Novel benzimidazolyl tetrahydroprotoberberines: Design, synthesis, antimicrobial evaluation and multi-targeting exploration

Article abstract of DOI:10.1016/j.bmcl.2017.02.071

A series of novel benzimidazolyl tetrahydroprotoberberines were conveniently designed and efficiently synthesized from berberine via direct cyclization of tetrahydroprotoberberine aldehyde and o-phenylene diamines under metal-free aerobic oxidation. All the new compounds were characterized by IR, ¹H NMR, ¹³C NMR and HRMS spectra. The antimicrobial evaluation revealed that the 5-fluorobenzimidazolyl derivative 5b was the most active antibacterial and antifungal molecule with broad spectrum in comparison to Berberine, Chloromycin, Norfloxacin and Fluconazole. It triggered almost no resistance development against MRSA even after 15 passages. Further studies demonstrated that compound 5b could not only effectively interact with Topo IA by hydrogen bonds, but also intercalate into calf thymus DNA and cleave pBR322 DNA, which might be responsible for its powerful bioactivities.

Full text of DOI:10.1016/j.bmcl.2017.02.071

Accepted Manuscript
Novel benzimidazolyl tetrahydroprotoberberines: Design, synthesis, antimicro-
bial evaluation and multi-targeting exploration
Ponmani Jeyakkumar, Han-Bo Liu, Lavanya Gopala, Yu Cheng, Xin-Mei Peng,
Rong-Xia Geng, Cheng-He Zhou
PII:
S0960-894X(17)30214-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.071
BMCL 24743
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
31 October 2016
20 February 2017
27 February 2017
Please cite this article as: Jeyakkumar, P., Liu, H-B., Gopala, L., Cheng, Y., Peng, X-M., Geng, R-X., Zhou, C-H.,
Novel benzimidazolyl tetrahydroprotoberberines: Design, synthesis, antimicrobial evaluation and multi-targeting
exploration, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.071
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Graphical Abstract
Novel benzimidazolyl tetrahydroprotoberberines:
Design, synthesis, antimicrobial evaluation and
multi-targeting exploration
Leave this area blank for abstract info.
Ponmani Jeyakkumara , Han-Bo Liua , Lavanya Gopalaa , Yu Chenga , Xin-Mei Pengb,* , Rong-Xia Genga and
Cheng-He Zhoua,*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Novel benzimidazolyl tetrahydroprotoberberines: Design, synthesis,
antimicrobial evaluation and multi-targeting exploration
Ponmani Jeyakkumara , Han-Bo Liua , Lavanya Gopalaa , Yu Chenga , Xin-Mei Pengb,* , Rong-Xia Genga
and Cheng-He Zhoua, ∗
aInstitute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China
bSchool of Chemistry and Chemical Engineering, Qiannan Normal University for Nationalities, Duyun 558000, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel benzimidazolyl tetrahydroprotoberberines were conveniently designed and
efficiently synthesized from berberine via direct cyclization of tetrahydroprotoberberine
aldehyde and o-phenylene diamines under metal-free aerobic oxidation. All the new compounds
were characterized by IR, 1H NMR, 13C NMR and HRMS spectra. The antimicrobial evaluation
revealed that the 5-fluorobenzimidazolyl derivative 5b was the most active antibacterial and
antifungal molecule with broad spectrum in comparison to Berberine, Chloromycin, Norfloxacin
and Fluconazole. It triggered almost no resistance development against MRSA even after 15
passages. Further studies demonstrated that compound 5b could not only effectively interact
with Topo IA by hydrogen bonds, but also intercalate into calf thymus DNA and cleave pBR322
DNA, which might be responsible for its powerful bioactivities.
