Determination of pKa values of some novel benzimidazole salts by using a new approach with 1H NMR spectroscopy

Article abstract of DOI:10.1002/mrc.4294

Benzimidazoles and their derivatives including imidazole are studied widely because they exist in the structure of natural products and different drugs. pK_a values are extremely important for drug discovery and improvement in order to determine pharmacokinetic and pharmacodynamic features such as permeation through biological barriers, interactions with the target area or side effects. Acid-base features (pK_a) have great importance not only for physiological characteristics but also for being used as a ligand or changing physico-chemical features by turning benzimidazoles into salts. Within the scope of this study, a variety of new benzimidazole salts were synthesized, and their characterizations were made by NMR spectroscopy, FTIR spectroscopy and element analysis techniques. The pK_a values of synthesized benzimidazole salts were determined by inflection point approach using integration values obtained with ¹H NMR spectroscopy and Henderson-Hasselbalch analysis. pK_a values of some benzimidazole salts were also determined by potentiometric methods in order to compare those of NMR spectroscopy results.

Full text of DOI:10.1002/mrc.4294

Research article
Received: 27 May 2015
Revised: 12 June 2015
Accepted: 18 June 2015
Published online in Wiley Online Library: 10 August 2015
(wileyonlinelibrary.com) DOI 10.1002/mrc.4294
Determination of pKa values of some novel
benzimidazole salts by using a new approach
1
with H NMR spectroscopy
Akın Mumcua,b and Hasan Küçükbaya*
ABSTRACT: Benzimidazoles and their derivatives including imidazole are studied widely because they exist in the structure of nat-
ural products and different drugs. pKa values are extremely important for drug discovery and improvement in order to determine
pharmacokinetic and pharmacodynamic features such as permeation through biological barriers, interactions with the target area
or side effects. Acid–base features (pKa) have great importance not only for physiological characteristics but also for being used as
a ligand or changing physico-chemical features by turning benzimidazoles into salts. Within the scope of this study, a variety of
new benzimidazole salts were synthesized, and their characterizations were made by NMR spectroscopy, FTIR spectroscopy
and element analysis techniques. The pKa values of synthesized benzimidazole salts were determined by inflection point approach
using integration values obtained with 1H NMR spectroscopy and Henderson–Hasselbalch analysis. pKa values of some benzimid-
azole salts were also determined by potentiometric methods in order to compare those of NMR spectroscopy results. Copyright ©
2015 John Wiley & Sons, Ltd.
Keywords: benzimidazole derivatives; determination of pKa; 1H NMR spectroscopy; potentiometric titration
benzimidazoles not only for physiologic characteristics but also for
being used as a ligand or for changing their physico-chemical features
Introduction
Nuclear magnetic resonance (NMR) is a powerful and common tool
used in a variety of disciplines, particularly to identify chemical
structures of compounds in synthetic chemistry.[1] NMR can also
be used to determine pKa value of weak acidic compounds and
to calculate the product yields comparing the peaks integral.
Acid–base properties are an important issue in living systems due
to many molecules working at certain pH values. On the other
hand, many heteroaromatic compounds take part in biological pro-
cesses such as providing energy, neurotransmission, metabolism
and genetic data, which are the primary sources of life.[2] Among
the heterocyclic compounds, benzimidazoles are interesting heterocy-
cles because they are present in many naturally occurring products
and various drugs.[3] They are also an important pharamcophore in
modern drug discovery and continue to be the most versatile class of
compounds possessing different pharmacological activities such as
antitumor,[4] anti-ulcer,[5] fungicidal,[6] bactericidal[7] and anthelmintic.[8]
Chemical stabilities and physiological activities are among the most im-
portant features of benzimidazole derivatives. Most of the physico-
chemical features are determinant in the transformation of drug candi-
datecompounds suchas benzimidazoles into pharmaceutical products.
