benzylamine (0.32 g, 0.32 mL, 2.99 mmol)
1
in toluene at room temperature (25 °C) and with a stirring time of 5 min. H-NMR (DMSO-d6):
δ = 8.42 (d, 4JH,H = 2.9 Hz, 1H, H3), 8.02 (dd, 3 JH,H = 9.5, 4JH,H = 3.0 Hz, 1H, H5), 6.65 (d, 3JH,H = 9.5 Hz,
1H, H6), 8.86 (s, 1H, H7), 4.84 (s, 2H, H9), 7.28–7.37 (m, 5H, H11-H15), 14.3 (broad signal, 1H,

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13
NHO) ppm. C-NMR (DMSO-d6): δ = 175.8 (s, 1C, C1), 115.0 (s, 1C, C2), 132.1 (s, 1C, C3), 136.9
(s, 1C, C4), 129.5 (s, 1C, C5), 122.2 (s, 1C, C6), 167.4 (s, 1C, C7), 57.2 (s, 1C, C9), 135.5 (s, 1C,
C10), 129.3 (s, 2C, C11, C15), 128.6 (s, 2C, C12, C14), 128.5 (s, 1C, C13) ppm. 15N-NMR (DMSO-d6):
δ = −162.1 (s, 1N, N8), −9.5 (s, 1N, N4) ppm.
(E)-2-((Benzylimino)methyl)-4-chlorophenol (2). Compound 2 was prepared by condensation of
5-chlorosalicylaldehyde (0.5 g, 3.19 mmol) with benzylamine (0.34 g, 0.34 mL, 3.19 mmol) in toluene
at room temperature (25 °C) and with a stirring time of 5 min. Yield 0.73 g (93%). m.p. 359–360 K.
FT-IR (ATR, cm−1): 1628 (C=N), 1573 (asymmetrical C=C-O-H stretch), 1479 (symmetrical C=C-O-H
stretch), 3069 (intramolecular hydrogen bonding N···H-O, as a weak broad band). LC-MS-TOF in
HPLC-methanol solution, m/z (%) calculated: 246.0686 (100); found: 246.0680 (100) [M+H]+,
empirical formula C14H13NOCl. 1H-NMR (DMSO-d6): δ = 7.53 (d, 4JH,H = 2.7 Hz, 1H, H3), 7.31 (dd,
3JH,H=8.7, 4JH,H = 2.7 Hz, 1H, H5), 6.87 (d, 3JH,H = 8.8 Hz, 1H, H6), 8.64 (s, 1H, H7), 4.76 (s, 2H, H9),
13
7.23–7.34 (m, 5H, H11-H15), 13.4 (broad signal, 1H, NHO) ppm. C-NMR (DMSO-d6): δ = 160.0
(s, 1C, C1), 120.2 (s, 1C, C2), 131.1 (s, 1C, C3), 122.5 (s, 1C, C4), 132.5 (s, 1C, C5), 119.0 (s, 1C,
C6), 165.8 (s, 1C, C7), 62.4 (s, 1C, C9), 138.8 (s, 1C, C10), 129.1 (s, 2C, C11, C15), 128.3 (s, 2C,
C12, C14), 127.7 (s, 1C, C13) ppm. 15N-NMR (DMSO-d6): δ = −80.5 (s, 1N, N8) ppm.
(E)-2-((Benzylimino)methyl)-4-bromophenol (3). Compound 3 was prepared as reported [73] by
