Design, Microwave-Assisted Synthesis and in Vitro Antibacterial and Antifungal Activity of 2,5-Disubstituted Benzimidazole

Article abstract of DOI:10.1002/cbdv.201800510

Seventeen novel 2,5-disubstituted benzimidazole derivatives were designed, synthesized and evaluated for their antibacterial activities. The tested compounds B1–B4 and C2–C6 exhibited not only good antifungal activity but also favorable broad-spectrum antibacterial activity. Also, the lowest MIC of antibacterial and antifungal activity was 2 μg/mL and 4 μg/mL, respectively. It suggested that the structure of compound including the different substituent and its sites directly affected the efficacy of the synthesized compounds.

Full text of DOI:10.1002/cbdv.201800510

Title: Design, microwave-assisted synthesis and in vitro antibacterial
and antifungal activity of 2,5-disubstituted benzimidazole
Authors: Yanpeng Shi, Kai Jiang, Ran Zheng, Jiaxu Fu, Liuqing Yan,
Qiang Gu, Yumin Zhang, and Feng Lin
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201800510
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800510

Chemistry & Biodiversity
10.1002/cbdv.201800510
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Design, microwave-assisted synthesis and in vitro antibacterial and antifungal
activity of 2,5-disubstituted benzimidazole
a
b
a
a
a
a
a
b
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Yanpeng Shi , Kai Jiang , Ran Zheng , Jiaxu Fu , Liuqing Yan , Qiang Gu , Yumin Zhang* , Feng Lin*
aCollege of Chemistry, Jilin University, Changchun 130012, P. R. China
b
5
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School of life Sciences, Jilin University, Changchun 130012, P. R. China
*Corresponding Author: Professor Yumin Zhang and Professor Feng Lin
Tel: (+86)-431-85168470 (3)
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Fax: (+86)-431-85168441
E-mail: zhang_ym@jlu.edu.cn
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Abstract Seventeen novel 2,5-disubstituted benzimidazole derivatives were designed, synthesized
and evaluated for their antibacterial activities. The tested compounds B1–B4 and C2–C6 exhibited
not only good antifungal activity but also favorable broad-spectrum antibacterial activity. Also, the
lowest MIC of antibacterial and antifungal activity was 2 μg/mL and 4 μg/mL, respectively. It
suggested that the structure of compound including the different substituent and its sites directly
affected on efficacy of the synthesized compound.
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Keyword: 2,5-disubstituted benzimidazole, microwave-assisted synthesis, antimicrobial activity
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Introduction
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Since the early 1900s, the incidence of systemic bacterial and fungal infections has been
increased in relatively high rates of morbidity and mortality.[1–3] This is because of an enhanced
number of immunocompromised patients who are impressionable to these infections. Several
research teams have indicated Candida albicans (C. albicans) is the most common fungal species in
these patients.[3–5] C. albicans is a commensal fungus resides in the skin and the human
gastrointestinal. It can cause serious mucosal infections along with fatal invasive infections in
potential individuals, who subjected to immunodeficiency result of chemotherapy for cancer,
immune suppression for the transplants of stem cells or solid organs, or HIV infections.[6–8] Recently,
although many new drugs have been used in clinical treatment, C. albicans sustained to dominate as
the predominant causative agent of systemic fungal infections.[9]
Additionally, it is well-known that the bacterium causing human diseases are mainly
gram-negative and positive bacteria all the time. [10][11] For example, Pseudomonas aeruginosa (P.
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Chemistry & Biodiversity
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aeruginosa) is one of the gram-negative opportunistic pathogens, which can extensively causes all
kind of clinical infections such as people getting severe burnt, AIDS, diabetes patients and cystic
fibrosis(CF) sufferers.[12][13] P. aeruginosa is one of the most epidemic and clinically significant
pathogens for CF of sufferers. All gram-positive bacteria infections were attributed to
multi-drug-resistant gram-positive bacteria expression a major public health professions problem in
the world, and are always related with high mortality rates.[14] It is estimated that Staphylococcus
aureus (S. aureus) as a gram-positive bacteria is in total found about 20–30% of the human
population for nasal passages, skin and mucous membranes.[15][16] S. aureus can lead to the infection
of the bloodstream, reduce respiratory track, destroy skin and soft tissue, further result in bacteremia,
ventilator associated pneumonia, as well as endocarditis and osteomyelitis illness.[17,18] Besides,
antimicrobial resistance, one of the all over worldwide threats, is a fearful matter under review by the
world health organization (WHO) and many countries in the globe.[19–22] Staphylococcus epidermidis
(S. epidermidis), one of the greatest important gram-positive bacterium, which is among important
compromise for the resistant strains[23–25], simultaneously is deemed to a significant nosocomial
pathogen influencing low immunity, such as athroplasty, central venous catheters, and analogues.
Currently, S. epidermidis has become a main clinical medicine problem, and led to vital morbidity
and mortality in both people biota group and those with potential complication.[23–25] The resistant
strains make complicated to the therapy, and significantly affect the get well of patients.[26]
Additionally, fungal resistance also increased. Therefore, it is imperative to design and synthesize
new antibacterial and antifungal agents.
It is reported that benzimidazole analogues have good biological and pharmaceutical activities
and drug-resistant properties[27][28] including antifungal, antimicrobial, anticancer, etc. because they
have a similar structure with a purine which play pivotal role in the synthesis of protein and nucleic
acid in the bacteria.[29] 2-Substituted benzimidazole derivatives are reported to exhibit good
antimicrobial activity,[30–33] for example, the 2-position of benzimidazole derivatives bearing
heterocyclic rings, such as thiabendazole, 2-(thiophen-2-yl)-1H-benzimidazole(Fig. 1) and
naphthalene ring.[34–36] Especially, thiabendazole without teratogenic, carcinogenic, and mutagenic
effects has been manufactured.[29][37] Also, it is reported that benzene ring of benzimidazole with an
inductive electron-withdrawing group can improve antibacterial activity.[38][39]
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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Thiabendazole
2-(Thiophen-2-yl)-1H-benzimidazole
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Fig. 1 The structure of Thiabendazole and 2-(Thiophen-2-yl)-1H-benzimidazole
Moreover, microwave irradiation technology is a rising organic synthesis technology in recent
two decades. It had attracted much attention because of its high yield, low waste, energy efficiency,
short reaction time and simple operation. With all this in mind and in view of the continuation of our
work,[40][41] 2,5-disubstituted benzimidazole compounds including heterocyclic ring or naphthalene
ring and electron-withdrawing group were designed and synthesized under microwave irradiation.
