Reevaluating the synthesis of 2,5-disubstituted-1H-benzimidazole derivatives by different green activation techniques and their biological activity as antifungal and antimicrobial inhibitor

Article abstract of DOI:10.1002/jhet.3801

A comparative study concerned with the preparation of diversely substituted-1H-benzimidazole under different green activation techniques and conventional methods is reported. Data are collected for infrared, ultrasound, microwave, and simultaneous irradiation with US and IR sources, as this last strategy shows an important improvement. Further, the small library of potentially bioactive benzimidazole 17-76 synthesized was screened as an antifungal and antimicrobial agent. Strong activity against Candida albicans and Staphylococcus aureus was observed. Remarkably, 2-(4-aminophenyl)-5-phenylamino-1H-benzimidazole 63 resulted better than that of reference drugs miconazole with a zone of inhibition up to 42 mm. Likewise, 2-(2-aminophenyl)-1H-benzimidazole 21 showed substantial antimicrobial activity against MRSA strain. When assayed by the microdilution method, this azaheterocyclic compound presented a minimum inhibitory concentration (MIC) ≥ 16.4 μg/100 mL and a bacterial percentage reduction of 96%.

Full text of DOI:10.1002/jhet.3801

Received: 8 January 2019
DOI: 10.1002/jhet.3801
Revised: 1 October 2019
Accepted: 14 October 2019
A R T I C L E
Reevaluating the synthesis of 2,5-disubstituted-1H-
benzimidazole derivatives by different green activation
techniques and their biological activity as antifungal and
antimicrobial inhibitor
José-Guillermo Penieres-Carrillo1 | Hulme Ríos-Guerra1 | Javier Pérez-Flores2
|
1
Braulio Rodríguez-Molina2 | Angeles Torres-Reyes | Francisco Barrera-Téllez1
|
ꢀ
Jessica González-Carrillo1 | Lessly Moreno-González1
|
Alejandro Martínez-Zaldívar1 | Juan-Jesús Nolasco-Fidencio1
Audifás-Salvador Matus-Meza3 | Ricardo-Alfredo Luna-Mora1
|
1Facultad de Estudios Superiores
Abstract
Cuautitlán, Departamento de Ciencias
Químicas, Sección de Química Orgánica,
UNAM, Mexico City, Mexico
2Instituto de Química, UNAM, Ciudad
Universitaria, Mexico City, Mexico
3Facultad de Química, Departamento de
Farmacia, UNAM, Ciudad Universitaria,
Mexico City, Mexico
A comparative study concerned with the preparation of diversely substituted-
1H-benzimidazole under different green activation techniques and conven-
tional methods is reported. Data are collected for infrared, ultrasound, micro-
wave, and simultaneous irradiation with US and IR sources, as this last
strategy shows an important improvement. Further, the small library of
potentially bioactive benzimidazole 17-76 synthesized was screened as an anti-
fungal and antimicrobial agent. Strong activity against Candida albicans and
Staphylococcus aureus was observed. Remarkably, 2-(4-aminophenyl)-
5-phenylamino-1H-benzimidazole 63 resulted better than that of reference
drugs miconazole with a zone of inhibition up to 42 mm. Likewise,
2-(2-aminophenyl)-1H-benzimidazole 21 showed substantial antimicrobial
activity against MRSA strain. When assayed by the microdilution method, this
azaheterocyclic compound presented a minimum inhibitory concentration
(MIC) ≥ 16.4 μg/100 mL and a bacterial percentage reduction of 96%.
Correspondence
Ricardo-Alfredo Luna-Mora, Facultad de
Estudios Superiores Cuautitlán,
Departamento de Ciencias Químicas,
Sección de Química Orgánica, UNAM,
Avenida 1ꢀ Mayo s/n, Sta. Ma. Gpe. Las
Torres Cuautitlán Izcalli, C.P. 54740,
Estado de México, Mexico City, Mexico.
Email: paulricardo1@yahoo.com.mx
Funding information
DGAPA-UNAM PAPIME, Grant/Award
Number: PE209518; DGAPA-UNAM,
Grant/Award Number: PAPIIT IN218515;
FES Cuautitlán-UNAM, Grant/Award
Number: PIAPI1618; CONACYT, Grant/
Award Number: 32841
1 | INTRODUCTION
pharmacological profile, such as anticancer,[1]
antimicrobial,[2] antiviral,[3] and antifungal.[4] In this
Benzimidazole motif belongs to a unique class of
privileged scaffold that forms an integral part of many
drugs and candidate drugs that exhibit a wide range of
regard, the benzo-fused 1,3-diazole skeleton has been
considered as pivotal building blocks for the discovering
of novel pharmaceutically important lead molecules
J Heterocyclic Chem. 2019;1–20.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
PENIERES-CARRILLO ET AL.
[12]
with a skeleton of pyrrolo[1,2-a]benzimidazole and
aziridinylpyrrolo[1,2-a]benzimidazolequinone
nitroanilines derivatives with Na2S2O4
has recently
emerged as important variants for the synthesis of 1H-
benzimidazoles where arenecarbaldehydes substrates
have nicely replaced the acid component. Notwith-
standing their well-known synthetic applications, this
one-pot strategy has remained scarcely explored by
eco-friendly reaction techniques as well as combined
green techniques despite their ever-increasing interest
as a frontline tool highly valued in organic synthesis.
In this context, we have reported the less know
combined use of the US with IR (simultaneous irradia-
tion with US and IR [SIUI]) as an improved tool to
promote SNAr reactions.[13] Our aim is to show how
these two important green techniques may be com-
bined to provide a simple, cost-effective, and reliable
method for application in organic chemistry. Thus,
herein, we present the results obtained in the synthesis
biologi-
cally active as DNA cleaving agent and alkylating agent
of DNA phosphate backbone.[5] Therefore, implementa-
tion and assessment of hi-tech synthetic protocols able to
afford concise preparation of structurally simple and
complex scaffolds, featuring mild reaction conditions,
high efficacy, short time, selectivity, broad substrate
scope, and low environmental charge continue attracting
the attention of medicinal and organic chemists.
Even though the reductive cyclization of N-
(4-methyl-2-nitrophenyl)acetamide with metallic reduc-
ing agents[6] has been known as one of the first con-
ventional approaches disclosed for the preparation of
benzimidazole scaffold, the condensation of o-
phenylenediamines with carboxylic acids along with
esters and lactones[7] stands as the most significant
strategy implemented in organic chemistry. Nonethe-
less, this strategy suffers from several drawbacks
wherein highlight handling of highly toxic solvent,[8,9]
harsh acid condition, high temperature, and prolonged
time of reaction that restrict the employment of acid-
sensitive inputs. By contrast, the o-phenylenediamine
approach catalyzed by I2/KI, FeCl3/O2, FeCl2/
FeCl3,[10,11] and nitroreductive cyclization of o-
of
a
small library of diversely decorated 1H-
mixture of o-
benzimidazoles 17-76 from
a
phenylenediamine 1-6 and arenecarbaldehyde 7-16 in
presence of sodium metabisulfite (Na2S2O5) dissolved
in a mixture of protic polar solvents (H2O/EtOH) using
SIUI sources, besides their comparison with three of
the most important nonconventional methodologies
(ultrasound [US], infrared [IR], and microwave [MW])
SCHEME 1 2-mono- and 2,5-disubstituted-1H-benzimidazole 17-76 synthesized by conventional and unconventional conditions in
presence of sodium metabisulfite

PENIERES-CARRILLO ET AL.
3
and thermal method (Δ). In addition to screening
their antifungal and antibacterial activity against Can-
dida albicans (ATCC 10231), Escherichia coli (ATCC
8739), and Staphylococcus aureus (ATCC 6538 and
ATCC43300) strain. Interestingly, benzimidazoles 58,
63, and 66 showed meaningful inhibitory activities
against C albicans, whereas compounds 22 and 21
exhibit notable activities against MRSA microorganism.
TABLE 1 Reaction times and yields of benzimidazole 17 and 18 estimated under conventional and nonconventional method
Entry
Product
Δe
IRe
USa
SIUIb
MWc
Mp (ꢀC) Exp/lit.
Time (h)/yield (%)
0.25/70
1
17
18
18
10/90
4.0/96
—
0.41/80
0.15/79
0.05/87
0.016/88
—
232-234/235-236[14]
287-299/288-290[15]
2
0.66/85
0.15/93
0.15/64 (42)d
0.03/90
2´
0.66/78(80)
0.03/86 (51)
Note. Reaction condition: o-phenylenediamine (5 mmol), benzaldehyde or formaldehyde (5.5 mmol), Na2S2O5 (5 mmol within 5-8 mL), and ethanol 5 mL were
subjected to react at 74 2ꢀC.
Abbreviations: IR, infared; MW, microwave; SIUI, simultaneous irradiation with US and IR; US, ultrasound.
aReactions were obtained by exposing the reactants to 2-minute cycling pulse sequences of US (with 5-min intermittent cooling).
bReactions were obtained by exposing the reaction to 1-minute cycling pulse sequences of US combined with IR irradiation (with 5-min intermittent cooling).
cReactions were subjected to one pulse sequences by the required time.
dReactions were submitted to react in presence of 1.8 mol% (1 equiv) of radical inhibitor 1,4-benzoquinone (Q).
eReactions were summited to react by the required time.
FIGURE 1 (A) Comparison of two feasible pathways of reaction (ionic-radical) induced by MW and SIUI and (B) overview of
cavitation bubble dynamics, their physical and chemical effects. In cavitation, bubbles collapse produce transient hot-spots that reach 5000 K
and 1000 atm in a short span of time capable to generate a heating/cooling rate of more than 1000 K/s.[17] Thereby, hot-spot drive high-
energy chemical reactions predominantly associated with the SET process. SET, single electron transfer

4
PENIERES-CARRILLO ET AL.
TABLE 2 Comparison of conditions and yields obtained in the preparation of 2,5-disubstituted-1H-benzimidazole 19-76
Entry
Product
Δd
IRd
Time (h)/yield (%)
0.58/80
USa
SIUIb
MWc
Mp (ꢀC) Exp./lit.
3
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
6.0/88
5.5/89
4.0/78
5.0/88
6.0/87
6.0/76
6.0/84
6.0/80
3.0/86
4.0/89
2.5/93
2.0/83
3.0/78
3.0/86
3.0/84
5.0/76
4.0/87
5.0/82
2.0/98
2.5/93
2.0/89
2.0/80
2.0/70
2.0/54
3.0/58
3.0/86
4.0/83
3.6/83
04/54
3.5/64
4.0/80
3.0/70
4.0/54
3.0/65
3.0/68
3.5/30
3.0/50
3.5/64
6.0/88
3.0/87
4.5/89
0.83/78
0.66/66
0.30/87
0.50/92
0.46/87
1.00/60
0.53/82
0.80/78
0.66/65
0.83/49
0.91/34
0.83/37
0.66/55
0.91/36
0.83/32
0.66/36
1.00/38
1.00/39
0.66/45
0.50/73
0.41/86
0.50/85
0.66/80
0.50/56
0.78/57
0.33/72
0.25/50
0.50/60
0.75/52
0.91/65
1.00/75
0.91/75
0.80/52
0.66/67
0.75/72
0.66/65
0.86/67
0.78/64
0.33/65
0.53/77
0.50/74
0.16/87
0.25/88
0.03/77
0.15/93
0.03/86
0.25/70
0.16/76
0.33/83
0.20/87
0.15/71
0.48/60
0.50/59
0.48/77
0.53/50
0.63/42
0.25/59
0.60/57
0.33/63
0.46/67
0.16/91
0.21/70
0.33/69
0.45/77
0.31/78
0.36/72
0.25/69
0.38/77
0.33/83
0.66/67
1.30/65
1.30/57
1.20/54
0.83/67
1.00/72
01.1/64
0.91/65
1.00/72
1.30/60
0.21/75
0.61/66
0.33/57
0.016/85
0.016/88
0.016/88
0.025/89
0.021/87
0.016/72
0.021/82
0.023/70
0.35/90
0.30/87
0.50/88
0.40/87
0.50/75
0.66/73
0.20/67
0.33/74
0.46/80
0.36/82
0.41/70
0.25/97
0.16/93
0.20/94
0.30/82
0.21/85
0.23/83
0.13/89
0.10/91
0.80/90
0.41/70
0.83/58
0.86/67
0.90/61
0.66/70
0.71/67
0.75/61
0.61/58
0.66/67
0.50/65
0.06/76
0.08/87
0.13/78
232-234/235-236[15]
287-299/288-290[22]
218-220/219[23]
257-259/258-260[24]
246-247/246-248[25]
233-235/232-235[24]
205-206/204-206[25]
299-301/298-300[25]
186-188[26]
206-208/207-208[27]
169-172/168-171[28]
199-201/198-199[29]
232-234
4
1.00/83
0.11/80
0.21/68
0.18/88
0.83/75
0.50/80
1.00/82
0.30/65
0.28/60
0.65/53
0.61/61
0.63/70
0.78/48
0.90/42
0.80/69
0.71/46
0.40/53
0.53/55
0.23/73
0.33/73
0.43/61
0.58/80
0.33/68
0.61/72
0.46/89
0.55/76
0.50/78
1.00/48
1.40/60
1.40/60
1.30/56
1.20/48
1.50/54
1.00/58
1.20/60
0.91/54
1.20/65
0.33/70
1.13/30
0.83/25
5
6
7
8
9
10
11
12
13
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
228-230
285-286/283-285
190-191/190-193[30]
263-265/265-267[31]
224-226/225-228[32]
130-133[33]
290-291[34]
166-169
203-205
178-180
184-186
210-211
223-225
230-231
210-213
131-133
160-163
122-124
155-157
150-152
165-167
132-134
142-144
145-148
160-162
259-261
197-199
145-147
(Continues)

