Tautomerism and isomerism in some antitrichinellosis active benzimidazoles: Morphological study in polarized light, quantum chemical computations

Article abstract of DOI:10.1016/j.molstruc.2017.08.073

The morphology of the crystal structure of some antitrichinellosis active benzimidazole derivatives including (1H-benzimidazol-2-ylthio)acetic acids, [1,3]thiazolo[3,2-a]benzimidazol-3(2H)-ones, 1H-benzimidazol-2-ylthioacetylpiperazines and starting 2-mercapto benzimidazoles, was studied by the use of Polarized Light Microscopy (PLM). Characterization of the crystal phase was complimented by Differential scanning calorimetry analysis (DSC) and spectroscopic data. DFT computations were performed in order to investigate the prototropic tautomerism and the geometry of the molecule of the synthesized compounds. One distinct type of crystal structure for each one of 5 or 6-methyl-(1H-benzimidazol-2-ylthio)acetic acid 6 was observed by PLM – dendritic and needle-shaped formations. Compound 14, containing a methyl substituent in the benzimidazole ring crystallized also into two phases; while for the unsubstituted compound 13 a separation of phases does not take place. The influence of the both solvents - chloroform and ethanol on the phase separation and the formation of the crystalline structure of compound 14 was investigated. The morphological study showed that the cyclization of 6 in the presence of acetic anhydride in pyridine medium led to a mixture of 6-methyl-[1,3]tiazolo[3,2-a]benzimidazol-3(2H)-one (10a) and 7-methyl-[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-one (10b), which crystallized in the form of fibrils and spherulites respectively. It was found that a difference in the crystal structures of substituted and unsubstituted benzimidazol-2-thiones, respectively benzimidazol-2-thiol derivatives exists, which may be due not only to the thiol-thione tautomerism but to the prototropic properties of the hydrogen atom in first position of the ring. The calculation results indicated that the thione form is more stable than the thiol tautomer by 51–55 kJ mol^?1. But at the same time ΔG for the two thiol tautomers is below 0.5 kJ mol^?1. In solid phase the 5(6)-substituted-1H-benzimidazol-2-thiols crystallized in two different crystal structures while the unsubstituted 1H-benzimidazol-2-thiol possess one type of crystal structure.

Full text of DOI:10.1016/j.molstruc.2017.08.073

Accepted Manuscript
Tautomerism and isomerism in some antitrichinellosis active benzimidazoles:
Morphological study in polarized light, quantum chemical computations
Kameliya Anichina, Anelia Mavrova, Denitsa Yancheva, Jordan Tsenov, Rasho
Dimitrov
PII:
S0022-2860(17)31142-0
10.1016/j.molstruc.2017.08.073
MOLSTR 24211
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 March 2017
Revised Date: 19 August 2017
Accepted Date: 22 August 2017
Please cite this article as: K. Anichina, A. Mavrova, D. Yancheva, J. Tsenov, R. Dimitrov, Tautomerism
and isomerism in some antitrichinellosis active benzimidazoles: Morphological study in polarized
light, quantum chemical computations, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.08.073.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Tautomerism and isomerism in some antitrichinellosis active benzimidazoles: Morphological
study in polarized light, quantum chemical computations
Kameliya Anichinaa, Anelia Mavrovaa, Denitsa Yanchevab,1 Jordan Tsenovb, Rasho Dimitrova
,
aUniversity of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str.,
build. 9, 1113 Sofia, Bulgaria
Abstract
The morphology of the crystal structure of some antitrichinellosis active benzimidazole
derivatives including (1H-benzimidazol-2-ylthio)acetic acids, [1,3]thiazolo[3,2-a]benzimidazol-
3(2H)-ones, 1H-benzimidazol-2-ylthioacetylpiperazines and starting 2-mercapto benzimidazoles,
was studied by the use of Polarized Light Microscopy (PLM). Characterization of the crystal
phase was complimented by Differential scanning calorimetry analysis (DSC) and spectroscopic
data. DFT computations were performed in order to investigate the prototropic tautomerism and
the geometry of the molecule of the synthesized compounds.