The synthesized results and the evaluation of antifungal activity against C. albicans(ATCC 1023), P.
aeruginosa(ATCC 27853), E. coli(ATCC 25922), Staphylococcus aureus(S. aureus ATCC 25923, S.
aureus ATCC 3933) and S. epidermidis(ATCC 12228) wish to be reported here.
Results and discussion
Design, synthesis and reaction conditions' optimization of 2,5-disubstituted benzimidazoles
According to the literature some 2-substituted benzimidazoles were synthesized using Na
2
S
O
2 5
as catalyst and oxidant starting from mono-substituted phenylenediamine under traditional heating
and water-ethanol solvent conditions. The method for synthesizing 2-substituted benzimidazoles
required not only long reaction time (12 h) but also a plenty of mixture solvent.[42] At the beginning
of the study, 6-methyl-2-(3-pyridyl)-1H-benzimidazole(A1) was designed and synthesized by
transforming traditional heating into microwave irradiation. Unfortunately, the desired prodAlso, it is
reported that the reaction time was evidently shortened when the reaction was carried out in the
[
43]
presence of Na
2
S
2
O catalyst under solvent-free and microwave radiation condition. uct was not
5
entirely obtained and always accompanied by byproduct bis-Schiff bases that was hard to be
separated completely, when the reaction started from 4-methylbenzene-1,2-diamine(a) and
3-nicotinaldehyde(D) using Al
2
O
3
, Na
2
S
2
O
5
or NaHSO
3
as catalysts and oxidants and EtOH as a
, Table 1,
, Table 1, entry 5). However, no
[PW12O40] as catalysts and oxidants. The main reason
o
solvent at 78 C. Also, the yields of the obtained product were respectively 46.2%( Al
2
O
3
entry 3), 68.6%( Na
S
2 2
O
5
, Table 1, entry 4) and 54.7% (NaHSO
3
product was obtained using FeCl
3
∙6H O and H
2
3
lied in that Na
2
S
2
O
5
could provide not only an acidic environment for the ring-closure, but also
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Chemistry & Biodiversity
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appropriate ability for in situ oxidizing 2,3-dihydrobenzimidazole to benzimidazole. Further, the
catalyst Na loading was explored in EtOH solvent (Table 1, entries 4, 6–9). It was observed that
2
S
2
O
5
the yields of A1 were gradually increased to 68.6% (1 mmol), followed sequentially decrease to
68.1% (1.25 mmol) and 68.0% (1.50 mmol), along with the reaction times was gradually shortened
o
by increasing Na
2
S
O
2 5
loading from 0.50 mmol to 1.50 mmol at 78 C. It is suspected that the low
yield was related to the low boiling point of ethanol and the solubility of reactants. Besides, the
reactants molar ratio (a:D) were investigated(Table 1, entries 4, 10–12). The highest yield (74.4%,
Table 1, entry 11) was obtained when the reactants molar ratio a:D was 1:1.1. Subsequently, four
other solvent applied in the reaction and the obtained yields in different condition are shown in Table
1 (entries 13–16). The yield used DMF (83.5%) as a solvent was highest, followed by EtOH (74.4%),
and then acetonitrile (47.3%) and ethyl acetate (46.5%). The target product A1 was not obtained
using THF as a solvent. These maybe were related to the solubility of reactants and the tangent value
of the solvent loss angle.[44] Because the above reaction that obtained target product was always
accompanied by byproduct bis-Schiff bases. In order to enhance the yield and purity, the reaction
1
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o
o
temperatures were increased from 100 C to 140 C (Table 1, entries 16–20). Exciting, not only the
by-product was disappeared, but the reaction time was gradually shortened from 15 min to 8 min.
o
The highest yield was up to 92.4 % at 130 C for 10 min. The possible reason was that the increase of
reaction temperature promoted the effective collision of reactants molecule and the ring closure
reaction. Therefore, taking into account the perspective of economic cost and environmental
protection, the optimized reaction condition was considered as follows: Catalyst select Na
2
S
2
O and
5
catalyst loaded 1.0 equivalent mol of a, The molar ratio of material was 1:1.1, DMF as a solvent and
o
1
1
10
11
microwave irradiation temperature 130 C (Table 1, entry 19).
Table 1 Some effect factors of the synthetic compound A1
o
Yielda
/ %
Entry
Reactant
a:D
Catalyst
Solvent
T/ C
Time/
min
Catalyst
equiv
1
1
1.0:1.2
H
3
[PW12
O
40
]
Ethanol
78
20
-
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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2
1.0:1.2
FeCl
Al
Na
NaHSO
3
∙6H
2
O
1
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
THF
78
25
18
15
22
18
15
13
13
13
13
13
25
25
20
15
15
12
10
8
-
1.0:1.2
1.0:1.2
1.0:1.2
1.0:1.2
1.0:1.2
1.0:1.2
1.0:1.2
1.0:1.0
1.0:1.1
1.0:1.3
1.0:1.1
1.0:1.1
1.0:1.1
1.0:1.1
1.0:1.1
1.0:1.1
1.0:1.1
1.0:1.1
2
O
3
1
78
46.2
68.6
54.7
59.2
63.4
68.1
68.0
66.8
74.4
67.6
-
2
S
2
O
5
1
78
3
1
78
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
Na
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
S
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0.5
0.75
1.25
1.5
1
78
78
78
78
0
1
2
3
4
5
6
7
8
9
0
78
1
78
1
78
1
68
1
CH
3
CN
82
47.3
45.6
83.5
85.3
87.9
92.5
91.4
1
EtOAc
DMF
DMF
DMF
DMF
DMF
98
1
100
110
120
130
140
1
1
1
1
1
1
1
1
1
1
1
1
12
13
14
15
16
17
18
19
a: isolated product yield; “-”: no product was obtained.