Keywords:
Tetrahydroprotoberberine
benzimidazole
antibacterial
antifungal
DNA
2016 Elsevier Ltd. All rights reserved.
The treatment of infectious diseases still remains a serious
problem for scientific community despite of the rapid progress of
biomedicinal science.1 Several limitations such as toxicity,
parenteral administration, and the decreased efficacy mainly due
to microbial drug resistance of clinically used drugs force an
immense need to search for new class of chemical entities with
strong activity, low toxicity and different modes of action from
currently clinical drugs.2
and papaveraceae etc.7b It plays a quite important role in the
treatment of intestinal infections such as cholera, bacillary
dysentery and acute gastroenteritis, which makes more and more
researchers be interesting in developing berberine derivatives
with antimicrobial ability. Tetrahydroprotoberberines, an
important class of berberine derivatives, containing an
isoquinoline backbone with methoxyl or hydroxyl group on
benzene ring have been demonstrated to possess various
biological activities.8 Benzimidazole is an important heterocyclic
structure in medicinal chemistry. It consists of a benzene ring
fused an imidazole ring, which possesses larger conjugated
system and higher electron-rich property than simple imidazole
ring. So far a lot of benzimidazole compounds have been
successfully developed as clinical drugs and have been widely
used in the treatment of various diseases. Recently, literature has
shown the great potentiality of benzimidazoles to inhibit the
growth of bacteria and fungi.9 Benzimidazole derivatives are able
to compete with purines due to their structural similarity. This
may inhibit the synthesis of nucleic acids, thereby killing
microbial strains.10 The different action mechanisms of
benzimidazole type of antimicrobial molecules have attracted
more and more attention to develop benzimidazoles as novel
antimicrobial agents. On the basis of our previous findings
In the design of new drugs, hybridization approach may
provide a more general method to obtain molecules with
improved biological activities.3 Especially the combination of
two or more bioactive fragments could afford hybrids capable of
overcoming multi-drug resistance.4 This strategy has already
given excellent results in drug discovery program5 to overcome
drug-resistance. Ciprofloxacin-1,2,4-triazole hybrids were
demonstrated as potential antibacterial agents against drug-
susceptible and drug-resistant bacteria.6 Furthermore, our group
has reported a series of berberine-azole hybrids as potential
antibacterial and antifungal agents.7
Berberine is a well-known natural isoquinoline alkaloid
p—re—sen—t in roots and rhizomes of berberidaceae, ranunculaceae
∗ Corresponding author. Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);
peng0703@sgmtu.edu.cn (Xin-Mei Peng)

regarding the antimicrobial properties of berberine derivatives
and in consideration of the fact that benzimidazole could
compete with purines and might inhibit the synthesis of nucleic
acids, both scaffolds could bind DNA in different ways and,
therefore, their combination could improve the efficacy as
antimicrobial agents.
Figure 1. Basic design of benzimidazolyl tetrahydroprotoberberines.
Considering the above observations, and in continuation of
our research in the field of heterocyclic compounds as novel
potential antimicrobial agents, a series of benzimidazolyl
tetrahydroprotoberberines were prepared via efficiently direct
cyclization of tetrahydroprotoberberine aldehyde and o-
phenylene diamines through metal-free aerobic oxidation in wet
organic solvent.11 Fluorine and chlorine atoms as well as
substituted benzyl groups were introduced into benzimidazole
ring with an aim to adjust bioactivities.12 Aliphatic chain was
incorporated to modulate molecular flexibility and regulate the
lipid-water partition coefficient.13 The modification by carbazole
moiety was explored to be as comparison in activity due to its
biological importance.14 The further transformation of the
prepared target compounds into their hydrochlorides was also
investigated since some researches have proved that
hydrochlorides could improve antimicrobial efficacy and broaden
antimicrobial spectrum due to reinforcing water solubility.15 The
design was shown in Figure 1.
o
o
Scheme 1. Reagents and conditions: i) 190 C/vacuum, EtOH/conc. HCl, 45 min; ii) NaBH4, MeOH, r.t., 5 h; iii) a. HMTA/TFA, 120 C, 5 h; b. 10% H2SO4,
90−100 oC, 2 h; iv) K2CO3, DMF, alkyl bromides or benzyl halides, r.t., 8 h; v) o-phenylene diamines/II/III, DMF/water, 80 oC, 12 h; vi) diethyl ether, HCl (g), 0
oC.