One distinct type of crystal structure for each one of 5 or 6-methyl-(1H-benzimidazol-2-
ylthio)acetic acid 6 was observed by PLM – dendritic and needle-shaped formations. Compound
14, containing a methyl substituent in the benzimidazole ring crystallized also into two phases;
while for the unsubstituted compound 13 a separation of phases does not take place. The
1
Corresponding author: Denitsa Yancheva, Tel.:+359 2 9606106; fax:+359 2 8700225; E-mail address:
deni@orgchm.bas.bg
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influence of the both solvents - chloroform and ethanol on the phase separation and the
formation of the crystalline structure of compound 14 was investigated.
The morphological study showed that the cyclization of 6 in the presence of acetic anhydride in
pyridine medium led to a mixture of 6-methyl-[1,3]tiazolo[3,2-a]benzimidazol-3(2H)-one (10a)
and 7-methyl-[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-one (10b), which crystallized in the
form of fibrils and spherulites respectively.
It was found that a difference in the crystal structures of substituted and unsubstituted
benzimidazol-2-thiones, respectively benzimidazol-2-thiol derivatives exists, which may be due
not only to the thiol-thione tautomerism but to the prototropic properties of the hydrogen atom in
first position of the ring. The calculation results indicated that the thione form is more stable than
the thiol tautomer by 51-55 kJ.mol-1. But at the same time ∆G for the two thiol tautomers is
below 0.5 kJ.mol-1. In solid phase the 5(6)-substituted-1H-benzimidazol-2-thiols crystallized in
two different crystal structures while the unsubstituted 1H-benzimidazol-2-thiol possess one type
of crystal structure.
Keywords: benzimidazole synthesis, tautomerism, polarized light microscopy, DSC, DFT
1. Introduction
The benzimidazole heterocyclic system is an essential pharmacophore in modern drug discovery
because of the broad spectrum of important biological and pharmacological properties exhibited
by benzimidazole compounds, as antimicrobial [1-4], antitumor [5-7], antiallergic [8-10] and
antiviral effects [11-15]. Benzimidazole derivatives albendazole, mebendazole, ciclobendazole,
febendazole, flubendazole, triclabendazole have achieved significant success in the treatment of
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many parasitic diseases due to their high efficiency, broad spectrum of antiparasitic activity and
low toxicity [16, 17].
Well-known specific features of the bezimidazole heterocycle are its prototropic properties,
which enable its easy interconvertion to different tautomers. The hydrogen atom attached to N-1
of the benzimidazole nucleus readily tautomerizes moving to N-3 atom; consequently, the
benzimidazoles may exist in two or even three tautomeric forms. The tautomerism of drug
structures plays an essential role in the interaction with biological systems [18-20]. Therefore,
after the establishment of the fact that the benzimidazole derivatives have antagonist effect
towards purine compounds many theoretical calculations and investigations were performed with
the purpose to predict and determined their conformational and tautomeric preferences [18, 21-
26].
Previously we reported a series of newly synthesized 5(6)-(un)substituted-1H-benzimidazol-2-
ylthioacetylpiperazine derivatives and also thiazolobenzimidazolones (Scheme 1) [27, 28]. Some
of these substances exhibited higher activity against Trichinella spiralis in vitro in comparison to
albendazole [27, 28]. The high biological activity of the above mentioned compounds and the
existing phenomenon of tautomerism observed in benzimidazole molecules prompted us to
explore the morphology of their crystal structure by polarized light microscopy (PLM). It should
be pointed out that there are very few data reported about the use of PLM for investigation of the
crystal structure of benzimidazole tautomers. PLM analysis is applied extensively in
pharmaceutical development and enables observation not only of the simple images of drug
substance but also the particle size and shape of the internal structure (morphology) of the
crystals of those substances [29, 30]. Having in view the range and specificity of the
crystallographic properties of pharmaceutical compounds, a PLM research on the synthesized
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benzimidazole derivatives will contribute to clarify the morphology of their crystals and the
possible presence of more than one tautomer. It will allow identifying the distinguishing
properties of the tautomeric forms. DSC analysis and spectroscopic data were used to assign the
different crystal phases to particular tautomer. The conformational and tautomeric preferences of
the molecules were studied by computational methods too. This study will complement the
characterization of the 5(6)-(un)substituted-1H-benzimidazol-2-ylthioacetylpiperazines and
thiazolobenzimidazolones for possible future application as antitrichinellosis agents.
