Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity

Article abstract of DOI:10.1021/acs.chemrestox.9b00256

This paper discusses antioxidative and biological activities of 25 novel amidino substituted benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with a variable number of methoxy or hydroxy substituents. Targeted compounds, bearing either amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel aromatic and heteroaromatic benzamide derivatives. The compounds were tested for antioxidative activity using in vitro spectrophotometric assays. Direct validation of 3D-QSAR approach for predicting activities of novel benzamide derivatives was carried out by comparing experimental and computationally predicted antioxidative activity. Experimentally determined activities for all novel compounds were found to be within a standard deviation of error of the models. Following this, structure-activity relationships among the synthesized compounds are discussed. Furthermore, antiproliferative activity in vitro against HeLa cells as well as antibacterial and antifungal activity was tested to confirm the other biological activities of the prepared compounds.

Full text of DOI:10.1021/acs.chemrestox.9b00256

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Article
Biological potential of novel methoxy and hydroxy substituted
heteroaromatic amides designed as promising antioxidative
agents: Synthesis, 3D-QSAR analysis and biological activity
Irena Sovi#, Maja Cindri#, Nataša Perin, Ida Bo#ek, Irena Novakovic, Ana Damjanovi#,
Tatjana Stanojkovi#, Mario Zlatovi#, Marijana Hranjec, and Branimir Bertosa
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00256 • Publication Date (Web): 05 Aug 2019
Downloaded from pubs.acs.org on August 7, 2019
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Biological potential of novel methoxy and hydroxy substituted heteroaromatic amides
designed as promising antioxidative agents: Synthesis, 3D-QSAR analysis and biological
activity
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Irena Sović , Maja Cindrić , Nataša Perin , Ida Boček , Irena Novaković , Ana Damjanović ,
d
c
a
e*
Tatjana Stanojković , Mario Zlatović , Marijana Hranjec , Branimir Bertoša
a
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia
b
Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12,
PO Box 815, 11000 Belgrade, Serbia
c
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
d
Institute of Oncology and Radiology, Pasterova 14, 11000 Belgrade, Serbia
e
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-
1
0000 Zagreb, Croatia
*Corresponding author: Dr. Branimir Bertoša, Assist. Prof., Department of Physical Chemistry,
Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia, Phone
No. +38514606132; Fax No. +38514606131; e-mail: bbertosa@chem.pmf.hr;
Dr. Marijana Hranjec. Assoc. Prof., Department of Organic Chemistry, Faculty of Chemical
Engineering and Technology, University of Zagreb, Marulićev trg 20, P.O. Box 177, HR-10000
Zagreb, Croatia, Phone No. +38514597245; Fax No. +38514597250; e-mail : mhranjec@fkit.hr
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Abstract
This paper discusses antioxidative and biological activity of 25 novel amidino substituted
benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with
a variable number of methoxy and/or hydroxy substituents. Targeted compounds, bearing either
amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano
precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel
aromatic and heteroaromatic benzamide derivatives. The compounds were tested for
antioxidative activity using in vitro spectrophotometric assays. Direct validation of 3D-QSAR
approach for predicting activities of novel benzamide derivatives was carried out by comparing
experimental and computationally predicted antioxidative activity. Experimentally determined
activities for all novel compounds were found to be within standard deviation of error of the
models. Following this, structure-activity relationships among the synthesized compounds are
discussed. Furthermore, antiproliferative activity in vitro against HeLa cells as well as
antibacterial and antifungal activity was tested to confirm the other biological activities of the
prepared compounds.
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Key words: amides, benzimidazoles, benzothiazoles, 3D-QSAR, antioxidative activity,
antiproliferative activity in vitro, antimicrobial activity
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. Introduction
Significant biological importance of nitrogen heterocycles, particularly benzimidazoles
and benzothiazoles, makes them very useful building blocks in organic and medicinal
chemistry.1,2 They are an essential structural part in numerous natural and synthetic bioactive
compounds possessing a wide range of biological characteristics including antibacterial,4,5
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anticancer,6,7 antifungal, antiviral, antioxidative
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etc. In the last decade, particular
attention and focus was given to their antioxidative potency. It is widely accepted that reactive
oxygen species (ROS) could cause the oxidative damage of important biomolecules including
nucleic acids, proteins or lipids. These reactive species could be generated in numerous
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pathophysiological and biochemical processes in the human body. ROS includes non-free
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radical species like ozone (O ), singlet oxygen ( O ), hydrogen peroxide (H O ), and free
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radicals including peroxyl radicals (ROO ), hydroperoxyl radicals (HOO ), hydroxyl radicals
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(OH ) and superoxide anion radicals (O ). Consequently, it could lead to the development of
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some chronic diseases like cancer, diabetes, inflammation, neurodegenerative syndrome or
aging.13,14 The proper usage of antioxidants could regulate the rate of reactive oxygen species
production, at even at low concentration, they prevent oxidation of easily oxidizable substrate.15
Thus, there is a constant growing interest for the study of antioxidative capacity of numerous
natural and synthetic organic molecules with the aim of prevention of the above mentioned
diseases.
Recently, our scientific research focused on the antiproliferative activity of versatile
benzimidazole and benzothiazole derivatives.16,17,18 A large number of active derivatives are
designed to have cationic amidino substituents at the termini of the molecule, which has allowed
additional interactions with the biological targets. Additional biological activity studies of
tetracyclic benzimidazole derivatives have been confirmed their strong interaction with DNA
as intercalators, while acyclic, either benzimidazole or benzothiazole, derivatives have been
proven to be selective DNA minor groove binders.16,17,19 Recent literature data suggests that
tumor cells are accompanied by a measurable emission of ROS which might be regulated by
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apropriate usage of antioxidant agents. Several publications described antioxidative activity
and potential of benzimidazole and benzothiazole derivatives, as well as a broad range of
salicylanilide and benzamide derivatives, as antioxidative agents.21,22,23
In order to explore the antioxidative potency of aromatic and heteroaromatic benzamide
derivatives bearing benzazole nuclei, we developed 3D-QSAR models and used them for
computational prediction of antioxidative activity of the presented compounds.
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Application of 3D-QSAR modeling for prediction of activity of novel compounds and
identification of molecular properties with the highest influence on the studied activity was
already proven as an useful method numerous times.24,25,26,27,28,29 In addition, antioxidative
activity of different classes of compounds has been studied using different approaches of QSAR
modeling.30,31,32,33 The main advantage of Volsurf+34,35 based approach is usage of molecular
descriptors that are independent of molecular alignment and have a clear chemical or physical
meaning. In this paper, Volsurf+ approach for generating 3D-QSAR models was
experimentally validated on prediction of antioxidative activity of the 25 novel compounds.
Combining the demonstrated biological potential of benzamides and amidino substituted
benzimidazoles and benzothiazoles, we have designed and synthesized novel derivatives
bearing a variable number of the methoxy and hydroxy groups substituted with different
amidino substituents. Prepared derivatives were explored for their antioxidative, antimicrobial
and antiproliferative activity. The obtained experimental results were compared to the predicted
results and a very good alignment was observed.
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. Results and Discussion
.1. 3D-QSAR models
Using a library of experimental data for antioxidative activity36,37,38 of already published
compounds, 3D-QSAR models were built. Models with the highest predictive ability, namely
model 1 and model 2, were chosen for the prediction of antioxidative activities of novel
compounds. Model 1 was generated using the results obtained from the activity testing against
DPPH stable radical while model 2 has been generated using activity from superoxide test.
Quality of the models validated by internal cross-validation procedure, using two procedures,
leave one out (LOO) and random groups (LMO), as well as by the external prediction (Table
1, Figure 1). Both models show similar predictive ability (Q2 , Q2LMO, SDEPEXT in Table 1).
LOO
Statistical properties of the models are shown in Table 1. Compounds for which activity was
expressed only as “≥400 µM” were not included in the training set of the model 2 due to the
possible largely decreased quality of model.
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Table 1. Statistical properties of 3D-QSAR models.
Model
nO LV
R2
SDEC Q2
SDEP LOO Q2
SDEP LMO
SDEPEXT
nOE
LOO
LMO
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2
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a
Number of objects which was used to build the model (training set). b Number of latent
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variables. SDEC - standard deviation of error of calculation. Q is the cross-validated
predictive performance, LOO stands for Leave One Out cross-validation procedure, LMO
e
stands for Leave Many Out (i.e. Random Groups) cross-validation procedure. SDEP -
f
standard deviation of error of prediction obtained by cross-validation. SDEP
stands for
EXT
standard deviation of error of prediction obtained by external validation. g Number of objects
used for external prediction validation (test set).
A)
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Figure 1. Predicted vs experimental antioxidative activity expressed as pIC50 – negative
logarithmic value of concentration that causes 50% of antioxidative activity measured: A)
against DPPH stable radical (model 1) and B) using superoxide test (model 2). Blue circles
present training set compounds; red squares present test set compounds.