The target compounds 5−8, 12 and 13 were synthesized
according to the route shown in Schemes 1 and 2. Commercially
available berberine chloride 1 was selected as starting material.
Selective demethylation of compound 1 at 9-position was carried
out in the presence of ethanol/hydrochloric acid solution at 190
Norfloxacin and Fluconazole. The antibacterial and antifungal
data as well as clogP values were depicted in Tables 2 and Table
S1. Minimal inhibitory concentration (MIC, µM) is defined as the
lowest concentration of the new compounds that completely
inhibited the growth of bacteria or fungi.
°C under vacuum (20 mm Hg) for 45 minutes to easily afford
The in vitro antibacterial evaluation (Table 2) demonstrated
that some title compounds showed moderate to good activities
against the tested strains. Amongst, compound 5b displayed good
antibacterial activities with MIC values ranging from 4.4 to 69.6
µM in comparison to reference drugs. It was more active against
B. subtilis (MIC = 4.4 µM), S. dysenteriae (MIC = 17.4 µM) and
B. typhi (MIC = 14.5 µM) than Chloromycin (MIC = 82.5 µM).
Moreover, the anti-MRSA (MIC = 17.4 µM), anti-E. coli JM109
(MIC = 8.7 µM) and anti-P. aeruginosa (MIC = 11.6 µM)
efficacies of compound 5b were comparative or superior to those
of Chloromycin (MIC = 41.3−99 µM) and Norfloxacin (MIC =
25.1−50.1 µM).
16
berberrubine hydrochloride 2 with 79% yield,
which was
further reduced by NaBH4 in methanol to produce compound 3 in
62% yield.17 The key intermediate 4 was obtained via direct
formylation of compound
3 by hexamethelene tetramine
(HMTA) in trifluoroacetic acid (TFA) and then hydrolysis with
10% H2SO4 at 90 °C for 5 h in excellent yield (82%) without
further column purification. The target benzimidazolyl
tetrahydroprotoberberines 5a−c, 6a−d and 7a−i were effectively
obtained in 25−48% yields by the cyclization of
tetrahydroprotoberberine aldehyde 4 with a series of o-phenylene
diamines under metal-free aerobic oxidation in DMF/water
system at 80 °C for 12 h. Amongst, compounds 7a, 7d, 7g and 7h
were further transformed into their corresponding hydrochlorides
8a−d (70−85% yields) in diethyl ether by using dry hydrogen
chloride gas at 0 °C.
Table 2 showed valuable effects of the substituted benzyl
groups at benzimidazole ring on biological activities. Most of the
benzyl modified benzimidazolyl tetrahydroprotoberberines were
more active towards the tested bacteria than aliphatic moiety
substituted ones. Compound 7b with 3-fluorobenzyl
benzimidazole gave better inhibition against MRSA (MIC = 12.2
µM) and E. coli JM109 (MIC = 29.1 µM) than Chloromycin
(MIC = 41.3 and 82.5 µM). Noticeably, compound 7d with 4-
chlorobenzyl benzimidazole showed remarkable antimicrobial
activity against B. subtilis, S. aureus, E. coli JM109, E. coli
DH52, S. dysenteriae, P. aeruginosa, B. thyphi and B. proteus in
comparison with Chloromycin. Compound 7e with 3-
chlorobenzyl benzimidazole and compound 7f with 2-
chlorobenzyl benzimidazole displayed excellent inhibitory
activity against E. coli JM109 with MIC value of 4.7 µM, which
was more active than Chloromycin (MIC = 82.5 µM) and
Norfloxacin (MIC = 50.1 µM). Regretfully, for hydrochlorides
8a−d, only compound 8c suggested good antibacterial ability
against the tested strains in contrast with reference drug
Chloromycin. Compound 12 exhibited more poor biological
activities than compound 13 because the formation of salt
improved the solubility of compound 13.