condensation of 5-bromosalicylaldehyde (0.5 g, 2.48 mmol) with benzylamine (0.26 g, 0.27 mL, 2.48
1
mmol) in toluene at room temperature of 25 °C and with a stirring time of 5 min. H-NMR
4
3
4
(DMSO-d6): δ = 7.64 (d, JH,H = 2.5 Hz, 1H, H3), 7.41 (dd, JH,H = 8.7 and JH,H = 2.7 Hz, 1H, H5),
6.82 (d, 3JH,H = 8.7 Hz, 1H, H6), 8.62 (s, 1H, H7), 4.75 (s, 2H, H9), 7.22–7.33 (m, 5H, H11-H15), 13.5
13
(broad signal, 1H, NHO) ppm. C-NMR (DMSO-d6): δ = 160.4 (s, 1C, C1), 120.9 (s, 1C, C2), 134.0
(s, 1C, C3), 109.8 (s, 1C, C4), 135.3 (s, 1C, C5), 119.5 (s, 1C, C6), 165.7 (s, 1C, C7), 62.4 (s, 1C, C9),
138.8 (s, 1C, C10), 129.1 (s, 2C, C11, C15), 128.3 (s, 2C, C12, C14), 127.8 (s, 1C, C13) ppm.
15N-NMR (DMSO-d6): δ = −81.8 (s, 1N, N8) ppm.
(E)-2-((Benzylimino)methyl)phenol (4). Compound 4 was prepared as reported [73] by condensation of
salicylaldehyde (0.5 g, 0.42 mL, 4.09 mmol) with benzylamine (0.43 g, 0.44 mL, 4.09 mmol) in
1
toluene at room temperature (25 °C) and with a stirring time of 5 min. H-NMR (DMSO-d6):
δ = 7.44 (dd, 3JH,H = 7.5, 4JH,H = 1.7 Hz, 1H, H3), 6.87 (td, 3JH,H = 7.4, 3JH,H = 8.3 and 4JH,H = 0.9, 1H, H4),
7.30 (t, d, 3JH,H = 7.4, 3JH,H = 8.2 and 4JH,H = 1.7 Hz, 1H, H5), 6.85 (dd, 3JH,H = 8.2 and 4JH,H = 1.0 Hz, 1H,
H6), 8.67 (s, 1H, H7), 4.76 (s, 2H, H9), 7.23–7.34 (m, 5H, H11-H15), 13.4 (s, 1H, NHO) ppm.
13C-NMR (DMSO-d6): δ = 161.2 (s, 1C, C1), 119.2 (s, 1C, C2), 132.3 (s, 1C, C3), 119.1 (s, 1C, C4),
132.9 (s, 1C, C5), 117.0 (s, 1C, C6), 166.8 (s, 1C, C7), 62.7 (s, 1C, C9), 139.0 (s, 1C, C10), 129.0 (s,
15
2C, C11, C15), 128.2 (s, 2C, C12, C14), 127.6 (s, 1C, C13) ppm. N-NMR (DMSO-d6): δ = −81.7
(s, 1N, N8) ppm.
S1-5 (E)-2-((Benzylimino)methyl)-4-methoxyphenol (5). Compound 5 was prepared as reported [73] by
condensation of 5-methoxysalicylaldehyde (0.5 g, 0.40 mL, 3.28 mmol) with benzylamine (0.35 g,
0.35 mL, 3.28 mmol) in toluene at room temperature (25 °C) and with a stirring time of 5 min.
1H-NMR (DMSO-d6): δ = 7.04 (d, JH,H = 3.1 Hz, 1H, H3), 6.95 (dd, JH,H = 9.0 and JH,H = 3.1 Hz,
4
3
4

