Synthesis and structure-activity relationship of amidine derivatives of 3,4-ethylenedioxythiophene as novel antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2014.11.003

9 new amidine derivatives of 3,4-ethylenedioxythiophene were synthesized.27 amidines were tested for their antibacterial activities.Bis-phenyl derivatives show highest activity against sensitive and resistant strains.Bis-benzimidazole derivative had the b

Full text of DOI:10.1016/j.ejmech.2014.11.003

Accepted Manuscript
Synthesis and structure-activity relationship of amidine derivatives of 3,4-
ethylendioxythiophene as novel antibacterial agents
Ivana Stolić, Hana Čipčić Paljetak, Mihaela Perić, Mario Matijašić, Višnja Stepanić,
Donatella Verbanac, Miroslav Bajić
PII:
S0223-5234(14)01025-3
10.1016/j.ejmech.2014.11.003
EJMECH 7496
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2014
Revised Date: 28 October 2014
Accepted Date: 1 November 2014
Please cite this article as: I. Stolić, H.Č. Paljetak, M. Perić, M. Matijašić, V. Stepanić, D. Verbanac, M.
Bajić, Synthesis and structure-activity relationship of amidine derivatives of 3,4-ethylendioxythiophene
as novel antibacterial agents, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.11.003.
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ACCEPTED MANUSCRIPT

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Highlights
9 new amidine derivatives of 3,4-ethylenedioxythiophene were synthesized.
27 amidines were tested for their antibacterial activities.
Bis-phenyl derivatives show highest activity against sensitive and resistant strains.
Bis-benzimidazole derivative had the best spectrum of activity and DNA binding.