2. Experimental section
2.1. Materials and methods
Melting points (mp) were determined on an Electrothermal AZ 9000 3MK4 apparatus and were
uncorrected. Thin layer chromatography (Rf values) was performed on silica gel 60 plates F254
(Merck, 0.2-mm thick) using mobile phase benzene-methanol, 3:1 and benzene-acetone, 8:1, and
1
visualized with ultraviolet light. H NMR spectra were obtained on a Bruker Avance II+ 250 MHz
and a Bruker Avance II+ 600 MHz NMR instrument using a dual 5-mm probe head and CDCl3 and
DMSO-d6 as solvents. The measurements were carried out at ambient temperature (300 K).
Chemical shifts were expressed relative to tetramethylsilan and reported as δ (ppm). Mass
spectrometric measurements were performed using an LCQ DECA instrument (Thermo Finnigan,
Palo Alto, CA,USA). The compounds solutions (10-6 M in CH3OH) were prepared and directly
infused into the ESI source. The ions were produced using spray voltage, capillary voltage and
capillary temperature of 4 kV, 8 kV and 220 °C, respectively.
Differential scanning calorimetry studies (DSC) were performed on Perkin Elmer DSC 7
differential scanning calorimeter in an argon atmosphere, in a temperature range from 50 to 250 º C
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and a heating rate of 5 º C min-1. The apparatus was calibrated using indium and lead as standards.
Samples were in an amount of 7 mg.
2.2. Chemistry
The synthesis of the studied benzimidazole derivatives was performed according to the procedure
described in [27, 28]. The methods are given in the Supporting material.
2.3. Optical polarizing microscopic assay
The morphology of the crystallizing benzimidazole derivatives was studied upon dissolving 2 mg of
the tested compound in a 0,1 ml CHCl3 (Sigma–Aldrich) at a room temperature. One drop of the
solution was applied on a glass plate and the solvent was allowed to volatilize, after which a cover
glass was placed over the sample and the preparation was observed using an Olympus BH2
Polarized Light Microscope, equipped with a camera.
2.4. Calculations:
All theoretical calculations were performed using the Gaussian 09 package [31] of programs.
Geometry and vibrational frequencies of the studied species were performed by analytical based
gradient technique without any symmetry constraint. All the results were obtained using the density
functional theory (DFT), employing the B3LYP (Becke’s three-parameter non-local exchange [32]
and Lee correlation potentials [31]. Incorporation of solvent effect was performed by Integral
Equation Formalism of Polrizable Continuum Model (IEFPCM) [33, 34].
Tautomeric interconversion in chloroform was simulated by potential energy surface (PES) scans
for hydrogen atom migration and rotation around the C-S bond. The moving of H (from S-H bond)
to benzimidazolium N was done at regular steps of 0.1 Å. The rotation around C-S bond was carried
out in steps of 3.0o. For both migration and rotation, geometry optimization was performed at each
step. Transition state was confirmed by optimization of the highest energy geometry identified by
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the H-atom migration scanning and vibrational analysis. The computations were done at B3LYP/6-
311+G** level of theory.
3. Results and discussion
The synthesis of the studied benzimidazole derivatives was carried out as depicted in the Scheme 1.
The preparation of the starting 1H-benzimidazole-2-thiols 1-4 was performed by refluxing of
ethanol/water solution of sodium hydroxide, carbon disulphide and 4-substituted-1,2-diamino-
benzenes. The 5(6)-(un)substituted-1H-benzimidazol-2-ylthioacetic acids 5-8 were obtained
through William’s reaction of 5(6)-(un)substituted-1H-benzimidazole-2-thiols 1-4 with chloroacetic
acid in the presence of sodium hydroxide under reflux. 1,3-Thiazolo[3,2-a]benzimidazol-3(2H)ones
9-12 were synthesized by heating 5(6)-(un)substituted-(1H-benzimidazol-2-ylthio)acetic acids 5-8
with acetic anhydride in pyridine medium on steam bath [28].
The next step comprised in the cleavage of the condensed thiazole ring in the [1,3]-thiazolo[3,2-
a]benzimidazol-3(2H)-ones with an appropriated N-monosubstituted piperazine in ethanol medium.
Refluxing for 3 h resulted in the piperazine derivatives of benzimidazol-2-ylthioacetic acids 13-15
[27].