2
.1.2. 3D-QSAR analysis
Principal component analysis (PCA) was performed on the overall dataset of 59
compounds. The first two principal components explained 95% of variance (the first three PCs
explained 98% of variance) of the descriptors (X-matrix). Molecular descriptors including
hydrophilic (W1-W3) and hydrophobic regions (D1) related to the polarizability and dispersion
forces, molecular volume (V), and possibility of accepting (WN1) and donating H-bonds
(
WO1) were identified, from the PCA loadings plot (Figure S63 in the Supplement), as the ones
with the highest variation among the dataset compounds
These results indicated to the mentioned descriptors as the ones with the highest
variation between the dataset compounds which might be related to the variation in
antioxidative activities of the dataset compounds. The PLS analysis could estimate the
quantitative impact of each descriptor on studied activities.
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The influence of each molecular descriptor to the compound’s antioxidative activity
could be evaluated from the analyses of the PLS coefficients of the obtained 3D-QSAR. Since
both models were generated using raw data, the real impact of a molecular descriptor on the
antioxidative activity is given as the product of the descriptor’s value and it’s PLS coefficient.
Such QSAR analysis performed for both models (Figure 2), enabled the finding of molecular
properties with the highest positive or negative impact on antioxidative activity.
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HSA
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WN1
Figure 2. Products of the descriptor’s average value calculated for the dataset used to derive
model and the associated PLS coefficient of 3D-QSAR model for: A) model 1 and B) model 2.
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Descriptors with the highest positive influence on antioxidative activity for both models
have proven to be hydrophilic volume related to polarizability (W1) and possibility of a
compound to donate H-bond (WO1). Consequently, their increase should lead to increase of
activity measured by both tests. Possibility of a compound to accept H-bond (WN1) and sum
of hydrophobic surface areas (HSA) are negatively correlated for both models and their
decrease should cause the increase of antioxidatve activity in both tests.
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.2. Chemistry
All chosen amide derivatives were synthesized following two synthetic procedures
shown in Scheme 1 and 2.39 Cyano substituted benzamides 4-6 and 2-benzimidazolyl/2-
benzothiazolyl substituted benzamides 20-25 were obtained from the methoxy substituted acyl-
halogenides 1-3 and p-cyanoaniline 2 or cyano substituted 2-aminobenzimidazole 18 or 2-
aminobenzothiazole 19 prepared by previously published synthetic procedures.40
R2
R1
R2
R1
O
Et N
3
R3
COCl + H N
CN
R3
2
Tolueneabs.
HN
CN
R4
2
R4
4
R = OCH ; R , R , R = H
1 3 2 3 4
1
2
R = OCH ; R , R , R = H
1 3 2 3 4
5 R , R = OCH ; R , R = H
6
1
3
3
2
4
R , R = OCH ; R , R = H
1
3
3
2
4
R = H; R , R , R = OCH
1 2 3 4 3
3 R = H; R , R , R = OCH
1
2
3
4
3
BCl or BBr3
3
CH Cl
2
2abs.
R2
R1
R2
R1
O
O
1. HCl(g)/EtOHabs.
R3
R3
2. NH (g)/EtOH
HN
R5
3
abs.
HN
CN
or
R4
EDA/EtOHabs.
R4
7 R = OH; R , R , R = H
1
2
3
4
1
1
1
1 R = OH; R , R , R = H
NH2+Cl-
1
2
3
4
8 R = OH; R = OCH , R , R = H
1 3 3 2 4
2 R = OH; R = OCH ; R , R = H R5 =
9 R , R = OH; R , R = H
1 3 2 4
1
3
3
2
4
NH2
10 R = H; R , R , R = OH
3 R , R = OH; R , R = H
1 2 3 4
1
3
2
4
H
N
14 R = OH; R , R , R = H
1
2
3
4
1
1
1
5 R = OH; R = OCH ; R , R = H
R =
5
1
3
3
2
4
6 R , R = OH; R , R = H
N
H Cl
1
3
2
4
+
-
7 R = H; R , R , R = OH
1
2
3
4
Scheme 1.
In order to obtain corresponding hydroxy substituted benzamides 7-10 and 26-29 bearing cyano
substituent, the removal of methoxy protecting groups was accomplished by using either boron
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2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
trichloride or boron tribromide in absolute dichloromethane at -75 °C. Reaction yields of all
compounds were moderate.
By using boron trichloride, only one methoxy group has been removed from the
compounds 5, 21 and 24 which allowed the synthesis of compounds 8, 27 and 29 with one
methoxy and one hydroxy group. 2D NOESY NMR spectra confirmed that the methoxy group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
at the position 2 (R ) was successfully deprotected. Target compounds with variable number of
1
hydroxy 11-17 and 30-37 and/or methoxy groups 38-47 bearing amidino or 2-imidazolinyl
substituent were prepared in acidic twostep Pinner reaction from corresponding cyano
precursors due to reaction with NH3(g) or ethylenediamine (EDA).
R2
R1
R2
R1
CN
X
N
O
Et N
3
R3
COCl + H2N
CN
R3
X
N
Tolueneabs.
HN
R4
18 X = NH
9 X = S
R4
1
20 X = NH; R = OCH ; R , R , R = H
1 X = NH; R , R = OCH ; R , R = H
2 X = NH; R = H; R , R , R = OCH
3
3 X = S; R = OCH ; R , R , R = H
1
R = OCH ; R , R , R = H
1
3
2
3
4
1
3
2
3
4
2
2
2
2
2 R , R = OCH ; R , R = H
1 3 3 2 4
1
3
3
2
4
3
R = H; R , R , R = OCH
1
2
3
4
1
2
3
4
3
1
3
2
3
4
4 X = S; R , R = OCH ; R , R = H
1
3
3
2
4
R2
R1
25 X = S; R = H; R , R , R = OCH
1 2 3 4 3
O
CN
BCl or BBr
3 3
R3
X
N
CH Cl
1. HCl(g)/EtOHaps
2. NH (g)/EtOH
2
2abs.
HN
3
aps
R4
or
2
2
2
2
6 X = NH; R = OH; R , R , R = H
EDA/EtOHaps
1
2
3
4
7 X = NH; R = OH; R = OCH ; R , R = H
1
3
3
2
4
8 X = S; R = OH; R , R , R = H
1
2
3
4
R2
R1
9 X = S; R = OH; R = OCH ; R , R = H
1
3
3
2
4
O
1
2
. HCl(g)/EtOHaps
R5
R3
X
. NH (g)/EtOH
3
aps
HN
or
EDA/EtOHaps
R4
N
R2
R1
3
3
40
8 X = NH; R , R = OCH ; R , R = H
1
3
3
2
4
O
9 X = NH; R = H; R , R , R = OCH
3
NH2+Cl-
1
2
3
4
R5
R3
X
N
X = S; R = OCH ; R , R , R = H
R =
5
1
3
2
3
4
NH2
HN
41 X = S; R , R = OCH ; R , R = H
1 3 3 2 4
4
2 X = S; R = H; R , R , R = OCH
1 2 3 4 3
R4
4
3 X = NH; R , R = OCH ; R , R = H
1 3 3 2 4
3
3
3
3
0 X = NH; R = OH; R , R , R = H
1
2
3
4
H
N
NH2+Cl-
44 X = NH; R = H; R , R , R = OCH
3
45 X = S; R = OCH ; R , R , R = H
1 X = NH; R = OH; R = OCH ; R , R = H
1
2
3
4
1
3
3
2
4
R =
R =
5
5
2 X = S; R = OH; R , R , R = H
1
3
2
3
4
1
2
3
4
NH2
N
H Cl
+
-
4
6 X = S; R , R = OCH ; R , R = H
3 X = S; R = OH; R = OCH ; R , R = H
1
3
3
2
4
1
3
3
2
4
47 X = S; R = H; R , R , R = OCH
3
1
2
3
4
3
3
3
3
4 X = NH; R = OH; R , R , R = H
1 2 3 4
5 X = NH; R = OH; R = OCH ; R , R = H
6 X = S; R = OH; R , R , R = H
7 X = S; R = OH; R = OCH ; R , R = H
H
N
1
3
3
2
4
R =
5
1
2
3
4
N
+
-
H Cl
1
3
3
2
4
Scheme 2.
The progress of the reaction was monitored by IR spectroscopy. In order to ensure their better
water solubility, all amidino substituted derivatives were prepared as hydrochloride salts. Due
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to some synthetic issues in the Pinner reaction, monomethoxy substituted analogues of amidino
compounds 38-47 have not been successfully synthesized.
2.3. Antioxidative activity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results obtained from DPPH test revealed that all tested compounds displayed improved
or similar activity in comparison to used standard, ascorbic acid (Table 2).
Table 2. Antioxidative activity of tested amidino substituted compounds.
Compound
11
DPPH (μM)
Superoxide test (μM)
59±4
66±5
39±3
137±4
158±8
123±4
1
1
2
3
1
1
1
1
4
5
6
7
89±6
70±6
51±6
42±5
170±8
156±7
138±5
133±7
3
3
3
0
1
2
42±3
38±3
49±4
46±7
56±5
65±6
80±3
58±3
59±6
52±3
135±4
130±5
139±5
141±6
148±6
163±4
166±3
154±5
142±8
145±4
33
3
4
35
3
3
3
3
6
7
8
9
40
66±7
73±4
57±2
149±7
171±7
151±6
4
4
1
2
4
4
3
4
71±5
85±4
163±7
180±9
45
82±4
94±2
73±4
79±3
169±5
195±9
172±8
95±2
4
4
6
7
Ascorbic acid
Interestingly, a regularly behavior was noticed in the pairs of tested bioisosteric compounds.