ACCEPTED MANUSCRIPT
Synthesis and structure-activity relationship of amidine derivatives of 3,4-
ethylendioxythiophene as novel antibacterial agents
Ivana Stolića,1, Hana Čipčić Paljetakb,1, Mihaela Perićb,*, Mario Matijašićb, Višnja Stepanićc,
Donatella Verbanacb, Miroslav Bajića,#
a
University of Zagreb, Faculty of Veterinary Medicine, Department of Chemistry and
Biochemistry, Heinzelova 55, 10000 Zagreb, Croatia
b University of Zagreb School of Medicine, Center for Translational and Clinical Research,
Department for Intercellular communication, Šalata 2, 10000 Zagreb, Croatia
c
Rudjer Bošković Institute, Division of Molecular Medicine, Laboratory for Epigenomics,
Bijenička 54, 10000 Zagreb, Croatia
* Corresponding author. Tel.: +385 1 4590070; fax: +385 1 4566724
# Corresponding author. Tel.: +385 1 2390300; fax: +385 1 2441390.
E-mail addresses: mihaela.peric@mef.hr (M. Perić), mbajic@vef.hr (M. Bajić).
1 These authors contributed equally to this work.
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ABSTRACT
Current antibacterial chemotherapeutics are facing an alarming increase in bacterial
resistance pressuring the search for novel agents that would expand the available therapeutic
arsenal against resistant bacterial pathogens. In line with these efforts, a series of 9 amidine
derivatives of 3,4-ethylenedioxythiophene were synthesized and, together with 18 previously
synthesized analogs, evaluated for their relative DNA binding affinity, in vitro antibacterial
activities and preliminary in vitro safety profile. Encouraging antibacterial activity of several
subclasses of tested amidine derivatives against Gram-positive (including resistant MRSA,
MRSE, VRE strains) and Gram-negative bacterial strains was observed. The bis-phenyl
derivatives were the most antibacterially active, while compound 19 from bis-benzimidazole
class exhibited the widest spectrum of activity (with MIC of 4, 2, 0.5 and ≤0.25 ꢀg/ml against
laboratory strains of S. aureus, S. pneumoniae, S. pyogenes, M. catarrhalis, respectively and
4-32 ꢀg/ml against clinical isolates of sensitive and resistant S. auresu, S. epidermidis and E.
faecium) and also demonstrated the strongest DNA binding affinity (ꢁTm of 15.4º C).
Asymmetrically designed compounds and carboxamide-amidines were, in general, less active.
Molecular docking indicated that the shape of the 3,4-ethylenedioxythiophene derivatives and
their ability to form multiple electrostatic and hydrogen bonds with DNA, corresponds to the
binding modes of other minor-groove binders. Herein reported results encourage further
investigation of this class of compounds as novel antibacterial DNA binding agents.
Key words: thiophene, amidine, minor groove binders, minimum inhibitory concentration,
antibacterial activity, DNA binding
Non-standard abbreviations: Cpd – compound, Ar – aromate, MGB – minor groove binder
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1. INTRODUCTION
Infectious diseases remain a major health problem worldwide and continue to
challenge both medical and pharmaceutical communities. Bacterial pathogens causing
infections in the hospital and community settings are continuously developing resistance to
the existing antibacterial therapeutics [1-5]. In particular, increasing drug resistance among
Gram-positive bacterial pathogens, including methicilline-resistant Staphyloccocus aureus
(MRSA) and vancomycin-resistant enterococci (VRE), represent a significant health issue [6].
As pathogens mutate, continued success in treating infectious diseases requires a steady
stream of new antimicrobial agents to which existing bacteria have not developed resistance.
Also, as the majority of the high level bacterial resistance mechanisms affect antibiotics with
related chemical structures, it is considered that the new antibacterial agents should possess
chemical characteristics which are clearly different from those of already known agents.
Most antibiotics in clinical use are designed in the way that their antibacterial activity
is achieved through one of the five possible mechanisms of action: inhibition of cell wall
synthesis, inhibition of protein synthesis, alteration of cell membranes, inhibition of enzymes
involved in nucleic acid synthesis and exhibiting antimetabolite activity. The introduction of
bacterial DNA as a therapeutic target has led to the design of novel classes of antibacterial
drugs [7]. Targeting bacterial DNA with new antibacterial agents is a promising direction of
research that could potentially result in the expansion of available arsenal to combat existing
bacterial drug resistance. This approach requires that bacterial DNA is preferentially targeted
over the human DNA, thus subsequently minimizing potential toxicity to host cells.
In addition, presence of the negatively charged phospholipids and other polyanionic
groups in prokaryotic membranes and their different composition compared to eukaryotic
membranes is used as a basis in the design of potential antibacterial peptide drugs [8-10].
Literature evidence for benzophenone-based tetraamide compounds also showed that positive
charge is a necessary requirement for selective interaction with bacterial over mammalian
membranes [11]. Thus, cationic compounds are strongly attracted by electrostatic interactions
to bacterial over mammalian membranes, and this characteristic is used in drug design in
order to increase selectivity of antibacterial agents.
During the last decade, a series of reports on new classes of compounds that bind to
DNA and display potent antibacterial activity were published [12-16]. These DNA binding
antibiotics are structurally based on natural products distamycin and netropsin. Mechanism of
action of antibacterial agents derived from distamycin, as well as other minor groove binders
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(MGBs), relies on inhibiting DNA function and RNA synthesis in bacteria [15] by binding to
duplex DNA specifically in the minor groove. In general, MGBs are molecules of diverse
structures composed of several subunits that have the ability to adopt a “crescent” shape
allowing the MGB moiety to fit into the minor groove of DNA. Bis-amidine type of MGBs is
known to have antibacterial, antiviral, antifungal and antiparasitic activities [12,17-19].
Intense research of bis-amidines as antibacterial drugs led to the synthesis of numerous
compounds that exhibit broad-spectrum in vitro activity against Gram-positive and Gram-
negative bacterial species [11,18,20-22]. With regard to the structural units’ arrangement
MGBs can generally be divided on head-to-tail (distamycin) and head-to-head (compounds I
and II) groups of compounds (Fig.1). Generally, diamidine MGBs of head-to-head type are
antibacterially more potent than the head-to-tail compounds with MICs against sensitive S.
aureus ≤ 0.5 ꢀg/mL for compounds I and II and 6.25 and 50 ꢀg/mL for compound III and
distamycin, respectivelly [21,23,24].
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Figure 1. Structures of a) head-to-head and b) head-to-tail linked MGBs with demonstrated
antibacterial activity. MICs against sensitive S. aureus are ≤ 0.5 ꢀg/mL for compounds I and
II and 6.25 and 50 ꢀg/mL for compound III and distamycin, respectively [12,21,23,25].
These findings prompted our investigation of diamidine derivatives of 3,4-
ethylenedioxythiophene, synthesized in our laboratory, as potential antibacterial agents [26-
28]. Here we report the antibacterial activities of compounds with 3,4-ethylenedioxythiophene
central unit combined with benzimidazole, phenyl, carboxamidophenyl and amidine building
blocks employed as already known pharmacophores.
3,4-ethylenedioxythiophene as a central unit in derivatives presented here was selected
to explore the chemical space around the already known thiophene central linker employed in
some other antimicrobially active MGBs [29,30]. Also, this moiety is considered chemically
very stable and robust. Specifically, 3,4-ethylenedioxythiophene is commonly used in
conducting polymers where its two electron-donating oxygen atoms adjacent to the thiophene
ring stabilize the positive charges generated in oxidized polymer [31,32]. We assumed that
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such electron donor capacity of 3,4-ethylenedioxythiophene will additionally stabilize
amidine end groups thus contributing to the overall stability of the studied molecules.
Benzimidazole ring is an important heterocyclic pharmacophore in drug discovery and
the compounds carrying different substituents on benzimidazole structure are associated with
a wide range of biological activities, including antibacterial properties [18,20,22,33]. A
number of dicationically substituted bis-benzimidazoles originally developed as DNA binding
agents have shown antibacterial activity [19,25,34,35]. In addition, compounds with
benzimidazol-phenyl moiety separated by different linkers exhibit high activity against drug–
resistant Gram-positive bacteria without cytotoxicity in their therapeutic concentrations [36].
Extensive biological studies of benzimidazoles and phenyl derivatives have confirmed that
presence of these structural units was crucial for antibacterial activity of compounds
[18,19,34,35]. Several bis-benzimidazoles have shown significant antibacterial activities
against drug-resistant bacteria (methicillin-resistant Staphylococcus aureus – MRSA and
methicillin resistant S. epidermidis – MRSE and vancomycin resistant enterococci – VRE
strains) [19,37]. In addition, diamidine derivatives of bis-indoles, as structural analogues of
benzimidazoles, were shown to exhibit in vitro activity against a broad spectrum of Gram-
negative and Gram-positive pathogens such as S. aureus (including MRSA) and E. faecalis
(including VRE) which additionally supports the use of mono- and bis-benzimidazole units in
3,4-ethylenedioxythiophene series [21,24].
Furthermore, bis-phenyl derivatives were synthesized in order to explore the influence
of aromatic phenyl unit on the compound DNA binding and antibacterial activity. Previous
reports by other research groups have suggested that planar and hydrophobic phenyl building
blocks contribute to increased antibacterial activity, especially against MRSA and VRE
strains [25]. Also, it is widely recognised that the introduction of phenyl group increases
lipophilicity. Literature data indicate that increased lipophilicity in some distamycin
derivatives was associated with improved DNA binding and higher activity against gram
positive bacteria [25,38].
In our compound design diamidines represent the main pharmacologically relevant
structural feature. Previously reported aromatic diamidines achieve their biological activities
through the interaction with DNA, acting as MGBs [39]. It is therefore generally accepted that
MGBs must have cationic end groups needed for electrostatic interactions with negatively
charged sugar-phosphate backbone of DNA [40,41]. In addition, acyclic and cyclic amidine
terminal groups with multiple positive charges show increased accumulation within cell
nuclei probably as a result of stronger DNA binding affinity [42].
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The present report describes the synthesis of nine new amidine derivatives (9–17) and
evaluation of antibacterial activity of 27 amidine derivatives from 3,4-ethylenedioxythiophene
class (including 18 previously synthesized analogues). The structure-activity relationship of
their antibacterial potencies and DNA binding affinities is examined.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The synthesis and characterization of compounds 18–35 have been described in our
previous reports [26-28]. In this study we extended our investigations towards evaluation of
the antibacterial activity. The preliminary testing of derivatives 18-35 demonstrated promising
in vitro antibacterial activity (Table 2). Based on the obtained results, our studies were further
expanded towards the synthesis of new amidine analogues. Thus, we designed and prepared
novel asymmetrical compounds (9–17) to investigate the structural features of this subclass
contributing to the biological activity.
Table 1. Structures of tested compounds illustrated by their major building blocks: 3,4-
ethylenedioxythiophene central unit, core groups (Ar1 and Ar2) and end groups (R1 and R2).
A. Structures of newly synthesized compounds. B. Structures of previously synthesized
compounds [26-28].
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A
Cpd
R1
Ar1
Ar2
R2
+
-
-
-
N
NH2 Cl
NC–
9
N
H
NH2
+
-
N
NH2 Cl
NC–
NC–
10
11
12
13
14
15
16
17
HN
N
H
- +
Cl H N
N
N
H
N
H
+
N
NH2 Cl
O2N–
O2N–
O2N–
N
H
NH2
+
-
N
NH2 Cl
HN
N
H
- +
Cl H N
N
N
H
N
H
+
N
NH2 Cl
+
Cl–H3 N–
N
H
NH2
+
-
N
NH2 Cl
Cl–H3 N–
+
HN
N
H
- +
Cl H N
N
+
Cl–H3 N–
N
H
N
H
B
Cpd
R1
Ar1
Ar2
R2
-
+
+
-
N
N
Cl H2N
NH2 Cl
18a
N
H
N
H
H2N
NH2
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-
+
+
-
Cl H2N
N
N
NH2 Cl
19a
HN
NH
N
H
N
H
- +
Cl H N
+
-
N
N
NH Cl
20a
21b
N
N
H
N
H
N
H
H
-
+
+
-
Cl H2N
NH2 Cl
H2N
NH2
-
+
+
-
Cl H2N
NH2 Cl
22b
HN
NH
+
-
-
+
NH2 Cl
Cl H2N
23b
24b
25b
26b
27c
NH
+
HN
NH2 Cl
-
+
-
Cl H2N
NH
HN
- +
Cl H N
+
-
NH Cl
N
N
H
H
+
-
- +
NH Cl
Cl H N
HN
NH
+
H
H
+
-
-
Cl H2N
NH2 Cl
N
N
O
O
O
O
H2N
NH2
-
+
+
-
H
N
H
N
Cl H2N
NH2 Cl
28c
HN
NH
H
N
H
N
+
-
-
+
NH2 Cl
Cl H2N
29c
30c
31c
32c
O
O
O
O
O
O
O
NH
HN
- +
H
N
H
N
+
-
Cl H N
NH Cl
N
N
H
H
H
N
H
N
+
-
- +
NH Cl
Cl H N
HN
NH
+
-
H
N
H
N
+
-
Cl H2N
NH2 Cl
O
H2N
NH2
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- +
Cl H N
+
-
H
N
H
N
NH Cl
33c
34c
35b
N
O
O
O
N
H
H
H
N
+
-
NH Cl
O
-
O
NH
-
+
+ -
Cl H2N
NH2 Cl
HN
HN
Synthesis presented in: a Ref. [26], b Ref. [27], c Ref. [28].
The synthesis of novel asymmetrical benzimidazoles 9–17 outlined in Scheme 1 was
achieved employing the similar approach developed to make the symmetrical bis-phenyl [27]
or bis-benzimidazole [26] analogues. The synthesis started with Still cross-coupling reactions
between 3,4-ethylenedioxythiophene (1) and 4-bromobenzonitrile or 1-bromo-4-nitrobenzene
to give the 2-aryl-3,4-ethylenedioxythiophene derivatives 2 or 3 in moderate yields.
Compounds 2 and 3 were converted to the aldehydes by reacting with POCl3. The target
benzimidazoles 9–17 were prepared by oxidative coupling of aldehydes 4 or 5 with the
appropriate 3,4-diaminobenzamidines (6–8) in 60–70 % yields. Catalytic hydrogenation of
nitro derivatives 12–14 gave corresponding amines 15–17 in good yields [43].
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H2N
H2N
R2
O
O
O
O
O
O
6−8
i
iii
ii
O
iv
S
S
S
R1
R1
H
1
2 R1 = CN
3 R1 = NO2
4 R1 = CN
5 R1 = NO2
O
O
N
S
R1
HN
R2
9−11 R1 = CN
12−14 R1 = NO2
15−17 R1 = NH2
v
H+
NH2+ Cl−
NH2+ Cl−
N
6, 9, 12, 15 R2 =
7, 10, 13, 16 R2 =
;
Cl−
;
8, 11, 14, 17 R2 =
NH2
HN
HN
Scheme 1. Reagents: i) 1. n-BuLi, THF; 2. Bu3SnCl; ii) Pd(PPh3)4, 4-bromobenzonitrile or 1-
bromo-4-nitrobenzene, THF; iii) POCl3, DMF; iv) 3,4-diaminobenzimidamide hydrochloride
(6), 3,4-diamino-N-isopropylbenzimidamide hydrochloride (7), 4-(4,5-dihydro-1H-imidazol-
2-yl)benzene-1,2-diamine hydrochloride (8), 1,4-benzoquinone, C2H5OH; v) H2, 10 % Pd/C,
CH3OH.
Different classes of amidine based minor groove binders exhibited activity against
Gram-positive bacteria while the related QSAR studies revealed benzoxazole, benzimidazole
and benzothiazole as necessary functionalities for adequate antibacterial activity [44-47]. For
that reason, we decided to use these pharmacophores in the design and synthesis of here
reported antibacterial compounds. Besides, terminal aliphatic amines are often employed in
the synthesis of biologically active compounds, due to their basic properties and strong
protonation at the physiological pH contributing to ionic interactions and hydrogen bonding
within groove [12]. In addition, compound curvature is an important factor for complex
formation and strong binding in the minor groove. The diamidines as well as other MGBs
either match the curvature of the DNA minor groove in their solution state or are able to adopt
a low energy conformation to complement the DNA groove shape in the complex [48].
Taking into account all mentioned, biological activity can be seen as a net result of van der
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Waals contacts, hydrogen bonding, and electrostatic interactions between investigated
compounds and DNA [12,49].
In order to explore the influence of structural features on antibacterial activity and
DNA binding affinity of here reported compounds all structural units (Ar1, Ar2, R1 and R2,
Table 1) except the linker (3,4-ethylenedioxythiophene) were modified. Previously conducted
research of influence of central linker on antibacterial activity of MGBs, including
benzimidazoles and phenyl core containing molecules, revealed that central linker is very
important for achieving potent antibacterial activity [19,25]. In this study we have chosen a
rigid 3,4-ethylenedioxythiophene linker to favour interactions within groove. We
hypothesized that the cumulative effects of steric factors and van der Walls contacts within
groove, the linker size and potential for hydrogen bonds formation as well as nonbonding
interactions of the rest of structure, will additionally contribute to DNA binding and
antibacterial activity as compared with often used small, cyclic aromatic linkers, such as
phenyl, pyrimidine, single atoms or aliphatic chains.
Structural modifications of 3,4-ethylenedioxthiophene compounds have included
aromatic cores Ar1 and Ar2 (phenyl-, benzimidazole-), end groups R1 and R2 (un-substituted
amidine, alkyl substitute amidines, cyclic amidines, nitro-, amino- and cyano- substituents)
and their combinations (Table 1). The influence of end group physical-chemical properties,
mainly charge and hydrophobicity on overall biological activity was explored. It has been
shown that enhancing the hydrophobicity of the cationic end groups could increase the
binding affinity of the molecule to DNA and aid in drug transport and antibiotic activity [11].
In order to investigate the influence of hydrogen bonding capacity on DNA binding and
biological activity two different aromatic scaffolds were introduced. Benzimidazole has the
capacity to form hydrogen bonds with DNA through NH group, while phenyl group
contributes to the stronger DNA binding through its hydrophobic interactions within the
groove. Additionally, asymmetrical design was investigated since some asymmetrical MGBs
exhibited high antimicrobial potencies [34].
Synthesized compounds are divided in eight structural groups: i) bis-phenyl- (21–26),
ii) bis-benzimidazole- (18–20), iii) bis-carboxamidophenyl- (para 27–31), iv) bis-
carboxamidophenyl- (meta 32,33), v) cyano-amidine (9–11), vi) nitro-amidine (12–14), vii)
amino-amidine (15–17) and viii) miscellaneous derivatives (34 and 35). Compound 34 is
derivative of compound 31 with Ar2 and R2 replaced by ethyl ester, while compound 35 is a
derivative of compound 21 with the end groups and aromates inversely arranged.
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2.2. UV-Vis spectroscopy
Since DNA binding studies are conducted by spectroscopic methods, the spectrometric
behaviour of aqueous solutions of newly synthesised compounds was studied by UV-Vis
spectroscopy. Linear dependence of UV-Vis spectra on the concentration of compounds 9–17
was in the range 1–5.5 × 10–5 mol dm–3 indicating the absence of intermolecular interactions
in their aqueous solutions. The UV-Vis spectra of compounds 9–17 were determined to be
temperature independent when measured in the 25–90 °C temperature range. Furthermore,
upon cooling to 25 °C excellent reproducibility of spectra was observed. Aqueous solutions of
all studied compounds were stable over several weeks at –18 °C.
2.3. Thermal denaturation experiments
Thermal melting enables the rapid qualitative evaluation of the relative binding
affinities of compounds for polynucleotides [50]. The stability of the calf thymus DNA
(ctDNA) double helix influences the DNA melting temperature (Tm), while the binding of
compounds to DNA alters the Tm due to the interactions formed at the interface. Thus, the
measurement of ꢁTm taking place after the formation of the small molecule-ctDNA complex
indicates the strength of compound-DNA interaction. To investigate this characteristic, the
changes in the Tm upon addition of amidines 9–17 to the ctDNA buffer solution were
measured at r ([amidines]/[DNA in base pair]) = 0.1 (Table 2). The obtained results were
compared to values for compounds 18–35 from previously published data set [26-28].
The ꢁTm values ranged from 0 (compounds 34 and 35 that do not bind to DNA) to
15.4 °C, with compound 19 having the highest ꢁTm. Previously conducted qualitative
estimation of the DNA binding affinities of amidines showed that the compounds from bis-
benzimidazole (18–20) and bis-phenyl (21–26) series had similar ꢁTm values (8.4–15.4 °C)
[26,27], even though benzimidazoles have additional hydrogen bonding site available. This
can be explained by the fact that even though hydrogen bonds established within the minor
groove are one of the key factors for formation of stable compound-DNA complex, the
number of hydrogen bonding sites on the molecule does not necessarily correlate with the
binding strength, since not all of the bonds will be formed [42]. This assumption is further
supported by the ꢁTm values of compounds from carboxamid-amidine series (27–33).
Introduction of amid bond between linker and phenyl units in bis-phenyl series, although
contributing additional hydrogen bonding sites, resulted in drastic decrease of ꢁTm values.
This can be attributed to their inability to assume appropriate conformation within groove,
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therefore failing to form maximum number of hydrogen bonds. Three newly synthesized
series of phenyl-benzimidazole compounds (9–11, 12–14 and 15–17), although having similar
molecular geometry as those from bis-benzimidazole and bis-phenyl series, bind to DNA
weakly (ꢁTm values of 1.1–3.8 °C). In addition, one amidino moiety was replaced with cyano-
, nitro-, amino-end group and this substitution led to the decrease in compounds' DNA
binding affinity. This is in accordance with the literature data claiming that basic nature and
protonated state of amidino group in biological conditions strongly contributes to DNA
binding [51].
2.4. Antibacterial activity and structure-activity relationship (SAR)
For the compounds from amidine series of 3,4-ethylenedioxythiophene, antibacterial
activity against five gram-positive (Streptococcus pneumoniae, Streptococcus pyogenes,
Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium) and three
gram-negative (Escherichia coli, Moraxella catarrhalis, Haemophilus influenzae) bacterial
species were determined. Activity against highly resistant strains of staphylococci
(methicillin-resistant S. aureus – MRSA and methicillin resistant S. epidermidis – MRSE), as
well as vancomycin resistant E. faecium (VRE) was also tested. Values for minimal inhibitory
concentrations (MICs) are given in Tables 2 and 3.
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Table 2. Biological activities of compounds 9–35: the influence of the compounds on the calf
thymus DNA stability expressed as the change in the melting temperature (∆Tm) and
inhibition of the in vitro bacterial growth expressed as the MIC (minimal inhibitory
concentration) values. ∆Tmvalues for compounds 18–35 are from [26-28].
MIC (µg/ml)
Cpd
9
∆Tm
(º C)
3.8
S. aureus
ATCC 13709
> 128
S. pneumoniae
ATCC 49619
> 128
S. pyogenes
ATCC 700294
> 128
M. catarrhalis
ATCC 23246
32
E. coli
ATCC25922
> 128
H. influenzae
ATCC 49247
> 128
2.2
1.1
4
4
64
4
4
0.5
0.5
32
> 128
128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
128
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
16
2.2
> 128
32
> 128
> 128
> 128
> 128
> 128
> 128
> 128
2
> 128
> 128
> 128
> 128
16
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
32
3.1
1
2.3
> 128
64
32
2.0
2
1.8
32
2
1.1
> 128
128
4
4
1
14.4
15.4
11.9
12.2
8.4
4
2
0.5
16
≤ 0.25
64
> 128
4
> 128
> 128
128
0.5
2
≤ 0.25
0.5
≤ 0.25
≤ 0.25
2
8
32
64
9
4
64
2
16
32
12.3
10.8
11.8
1.3
2
64
0.5
2
8
64
16
64
128
> 128
32
16
> 128
> 128
> 128
> 128
> 128
> 128
4
1
32
8
4
8
64
> 128
> 128
> 128
> 128
> 128
1.6
> 128
> 128
64
> 128
> 128
4
> 128
> 128
16
> 128
> 128
>128
>128
2.1
1.2
1.4
64
32
1
0.8
1.8
128
64
> 128
128
32
16
2
> 128
> 128
> 128
> 128
32
33
32
0
0
> 128
> 128
> 128
64
> 128
> 128
34
> 128
1
> 128
0.25
> 128
0.125
> 128
0.06
> 128
8
> 128
2
35
azithromycin
15