Compound 10 was synthesized from carbazole 9 and 1,6-
dibromohexane in DMF in the presence of KOH at room
temperature for 12 h in 59% yield,18 which was reacted with o-
phenylene diamine to give compound 11 in the yield of 52%
using DMF as solvent and K2CO3 as base at room temperature
for 8 h. The target molecule 12 was obtained by the cyclization
of compound 11 and intermediate 4 in DMF/water system at 80
°C for 12 h with 28% yield. Further hydrochloride 13 was
prepared in 80% yield by using dry hydrogen chloride gas at 0 °C
in CHCl3/diethyl ether.
Table 1. Screening of the optimal formylation conditions for
intermediate 4
HMTA
Eq.a
Entry
Solvent
Temperature
Time
Yield
1
2
3
4
5
6
Water
25% AcOH
50% AcOH
80% AcOH
AcOH
80 °C
80 °C
80 °C
80 °C
Reflux
Reflux
2
5h
6h
8h
8h
5h
5h
-
2
11%
20%
37%
44%
82%
1
1
i
ii
1.2
1.2
N
H
N
N
H
N
TFA
Br
10
11
9
aHMTA Eq., HMTA Equivalent.
5
5
NH2
We here make the first report on an efficiently direct synthesis
of tetrahydroprotoberberrubine-12-carbaldehyde 4. Formylation
of compound 3 was investigated by different conditions such as
solvent, temperature, loading amount of HMTA, and reaction
time (Table 1). Initially, the reaction was conducted under water
medium, no reaction was observed. In the presence of acetic acid
(different concentration), the formylation initiated but product 4
gave low yield. According to the overall observations, strong
acid TFA made the formylation reaction go effectively (with
product in 82% yield) under reflux for 5 h using 1.2 equivalent
HMTA. In this reaction, TFA acted as not only the optimal
solvent but also strong protonating agent.
iii
O
O
O
HCl
N
N
O
OH
OCH3
OH
iv
N
N
OCH3
N
N
5
N
N
5
13
12
Scheme 2. Reagents and conditions: i) KOH, 1,6-dibromohexane, DMF, r.t.,
12 h; ii) K2CO3, DMF, o-phenylene diamine, r.t., 8 h; iii) compound 4,
DMF/water, 80 oC, 12 h; iv) CHCl3/diethylether, HCl (g), 0 oC.
The structures of all the new compounds were confirmed by
1
IR, H NMR, 13C NMR and HRMS (Supporting Information for
The in vitro antifungal data in Table S1 indicated that most of
the synthesized intermediates and target compounds displayed
effective potency against the tested five fungi. Compound 7d was
demonstrated to be the most active antifungal molecule against
all the tested fungal strains in contrast with the clinically used
Fluconazole. From the overall observations, the antifungal
details). The in vitro antimicrobial activities for intermediate 4
and all the target compounds were evaluated against four Gram-
positive bacteria, six Gram-negative bacteria and five fungi using
two fold serial dilution technique recommended by National
Committee for Clinical Laboratory Standards (NCCLS)19 with
the positive control of clinical antimicrobial drugs Chloromycin,

activities of benzyl substituted benzimidazoles were more active
than those of alkyl ones. Except for compounds 5c, 8c−d, 12 and
13, all the tested compounds gave better inhibitory potency
against A. flavus (MIC = 14.1−293.3 µM) than Fluconazole (MIC
= 835.9 µM). Compounds 5b, 6c and 6d showed the strongest
inhibition towards C. mycoderma with MIC values ranging from
3.3 to 8.7 µM in comparison with Fluconazole (MIC = 26.1 µM).
Compounds 5b and 7d showed strong activity towards S.
cerevisia with MIC values of 8.7 and 14.1 µM respectively as
compared to Fluconazole (MIC = 52.2 µM). The in vitro
antifungal abilities of compounds 5c, 8d, 12 and 13 were quite
poor, which were in accordance with their antibacterial activities.
the absorption values of simply sum of free DNA and free
compound 5b, which was observed in the inset of Figure 4.
These indicated that a weak hyperchromic effect existed between
DNA and compound 5b, which suggested the strong interaction
of compound 5b and DNA. The binding constant (K) of
compound 5b interacting with DNA was calculated to be K =
2.87 × 104 L/mol, R = 0.995, SD = 0.17 (R is the correlation
coefficient. SD is standard deviation).