PENIERES-CARRILLO ET AL.
5
TABLE 2 (Continued)
Entry
44
45
45
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Product
60
Δd
IRd
USa
SIUIb
MWc
Mp (ꢀC) Exp./lit.
210-212
195-197
166-168
133-134
207-209
232-234
245-247
249-251
210-212
268-269
259-261
250-251
234-236
240-242
241-243
226-228
230/229
4.0/92
3.0/79
3.5/73
4.0/80
2.0/62
2.0/47
1.0/52
3.0/73
2.0/80
6.0/92
5.5/91
2.5/82
2.0/70
2.5/68
5.0/82
4.0/78
3.5/62
0.83/69
0.45/82
0.46/87
0.75/70
0.53/52
0.75/47
0.61/42
0.91/67
0.75/77
0.41/80
0.58/76
0.91/88
1.16/67
1.00/76
0.58/78
0.66/81
0.63/77
1.00/32
0.70/26
0.83/22
1.00/26
0.86/27
1.03/24
1.00/23
1.00/21
1.50/27
0.80/30
1.00/58
0.83/29
1.16/22
1.50/23
0.91/38
0.96/28
1.00/27
0.75/59
0.50/67
0.58/70
0.66/66
0.75/56
0.83/47
1.00/52
0.46/38
0.76/47
0.28/73
0.25/78
0.38/61
0.91/32
0.83/28
0.56/56
0.60/53
0.66/58
0.13/80
0.25/90
0.23/86
0.20/82
0.30/78
0.26/62
0.30/80
0.15/77
0.15/68
0.15/83
5.50/91
0.25/79
0.23/68
0.30/57
0.20/86
0.30/83
0.20/78
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Note. Reaction condition: o-phenylenediamine derivatives (5 mmol), corresponding arenecarbaldehyde (5.5 mmol), Na2S2O5 (5 mmol within 5-8 mL), and
ethanol 5 mL were subjected to react at 74 2ꢀC.
Abbreviations: IR, infared; MW, microwave; SIUI, simultaneous irradiation with US and IR; US, ultrasound.
aReactions were obtained by exposing the reactants to 2-minute cycling pulse sequences of US (with 5-min intermittent cooling).
bReactions were obtained by exposing the reaction to 1-minute cycling pulse sequences of US combined with IR irradiation (with 5-min intermittent cooling).
cReactions were subjected to one pulse sequences by the required time.
dReactions were summited to react by the required time.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
which remains without substantial variation. Notably,
the assembling of compound 18 at 74 2ꢀC under SIUI
method proceeds fivefold faster (90% yield within
1.8 min) than that seen in the formation of 17 (79% yield
within 9 min) under similar condition, although the dif-
ferences of yield do not exceed 11%. Surprisingly, the con-
trasting effect was found with the MW method, as for
this dielectric heating slow down the reaction rate by
approximately 1.9 orders of magnitude, despite its evident
superior activation capacity. In this regard, the ration rate
enhancement of the SIUI-assisted reactions does not
exceed approximately 38%; but remarkably, it increases
up to 50% (2.5-fold faster) as contrasted with either US or
Δ alone. But, more importantly, the reaction exposed to
IR irradiation results as effective as the US but reversed
in order of magnitude.
On the other hand, Δ/SIUI rate ratio that involves the
of use aromatic and aliphatic inputs was deemed in 134:1
(4 h vs 1.8 min) and 67:1 (10 h vs 9 min), respectively.
This sizeable increase in the reaction rate acquires even
greater significance when the SIUI/MW ratio was com-
pared. In this case, we found that the MW method
exceeds the SIUI effect nearly by a factor of 1.9 to 3.
Over the years, several papers have documented the
importance of the synergism effect in organic synthesis
mainly by a combination of MW with US sources.[13]
Notwithstanding, other conjunctiva green techniques
such as SIUI has little practical application in the organic
chemistry field. Consequently, in an attempt to compare
their reliability and efficiency with conventional method
(Δ) and two of the most important green activation tech-
niques (viz, MW and the US) in the synthesis of diversely
substituted-1H-benzimidazoles 18-76, 4-substituted o-
phenyendiamine 2-6 were reacted with formaldehyde 7,
and nine arenecaraldehydes derivatives 8-16 in presence
of Na2S2O5 dissolved in polar media as depicted in
Scheme 1.
Data gathering in Table 1 shows that replacement of
the aldehyde aliphatic (R1 = H, entry 1) by aromatic one
(R1 = Ph, entry 2) dramatically reduces reaction times,
except for IR method. Nonetheless, this factor seems to
impact to a less extent on the efficiency of reaction,

6
PENIERES-CARRILLO ET AL.
TABLE 3 Inhibitory activity of compounds 17-76 against
C albicans, E coli, and S aureous strains
The increased acceleration induced by SIUI was
interpreted in terms of excitation of vibrational modes[16]
and acoustic cavitation. Particularly, hot-spot and
associated chemical effects (such as sonolysis of water:
H2O ! HO˙ + H˙, Figure 1B)[18] that occur during the
transient cavitation effect (Figure 1B) might trace
alternative pathways of reaction with lowest energy (viz,
selectivity changes), eg, mechanistic shifting favored by
single electron transfer (SET) process associated with
Bacteria
Gram (−)
E coli
Fungus
Gram (+)
C albicans
S aureus
Compound (ATCC 10231) (ATCC 8739) (ATCC 6538)
17
28.3
13.0
—
21.0
15.0
—
32.0
21.0
27.0
28.0
40.0
33.0
20.0
18.0
28.0
23.0
22.0
23.0
18.0
22.0
21.0
18.0
18.0
25.0
26.0
27.0
23.0
23.0
18.0
20.0
—
18
ǂ
radical transition state (TSR ) highly stabilized. Various
20
radical intermediates have recently been postulated as
key species in the benzimidazole synthesis exposure to
visible light irradiation (450 nm) in the presence of
molecular oxygen.[19] Accordingly, our explanation is
connected to mechanistic pathway changes associated
with the postulation of highly stabilized benzyl-type
radical species III generation, presumably evolving from
unstable intermediate benzimidazole II[20] prior removal
of H˙ by interaction with radical entities formed during
the transient cavitation effect (Figure 1). Succeeding
losses of H˙ from the incipient radical III provides the
driven force to the formation of the thermodynamically
stable benzimidazole product as well as H2O and H2 as a
byproduct.
21
13.0
9.60
20.6
18.3
10.3
8.30
7.00
—
14.0
15.0
30.0
—
22
24
26
27
14.0
—
28
30
9.00
—
31
32
—
17.0
—
33
7.60
8.00
10.0
—
34
—
35
13.0
29.0
31.0
—
To prove the radical intermediate, we have carried
out the same reaction under IR, SU, and SIUI in the pres-
ence of 1.8 mol% and 1.0 equiv of Q as a radical inhibitor.
The two latter methods afford a significant decrease in
the yield of the corresponding benzimidazole (Table 1,
entry 20), suggesting the possibility of a radical intermedi-
ate in the course of the reaction (Figure 1A). On the basis
of this observation, we assumed that the stabilization of
37
38
16.6
9.30
—
39
40
—
41
—
—
44
10.3
—
—
45
—
ǂ
the postulated TSR pathway should be more effective
58
38.0
—
28.0
8.00
8.00
—
than the TSǂ
pathway because of the extended
MW
60
conjugation effects inherent to the benzyl-type radical
species proposed. Therefore, the presumption is that
61
14.6
42.0
15.6
38.0
9.30
—
GǂSIUI < < Gǂ
supports the enhancement of reactivity
63
—
MW
by a decrease in the activation energy. Thus, the differ-
ences of reactivity observed between SIUI and MW are
attributed to that nature and effect of the US are intrinsi-
cally different from MW, as for the latter might benefits
mostly from highly stabilized ionic transition state (TSIǂ)
induced by rapid dielectric heating, eg, the tendency of a
polarized transition state (or dipole species) to follow the
inversion of an alternating electric field.[21] The logical
inference from these preliminary findings suggests that
aldehydes that fulfill Hückel's (4n + 2) π-electron rule
should mostly increase, rather than decrease, the reaction
rate of 2-arenebenzimidazole promoted under SIUI con-
dition, as of that the magnitude of the elicited effects
depend on the kind of the source of energy used. These
results lead to identifying two different relative order of
activation depending on the nature of the aromatic and
65
—
—
66
13.0
15.0
6.00
—
24.0
33.0
—
67
72
73
8.00
11.3
—
—
74
13.0
7.00
—
27.0
—
75
76
7.30
20.0
ND
ND
—
Miconazole
TMP/SMX
Ampicillin
ND
25.0
ND
ND
ND
30.0
Note. The values of inhibition are expressed in mm.; compounds 19, 23, 25,
29, 36, 42, 43, 46-57, 59, 62, 64, and 68-71 shown none activity.
Abbreviations: C albicans, Candida albicans; E coli, Escherichia coli;
S aureous, Staphylococcus aureus; Not determined.

PENIERES-CARRILLO ET AL.
7
aliphatic aldehyde used, that is, MW > SIUI > the US >
IR > Δ vs MW > SIUI > IR > the US > Δ, respectively.
By contrast, it was found that p-monosubstituted phe-
nylenediamines inputs 2-3 bearing electron-withdrawing
group (EWG, R = NO2,) or electron-donating group
(EDG, R = NH2) adversely affect the reaction rate of
Highlight that whereas the synthesis of 28 (entry 12)
and 27 showed small changes in the rate and yield of the
reaction, a noticeable improvement was observed in the
preparation of 38 (entry 22), which even resulted better
than the preparation of 37, whatever the non-
conventional source of energy used. Consequently, the
5-monosubstituted-
and
2,5-disubstituted-1H-
preparation of 38 by SIUI exposure at 74
2ꢀC for
benzimidazole 27, 28, 37, and 38 when they were sub-
jected to react under nonconventional conditions
(Table 2, entries 11, 12, 21, and 22). Thus, when the elec-
tron richer 4-aminophenylendiamine 3 reacts with,
7 afford 37 in lower to moderate yield (45-70%, entry 21)
despite the longer time of reaction (approximately
25-40 min), wherein the IR effect results to be the least
efficient promoter (45% for 40 min, entry 21). Whereas
the Δ effect gives excellent yield (98%) with improved
times of reaction (2.0 h) as compared with 17. These con-
trasting findings are reasonably comparable with the
result obtained in the synthesis of 27 (entry 11). Never-
theless, it is evident that the presence of the strong EWG
on the phenylendiamine ring mostly improves the yield
and rate of reaction of 27 as contrasted with 37. In this
regard, the SIUI effect not only provides an excellent
yield (87%) at 74 2ꢀC but also proceeds with a better
time of reaction (12 min) that heavily contrasts with Δ,
which requires 3 hours of continuous exposure to furnish
27 in similar yield (86%). But more importantly, the MW
increases their reaction time 9 minutes more, whereas
the US and IR alone lessen the efficiency of the reaction,
even with a larger time of reaction. Despite it, the reac-
tion times are reduced from 40 minutes with IR at
74 2ꢀC to 18 minutes with the US, whereas the yields
remain unchanged (65%). Note that in the case of Δ, the
EDG revert significantly the results, that is, 98% for
2 hours vs 86% for 3 hours as compared with 37 and 27,
respectively.
approximately 9.0 minutes furnish 91% of yield versus
71% of the compound 28 at comparable time of reaction,
whereas their promotion under Δ affords 93% for
2.5 hours. Highlight that while the latter method requires
at least 2.5 to 4 hours of reaction under continuous
heating exposure at 74 2ꢀC, the SIUI-assisted reactions
were readily accomplished within 10 minutes when phe-
nylendiamine inputs bearing EDG was used. Remark-
ably, these results differ significantly from the relative
order of reactivity observed in the synthesis of 27 and 37
(R1 = H). But, more importantly, the reaction times of
such reactions get improved significantly under the SIUI
effect as compared with MW, SU, IR, or Δ. Thus, the
order of activation deduced from the different kinds of
energies explored appear as SIUI > the US > MW > IR > Δ,
which seems to be dependent on the nature of the groups
R1 and R installed on the benzimidazole core
In an attempt to assess the role of the electronic
effects induced by mono- and disubstituted
arenecarbaldehyde 9-16 bearing EWG or EDG (R1 = 4-N
(Me)2Ph, 3,4-(OMe)2Ph, and NO2Ph) in the synthesis of
the benzimidazoles derivatives 39-46 (Table 2, entries
23-30); further reactions were explored. The experimental
results show no appreciable differences upon yields and
reaction times when those reactions were exposed to Δ
and nonconventional effects at comparable conditions.
Besides, 2-phenyl-5-substituted benzimidazoles analogs
bearing either phenyldiazenyl (R = -N=NPh, 48), N-
phenylamino (R = -NHPh, 58), or N-hydroxymethyl
FIGURE 2 Schematic setup of the devices
used for the SIUI-assisted organic synthesis.
SIUI, simultaneous irradiation with US and IR