Subsequently, having been optimized the reaction conditions for the model system (Table 1,
entry 19) was chosen to synthesize the designed compounds(Table 2). A sequence of novel
2,5-disubstituted benzimidazoles including heterocyclic and naphthyl ring had been synthesized in
50.2%–92.5% yields. Seventeen different 2,5-disubstituted benzimidazoles(A1–A4, B1–B4 and
C1–C9) have been synthesized using Na
2
S
2
O as a catalyst and oxidant under microwave
5
irradiation.(Scheme 1). Various 4-substituted phenylenediamine with 3-pyridylaldehyde (to
Scheme 1 The route for synthesizing 2,5-disubstituted-benzimidazole.
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Chemistry & Biodiversity
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synthesize A series products) or 2-hydroxyl-1-naphthaldehyde (to synthesize B series products) or
various 2-thiophenecarboxaldehyde (to synthesize C series products) were carried out by dissolving
both reactants and Na
2
S
2
O in DMF in 1:1.1:1 molar ratio and heating them in microwave reactor at
5
o
130 C for 10–15 min. Structure, irradiation time, yield and Mp. of the synthesized products are
listed in Table 2. In this reaction, a nucleophilic amino group of 4-substituted phenylenediamine
firstly attacked carbon on aldehyde group of the other reactant giving rise to formation of a single
[
45]
Schiff-base which then further underwent cyclization to afford the target products . It is seen from
Table 2 that the yield of the synthesized 2,5-disubstituted benzimidazoles were 55.5–92.5% (Table 2,
entries A1–A4), 75.7–89.5% ( Table 2, entries B1–B4) and 50.2–92.1% (Table 2, entries C1–C9),
respectively. When 2-substituted group of the obtain 2,5-disubstituted benzimidazoles was the same,
the yield of the obtained target product was decreased with the electron-donating ability of
5-substituted group(–CH , –H, –Cl and –Br) on o-phenylenediamines decreasing (Table 2, entries
3
A1–A4). The reason maybe lied in the fact that the electron-donating groups increased the electron
cloud density of amino N on substituted o-phenylenediamine. It is helpful that the nucleophilic
amino group of 4-substituted phenylenediamine attacks carbon on carbonyl group of the
corresponding aldehyde and promoting nucleophilic addition reaction. When nicotinaldehyde was as
a reactant material, the yield was much higher than that of thiophene-2-carbaldehyde as a reactant
(Table 2, entries A1, C1 or A2, C2 or A3, C3 and so all), because electron-poor π feature of pyridine
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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39
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ring with higher conjugation energy enhanced nucleophilicity of carbonyl carbon. Meanwhile, when
2-hydroxy-1-naphthaldehyde as a reactant, the yield was slightly lower than that of
thiophene-2-carbaldehyde as a reactant, but slightly higher with nicotinaldehyde as a reactant (Table
2, entries B1, C1, A1 or B3, C2, A3 and so all). Additionally, it is also seen from Table 2 that the
reaction time shortened with the enhancement of the electron-donating ability of substituted
o-phenylenediamine. It is not difficult to find that the method proposed in this paper has good
applicability by combining the synthesis of the above benzimidazole derivatives and the effect of
different substituents on the yield of the target product.
Table 2 The synthesized compounds A1–A4, B1–B4 and C1–C9
a
o
Compound
Product
Reaction Time (min)
Yield (%)
Mp. ( C)
A1
10
92.5
80.6
70.9
55.5
218-220
218-220
244-246
238-240
A2
A3
A4
10
13
15
B1
B2
B3
13
13
15
89.5
85.1
85.1
230-232
224-225
234-236
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Chemistry & Biodiversity
10.1002/cbdv.201800510
B4
15
75.7
130-132
C1
C2
C3
10
12
15
90.6
67.8
51.4
238-240
226-228
196-198
C4
C5
C6
10
12
15
85.3
69.7
50.2
214-216
216-218
261-264
C7
C8
10
12
15
92.1
89.3
54.7
218-220
266-268
271-273
C9
1
1
48
49
a:Isolated product yield.
The antimicrobial activity analysis
1
1
1
1
50
51
52
53
Antimicrobial activity evaluation of the obtained compounds A1–A4, B1–B4 and C1–C9 was
carried out using 96-well tissue culture plates. Compounds A1–A4, B1–B4 and C1–C9 were
screened for biological antifungal activities against fungi C. albicans ATCC 10231, and resist
bacteria l P. aeruginosa ATCC 27853, E. coli ATCC 25922，S. aureus ATCC 25923，ATCC 3933 and
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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S. epidermidis ATCC 12228, respectively. Herein, ciprofloxacin (anti-bacteria), fluconazol and
itraconazole (anti-fungus) were respectively representatives as positive controls. The results of each
tested compound were recorded by the average diameter of inhibition zones for bacteria or fungi
cultivated around the discs in mm respectively. Then, the minimum inhibitory concentration (MIC)
was measured with the two-fold serial dilution by the method of Clinical and Laboratory Standards
Institute (CLSI).[46] All synthesized compounds A1–A4, B1–B4 and C1–C9 were evaluated under
concentrations rang of 0.125–64 μg/mL and the level of the growth inhibition microorganisms had
been determined by MIC compared with ciprofloxacin, fluconazol and itraconazole. The data of all
the compounds about antifungal and antibacterial activities are displayed in Table 3. It is seen from
Table 3 that some tested compounds exhibited not only antifungal activity but also favorable
broad-spectrum antibacterial activity excepted that had no inhibitory effect on P. aeruginosa. The
tested compounds A1–A4 were almost no inhibition to the tested microorganism when 2-position of
thiabendazole or 2-(thiophen-2-yl)-1H-benzimidazole(Fig. 1) was replaced with 3-pyridine ring
except from compound A3 (MIC against C. albicans was 64 µg/mL). Besides, the tested compounds
B1–B4 showed favorable abilities to inhibit bacteria when 2-position of thiabendazole or
2-(thiophen-2-yl)-1H-benzimidazole was replaced with 2-oxhydryl-naphtalene ring, the MIC of
compound B1 and B4 against S. aureus ATCC 25923 and ATCC 3933, and S. epidermidis ATCC
12228 respectively were all 16 µg/mL(B1) and 64 µg/mL(B4), the MIC of compound B2 and B3
against S. aureus ATCC 3933 and S. epidermidis ATCC 12228 were both 32 µg/mL (B2 and B3) and
16 µg/mL (B2 and B3). Only the MIC of compound B4 against C. albicans was 64 µg/mL in B1–B4.