As is known, the interactions between drugs or bioactive small
molecules and biomacromolecules are helpful to study the
absorption, transportation, distribution, metabolism and possible
action mechanism of agents.21 Literature has reported that
berberine and its derivatives could effectively interact with
DNA.22 Therefore, this work selected calf thymus DNA as a
model to investigate the in vitro binding behavior of the most
active compound 5b with DNA at molecular level by UV-vis and
fluorescence spectroscopic methods.23
To rationalize the observed antimicrobial activity and to
understand the possible mechanism of the hybrids, molecular
docking study of active compound 5b with DNA topoisomerase
IA (Topo IA) was undertaken. The crystal structure data was
obtained from the Protein Data Bank. Compound 5b possessed
lowest binding energy (−13.74 KJ/mol). As shown in Figure 2,
the results indicated that compound 5b could interact with Topo
IA through the formation of hydrogen bond between the
hydroxyl group of compound 5b and ASN 555 of Topo IA. On
the other hand, HIS 365 and ASP 323 could form two hydrogen
bonds with nitrogen atom at benzimidazole ring and –O-CH2-O-
group of compound 5b, respectively. Furthermore, this molecule
could also form electrostatic interactions by benzimidazole and
berberine aromatic skeleton with ALA 554, ARG 161, ARG 114
and ASP 551. These hydrogen bonds might be the crucial reason
that compound 5b displayed strong inhibitory efficacy against the
tested strains.
(B)
300
MRSA
5b
Chloromycin
Norfloxacin
200
100
0
0
2
4
6
8
Day
10
12
14
16
Figure 3. Evaluation of resistant development against compound 5b in
bacterial strain MRSA.
1.2
0.8
5b-DNA
h
5b+DNA
1.0
0.7
0.6
0.8
a
0.5
Figure 2. Docking results of compound 5b with Topo IA (PDB code:
3PWT).
0.4
0.6
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
10-5[5b] (mol/L)
0.4
0.2
0.0
Because of the emergence of drug resistant pathogens, the
efficacy of current antibiotics against infections has been
diminished.20 To investigate the drug resistance development of
the tested strains against compound 5b, the susceptible pathogen
MRSA were employed. The new MIC values were determined
every 24 h after propagation of bacterial cultures with fresh
media and serially diluted concentrations of compound 5b. To
make comparative analysis, parallel cultures were exposed to 2-
fold dilutions with the reference drugs Chloromycin and
Norfloxacin as positive control. The experiment was repeated for
15 days. Results in Figure 3 showed the low propensity of
bacterial pathogens to develop resistance towards compound 5b
as there was almost no change in the MIC values even after 15
passages.
DNA
260
220
240
280
300
320
340
360
380
Wavelength (nm)
Figure 4. UV absorption spectra of DNA with different concentrations of
compound 5b (pH = 7.4, T = 290 K). Inset: comparison of absorption at 260
nm between the 5b–DNA complex and the sum values of free DNA and free
compound 5b. c(DNA) = 4.52 × 10-5 mol/L, and c(compound 5b) = 0–1.4 ×
10-5 mol/L for curves a–h respectively at increment 0.2 × 10-5.
Neutral Red (NR) is a planar phenazine dye, whose binding
mode with DNA is demonstrated to be intercalation. This work
employed NR as a spectral probe to investigate the binding mode
of 5b with DNA. The absorption spectra of NR upon the addition
of DNA were shown in Supporting Information. It was apparent
that the absorption peak of the NR at around 460 nm gave
gradual decrease with the increasing concentration of DNA, and
a new band at around 530 nm developed. This was attributed to
the formation of the new DNA–NR complex. An isosbestic point
at 504 nm provided evidence of the formation of DNA–NR
complex (Figure S3). The competitive binding between NR and
The application of absorption spectroscopy is one of the most
important techniques in DNA-binding studies. Generally,
hyperchromism and hypochromism are important spectral
features to distinguish the change of DNA double-helical
structure.24 In this work, with a fixed concentration of DNA, UV-
vis absorption spectra were recorded with the increasing amount
of compound 5b. Figure 4 showed that the maximum absorption
of DNA at 260 nm displayed proportional increase with the
increasing concentration of compound 5b. Simultaneously, the
measured values of 5b–DNA complex was a little greater than

Table 2 ClogP values and antibacterial data as MIC (µM) for intermediate 4 and berberine-derived benzimidazolesa,b,c
Gram-positive bacteria
Gram-negative bacteria
Compds
clogP
B.