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O
O
Cl
AC2O/Pyr
N
N
OH
SH
R
S
OH
R
N
H
NaOH
N
H
1 R = H
5 R = H
5- 8
1- 4
2 R = CH3
3 R = NO2
4 R = Cl
6 R = CH3
7 R = NO2
8 R = Cl
O
8
5
8
R
N
N
R
HN
N
CH3
N
7
6
6
S
S
+
N
7
5
a
b
O
9 R = H
10 a-b R = CH3
11 a-b R = NO2
12 a-b R = Cl
9 -12
CH3
N
13 R = H
14 R = CH3
15 R = NO2
N
O
N
S
R
N
H
13 - 15
Scheme 1. Synthesis of 1H-benzimidazol-2-ylthioacetylpiperazine derivatives.
The structures of all compounds were established by IR, 1H NMR as well as elemental analysis [27,
28]. The data of some 1H NMR, 13C NMR and MS spectra of the synthesized compounds are given
as supplementary materials (Figures 1S-12S). The mass spectral data of thioacetoamides are
presented in Table 1.
Table 1. Multi-stage mass spectrometry (MSn) data of compounds 13 and 14.
№
MH+
MS2
MS3
MS4
m/z 367
m/z 177 (100)
m/z 205 (26)
m/z 134
m/z 119 (100)
13
→
→
→
m/z 381
m/z 177 (100)
m/z 133 (100)
14
→
→
→
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m/z 177(100)
m/z 134(100)
m/z 119 (100)
→
→
→
For compound 14 the 2D heteronuclear 1H/13C correlation spectra (HMQC technique -
heteronuclear multiple quantum correlation) were recorded to confirm the assignment of the signals
in 1H and 13C spectra and the purity of the compounds (Fig. 9S).
Thus four groups of benzimidazoles were synthesized arising different possibilities for prototropic
tautomerism and positional isomerism.
Due to prototropic hydrogen atom at the benzimidazole ring it should be expected the cyclization of
thioacetic acid would give two isomers of thiazolobenzimdazolones.
Tanaka and co-workers reported that the cyclization of 5(6)-substituted-(1H-benzimidazol-2-
ylthio)acetic acid in the eutectic mixture Dewtherm A afforded two positional isomers – 6-th and 7-
1
th isomer [35]. The authors determined their structure by means of H NMR spectra. Carrying out
the experiment by heating the thioacetic acid with acetic anhydride in pyridine medium they
established the presence of the two isomers in the reaction solution through 1H NMR spectroscopy,
but they succeeded to isolate only the 6-substituted isomer. Tanaka and co-workers [35] have
attributed this to the fact that probably 6-substituted thiazolobenzimdazole is thermodynamically
more stable than the 7-substituted isomer.
The formation of only 6-substituted thiazolobenzimdazolones as products was reported for the
cyclization of 5(6)-methoxy- [36], 5(6)-methyl- [37], 5(6)-chloro- [38] and 5(6)-nitro-(1H-
benzimidazol-2-ylthio) acetic acid with acetic anhydride / pyridine [39].
In our study the cyclization of the 5(6)-methyl-(1H-benzimidazol-2-ylthio)acetic acid 6 with acetic
anhydride was achieved in pyridine medium in accordance with the method described in [27]. After
recrystallization of the crude product we obtained a substance with melting point 166.9ºC and Rf
value 0.500 (mobile phase - benzene-acetone = 8:1).
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To determine whether the product is a mixture of two positional isomers, we carried out an
examination on the morphological crystalline structure in polarized light. The studies demonstrated
the existence of two types of crystal structures (Fig. 1), which is a proof that the obtained solid is a
mixture of two positional isomers. On Fig. 1a there can be seen clearly distinct bundles of colored
needles (fibril structure) – first phase and radial structures with pronounced "Maltese Cross" shape
– the second phase. The size of the first phase was larger in comparison to that of the second phase.
The substance had a great rate of crystallization. The total crystallization process was completed
during the evaporation of the solvent, while the phase which formed a spherulite structure was
slightly lagging in time from the phase constituting the needle-shaped crystals. The analysis of the
1H-NMR data for the obtained product indicated the presence of two isomers in ratio 1:1.