Surprisingly, when the different bromo-substituted thiazole rings were introduced to 2-position of
different substituted benzimidazoles, the antifungal and antibacterial activities of compounds C1–C9
were remarkably improved compared with that of compounds A1–A4 and B1–B4. In the initial
exploration, compounds C3 was found to exhibit the favorable broad-spectrum antimicrobial
activities, also, the MICs of compounds C3 against C. albicans, E. Coli, S. aureus ATCC 25923 and
ATCC 3933, and S. epidermidis ATCC 12228 were 8 μg/mL, 32 μg/mL, 32 μg/mL, 32 μg/mL and 32
μg/mL, respectively. It was reported halogen substituent on aromatic ring might improve
antimicrobial activity.[29][39] Herein, halogen groups were designed to introduce onto benzene ring of
benzimidazole and thiazole rings to synthesize new 2,5-disubstituted benzimidazoles(C4–C9). It was
found that the antifungal and antibacterial activities of compounds(C5 and C6) dramatically
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
enhanced when 3-position of thiazole on 2-position of benzimidazole was substituted by bromo
group. Also, the determined MIC of compound C5 against S. aureus ATCC 25923 and ATCC 3933,
and S. epidermidis ATCC 12228 were respectively 2 μg/mL, 4 μg/mL and 4 μg/mL without
antifungal activity compared with that of compound C3. This could be because the interaction
between chloro substituent on benzene ring of benzimidazole and bromo group on thiazole rings led
to no antifungal activity. However, compound C6 exhibited not only good antifungal activity but also
remarkable broad-spectrum antibacterial activity. The MIC against C. albicans, S. aureus ATCC
25923 and ATCC 3933, and S. epidermidis ATCC 12228 respectively were 4 μg/mL, 2 μg/mL, 2
μg/mL and 4 μg/mL. The reason maybe lied in the fact that bromo group on benzene ring of
benzimidazole and thiazole rings had good synergistic effect. Unfortunately, the tested compounds
C7, C8 and C9 almost no antimicrobial activities except for the MIC of compound C8 against S.
aureus ATCC 25923 when bromo group bearing 4-prisition of thiazole on 2-position of
benzimidazole. It suggested that different substituents and their synergies play important roles in the
structure-activity relationship of the drug molecular design.
Table 3 MIC of the synthetic compounds
MIC (µg/mL)
C.
P.
E.
S.
S.
Compound
No.
albicans
ATCC
aeruginosa
ATCC
coli
aureus
epidermidis
ATCC
ATCC
25922
ATCC
25923
ATCC
1
0231
27853
3933
12228
A1
A2
A3
A4
B1
B2
B3
B4
C1
C2
C3
C4
C5
C6
C7
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
16
>64
>64
>64
>64
16
>64
>64
>64
>64
16
>64
>64
>64
>64
64
>64
>64
64
32
16
32
16
64
64
>64
16
>64
>64
32
>64
64
>64
32
8
32
32
32
32
>64
>64
>64
>64
16
16
16
>64
4
2
4
4
2
2
4
>64
>64
>64
>64
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Chemistry & Biodiversity
10.1002/cbdv.201800510
C8
>64
>64
-
>64
>64
0.125
-
>64
>64
0.125
-
4
>64
0.25
-
>64
>64
0.25
-
>64
>64
0.5
-
C9
Ciprofloxacin
Fluconazole
Itraconazole
0.5
0.125
-
-
-
-
-
1
2
99
00
Note：Values are presented as mean ± SD，n=3
Conclusion
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
In a word, a series of 2,5-disubstituted benzimidazole derivatives containing halogen and
heterocyclic ring structures using Na as the catalyst and oxidant under microwave assisted
2
S
O
2 5
condition were conveniently synthesized in 50.2–92.5% yields. The effect of various substituted
group on the yield of the target products was discussed. The structure of the obtained compounds
1
13
was confirmed by various spectral such as FT-IR, ESI-MS, H NMR, C NMR. Further, the test of
the antimicrobial activity showed that the synthesized B1–B4 and C2–C6 compounds exhibited not
only good antifungal activity but also favorable broad-spectrum antibacterial activity excepted that
had no inhibitory effect on P. aeruginosa. The MIC of compound C5 and C6 against the tested
bacterials were both less than 4 μg/mL, and the lowest one was 2 μg/mL. Besides, The MIC of
compound C6 against C. albicans was 4 μg/mL. It suggested that different substituent and their
synergies play important role in the structure activity relationship of the drug molecular design. In
addition, the study for the synthesis of novel benzimidazoles using microwave radiated method and
application in antimicrobial and bacterial quorum sensing field is currently in progress in our
laboratory.