M.
S.
E. coli
E. coli
S.
P.
B.
B.
MRSA
subtilis
luteus
aureus
JM109
DH52
dysenteriae
aeruginosa
typhi
proteus
3
2.91
2.66
3.74
3.90
4.30
4.31
4.80
6.89
8.56
5.87
5.87
5.87
7.27
7.27
7.27
6.82
6.82
5.58
6.19
6.59
7.14
7.14
7.99
8.31
–0.77
–0.23
1.37
1311.3±4.3
301.8±4.6
120.8±2.9
17.4±0.5
1075.8±7.8
14.2±4.9
55.2±1.1
231.2±2.5
174.9±2.6
50.2±0.4
12.2±0.2
24.3±0.4
18.9±1.2
56.5±0.6
23.6±2.2
53.3±1.2
22.2±1.7
57±1.6
1573.6±2.1
482.9±9.1
48.3±1.9
4.4±0.8
1573.6±6.1
301.8±4.6
241.6±1.7
46.4±1.1
896.5±6.6
28.4±2.8
110.3±1.2
48.2±1.7
105±0.9
786.8±1.7
724.4±0
579.9±2.4
58±0.6
655.7±7.1
241.5±4.6
60.4±1.9
8.7±0.8
1573.6±1.7
241.5±4.6
96.7±0.9
23.2±1.9
896.5±8.6
136.3±1.6
110.3±1.2
24.1±1.3
87.5±1.3
100.3±2.8
38.8±2.8
38.8±1.8
56.5±0.7
75.4±2.6
23.6±1.2
106.6±2.0
44.4±2.4
95±1.9
1573.6±2.3
120.7±2.3
96.7±1.9
17.4±0.6
1075.8±9.9
136.3±1.6
264.7±2.7
77.1±1.4
174.9±2.6
60.2±0.9
116.4±0.7
48.5±1.8
18.9±1.2
23.6±2.2
28.3±0.7
71.1±1.8
44.4±1.4
57±0.8
786.8±1.5
301.8±4.6
145±0.3
11.6±0.7
358.6±5.3
68.2±1.7
66.2±1.8
38.5±2.7
17.5±0.6
25.1±0.7
48.5±1.8
58.2±0.7
23.6±1.2
28.3±0.5
23.6±1.2
88.8±2.8
35.5±1.4
38±1.5
786.8±1.5
301.8±4.6
48.3±1.9
14.5±0.9
448.3±5.3
14.2±0.9
27.6±1.6
24.1±1.3
8.7±0.8
1311.3±4.3
482.9±9.1
241.6±2.7
69.6±0.5
896.5±8.6
56.8±1.7
22.1±1.6
9.6±0.3
4
5a
5b
5c
717.2±5.6
34.1±1.5
110.3±1.2
57.8±1.7
35±1.2
1075.8±8.8
28.4±1.8
44.1±1.1
14.4±1.2
26.2±0.7
60.2±1.5
48.5±1.8
58.2±0.7
9.4±1.1
896.5±3.6
28.4±2.8
110.3±1.2
115.6±1.7
26.2±0.3
25.1±1.5
29.1±0.5
29.1±0.5
37.7±1.3
4.7±0.4
6a
6b
6c
6d
5.5±0.9
7a
20.1±1.7
19.4±1.4
48.5±1.8
11.8±1.1
47.1±1.3
188.4±8.3
11.1±1.0
22.2±1.7
19±1.2
40.1±1.4
19.4±0.8
29.1±0.6
28.3±0.9
28.3±0.5
23.6±1.2
44.4±1.4
106.6±3.3
57±0.9
60.2±0.7
48.5±1.8
24.3±0.4
9.4±1.1
25.1±1.7
38.8±1.8
38.8±1.8
11.8±1.1
47.1±2.3
47.1±1.3
17.7±0.7
22.2±2.7
47.5±1.5
182±3.6
88.5±2.7
20.9±1.2
535.9±9.6
617.6±2.9
88±1.6
7b
7c
7d
7e
23.6±1.2
9.4±0.8
4.7±2.3
7f
4.7±0.7
37.7±2.3
22.2±1.7
53.3±1.8
47.5±2.5
218.4±8.9
106.2±1.8
6.3±0.6
7g
22.2±0.7
35.5±2.4
47.5±1.5
109.2±6.0
70.8±1.7
20.9±1.2
267.9±1.9
617.6±2.9
146.7±2.8
1376.8±5.1
41.3±1.3
1.1±0.5
35.5±2.4
44.4±1.4
38±1.5
7h
7i
8a
218.4±2.9
35.4±1.3
20.9±0.9
535.9±2.0
617.6±2.9
586.7±2.2
229.5±1.4
41.3±2.3
25.1±0.2
182±4.1
88.5±1.7
33.5±1.5
134±2.5
741.1±3.7
704±2.5
1147.3±5.4