8
PENIERES-CARRILLO ET AL.
amino (R = -NHCH2CH2OH, 68) moiety at this position
generally failed to improve the above parameters as com-
pared with the parent compound 38 (R = NH2). Similar
results were also observed in the preparation of com-
pound 49-56, 59-66, and 59-66 (Table 2, entries: 33-40,
43-50, and 63-60, respectively). Consequently, the general
order of activation deduced from these activating groups,
regardless of the approach used appeared as NH2 > NHPh
> NHCH2CH2OH > N = NPh (entries 22, 32, 42, and 52).
Notably, the MW technology gets improved in terms of
time and yield of reaction over the remaining methods.
On the basis of this result, it seems that the reaction
times depend mostly on the electronic effects induced by
the R-group attached to the phenylendiamine core rather
than on the nature and pattern of substitution of the phe-
nyl ring installing on C-2 of the imidazole motif.
2.2 | Antifungal and antibacterial
evaluation
Antimicrobial and antifungal screening of test com-
pounds 17-76 carried out by the disk diffusion test is
summarized in Table 3. Unfortunately, most synthesized
FIGURE 3 MIC values of TMP, triclabendazole, 21 and 24 against MRSA microorganisms (middle) and UFCs observed after re-
incubating TMP (A1-A4, up) and 24 (D1-D4, bottom) with MRSA in a concentration of 16.4 μg/100 μL to 131.5 μg/100 μL. MIC, minimum
inhibitory concentration

PENIERES-CARRILLO ET AL.
9
compounds did not show promising activity against wild
type E coli (ATCC 8739), at best compounds 24, 37-38,
and 58 shown activities comparable with trimethoprim-
sulfamethoxazole formulation (TMP/SMX) used as refer-
ence drug.
determine their MIC values. The results show that the
MRSA ATCC43300 strain presented high sensibility to
the compound 21, which exhibits a MIC greater than
16.4 μg/100 μL comparable with TMP used as the refer-
ence drug (Figure 3). Unfortunately, decreased sensibility
was found for compound 24 (MIC ≥ 65.7 μg/100 μL).
Notably, none of the screened compounds were active
against E coli at a concentration range from 0.12 μg/100 μL
to 131.5 μg/100 μL; however, TMP shows a MIC greater
than 1.02 μg/100 μL. As evidenced by these results, the
benzimidazole 21 exhibits a high selectivity toward
Gram(+) microorganism and bacterial percentage reduc-
tion of 96%, which contrasts with 0.0% of TMP.
Notably, the unsubstituted benzimidazole 17 exhibits
improved antifungal activity than Miconazole at the
highest tested concentration (4 mg/100 μL) explored.
Whereas compound 63 with 4-aminophenyl and N-
phenylamino moieties (R1 = 4-NH2Ph, R = -NHPh) on
C2 and C5 positions improve twice the biological activity.
Interestingly, the replacement of the 4-NH2Ph group with
a hydrogen atom, as in compound 57, induces complete
loss of activity. However, significant antifungal activity
was observed when Ph substituent was installed on such
a position while maintaining the -NHPh moiety (58). In
this context, replacement of the 4-NH2 group on the phe-
nyl ring attached to C2 position with other polar groups
such as NO2, OMe, and -N (Me)2 (compounds 66 and
59-60), shows unfavorable effect, even loss of activity.
Such results suggested that the hydrophobic nature of the
substituent at C2 position and the capacity of their polar
group to form additional hydrogen bonding become
determinant to the biological activity.
On the other hand, compound 22 with a 3-NH2Ph
group installed on the C2 position showed promising
activity against wild type S aureus, whereas the com-
pound 25 bearing a more polar 3-NO2Ph group on this
position loses their microbial activity. In this connection,
the compounds bearing those polar groups either on C2
or C4 position of the phenyl ring, that is, compounds 21,
24 and 26, display decreasing activity, whereas 23 results
were inactive. The substantial activity exhibited for 22,
allegedly due to the better capacity of the -NH2 group for
hydrogen bonding, argued for additional SAR develop-
ment around the 5-positions of the benzimidazole core
with polar groups able to form additional hydrogen bond-
ing that could impart even greater hydrophilic capacity.
This assumption prompts us to synthesize compound 72
bearing 2-hydroxyethyl amino group on C5 position
while maintaining the 3-NH2Ph moiety on C2 position.
However, the presence of this more hydrophilic group
gives complete loss of activity. Likewise, the removal of
the 3-NH2 group on the phenyl ring attached to C2 (com-
pound 68) elicit a lack of biological activity. However, the
replacement of 3-NH2Ph moiety by hydrogen atom while
maintaining the 2-hydroxyethyl amino group on the C5
position (compound 67) shows activity comparable with
ampicillin drug.
3 | CONCLUSION
We demonstrated that combined US/IR irradiation
can accelerate significantly the formation of
2-arenesubstituted benzimidazoles depending on the
chemical nature of the reactants used. But more impor-
tantly, the different nature of the combined tools may
impact significantly on the pathway of reaction, which
affects the rate and efficiency of the reaction. Although
we did not fully understand how the combination of cavi-
tation and IR effect influence the course of the reaction, a
process requiring ionic-radical sequential mechanism
might benefit from these green methods. Thus, this strat-
egy may be considered a promising and cost-effective
new application, but after all, an emerging technological
innovation deserves further attention in organic
synthesis.
4 | EXPERIMENTAL
4.1 | Materials and general methods
All commercial reagents and solvents were of reagent
grade from Sigma-Aldrich and used as received without
further purification. Analytical thin layer and preparative
chromatography were performed on precoated Kieselgel
60F254 plates and Gel silice MN-Kieselgel G/UV254
,
respectively; spots were located using UV (254 nm). All
melting points were measured in a MEL-TEMP II Elec-
trothermal melting point apparatus and remain
uncorrected. 1H and 13C NMR spectra were obtained
either in DMSO-d6 or CDCl3 using a 300-MHz spectrome-
ter. Chemical shifts (δ) are expressed in parts per million
relative to the tetramethylsilane peak used as an internal
standard. The J values are in hertz, and the splitting pat-
terns are designated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet; and bs, broad singlet. IR spectra
Even though most of the screened compounds
showed good qualitative activity by disk diffusion test,
only the compound giving excellent growth inhibition
zones was further tested by microdilution assay[35,36] to

10
PENIERES-CARRILLO ET AL.
were recorded on an FTIR Bruker spectrophotometer
using attenuated total reflection method. Mass spectra
(MS) and high-resolution mass spectra (HRMS) were
obtained using a JEOLJMS-AX505 spectrometer.
4.3 | General procedure for the synthesis
of 1H-benzimidazole derivatives (17-76)
4.3.1 | MW reactions
A freshly prepared aqueous solution of Na2S2O5 (5 mmol,
0.95 g, 5-8 mL) was added to a stirring solution of o-
phenylenediamine derivate 1-6 (5 mmol, 0.554 g) and the
corresponding aldehyde 7-16 (5.5 mmol) in ethanol
(5 mL). The mixture was transferred to a 20-mL vial,
sealed and exposed to MW irradiation in monowave of
300 MW of reactors, Anton Paar at 850 W of power in the
required time (refer to Tables 1 and 2) at 74 2ꢀC. The
progress of reactions was monitored by TLC. After the
crude of the reaction was cooled, it was poured onto
stirred ice water. The precipitate was collected, dried, and
purified by preparative chromatography using an eluting
system of hexane/ethyl acetate (7:3). The scraping spot
was extracted with acetone and dried to obtain the spec-
troscopically pure compounds 17-76.
4.2 | Experimental equipment
4.2.1 | US-IR coupling
The devices used herein is composed of two independent
blocks: a Cole Parmer 500-W 20 kHz 50 Hz ultrasonic
processor and a homemade IR device adapted with a
THERA-TERM OSRAM 250-W Red IR bulb, 125 V which
emit a wavelength of 1100 nm (9.09 cm−1) and a thermo-
stat to regulate the power output. Both of these devices
were assembled as shown in Figure 2. All reactions were
submitted to react after locating the IR device at 10 cm
away of the reaction flask while holding the ultrasonic
horn at approximately 1.4 cm within the reactant system
with the US equipment working at 80% of potency and
50% of amplitude at an irradiation frequency of
20 kHz
50 Hz, while the reaction temperature was
4.3.2 | Thermic reactions
monitored with an InfraPro IR thermometer.
A 25-mL flask charged with a stirring bar and the
corresponding reactant mixture prepared as previously
described was submitted to react in the required time
(refer to Tables 1 and 2) at 74 2ꢀC using an ordinary
chemical heating mantle. Afterward, isolation and purifi-
cation were carried out as described in Section 4.3.1.
4.2.2 | Ultrasound
A Cole Parmer 500-W 20 kHz 50 Hz ultrasonic proces-
sor, 120 VAC was used in the standard configuration as
delivered, and the reaction temperature was monitored
with an InfraPro IR thermometer.
4.3.3 | IR reactions
4.2.3 | Infrared
The corresponding reactant mixture prepared as in
Section 4.3.1 was exposed to IR irradiation using a
THERA-TERM OSRAM 250-W Red IR bulb in the
required time (refer to Tables 1 and 2) at 74 2ꢀC. All
reactions were promoted by placing the IR bulb at
approximately 10 cm away from the reaction flask. The
resulting crude of the reaction was processed according
to Section 4.3.1.
An electric homemade metallic cylindrical can (29.5 cm
in length with a diameter of 15.3 cm of width) designed
to fix and adjust the position of an IR emission bulb was
assembled with a THERA-TERM OSRAM 250-W Red IR
bulb, 125 V which emits a wavelength of 1100 nm
(9.09 cm−1). The device is adapted with a varicap diode to
regulating the power output. The reaction temperature
was monitored with an InfraPro IR thermometer.
Ultrasonic reactions
Method A. The corresponding reactant mixture prepared
as before was exposed to a Cole Parmer
500-W 20 KHz 50 Hz ultrasonic processor
in the required time (refer to Tables 1 and 2)
at 74 2ꢀC. The reactions were obtained by
exposing the mixture to 2-minute cycling
pulse sequences with 5-minute intermittent
cooling, while holding the ultrasonic horn at
1.4 cm within the reactant system with the
4.2.4 | Microwave
A monowave of 300 MW synthesis reactor by Anton Paar
was used in the standard configuration as delivered. All
experiments we carry out in a sealed MW vial (20 mL)
and the reaction temperature were monitored with an
immersing ruby thermometer.