For instance, many drug candidates fail because of low solubility or low
determination. In the discovery of new drugs, prevalently, this type of
failure is disposed without changing the structure of drug candidate
compounds with the method of changing the physico-chemical fea-
tures by means of salt formation.[9] Acid–base feature is one of the
key parameters in the development of drug candidates. The reason is
that this feature controls the solubility, absorption, distribution and elim-
ination. Also, when a drug candidate is being carried through the body,
transition between and inside the cell is a function of physico-chemical
parameters.[10] Acid–base (pKa) features have a vital importance for
by turning them into salts. pKa values play an important role in many
research fields such as the stereochemical and conformational structure
determination, orientation of nucleophilic and electrophilic attacks, the
stabilities of intermediates, the magnitude of activation energy in
organic reactions and the detection of active centers of biochemical
enzymes.[2,11] Besides these, pKa values are important thermodynamic
parameters for biochemical interactions, industrial processes or the
environmental fate of polyprotic molecules.[12] A variety of methods
are used for the determination of pKa.[10] Potentiometry and UV–Visible
spectrophotometry (VIS) are the most practical methods for the
determination of equilibrium constants because of accuracy and
repeatability.[13–16] In recent years, alternative techniques depending
on separation techniques such as liquid chromatography (LC) and
capillary electrophoresis (CE) have been developed. In these methods,
a physical feature of the analyte is measured as a function of the
solution’s pH, and the obtained data are used in the determination of
dissociation constants (Ka, pKa).[17,18] Also, pKa values can be determined
by the procedure using LC and CE methods combined with a diode-
array detector for the absorption measurements. In this case, the pKa
values are determined by the absorbance spectrum observed in the
*
Correspondence to: H. Küçükbay, Department of Chemistry, Faculty of Arts and
Sciences, İnönü University, Malatya 44280, Turkey. E-mail: hasan.kucukbay@
inonu.edu.tr
a Department of Chemistry, Faculty of Arts and Sciences, İnönü University, Malatya
44280, Turkey
b The Scientific and Technological Research Center, İnönü University, Malatya
44280, Turkey
Magn. Reson. Chem. 2015, 53, 1024–1030
Copyright © 2015 John Wiley & Sons, Ltd.

pKa determination using new approches in NMR
situation of the biggest chromatographic or electrophoretic
peaks.[19] Moreover, pKa values can be predicted by the computa-
tional methods on the basis of the molecular structure.[20] NMR
spectroscopy is an important alternative method in order to de-
termine pKa values. This method is a technique about NMR active
nucleus chemical shift value for the protonation of adjacent
center.[21] In the determination of pKa, special approaches based
on 31P, 15N, 19F or 33S NMR spectroscopy are also used.[12,22–24]
However, pH-dependent chemical shift values of 13C and especially
1H are more common, and they are used successfully in determin-
ing pKa values.[25,26] The pKa determination with NMR spectroscopy
is based on analysis of protonation degree of a molecule with
changing the pH. The protonation degree is monitored with the
chemical shift changes close to the acidic or basic regions of
molecules.[27] NMR titration observes every nucleus in a compound,
and in this way, the protonation area is specified clearly.[28] Due to
low signal/noise ratio for many nuclei, the pKa studies have concen-
Scheme 1. Synthesis pathways of the new benzimidazole derivatives.
bromide, VII were synthesized according to literature method[33]
(Scheme 1).
1
trated on H NMR.[27] In the studies, the chemical shifts obtained
Synthesis of 1-benzyl-3-[2-(4-methoxyphenyl)ethyl]benzimida-
zolium chloride, 1
with NMR spectrums were analyzed with inflection-point graphical
analysis and Henderson–Hasselbalch method.[29,30]
The aim of this study is to synthesize new benzimidazole salts
and determine the pKa values by using a newly developed NMR-
supported method.
A mixture of 1-benzylbenzimidazole (III) (1.50 g, 7.20 mmol) and 2-(4-
methoxyphenyl)ethyl chloride (1.13 ml, 7.20 mmol) in dimethyl-
formamide (3 ml) was refluxed for 5 h. The mixture was then
cooled, and volatiles were removed under vacuum. The residue
was crystallized from ethanol/diethyl ether (1 : 5). White crystals
of the title compound 1 (1.65 g, 60%) were obtained, mp
173–175 °C; 1H NMR (DMSO-d6, 300 MHz) δ 9.89 (s, 1H, NCHN),
8.12–7.31 (m, 9H, C6H4, C6H5), 7.11–6.79 (AA’BB’ system, 4H,
CH2CH2C6H4), 5.74 (s, 2H, CH2C6H5), 4.79 (t, 2H, CH2CH2C6H4,
J = 6.90 Hz), 3.70 (s, 3H, OCH3), 3.21 (t, 2H, CH2CH2C6H4,
J = 6.90 Hz).13C NMR (DMSO-d6, 300 MHz) δ 142.7 (NHCN), 158.6,
134.3, 131.6, 131.1, 130.3, 129.4, 129.1, 128.9, 128.4, 127.2, 127.1,
114.4 and 114.2 (C6H4, CH2C6H5, CH2CH2C6H4), 55.5 (OCH3), 50.1
(CH2C6H5), 48.5 (CH2CH2C6H4), 33.9 (CH2CH2C6H4). IR υmax (ATR)
1559 cmꢀ1 (CN). Anal. Calculated for C23H23N2OCl.H2O (396.91):
C, 69.60; H, 6.35; N, 7.06. Found: C, 68.96; H, 6.39; N, 7.04.