Experimental
All the chemical reagents used in the synthesis reactions were available from commercial
companies (Tianjin Fuyu Chemical Co. Ltd, China; and Beijing Chemical Plant, China;
Aladdin-reagent Co., China) and without further purification. All kinds of analytical-reagent grade of
substituted o-phenylenediamine and were from Shanghai Darui Chemical Co. Ltd, China and used
without further purification. Thin Layer Chromatography (TLC) was used to tracking the completion
of the reaction and silica gel plate (GF 254) was purchased from Qingdao Ocean Chemcial Reagent
Company. Silica gel (200-300 mesh) was supplied by Tianjin Fuyu Chemcial Reagent Company. All
the melting points were detected by a XT−4 melting point apparatus and were uncorrected. FT-IR
spectra were collected, KBr pellets as a reference, using a Shimadzu IRAffinity–1 instrument in the
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Chemistry & Biodiversity
10.1002/cbdv.201800510
–
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
range of 500−3500 cm , HRMS were recorded on an Agilent1290–micrOTOF Q II spectrometer.
1H NMR and 13C NMR spectra was measured using a Bruker AVANCE–600 (Germany) and a
Zhongke-Niujin Q. One–AS 400 (China) NMR spectrometer with TMS regard as a reference. The
chemical shift δ concerned with TMS. A XO-50N microwave reactor with a thermocouple
thermometer purchased from NanJing Xianou instruments manufacture company was used to
synthesize the desired products.
General synthetic procedures
A mixture of 4-substituted o-phenylenediamine (1.0 mmol), 3-pyridinecarboxaldehyde (1.1 mmol)
or 2-hydroxy-1-naphthaldehyde (1.1 mmol) or substituted thiophene-2-carbaldehyde(1.1 mmol),
Na
2
S
2
O ( 1.0 mmol) were dissolved in DMF (15 mL) or EtOH (15 mL) and fully transferred to a
5
250 mL microwave reaction bottle equipped with a magnetic stir bar, reflux condenser and
o
thermocouple thermoelement, then heated with microwave irradiation at 130 C for 10–15 min. The
reaction progress was monitored by TLC. When the microwave-assisted reaction was over, the
reaction bottle was cooled to room temperature. Further, the residue was poured into beaker filled
with 80–100 mL ice water. The crude product was filtrated after it was completely precipitated. The
desired pure products A1-A4, B1-B4, C1-C9 were obtained by the purified with column
chromatography (silica gel, 200-300 mesh) using petroleum ether/ethyl acetate as a developing
solvent.
2
43
Representative spectral data
1
2
2
2
2
2
2
2
2
2
2
44
45
46
47
48
49
50
51
52
53
5-methyl-2-(pyridin-3-yl)-1H-benzimidazole(A1) Creamy white solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 12.95 (s, 1H,-NH), 9.35 (d, J = 1.8 Hz, 1H, ArH), 8.71 – 8.63 (m, 1H, ArH), 8.49
6
1
3
(d, J = 8.0 Hz, 1H, ArH), 7.63 – 7.34 (m, 3H, ArH), 7.07 (s, 1H, ArH), 2.45 (s, 3H, –CH
3
). C NMR
(101 MHz, DMSO) δ, ppm: 150.80, 148.97, 147.88, 134.06, 126.77, 124.45, 21.81. HRMS, m/z:
+
–1
(M+H) calcd for C13
H
11
N H 210.1026, found 210.1106. IR (KBr) υ (cm ): 3120, 3039, 3001, 2947,
3
2866, 1624, 1581, 1527, 1492, 1450, 1315, 1226, 1273, 952, 810, 705.
1
2-(pyridin-3-yl)-1H-benzimidazole(A2) Yellow solid. H NMR (600 MHz, DMSO-d
6
) δ,
ppm:13.12 (s, 1H, -NH), 9.38 (d, J = 2.3 Hz, 1H, ArH), 8.69 (dd, J = 4.8, 1.5 Hz, 1H, ArH), 8.52 (dd,
1
3
J = 8.0, 1.9 Hz, 1H, ArH), 7.71 – 7.57 (m, 3H, ArH), 7.28 – 7.23 (m, 2H, ArH). C NMR (101 MHz,
+
DMSO) δ, ppm: 151.00, 149.34, 148.01, 134.23, 126.65, 124.50. HRMS, m/z: (M+H) calcd for
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Chemistry & Biodiversity
10.1002/cbdv.201800510
–
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
C
12
H
9
N
3
H 196.0869, found 196.0958. IR (KBr) υ (cm ): 3441, 3043, 2993, 2912, 2877, 1573, 1539,
1489, 1446, 1273, 1226, 1188, 1022, 867, 810, 740, 698.
5-chloro-2-(pyridin-3-yl)-1H-benzimidazole(A3) Creamy white solid. H NMR (600 MHz,
DMSO-d ) δ, ppm:13.32 (s, 1H, –NH), 9.36 (dd, J = 2.3, 0.9 Hz, 1H, ArH), 8.71 (dd, J = 4.8, 1.6 Hz,
1H, ArH), 8.50 (dt, J = 7.9, 1.9 Hz, 1H, ArH), 7.72 (d, J = 8.7 Hz, 1H, ArH), 7.69 – 7.63 (m, 1H,
1
6
1
3
ArH), 7.61 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H, ArH), 7.27 (dd, J = 8.5, 2.0 Hz, 1H, ArH). C NMR (101
+
MHz, DMSO) δ, ppm: 151.31, 150.77, 148.10, 134.40, 126.22, 124.53. HRMS, m/z: (M+H) calcd
–
1
for C12
H
8
ClN
1446, 1396, 1276, 1222, 1188, 1056, 844, 810, 702.