(a)
(b)
Fig. 1. Microscopic pictures of the mixture of 6- and 7-methyl-[1,3]thiazolo[3,2-a]-
benzimidazole-3(2H)-one 10a,b (a) and 6- and 7-chloro-[1,3]thiazolo[3,2-a]-benzimidazole-
3(2H)-ones 12a,b (b) in polarized light.
The two phases were mechanically separated with the help of a microscope and for each of them
1
there was recorded a H-NMR spectrum (the NMR-spectra are given in Fig. 4S and 5S in the
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Supplementary material). The chemical shifts δ (ppm) recorded on a Bruker Avance II+ 600 MHz
NMR instrument were as follows:
1
the fibril structure: H NMR (DMSO-d6) δ (ppm): 2.428(s, 3H, 6-CH3), 4.599(s, 2H, CH2),
7.199(ddd, J = 0.6, 1.6, 8.2 Hz, 1H, 7-H), 7.462(d, J = 8.2 Hz, 1H, 8-H), 7.686(m, 1H, 5-H);
the spherulitic structure: 1H NMR (DMSO-d6) δ (ppm): 2.410(s, 3H, 6-CH3), 4.591(s, 2H, CH2),
7.199(dd, J = 0.8, 8.1 Hz, 1H, 6-H), 7.403(m, 1H, 8-H), 7.729(d, J = 8.1 Hz, 1H, 5-H);
Differential scanning calorimetry (DSC) studies confirmed the suggestion that the synthesized
product is a mixture of 6-the and the 7-th isomer, since in the DSC-diagrams two peaks appeared for
the melting point, the one at the 120.7º C and the other one at 185.7º C for compounds 10a and 10b,
respectively (Table 2). The diagrams are given in Fig. 13S in the Supplementary material. When
using fractional crystallization with ethanol it was possible to separate the 6- and the 7-isomer.
Table 2. Differential scanning calorimetry (DSC) investigations of [1,3]thiazolol[3,2-a]-
benzimidazole-3(2H)-ones.
Compound
№
Mp ˚С
∆ Н, J/g
Weight loss on heating, %
183.8
185.7
120.7
228.5
223.0
139.6
145.2
10
4.7
9a
10a
10b
11a
11b
106.7
1.4
Based on these results it can be concluded that 6-methyl[1,3]-thiazolo[3,2-a]-benzimidazole-3 (2H)-
one 10a crystallized in the form of fibrils, and 7-methyl[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-
one 10b in the form of spherulites.
The mixture of 6- and 7-chloro[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-ones 12, whose crystal
structures are shown in Fig. 1b crystallized in significantly finer formations in comparison to that of
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6- and 7-methyl[1,3]thiazolol[3,2-a]-benzimidazole-3(2H)-ones 10, the first phase having a
markedly acicular nature, and the second, which was localized in a narrow area (the lighter zone in
Fig. 1b) forming a multitude of polycrystalline grains.
Compounds 10 and 11, substituted in the benzimidazole ring, showed presence of two types of
crystal structures and in their DSC-diagrams appeared two peaks for the melting points in contrast
to the unsubstituted compounds, where the separation of the crystalline phase was not observed and
there was only one value of the melting point. In the DSC diagram of compound 9 there is only one
peak at 183.4°C.
Having in view the observation of two crystal phases in the substituted derivatives 10-12 we
undertook investigation of the precursor compounds.
Our observation by PLM showed that 1 (Fig. 2) formed a crystalline phase having a dendritic
structure with orientation, affected by the formation of the dendrites.
Fig. 2. Microscopic picture of compound 1H-benzimidazol-2-thione 1 in polarized light.
For compound 1 earlier XRD studies revealed that it is crystallizing in thione form [40].
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The PLM observation of the benzimidazole derivative 5(6)-methyl-1H-benzimidazol-2-thiol 2
showed that the compound builds two kinds of crystal structures (Fig. 3a) - grained
polycrystalline layer and a leaf-like structure. The second phase was markedly perfect and
imitated elements of spherulites, which at their borders were formed as an amorphous phase that
did not crystallized due to the lack of space for the formation of spherulites (Fig. 3b).
(a)
(b)
Fig. 3. Microscopic pictures of compound 5(6)-methyl-(1H)-benzimidazol-2-thiol 2 in polarized
light, scale 100µm (a) and scale 20µm (b).