The radical scavenging test for the compounds with amidino moiety always resulted in ehanced
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activity when compared to compound having imidazole moiety in the same position (pairs are
as goes: 11 and 14, 30 and 34, 32 and 36, 40 and 45, 12 and 15, 31 and 35, 33 and 37, 38 and
4
3, 41 and 46, 13 and 16, 39 and 44, 42 and 47).
According to the presented results, the compound with the highest antioxidative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
potential has proven to be 13, with the activity almost 50% better in comparison to the control
compound (ascorbic acid). The pair of compounds including 13 and 16, is the only one having
two hydroxy groups in positions 2 and 4, and thus has a highest potential for radical transfer to
stable DPPH radical and still maintain his stability by resonance stabilization. Compared to
compound 13, its bioisosteric compounds 11, having hydroxy group on first phenyl ring in
position 2, and compound 12, with hydroxy and methoxy groups placed in positions 2 and 4
respectively, showed significantly lower activity, although still better than control compound.
From this result we can conclude that hydroxy groups are important for activity in order to
promote radical scavenging activity. This is in accordance with QSAR analysis showing that
the values of descriptors WO1 and W1 are higher for compounds 11-13 comparing to ascorbic
acid, while the opposite is true for the descriptors WN1 and HSA (Figure 3).
In second performed antioxidative test, which included measuring capability of compounds for
superoxide anion radical scavenging, all compounds showed lower activity compared to
ascorbic acid. Nevertheless, compound 13 is the most active in this test as well.
1400
average
13
11
12
Ascorbic acid
1
1
200
000
8
6
00
00
400
2
00
0
W1
WO1
WN1
HSA
Figure 3. Comparison of values of the descriptors with the highest influence on activity for the
compounds 11-13, average value and ascorbic acid.
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2
.4. Comparison of predicted and experimental antioxidative activities
Internal validation using cross validation procedure, as well as validation by external
prediction, are the most often used methods for validation of QSAR models.
A)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
60
40
120
100
80
6
4
2
0
0
0
0
1
1 12 13 14 15 16 17 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Compound
B)
250
2
1
00
50
100
5
0
0
1
1 14 30 34 32 36 40 45 12 15 31 35 33 37 38 43 41 46 13 16 17 39 44 42 47
Compound
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 4. Comparison of experimental (orange squares) and computationally predicted (blue
circles) antioxidative activities of the synthetized compounds expressed as IC . Error bars
5
0
coloured blue on the blue circles present standard deviation of error of prediction (SDEP) of
the 3D-QSAR model used to predict activities: A) model 1 and B) model 2, while orange error
bars on orange squares present standard deviation of experimental measurements.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
However, the experimental measurements of the activities of novel compounds whose activities
were previously predicted by the QSAR model should be the most direct and reliable method
for validation of prediction ability of built QSAR model. The antioxidative activities of 25 novel
compounds (11-17 and 30-47), were predicted by using model 1 and model 2. Therefore,
experimental measurements of their antioxidative activity enabled direct validation of the
obtained 3D-QSAR models (Table 3 and Figure 4). Antioxidative activities of all 25 newly
synthesised compounds are correctly predicted by both models i.e. experimentally measured
activities for all novel compounds are within standard deviation of error of prediction (SDEP)
of the models estimated by cross-validation procedure (Figure 3). Correlation coefficients (R2)
between experimental and predicted values of antioxidative activities were 0.82 and 0.78 for
the models 1 and 2, respectively. Furthermore, comparison of experimental and predicted
activities shows that for majority of the novel compounds (18 out of 25) predicted antioxidative
activity is within experimental error (Figure 4). For 7 compounds for which discrepancy of
predicted and experimental activity is larger than standard deviation of experimental
measurements, predicted values are smaller than experimental, showing that models slightly
underestimated antioxidative activity of these compounds.
2
.4. Antiproliferative, antibacterial and antifungal activity
Additionally, to explore and confirm the biological potential of newly prepared
compounds, antiproliferative activity in vitro, antibacterial and antifungal activities were
evaluated. As the antiproliferative potency of suchlike compounds was previously confirmed
in our published papers, the synthesized compounds were tested against HeLa cancer cells.
Obtained results are depicted in the Table 3 while cisplatin was used as a standard anticancer
drug. Obtained results indicated that the majority of tested compounds showed moderate to low
activity at micromolar inhibition concentrations.
In general, it could be concluded that benzothiazole derivatives showed stronger
antiproliferative activity in comparison to their benzimidazole analogues with the exception of
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2
,4-dimethoxy substituted benzimidazole 43 bearing 2-imidazolinyl group. Thus, the strongest
antiproliferative activity against HeLa cells displayed 2-hydroxy 36 (IC = 23.0 μM) and 2,4-
5
0
dimethoxy 45 (IC = 13.6 μM) substituted benzothiazoles bearing 2-imidazolinyl group and
5
0
2
-methoxy 40 (IC50 = 20.1 μM) and 3,4,5-trimethoxy 42 (IC50 = 32.8 μM) substituted
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzothiazoles bearing amidino substituent. All tested amidino substituted benzamides 11-17
showed very low antiproliferative activity.
Table 3. Antiproliferative activity in vitro against HeLa cells.
Comp.
IC50 (µM)
>200
1
1
1
2
>200
13
172.9±15.44
>200
1
4
15
>200
1
1
3
3
6
7
0
1
195.1±6.99
78.0±2.22
167.2±10.93
133.6±12.25
192.0±7.99
41.4±5.15
194.6±7.65
>200
32
3
3
3
3
3
4
5
6
23.0±0.40
>200
37
3
3
8
9
129.2±5.61
175.6±1.28
20.1±5.71
>200
4
4
0
1
42
32.8±0.21
40.9±7.57
117.4±10.90
13.6±1.24
>200
4
3
44
4
4
4
5
6
7
55.4±0.54
2.5±0.24
Cisplatin
Furthermore, the in vitro antibacterial activity of the prepared compounds was evaluated against
eight different bacterial strains fully sensitive to antibiotics. Gram-positive bacterial strains
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
included key representatives of human pathogens including S. aureus, M. luteus, B. subtilis and
C. sporogenes, while the panel of Gram-negative bacteria included E. coli, P. hauseri, P.
aeruginosa and K. pneumonia. Obtained results were displayed in Table 4 as the minimal
inhibitory concentrations (MICs) and are compared to antibiotic amikacin used as a standard.
In Table 4 are shown only results for compounds which showed some activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Antibacterial activity of tested compounds.
MIC (mM)
Comp.
11
P.
P.
K.
M.
S.
B.
C.
E. coli
hauseri aeruginosa
pneumoniae luteus aureus subtilis sporogenes
4.23
2.03
0.89
3.46
7.17
6.99
0.37
3.11
1.39
0.012
2.14
2.03
0.89
3.46
7.17
3.50
0.37
6.22
1.39
0.085
2.14
2.03
1.77
3.46
7.17
6.99
0.77
6.22
0.70
0.009
2.14
4.06
0.89
3.46
7.17
6.99
0.77
6.22
1.39
0.003
4.23
4.06
1.77
6.91
7.17
6.99
0.77
6.22
1.39
0.014
2.14
2.03
1.77
6.91
7.17
6.99
0.77
3.11
1.39
0.019
0.13
1.02
0.89
3.46
7.17
6.99
0.37
1.56
0.70
0.072
0.27
2.03
0.89
3.46
7.17
3.50
1.54
0.78
1.39
0.026
1
1
3
7
31
3
2
34
3
4
4
9
3
7
Amikacin
The obtained results revealed that the compound with the most significant antibacterial
activity proved to be 3,4,5-trimethoxy substituted benzimidazole derivative 39 bearing amidino
group (MICs value 0.37-1.54 µg/mL) without conspicuous selectivity between Gram-negative
and Gram-positive bacteria. The highest activity was observed against P. hauseri, P. aeruginosa
and B. subtilus bacteria with MIC value 0.37 µg/mL. 3,4,5-trihydroxy substituted benzamide
derivative 17 bearing 2-imidazolinyl group showed also promising antibacterial activity with
MICs values 0.89-1.77 µg/mL. Interestingly, compounds 36, 40, 42 and 45 which showed the
most pronounced antiproliferative activity were not active on tested bacterial strains at all. In
addition, the antifungal activity of prepared compounds was also tested against tree strains
including C. albicans, A. brasiliensis and S. cerevisiae. The obtained results for compounds
which displayed some activity are presented in Table 5 including the nystatin which was used
as a standard antifungal drug.
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Table 5. Antifungal activity of tested compounds.
MIC (mM)
Comp.