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Table 3. Antibacterial activity of selected active compounds against additional clinical strains
of Gram-positive organisms.
MIC (µg/ml)
S. aureus
B0008
S. aureus
B0967
S. epidermidis
B0423
S. epidermidis
B0674
E. faecium
B0557
E. faecium
B0085
Compound
MRSA
MRSA
MSSE
MRSE
VSE
VRE
8
4
>128
128
>128
>128
>128
>128
>128
>128
32
32
16
64
16
128
32
32
16
10
11
>128
16
>128
128
128
64
128
128
128
128
>128
64
128
128
128
128
>128
32
64
12
64
13
128
64
128
128
128
32
15
16
>128
128
16
>128
128
16
17
18
16
4
8
19
128
8
128
64
128
8
128
4
32
32
20
2
16
21
16
128
32
4
64
32
>128
64
22
8
16
16
32
23
8
32
8
16
2
32
24
32
32
16
32
32
32
25
64
64
32
32
32
128
32
26
8
32
4
1
128
64
27
30
64
16
32
16
32
32
128
128
>128
>128
128
2
64
0.5
32
128
128
128
32
64
31
128
64
16
64
32
32
32
64
33
>128
128
1
≤0.25
≤0.25
2
>128
64
>128
64
oxacillin
ampicillin
vancomycin
azithromycin
0.5
0.25
≤0.25
1
>16
32
>128
>128
1
1
16