Two kinds of crystal structures – dendritic and needle-shaped formations – were observed in
polarized light also for 5(6)-methyl-(1H-benzimidazol-2-ylthio)acetic acid 6 (Fig. 4a). The survey
of that compound revealed that together with the formation of different crystallites, the influence of
the “time” factor was very important. The crystal formations obtained in two hours are shown in
Fig. 4b. After 24 hours the same sample showed a different appearance – a second crystalline phase
with a strong needle character appeared together with the previously mentioned formations (Fig.
4c).
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(a)
(b)
(c)
Fig. 4. Microscopic pictures of compound 5(6)-methyl-(1H-benzimidazol-2-ylthio)acetic acid 6
in polarized light 2 hours after preparation of sample; scale 100µm (a); scale 20 µm (b); and 24
hours after preparation of sample; scale 30 µm (c).
The tests were extended by conducting a microscopic analysis of the crystal structures of the two
structurally similar products - 13 and 14, which differ only by one methyl group as a substituent at
the benzimidazole nucleus. For compound 13 characteristic was a continuous process of formation
of the crystal structure. The observations were carried out for nearly two months, through which we
registered a steady increase in the crystalline phase. As it can be seen from Fig. 5a small spaced
apart areas of crystal formations were visible 24 hours after the sample preparation. After one
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month, a homogeneous crystal structure of miniature dendrites was formed, whereat the dendrites
were densely arranged on one another in a part of the sample, and with an emphasized loose
placement adjacent to the described zone, where the compound was in a small amount (Fig. 5b).
(a)
(b)
(c)
Fig. 5. Microscopic pictures of compound 13 in polarized light 24 hours after preparation of the
sample (a); 1 month after preparation of the sample (b) and 2 month after preparation of the
sample (c).
On Fig. 5c there can be seen dendrites of the test compound, well formed in a period of two months.
Taking into account that free (empty) areas as in Fig. 5b were not observed, it could be assumed
that the crystallization process is fully completed at that time.
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The influence of both solvents - chloroform (Fig. 6a) and ethanol (Fig. 6b) on the phase separation
and the formation of the crystalline structure of compound 14 was investigated. It is noteworthy,
that in each of the two solvents a preventing of phase separation was not observed. In the sample
dissolved in ethanol a single phase structure was formed after two hours, consisting of a great
number of spherical particles. But after a period of 72 hours already a separation of two phases was
observed in the same sample (Fig. 6a). The one phase consisted of coacervates with significant
sizes, while in the other remained spherical particles having a size almost equal to the size of the
particles generated at the moment of preparation.
(a)
(b)
Fig. 6. Microscopic pictures of compound 14 in polarized light by use of ethanol as solvent (a)
and chloroform as solvent (b).
In consequence of the use of chloroform, the first phase consisted of a grain structure, while for the
second phase there were characteristic well defined, although deformed, spherulites with typical
"Maltese Cross" shape (Fig. 6b). Based on these results it could be concluded that irrespectively of
the differences in the crystal structures, obtained by the use of various solvents, compound 14,
containing a substituent at the benzimidazole ring, crystallizes into two phases, while for the
unsubstituted compound 13 a separation of the phases does not occur.
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According to Katritzky [19] there is no clear dividing line between tautomerism and isomerism. The
tautomers are simply isomers that interconvert with a relatively low activation energy – below ca 20
kcals.mol-1. The rate of interconversion is controlled by the free energy of activation between the
interconverting species. In order to investigate the prototropic tautomerism and the geometry of the
synthesized compounds (Scheme 2) DFT computations were performed at B3LYP/6-311+G** level
of theory.
H
N
N
SH
S
S
N
H
N
H
1a
2a
1b
H
N
H
H3C
H3C
H3C
N
N
SH
SH
N
H
N
H
N
2b
2c
6c
O
S
OH
HO
N
H
N
O
H3C
H
N
H3C
H3C
N
H3C
S
S
S
N
HO
N
H
N
H
N
O
O
6b
OH
6d
6a
CH3
H3C
N
S
N
H
O
N
N
N
N
O
H
H3C
N
S
N
CH3
14b
14a
Scheme 2. Tautomeric equilibria for 1H-benzimidazole derivatives.