11
C. albicans
A. brasiliensis
S. cerevisiae
0.54
0.25
0.26
0.90
0.12
0.19
0.22
0.90
0.82
0.11
0.21
0.19
0.11
0.20
0.18
0.20
0.69
2.70
1.07
1.94
8.12
1.80
0.94
0.94
1.73
1.79
3.30
1.75
0.83
1.54
3.45
0.78
1.44
3.21
1.34
1.35
0.54
0.25
0.51
0.45
0.12
0.19
0.11
1.79
0.41
0.11
0.11
0.39
0.43
0.20
0.18
0.10
0.35
1.35
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
3
1
1
5
6
17
3
1
32
3
3
3
3
3
4
8
9
40
4
4
3
4
4
4
5
7
Nystatin
In general, obtained results revealed that some tested compounds showed better and
improved antifungal activity in comparison to antibacterial activity. The most pronounced and
significantly improved antifungal activity of tested compounds was observed against C.
albicans as well as against S. cerevisiae strains compared to the standard drug nystatin. Only
2-hydroxy substituted benzothiazole derivative bearing amidino group 32 showed weaker
antifungal activity against S. cerevisiae in comparison to nystatin. The most active compounds
with high antifungal potential have proven to be 2-hydroxy substituted benzimidazole 34 and
2
,4-dihydroxy substituted benzamide 16 both bearing 2-imidazolinyl group with MICs values
0.11 and 0.12 µg/mL. Also, among all compounds showing antifungal activity, benzimidazole
derivatives displayed more pronounced antifungal activity in comparison to their benzothiazole
analogues. In conclusion, it is obvious that this type of amidino substituted benzimidazole and
benzothiazole derivatives have a high potential for further optimization and design of very
efficient antifungal agents.
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. Conclusion
Within this paper, we present the design and synthesis of 25 novel amidino substituted
benzamides that differ in the heteroaromatic nuclei attached to the benzamide moiety as well
as in the number of methoxy and hydroxy groups. All synthesized benzamide derivatives, were
also, besides with methoxy and hydroxy groups, substituted with either amidino or 2-
imidazolinyl group and are prepared within the acidic Pinner reaction from corresponding
cyano substituted precursors. In order to ensure better solubility, all amidino derivatives were
prepared as their hydrochloride salts.
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For prediction of the antioxidative activities of 25 novel compounds, 3D-QSAR models
were generated and validated. Comparison of computationally predicted and experimentally
obtained activities indicated that 3D-QSAR method can be successfully applied for design of
novel methoxy and hydroxy substituted heteroaromatic amides as well as to successfully predict
their antioxidative potential.
The experimental evaluation of antioxidative activity revealed that hydroxy groups are
essential and promote radical scavenging activity. The most pronounced antioxidative activity
was observed for the compound 13. DPPH test showed that all novel compounds showed
improved or similar antioxidative activity compared to standard, ascorbic acid. Furthermore,
the majority of tested compounds displayed moderate to low antiproliferative activity at
micromolar inhibitory concentrations. In general, it could be concluded that benzothiazole
derivatives showed stronger antiproliferative activity in comparison to their benzimidazole
analogues. The strongest antiproliferative activity against HeLa cancer cells showed the
compound 45. Additionally, to confirm the great biological potential of suchlike derivatives,
the antibacterial and antifungal activities were evaluated. The compound with the most
significant in vitro antibacterial activity proved to be 3,4,5-trimethoxy substituted
benzimidazole derivative 39. Antifungal activity of the tested compounds was significantly
better than their antibacterial activity, while the majority of tested compounds showed improved
activity in comparison to standard.
To conclude, all presented results of biological activities evaluation, confirmed the high
biological potential of amidino substituted benzimidazole and benzothiazole derivatives, with
several compounds which could be chosen as lead compounds for a further structure
modification and optimization of suchlike scaffold in order to developed more efficient
biologically active molecules with enhanced antioxidant potential.
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. Experimental part
4.1. 3D-QSAR modelling
.1.1. Dataset
D-QSAR models were generated using the data on antioxidative activities of methoxy
4
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and hydroxy substituted heteroaromatic amides found in the literature.41,42 For each compound,
antioxidative activities were measured using two tests, DPPH stable radical and superoxide
tests, and expressed as IC values i.e. concentration that causes 50% antioxidative activity. For
5
0
purpose of generating 3D-QSAR models, pIC (negative logarithm of IC value expressed in
5
0
50
-3
mol dm ) were calculated. Dataset for model 1, generated using activities against DPPH stable
radical, consisted of 59 compounds from the literature, while dataset for model 2, generated
using activities obtained with superoxide test, consisted of 32 compounds from the literature.
4
.1.2. Molecular descriptors
Molecular descriptors for each compound were generated with program Volsurf+.33,34
Starting from the SMILES codes, 3D structures were generated using VolSurf+ 3D structure
generator. Considering high rigidity of the dataset compounds, VolSurf+ 3D structure generator
was considered to be powerful enough to produce the most relevant conformers and no
additional conformational search was applied. Using following probes: H2O (the water
2
molecule), O (sp carbonyl oxygen atom), N1 (neutral NH group (e.g. amide)), and DRY (the
hydrophobic probe), Molecular Interaction Fields (MIFs) were calculated. From the MIFs, 128
descriptors with clear chemical and physical meaning were calculated for each compound. The
detailed description of all 128 VolSurf+ descriptors is given in the VolSurf+ manual.35,36
4
.1.3. Partial Least Square and Principal Component analysis
Partial Least Square (PLS) analysis was applied with goal of finding the relationship
between the 3D structure-based molecular descriptors and antioxidative activities. The number
of significant latent variables (nLV) and quality of the models were determined using the leave-
one-out (LOO) cross-validation procedures and leave many out (LMO) cross-validation
procedure with eight random groups. Also, for each model, standard deviation of error of
calculation (SDEC) and standard deviation of error of prediction (SDEP) were calculated.
Validation of predictive ability of each model was also performed using external validation.
For that purpose, dataset was divided into the training set, that was used to build the model, and
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the test set, that was used to validate predictive ability of the model. Standard deviation of error
of prediction (SDEP) for external prediction was calculated for each model.
In order to identify molecular descriptors with the highest positive or negative impact
on antioxidative activities of studied compounds, analyses of PLS coefficients of the 3D-QSAR
models were performed. Models were built using raw data (without autoscaling pretreatment
of descriptors) and the quantitative influence of each descriptor on the anti-proliferative activity
is given as product of the descriptor’s value and it’s PLS coefficient.
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Principal Component Analysis (PCA) was performed on the molecular descriptors of
the complete dataset (all 59 compounds). PCA loadings were used for identification of the
descriptors with the highest contribution to the overall variance in the X-space.
4
.2. Synthesis
4.2.1. General Methods
All chemicals and solvents were purchased from commercial available suppliers
including Aldrich, Fluka and Acros. Melting points were recorded on an Original Keller
Mikroheitztisch apparatus (Reichert, Wien), SMP11 Bibby and Büchi 535 apparatus and are
1
13
not corrected. H and C NMR spectra were recorded on a Varian Gemini 300 or Varian Gemini
00 spectrophotometers at 300, 600, 150 and 75 MHz, respectively. All NMR spectra were
6
measured in DMSO-d solutions using TMS as an internal standard. Chemical shifts are
6
reported in ppm (δ) relative to TMS. All compounds were routinely checked by thin layer
chromatography (TLC) using precoated Merck silica gel 60F-254 plates and the spots were
detected under UV light (254 nm). Column chromatography (CC) was performed using silica
gel (0.063–0.2 mm) Fluka; glass column was slurry-packed under gravity.
4
.2.2. General method for the preparation of carboxamides 20-25
To a solution of mono-, di- or tri-methoxy substituted benzoyl-chloride in dry toluene
corresponding amine was added followed by the addition of Et N. The reaction mixture was
3
refluxed for 24 hours. After cooling the resulting product was filtered off and washed with
diluted HCl and water to obtain pure solid.
2-Methoxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide 20
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Following the general method, from 0.32 g (1.9 mmol) 2-methoxybenzoylchloride 1, 0.30 g
(1.9 mmol) 2-amino-5(6)-cyano-1H-benzimidazole 18 and 0.27 g (2.7 mmol) Et N in dry
3
1
toluene (15 ml), 0.47 g (86%) of white powder was obtained; mp 234–237 °C; H NMR (300
MHz, DMSO-d ) (δ/ppm): 11.55 (s, 1H, NHamide), 7.92 (s, 1H, Harom), 7.79 (dd, J = 7.6 Hz, J
2
6
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1
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=
1.6 Hz, 1H, Harom), 7.63 (t, J = 7.2 Hz, 2H, Harom), 7.55 (t, J = 8.2 Hz, 1H, Harom), 7.25 (d, J
1
3
= 8.4 Hz, 1H, Harom), 7.13 (t, J = 7.5 Hz, 1H, Harom), 3.96 (s, 3H, CH ); C NMR (150 MHz,
3
DMSO-d ) (δ/ppm): 165.1, 157.1, 148.6, 133.6, 130.2, 125.1, 121.9, 120.7 (2C), 120.2, 112.3
6
(2C), 56.2.