ACCEPTED MANUSCRIPT
The results of antibacterial screening revealed that, in general, the bis-phenyl
derivatives (21–27) were the most active compound subset with promising activity against
vancomycin resistant E. faecium strain (16–64 µg/ml). This subclass also exhibited high
binding affinity towards DNA (compounds 21, 24, 25, 26 all had ꢁTm values >10 °C). Within
the bis-phenyl series, compound 24 displayed the best overall antibacterial activity. The most
potent effect of compound 24 was manifested against E. coli and S. aureus (MICs of 8 and 2
µg/ml, respectively) approaching the activity of standard antibiotic azithromycin. In addition,
compound 24 showed marked activity toward S. pyogenes and M. catarrhalis (MICs of 0.5
and ≤0.25 µg/ml, respectively). The MICs of 64 µg/ml against S. pneumoniae and H.
influenzae indicated much lower potency against these species. Two additional compounds
from bis-phenyl series, compounds 21 and 23, showed similar trends in antibacterial activity
(Table 2). Compounds from bis-phenyl class also showed good antibacterial activity against
clinical strains of staphylococci and enterococci, especially compound 21 with MIC of 16
µg/ml against VRE.
In bis-benzimidazole analogues 18–20, replacement of both phenyl moieties with
benzimidazole groups did not improve antibacterial activity. Although all three analogues
retained high DNA binding affinities, only compound 19 exhibited significant antibacterial
potency. Compound 19 showed substantial activity against Gram-positive bacterial species,
including VRE (with the lowest MIC in this set of compounds, 8 µg/ml), and promising
activity against MRSA and MRSE strains (MICs 16–32 µg/ml). It was the only compound
with noteworthy activity against S. pneumoniae, impeding its growth at 2 µg/ml and at the
same time inhibiting S. pyogenes strain at 0.5 µg/ml. In addition, compound 19 showed the
strongest DNA binding affinity of all tested compounds (ꢁTm 15.4 °C).
Replacement of one amidino-benzimidazole group in symmetrical compounds
–
18 20
with substituted phenyl unit yielded asymmetrical compounds – and generally resulted in
9 17
the decrease of antibacterial activity. Among the three groups: cyano- ( – ), nitro- ( – )
9 11 12 14
and amino-amidine derivatives ( – ), only two compounds from the nitrile class, and
15 17
10
demonstrated somewhat wider antibacterial spectrum with prominent activity against M.
11,
catarrhalis (MICs of 0.5 g/ml), S. pyogenes and sensitive strain of S. aureus (MICs of 4
µ
g/ml for both species). This finding is in accordance with the wide use of nitrile groups in
µ
many commercial drugs since nitriles often play a key role as hydrogen bond acceptors and
polarize the aromatic π-system to optimize π–π interactions between compounds and DNA
[52]. Compounds
–
from the amino-amidine subclass, even though bearing amine groups
15 17
17