5-bromo-2-(pyridin-3-yl)-1H-benzimidazole(A4) Brown schistose solid. H NMR (600 MHz,
DMSO-d ) δ, ppm:13.31 (d, J = 24.0 Hz, 1H, -NH), 9.35 (dd, J = 2.3, 0.9 Hz, 1H, ArH), 8.71 (dd, J
= 4.8, 1.6 Hz, 1H, ArH), 8.50 (ddd, J = 8.0, 2.3, 1.7 Hz, 1H, ArH), 7.69 (d, J = 8.17 Hz, 1H, ArH),
3
H 230.0480, found 230.0483. IR (KBr) υ (cm ): 3491, 3124, 3032, 2954, 1577, 1523,
1
6
1
3
7.60 (dd, J = 4.8, 0.9 Hz, 1H, ArH), 7.42 – 7.35 (m, 1H, ArH). C NMR (101 MHz, DMSO) δ, ppm:
+
151.33, 150.75, 148.13, 134.43, 126.19, 124.53. HRMS, m/z: (M+H) calcd for C12
H
8
BrN H
3
–1
273.9974, found 273.9990. IR (KBr) υ (cm ): 3444, 3124, 2981, 2951, 2900, 1573, 1539, 1485,
1442, 1276, 1222, 1184, 1041, 910, 806, 752, 698.
1
1-(5-methyl-1H-benzimidazol-2-yl)naphthalen-2-ol(B1)
MHz, DMSO-d
Faint yellow sliod. H NMR (600
6
) δ, ppm: 12.29 (s, 1H, –NH), 8.26 (dd, J = 8.5, 1.0 Hz, 1H, ArH), 7.99 – 7.93 (m,
1H, ArH), 7.93 – 7.88 (m, 1H, ArH), 7.59 – 7.51 (m, 2H, ArH), 7.46 (s, 1H, ArH), 7.39 (ddd, J = 8.0,
6.8, 1.1 Hz, 1H, ArH), 7.33 (d, J = 8.9 Hz, 1H, ArH), 7.09 (dd, J = 8.1, 1.7 Hz, 1H, ArH), 2.47 (s, 3H,
1
3
–CH
3
). C NMR (101 MHz, DMSO) δ, ppm: 155.14, 142.13, 137.52, 135.85, 133.31, 129.70,
128.34, 127.29, 123.75, 121.65, 120.85, 119.32, 118.64, 116.80, 109.72, 21.42. HRMS, m/z: (M+H)+
–
1
calcd for C18
H
14
N OH 275.1179, found 275.1157. IR (KBr) υ (cm ):3325, 3132, 3045, 2993, 2984,
2
1615, 1530, 1466, 1345, 1268, 889, 856, 744.
1
1-(1H-benzimidazol-2-yl)naphthalen-2-ol(B2)
Faint yellow sliod. H NMR (600 MHz,
DMSO-d
6
) δ, ppm: 12.24 (s, 2H, –NH, –OH), 8.24 (d, J = 8.6, 1H, ArH), 7.96 (d, J = 8.9 Hz, 1H,
ArH), 7.91 (d, J = 7.9 Hz, 1H, ArH), 7.67 (dd, J = 5.1, 3.3 Hz, 2H, ArH), 7.53 (ddd, J = 8.4, 6.8, 1.4
1
3
Hz, 1H, ArH), 7.38 (m, 2H, ArH), 7.28 (dd, J = 8.5, 2.1 Hz, 1H, ArH). C NMR (101 MHz, DMSO)
δ, ppm: 156.12, 149.84, 132.52, 132.28, 128.88, 128.41, 127.86, 124.58, 123.72, 122.54, 119.04,
+
12ClN
OH 261.1022, found 261.1162. IR (KBr) υ (cm–1):
108.93. HRMS, m/z: (M+H) calcd for C17
H
2
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Chemistry & Biodiversity
10.1002/cbdv.201800510
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
3322, 3122, 3040, 1615, 1521, 1456, 1334, 1268, 893, 856, 734.
1
1-(5chloro-1H-benzimidazol-2-yl)naphthalen-2-ol(B3)
Yellow solid. H NMR (600 MHz,
DMSO-d6) δ, ppm: 12.04 (s, 2H, –NH, –OH), 8.24 (dd, J = 8.6, 1.1 Hz, 1H, ArH), 7.97 (d, J = 8.9
Hz, 1H, ArH), 7.90 (dd, J = 8.2, 1.3 Hz, 1H, ArH), 7.71 (dd, J = 36.1, 9.1 Hz, 2H, ArH), 7.51 (ddd, J
13
= 8.4, 6.8, 1.4 Hz, 1H, ArH), 7.40 – 7.34 (m, 2H, ArH), 7.28 (dd, J = 8.5, 2.1 Hz, 1H, ArH). C
NMR (101 MHz, DMSO) δ, ppm: 153.47, 150.11, 147.19, 126.45, 122.89, 122.36, 118.62, 114.27,
+
12ClN
OH 295.0633, found 295.0762. IR (KBr) υ (cm–1):
112.62. HRMS, m/z: (M+H) calcd for C17
H
2
3342, 3162, 3047, 1615, 1528, 1456, 1354, 1278, 890, 876, 831, 740.
1-(5-bromo-1H-benzimidazol-2-yl)naphthalen-2-ol(B4) Yellow solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 11.99 (s, 1H, –NH), 8.27 – 8.19 (m, 1H, ArH), 7.97 (d, J = 8.9 Hz, 1H, ArH),
7.90 (dd, J = 8.2, 1.4 Hz, 1H, ArH), 7.73 (s, 1H, ArH), 7.67 (d, J = 8.5 Hz, 1H, ArH), 7.51 (ddd, J =
1
6
1
3
8.4, 6.8, 1.4 Hz, 1H, ArH), 7.40 – 7.34 (m, 2H, ArH), 7.28 (dd, J = 8.5, 2.1 Hz, 1H, ArH). C NMR
(101 MHz, DMSO) δ, ppm: 155.78, 150.99, 132.80, 132.45, 128.82, 128.41, 127.87, 126.73, 124.72,