2-Methoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 23
Following the general method, from 0.29 g (1.7 mmol) 2-methoxybenzoylchloride 1, 0.30 g
(
1.7 mmol) 2-amino-6-cyanobenzothiazole 19 and 0.24 g (2.4 mmol) Et N in dry toluene (15
3
1
ml), 0.40 g (77%) of white powder was obtained; mp 238–241 °C; H NMR (300 MHz, DMSO-
d6) (δ/ppm): 12.40 (s, 1H, NHamide), 8.62 (d, J = 1.1 Hz, 1H, Harom), 7.92 (d, J = 8.4 Hz, 1H,
Harom), 7.86 (dd, J = 8.4 Hz, J = 1.6 Hz, 1H, H ), 7.75 (dd, J = 7.7, J = 1.7 Hz, 1H, Harom),
1
2
arom
1
2
7
3
1
2
.62 (t, J = 7.0 Hz, 1H, Harom), 7.25 (d, J = 8.3 Hz, 1H, Harom), 7.13 (t, J = 7.5 Hz, 1H, Harom),
1
3
.95 (s, 3H); C NMR (75 MHz, DMSO-d ) (δ/ppm): 165.9, 162.1, 157.8, 152.2, 134.4, 132.8,
6
30.8, 130.1, 127.6, 121.7, 121.2, 119.6, 112.8, 105.9, 56.6.
,4-Dimethoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 24
Following the general method, from 1.94 g (9.7 mmol) 2,4-dimethoxybenzoylchloride 2, 1.7 g
(9.7 mmol) 2-amino-6-cyanobenzothiazole 19 and 1.37 g (13.6 mmol) Et N in dry toluene (30
3
1
ml), 2.58 g (78%) of white powder was obtained; mp > 280 °C; H NMR (300 MHz, DMSO-
d6) (δ/ppm): 11.89 (s, 1H, NHamide), 8.60 (d, J = 1.0 Hz, 1H, Harom), 7.93 – 7.82 (m, 3H, Harom),
13
6
.78 – 6.70 (m, 2H, Harom), 4.00 (s, 3H, CH ), 3.88 (s, 3H, CH ); C NMR (150 MHz, DMSO-
3
3
d6) (δ/ppm): 164.5, 164.1, 161.6, 159.4, 151.7, 132.7, 132.4, 129.6, 127.0, 121.1, 119.2, 112.5,
1
06.5, 105.3, 98.7, 56.5, 55.8.
3
,4,5-Trimethoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 25
Following the general method, from 2.95 g (13.0 mmol) 3,4,5-trimethoxybenzoylchloride 3,
2
.24 g (13.0 mmol) 2-amino-6-cyanobenzothiazole 19 and 1.88 g (13.0 mmol) Et N in dry
3
1
toluene (30 ml), 3.30 g (69%) of white powder was obtained; mp 251–253 °C; H NMR (600
MHz, DMSO-d ) (δ/ppm): 13.16 (brs, 1H, NHamide), 8.62 (s, 1H, Harom), 7.92 (d, J = 8.3 Hz,
6
1H, Harom), 7.87 (dd, J = 8.4 Hz, J = 1.5 Hz, 1H, Harom), 7.54 (s, 2H, Harom), 3.90 (s, 6H,
1
2
1
3
OCH ), 3.77 (s, 3H, OCH ); C NMR (75 MHz, DMSO-d ) (δ/ppm): 165.7, 163.3, 153.3 (2C),
3
3
6
1
42.0, 130.2, 127.6, 126.5, 121.6, 119.7, 106.5 (2C), 105.9, 60.6, 56.6 (2C).
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.2.3. General method for the preparation of hydroxy substituted compounds 7-10 and
26-29
Corresponding methoxy substituted compound was dissolved in dry dichloromethane,
cooled to -78 °C and BCl or BBr was added under argon atmosphere. The reaction mixture
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was stirred on that temperature for several hours and kept refrigerated overnight. The reaction
was quenched by the addition of methanol, solvent was evaporated to dryness and the residue
was purified by column chromatography (CH Cl /CH OH).
2
2
3
2-Hydroxy-N-(4-cyanophenyl)benzamide 7
Following the general method, from 0.37
g
(1.5 mmol) 2-methoxy-N-(4-
cyanophenyl)benzamide 4 and 5.0 ml BCl (2.2 mmol) in absolute dichloromethane (20 ml)
3
1
under the argon to obtain 0.31 g (85%) of white powder; mp 177–180 °C; H NMR (300 MHz,
DMSO-d ) (δ/ppm): 11.38 (brs, 1H, OH), 10.65 (s, 1H, NHamide), 7.94 (d, J = 8.8 Hz, 2H, Harom),
6
7
.88 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H, Harom), 7.83 (d, J = 8.8 Hz, 2H, Harom), 7.45 (m, 1H,
1 2
13
Harom), 7.02 – 6.95 (m, 2H, Harom); C NMR (150 MHz, DMSO-d ) (δ/ppm): 166.5, 157.5,
6
1
42.7, 133.7, 133.2 (2C), 129.5, 120.4 (2C), 119.2, 119.0, 118.5, 117.1, 105.6.
2
-Hydroxy-4-methoxy-N-(4-cyanophenyl)benzamide 8
Following the general method, from 0.50 g (1.8 mmol) 2,4-dimethoxy-N-(4-
cyanophenyl)benzamide 5 and 10.0 ml BCl (5.3 mmol) in absolute dichloromethane (30 ml)
3
1
under the argon to obtain 0.21 g (44%) of beige powder; mp 222–225 °C; H NMR (600 MHz,
DMSO-d ) (δ/ppm): 11.99 (brs, 1H, OH), 10.50 (s, 1H, NHamide), 7.95 (d, J = 8.0 Hz, 1H, Harom),
6
7
.92 (d, J = 9.1 Hz, 1H, Harom), 7.82 (d, J = 8.8 Hz, 1H, Harom), 6.58 (dd, J = 8.9 Hz, J = 2.4
1 2
13
Hz, 1H, Harom), 6.52 (d, J = 2.4 Hz, 1H, Harom), 3.80 (s, 3H, OCH ); C NMR (150 MHz,
3
DMSO-d ) (δ/ppm): 167.0, 164.0, 161.0, 142.7, 133.1 (2C), 130.7, 120.7 (2C), 119.0, 109.5,
6
1
06.5, 105.5, 101.3, 55.5.
2
,4-Dihydroxy-N-(4-cyanophenyl)benzamide 9
Following the general method, from 0.50 g (1.8 mmol) 2,4-dimethoxy-N-(4-
cyanophenyl)benzamide 5 and 10.0 ml BBr (2.7 mmol) in absolute dichloromethane (30 ml)
3
1
under the argon to obtain 0.06 g (13%) of beige powder; mp 250–253 °C; H NMR (300 MHz,
DMSO-d ) (δ/ppm): 11.87 (brs, 1H, OH), 10.44 (s, 1H, NHamide), 10.26 (brs, 1H, OH), 7.91 (d,
6
J = 8.8 Hz, 2H, Harom), 7.87 (d, J = 8.7 Hz, 1H, Harom), 7.81 (d, J = 8.7 Hz, 2H, Harom), 6.41 (dd,
1
3
J = 8.6 Hz, J = 2.3 Hz, 1H, Harom), 6.38 (d, J = 2.3 Hz, 1H, Harom); C NMR (75 MHz, DMSO-
1
2
d6) (δ/ppm): 167.5, 163.3, 161.4, 143.3, 133.6 (2C), 131.5, 121.0 (2C), 119.5, 108.7, 108.2,
05.8, 103.3.
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,4,5-Trihydroxy-N-(4-cyanophenyl)benzamide 10
Following the general method, from 0.50 g (1.6 mmol) 3,4,5-trimethoxy-N-(4-
cyanophenyl)benzamide 6 and 15.0 ml BBr (15.0 mmol) in absolute dichloromethane (30 ml)
3
1
under the argon to obtain 0.31 g (71%) of white powder; mp 273–275 °C; H NMR (300 MHz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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3
4
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7
8
9
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2
3
4
5
6
7
8
9
0
DMSO-d ) (δ/ppm): 10.30 (s, 1H, NHamide), 9.22 (s, 2H, OH), 8.91 (s, 1H, OH), 7.97 (d, J = 8.8
6
1
3
Hz, 2H, Harom), 7.78 (d, J = 8.7 Hz, 2H, Harom), 6.97 (s, 2H, Harom); C NMR (75 MHz, DMSO-
d6) (δ/ppm): 166.6, 146.0, 144.4, 137.8, 133.5 (2C), 124.7, 120.4 (2C), 119.7, 107.9 (2C), 105.2.
2
-Hydroxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide 26
Following the general method, from 0.50 g (1.7 mmol) 2-methoxy-N-[5(6)-cyano-1H-
benzimidazol-2-yl]benzamide 20 and 10.0 ml BBr (10.0 mmol) in absolute dichloromethane
3
1
(25 ml) under the argon to obtain 0.35 g (74%) of white powder; mp > 280 °C; H NMR (300
MHz, DMSO-d ) (δ/ppm): 12.91 (brs, 2H, NH
, OH), 8.02 (dd, J = 7.7 Hz, J = 1.3
1 2
6
benzimidazole
Hz, 1H, Harom), 7.90 (s, 1H, Harom), 7.69 – 7.57 (m, 2H, Harom), 7.44 (t, J = 7.0 Hz, 1H, Harom),
1
3
6
1
2
.94 (m, 2H, Harom); C NMR (75 MHz, DMSO-d ) (δ/ppm):200.7, 173.8, 165.9, 164.3, 150.2,
6
24.2, 116.1, 108.9.