ACCEPTED MANUSCRIPT
as hydrogen bond donors, were found to be less potent. Substitution of amine group present in
–
with electron-acceptor dipolar nitro group yielded compounds
–
that were
15 17
12 14
biologically less active.
Expansion of the core by introduction of carboxamide bond between the phenyl group
and the central 3,4-ethylenedioxythiophene unit yielded compounds 27–34 and resulted in the
substantial decrease of the antibacterial activity as well as in the affinity towards DNA.
Compounds 31 and 27 (obtained by substitution of 1,4,5,6-tetrahydropyrimidine substituents
of compound 31 with amidine groups) showed excellent activities against methicillin-resistant
S. epidermidis strain with MICs of 0.5 and 1 µg/ml, respectively. Shifting the amidino moiety
on the phenyl ring from para (compounds 21 and 25) to meta position (compounds 32 and 33)
significantly impaired antibacterial activity against all the tested strains. Compounds 34 and
35, showing no changes in ꢁTm (Table 2), were antibacterially inactive.
In this study it was observed that in all derivative groups, except carboxamid-
amidines, amidine derivatives with alkyl end groups appeared to be more active than their
counterparts containing unsubstituted amidine moiety or cyclic amidines as end groups. This
might be due to the fact that hydrophobic groups, which are often present in DNA binding
agents, maximize contacts with deoxyribose in DNA and thus increase the agent-DNA
binding strength [11]. Therefore, structural modifications of the end groups have a decisive
effect on the biological activity.
To additionally verify suggested interactions, the binding of here investigated
compounds into the DNA minor groove was analysed in silico by utilizing the most active
compound
as a representative. This compound has both the best antibacterial profile and
19
the highest relative binding affinity for DNA. The binding of the molecule
to DNA has
19
been suggested by using simple molecular docking approach and the crystal structure of the
complex available in the RCSB Protein and Nucleic Acid Data Banks with
-
DB818 DNA
accession number
[53].
binds to AT rich minor groove region of the duplex B-
1VZK
DB818
1VZK
utilised B-DNA duplex model structure with AATT minor groove site. We supposed that due
DNA [30,39] DNA model used in
structure has been d(CGCGAATTCGCG) , often
2
to structural similarity with
(Figures 2 (A) and (B)).
compound
is likely to share similar binding mode
DB818,
19
The thiophene compounds
and
19
have very similar concave inner radius
DB818
(Figure 2 (B)). Their central phenyl/benzimidazole-thiophene-benzimidazole building blocks
18

ACCEPTED MANUSCRIPT
are nearly planar allowing their optimal fit against the five base-pair edges at the floor of the
groove. Both compounds form hydrogen bond contacts primarily with the groove floor,
specifically AT bases of the DNA model molecule [30]. Their terminal amidine groups are
conformationally flexible. In the case of
these groups make hydrogen bonds with AT
DB818
bases of DNA [30], while in the case of the larger compound
they face sugar-phosphate
19
backbone for hydrogen bonding and electrostatic interactions (Figure 2 (C)). Although
compounds - are structurally more similar to than compound the presence of
9 17
DB818
19,
structural groups less prone to form hydrogen bonds such as cyano, amino and nitro, limits
their hydrogen bonding capacity and consequently results in a lower DNA binding affinity
(i.e. lower T values).
∆
m
19

ACCEPTED MANUSCRIPT
20

ACCEPTED MANUSCRIPT
Figure 2. (A) 2D structures of compounds DB818 and 19. (B) Alignment of the crystal
structure of DB818 (magenta) and the optimized, B3LYP/6-31+G** structure of 19. The
more stable conformer of 19 with two intramolecular hydrogen bonds between imidazoles and
3,4-ethylenedioxy fragment, is shown. (C) Overlay of diprotonated structures of DB818 and
19 (shown in (B)) within the AATT binding site of d(CGCGAATTCGCG)2 [30]. Possible
interactions between compound 19 and DNA are shown in yellow dots.
From the results of this study, it could be generally suggested that DNA binding
represents the mechanism of antibacterial action of here reported compounds. Nevertheless,
no direct correlation between ꢁTm values and antibacterial activity for all the compounds
could be observed. A number of possible explanations for this could be proposed. Additional
biological and cellular aspects including different ability of the compounds to passively cross
bacterial cell wall and/or engagement of different influx/efflux transport mechanisms might
lead to poor accumulation within the cell and consequently impede DNA-compound
interactions. The potential for the distinct substrate specificity for closely related bis-indoles
was already suggested for Multidrug and Toxic Compound Extrusion (MATE) family of
efflux pumps [24] indicating that altered compound cell accumulation could influence the
discrepancies observed between target and whole-cell activities. Previous studies of MGBs
have made similar observations and conclusions, mainly emphasizing that target activity is
just one of the factors influencing biological activity and that physicochemical properties,
membrane interactions and transport play significant part in the overall drug potency [11,12].
2.5. In vitro cytotoxic activity
Nine new amidine derivatives 9–17 were assessed on their cytotoxic effect against two human
cell lines: HepG2 (hepatocellular carcinoma) and THP-1 (acute monocytic leukemia) to
determine whether the new compounds have inhibitory influence on host cells. By using MTS
assay, metabolic activity of the cells was evaluated and none of the nine compounds inhibited
cell proliferation i.e. IC50 values for all compounds and for both cell lines were >95 ꢀM. New
derivatives 9-17, in general, affected eukaryotic cells to a lesser extent than previously
synthesized compounds bis-benzimidazoles 18–20 [26], bis-phenyls 21–26 [27] and
carboxamid-amidines 27–34 [28] that showed cytotoxic activity against several different
human cell lines. Out of the subsets of previously synthesized molecules, compounds 25 and
27 were more cytotoxic than their counterparts displaying cytotoxic effect in the low
21