Experimental section
All NMR experiments were performed on a Bruker Avance 300 MHz
spectrometer (Bruker Billerica-Massachusetts, USA) equipped with a
5-mm BroadBand Observe (BBO) probehead with Z gradients at a
temperature of 298 K. The following parameters were used to
record the 1H NMR spectra in pKa determination experiments: pulse
90 °, relaxation delay 1 s, number of scans 64 and resolution
0.01 ppm per point. Computer processing was performed with
Bruker TOPSPIN 2.1 software (Bruker). Infrared spectra were recorded
with attenuated total reflection (ATR) equipment in the range
4000–650 cmꢀ1 on a Perkin Elmer spectrum one FTIR spectropho-
tometer (Perkin Elmer, Waltham-Massachusetts, USA). Elemental
analyses were performed by Leco CHNS-932 elemental analyzer
(Leco Corporation, St. Joseph, Michigan, USA). Melting points were
recorded using an electrothermal-9200 melting point apparatus
and are uncorrected. The pH values of the solutions at 298 K were
measured using a glass electrode (Hanna HI 221 microprocessor).
The same temperature was used for the NMR experiments.
General method for the synthesis of compounds 2–5
Equivalent amount of the 1-[2-(4-methoxyphenyl)ethyl]benzimid-
azole (I) and appropriate alkyl halide were refluxed in dimethyl-
foramide (3ml) for 5 h. Then, the mixture was cooled to room
temperature, and the volatiles were removed under reduced pres-
sure. The residue was crystallized from ethanol/diethyl ether (1: 5).
The starting materials and reagents used in the new
benzimidazolium salts synthesis were supplied commercially by
Aldrich, Acros, ABCR and Merck. Deuterated d6-dimethyl sulfoxide
(DMSO-d6), DMSO, isopropanol and tetrabutylammonium hydrox-
ide solution (TBAH) were purchased from Merck. 3-(trimethylsilyl)-
1-propanesulfonic acid sodium salt (DSS) was obtained from Sigma.
1-[2-(4-methoxyphenyl)ethyl]-3-methylbenzimidazolium iodide, 2
Yield, 2.51 g (white crystals), 64%; mp 160–161 °C; 1H NMR (DMSO-
d6, 300 MHz) δ 9.68 (s, 1H, NCHN), 8.06–7.64 (m, 4H, C6H4), 7.19–6.83
(AA’BB’ system, 4H, CH2CH2C6H4), 4.72 (t, 2H, CH2CH2C6H4,
J = 7.17 Hz), 4.08 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 3.18 (t, 2H,
CH2CH2C6H4, J = 7.17Hz).13C NMR (DMSO-d6, 300 MHz) δ 143.1
(NHCN), 158.7, 132.1, 131.3, 130.4, 129.2, 126.9, 114.5, 114.1 and
114.0 (C6H4, CH2CH2C6H4), 55.5 (OCH3), 48.4 (CH2CH2C6H4), 34.3
(CH2CH2C6H4), 33.8 (CH3). IR υmax (ATR) 1573 cmꢀ1 (CN). Anal. Calcu-
lated for C17H19N2OI (394.25): C, 51.79; H, 4.86; N, 7.11. Found: C,
50.84; H, 4.89; N, 7.15.
Synthesis of benzimidazolium salts
1-[2-(4-Methoxyphenyl)ethyl]benzimidazole (I), 1-(phthalimide-2-yl)
methylbenzimidazole (II), 1-benzylbenzimidazole (III), 5(6)-methyl-
1-(3-phenylpropyl)benzimidazole (IV) and 5(6)-nitro-1-(3-phenyl
propyl)benzimidazole (V) used in this work as a starting compound
were prepared by treating benzimidazole and 2-(4-methoxyphenyl)
ethyl chloride, (phthalimide-2-il)methyl chloride, benzyl chloride
and 3-bromopropylbenzene, respectively, similar to the literature
procedure.[31,32] 5(6)-Methyl-1,3-di(3-phenylpropyl)benzimidazolium
bromide, VI and 5(6)-Nitro-1,3-di(3-phenylpropyl)benzimidazolium
1-(4-chlorobenzyl)-3-[2-(4-methoxyphenyl)ethyl]benzimidazo-
lium chloride, 3
Yield, 2.77 g (white crystals), 85%; mp 154–156 °C; 1H NMR (DMSO-
d6, 300MHz) δ 10.09 (s, 1H, NCHN), 8.15–7.41 (m, 8H, C6H4,
CH2C6H4Cl), 7.14–6.80 (AA’BB’ system, 4H, CH2CH2C6H4), 5.79
Magn. Reson. Chem. 2015, 53, 1024–1030
Copyright © 2015 John Wiley & Sons, Ltd.