-Hydroxy-4-methoxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide 27
Following the general method, from 0.20 g (0.6 mmol) 2,4-dimethoxy-N-[5(6)-cyano-1H-
benzimidazol-2-yl]benzamide 21 and 3.6 ml BBr (3.6 mmol) in absolute dichloromethane (15
3
1
ml) under the argon to obtain 0.14 g (59%) of white powder; mp > 280 °C; H NMR (300 MHz,
DMSO-d ) (δ/ppm): 12.05 (brs, 2H, NHbenzimidazole, NHamide), 8.58 (s, 1H, Harom), 8.01 (d, J =
6
8
.9 Hz, 1H, Harom), 7.86 (s, 2H, Harom), 6.63 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H, Harom), 6.58 (d, J
1
2
1
3
=
2.3 Hz, 1H, Harom), 6.47 (s, 1H, Harom), 3.82 (s, 3H, CH ); C NMR (150 MHz, DMSO-d )
3
6
(δ/ppm): 167.6, 164.2, 131.7 (2C), 129.6, 126.5, 119.6 (2C), 106.5, 104.1, 101.0 (2C), 55.4.
2-Hydroxy-N-(6-cyanobenzothiazol-2-yl)benzamide 28
Following the general method, from 0.50 g (1.6 mmol) 2-methoxy-N-[5(6)-cyano-1H-
benzothiazol-2-yl]benzamide 23 and 10.0 ml BBr (10.0 mmol) in absolute dichloromethane
3
1
(25 ml) under the argon to obtain 0.37 g (78%) of white powder; mp > 280 °C; H NMR (300
MHz, DMSO-d ) (δ/ppm): 12.30 (brs, 1H, NHamide), 11.77 (brs, 1H, OH), 8.61 (s, 1H, Harom),
6
7
.99 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H, Harom), 7.93 – 7.84 (m, 2H, Harom), 7.53 (t, J = 7.7 Hz,
1 2
13
1
H, Harom), 7.13 – 6.99 (m, 2H, Harom); C NMR (150 MHz, DMSO-d ) (δ/ppm): 144.0, 134.9,
6
130.6, 129.8, 127.2, 119.9, 119.1, 117.6, 117.2, 105.5.
2
-Hydroxy-4-methoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 29
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Following the general method, from 0.50 g (1.5 mmol) 2,4-dimethoxy-N-(6-
cyanobenzothiazol-2-yl)benzamide 24 and 0.65 g (5.6 mmol) BCl in dichloromethane (30 ml)
3
1
under the argon to obtain 0.12 g (25%) of yellow powder; mp > 280 °C; H NMR (300 MHz,
DMSO-d ) (δ/ppm): 12.21 (brs, 2H, OH, NHamide), 8.60 (s, 1H, Harom), 8.03 (d, J = 8.8 Hz, 1H,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Harom), 7.88 (s, 2H), 6.65 (dd, J = 8.9 Hz, J = 2.2 Hz, 1H, Harom), 6.59 (d, J = 2.1 Hz, 1H,
1
2
1
3
Harom), 3.84 (s, 3H); C NMR (75 MHz, DMSO-d ) (δ/ppm): 165.2, 132.7, 130.3, 127.6, 119.6,
6
1
07.7, 105.9, 101.8, 56.0.
4.2.4. General method for the preparation of amidines
Dry HCl gas was bubbled for 4 h into a cooled suspension (0 °C) of nitrile compound
in anhydrous ethanol or methoxyethanol. The suspension was stirred at room temperature until
the –CN band was undetectable (IR). After anhydrous diethylether was added, the
corresponding imidate ester was filtered off and dried in vacuo. The product was then
suspended in anhydrous ethanol and the corresponding amine was added. For the synthesis of
unsubstituted amidines, the suspension was treated with anhydrous ammonia gas and stirred for
2
4 h at room temperature. For the synthesis of imidazolinyl-substituted amidines, excess of
ethylenediamine (3.5 molar equivalents) was added to a suspension of imidate ester and the
reaction mixture was stirred at reflux for 24 h. The crude product was then filtered off and
washed with diethylether to give powder products which were suspended in anhydrous ethanol
and saturated with HCl gas. The reaction mixture was stirred at room temperature for 24 h. The
products were filtered off.
2
-Hydroxy-N-(4-amidinophenyl)benzamide 11
The compound was prepared following the general method. A suspension of 0.19 g (0.8 mmol)
2-hydroxy-N-(4-cyanophenyl)benzamide 7 in anhydrous ethanol (15 ml) was saturated with dry
HCl gas and stirred for 5 days. The crude imidate was filtered off, suspended in absolute ethanol
(
8 ml) and dry NH was bubbled into the suspension. The mixture was worked up as it is
3
1
described to give 0.98 g (87%) of light yellow powder; mp 270–273 °C; H NMR (300 MHz,
DMSO-d ) (δ/ppm): 15.69 (brs, 1H, NHamide), 8.92 (brs, 4H, NHamidine), 7.84 (s, 4H, Harom), 7.2
6
(
dd, J = 7.8 Hz, J = 1.8 Hz, 1H, Harom), 7.07 – 7.01 (m, 1H, Harom), 6.49 (d, J = 8.2 Hz, 1H,
1 2
13
Harom), 6.27 (t, J = 7.3 Hz, 1H, Harom), 4.35 (brs, 1H, OH); C NMR (150 MHz, DMSO-d )
6
(δ/ppm): 169.9, 167.3, 164.8, 145.8, 132.6, 129.6, 129.2 (2C), 121.0, 120.9, 120.3, 118.9 (2C),
1
18.0, 111.2.
2
-Hydroxy-4-methoxy-N-(4-amidinophenyl)benzamide 12
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The compound was prepared following the general method. A suspension of 0.12 g (0.4 mmol)
2-hydroxy-4-methoxy-N-(4-cyanophenyl)benzamide 8 in anhydrous ethanol (10 ml) was
saturated with dry HCl gas and stirred for 4 days. The crude imidate was filtered off, suspended
in absolute ethanol (10 ml) and dry NH was bubbled into the suspension. The mixture was
3
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
worked up as it is described to give 0.07 g (51%) of light rose powder; mp 256–259 °C; H
NMR (600 MHz, DMSO-d ) (δ/ppm): 15.47 (brs, 1H, NHamide), 9.13 (brs, 4H, NHamidine), 7.84
6
(
d, J = 8.8 Hz, 2H, Harom), 7.81 (d, J = 8.8 Hz, 2H, Harom), 7.61 (d, J = 8.7 Hz, 1H, Harom), 5.94
d, J = 2.1 Hz, 1H, Harom), 5.87 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H, Harom), 4.36 (brs, 1H, OH),
(
1
2
1
3
3.67 (s, 3H, OCH ); C NMR (150 MHz, DMSO-d ) (δ/ppm): 172.8, 167.3, 164.9, 163.6,
3
6
1
46.6, 130.5, 129.2 (2C), 119.4, 118.6 (2C), 111.9, 103.5, 99.3, 54.4.
2
,4-Dihydroxy-N-(4-amidinophenyl)benzamide 13
The compound was prepared following the general method. A suspension of 0.30 g (1.2 mmol)
2,4-dihydroxy-N-(4-cyanophenyl)benzamide 9 in anhydrous ethanol (15 ml) was saturated with
dry HCl gas and stirred for 9 days. The crude imidate was filtered off, suspended in absolute
ethanol (15 ml) and dry NH was bubbled into the suspension. The mixture was worked up as
3
1
it is described to give 0.06 g (17%) of light brown powder; mp > 280 °C; H NMR (600 MHz,
DMSO-d ) (δ/ppm): 15.67 (brs, 1H, NHamide), 8.94 (brs, 6H, NHamidine, OH), 7.82 – 7.78 (m,
6
4
H, Harom), 7.53 (d, J = 8.6 Hz, 1H, Harom), 5.82 (s, 1H, Harom), 5.78 (d, J = 8.5 Hz, 1H, Harom);
13
C NMR (150 MHz, DMSO-d ) (δ/ppm): 172.8, 167.7, 165.2, 162.4, 146.9, 131.2, 129.7 (2C),
6
120.0, 119.0 (2C), 111.5, 105.8, 101.6.
-Hydroxy-N-[4-(imidazolin-2-yl)phenyl]benzamide 14
The compound was prepared following the general method. A suspension of 0.19 g (0.8 mmol)
-hydroxy-N-(4-cyanophenyl)benzamide 7 in anhydrous ethanol (15 ml) was saturated with dry
2
2
HCl gas and stirred for 5 days. The crude imidate was filtered off and suspended in absolute
ethanol. EDA (0.09 ml, 1.4 mmol) was added and the mixture was stirred at reflux for 24 h.