PENIERES-CARRILLO ET AL.
11
US equipment working at 80% of potency
and 50% of amplitude at an irradiation fre-
quency of 20 kHz 50 Hz. The resulting
122.1, 123.0, 126.9, 129.4, 130.3, 130.6, 151.7 ppm. HR-
DART-MS m/z ([M + 1]+): Calcd. for C13H11N2:
195.09222, found: 195.09209.
crude of the reaction was processed as
described previously.
2-(4-N,N-Dimethylaminophenyl)-1H-benzimidazole (19)
White powder; yield: 89%; [lit. 16]; mp: 287ꢀC to 299ꢀC;
IR (ATR): νmax in cm−1: 3421, 3279 (NH), 3051, 2921,
Method B. (synthesis of 18 in the presence of a radical
inhibitor agent). Following method A, the
corresponding mixture of the reaction was
subjected to react in the presence of 1.8 mol%
and 1 equiv of 1,4-benzoquinone to afford
67% and 37% of yield. The resulting crude of
the reaction was processed as described
previously.
1
2805, 2666 (CH's CH3), 1609 (CN), 1469 (C C). H NMR
(300 MHz, DMSO-d6 + CDCl3/TMS) δ: 3.00 (s, 3H), 4.38
(s, 1H, NH), 6.67 (s, J = 9.0 Hz, 2H), 7.18 (q, J = 5.7 and
J = 3.3 Hz, 2H), 7.55 (q, J = 6.0 and J = 3.0 Hz, 2H), 7.89
(d, J = 8.7 Hz, 2H) ppm. 13C NMR (75 MHz, DMSO-
d6 + CDCl3/TMS) δ: 39.5, 111.9, 117.5, 121.5, 124.7,
127.7, 129.4, 151.3, 152.4 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. For C15H15N3: 238.13442, found:
238.13449.
4.3.4 | US-IR coupling
The corresponding reactant mixture was exposed simul-
taneously to a Cole Parmer 500-W 20 kHz 50 Hz ultra-
sonic processor and a homemade IR devise in the
required time (refer to Tables 1 and 2) at 74 2ꢀC. The
reactions were summited to react by exposing the reac-
tion to repetitive 1-minute cycling pulse sequences of US
combined with IR irradiation (with 5-min intermittent
cooling). Afterward, the compounds were isolated and
purified as described in the MW section.
The known products were identified by comparing
their spectral parameters and physical constants with
those reported in the literature, and the new ones were
fully characterized using spectrometric and spectroscopic
methods. Only physical data of some selected 1H-
benzimidazoles are included here, for comprehensive
information refer to Data S1.
2-(3,4-Dimethoxyphenyl)-1H-benzimidazole (20)
White powder; yield: 88%; [lit. 17]; mp: 232ꢀC to 234ꢀC;
IR (ATR): νmax in cm−1: 3112, 3100 (NH), 3053, 2974,
2939, 2886, 2845, 2797 (CH's, CH3), 1604 (CN), 1443
(C C), 1020 (C O). 1H NMR (300 MHz; DMSO-d6/TMS)
δ: 3.63 (s, 3H), 3.67 (s, 1H, NH), 6.7 (d, J = 8.4 Hz, 1H),
6.95-7.01 (m, 2H), 7.35-7.40 (m, 2H), 7.54-7.58 (m, 1H),
7.61 (t, J = 9.0 Hz, 1H) ppm. 13C NMR (75 MHz, DMSO-
d6/TMS) δ: 39.1, 39.4, 109.5, 110.5, 114.1, 119.4, 121.5,
121.9, 137.9, 148.5, 150.1, 151.2 ppm. DART-MS m/z
([M + 1]+): Calcd. for C15H15N2O2: 255.11335, found:
255.11347.
2-(2-Aminophenyl)-1H-benzimidazole (21)
Gray light powder; yield: 88%; [lit. 18]; mp: 218ꢀC to
220ꢀC; IR (ATR): νmax in cm−1: 3429, 3376 (NH), 3325,
3209 (NH), 3050, 3045 (CH's), 1632 (CN), 1464 (C C),
1
1H-Benzimidazole (17)
761, 713, 646. H NMR (300 MHz, DMSO-d6/TMS) δ:
White crystals; yield: 90%; [lit. 14]; mp: 170ꢀC to 171ꢀC;
IR (ATR): νmax in cm−1: 1770 (CN), 1477 (C C), 3113
(NH), 3038, 3061, 3000, 2942, 2862, 2792, 2540 (CH's). 1H
NMR (300 MHz, DMSO-d6/TMS) δ: 7.19 (q, J = 6.0 and
J = 3.0 Hz, 2H), 7.62 (q, J = 6.0 and J = 3.3 Hz, 2H), 8.27
(s, 1H) ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ:
115.7, 122.2, 138.5, 142.4 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C7H7N2: 119.06092, found:
119.06055.
3.66 (s, 3H, NH), 6.68 (t, J = 7.5 Hz, 1H), 7.09 (t,
J = 7.5 Hz, 1H), 7.48 (t, J = 7.2 Hz, 1H), 7.59-7.62 (m,
2H), 7.71 (t, J = 7.8 Hz, 2H), 7.80 (d, J = 7.8 Hz, 1H)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ: 110.5, 114.9,
115.5, 116.6, 122.4, 127.8, 130.9, 138.6, 148.7, 153.0 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C13H12N3,
210.10312; found: 210.10258.
2-(3-Aminophenyl)-1H-benzimidazole (22)
Gray light powder; yield: 93%; [lit. 19]; mp: 257ꢀC to
2-Phenyl-1H-benzimidazole (18)
259ꢀC; IR (ATR): νmax in cm−1: 3184 (NH), 3058 (CH's),
1
White powder; yield: 96%; [lit. 15]; mp: 290ꢀC to 291ꢀC;
IR (ATR): νmax in cm−1: 3372 (NH), 3046, 2961, 2920,
2853, 2750, 2716, 2672, 2584 (CH's), 1588 (CN), 1441
1611 (CN), 1443 (C C). H NMR (300 MHz, DMSO-d6/
TMS) δ: 4.09 (s, 3H), 6.76 (d, J = 7.8 Hz, 1H), 6.97 (d,
J = 7.8 Hz, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.35 (d,
J = 1.8 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.51 (d,
J = 7.8 Hz, 1H), 8.62 (d, J = 7.8 Hz, 1H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 111.2, 112.4, 118. 0, 119.2,
121.0, 121.3, 131.2, 132.9, 144.9, 148.8, 153.1 ppm. HR-
1
(C C). H NMR (300 MHz, DMSO-d6/TMS) δ: 7.24 (d,
J = 6 Hz, 2H), 7.48-7.61 (m, 4H), 7.71 (d, J = 6 Hz, 1H),
8.22 (dd, J = 6 and J = 3 Hz, 2H), 12.98 (s, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS) δ: 111.8, 119.3,

12
PENIERES-CARRILLO ET AL.
DART-MS m/z ([M + 1]+): Calcd. for C13H12N3:
149.5, 164.8 ppm. HR-DART-MS m/z ([M + 1]+): Calcd.
210.10312, found: 210.10343.
for C13H10N3O2: 240.08132, found: 240.08071.
2-(4-Aminophenyl)-1H-benzimidazole (23)
5(6)-Nitro-1H-benzimidazole (27)
Gray light powder; Yield: 88%; [lit. 20]; mp: 246ꢀC to
247ꢀC; IR (ATR): νmax in cm−1: 3291 (NH), 3068, 2825
(CH's), 1681 (CN), 1452 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 6.73 (d, J = 9.0 Hz, 2H), 7.22 (d,
J = 9.0 Hz, 2H), 7.32 (m, 3H, NH2, NH), 7.85 (d,
J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H). 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 123.4, 126.3, 127.4, 127.6,
131.5, 144.7, 150.0, 150.7 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C13H12N3: 210.10312, found:
210.10330.
Yellow light powder; yield: 98%; [lit. 21]; mp: 202ꢀC
to204ꢀC; IR (ATR): νmax in cm−1: 3100 (NH), 2918, 2851,
2813, 2723 (CH's), 1623, 1590 (CN), 1513, 1339 (NO2),
1
1457 (C C). H NMR (300 MHz, DMSO-d6/TMS) δ: 7.76
(d, J = 9.3 Hz, 1H), 8.10 (q, J = 2.4 and 9.3 Hz, 1H), 8.50
(d, J = 2.1 Hz, 1H), 8.55 (s, 1H), 13.09 (sb, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS) δ: 112.7, 114.8, 117.5,
138.5, 142.6, 146.7 ppm. HR-DART-MS m/z ([M + 1]+):
Calcd. for C7H6N3O2: 164.04600, found: 164.04612.
5(6)-Nitro-2-phenyl-1H-benzimidazole (28)
2-(2-Nitrophenyl)-1H-benzimidazole (24)
Red light powder; yield: 87%; [lit. 22]; mp: 208ꢀC to
210ꢀC; IR (ATR): νmax in cm−1: 3282 (NH), 3055, 2994,
2924, 2858, 2803, 2729 (CH's), 1628, 1597 (CN), 1521,
Yellow light powder; yield: 76%; [lit. 19]; mp: 233ꢀC to
235ꢀC; IR (ATR): νmax in cm−1: 3535, 3476 (NH), 3440,
3239 (NH), 3108, 2850 (CH's), 1601 (CN), 1517, 1344
1
1338 (NO2), 1474 (C C). H NMR (300 MHz, DMSO-d6/
1
(NO2), 1454 (C C). H NMR (300 MHz, DMSO-d6/TMS)
TMS) δ: 7.62 (d, J = 6.6 Hz, 3H), 7.78 (s, 1H), 8.23-8.14
(m, 3H), 8.51 (s, 1H), 13.64 (s, 1H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 118.44, 127.46, 129.51,
129.62, 131.43, 143.18 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C13H10N3O2: 240.07730, found:
240.07664.
δ: 7.25 (dd, J = 5.4 and J = 2.4 Hz, 2H) 7.63 (s, 2H), 7.73
(t, J = 7.7 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 8.01 (dd,
J = 7.8 and J = 2.6 Hz, 2H), 13.10 (s, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 112.6, 119.3, 123.0,
124.7, 131.3, 131.4, 133.1, 135.7, 143.9, 147.8, 149.4 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C13H10N3O2:
240.08132, found: 240.08043.
5(6)-Nitro-2-(4-N,N-dimethylaminophenyl)-1H-
benzimidazole (29)
2-(3-Nitrophenyl)-1H-benzimidazole (25)
Red light powder; yield: 88%; [lit. 23]; mp: 170ꢀC to
172ꢀC; IR (ATR): νmax in cm−1: 3434, 3378 (NHas), 3340,
3292, 3223 (NHsim), 3100, 3078, 2900 (CH's), 1638, 1606
(CN), 1520, 1284 (NO2), 1468 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 3.05 (s, 6H, 2CH3), 6.90 (d, J = 6.9 Hz,
2H), 7.75 (d, J = 9.0 Hz, 1H), 8.07 (d, J = 9.0 Hz, 2H),
8.17 (dd, J = 9.0 and J = 3.0 Hz, 1H), 8.38 (d, J = 3.0 Hz,
1H), 8.98 (sbb, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-
d6/TMS) δ: 46.7, 112.2, 112.8, 119.2, 122.0, 129.2, 139.4,
146.9, 152.8, 158.1 ppm. HR-DART-MS m/z ([M + 1]+):
Calcd. for C15H15N4O2: 283.11950, found: 283.11976.
Yellow light powder; yield: 84%; [lit. 20]; mp: 205ꢀC to
206ꢀC; IR (ATR): νmax in cm−1: 3208, 3105 (NH), 2992,
2856 (CH's), 1621 (CN), 1520, 1348 (NO2), 1475 (C C).
1H NMR (300 MHz, DMSO-d6 + CDCl3/TMS) δ: 2.60 (s,
1H, NH), 7.27 (q, J = 6.0 and J = 3.3 Hz, 2H), 7.66 (t,
J = 8.1 Hz, 1H), 8.26 (dq, J = 5.7 and J = 1.8 and
J = 0.9 Hz, 1H), 8.27 (dq, J = 5.7 and J = 1.8 and
J = 0.9 Hz, 1H), 8.63 (t, J = 1.2 Hz, 1H), 8.66 (t,
J = 1.2 Hz, 1H), 9.08 (t, J = 2.1 Hz, 1H) ppm. 13C NMR
(75 MHz, DMSO-d6 + CDCl3/TMS) δ: 120.8, 122.3, 123.4,
129.4, 131.7, 132.2, 148.0, 148.9 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C13H10N3O2: 240.08132, found:
240.08133.
5(6)-Nitro-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole (30)
Red light powder; yield: 87%; [lit. 24]; mp: 229ꢀC to
231ꢀC; IR (ATR): νmax in cm−1: 3227 (NH), 2934, 2837
(CH's), 1639, 1600 (CN), 1502, 1264 (NO2), 1460 (C C),
2-(4-Nitrophenyl)-1H-benzimidazole (26)
Yellow light powder; yield: 83%; [lit. 20]; mp: 299ꢀC to
301ꢀC; IR (ATR): νmax in cm−1: 3442 (NH), 2922, 2853,
2744 (CH's), 1603 (CN), 1513, 1339 (NO2), 1433 (C C).
1H NMR (300 MHz, DMSO-d6 + CDCl3/TMS) δ: 2.92 (s,
1H, NH), 7.29 (q, J = 6.7 and J = 3.3 Hz, 2H), 7.67 (q,
J = 5.7 and J = 3.0 Hz, 2H), 8.31 (t, J = 2.1 Hz, 1H), 8.34
(t, J = 2.1 Hz, 1H), 8.41 (t, J = 2.1 Hz, 1H), 8.44 (t,
J = 1.8 Hz, 1H) ppm. 13C NMR (75 MHz, DMSO-
d6 + CDCl3/TMS) δ: 122.7, 123.6, 127.0, 136.5, 148.2,
1
1065, 1022 (C O). H NMR (300 MHz, DMSO-d6/TMS)
δ: 3.85 (s, 3H, CH3), 3.88 (s, 3H, CH3), 7.15 (d, J = 9.0 Hz,
2H), 7.72 (d, J = 6.0 Hz, 1H), 7.77 (s, 1H), 7.80 (s, 1H),
8.09 (d, J = 9.0 Hz, 1H), 8.42 (s, 1H), 15.43 (s, 1H, NH)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ: 46.1, 46.4,
105.1, 111.3, 112.3, 115.9, 129.1, 134.5, 137.3, 144.5, 148.5,
149.2, 150.9, 153.0 ppm. HR-DART-MS m/z ([M + 1]+):
Calcd. for C15H14N3O4: 300.09843, found: 300.09907.