ACCEPTED MANUSCRIPT
micromolar concentrations across all the tested cell lines, while compound 19, although being
the most effective in its subset, was not as potent and consistent against all the cell lines.
Overall, SAR between different biological effects, primarily with regard to cytotoxicity and
antibacterial potency, could not be explicitly claimed.
3. CONCLUSIONS
In this study 9 new amidine derivatives of 3,4-ethylenedioxythiophene (9–17) were
synthesized. Their DNA binding properties and cytotoxicity were measured. Here presented
new derivatives, together with our previously reported compound classes, were for the first
time evaluated for their antibacterial potency against sensitive and resistant bacterial strains.
Most of the compounds have shown moderate activity against selected bacterial
species with antibacterial spectrum extended against resistant gram positive strains (MRSA,
MRSE, VRE). The bis-phenyl derivatives (21–27) were the most active subset of compounds
with promising activity against both sensitive and resistant strains and compounds 21–24
being the most potent. Compound 19 from bis-benzimidazole class exhibited the widest
antibacterial spectrum of activity (with MIC of 4, 2, 0.5 and ≤0.25 ꢀg/ml against laboratory
strains S. aureus, S. pneumoniae, S. pyogenes, M. catarrhalis, respectively and 4-32 ꢀg/ml
against clinical isolates of sensitive and resistant S. aureus, S. epidermidis and E. faecium)
and demonstrated the strongest DNA binding affinity (ꢁTm of 15.4 °C). Asymmetrically
designed compounds and carboxamid-amidines were, in general, less active although nitriles
10 and 11 demonstrated wider antibacterial spectrum of activity. Also, in this class of
asymmetric compounds, alkyl derivatives have shown higher potency. Simple molecular
docking indicated that the shape of here presented compounds and their ability to form
multiple electrostatic and hydrogen bonds correspond to the DNA binding mode of other
MGBs. For compounds, although no direct correlation between DNA binding and
antibacterial action was established, it can be suggested that DNA binding plays an important
role in the mechanism of action.
Herein reported results encourage further derivatization and investigation of this
compound class as novel antibacterial MGBs.
22

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4. EXPERIMENTAL SECTION
4.1. Chemistry
The compounds 18–35 have been described previously [26-28]. The synthesis and
physical properties of novel compounds are given as follows.
Solvents were distilled from appropriate drying agents shortly before use. TLC was
carried out on DC-plastikfolien Kieselgel 60 F254, Merck. Melting points were determined on
a Büchi 510 melting point apparatus and were uncorrected. IR spectra [νmax/cm–1] were
1
obtained on a Bruker Vertex 70 spectrophotometer. The H and 13C NMR spectra were
recorded on a Bruker Avance 300 MHz spectrometer. Chemical shifts (δ/ppm) were referred
to TMS. Mass spectra were recorded on a Waters Micromass Q-ToF micro. Elemental
analyses were performed by the Applied Laboratory Research Department at Ina, d.d.,
Research and Development Sector, Central Analytical Laboratory.
4.1.1. 5-(4-cyanophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine (2)
To a stirred solution of 3,4-ethylenedioxythiophene (1) (0.27 g, 1.90 mmol) in freshly
distilled THF (20 mL), 2.5 M n-BuLi in hexane (1 mL, 2.50 mmol) was slowly added under
the nitrogen atmosphere at –78 °C. The resulting solution was stirred until it reached
temperature of –40 °C and Bu3SnCl (0.7 mL, 0.84 g, 2.59 mmol) was added dropwise. The
vigorously stirred mixture was left overnight at room temperature. The solvent was removed
under reduced pressure, the residue suspended in anhydrous hexane and the suspension was
filtered off. The filtrate was concentrated to dryness under reduced pressure and the obtained
yellow liquid (0.81 g, 2.79 mmol) was dissolved in freshly distilled THF (20 mL) under a
nitrogen atmosphere. 4-Bromobenzonitrile (0.34 g, 2.86 mmol) and [Pd(PPh3)4] (0.11 g, 0.1
mmol) were added to the solution. The vigorously stirred mixture was refluxed for 7 days
under a nitrogen atmosphere. The solvent was partially evaporated, and obtained product was
purified by column chromatography (CH2Cl2:petrolether 1:1) to yield 0.39 g (85.7 %) of
white powder, mp 100–101 °C; IR (KBr) (νmax/cm–1): 2927, 2856, 2224, 1606, 1511, 1485,
1179, 1066, 910, 845, 819, 711, 681, 554; 1H NMR (DMSO-d6) δ/ppm: 7.75 (d, 2H, J = 8.60
Hz, ArH), 7. 77 (d, 2H, J = 8.60 Hz, ArH), 6.36 (s, 1H, ArH), 4.38 + 4.27 (m + m, 4H,
OCH2CH2O). MS m/z 244.00 (M+H)+.
4.1.2. 5-(4-nitrophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine (3)
23