The mixture was worked up as it is described to give 0.09 g (37%) of beige powder; mp 290–
1
293 °C; H NMR (300 MHz, DMSO-d ) (δ/ppm): 11.52 (brs, 1H, NHamide), 10.79 (s, 1H, OH),
6
1
0.64 (brs, 2H, NHamidine), 8.07 (d, J = 8.8 Hz, 2H, Harom), 8.01 (d, J = 8.7 Hz, 2H, Harom), 7.93
(d, J = 7.7 Hz, 1H, Harom) 7.45 (t, J = 7.6 Hz, 1H, Harom), 7.07 (d, J = 8.2 Hz, 1H, Harom), 6.98
13
(
t, J = 7.5 Hz, 1H, Harom), 3.99 (s, 4H, CH ); C NMR (75 MHz, DMSO-d ) (δ/ppm): 166.4,
2
6
164.1, 157.5, 144.0, 133.7, 129.8 (2C), 129.7, 120.0 (2C), 119.2, 118.5, 117.1, 116.7, 44.2 (2C).
2
-Hydroxy-4-methoxy-N-[4-(imidazolin-2-yl)phenyl]benzamide 15
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The compound was prepared following the general method. A suspension of 0.20 g (0.8 mmol)
2-hydroxy-4-methoxy-N-(4-cyanophenyl)benzamide 8 in anhydrous ethanol (35 ml) was
saturated with dry HCl gas and stirred for 4 days. The crude imidate was filtered off and
suspended in absolute ethanol. EDA (0.07 ml, 1.1 mmol) was added and the mixture was stirred
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
at reflux for 24 h. The mixture was worked up as it is described to give 0.06 g (24%) of white
1
powder; mp 292–295 °C; H NMR (600 MHz, DMSO-d ) (δ/ppm): 12.13 (s, 1H, NHamide),
6
1
0.72 (s, 3H, NHamidine, OH), 8.09 (d, J = 8.7 Hz, 2H, Harom), 8.06 (d, J = 8.8 Hz, 1H, Harom),
.01 (d, J = 8.7 Hz, 2H, Harom), 6.61 (d, J = 1.9 Hz, 1H, Harom), 6.58 (dd, J = 8.9 Hz, J = 1.9
8
1
2
1
3
Hz, 1H, Harom), 3.99 (s, 4H, CH ), 3.81 (s, 3H, OCH ); C NMR (75 MHz, DMSO-d ) (δ/ppm):
2
3
6
1
5
2
67.4, 164.5, 164.4, 161.3, 144.5, 131.5, 130.2 (2C), 120.7 (2C), 117.1, 110.1, 106.9, 101.8,
5.9, 44.7 (2C).
,4-Dihydroxy-N-[4-(imidazolin-2-yl)phenyl]benzamide 16
The compound was prepared following the general method. A suspension of 0.14 g (0.6 mmol)
,4-dihydroxy-N-(4-cyanophenyl)benzamide 9 in anhydrous ethanol (10 ml) was saturated with
2
dry HCl gas and stirred for 10 days. The crude imidate was filtered off and suspended in
absolute ethanol. EDA (0.16 ml, 2.4 mmol) was added and the mixture was stirred at reflux for
2
4 h. The mixture was worked up as it is described to give 0.10 g (53%) of beige powder; mp
1
>
280 °C; H NMR (300 MHz, DMSO-d ) (δ/ppm): 11.96 (s, 1H, OH), 10.63 – 10.62 (m, 3H,
6
NHamidine, OH), 10.38 (s, 1H, NHamide), 8.06 (d, J = 8.7 Hz, 2H, Harom), 7.99 (d, J = 8.8 Hz, 2H,
1
3
Harom), 7.94 (d, J = 8.6 Hz, 1H, Harom), 6.45 – 6.41 (m, 2H, Harom), 3.99 (s, 4H, CH ); C NMR
2
(
75 MHz, DMSO-d ) (δ/ppm): 167.4, 164.6, 163.4, 161.2, 144.6, 131.7, 130.1 (2C), 120.6 (2C),
6
1
16.9, 108.7, 108.3, 103.3, 44.7 (2C).
3
,4,5-Trihydroxy-N-[4-(imidazolin-2-yl)phenyl]benzamide 17
The compound was prepared following the general method. A suspension of 0.14 g (0.5 mmol)
,4,5-trihydroxy-N-(4-cyanophenyl)benzamide 10 in anhydrous ethanol (15 ml) was saturated
3
with dry HCl gas and stirred for 10 days. The crude imidate was filtered off and suspended in
absolute ethanol. EDA (0.05 ml, 0.8 mmol) was added and the mixture was stirred at reflux for
2
4 h. The mixture was worked up as it is described to give 0.03 g (24%) of yellow powder; mp
1
>
280 °C; H NMR (300 MHz, DMSO-d ) (δ/ppm): 10.50 (s, 2H, NHamidine), 10.38 (s, 1H,
6
NHamide), 9.24 (s, 2H, OH), 9.01 (s, 1H, OH), 8.02 (d, J = 9.2 Hz, 2H, Harom), 7.98 (d, J = 9.2
1
3
Hz, 2H, Harom), 6.99 (s, 2H, Harom), 3.98 (s, 4H, CH2); C NMR (75 MHz, DMSO-d ) (δ/ppm):
6
1
66.6, 164.8, 146.01, 145.8, 138.0, 130.0 (2C), 124.6, 120.0 (2C), 116.3, 107.9 (2C), 44.7 (2C).
2
-Hydroxy-N-[5(6)-amidino-1H-benzimidazol-2-yl]benzamide 30
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The compound was prepared following the general method. A suspension of 0.14 g (0.5 mmol)
2-hydroxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide 26 in anhydrous ethanol (15 ml)
was saturated with dry HCl gas and stirred for 9 days. The crude imidate was filtered off,
suspended in absolute ethanol (10 ml) and dry NH was bubbled into the suspension. The
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mixture was worked up as it is described to give 0.04 g (58%) of light yellow powder; mp >
1
280 °C; H NMR (600 MHz, DMSO-d ) (δ/ppm): 17.49 (brs, 1H, OH), 12.44 (brs, 1H, NHamide),
6
9
7
.08 (s, 2H, NHamidine), 8.53 (s, 2H, NHamidine), 7.87 – 7.80 (m, 2H, Harom), 7.50 (s, 1H, Harom),
.45 (d, J = 8.0 Hz, 1H, Harom), 7.15 (t, J = 7.5 Hz, 1H, Harom), 6.63 (d, J = 8.2 Hz, 1H, Harom),
1
3
6.53 (t, J = 7.2 Hz, 1H, Harom); C NMR (150 MHz, DMSO-d ) (δ/ppm): 170.6, 166.9 (2C),
6
1
66.4, 132.8, 129.5 (2C), 120.6, 119.4, 119.1 (2C), 114.4.
2
-Hydroxy-4-methoxy -N-[5(6)-amidino-1H-benzimidazol-2-yl]benzamide 31
The compound was prepared following the general method. A suspension of 0.08 g (0.3 mmol)
27 in anhydrous ethanol (10 ml) was saturated with dry HCl gas and stirred for 9 days. The
crude imidate was filtered off, suspended in absolute ethanol (10 ml) and dry NH was bubbled
3
into the suspension. The mixture was worked up as it is described to give 0.05 g (57%) of light
1
brown powder; mp 255–260°C; H NMR (300 MHz, DMSO-d ) (δ/ppm): 17.12 (brs, 1H, OH),
6
1
7
2.23 (brs, 1H, NHbenzimidazole), 8.76 (brs, 4H, NHamidine), 7.83 (s, 1H, Harom), 7.70 (s, 1H, Harom),
1
3
.48 (s, 2H, Harom), 6.09 (s, 2H, Harom), 3.70 (s, 3H, CH ); C NMR (150 MHz, DMSO-d )
3
6
(
δ/ppm): 166.1, 165.5, 163.9, 163.1, 154.8, 132.9, 131.4, 130.5, 121.4, 121.1, 119.3, 118.4,
101.2, 55.2.
-Hydroxy-N-(6-amidinobenzothiazol-2-yl)benzamide 32
The compound was prepared following the general method. A suspension of 0.20 g (0.7 mmol)
8 in anhydrous ethanol (15 ml) was saturated with dry HCl gas and stirred for 10 days. The
2
2
crude imidate was filtered off, suspended in absolute ethanol (10 ml) and dry NH was bubbled
3
into the suspension. The mixture was worked up as it is described to give 0.03 mg (12%) of
1
beige powder; mp > 280 °C; H NMR (300 MHz, DMSO-d ) (δ/ppm): 15.75 (brs, 1H, OH),
6
9.14 (brs, 2H, NHamidine), 8.63 (brs, 2H, NHamidine), 8.26 (d, J = 1.3 Hz, 1H, Harom), 7.88 (dd, J1
=
7.7 Hz, J = 1.7 Hz, 1H, Harom), 7.73 – 7.62 (m, 2H, Harom), 7.25 (t, J = 8.5 Hz, 1H, Harom),
2
1
3
6
1
.83 – 6.69 (m, 2H, Harom); C NMR (75 MHz, DMSO-d ) (δ/ppm): 171.3, 169.4, 165.8, 162.2,
6
55.4, 133.6, 132.9, 129.8, 125.4, 122.0, 120.3, 119.9, 119.0, 117.5, 117.4.