PENIERES-CARRILLO ET AL.
13
5(6)-Nitro 2-(2-aminophenyl)-1H-benzimidazole (31)
Orange light powder; yield: 67%; mp: 285ꢀC to 286ꢀC; IR
(ATR): νmax in cm−1: 3434, 3379, 3338, 3293 (NH), 3099,
3077 (CH's), 1619, 1594 (CN), 1520, 1290 (NO2), 1468
(C C), 809, 746 (sust 1,2,4), 879 (sust 1,4). 1H NMR
(300 MHz, DMSO-d6/TMS) δ: 5.92 (sb, 2H, NH2), 6.52 (d,
J = 8.4 Hz, 1H), 6.68 (t, J = 7.8 Hz, 1H), 6.87 (d,
J = 7.8 Hz, 1H), 7.22 (t, J = 2.1 Hz, 1H), 7.84 (d,
J = 7.8 Hz, 1H), 8.13 (q, J = 2.1 and 9.0 Hz, 1H), 8.44 (s,
1H), 12.51 (s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-
d6/TMS) δ: 109.3, 113.2, 116.3, 117.3, 118.8, 119.4, 126.0,
130.5, 139.8, 142.3, 145.8, 149.5, 154.8 ppm. HR-DART-
MS m/z ([M + 1]+): Calcd. for C13H11N4O2: 255.08820,
found: 255.08809.
5(6)-Nitro-2-(3-nitrophenyl)-1H-benzimidazole (35)
Red light powder; yield: 87%; [lit. 26]; mp: 282ꢀC to
284ꢀC; IR (ATR): νmax in cm−1: 3495, 3437, 3380, 3223
(NH), 3101, 3076 (CH's), 1626, 1606 (CN), 1519, 1346
1
(NO2), 1431 (C C). H NMR (300 MHz, DMSO-d6/TMS)
δ: 6.92-7.16 (m, 3H), 784 (d, J = 8.0 Hz, 1H), 8.04-8.15
(m, 1H), 8.72 (d, J = 7.8 Hz, 2H), 11.92 (s, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS) δ: 111.0, 117.4,
118.0, 122.6, 123.2, 129.5, 129.7, 130.2, 139.0, 144.6, 149.6,
150.1, 150.2 ppm. HR-DART-MS m/z ([M + 1]+): Calcd.
for C13H9N4O4: 285.06238, found: 285.06111.
5(6)-Nitro-2-(4-nitrophenyl)-1H-benzimidazole (36)
Red light powder; yield: 82%; [lit. 27]; mp: 298ꢀC to
300ꢀC; IR (ATR): νmax in cm−1: 3338, 3221 (NH), 3099,
2864 (CH's), 1625, 1605 (CN), 1507, 1337 (NO2), 1469
5(6)-Nitro 2-(3-aminophenyl)-1H-benzimidazole (32)
Orange light powder; yield: 73%; mp: 228ꢀC to 230ꢀC; IR
(ATR): νmax in cm−1: 3507, 3362 (NH), 3097, 2919, 2880
(CH's), 1629, 1592 (CN), 1521, 1339 (NO2), 1475 (C C),
1
(C C). H NMR (300 MHz, DMSO-d6/TMS) δ: 7.32 (d,
J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.66-7.71 (m, 3H), 8.17 (d,
J = 7.5 Hz, 2H), 12.02 (sb, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 112.1, 115.1, 117.9, 124.7,
128.8. 134.4, 140.4, 144.0, 147.0, 147.7, 152.9 ppm. HR-
DART-MS m/z ([M + 1]+): Calcd. for C13H9N4O4:
285.062381, found: 285.06220.
1
818, 739 (sust 1,2,4), 843, 739, 689 (sust 1,3). H NMR
(300 MHz, DMSO-d6/TMS) δ: 4.38 (sb, 2H, NH2), 6.76 (d,
J = 8.1 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.31 (d,
J = 7.8 Hz, 1H), 8.15 (d, J = 8.7 Hz, 1H), 8.30-8.40 (m,
1H), 8.62 (d, J = 7.8 Hz, 1H), 9.01 (d, J = 1.5 Hz, 1H),
13.94 (sb, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS) δ: 111.6, 114.6, 1157, 116.5, 117.9, 121.6, 129.3,
136.9, 143.4, 147.9, 149.2, 158.8 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C13H11N4O2: 255.08820, found:
255.08756.
5(6)-Amino-1H-benzimidazole (37)
White powder; Yield: 90%; [lit. 28]; mp: 107ꢀC to 109ꢀC;
IR (ATR): νmax in cm−1: 3276, 3151 (NH), 2930, 2898
(CH's), 1635 (CN), 1506 (C C). 1H NMR 300 MHz,
DMSO-d6/TMS) δ: 5.20 (sb, 2H, NH2), 6.51 (d, J = 3.0 Hz,
1H), 6.67 (s, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.86 (s, 1H)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ: 97.0, 111.7,
117.3, 133.2, 137.2, 139.8, 144.8 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C7H8N3: 134.07182, found:
134.07281.
5(6)-Nitro 2-(4-aminophenyl)-1H-benzimidazole (33)
Orange light powder; yield: 77%; mp: 232ꢀC to 234ꢀC; IR
(ATR): νmax in cm−1: 3597, 3343, 3211 (NH), 3108 (CH's),
1626, 1598 (CN), 1496, 1337 (NO2), 1411 (C C), 858, 726
(sust 1,2,4), 818 (sust 1,4). 1H NMR (300 MHz, DMSO-d6/
TMS) δ: 5.05 (s, 2H, NH2), 6.53 (d, J = 9 Hz, 2H), 6.80 (d,
J = 9 Hz, 1H), 7.37-7.42 (m, 3H), 7.96 (d, J = 9 Hz), 13.24
(s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ:
111.8, 113.7 113.8 116.0, 124.2 135.0 136.3 142.1 143.7
157.9 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C13H11N4O2: 255.08820, found: 255.08779.
5(6)-Amino-2-phenyl-1H-benzimidazole (38)
White powder; yield: 97%; [lit. 29]; mp: 190ꢀC to 192ꢀC;
IR (ATR): νmax in cm−1: 3421, 3263 (NH), 3066, 2949,
2782 (CH's), 1641 (CN), 1477 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 5.02 (s, 1H, NH2), 6.86 (q, J = 9.0 and
3.0 Hz, 1H), 7.11 (d, J = 3.0 Hz, 1H), 7.57-7.60 (m, 4H),
8.15 (q, J = 6.0 and 3.0 Hz, 2H), 12.47 (s, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS) δ: 104.7, 110.7,
113.8, 126.7, 127.0, 129.2, 131.6, 142.4, 144.0, 155.0,
162.6 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C13H12N3: 210.10312, found: 210.10372.
5(6)-Nitro-2-(2-nitrophenyl)-1H-benzimidazole (34)
Red light powder; yield: 86%; [lit. 25]; mp: 129ꢀC to
131ꢀC; IR (ATR): νmax in cm−1: 3553 (NH), 3098, 2922,
2859, 2800 (CH's), 1625, 1593, (CN), 1528, 1339 (NO2),
1432 (C C). 1H NMR (300 MHz, DMSO-d6/TMS) δ:
7.49-7.63 (m, 2H), 7.70-7.84 (m, 2H), 7.90-8.00 (m, 3H),
12.60 (s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS) δ: 116.5, 117.4, 119.6, 124.7, 127.1, 128.8, 133.2,
135.2, 139.5, 143.7, 147.9, 149.0, 152.1 ppm. HR-DART-
MS m/z ([M + 1]+): Calcd. for C13H8N4O4: 285.06238,
found: 285.06113.
5(6)-Amino-2-(4-N,N-dimethylaminophenyl)-1H-
benzimidazole (39)
White powder; yield: 93%; mp: 260ꢀC to 264ꢀC; IR (ATR):
νmax in cm−1: 3251 (NH), 3072, 2935, 611 (CH's, CH3),
1
1634 (CN), 1607(C C). H NMR (300 MHz, DMSO-d6/
TMS) δ: 3.94 (sb, 2H, NH2), 4.18 (s, 6H, 2CH3), 6.32 (s,

14
PENIERES-CARRILLO ET AL.
2H), 6.53 (s, 1H), 6.66 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 7.5,
1H), 7.31 (d, J = 8.4 Hz, 2H), 11.08 (s, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 56.2, 96.1, 108.8,
113.7, 113.8, 114.4, 123.7, 129.0, 133.2, 146.1, 148.0,
153.4 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C15H17N4: 253.14085, found: 253.12518.
2H), 10.22 (s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-
d6/TMS) δ: 97.5, 112.0, 115.1, 116.0, 116.1, 128.2, 130.0,
131.0, 135.1, 139.7, 144.3, 145.1, 152.4 ppm. HR-DART-
MS m/z ([M + 1]+): Calcd. for C13H13N4: 225.11402,
found: 225.11447.
5(6)-Amino-2-(2-nitrophenyl)-1H-benzimidazole (44)
Yellow powder; yield: 78%; mp: 210ꢀC to 213ꢀC; IR
(ATR): νmax in cm−1: 3281, 3198 (NH), 3062 (CH's), 1644
(CN), 1601 (C C), 1524, 1344 (NO2), 693, 615 (sust
1,2,4), 800 (sust 1,2). 1H NMR (300 MHz, DMSO-d6/TMS)
δ: 5.77 (s, 2H, NH2), 6.56 (d, 1H, J = 9.0 Hz), 6.57 (s, 1H),
7.53 (d, J = 7.5 Hz, 1H), 7.73-7.78 (t, 1H), 7.95-8.02 (m,
1H), 8.07-8.13 (m, 2H), 11.52 (sb, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 98.1, 110.2, 117.4,
121.7, 122.3, 130.3, 132.2, 133.7, 134.2, 137.2, 145.6, 148.2,
148.8 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C13H11N4O2: 255.08820, found: 255.08797.
5(6)-Amino-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole (40)
White powder; yield: 94%; mp: 203ꢀC to 206ꢀC; IR (ATR):
νmax in cm−1: 3238 (NH), 3064, 2929, 2837 (CH's, CH3),
1
1665 (CN), 1509 (C C). H NMR (300 MHz, DMSO-d6/
TMS) δ: 3.50(s, 3H, CH3), 3.98 (s, 3H, CH3), 5.61 (sb, 2H,
NH2), 6.82 (d, J = 7.5 Hz,1H), 7.32 (d, J = 8.4 Hz, 1H),
7.42 (s, 1H), 7.57 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 8.17 (d,
J = 7.5 Hz, 1H), 10.81 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 56.0, 95.6, 111.6, 112.5,
115.6, 119.2, 123.5, 134.6, 140.2, 143.2, 148.0, 153.2 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C15H16N3O2:
270.12425, found: 270.12413.
5(6)-Amino-2-(3-nitrophenyl)-1H-benzimidazole (45)
Yellow powder; yield: 76%; mp: 230ꢀC to 231ꢀC; IR
(ATR): νmax in cm−1: 3210 (NH), 3063 (CH's), 1645 (CN),
1602 (C C), 1520, 1347 (NO2), 703, 614 (sust 1,2,4), 860,
5(6)-Amino-2-(2-aminophenyl)-1H-benzimidazole (41)
Gray light powder; yield: 82%; mp: 278ꢀC to 280ꢀC; IR
(ATR): νmax in cm−1: 3344, 3240 (NH), 3063, 2976, 2933
(CH's), 1622 (CN), 1591 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 5.05 (s, 2H, NH2), 6.0.3 (s, 2H, NH2),
7.48 (d, J = 7.2 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.85 (t,
J = 7.5 Hz, 1H), 7.98 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.43
(d, J = 7.5 Hz, 1H, 11.04 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 99.1, 108.4, 11.8, 113.7,
113.8, 116.0, 125.5, 130.5, 134.3, 136.3, 142.1, 143.7,
157.9 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C13H13N4: 225.11402, found: 225.11385.
1
801, 699 (sust. 1,3). H NMR (300 MHz, DMSO-d6/TMS)
δ: 5.29 (sb, 2H, NH2), 6.68 (t, J = 7.5 Hz, 1H), 6.88 (d,
J = 8.1 Hz, 1H), 7.15-7.24 (m, 3H), 7.60-7.63 (m, 2H), 7.88
(d, J = 7.8 Hz, 1H), 13.87 (sb, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 98.2, 98.5, 110.5, 115.5,
116.6, 122.4, 127.8, 130.9, 132.7, 135.0, 138.6, 144.0, 148.7,
153.0 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C13H11N4O2: 255.08820, found: 255.08822.
5(6)-Amino-2-(4-nitrophenyl)-1H-benzimidazole (46)
Yellow powder; yield: 72%; mp: 223ꢀC to 225ꢀC; IR
(ATR): νmax in cm−1: 3454, 3330, 3224 (NH), 3059, 2875,
2777 (CH's), 1640 (CN), 1606 (C C), 695, 610 (sust 1,2,4),
5(6)-Amino-2-(3-aminophenyl)-1H-benzimidazole (42)
Gray powder; yield: 86%; mp: 284ꢀC to 286ꢀC; IR (ATR):
νmax in cm−1: 3397, 3222, 3218 (NH), 3131, 2995, 2893
(CH's), 1639 (CN), 1610 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 5.06 (s, 2H, NH2), 6.03 (s, 2H, NH2),
6.52 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 9.0 Hz 1H), 6.90 (s,
1H), 7.38–7.41 (t, 2H), 7.94 (d, J = 9.0 Hz, 2H), 8.34
(d,J = 8.1 Hz, 1H), 10.94 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 96.1, 112.3, 114.8, 116.3,
117.5, 118.2, 124.3, 127.1, 130.0, 131.0, 133.1, 139.5, 143.2,
148.1, 154.4 ppm. HR-DART-MS m/z ([M + 1]+): Calcd.
for C13H13N4: 225.11402, found: 225.11471.
1
801 (sust. 1,4). H NMR (300 MHz, DMSO-d6/TMS) δ:
5.55 (sb, 2H, NH2), 7.13 (d, J = 9.3 Hz, 1H), 7.79 (d,
J = 6.9 Hz, 1H), 7.93 (d, J = 6.9 Hz, 1H), 8.01 (s, 2H), 8.50
(d, J = 7.2 Hz, 2H), 11.08 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 99.5, 111.8, 120.6, 122.6,
129.6, 135.7, 141.8, 145.4, 148.6, 152.2 ppm. HR-DART-
MS m/z ([M + 1]+): Calcd. for C13H11N4O2: 255.08820,
found: 255.08834.
5(6)-(E)-(Phenylazo)-1H-benzimidazole (47)
Violet powder; yield: 47%; mp: 245ꢀC to 248ꢀC; IR (ATR):
νmax in cm−1: 3404, 3281 (NH), 3165 (CH's), 1687, 1635
(CN), 1544, 1406 (N N), 1482 (C C). 1H NMR
(300 MHz, DMSO-d6/TMS) δ: 6.89 (s, 1H), 7.07 (s, 3H),
7.23 (s, 4H), 8.03 (s, 1H), 10.96 (s, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 113.23, 115.4, 117.9,
112.5, 125.4, 129.5, 129.9, 132.6, 139.2, 142.3, 142.9 ppm.
5(6)-Amino-2-(4-aminophenyl)-1H-benzimidazole (43)
Gray powder; yield: 84%; mp: >350ꢀC; IR (ATR): νmax in
cm−1: 3454, 3330, 3224 (NH), 3059, 2875, 2777 (CH's),
1640 (CN), 1606 (C C). H NMR (300 MHz, DMSO-d6/
TMS) δ: 5.46 (sb, 4H, 2NH2), 6.72-6.78 (m, 3H), 6.84 (d,
J = 1.8 Hz, 1H), 7.19-7.21 (m, 2H), 7.38 (d, J = 8.4 Hz,
1