The stability order for the tautomers depicted in Scheme 2 is presented in Table 3 based on the
calculated free energies of the species. The energy preferences regarding the positional
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isomerism in the substituted 1,3-thiazolo[3,2-a]benzimidazol-3(2H)ones 9-12 were estimated by
the two isomers (a and b) of 10.
Table 3. DFT (B3LYP/6-311+G**) Total (Gtot) and relative (ꢀG) free energies of the studied
species
No.
Chloroform
Gtot
DMSO
∆Ga
∆Ga
Gtot
(kJ.mol-1)
(kJ.mol-1)
(a.u.)
(a.u.)
1.
-778.127325
-778.131668
1a
2.
-778.107821
-817.431217
-817.410805
-817.410544
-1045.335400
-1045.335057
-1045.329259
-1045.329130
-968.878772
-968.878687
51.21
-778.111107
-817.435139
-817.414078
-817.413651
53.98
1b
2a
3.
4.
53.59
0.69b
55.30
1.12b
2b
2c
5.
6.
-1045.340926
-1045.340483
-1045.333538
-1045.333721
-968.88235
6a
7.
0.90
16.12
0.34c
1.16
0.48d
18.92
6b
6c
8.
9.
6d
10a
10b
10.
11.
0.22
-968.88190
1.18
3.26
12.
13.
-1507.109035
-1507.115514
-1507.114274
14a
14b
-1507.108261
2.03
b
a ∆G=Gnb - Gna; ∆G=G2c – G2b; c ∆G=G6d – G6c; d ∆G=G6c – G6d;
As the tautomeric equilibrium in heterocyclic compounds depends strongly on the dielectric
constant of the medium and the ability of the solvents to form hydrogen bonds with each one of the
17

ACCEPTED MANUSCRIPT
tautomers, the optimization of molecular structures 1-14 (Scheme 2) was carried out in chloroform
and dimethyl sulfoxide. The respective total (Gtot) and relative (ꢀG) energies are summarized in
Table 3. The Gtot of compounds 1-2 in chloroform and DMSO indicated that the thione form is
preferred. In order to estimate the kinetic barrier (free energy of activation) for thione-thiol
interconversion, a potential energy scan for H-atom migration in 1a was carried out (Fig. 17S in
Supplementary material). The highest energy geometry identified by the scanning was then
optimized to obtain Gtot of the transition state (Fig. 18S in Supplementary material). Thus the free
energy of activation in chloroform was found to be 122.33 kJ.mol-1 (29.24 kcal.mol-1). On the other
hand, thiol 2b would easily convert into thiol 2c as according to the calculations the free energy
difference between the two forms is within the range 0.69-1.12 kJ.mol-1. The thiol 2b with
substituent in position 5 of the benzimidazole ring is more favorable in comparison to thiol 2c with
substituent in position 6. The IR spectra of derivatives 2-4, recorded in KBr disks, showed the
simultaneous presence of thione and thiol form (IR-spectra of 2 are given in Fig. 15S and 16S in the
Supplementary material). The existence of two different crystalline phases cannot be explained
simply by the presence of thiol-thione tautomers, but also with the prototropic properties of the
imide hydrogen in the benzimidazole ring.
Taking into consideration the calculation results for the 1H-benzimidazol-2-yltioacetic acids 6 and
14, it should be pointed out that the preferred tautomers are those where the substituent is in
position 6-th of the benzimidazole ring. For 6 the free energy difference ꢀG between the 6-H (6a)
and 7-H (6b) tautomer is 0.9-1.16 kJ.mol-1. The free energy of activation for interconversion
between the tautomeric forms of 1H-benzimidazol-2-yltioacetic acid 6 was modeled by calculating
the energy requirements for rotation around the C-S bond and migration of the H-atom from the
carboxylic group to the benzimidazolium N-atom in chloroform. H-atom migration to
18

ACCEPTED MANUSCRIPT
benzimidazolium N-atom is unfavorable and requires 27.56 kJ.mol-1 (6.59 kcal.mol-1) according to
the free energy difference ꢀG between 6a and the respective thione/carboxylate form (Fig. 19S in
Supplementary material). The rotation is associated to 36.54 kJ.mol-1 (8.73 kcal.mol-1) based on ꢀG
between 6a and the highest energy conformer identified by the PES for rotation around the C-S
bond (Fig. 20S in Supplementary material). Having in mind the small energy difference between the
tautomeric forms of 6 and low activation barrier, it can be concluded that the presence of two
crystal structures demonstrated by the PLM analysis is due to the prototropic properties of N1-
hydrogen atom in the benzimidazole ring. The molecular structure is stabilized by the formation of
a hydrogen bond between the N-atom from the benzimidazole ring and the hydroxy group (Fig. 7a).