ACCEPTED MANUSCRIPT
To a stirred solution of 3,4-ethylenedioxythiophene (1) (3 g, 21.1 mmol) in freshly
distilled THF (230 mL), 2.7 M n-BuLi in hexane (8 mL, 21.6 mol) was slowly added under
the nitrogen atmosphere at –78 °C. The resulting solution was stirred till –40 °C when
Bu3SnCl (6 mL, 7.2 g, 22.2 mol) was added dropwise. The vigorously stirred mixture was left
over night at room temperature. The solvent was removed under reduced pressure. The
residue was suspended in anhydrous hexane and suspension was filtered off. The filtrate was
concentrated to dryness under reduced pressure and the was dissolved in freshly distilled THF
(200 mL) under a nitrogen atmosphere and then 4-bromo-1-nitrobenzen (4.26 g, 21.1 mmol)
and [Pd(PPh3)4] (1.22 g, 1.06 mmol) were added. The vigorously stirred mixture was refluxed
for 3 days under a nitrogen atmosphere. The solvent was evaporated, and the precipitate was
dissolved in mixture of CH2Cl2:petrolether 1:1, and purified by column chromatography.
Yield 2.74 g (49.4 %) of yellow powder, mp 189–190 °C; IR (νmax/cm–1): 1587, 1498, 1474,
1
1336, 1175, 1060, 851, 753, 705, 664, 526, 461; H NMR (CDCl3) δ/ppm: 8.25 (d, 2H, J =
9.01 Hz, ArH), 7.89 (d, 2H, J = 9.05 Hz, ArH), 6.88 (s, 1H, thiophene), 4.41 + 4.29 (m + m,
4H, OCH2CH2O); 13C NMR (DMSO-d6) δ/ppm: 145.4, 142.9, 141.6, 139.9, 125.9, 124.8,
114.1, 102.3, 65.6, 64.5.
4.1.3. 7-(4-cyanophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-carbaldehyde (4)
The solution of phosphorous oxychloride (16 mL, 26.40 g, 0.17 mol) and
dimethylformamide (13 ml, 12.2 g, 0.17 mol) was stirred at 0 °C for 1.5h. Then solution of
compound 2 (8.14 g, 0.03 mmol) in dry DMF (150 mL) was added and the reaction mixture
was stirred under reflux for 5 h. After cooling, the solution was poured into ice and Na2CO3 is
added until pH 8. Resulting solid is filtered off and washed with water till pH 7.
Recrystallization from ethanol yield 6.02 g (66.9 %) of product, mp 262–263 °C; IR (KBr)
(νmax/cm–1): 2925, 2855, 2229, 1658, 1606, 1516, 1488, 1452, 1371, 1277, 1082, 834, 672,
538; 1H NMR (DMSO-d6) δ/ppm: 9.97 (s, 1H, CHO), 7.88 (d, 2H, J = 8.37 Hz, ArH), 7.67 (d,
2H, J = 8.33 Hz, ArH), 4.43 (s, 4H, OCH2CH2O).; 13C NMR (DMSO-d6) δ/ppm: 179.6,
149.2, 140.0, 135.7, 132.9, 126.9, 123.5, 118.4, 116.4, 110.7, 65.3, 65.0; MS m/z 272.1
(M+H)+.
4.1.4. 7-(4-nitrophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-carbaldehyde (5)
The solution of phosphorous oxychloride (0.4 mL, 0.66 g, 4.31 mmol) and
dimethylformamide (0.33 ml, 0.31 g, 4.31 mmol) was stirred at 0 °C for 1.5 h. Then solution
of compound 3 (0.19 g, 0.72 mmol) in dry DMF (8 mL) was added and the reaction mixture
24

ACCEPTED MANUSCRIPT
was stirred under reflux for 5 h. After cooling, the solution was poured into ice and Na2CO3 is
added until pH 8. Resulting solid is filtered off and wash with water till pH 7.
Recrystallization from ethanol yield 0.14 g (69 %) of product, mp 253–254 °C; IR (KBr)
(νmax/cm–1): 1652, 1595, 1498, 1449, 1336, 1077, 842, 681, 461; 1H NMR (DMSO-d6) δ/ppm:
9.94 (s, 1H, ArH), 8.31 (d, 2H, J = 9.02 Hz, ArH), 8.04 (d, 2H, J = 9.03 Hz, ArH), 4.5 (s, 4H,
13
OCH2CH2O); C NMR (DMSO-d6) δ/ppm: 180.4, 149.7, 147.0, 141.0, 138.1, 127.7, 124.9,
123.4, 117.1, 65.8, 65.5;
4.1.5. 2-(7-(4-cyanophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-yl)-1H-
benzo[d]imidazole-6-carboximidamide hydrochloride (9)
A solution of compound 4 (0.07 g, 0.26 mmol), 3,4-diaminobenzimidamide
hydrochloride (6) (0.05 g, 0.27 mmol), and 1,4-benzoquinone (0.03 g, 0.28 mmol) in ethanol
(30 mL) was heated at reflux for 4h (under nitrogen). The reaction mixture was cooled to
room temperature. Dark solid collected by filtration was washed with anhydrous ether. Crude
solid was dissolved in water and by addition of acetone precipitate is formed. It was dissolved
in HCl-saturated ethanol and stirring overnight. Green solid was collected by filtration,
washed with anhydrous ether and dried under vacuum to yield 0.04 g (11.1 %) of brown
powder, mp > 290 °C; IR (νmax/cm–1): in KBr: 3099, 2219, 1596, 1481, 1315, 1083, 815, 538;
1H NMR (DMSO-d6) δ /ppm: 12.63 (s, 1H, NH), 9.27 (s, 2H, NH), 8.93 (s, 2H, NH), 8.09 (s,
1H, ArH), 7.93 (d, 4H, J = 1.26 Hz, ArH), 7.75 (d, 1H, J = 7.89 Hz, ArH), 7.65 (d, 1H, J =
13
8.09 Hz, ArH), 4.58 + 4.54 (s + s, 4H, OCH2CH2O); C NMR (DMSO-d6) δ /ppm: 166.1,
142.1, 140.2, 136.3, 132.9, 126.0, 122.1, 121.4, 118.8, 116.6, 109.2, 106.5, 65.1, 65.0.
HRMS: calcd. for C21H16N5O2S (M+H)+: 402.1025; found: 402.1000. Anal. calcd. for
C21H15N5O2S × HCl × 2.5H2O (Mr = 482.95): C 52.23, H 4.38, N 14.50; found: C 52.51, H
4.29, N 14.11%.
4.1.6. 2-(7-(4-cyanophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-yl)-N-isopropyl-
1H-benzo[d]imidazole-6-carboximidamide hydrochloride (10)
A
solution of compound
4
(0.1385 g, 0.51 mmol), 3,4-diamino-N-
isopropylbenzimidamide hydrochloride (7) (0.117 g, 0.51 mmol), and 1,4-benzoquinone (0.05
g, 0.51 mmol) in ethanol (50 mL) was heated at reflux for 9 h (under nitrogen). The reaction
mixture was cooled to room temperature. Dark precipitate collected by filtration was washed
with anhydrous ether. Crude solid was dissolved in water and by addition of acetone
precipitate was formed. It was dissolved in HCl-saturated ethanol and stirred overnight. Green
25