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3
1H, H5), 6.87 (d, JH,H = 9.0, 1H, H6), 8.58 (s, 1H, H7), 4.74 (s, 2H, H9), 7.22–7.34 (m, 5H,
13
H11-H15), 3.70 (s, 3H, H16), 12.8 (s, 1H, NHO) ppm. C-NMR (DMSO-d6): δ= 155.0 (s, 1C, C1),
119.0 (s, 1C, C2), 115.4 (s, 1C, C3), 152.2 (s, 1C, C4), 119.8 (s, 1C, C5), 117.7 (s, 1C, C6), 166.6 (s,
1C, C7), 62.9 (s, 1C, C9), 139.1 (s, 1C, C10), 129.0 (s, 2C, C11, C15), 128.2 (s, 2C, C12, C14), 127.6 (s,
1C, C13), 55.9 (s, 1C, C16) ppm. 15N-NMR (DMSO-d6): δ = −79.8 (s, 1N, N8) ppm.
(E)-2-((Benzylimino)methyl)benzene-1,4-diol (6). Compound 6 was prepared by condensation of
5-hydroxysalicylaldehyde (0.5 g, 3.62 mmol) with benzylamine (0.38 g, 0.39 mL, 3.62 mmol) in
toluene at room temperature of 25 °C and with a stirring time of 5 min. Yield 0.73 g (89%). m.p.
397–399 K. FT-IR (ATR, cm−1): 1641 (C=N), 1601 (asymmetrical C=C-O-H stretch), 1496
(symmetrical C=C-O-H stretch), 3311 (free phenolic O-H medium broad band), 3054 (intramolecular
hydrogen bonding N···H-O, as a weak broad band). LC-MS-TOF in HPLC-grade methanol, m/z (%)
calculated: 228.1025 (100); found: 228.1022 (100) [M+H]+, empirical formula C14H14NO2. 1H-NMR
(DMSO-d6): δ= 6.83 (d, 4JH,H = 3.0 Hz, 1H, H3), 6.76 (dd, 3JH,H = 8.8, 4JH,H = 3.0 Hz, 1H, H5), 6.68 (d,
3JH,H = 8.9, 1H, H6), 8.58 (s, 1H, H7), 4.74 (s, 1H, H9), 7.23–7.34 (m, 5H, H11-H15), 9.00 (s, 1H,
C4-OH), 12.5 (s, 1H, NHO) ppm. 13C-NMR (DMSO-d6): δ= 153.5 (s, 1C, C1), 119.1 (s, 1C, C2),
117.0 (s, 1C, C3), 149.9 (s, 1C, C4), 120.5 (s, 1C, C5), 117.4 (s, 1C, C6), 166.8 (s, 1C, C7), 62.8 (s,
1C, C9), 139.3 (s, 1C, C10), 129.1 (s, 2C, C11, C15), 128.3 (s, 2C, C12, C14), 127.6 (s, 1C, C13) ppm.
15N-NMR (DMSO-d6): δ= −79.7 (s, 1N, N8) ppm.
3.2. Sample Preparation, Titrant Solution and pH Meter
Solutions of compounds 1–6 (0.06–0.10 M) in CD3OD (0.4–0.5 mL) and 1,4-dioxane as internal
reference (0.5–1.5 μL, δH 3.53), were prepared in resonance tubes. The NaOD titrant solution, was
prepared to 1.4 and 4.8% (v/v) from NaOD/D2O (40%) in CD3OD, while the DCl solution was
prepared to 5% from DCl/D2O (70%) in CD3OD. The glass electrode was filled with a KCl standard
solution and calibrated with phosphate buffer pH 7.0 and 4.0.
3.3. NMR Spectrometric Titration
1
The H-NMR spectra were recorded in CD3OD on a JEOL ECA-500 spectrometer at room
1
temperature of 295.15 ± 1 K (22 ± 1 °C). An initial H-NMR spectrum of the solutions was recorded
and assigned as initial value for the titration. Subsequently the solutions were titrated with aliquots of
the NaOD/D2O solution base (3.0 μL), until invariant changes in the chemical shifts were observed;
1
each H-NMR spectrum as well as the corresponding pH reading were recorded simultaneously, after
the addition of the base. Only compound 2 was further titrated with DCl (5%), to observe the behavior
of the system at acidic pH.
3.4. NMR Titration Graphics (Figures 6–11)