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A. Mumcu and H. Küçükbay
(s, 2H, CH2C6H4Cl), 4.79 (t, 2H, CH2CH2C6H4, J = 7.05 Hz), 3.71 (s, 3H,
OCH3), 3.22 (t, 2H, CH2CH2C6H4, J = 7.05Hz).13C NMR (DMSO-d6,
300 MHz) δ 142.9 (NHCN), 158.6, 133.9, 133.4, 131.5, 131.1, 130.9,
130.5, 130.3, 129.4, 128.9, 127.3, 127.2, 114.5 and 114.2 (C6H4,
CH2C6H4Cl, CH2CH2C6H4), 55.5 (OCH3), 49.3 (CH2C6H4Cl), 48.5
(CH2CH2C6H4), 33.9 (CH2CH2C6H4). IR υmax (ATR) 1565 cmꢀ1 (CN).
Anal. Calculated for C23H22N2OCl2.H2O (431.35): C, 64.04; H, 5.61;
N, 6.49. Found: C, 63.77; H, 5.85; N, 6.24.
1-methyl-3-(phthalimide-2-yl)methylbenzimidazolium iodide, 7
Yield, 0.92 g (yellow crystals), 61%; mp 270–273 °C; 1H NMR (DMSO-
d6, 300MHz) δ 9.83 (s, 1H, NCHN), 8.22–7.70 (m, 8H, C6H4), 6.36 (s,
2H, NCH2N), 4.13 (s, 3H, CH3). 13C NMR (DMSO-d6, 300MHz) δ
167.5 (CO), 144.5 (NHCN), 135.5, 132.1, 131.8, 130.9, 127.3, 127.1,
124.2, 114.2 and 114.0 (C6H4), 47.4 (NCH2N), 33.8 (CH3). IR υmax
(ATR) 1561 cmꢀ1 (CN), 1717 cmꢀ1 (CO). Anal. Calculated for
C17H14N3O2I (419.22): C, 48.71; H, 3.37; N, 10.02. Found: C, 48.80; H,
3.22; N, 9.99.
1-(4-nitrobenzyl)-3-[2-(4-methoxyphenyl)ethyl]benzimidazo-
lium chloride, 4
1-ethyl-3-(phthalimide-2-yl)methylbenzimidazolium iodide, 8
Yield, 2.12 g (yellow crystals), 64%; mp 194–196 °C; 1H NMR (DMSO-
d6, 300 MHz) δ 10.10 (s, 1H, NCHN), 8.27–7.59 (m, 8H, C6H4,
CH2C6H4NO2), 7.14–6.83 (AA’BB’ system, 4H, CH2CH2C6H4), 5.97 (s,
2H, CH2C6H4NO2), 4.81 (t, 2H, CH2CH2C6H4, J = 7.05 Hz), 3.69 (s, 3H,
OCH3), 3.23 (t, 2H, CH2CH2C6H4, J = 7.05Hz).13C NMR (DMSO-d6,
300 MHz) δ 143.2 (NHCN), 158.6, 147.9, 143.2, 141.9, 131.5, 131.1,
130.3, 129.6, 128.9, 127.4, 127.3, 124.4, 114.6, 114.5 and 114.1
(C6H4, CH2C6H4NO2, CH2CH2C6H4), 55.5 (OCH3), 49.2 (CH2C6H4NO2),
48.6 (CH2CH2C6H4), 33.8 (CH2CH2C6H4). IR υmax (ATR) 1562 cmꢀ1
(CN). Anal. Calculated for C23H22N3O3Cl.H2O (441.91): C, 62.51; H,
5.47; N, 9.51. Found: C, 62.62; H, 5.23; N, 9.91.
Yield, 1.10 g (yellow crystals), 71%; mp 202–205 °C; 1H NMR (DMSO-
d6, 300MHz) δ 9.87 (s, 1H, NCHN), 8.21–7.69 (m, 8H, C6H4), 6.33 (s,
2H, NCH2N), 4.59 (q, 2H, CH2, J = 7.23 Hz), 1.51 (t, 3H, CH3,
J = 7.23 Hz).13C NMR (DMSO-d6, 300 MHz) δ 167.4 (CO), 143.8
(NHCN), 135.5, 131.9, 131.2, 131.1, 127.3, 127.1, 124.2, 114.4 and
114.1 (C6H4), 47.5 (NCH2N), 42.7 (CH2) 14.9 (CH3). IR υmax (ATR)
1565 cmꢀ1 (CN), 1716cmꢀ1 (CO). Anal. Calculated for C18H16N3O2I
(433.24): C, 49.90; H, 3.72; N, 9.70. Found: C, 49.31; H, 3.43; N, 9.62.