ACCEPTED MANUSCRIPT
solid was collected by filtration, washed with anhydrous ether and dried under vacuum to
yield 0.12 g (54.6 %) of green powder, mp 271–272 °C; IR (νmax/cm–1): 3093, 227, 2595,
1
1482, 1441, 1352, 1077, 535; H NMR (DMSO-d6) δ /ppm: 9.50 (s, 1H, NH), 9.37 (s, 1H,
NH), 8.96 (s, 1H, NH2), 8.00 (s, 1H, ArH), 7.93 (s, 4H, ArH), 7.74 (d, 1H, J = 7.94 Hz, ArH),
7.54 (d, 1H, J = 7.64 Hz, ArH), 4.59 + 4.57 (br.s + br s, 4H, OCH2CH2O) 4.07 (m, 1H, CH),
13
1.30 (d, 6H, J = 6.35 Hz, CH3); C NMR (DMSO-d6) δ /ppm: 162.9, 147.5, 142.6, 140.7,
136.8, 133.4, 126.5, 123.3, 122.8, 119.3, 117.0, 109.7, 107.1, 65.6, 65.5, 45.5, 21.8. HRMS:
calcd. for C24H22N5O2S (M+H)+: 444.1494; found: 444.1489. Anal. calcd. for C24H21N5O2S ×
HCl × 3H2O (Mr = 534.04): C 53.98, H 5.28, N 13.11; found: C 53.73, H 5.14, N 13.12 %.
4.1.7. 2-(7-(4-cyanophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-yl)-6-(4,5-
dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole hydrochloride (11)
A solution of compound 4 (0.05 g, 0.18 mmol), 4-(4,5-dihydro-1H-imidazol-2-
yl)benzene-1,2-diamine hydrochloride (8) (0.04 g, 0.18 mmol), and 1,4-benzoquinone (0.02 g,
0.18 mmol) in ethanol (40 mL) was heated at reflux for 13 h (under nitrogen). The reaction
mixture was cooled to room temperature and the dark solid collected by filtration was washed
with anhydrous ether. It was dissolved in water and by addition of acetone precipitate was
formed. The precipitate was dissolved in HCl-saturated ethanol and stirred overnight. Green
solid was collected by filtration, washed with anhydrous ether and dried under vacuum to
yield 0.06 g (81.8 %) of green powder, mp 274–275 °C; IR (νmax/cm–1): in KBr: 3091, 2221,
1
1587, 1355, 1271, 1078, 1006, 849, 702, 538; H NMR (DMSO-d6) δ/ppm: 10.42 (s, 2H,
NH), 7.93 (s, 1H, NH), 7.93 (s, 4H, ArH), 7.77 (s, 3H, ArH), 4.60 + 4.56 (br s + br s, 4H,
13
OCH2CH2O), 4.01 (s, 4H, NCH2CH2N); C NMR (DMSO-d6) δ/ppm: 166.1, 147.0, 143.0,
142.8, 140.7, 140.5, 136.8, 133.4, 126.6, 123.1, 119.2, 115.9, 109.8, 106.9, 65.6, 65.5, 44.7,
40.5. HRMS: calcd. for C23H18N5O2S (M+H)+: 428.1181; found: 428.1197. Anal. calcd. for
C23H17N5O2S × HCl × 2.5H2O (Mr = 508.99): C 54.28, H 4.55, N 13.76; found: C 54.46, H
4.20, N 13.87.
4.1.8. 2-(7-(4-nitrophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-yl)-1H-
benzo[d]imidazole-6-carboximidamide hydrochloride (12)
A solution of compound 5 (0.2186 g, 0.75 mmol), 3,4-diaminobenzimidamide
hydrochloride (6) (0.1686 g, 0.75 mmol), and 1,4-benzoquinone (0.09 g, 0.75 mmol) in
absolute ethanol (200 mL) was heated at reflux for 2 days (under nitrogen). The reaction
mixture was cooled to room temperature and the dark solid was collected by filtration and
26

ACCEPTED MANUSCRIPT
washed with anhydrous ether. The solid was suspended in HCl-saturated ethanol and stirred
overnight. Green solid was collected by filtration, washed with anhydrous ether, and dried
under vacuum to yield 0.22 g (64.4 %) of green powder, mp > 290 °C; IR (νmax/cm–1): in KBr:
1
3077, 1685, 1588, 1482, 1442, 1337, 1086, 851; H NMR (DMSO-d6) δ /ppm: 9.35 (s, 2H,
NH), 9.11 (s, 2H, NH), 8.31 (d, 2H, J = 8.80 Hz, ArH), 8.11 (s, 1H, ArH), 8.00 (d, 2H, J =
8.79 Hz, ArH), 7.76 (d, 1H, J = 7.52 Hz, ArH), 7.67 (dd, 1H, J1 = 8.38 Hz, J2 = 0.84 Hz ArH),
4.61 + 4.58 (br s+ br s, 4H, OCH2CH2O); 13C NMR (DMSO-d6) δ /ppm: 166.2, 147.0, 145.6,
142.4, 140.7, 138.3, 126.2, 124.4, 122.3, 121.6, 116.4, 106.9, 65.1; HRMS: calcd. for
C20H16N5O4S (M+H)+: 422.0918; found: 422.0917. Anal. calcd. for C20H15N5O4S × HCl ×
2H2O (Mr = 493.93): C 48.63, H 4.08, N 14.18; found: C 48.87, H 4.10, N 14.22.
4.1.9. 2-(7-(4-nitrophenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-yl)-N-isopropyl-
1H-benzo[d]imidazole-6-carboximidamide hydrochloride (13)
A
solution of compound
5
(0.098 g, 0.34 mmol), 3,4-diamino-N-
isopropylbenzimidamide hydrochloride (7) (0.077 g, 0.34 mmol), and 1,4-benzoquinone (0.04
g, 0.34 mmol) in ethanol (110 mL) was heated at reflux for 3 days (under nitrogen). The
reaction mixture was cooled to room temperature and the dark solid was collected by filtration
and washed with anhydrous ether. The solid was dissolved in HCl-saturated ethanol and
stirred overnight. Green solid was collected by filtration, washed with anhydrous ether, and
dried under vacuum to yield 0.12 g (70.6 %) of green powder, mp > 290 °C; IR (νmax/cm–1): in
1
KBr: 3085, 2357, 1580, 1482, 1337, 1087, 851, 673; H NMR (DMSO-d6) δ /ppm: 9.49 (d,
1H, J = 7.61 Hz, NH), 9.35 (s, 1H, NH), 8.94 (s, 1H, NH), 8.31 (d, 2H, J = 8.94 Hz, ArH),
8.02 (d, 2H, J = 8.95 Hz, ArH), 7.98 (s, 1H, ArH), 7.76 (d, 1H, J = 8.34 Hz, ArH), 7.54 (dd,
1H, J1 = 8.37 Hz, J2 = 1.40 Hz, ArH), 4.61 + 4.59 (br s+ br s, 4H, OCH2CH2O), 4.16 (m, 1H,
13
CH), 1.31 (d, J = 6.39 Hz, 6H); C NMR (DMSO-d6) δ /ppm: 163.2, 147.4, 146.4, 143.3,
141.5, 139.1, 127.1, 125.2, 123.9, 123.4, 117.3, 107.5, 65.9, 45.8, 22.1. HRMS: calcd. for
C23H22N5O4S (M+H)+: 464.1387; found: 464.1341. Anal. calcd. for C23H21N5O4S × HCl ×
2.5H2O (Mr = 545.02): C 50.69, H 4.99, N 12.85; found: C 50.35, H 4.92, N 12.83.
4.1.10. 6-(4,5-dihydro-1H-imidazol-2-yl)-2-(7-(4-nitrophenyl)-2,3-dihydrothieno[3,4-
b][1,4]dioxine-5-yl)-1H-benzo[d]imidazole hydrochloride (14)
A solution of compound 5 (0.087 g, 0.29 mmol), 4-(4,5-dihydro-1H-imidazol-2-
yl)benzene-1,2-diamine hydrochloride (8 (0.062 g, 0.29 mmol), and 1,4-benzoquinone (0.03
g, 0.29 mmol) in ethanol (110 mL) was heated at reflux for 3 days (under nitrogen). The
27