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Figure 6. (A) titration curve (pH vs. δ1H9); (B) δ-diagram with Polster-Lachmann analysis
(δ1H6 vs. δ1H9) and (C) plot of the semilogarithmic Henderson-Hasselbalch equation
(pH vs. log[(δH9max − δH9obs)/(δH9obs − δH9min)]) of compound 1 (R = NO2).
pH δ1H6 δ1H9 pH δ1H6 δ1H9
5.72 6.661 4.481 8.05 6.481 4.666
6.57 6.664 4.781 8.09 6.483 4.666
7.50 6.630 4.788 8.14 6.474 4.660
7.69 6.572 4.724 8.15 6.471 4.660
7.81 6.549 4.710 8.15 6.445 4.641
7.91 6.503 4.685 8.16 6.410 4.619
7.95 6.506 4.681 8.78 6.408 4.619
8.03 6.498 4.676 9.90 6.407 4.618

7
3
6
15
12
9
4
2
O2N
14
13
N
8
10
11
5
OH
1

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Figure 7. (A) titration curve (pH vs. δ1H9); (B) δ-diagram with Perrin model analysis
471
(δ1H6 vs. δ1H9) and (C) plot of the semilogarithmic Henderson-Hasselbalch equation
(pH vs. log[(δH9max − δH9obs)/(δH9obs − δH9min)]) of compound 2 (R = Cl). Only compound 2
was titrated with DCl solution.
pH
δ1H6
6.777
6.757
6.733
6.731
6.717
6.717
6.693
6.669
6.643
δ1H9
4.793
4.729
4.702
4.697
4.679
4.677
4.668
4.659
4.649
pH
δ1H6
6.616
6.587
6.560
6.530
6.508
6.499
6.496
δ1H9
4.638
4.627
4.615
4.605
4.597
4.594
4.592
4.591
5.11
5.16
5.27
5.44
6.72
7.30
8.18
8.37
8.55
8.65
8.78
8.84
9.01
9.49
9.68
9.96
7
3
6
15
12
9
4
2
Cl
14
13
N
8
10
11
5
OH
1
10.05 6.494
Initial state of
compound 2
Titration region
with DCl
Titration region
with NaOD

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Figure 8. (A) titration curve (pH vs. δ1H9); (B) δ-diagram with Perrin model analysis
(δ1H6 vs. δ1H9) and (C) plot of the semilogarithmic Henderson-Hasselbalch equation (pH
vs. log[(δH9max − δH9obs)/(δH9obs − δH9min)]) of compound 3 (R = Br).
pH δ1H6
δ1H9
pH
δ1H6 δ1H9
7.79 6.667 7.269
8.53 6.639 7.234
8.67 6.608 7.194
9.48 6.474 7.027
9.69 6.455 7.004
9.94 6.450 6.998
7
3
6
15
12
9
4
2
Br
14
13
N
8
10
11
5
8.82 6.576 7.154 10.12 6.448 6.995
8.98 6.543 7.114 10.22 6.446 6.993
9.10 6.505 7.066
OH
1

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Figure 9. (A) titration curve (pH vs. δ1H9); (B) δ-diagram with Perrin model analysis
(1H6 vs. 1H9) and (C) plot of the semilogarithmic Henderson-Hasselbalch equation (pH vs.
log[(δH9max − δH9obs)/(δH9obs − δH9min)])of compound 4 (R = H).
pH
δ1H6
6.746
6.730
6.720
6.711
6.703
6.693
6.683
6.673
6.663
6.650
δ1H9
4.654
4.651
4.648
4.646
4.645
4.640
4.638
4.636
4.633
4.629
pH
δ1H6
6.640
6.628
6.617
6.607
6.598
6.591
6.586
6.581
6.576
δ1H9
4.626
4.624
4.619
4.617
4.613
4.611
4.610
4.608
4.606
7.66
7.93
8.23
8.39
8.44
8.50
8.56
8.72
8.93
9.13
9.32
9.51
9.76
7
3
6
15
12
9
4
2
H
14
13
9.90
N
8
10
11
10.14
10.41
10.52
10.54
10.87
5
OH
1

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Figure 10. (A) titration curve (pH vs. δ1H9); (B) δ-diagram with Polster-Lachmann
analysis (δ1H6 vs. δ1H9) and (C) plot of the semilogarithmic Henderson-Hasselbalch
equation (pH vs. log[(δH9max − δH9obs)/(δH9obs − δH9min)]) of compound 5 (R = OMe).
pH δ1H6 δ1H9 pH δ1H6 δ1H9
7.55 6.675 4.669 9.02 6.569 4.622
8.27 6.661 4.655 9.01 6.552 4.615
8.53 6.646 4.649 9.20 6.540 4.611
8.70 6.631 4.644 9.35 6.531 4.607
8.85 6.615 4.638 9.47 6.525 4.605
8.95 6.599 4.633 9.58 6.521 4.603
9.00 6.582 4.627 9.63 6.518 4.606
7
3
6
15
12
9
4
2
MeO
14
13
N
8
10
11
5
OH
1