1-(4-chlorobenzyl)-3-(phthalimide-2-yl)methylbenzimidazolium
iodide, 9
1-(4-bromobenzyl)-3-[2-(4-methoxyphenyl)ethyl]benzimidazo-
lium chloride, 5
Yield, 1.48 g (white crystals), 73%; mp 256–258 °C; 1H NMR (DMSO-
d6, 300MHz) δ 10.10 (s, 1H, NCHN), 8.21–7.47 (m, 12H, C6H4,
CH2C6H4Cl), 6.37 (s, 2H, NCH2N), 5.85 (s, 2H, CH2C6H4Cl). 13C NMR
(DMSO-d6, 300 MHz) δ 167.4 (CO), 144.8 (NHCN), 135.5, 133.9,
133.3, 131.9, 131.4, 130.9, 129.3, 127.4, 127.2, 124.2, 114.6 and
114.3, (C6H4, CH2C6H4Cl), 49.6 (NCH2N), 47.6 (CH2C6H4Cl). IR υmax
(ATR) 1561 cmꢀ1 (CN), 1720 cmꢀ1 (CO). Anal. Calculated for
C23H17N3O2Cl2.H2O (456.32): C, 60.54; H, 4.20; N, 9.21. Found: C,
61.04; H, 4.31; N, 9.23.
Yield, 1.51g (white crystals), 75%; mp 175–176 °C; 1H NMR (DMSO-
d6, 300 MHz) δ 9.85 (s, 1H, NCHN), 8.14–7.12 (m, 8H, C6H4,
CH2C6H4Br), 7.12–6.81 (AA’BB’ system, 4H, CH2CH2C6H4), 5.74 (s,
2H, CH2C6H4Br), 4.79 (t, 2H, CH2CH2C6H4, J = 6.99Hz), 3.71 (s, 3H,
OCH3), 3.22 (t, 2H, CH2CH2C6H4, J = 6.99Hz).13C NMR (DMSO-d6,
300 MHz) δ 142.8 (NHCN), 158.6, 133.8, 132.3, 131.5, 131.1, 130.8,
130.3, 128.9, 127.3, 127.2, 122.5, 114.6, 114.5 and 114.3 (C6H4,
CH2C6H4Br, CH2CH2C6H4), 55.5 (OCH3), 49.4 (CH2C6H4Br), 48.6
(CH2CH2C6H4), 33.9 (CH2CH2C6H4). IR υmax (ATR) 1563 cmꢀ1 (CN).
Anal. Calculated for C23H22N2OBr.H2O (529.26): C, 53.10; H, 4.65; N,
5.38. Found: C, 53.47; H, 5.06; N, 5.17.
1-(4-bromobenzyl)-3-(phthalimide-2-yl)methylbenzimidazo-
lium iodide, 10
Yield, 1.32 g (white crystals), 70%; mp 265–266 °C; 1H NMR (DMSO-
d6, 300MHz) δ 10.09 (s, 1H, NCHN), 8.22–7.48 (m, 12H, C6H4,
CH2C6H4Br), 6.38 (s, 2H, NCH2N), 5.84 (s, 2H, CH2C6H4Br). 13C NMR
(DMSO-d6, 300 MHz) δ 167.4 (CO), 144.7 (NHCN), 135.5, 133.7,
132.3, 131.9, 130.9, 127.4, 127.2, 122.5, 114.6 and 114.2, (C6H4,
CH2C6H4Br), 49.6 (NCH2N), 47.6 (CH2C6H4Br). IR υmax (ATR)
1561 cmꢀ1 (CN), 1718 cmꢀ1 (CO). Anal. Calculated for
C23H17N3O2Br2.H2O (545.22): C, 50.71; H, 3.51; N, 7.71. Found: C,
51.32; H, 3.58; N, 7.69.
Synthesis of 1-benzyl-3-(phthalimide-2-yl)methylbenzimidazo-
lium chloride, 6
A mixture of 1-benzylbenzimidazole (III) (1.50g, 7.20 mmol) and
(phthalimide-2-yl)methyl chloride (1.45 g, 7.20mmol) in
dimethylformamide (3ml) was refluxed for 5 h. The mixture was
then cooled, and volatiles were removed under vacuum. Solid
was crystallized from ethanol/diethyl ether (1: 5). White crystals of
the title compound 6 (2.23 g, 77%) were obtained, mp 276–
1
278 °C; H NMR (DMSO-d6, 300 MHz) δ 10.15 (s, 1H, NCHN), 8.20–
7.38 (m, 13H, C6H4, CH2C6H5), 6.38 (s, 2H, NCH2N), 5.86 (s, 2H,
CH2C6H5). 13C NMR (DMSO-d6, 300MHz) δ 167.4 (CO), 144.5 (NHCN),
135.5, 134.2, 131.9, 131.4, 131.0, 129.4, 129.2, 128.7, 127.4, 127.2,
124.2, 114.6 and 114.1 (C6H4, CH2C6H5), 50.1 (NCH2N), 47.7
(CH2C6H5). IR υmax (ATR) 1563 cmꢀ1 (CN), 1720 cmꢀ1 (CO). Anal. Cal-
culated for C23H18N3O2Cl (403.86): C, 68.40; H, 4.49; N, 10.40. Found:
C, 67.31; H, 4.79; N, 10.18.