+
123.78, 122.61, 118.90, 109.16. HRMS, m/z: (M+H) calcd for C17
H
12BrN OH 339.0128, found
2
–1
339.0154. IR (KBr) υ (cm ): 3339, 3146, 3030, 1635, 1551, 1436, 1324, 1265, 893, 866, 737, 660.
1
5-methyl-2-(thiophen-2-yl)-1H-benzimidazole(C1) Faint yellow solid. H NMR (600 MHz,
DMSO-d
6
) δ, ppm: 12.79 (d, J = 13.3 Hz, 1H,–NH), 7.81 (d, J = 3.6 Hz, 1H, ArH), 7.70 (d, J = 5.0
Hz, 1H, ArH), 7.53 – 7.26 (m, 2H, ArH), 7.22 (dd, J = 5.0, 3.7 Hz, 1H, ArH), 7.06 – 6.97 (m, 1H,
1
3
ArH), 2.42 (s, 3H, –CH
3
). C NMR (101 MHz, DMSO) δ, ppm: 147.14, 134.41, 128.92, 128.69,
+
126.85, 123.73, 111.33, 21.81. HRMS, m/z: (M+H) calcd for C12
H
10
2
N SH 215.0637, found
–1
215.0731. IR (KBr) υ (cm ): 3336, 3035, 3016, 2924, 1566, 1492, 1462, 1419, 1280, 1230, 1130,
1072, 941, 852, 798, 713, 597.
1
5-chloro-2-(thiophen-2-yl)-1H-benzimidazole(C2) Yellow schistose solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 13.13 (s, 1H, –NH), 7.85 (dd, J = 3.7, 1.1 Hz, 1H, ArH), 7.75 (dd, J = 5.0, 1.1 Hz,
6
1H, ArH), 7.55 (s, 2H, ArH), 7.23 (dd, J = 5.0, 3.7 Hz, 1H, ArH), 7.20 (dd, J = 8.6, 2.0 Hz, 1H, ArH).
13C NMR (101 MHz, DMSO) δ, ppm: 145.09, 135.97, 133.61, 128.87, 123.12, 112.88. HRMS, m/z:
+
–1
(M+H) calcd for C11
1566, 1492, 1465, 1438, 1338, 1296, 1230, 1060, 852, 798, 597.
5-bromo-2-(thiophen-2-yl)-1H-benzimidazole(C3) Creamy white schistose solid. H NMR
H
7
ClN
2
SH 235.0091, found 235.0109. IR (KBr) υ (cm ): 3325, 3062, 3001,
1
(600 MHz, DMSO-d
6
) δ, ppm: 13.14 (s, 1H, –NH), 7.86 (dd, J = 3.7, 1.2 Hz, 1H, ArH), 7.77 (dd, J =
This article is protected by copyright. All rights reserved.

Chemistry & Biodiversity
10.1002/cbdv.201800510
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
5.0, 1.2 Hz, 1H, ArH), 7.52 (s, 1H, ArH), 7.33 (dd, J = 8.5, 1.9 Hz, 1H, ArH), 7.25 (dd, J = 5.0, 3.7
1
3
Hz, 1H, ArH). C NMR (101 MHz, DMSO) δ, ppm: 145.62, 136.47, 134.32, 133.53, 128.87, 114.39.
+
–1
HRMS, m/z: (M+H) calcd for C11
H
7
BrN SH 278.9586, found 278.9937. IR (KBr) υ (cm ): 3305,
2
3062, 2993, 2912, 1566, 1492, 1462, 1415, 1276, 1234, 1095, 941, 914, 852, 802, 709, 590.
1
2-(3-bromothiophen-2-yl)-5-methyl-1H-benzimidazole(C4)
Creamy white solid. H NMR
(500 MHz, DMSO-d ) δ, ppm: 12.87 (s, 1H, –NH), 7.62 (d, J = 3.9 Hz, 1H, ArH), 7.45 (d, J = 8.9 Hz,
6
1
3
1H, ArH), 7.35 (d, J = 3.9 Hz, 2H, ArH), 7.03 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 2.42 (s, 3H, –CH
3
). C
NMR (101 MHz, DMSO) δ, ppm: 146.07, 136.16, 132.13, 127.28, 124.67, 114.35, 21.81. HRMS,
+
–1
m/z: (M+H) calcd for C12
H
9
BrN SH 292.9743, found 292.9750. IR (KBr) υ (cm ): 3329, 3039,
2
3026, 2934, 1556, 1482, 1452, 1429, 1278, 1235, 1125, 1062, 931, 842, 788, 723, 607.
1
2-(3-bromothiophen-2-yl)-5-chloro-1H-benzimidazole(C5) Yellow solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 13.22 (s, 1H, –NH), 7.67 (d, J = 4.0 Hz, 1H, ArH), 7.63 (s, 1H, ArH), 7.58 (d, J =
6
1
3
8.5 Hz, 1H, ArH), 7.38 (d, J = 4.0 Hz, 1H, ArH), 7.23 (dd, J = 8.6, 2.1 Hz, 1H, ArH). C NMR (101
MHz, DMSO) δ, ppm: 147.70, 135.32, 132.29, 128.22, 127.14, 123.21, 115.29. HRMS, m/z: (M+H)+
–
1
calcd for C11
1502, 1455, 1448, 1348, 1292, 1220, 1050, 842, 795, 590.
5-bromo-2-(3-bromothiophen-2-yl)-1H-benzimidazole(C6) Brown solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 13.22 (s, 1H, –NH), 7.79 – 7.74 (m, 1H, ArH), 7.67 (d, J = 3.9 Hz, 1H, ArH),
H
6
BrClN
2
SH 312.9196, found 312.9206. IR (KBr) υ (cm ): 3324, 3082, 3021, 1586,
1
6
1
3
7.53 (d, J = 8.4 Hz, 1H, ArH), 7.38 (d, J = 4.0 Hz, 1H, ArH), 7.35 (dd, J = 8.5, 1.9 Hz, 1H, ArH). C
NMR (101 MHz, DMSO) δ, ppm: 147.53, 135.28, 132.31, 128.28, 125.78, 115.33. HRMS, m/z:
+
–1
(M+H) calcd for C11
2922, 1566, 1482, 1453, 1410, 1266, 1235, 1085, 945, 916, 855, 812, 719, 595.