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Figure 11. (A) titration curve (pH vs. δ1H5); (B) δ-diagram with Polster-Lachmann
475
analysis (δ1H6 vs. δ1H5) and (C) plot of the semilogarithmic Henderson-Hasselbalch
equation (pH vs. log[(δH5max – δH5obs)/(δH5obs – δH5min)])of compound 6 (R = OH)
calculating the pKa2. The pKa2 value belong to deprotonation of intramolecular hydrogen bond
N···H-O, whereas pKa3 correspond to free phenolic C4-OH. For this compound the plot of
δ-diagram was done with 1H6 vs. 1H5 chemical shifts, since only this correlation showed a
system with three slope changes similar to polyprotic system.
pH
δ1H6
6.603
6.602
6.601
6.596
6.595
6.590
6.587
6.583
6.580
6.577
6.573
δ1H5
6.690
6.689
6.686
6.684
6.683
6.681
6.678
6.676
6.673
6.670
6.667
pH
δ1H6
6.569
6.566
6.566
6.562
6.558
6.555
6.551
6.546
6.545
6.539
6.534
δ1H5
6.664
6.662
6.662
6.659
6.655
6.653
6.650
6.646
6.644
6.640
6.635
6.41
8.85
9.58
9.76
9.80
9.83
9.84
9.85
9.84
9.68
9.68
9.69
9.77
9.92
7
3
6
15
12
9
9.99
4
2
HO
14
13
N
8
10
11
10.04
10.04
10.03
10.02
10.02
10.03
10.01
5
OH
1

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3.5. Data Analysis
The calculations were performed on a Microsoft Excel worksheet. The data analysis of the
semilogarithmic Henderson-Hasselbalch (Equation (3)) was applied to data that have adapted to this
analysis method, using a dependent variable log[(δHmax − δHobs)/(δHobs − δHmin)]; H9 for compounds 1–5
and H5 for compound 6) (HX = H5 or H9) and pH as an independent variable for titration curves to
find the pKa values.
Data analysis for the δ-diagram by both Perrin and Polster-Lachmann analysis used H6 and H9
proton chemical shifts for the analysis in δ-diagram, since they are the proton chemical shifts adjacent
to NHO intramolecular hydrogen bond and the most affected by deprotonation. Thus in the Perrin
Analysis Equation (5) can be written as follows (Equation (7)):

H 9 H 9

H 6e H 6

 KNHO

H 6 H 6

H 9e H 9

(7)
where δH9° and δH6° are the chemical shifts from the species at the beginning of the titration, δH9 and
δH6 the chemical shifts observed in the course of the titration, δH9e and δH6e are the chemical shifts from
species at the end of the titration. Finally in the Polster-Lachmann analysis, the ratio of distances to
calculate the ΔKNHO value is established by the graphic method described by the Gibbs triangle [62,68].
4. Conclusions
The study of compounds 1–6 by NMR titration in methanol solution, confirmed the predominant
tautomeric forms in solution, noting that the NHO prototropic equilibrium is dependent of the
substituent and the solvent. The pKa values obtained using the Henderson-Hasselbalch analysis showed
that all compounds are weak acids. The strength and lability of the NHO intramolecular hydrogen
bond are consequently affected by the mesomeric and inductive effects exerted by the substituents. The
values of the KNHO equilibrium constant indicate that the equilibrium is slightly shifted to the nitrogen
atom when the substituent in the phenyl ring exerts a strong electronic effect, either ED or EW
(R = NO2, Cl, OMe and OH), and to the oxygen atom when Br or H in CD3OD solutions. Nevertheless
the ΔKNHO values close to the unit, highlight that the proton is in the middle of both basic sites
(O−···H···N+), in contrast to what is found in DMSO-d6 solutions, where NMR data is in agreement with
the neutral N···H–O tautomer for most of the compounds except for the nitro derivative which is in the
zwitterion +N–H···O form. Finally, we have demonstrated the simplicity, accuracy and versatility of both
the Perrin and Polster-Lachmann analysis applied to the study of intramolecular hydrogen bonds.
Acknowledgments
The authors are grateful for financial support from Conacyt (Grants 204648), FRABA Universidad
de Colima (797/12) and PROMEP. ORD is greatfully acknowledged to Conacyt grant number 209273
for PhD formation.
Conflicts of Interest
The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–6 are available from the authors.
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