PENIERES-CARRILLO ET AL.
15
HR-DART-MS m/z ([M + 1]+): Calcd. for C13H11N4:
223.09837, found: 223.09695.
126.3, 128.4, 128.7, 130.1, 142.7, 143.5, 144.3, 154.2,
156.3 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C19H16N5: 314.13275, found: 314.13197.
5(6)-(E)-(Phenylazo)-2-phenyl-1H-benzimidazole (48)
Violet powder; yield: 52%; mp: 160ꢀC to 162ꢀC; IR (ATR):
νmax in cm−1: 3335 (NH), 3198, 3062 (CH's), 1639, 1629
(CN), 1495 (N N), 1400 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 7.60-7.97 (m, 13H), 9.79 (1H, NH)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ: 109.7, 112.6,
114.5, 115.3, 125.4, 127.4, 128.3, 128.5, 129.8, 134.2, 138.5,
142.9, 146.5, 153.8, 154.4 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C19H15N4: 299.12967, found:
299.12970.
5(6)-(E)-(Phenylazo)-2-(3-aminophenyl)-1H-
benzimidazole (52)
Violet powder; yield: 61%; mp: 255ꢀC to 257ꢀC; IR (ATR):
νmax in cm−1: 3428, 3338 (NH), 2918, 2845 (CH's), 1630
(CN), 1505, 1494 (N N), 1381 (C C). 1H NMR
(300 MHz, DMSO-d6/TMS) δ: 4.81 (s, 2H, NH2), 6.74 (d,
J = 7.5 Hz, 1H), 6.94 (s, 1H), 7.07 (q, J = 7.2 Hz, 1H), 7.14
(t, J = 3.3 Hz, 1H), 7.22 (s, 2H), 7.32 (t, J = 7.5 Hz, 2H),
7.42 (t, J = 7.5 Hz, 2H), 7.48-7.62 (m, 6H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 108.0, 114.2, 114.4, 114.6,
115.2, 116.3, 126.4, 126.9, 130.0, 130.5, 131.6, 137.5, 138.2,
144.0, 148.5, 149.5, 150.2 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C19H16N5: 314.13275, found:
314.13205.
5(6)-(E)-(Phenylazo)-2-(4-N,N-dimethylaminophenyl)-
1H-benzimidazole (49)
Violet powder; yield: 58%; mp: 242ꢀC to 244ꢀC; IR (ATR):
ν
max in cm−1: 3469 (NH), 3060 (CH's), 2918, 2850 (CH3's),
1607 (CN), 1453, 1428 (N N). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 2.99 (s, 6H, 2(CH3)), 6.65 (d,
J = 6.0 Hz, 2H), 6.87 (q, J = 6.0 Hz, 4H), 7.17-7.23 (m,
2H), 7.40 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 6.0 Hz, 2H) 7.66
(d, J = 9.0 Hz, 1H), 10.98 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 63.4, 104.0, 112.3, 114.1,
117.6, 122.3, 125.5, 127.9, 128.0, 129.7, 135.7, 144.5, 145.3,
151.6, 153.7, 154.2 ppm. HR-DART-MS m/z ([M + 1]+):
Calcd. for C21H20N5: 342.17187, found: 342.17186.
5(6)-(E)-(Phenylazo)-2-(4-aminophenyl)-1H-
benzimidazole (53)
Violet powder; yield: 60%; mp: 260ꢀC to 263ꢀC; IR (ATR):
νmax in cm−1: 3350, 3221 (NH), 2900 (CH's), 1606 (CN),
1505, 1478 (N N), 1368 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS + CDCl3/TMS) δ: 4.15 (s, 2H, NH2),
6.50-6.56 (m, 2H), 6.92-7.19 (m, 4H), 7.83-8.24 (m, 6H)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS + CDCl3/TMS)
δ: 111.7, 115.3, 115.6, 119.9, 120.5, 122.6, 127.3, 129.6,
130.1, 137.9, 141.8, 144.6, 147.5, 151.9, 152.3, 154.2 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C19H16N5:
314.13275, found: 314.13189.
5(6)-(E)-(Phenylazo)-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole (50)
Violet powder; yield: 64%; mp: 232ꢀC to 235ꢀC; IR (ATR):
νmax in cm−1: 3447, 3337 (NH), 3228, 3201 (CH's), 2889
1
(CH3), 1638 (CN), 1492, 1480 (N N), 1386 (C C). H
5(6)-(E)-(Phenylazo)-(2-nitrophenyl)-1H-
NMR (300 MHz, DMSO-d6/TMS) δ: 3.62 (s, 3H, CH3),
3.85 (s, 3H, CH3), 6.95 (d, J = 8.1 Hz, 1H), 7.10-7.34 (m,
6H), 7.51 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 7.2 Hz, 2H),
7.68 (d, J = 7.5 Hz), 12.15 ppm. 13C NMR (75 MHz,
DMSO-d6/TMS) δ: 55.8, 56.1, 110.1, 111.9, 112.2, 116.7,
122.3, 122.9, 124.5, 129.5, 131.6, 140.75, 140.78, 145.1
149.5, 150.2, 152.1 152.2 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C21H19N4O2: 359.14298, found:
359.14284.
benzimidazole (54)
Dark Violet powder; yield: 60%; mp: 260ꢀC to 262ꢀC; IR
(ATR): νmax in cm−1: 3457, 3333 (NH), 3176, 3060 (CH's),
1626 (CN), 1567, 1343 (NO2), 1519, 1497 (N N), 1457
1
(C C). H NMR (300 MHz, DMSO-d6/TMS + CDCl3/
TMS) δ: 7.10 (t, J = 6.0 Hz, 1H), 7.15–7.29 (m, 5H),
7.37-7.53 (m, H), 12.17 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 112.0, 113.2,
113.8, 119.0, 119.2, 123.8, 126.8, 127.5, 28.7, 128.8, 130.0,
132.0, 134.4, 139.9, 142.0, 145.2, 148.0, 149.0, 151.1 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C19H14N5O2,
344.10692; found: 344.10612.
5(6)-(E)-(Phenylazo)-2-(2-aminophenyl)-1H-
benzimidazole (51)
Violet powder; yield: 67%; mp: 222ꢀC to 224ꢀC; IR (ATR):
νmax in cm−1: 3458, 3328 (NH), 3186 (CH's), 1635 (CN),
1510, 1455 (N N), 1340 (C C) ppm. 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 5.39 (s, 2H, NH2), 7.50-7.57 (m, 2H),
7.58-7.21 (m, 2H), 7.68 (d, J = 7.2 Hz, 2H), 7.78-7.84 (m,
4H), 7.92 (d, J = 7.2 Hz, 2H), 8.03 (d, J = 8.1 Hz and
J = 1.2 Hz, 2H) ppm. 13C NMR (75 MHz, DMSO-d6/TMS)
δ: 108.0, 112.0, 114.1, 114.2, 114.3, 119.1, 122.0, 123.0,
5(6)-(E)-(Phenylazo)-(3-nitrophenyl)-1H-
benzimidazole (55)
Dark Violet powder; yield: 67%; mp: 245ꢀC to 248ꢀC; IR
(ATR): νmax in cm−1: 3455, 3331, (NH) 3135, 3056, 3093
(CH's) 1626 (CN) 1567, 1443 (NO2) 1508, 1456 (N N)
1567 (C C). 1H NMR (300 MHz, DMSO-d6/
TMS + CDCl3/TMS) δ: 6.50-6.60 (m, 4H), 6.92-7.19 (m,

16
PENIERES-CARRILLO ET AL.
5H), 8.04-8.15 (m, 3H), 10.41 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 101.9, 103.6,
108.3, 108.4, 110.4, 11.0, 129.5, 129.7, 130.2, 139.2, 149.6,
150.1, 159.9, 160.0, 161.2 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C19H14N5O2, 344.10692; found:
344.10603.
CH3), 1607 (CN), 1518 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 2.99 (s, 3H), 3.03 (s, 3H), 3.82 (s, 1H,
NH), 6.88 (d, J = 8.7 Hz, 2H), 7.05 (t, J = 4.5 Hz, 2H),
7.14 (d, J = 7.5 Hz, 1H), 7.27 (t, J = 7.5 Hz, 2H), 7.37 (d,
J = 8.4 Hz, 1H), 7.57 (d, J = 8. 7 Hz, 1H), 7.94 (d,
J = 8.4 Hz, 2H), 8.45 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 21.5, 112.5, 117.0, 122.5,
122.7, 123.0, 125.4, 128.4, 129.3, 129.7, 130.2, 132.6, 143.9,
148.1, 152.0, 152.5 ppm. HR-DART-MS m/z ([M + 1]+):
Calcd. for C21H21N4: 3289.17662, found: 329.17549.
5(6)-(E)-(Phenylazo)-(4-nitrophenyl)-1H-
benzimidazole (56)
Dark Violet powder; yield: 60%; mp: 242ꢀC to 244ꢀC; IR
(ATR): νmax in cm−1: 3465, 3343 (NH), 3140, 3021 (CH's),
1629 (CN), 1565, 1385 (NO2), 1508, 1457 (N N), 1565
5(6)-Aminophenyl-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole (60)
1
(C C). H NMR (300 MHz, DMSO-d6/TMS + CDCl3/
TMS) δ: 7.13 (d, J = 9 Hz, 1H), 7.48-7.52 (m, 4H), 7.80 (d,
J = 9 Hz, 2H), 7.91 (d, J = 3 Hz, 1H), 8.01 (s, 2H), 8.50(s,
1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS + CDCl3/TMS) δ: 120.6, 122.6, 124.6, 127.4, 129.6,
130.1, 131.0, 139.6, 140.2, 140.9, 141.8, 145.6, 147.1, 148.6,
152.3 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C19H14N5O2, 344.10692; found: 344.10614.
Light green powder; yield: 69%; mp: 203ꢀC to 205ꢀC; IR
(ATR): νmax in cm−1: 3369 (NH), 3001, 2933, (CH's, CH3),
1632, 1598 (CN), 1176, 1023 (C O), 1496 (C C). 1H
NMR (300 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 3.82
(s, 3H), 3.86 (s, 3H), 6.75 (t, J = 7.2 Hz, 1H), 6.97 (s, 2H),
7.01 (d, J = 7.8 Hz, 1H), 7.19 (t, J = 8.1 Hz, 2H), 7.39 (d,
J = 1.8 Hz, 1H), 7.43 (d, J = 9 Hz, 1H), 7.56 (dd, J = 8.4
and J = 1.8 Hz, 1H), 8.01 (s, 1H), 9.79 (s, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS + CDCl3/TMS) δ:
55.9, 56.3, 109.8, 111.2, 111.7, 116.1, 118.3, 118.3, 119.3,
120.8, 126.6, 129.6, 129.6, 129.9, 132.6, 137.1, 143.7, 156.2,
159.3 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C21H20N3O2: 346.15555, found: 346.15558.
5(6)-Aminophenyl-1H-benzimidazole (57)
White powder; yield: 65%; mp: 232ꢀC to 233ꢀC; IR (ATR):
νmax in cm−1: 3425 (NH), 2929 (CH's), 1631 (CN), 1523
1
(C C). H NMR (300 MHz, DMSO-d6/TMS + CDCl3/
TMS) δ: 4.33 (s, 1H, NH), 7.78 (d, J = 9.0 Hz, 1H), 6.95(s,
1H, NH), 7.16 (d, J = 6.0 Hz, 1H), 7.22 (d, J = 6.0 Hz,
1H), 7.28 (t, J = 6.0 Hz, 2H), 7.33 (d, J = 6.0 Hz, 2H), 8.0
(s, 1H), 9.42 (sb, 1H, NH) ppm. 13C NMR (75 MHz,
DMSO-d6/TMS + CDCl3/TMS) δ: 108.5, 111.9, 116.0,
127.4, 129.4, 129.6, 131.4, 134.3, 137.2, 143.2, 143.8 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C13H12N3:
210.10312, found: 210.10372.
5(6)-Aminophenyl-2-(2-aminophenyl)-1H-
benzimidazole (61)
Light yellow powder; yield: 72%; mp: 210ꢀC to 212ꢀC; IR
(ATR): νmax in cm−1: 3468, 3306 (NH), 3065, 2922, 2852
(CH's), 1660 (CN), 1591 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS + CDCl3/TMS) δ: 5.81 (s, 2H, NH2),
7.54-7.59 (m, 6H), 7.72-7.94 (m, 3H), 7.97-8.01 (m, 2H),
8.2 (s, 1H), 9.10 (s, 1H, NH), 10.2 (s, 1H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 106.0, 112.1,
114.7, 117.0, 117.6, 120.7, 124.1, 127.6, 128.4, 132.3, 134.9,
135.1, 135.5, 148.3, 149.2, 155.6 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C19H17N4: 301.17903, found:
301.17987.
5(6)-Aminophenyl-2-phenyl-1H-benzimidazole (58)
White powder; yield: 73%; mp: 290ꢀC to 291ꢀC; IR (ATR):
νmax in cm−1: 3335 (NH), 3216 (CH's), 1598 (CN), 1516,
1
1479 (C C). H NMR (300 MHz, DMSO-d6/TMS) δ: 7.31
(t, J = 7.5 Hz, 1H), 7.41 (q, J = 7.5 and J = 1.2 Hz, 3H),
7.44 (s, 1H), 7.48 (q, J = 7.8 and J = 2.4 Hz, 1H), 7.55 (q,
J = 7.2 and J = 2.5 Hz, 2H), 7.65 (q, J = 7.8 and
J = 4.5 Hz, 1H), 8.08 (dd, J = 8.7 and J = 1.5 Hz, 1H),
8.26 (dd, J = 8.4 and J = 1.2 Hz, 1H), 8.30 (d, J = 2.4 Hz,
2H), 8.32 (d, J = 1.5 Hz, 2H) ppm. 13C NMR (75 MHz,
DMSO-d6/TMS) δ: 109.2, 115.6, 119.2, 123.58, 127.6,
128.4, 128.6, 129.1, 129.5, 130.2, 131.5, 134.6, 143.2, 145.2,
153.2 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C19H16N3: 286.13442, found: 286.13434.
5(6)-Aminophenyl-2-(3-aminophenyl)-1H-
benzimidazole (62)
Light yellow poder; yield: 87%; mp: 166ꢀC to 168ꢀC. IR
(ATR): νmax in cm−1: 3429 (NH), 2900 (CH's), 1605 (CN),
1
1518 (C C). H NMR (300 MHz, DMSO-d6/TMS) δ: 5.18
(s, 2H, NH2), 7.01 (d, J = 7.2 Hz, 1H), 7.39 (s, 1H),
7.51-7.58 (m, 4H), 7.95-8.41 (m, 6H), 10.22 (s, 1H, NH),
10.62 (s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS) δ: 108.1, 115.6, 116.7, 119.3, 122.117, 122.119, 124.0,
124.3, 129.2 130.2, 130.8., 134.7, 135.1 143.2, 147.4, 148.9,
155.6, ppm. HR-DART- MS m/z ([M + 1]+): Calcd. for
C19H17N4: 301.17903, found: 301.17987.
5(6)-Aminophenyl-2-(4-N,N-dimethylaminophenyl)-1H-
benzimidazole (59)
Light green powder; yield: 86%; mp: 266ꢀC to 269ꢀC; IR
(ATR): νmax in cm−1: 3350, 3215 (NH), 3071, 2887 (CH's,