Determination of pKa values by NMR spectroscopy
The chemical shift changes close to acidic or basic area of the mole-
cule are examined for the determination of organic compounds,
which include carbonyl groups, hydroxyl groups, amino acids, in its
1
structure, and pKa values by H NMR spectroscopy depending on
the change of pH.[25–27] In the conducted studies, pKa values can
be determined by the inflection point of the sigmoidal curve drawn
by the chemical shift values showing differences according to chang-
ing pH values. The pKa values can be also determined by the analysis
of NMR data obtained with different pH with Henderson–Hasselbach
formula.[29,34] In some NMR studies, internal or external standard
(2,4,6-triiodophenol, 3,4,5-trichloropyridine, fumaric acid, DSS, 1,4-
dinitrobenzene, dimethyl sulfone, trimethylsilylpropionic acid etc.)
substances are used. It is necessary that the compounds that are
General method for the synthesis of compounds 7–10
Equivalent amount of the 1-(phthalimide-2-yl)methylbenzimidazole
(II) and appropriate alkyl halide were refluxed in dimethylforamide
(3 ml) for 5 h. Then, the mixture was cooled to room temperature,
and the volatiles were removed with reduced pressure. The residue
was crystallized from ethanol/diethyl ether (1 : 5).
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Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 1024–1030

pKa determination using new approches in NMR
used should have good solubility in the applied solvent system, be
not reactive, possess characteristic and constant signals, be not vol-
atile and stay stable for a long time.[35,36] In determining the pKa
values of benzimidazolium salts depending on these situations,
due to the structural features of these compounds instead of the
chemical shift changes in the related peaks, the NMR data of the
decreasing integration value of benzimidazolium salts’ changing
pH value of one-proton singlet signal-bonding carbon number 2
are considered as NMR data. In order to measure the integration
values, DSS is used as a reference substance.
solvent can be also used in case of the insufficient samples solubil-
ity. However, except for the methods depending upon LC and CE
separation techniques, the studied samples should not include con-
tamination. Besides this, if UV–VIS method is used, the sample
should include UV-active chromophore. Another shortcoming of
these methods is that all protonation zones cannot be observed
clearly at the same time.
NMR spectroscopy as a function of pH reflects the protonation
level of each group in the molecule progressively. Moreover, espe-
cially the 1H NMR experiment enables to work with sample amounts
in microgram levels. It can use dual or ternary solvent combinations
in case the sample’s solubility is inadequate and because it is not a
disruptive technique the samples can be regained. In this study,
some new benzimidazolium salts (1–10) were synthesized by using
starting compounds such as 2-(4-methoxyphenyl)ethyl chloride,
(phthalimide-2-yl)methyl chloride, benzyl chloride and 3-bromo-
propylbenzene. It was seen that the hydrogen atom at the two
positions of the benzimidazole compounds that turned into salts
showed acidic feature (Scheme 2).
Firstly, the pKa values were identified by the inflection point of
the sigmoidal curve that was obtained through passing on the de-
creasing integration values of the hydrogen atom attached to the
two positions of benzimidazolium salt after each pH measurement
in the 1H NMR analyses against pH. The data obtained for 1-[2-(4-
methoxyphenyl)ethyl]-3-methylbenzimidazolium iodide (2) com-
pound for H-2 proton 2-propanol: DMSO-d6 (4 : 1) for the solvent
system the change in 1H NMR integral values were shown in Fig. 1,
and for compound number 2, the data acquired of pH-NMR titra-
tion were given in Table 1.
pKa values of all the synthesized benzimidazolium salts were de-
termined by 1H NMR spectroscopic pKa determination method, but
pKa values of some of these benzimidazolium salts (2, 7, 11 and 12)
were also determined by the potentiometric titration method for
the comparison purpose. The acidic properties of benzimidazolium
salts were examined through the hydrogen atom bonding these
compounds on 2 positions. In all compounds, 1H NMR integration
values observed in different pH values were analyzed by using
two different approaches, and the pKa values were determined.
The obtained results are illustrated in Table 2.
Preparation of samples and 1H NMR experiments
In general, in NMR spectroscopy, insufficient sample amounts and
especially the limited solubility of samples in an appropriate
solvent are a disadvantage. Due to this reason, NMR analyses are
prepared with dual or triple solvent systems.[29] Therefore, the
samples were prepared in 2-propanol: DMSO-d6 (4: 1) solvent
system in this study because the solubility of synthesized
benzimidazolium salts in H2O is very limited. The analyte solution
was prepared in 2-propanol and DMSO-d6 solvent mixture that
consists of 10 mM of benzimidazolium salt and DSS. The stock
solutions prepared was divided to 8–10 parts and added to the
appropriate test tubes. The pH value of samples was adjusted by
adding in increasing amounts of TBAH prepared in 10 and
50 mM diluted in 2-propanol : DMSO-d6 (4: 1) solvent system to
the stock solution, which was in test tubes in equal amounts. The
pH values of the samples that were constant at 296 K point were
recorded. After the pH measurement was completed, 600 μl of
the analyte solution was taken and put into a 5-mm diameter
1
NMR tube, and the H NMR spectrum was recorded at the same
temperature. In this study, the comparison ratio between integral
value of the 9-proton singlet signal that has the chemical shift
value of DSS standard substance in approximately 0 pmm
recorded after each pH measurement in NMR spectrum with
integral value of the 1-proton singlet signal on the two positions
of the benzimidazole is taken into consideration for NMR data for
the benzimidazole compounds (Fig. 1).