2-(4-bromothiophen-2-yl)-5-methyl-1H-benzimidazole(C7). Creamy white solid. H NMR (600
MHz, DMSO-d ) δ, ppm: 12.88 (s, 1H, –NH), 7.84 (d, J = 1.5 Hz, 1H, ArH), 7.79 (d, J = 1.5 Hz, 1H,
H
6
Br
2
N
2
SH 356.8691, found 356.8699. IR (KBr) υ (cm ): 3320, 3052, 2995,
1
6
ArH), 7.47 (d, J = 7.6 Hz, 1H, ArH), 7.37 (s, 1H, ArH), 7.06 – 7.03 (m, 1H, ArH), 2.43 (s, 3H, –CH
3
).
13C NMR (101 MHz, DMSO) δ, ppm: 145.79, 135.91, 128.57, 126.42, 124.48, 109.81, 21.80. HRMS,
+
–1
m/z: (M+H) calcd for C12
H
9
BrN SH 292.9743, found 292.9754. IR (KBr) υ (cm ): 3340, 3039,
2
3010, 2934, 1556, 1497, 1472, 1423, 1275, 1225, 1130, 1062, 972, 842, 786, 723, 591.
1
2-(4-bromothiophen-2-yl)-5-chloro-1H-benzimidazole(C8) Faint yellow solid. H NMR (600
6
MHz, DMSO-d ) δ, ppm: 13.21 (s, 1H, –NH), 7.89 (d, J = 1.5 Hz, 1H, ArH), 7.84 (d, J = 1.5 Hz, 1H,
This article is protected by copyright. All rights reserved.

Chemistry & Biodiversity
10.1002/cbdv.201800510
3
3
3
3
3
3
3
3
3
3
44
45
46
47
48
49
50
51
52
53
ArH), 7.67 – 7.64 (m, 1H, ArH), 7.60 (d, J = 8.6 Hz, 1H, ArH), 7.25 (dd, J = 8.6, 2.1 Hz, 1H, ArH).
13C NMR (101 MHz, DMSO) δ, ppm: 147.43, 135.11, 129.45, 127.25, 123.18, 109.98. HRMS, m/z:
+
–1
(M+H) calcd for C11
1567, 1482, 1455, 1408, 1331, 1285, 1220, 1051, 852, 799, 587.
5-bromo-2-(4-bromothiophen-2-yl)-1H-benzimidazole(C9) Brown solid. H NMR (600 MHz,
DMSO-d ) δ, ppm: 13.22 (s, 1H, –NH), 7.90 (d, J = 1.4 Hz, 1H, ArH), 7.85 (d, J = 1.5 Hz, 1H, ArH),
H
6
BrClN SH 312.9196, found 312.9210. IR (KBr) υ (cm ): 3325，3052，3005，
2
1
6
1
3
7.79 (s, 1H, ArH), 7.55 (d, J = 8.5 Hz, 1H, ArH), 7.36 (dd, J = 8.6, 1.9 Hz, 1H, ArH). C NMR (101
+
MHz, DMSO) δ, ppm: 147.25, 135.05, 129.49, 127.29, 125.94, 109.99. HRMS, m/z: (M+H) calcd
–1
6
for C11H Br
2
N SH 356.8691, found 356.8699. IR (KBr) υ (cm ): 3315, 3062, 2990, 2914, 1564,
2
1498, 1465, 1418, 1274, 1224, 1090, 944, 916, 855, 805, 709, 595.
3
3
54
55
Biological activity analysis
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The antibacterial activity was evaluated by the antimicrobial susceptibility test method of
[
46]
Clinical and Laboratory Standards Institute (CLSI) in vitro . Bacteria strains were cultured to 6-8 h
o
at 37 C in Mueller-Hinton broth (MHB) for bacteria strains. Meanwhile, yeast strains were
o
incubated to 24 h at 35 C in yeast extract peptone dextrose medium (YEPD). The range of inoculum
8
density was limited to 2–4×10 CFU/mL (about two times of the final inoculum concentration) for
3
3
bacteria, while yeast was range from 1.0×10 to 5.0×10 CFU/mL(two times about the final inoculum
concentration). Herein, fluconazole and itraconazole were chosen as positive controls against fungi
and ciprofloxacin was chosen as positive controls against bacteria.
Compounds were dissolved in dimethylsulfoxide(DMSO) solution at 5.12 mg/mL. Tests were
accomplished in sterile 96-well plates, which were added to 100 μL prepared inoculum into each
well. Meanwhile, another 100 μL inoculum containing 2 μL of test compound was added into the
first well and mixed well. Then obtained 100 μL mixed inoculum from the first well was transfered
into the second well. Therefore, this two-fold dilution method was sustained from the second to the
ninth well, and so on. The 100μL inoculum of the ninth column was discarded. The tenth well was
served as blank control (without inoculation), the eleventh column was as negative growth control
(with standard antimicrobial medicines) and the twelfth column was as positive growth control
(without medicines or test compounds). The final concentrations of the compounds were ranged from
64 μg/mL to 0.125 μg/mL and DMSO concentration was not exceeding 1% V/V in each microplate.
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Chemistry & Biodiversity
10.1002/cbdv.201800510
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The bacteria strains of plates were covered and cultivated for 18 h at 37 C, while yeast for 24 h at 30
oC. The MIC was defined as the lowest concentration which inhibits the visible growth of a microbe.
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76 Acknowledgements
77
We are grateful to Dr. Ch. Y. Wang for NMR spectra, Dr. Zh. L. Wei for Ms spectrum.
78 The Author Contribution Statement
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85
Yanpeng Shi performed the synthesis of experiments, analyzed the data and wrote the paper.
Kai Jiang performed the antimicrobial activity evaluation of the obtained compounds.
Ran Zheng, Jiaxu Fu and Liuqing Yan performed the synthesis of some compounds.
Qiang Gu designed some experiments.
Yumin Zhang conceived and designed the experiments, explained experimental phenomena and
laws, and revised the manuscript.
Feng Lin conceived and designed the antimicrobial experiments
3
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