2-Hydroxy-4-methoxy-N-(6-amidinobenzothiazol-2-yl)benzamide 33
The compound was prepared following the general method. A suspension of 0.08 g (0.2 mmol)
2
-hydroxy-4-methoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 29 in anhydrous ethanol (10
ml) was saturated with dry HCl gas and stirred for 9 days. The crude imidate was filtered off,
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2
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suspended in absolute ethanol (10 ml) and dry NH was bubbled into the suspension. The
3
mixture was worked up as it is described to give 0.04 mg (40%) of white powder; mp > 280 °C;
1
H NMR (300 MHz, DMSO-d ) (δ/ppm): 13.02 (brs, 2H, OH, NHamide), 9.29 (brs, 2H,
6
NHamidine), 8.99 (brs, 2H, NHamidine), 8.47 – 8.43 (m, 1H, Harom), 7.96 – 7.88 (m, 1H, Harom), 7.85
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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3
4
5
6
7
8
9
0
–
7.75 (m, 2H, Harom), 6.52 (s, 2H, Harom), 3.80 (s, 3H, CH ); C NMR (150 MHz, DMSO-d )
3 6
(δ/ppm): 165.5, 164.4, 160.6, 131.9, 129.6, 126.8, 126.0, 122.6, 122.3, 119.3, 109.5, 106.7,
1
04.9, 102.8, 101.4, 55.4.
2
-Hydroxy-N-[5(6)-(imidazolinil-2-yl)-1H-benzimidazol-2-yl]benzamide 34
The compound was prepared following the general method. A suspension of 0.14 g (0.5 mmol)
-hydroxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide 26 in anhydrous methoxyethanol
15 ml) was saturated with dry HCl gas and stirred for 9 days. The crude imidate was filtered
2
(
off and suspended in absolute ethanol (10 ml). EDA (0.05 ml, 0.8 mmol) was added and the
mixture was stirred at reflux for 24 h. The mixture was worked up as it is described to give 0.05
1
g (68%) of white powder; mp > 280 °C; H NMR (600 MHz, DMSO-d ) (δ/ppm): 17.36 (brs,
6
1
H, OH), 12.41 (brs, 1H, NHbenzimidazole), 9.63 (brs, 2H, NHamidine), 7.92 (s, 1H, Harom), 7.86 (dd,
J = 7.7, J = 1.5 Hz, 1H, Harom), 7.57 (d, J = 8.3 Hz, 1H, Harom), 7.51 (d, J = 8.0 Hz, 1H, Harom),
1
2
7
3
1
.17 (t, J = 7.6 Hz, 1H, Harom), 6.65 (d, J = 8.2 Hz, 1H, Harom), 6.56 (t, J = 7.4 Hz, 1H, Harom),
1
3
.94 (s, 4H, CH ); C NMR (75 MHz, DMSO-d ) (δ/ppm): 165.7, 134.7, 130.7, 123.6, 119.4,
2
6
17.7 (2C), 116.4, 113.6, 44.8 (2C).
2-Hydroxy-4-methoxy -N-[5(6)-(imidazolinil-2-yl)-1H-benzimidazol-2-yl]benzamide 35
The compound was prepared following the general method. A suspension of 0.08 mg (0.02
mmol)
2-hydroxy-4-methoxy-N-[5(6)-cyano-1H-benzimidazol-2-yl]benzamide
27
in
anhydrous metoxyethanol (10 ml) was saturated with dry HCl gas and stirred for 9 days. The
crude imidate was filtered off and suspended in absolute ethanol (10 ml). EDA (0.02 ml, 0.4
mmol) was added and the mixture was stirred at reflux for 24 h. The mixture was worked up as
1
it is described to give 0.02 g (38%) of white powder; mp > 280 °C; H NMR (300 MHz, DMSO-
d6) (δ/ppm): 10.02 (brs, 2H, NHamidine), 7.92 (s, 1H, Harom), 7.74 (d, J = 8.4 Hz, 1H, Harom), 7.59
1
3
–
7.49 (m, 2H, Harom), 6.13 – 6.09 (m, 2H, Harom), 3.95 (s, 4H, CH ), 3.71 (s, 3H, CH ); C
2 3
NMR (75 MHz, DMSO-d ) (δ/ppm): 169.7, 166.5, 163.9, 155.8, 154.8, 133.7, 130.7, 125.6,
6
1
21.9, 121.1, 119.0, 114.6, 113.4, 112.1, 102.4, 55.2, 45.0 (2C).
2-Hydroxy-N-[6-(imidazolinil-2-yl)benzothiazol-2-yl]benzamide 36
The compound was prepared following the general method. A suspension of 0.20 g (0.7 mmol)
2
8 in anhydrous ethanol (15 ml) was saturated with dry HCl gas and stirred for 9 days. The
crude imidate was filtered off and suspended in absolute ethanol (10 ml). EDA (0.08 ml, 1.2
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mmol) was added and the mixture was stirred at reflux for 24 h. The mixture was worked up as
1
it is described to give 0.08 g (56%) of beige powder; mp > 280 °C; H NMR (600 MHz, DMSO-
d6) (δ/ppm): 15.69 (brs, 1H, OH), 9.49 (brs, 2H, NHamidine), 8.37 (s, 1H, Harom), 7.90 (d, J = 6.9
Hz, 1H, Harom), 7.82 (d, J = 8.0 Hz, 1H, Harom), 7.70 (d, J = 8.5 Hz, 1H, Harom), 7.28 (t, J = 6.7
1
3
1
1
1
1
1
1
1
1
1
1
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2
2
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5
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6
0
1
2
3
4
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6
7
8
9
0
1
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7
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9
0
1
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0
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0
Hz, 1H, Harom), 6.80 – 6.76 (m, 2H, Harom), 3.99 (s, 4H, CH ); C NMR (75 MHz, DMSO-d )
2
6
(δ/ppm): 171.2, 165.6, 161.8, 155.7, 133.7, 133.1, 129.9, 125.7, 122.3, 120.1, 119.4, 118.0,
1
17.7, 117.5, 114.2, 44.7 (2C).
2
-Hydroxy-4-methoxy-N-[6-(imidazolinil-2-yl)benzothiazol-2-yl]benzamide 37
The compound was prepared following the general method. A suspension of 0.08 g (0.2 mmol)
-hydroxy-4-methoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 29 in anhydrous ethanol (10
2
ml) was saturated with dry HCl gas and stirred for 9 days. The crude imidate was filtered off
and suspended in absolute ethanol (10 ml). EDA (0.03 ml, 0.5 mmol) was added and the mixture
was stirred at reflux for 24 h. The mixture was worked up as it is described to give 0.02 g (23%)
1
of white powder; mp > 280 °C; H NMR (600 MHz, DMSO-d ) (δ/ppm): 16.21 (brs, 1H, OH),
6
9
1
.69 (brs, 2H, NHamidine), 8.33 (s, 1H, Harom), 7.81 – 7.77 (m, 2H, Harom), 7.66 (d, J = 8.4 Hz,
H, Harom), 6.33 (d, J = 8.7 Hz, 1H, Harom), 6.31 (s, 1H, Harom), 3.98 (s, 4H, CH ), 3.75 (s, 3H,
2
CH3).
2
-Methoxy-N-(6-amidinobenzothiazol-2-yl)benzamide 40
The compound was prepared following the general method. A suspension of 0.15 g (0.5 mmol)
2-methoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 23 in anhydrous ethanol (20 ml) was
saturated with dry HCl gas and stirred for 9 days. The crude imidate was filtered off, suspended
in absolute ethanol (10 ml) and dry NH was bubbled into the suspension. The mixture was
3
1
worked up as it is described to give 0.14 g (81%) of white powder; mp 258–261°C; H NMR
(300 MHz, DMSO-d ) (δ/ppm): 8.76 (brs, 4H, NHamidine), 8.22 (s, 1H, Harom), 7.70 (d, J = 7.7
6
Hz, 1H, Harom), 7.58 – 7.53 (m, 2H, Harom), 7.32 (t, J = 7.1 Hz, 1H, Harom), 7.03 (d, J = 8.2 Hz,
13
1
H, Harom), 6.95 (t, J = 7.3 Hz, 1H, Harom), 3.79 (s, 3H, OCH ); C NMR (75 MHz, DMSO-d )
3
6
(δ/ppm): 165.7, 157.4, 133.5, 130.0, 125.1, 121.7, 120.3, 118.3, 112.5, 56.0.
2
,4-Dimethoxy-N-(6-amidinobenzothiazol-2-yl)benzamide 41
The compound was prepared following the general method. A suspension of 0.23 g (6.6 mmol)
,4-dimethoxy-N-(6-cyanobenzothiazol-2-yl)benzamide 24 in anhydrous methoxyethanol (20
2
ml) was saturated with dry HCl gas and stirred for 27 days. The crude imidate was filtered off,
suspended in absolute ethanol (15 ml) and dry NH was bubbled into the suspension. The
3
29
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