PENIERES-CARRILLO ET AL.
17
5(6)-Aminophenyl-2-(4-aminophenyl)-1H-
benzimidazole (63)
139.5, 141.0, 149.9, 155.1 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C19H15N4O2: 331.12026, found:
331.13750.
Light yellow powder; yield: 77%; mp: 178ꢀC to 180ꢀC;
IR (ATR): νmax in cm−1: 3335, 3216 (NH), 3105 (CH's),
1
1598 (CN), 1580 (C C). H NMR (300 MHz, DMSO-d6/
5(6)-Ethanolamine-1H-benzimidazole (67)
TMS) δ: 5.06 (s, 2H, NH2), 7.04 (d, J = 6.0 Hz, 2H),
7.14-7.22 (m, 4H), 7.61-7.67 (m, 4H), 8.02 (d, J = 9.0 Hz,
2H, NH), 10.26 (s, 1H, NH), 12.56 (s, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 110.5, 114.1, 114.5,
119.6, 120.1, 122.0, 127.6, 128.4, 130.0, 131.6, 132.5,
136.1, 144.1 146.1, 158.4 ppm. HR-DART- MS m/z
([M + 1]+): Calcd. for C19H17N4: 301.17903, found:
301.18937.
Violet powder; yield: 67%; mp: 320ꢀC to 321ꢀC; IR
(ATR): νmax in cm−1: 3266 (NH), 3168, 2897 (CH's,
CH2), 1665 (CN), 1561 (C C), 1100 (OH), 1000 (C O).
1H NMR (300 MHz, DMSO-d6/TMS) δ: 3.23 (q,
J = 5.4 Hz, 2H), 3.42 (q, J = 5.4 Hz, 2H), 3.56 (d, 1H,
OH), 3.73 (s, 1H, NH), 4.69 (s, 1H, NH), 7.38 (d,
J = 9.0 Hz, 1H), 7.81 (d, J = 9 Hz, 1H), 8.60 (d,
J = 1.8 Hz, 1H), 8.88 (d, J = 1.8 Hz, 1H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 46.0, 60.1, 109.6, 113.1,
118.3, 129.0, 136.8, 139.8, 143.2 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C9H11N3O: 177.09021, found:
177.08951.
5(6)-Aminophenyl-2-(2-nitrophenyl)-1H-
benzimidazole (64)
Dark violet powder; yield: 78%; mp: 210–213ꢀC; IR
(ATR): νmax in cm−1: 3287, 3253 (NH), 3109, 3084 (CH's),
1593 (CN), 1555 (C C), 1505, 1336 (NO2). 1H NMR
(300 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 6.73-7.00
(m, 4H), 7.54-7.90 (m, 8H), 8.50 (s, 1H), 10.40 (s, 1H)
ppm. 13C NMR (75 MHz, DMSO-d6/TMS + CDCl3/TMS)
δ: 113.3, 115.2, 115.4, 124.6, 125.4, 126.0, 127.6, 128.3,
130.1, 132.8, 136.4, 139.5, 141.2, 148.9, 150.0 ppm. HR-
DART-MS m/z ([M + 1]+): Calcd. for C19H17N4:
331.11950, found: 331.12026.
5(6)-Ethanolamine-2-phenyl-1H-benzimidazole (68)
White powder; yield: 68%; mp: 210ꢀC to 212ꢀC; IR
(ATR): νmax in cm−1: 3244 (NH), 3107, 3051, 2922 (CH's,
CH2), 1663 (CN), 1513 (C C), 1181 (C O), 1107 (OH).
1H NMR (300 MHz, DMSO-d6/TMS) δ: 3.255 (q,
J = 5.4 Hz, 2H), 3.60 (q, J = 5.4 Hz, 2H), 5.43 (s, 1H,
OH), 6.81 (d, J = 6.9 Hz, 2H), 7.24 (d, J = 1.5 Hz, 1H),
7.89-8.02 (m, 1H), 8.03-8.05 (m, 3H), 8.15 (d, J = 6.9 Hz,
1H), 8.26 (d, J = 6.9 Hz, 2H), 9.05 (s, 1H, NH), 10.94 (s,
1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ:
45.5, 59.5, 113.3, 124.2, 124.4, 124.6, 127.2, 128.9, 130.4,
131.0, 140.4, 149.6, 151.0 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C15H16N3O: 254.12934, found:
254.12820.
5(6)-Aminophenyl-2-(3-nitrophenyl)-1H-
benzimidazole (65)
Dark violet powder; yield: 77%; mp: 230ꢀC to 231ꢀC; IR
(ATR): νmax in cm−1: 3376, 3200 (NH), 3085, 2926, 2865
1
(CH's), 1665 (CN), 1598 (C C), 1526, 1348 (NO2). H
NMR (300 MHz, DMSO-d6/TMS + CDCl/TMS) δ: 5.61 (s,
1H, NH), 7.02 (d, J = 6.0 Hz, 1H), 7.23-7.31 (m, 4H),
7.48-7.56 (m, 4H), 7.75 (dd, J = 6.0 and 3.0 Hz, 2H), 8.20
(d, J = 6.0 Hz, 1H), 12.94 (s, 1H, NH) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 109.9, 113.9,
117.9, 122.1, 123.1, 123.6, 124.1, 127.9, 128.9, 130.1, 131.6,
134.8, 135.1, 135.7, 141.9, 147.5, 147.7 ppm. HR-DART-
MS m/z ([M + 1]+): Calcd. for C19H15N4O2: 331.11950,
found: 331.12026.
5(6)-(Ethanolamino)-2-(4-N,N-dimethylaminophenyl)-
1H-benzimidazole (69)
Green light powder; yield: 73%; mp: 268ꢀC to 269ꢀC; IR
(ATR): νmax in cm−1: 3331 (NH), 3059, 2857 (CH's, CH3),
1640, 1608 (CN), 1506 (C C), 1174, 1110 (C O), 1062
(OH). 1H NMR (300 MHz, DMSO-d6/TMS) δ: 2.80 (s,
1H, OH), 3.12 (t, J = 5.7 Hz, 2H), 3.61 (t, J = 5.7 Hz,
2H), 5.87 (s, 2H, NH), 6.65 (s, 1H), 6.67 (s, 1H), 6.80 (d,
J = 8.7 Hz, 2H), 7.33 (d, J = 9 Hz, 1H), 7.91 (d,
J = 8.7 Hz, 2H) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS) δ: 42.2, 46.3, 59.5, 93.2, 111.8, 113.9, 114.9, 127.5,
128.7, 136.3, 146.2, 148.7, 151.5 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C17H21N4O: 297.17154, found:
297.17161.
5(6)-Aminophenyl-2-(4-nitrophenyl)-1H-
benzimidazole (66)
Dark violet powder; yield: 72%; mp: 223ꢀC to 225ꢀC; IR
(ATR): νmax in cm−1: 3382 (NH), 3100, 3067, 2922, 2852
1
(CH's), 1704 (CN), 1598 (C C), 1518, 1344 (NO2). H
NMR (300 MHz, DMSO-d6/TMS + CDCl3/TMS) δ: 4.81
(s, 1H, NH), 6.61 (s, 1H), 6.69 (d, J = 9 Hz, 1H), 6.70
(d, J = 9 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 8.27-8.37
(m, 6H), 8.41 (s, 1H, NH) ppm. 13C NMR (75 MHz,
DMSO-d6/TMS + CDCl3/TMS) δ: 113.1, 115.2, 115.5,
124.69, 125.2, 125.6, 125.9, 130.2, 130.7, 132.2, 135.5,
5(6)-(Ethanolamino)-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole (70)
Dark green powder; yield: 78%; mp: 259ꢀC to 261ꢀC; IR
(ATR): νmax in cm−1: 3375 (NH), 2933, 2839 (CH's,
CH3), 1636 (CN), 1510 (C C), 1177, 1153 (C O), 1105