Potentiometric titrations
Secondly, the NMR data are analyzed by using Henderson–
Hasselbalch equation (Eqn. (1)). This logarithmic analysis shows
the interdependency of the integral value obtained linearly after
each pH measurement to the pH. In the line graphic that was ob-
tained as a result of this analysis, the place where the line graphic
cuts x-axis was recorded as the pKa for the related compound.
In this study, a Hanna HI 221 model pH-ion meter was used for
potentiometric titrations. Before the potentiometric titrations, pH
meter was adjusted with the help of 7.00 0.01 and 10.00 0.01
buffer solutions. The 20 ml of the stock solutions that was prepared
from benzimidazolium salts in 2-propanol : DMSO (4: 1) solvent
was taken into a beaker. After that, it was made homogenous by
stirring it in 300 rpm rotational speed with a magnetic stirrer. Each
time, 0.05ml 50 mM TBAH solution was added to the analyte
solution with the help of a micropipette. The fixed pH and mV
values were recorded after the titrant addition. Finally, the half-
neutralization potential values were determined by drawing the
ml (TBAH)-mV graphic, and corresponding pKa values were
calculated.[37,38]
pH ¼ pKa þ log ½intðmax:Þ ꢀ intꢁ=½int ꢀ intðmin:Þꢁ
(1)
The sigmoidal curve graphic drawn by using integral values
obtained from different pH value 1H NMR experiments for 1-[2-(4-
methoxyphenyl)ethyl]-3-methylbenzimidazolium iodide (2) com-
pound and for H-2 proton 2-propanol: DMSO-d6 (4: 1) solvent
system and the graphical presentation of Henderson–Hasselbalch
analysis are given in Figs 2 and 3.
When pKa values determined by NMR method of benzimidazole
salts including 2-(4-metoksifenil)ethyl group at position 1 were ex-
amined, it was noticed that compound 2 with an electron-releasing
methyl at position 3 of the ring showed a weaker acidic feature in
comparison with the other benzimidazole salts including 2-(4-
metoksifenil)ethyl group. The acidic strength of other benzimid-
azole salts including 2-(4-metoksifenil)ethyl group at position 1
Results and discussion
For the determination of pKa values, some spectroscopic methods
such as potentiometric titration,[14] UV–VIS spectroscopy,[15] CE[17]
and LC[18] were used. These methods were used commonly be-
cause of giving high certainty results, being available commercially
and automation concurrency. In these methods, an appropriate
Magn. Reson. Chem. 2015, 53, 1024–1030
Copyright © 2015 John Wiley & Sons, Ltd.
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A. Mumcu and H. Küçükbay
Figure 1. Different pH value 1H NMR spectrums obtained by the 10 mM tetrabutylammonium hydroxide solution (TBAH) titration of 1-[2-(4-methoxyphenyl)
ethyl]-3-methylbenzimidazolium iodide (2) compound in 10 mM solution and the sigmoidal curve that was acquired with the passing of integral values
obtained from these spectrums to the against pH values.
In this acidity ranking, except for compounds 3 and 5, it was
observed that the inflection point of all salts including 2-(4-
metoksifenil)ethyl group in pKa results at position 1 and the
Henderson–Hasselbalch estimations were in conformity with
each other.
It was seen that the pKa values of the benzimidazole salts
including (phthalimide-2-yl)methyl at position 1 were found to be
lower in comparison with benzimidazole salts including 2-(4-
metoksifenil)ethyl at position 1 due to the formation of a likely
O–H bond between carbonyl groups in phthalimide and
hydrogen atom attached to the 2 positions of the benzimidazolium
salt. When the pKa values of the benzimidazole salts containing
(phthalimide-2-yl)methyl group determined by the NMR method
were examined, it was seen that compounds 7 and 8 containing
electron providing groups to the ring showed a weaker acidic
properties when compared with others. At position 1, the acidity
strength in other benzimidazole salts including (phthalimide-2-yl)
methyl group increases according to the electron-withdrawing
group attached to the ring. According to this, the acidity
strength of the compounds has been determined with regard to in-
flection point approach as 9 > 10> 6 > 7 > 8, and with respect to
Henderson–Hasselbalch analysis, it has been identified as
10> 6 > 9 > 7 > 8. When the pKa values of the benzimidazole salts
VI and VII determined by NMR method were examined, it was seen
that the pKa value of VI possessing electron-withdrawing nitro
group was lower than the pKa value of VI having methyl group that
provides electron to the ring both with the inflection point and
Henderson–Hasselbalch estimations. It has been identified that
acidity strength of these salts in the environment of 2-propanol:
DMSO-d6 (4: 1) as VII > VI.