Total (Mulliken) atomic charges: O = -0.327; H = 0.370;
H-bond RO...H = 1.91 Å
Total (Mulliken) atomic charges:
N = -0.173; H = 0.421;
H-bond RN...H = 1.75 Å.
(a)
(b)
Fig. 7. Hydrogen bonding in tautomer 6a (a) and tautomer 14a (b).
Formation of a hydrogen bond C=O…H-N is also possible, but less favorable. Nevertheless, ꢀG
between the corresponding 6-H (6c) and 7-H (6d) tautomer remains low 0.34-0.48 kJ.mol-1. Similar
results were also seen for 14a and 14b (ꢀG 2.03-3.26 kJ.mol-1). In this case the molecular structure
is stabilized by C=O…H-N interaction. The molecular configuration of the lower-energy tautomer
14a is presented in Fig. 7b.
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Based on the computational results, non-planar geometry of the investigated compounds was
identified. The computed distances from the carbonyl oxygen to the hydrogen atom of 1(H)-
benzimidazole cycle and from the hydroxyl group to the benzimidazolyl nitrogen atom are in the
range of 1.75 – 1.91 Å, hence the possibility for H-bond forming is obvious. The presence of H-
bond allows the formation of seven-member-pseudo-cycle, which additionally stabilizes the
preferred configuration.
The energy difference between the positional isomers of 10 is almost negligible, with 10a being
slightly more favorable in nonpolar medium, while 10b being preferred in polar medium.
4. Conclusions
Benzimidazolthiol derivatives were synthesized and the morphology of their crystal structure was
investigated microscopically in polarized light. The structures of the compounds were confirmed by
IR, 1H NMR, 13C NMR and MS spectral data and studied by use of Density Functional Theory. As a
result, the following conclusions were determined:
- According to DFT calculations the thione form of compounds 1 and 2 is more stable in nonpolar
and polar medium than the thiol form. By the use of polarizing microscopy two different crystal
structures were observed in solid phase for the compounds substituted in the benzimidazole ring;
- One distinct type of crystal structure was observed for each one of 5 or 6-methyl-(1H-
benzimidazol-2-ylthio)acetic acid 6 - dendritic and needle-shaped formations were determined in
polarized light. The derivative unsubstituted in the ring crystallized only in one phase;
- The compound 14, containing a substituent in the benzimidazole ring crystallized in two phases
(independent of the differences in the crystal structures obtained by the use of various solvents),
while by the unsubstituted compound 13 a separation of phases does not take place.
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- By the use of polarized light microscopy and the differential scanning calorimetry it was
confirmed that the cyclization of the thioacetic acids in pyridine medium resulted in a mixture of
two isomers 6 - and 7-substituted thiazolobenzimidazolones.
- The results of the examinations through microscopy in polarized light revealed that tautomeric
benzimidazoles have a different crystal structure.
6. Acknowledgement
This study was supported by the Science Fund of the University of Chemical Technology
and Metallurgy in Sofia, Grant number N 11252, and by the National Science Fund of Bulgaria,
Contract RNF01/0110. The authors declare that they have no conflict of interest.
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Tautomerism and isomerism in some antitrichinellosis active benzimidazoles: Morphological
study in polarized light, quantum chemical computations
Kameliya Anichina,a Anelia Mavrova,a Denitsa Yancheva,b,1 Jordan Tsenov,b Rasho Dimitrova
aUniversity of Chemical Technology and Metallurgy, 8Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113
Sofia, Bulgaria
Highlights
> Tautomeric benzimidazol-2-thiones crystallize in two different crystal structures
> The thione forms are more stable (∆E 51-55 kJ.mol-1) than the thiol forms
> Dendritic and needle-shaped formations are observed for benzimidazolthioacetic acids
> 6-Methyl-[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one crystallizes as fibrils
>7-Methyl-[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-one crystallizes as spherulites