18
PENIERES-CARRILLO ET AL.
(OH). 1H NMR (300 MHz, DMSO-d6/TMS) δ: 3.11 (t,
J = 6 Hz, 2H), 3.22 (s, 1H, OH), 3.60 (t, J = 6 Hz, 2H),
3.81 (s, 3H), 3.85 (s, 3H), 5.68 (s, 1H, NH), 6.57 (d, J = 8.7
and J = 2.1 Hz, 1H), 6.60 (s, 1H), 7.07 (d, 1H, J = 8.7 Hz),
7.28 (d, J = 8.4 Hz, 1H), 7.61 (dd, J = 8.1 and J = 8.1 Hz,
1H), 7.67 (d, J = 1.8 Hz, 1H), 9.89 (s, 1H, NH) ppm. 13C
NMR (75 MHz, DMSO-d6/TMS) δ: 46.7, 55.9, 56.1, 60.0,
104.1, 109.9, 110.7, 112.2, 112.4, 119.9, 122.4, 128.3, 140.2,
142.9, 149.2, 149.4, 151.5 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C17H20N3O3: 314.14264, found:
314.14178.
J = 2.7 Hz, 1H), 8.13 (s, 2H), 8.19 (d, J = 3.2 Hz, 1H), 8.37
(s, 2H), 9.56 (s, 1H, NH) ppm. 13C NMR (75 MHz,
DMSO-d6/TMS) δ: 45.5, 55.5, 101.4, 113.8, 113.9, 114.9,
116.1, 128.4, 133.1, 137.1, 141.9, 145.8, 153.7 ppm. HR-
DART-MS m/z ([M + 1]+): Calcd. for C15H17N4O:
269.14024, found: 269.14076.
5(6)-(Ethanolamino)-2-(2-nitrophenyl)-1H-
benzimidazole (74)
Dark violet powder; yield: 62%; mp: 230ꢀC to 233ꢀC; IR
(ATR): νmax in cm−1: 3347 (NH), 3105, 2922 (CH's, CH2),
1701 (CN), 1595 (C C), 1520, 1344 (NO2), 1159, 1105
1
5(6)-(Ethanolamino)-2-(2-aminophenyl)-1H-
benzimidazole (71)
(C O), 1067 (OH). H NMR (300 MHz, DMSO-d6/TMS)
δ: 3.13 (q, J = 6.8 and 1.3 Hz, 2H), 3.55 (q, J = 6.8 and
1.3 Hz, 2H), 3.91 (s, 1H, OH), 4.92 (sb, 2H, NH), 6.65 (d,
J = 9.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.75-7.88 (m,
3H), 8.22 (d, J = 7.8 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H),
8.88 (s, 1H, NH) ppm. 13C NMR (75 MHz, DMSO-d6/
TMS) δ: 45.9, 62.0, 104.0, 115.2, 115.5, 124.9, 125.2, 125.6,
130.2, 130.7, 132.2, 135.5, 139.5, 141.0, 149.9, 155.1 ppm.
HR-DART-MS m/z ([M + 1]+): Calcd. for C15H15N4O3:
299.11441, found: 299.11361.
Green-yellow light powder; yield: 57%; mp: 240ꢀC to
242ꢀC; IR (ATR): νmax in cm−1: 3479, 3366 (NH), 3000,
2920 (CH's, CH2), 1634 (CN), 1606 (C C), 1160, 1100
1
(CO), 1092 (OH). H NMR (300 MHz, DMSO-d6/TMS)
δ:2.8 (s, 1HH, OH), 3.23 (q, J = 6.9 Hz, 2H), 3.43 (q,
J = 6.9 Hz, 2H), 6.76 (d, J = 3.0 Hz, 1H), 6.87-6.95 (m,
2H), 7.29-7.31 (m, 1H), 7.34 (q, J = 9.3 and J = 2.4 Hz,
1H), 7.72 (d, J = 9.0 Hz, 1H), 8.45 (s, 1H, NH), 8.61 (s,
2H, NH2), 9.8 (s, 1H, NH) ppm. 13C NMR (75 MHz,
DMSO-d6/TMS) δ: 40.8, 60.4, 91.1, 101.3, 112.8, 113.7,
116.8, 121.5, 124.7, 130.3, 134.6, 134.7, 144.6, 153.5,
154.2 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C15H17N4O: 269.14024, found: 269. 14076.
5(6)-(Ethanolamino)-2-(3-nitrophenyl)-1H-
benzimidazole (75)
Dark violet powder; yield: 78%; mp: 226ꢀC to 229ꢀC; IR
(ATR): νmax in cm−1: 3358, 3226 (NH), 3085, 2930, 2868
(CH's, 5CH), 1720 (CN),1635 (C C), 1525, 1348 (NO),
1
5(6)-(Ethanolamino)-2-(3-aminophenyl)-1H-
1196, 1099 (C O), 1061 (OH) ppm. H NMR (300 MHz,
benzimidazole (72)
DMSO-d6/TMS) δ: 2.60 (s, 1H, OH), 3.14 (d, J = 5.7 Hz,
2H), 3.62 (d, J = 5.7 Hz, 2H), 4.72 (s, 2H, NH), 6.63-6.67
(m, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H),
8.22 (d, J = 7.8 Hz, 1H), 8.48 (d, J = 7.8 Hz, 1H), 8.89 (s,
1H) ppm. 13C NMR (75 MHz, DMSO-d6/TMS) δ: 44.1,
55.5, 102.9, 113.9, 116.1, 122.4, 124.6, 130.1, 130.5, 130.6,
130.9, 137.1, 140.8, 148.7, 153.7 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C15H15N4O3: 299.11441, found:
299.11423.
Green-yellow light powder; yield: 67%; mp: 234ꢀC to
236ꢀC; IR (ATR): νmax in cm−1: 3252 (NH), 3108, 2925,
(CH's, CH2), 1664 (CN), 1516 (C C), 1181 (C O), 1109
(OH). 1H NMR (300 MHz, DMSO-d6/TMS) δ: 3.24 (t,
J = 5.7 Hz, 2H, OH), 3.63 (t, J = 5.7 Hz, 2H), 5.05 (s, 2H,
NH2), 6.03 (s, 1H, OH), 6.50 (d, J = 2.4 Hz, 1H),
6.52-6.80 (q, J = 8.4 and J = 2.1 Hz, 1H), 7.22 (s, 1H),
7.26 (s, 1H), 7.64-7.79 (m, 2H), 7.96 (q, J = 7.8 and
J = 1.5 Hz, 1H), 9.04 (s, 1H, NH), 13.14 (s, 1H, NH) ppm.
13C NMR (75 MHz, DMSO-d6/TMS) δ: 47.9, 59.0, 106.5,
116.1, 116.5, 117.4, 119.6, 124.2, 124.7, 131.4, 131.9,
133.2, 134.9, 135.2, 147.9, 157.0 ppm. HR-DART-MS m/z
([M + 1]+): Calcd. for C15H17N4O: 269.14024, found:
269.14076.
5(6)-(Ethanolamino)-2-(4-nitrophenyl)-1H-
benzimidazole (76)
Dark violet powder; yield: 78%; mp: 241ꢀC to 245ꢀC; IR
(ATR): νmax in cm−1: 3269, 3231 (NHas), 3188 (NHsim),
2933, 2877 (CH's, CH2), 1724 (CN), 1634 (C C), 1514,
1340 (NO2), 1192, 1135 (C O), 1108 (OH). 1H NMR
(300 MHz, DMSO-d6/TMS) δ: 2.83 (s, 1H, OH), 3.28 (q,
J = 6.6 and 3.2 Hz, 2H), 3.75 (d, J = 6.6 Hz, 2H), 4.68 (s,
1H, NH), 5.23 (s, 1H, NH), 7.12 (d, J = 8.1 Hz, 1H), 7.24
(s, 1H), 7.41 (s, 1H), 7.53–7.69 (m, 4H) ppm. 13C NMR
(75 MHz, DMSO-d6/TMS) δ: 45.9, 59.2, 100.3, 107.1,
108.6, 115.2, 127.7, 131.3, 136.9, 139.0, 145.8, 150.0,
153.2 ppm. HR-DART-MS m/z ([M + 1]+): Calcd. for
C15H15N4O3: 299.11441, found: 299.11472.
5(6)-(Ethanolamino)-2-(4-aminophenyl)-1H-
benzimidazole (73)
Green-yellow light powder; yield: 61%; mp: 250ꢀC to
252ꢀC; IR (ATR): νmax in cm−1: 3353 (NH), 3106, 2939,
(CH's, CH2), 1621 (CN), 1596 (C C). 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 3.12 (q, J = 5.7 Hz, 2H), 3.27 (s, 1H,
OH), 3.56 (d, J = 4.5 Hz, 2H), 3.84 (s, 1H, NH), 4.82 (s,
2H, NH2), 6.62 (d, J = 8.4 Hz, 1H), 7.69 (q, J = 9.6 and

PENIERES-CARRILLO ET AL.
19
5 | BIOLOGICAL ASSAYS
11th well. Well, the 12th is the growth control and con-
tains no drug.
5.1 | Materials and general methods
5.1.1 | Preparation of drugs and test
compounds
5.1.6 | Preparation of cultures and assay
plates
The synthesized compounds and reference drugs were
dissolved in 1 mL of DMSO and diluted to 500 μg/100 mL
with a culture medium.
Using a sterile inoculating loop several colonies wire
transfer from an overnight plate culture into 5 mL of SSS.
Inoculum density was adjusted to tube 0.5 of McFarland
nephelometer (1-2 × 108 UFC mL−1). All test and control
strains were handled in the same manner. The above sus-
pension was diluted by adding 200 μL into 28 mL of
Mueller Hinton broth. Afterward, 100 μL of the resulting
inoculum suspension was added to all 96-wells plates.
Additionally, 100 μL of DMSO solution prepared by dis-
solving 100 μL of DMSO into 1.900 mL of Mueller Hinton
broth, as no-drug containing well control was added to
the 12th well. These plates were incubated in an ambient
air incubator for 24 hours at 35ꢀC to 37ꢀC (85% relative
humidity). After, the minimum inhibitory concentration-
MIC values were read and recorded.
5.1.2 | Microorganisms
S aureus ATCC 6538 and ATCC 43300, E coli ATCC
8739, and C albicans ATCC 10231 strains were used. The
qualitative antimicrobial activity technique was carried
out according to the literature method.[37]
5.1.3 | MIC assay by microdilution and
viable count
The MIC values were determined by the microdilution
test using a methicillin-resistant MRSA strain (ATCC
43300). The biological assay was carried out regarding a
reported method[37] with slight modification.
5.1.7 | Determination of microbial
reduction percentage
After the MIC value was recorded; the 96-well MIC plates
were shaking and re-incubated for an additional 4 hours
at 35ꢀC to 37ꢀC. Then, a 100-μL sample from each well
with no visible growth in the MIC assay was removed
and spread on a sheep blood agar plate (in duplicate).
Afterward, the inverted agar plates were incubating for
24 hours at 35ꢀC to 37ꢀC. Elapsed this time, colonies
were counted on the agar plate and reported as the %
reduction (% R) using the following equation:
5.1.4 | Preparation of cultures and
inoculum
Two days before the experiment, the test strains were
cultivated on sheep blood agar plate and incubated for 20
to 24 hours at 35ꢀC to 37ꢀC. All bacterial strains
were treated in the same manner from this point on.
From the resulting growth on each plate, selected and
isolated colonies were spread, using a sterile disposable
loop, onto a fresh Mueller Hinton agar plate and incubate
for 20 to 24 hours at 35ꢀC to 37ꢀC.
UFC1−UFC2
%R =
× 100:
ð1Þ
UFC1
where UFC1 = UFC of 100 μL of no-drug control
well and.
5.1.5 | Preparation of assay plates with
UFC2 = UFC of 100 μL of drug-containing well.
medium and drugs
One hundred microliter of Mueller Hinton broth was
added to each well of 96-well microtiter plate at room
temperature. Then, using a multichannel pipet, 100 μL of
the appropriate compound stock solution was added to
the first well of each row and serially diluted each row
twofold across the plate to the 11th well, discarding the
remaining 100 μL to obtain compound concentrations
ranging from 131.5 μg in the first well to 0.12 μg in the
5.1.8 | Determination of viable count
A
1:100 dilution of the 0.5 McFarland solution
(0.1 mL + 9.9 mL of standard saline solution SSS) was
made. Once the concentration was adjusted to 106, 105,
104 UFC, and 100 μL of each solution was sown onto the
agar plates, and after their incubation for 24 hours at
35ꢀC, the number of colonies were counted and recorded.

20
PENIERES-CARRILLO ET AL.
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ACKNOWLEDGMENTS
[22] S. E. López, J. Restrepo, B. Pérez, S. Ortiz, J. Salazar, Bull.
Korean Chem. Soc. 2009, 30, 1628.
[23] C. Cimarelli, M. D. Nicola, S. Diomedi, R. Giovannini,
D. Hamprecht, R. Properzi, F. Soranaac, E. Marcantoni, Org.
Biomol. Chem. 2015, 13, 11687.
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S. Scholz, A. Giannis, Bioorg. Med. Chem. 2009, 15, 4943.
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Res Chem Intermed 2013, 39, 2917.
[26] J. Liu, Q. Liu, W. Xu, W. Wang, Chinese J Chem 2011, 29,
1739.
José G. Penieres-Carrillo and Ricardo A. Luna-Mora
acknowledge to DGAPA-UNAM the financial support to
PAPIIT IN218515 and FES Cuautitlán-UNAM PIAPI1618
and DGAPA-UNAM PAPIME PE209518 programs.
Ricardo A. Luna-Mora thanks to CONACYT for the
Grant 32841.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
[27] K. Bougrin, M. Soufiaoui, Tetrahedron Lett. 1995, 36, 3683.
[28] R. H. Datani, S. G. Kini, M. Mubeen, J Comp Meths Mol des
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[29] D. T. Nannapaneni, V. S. S. S. Atyam, M. I. Reddy, C. Raidu,
J. Young Pharm. 2010, 2, 273.
ORCID
Ricardo-Alfredo Luna-Mora https://orcid.org/0000-
0001-7752-7562
[30] Z. Mao, Z. Wang, J. Li, X. Song, Y. Luo, Synth Comm 2010, 40,
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How to cite this article: Penieres-Carrillo J-G,
Ríos-Guerra H, Pérez-Flores J, et al. Reevaluating
the synthesis of 2,5-disubstituted-1H-
benzimidazole derivatives by different green
activation techniques and their biological activity
as antifungal and antimicrobial inhibitor.
J Heterocyclic Chem. 2019;1–20. https://doi.org/10.
1002/jhet.3801