Scheme 2. The balance between benzimidazolium salt and its resonance
stabilized conjugate.
Table 1. The pH-NMR titration data of 1-[2-(4-methoxyphenyl)ethyl]-3-
methylbenzimidazolium iodide (2)
TBAH (ml)
pH
NMR (int data)
log[(int(max.) ꢀ int)/
(int ꢀ int(min.))]
0.040
0.060
0.080
0.090
0.095
0.100
0.120
0.150
0.250
0.350
9.90
11.05
11.66
12.35
12.90
13.10
13.30
13.53
13.73
14.05
0.09276
0.09248
0.09207
0.08474
0.06621
0.03850
0.01488
0
—
ꢀ2.386
ꢀ2.066
ꢀ1.018
ꢀ0.395
0.147
0.719
—
0
—
0
—
TBAH, tetrabutylammonium hydroxide solution.
aDetermined from the inflection point.
bDetermined by Henderson–Hasselbalch analysis.
cDetermined from potentiometric titration.
For the purpose of comparison, the pKa values of compounds 2, 7,
VI and VII were determined not only by NMR method but also with
potentiometric method (half neutralization method). According to
this method, the acidity strength of these salts was identified in
the environment of 2-propanol : DMSO-d6 as 7 > VII > 2> VI,
respectively, in the conducted studies. The reason behind the order
increased according to the strength of the electron withdrawing
group attached to the ring. Accordingly, ranking of the acidity
strength of these compounds was identified as 4 > 3 > 5 > 1 > 2.
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Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 1024–1030

pKa determination using new approches in NMR
Table 2. The pKa values of synthesized benzimidazole compounds at
(VI, VII, 1–10) 23 °C
No Compound Proton Inflection
H–H
Potentiometric
method
structure
point
—
1
2
H-2
H-2
13.07 0.12
12.96 0.15
13.10 0.09
12.92 0.15
13.00 0.11
12.68 0.08
12.23 0.07
—
3
H-2
Figure 2. The pH-dependent integration curve for 1-[2-(4-methoxyphenyl)
ethyl]-3-methylbenzimidazolium iodide (2).
—
4
5
H-2
H-2
12.85 0.16
12.95 0.12
12.45 0.11
12.50 0.09
—
—
6
7
H-2
H-2
10.37 0.11
10.40 0.09
10.34 0.08
10.44 0.12
9.51 0.11
Figure 3. Graphical presentation of Henderson–Hasselbalch (H–H) analysis
for 1-[2-(4-methoxyphenyl)ethyl]-3-methylbenzimidazolium iodide (2).
—
8
9
H-2
H-2
10.45 0.08
10.18 0.12
10.60 0.09
10.40 0.09
Conclusion
—
In this study, pKa values of some new benzimidazole salts were de-
termined by using pH-dependent 1H NMR spectroscopy. In deter-
mining the acidity constants (pKa), distinctively from classic NMR
pKa determination method that uses the peak shift to another
new NMR spectroscopic method using the peak losses was used.
In the conducted studies, it was seen that pKa values depend to a
large extent on the compounds structure and the electron with-
drawing and providing groups in the molecule.
—
10
11
12
H-2
H-2
H-2
10.23 0.13
13.48 0.18
11.30 0.07
10.30 0.08
13.08 0.15
11.35 0.09
12.43 0.05
10.36 0.18
In conclusion, 1H NMR spectroscopy is a fast, sensitive technique
that is used for the determination of pKa values in a large pH work-
ing range. Besides this, it has advantages such as applicability on
very low amounts of analyte and regaining compound because it
is not a disruptive technique. Moreover, it enables to work with dif-
ferent solvent systems even in low sample solubility. This method
can be accepted as an alternative new method to determine the
pKa values of benzimidazole salts. The acidity properties of other
substances in salt structure can also be examined by using this
method.
H–H, Henderson–Hasselbalch.
of the acidity strength is the electron withdrawing and providing
groups attached directly to the imidazole ring of the benzimid-
azoles. In accordance with these results, it has been observed that
the acidity strength sequencing according to pKa values determined
by both NMR and potentiometric methods for four benzimidazole
salts showed consistency.
Acknowledgements
We wish to thank İnönü University Research Fund (BAPB-2011/137)
for its financial support of this study. We also thank Associate
Prof. F. Zehra Küçükbay for her helpful discussion and advice
during the potentiometric titration works.
Magn. Reson. Chem. 2015, 53, 1024–1030
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A. Mumcu and H. Küçükbay
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