82.5±1.7
3.1±.03
182±4.2
182±2.5
70.8±1.7
20.9±1.2
535.9±2.1
617.6±2.9
469.3±6.2
1376.8±3.6
82.5±2.6
50.1±0.9
182±2.5
141.6±1.3
20.9±1.2
669.8±2.5
617.6±2.9
88±1.6
145.6±5.1
106.2±1.6
50.2±2.1
535.9±2.0
617.6±8.9
146.7±1.8
1147.3±5.4
99±1.9
182±3.7
212.4±1.9
67±2.3
8b
70.8±1.7
20.9±1.2
535.9±2.9
741.1±2.3
293.3±5.6
1376.8±5.1
20.7±1.2
4.2±1.8
8c
8d
535.9±2.0
741.1±3.7
469.3±4.2
573.7±3.7
99±1.5
201±2.0
617.6±2.9
73.3±1.4
458.9±2.7
82.5±1.6
10.4±3.6
12
13
Berberine
Chloromycin
Norfloxacin
1147.3±7.4
66±2.6
688.4±2.4
99±1.8
50.1±0.9
5.3±1.8
41.8±0.8
12.5±0.5
aMIC, minimal inhibitory concentration. bS. aureus, Staphylococcus aureus (ATCC25923); MRSA, methicillin-resistant Staphylococcus aureus (N315); S. dysenteriae, Shigella dysenteriae; B. subtilis, Bacillus subtilis; M. luteus,
Micrococcus luteus (ATCC4698); B. proteus, Bacillus proteus (ATCC13315); E. coli, Escherichia coli (JM109); E. coli, Escherichia coli (DH52); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi. cclogP values
were calculated using ChemBioDraw Ultra 14.0.

5b with DNA was observed in Figure S4. With the increasing
concentration of compound 5b, an apparent intensity increase
was observed around 460 nm, which was opposite with the
absorption of NR–DNA complex at the same wavelength. The
various spectral changes suggested that compound 5b could
substitute NR in the DNA–NR complex.
In conclusion,
a
series of novel benzimidazolyl
tetrahydroprotoberberines have been successfully and efficiently
prepared via efficient cyclization of tetrahydroprotoberberine
aldehyde and o-phenylene diamines under metal-free aerobic
oxidation. All the new compounds were confirmed by 1H NMR,
13C NMR, IR and HRMS spectra. The in vitro antimicrobial
evaluation revealed that some title compounds showed good
bioactivities against the tested strains. Compound 5b bearing 5-
fluorobenzimidazole moiety was found to be the most active
antibacterial and antifungal molecule with broad spectrum in
comparison with standard drugs Chloromycin, Norfloxacin and
Fluconazole. More importantly, there was almost no MRSA
resistance development even after 15 passages of bacterial
incubation against compound 5b. Further studies showed that this
compound not only was able to effectively interact with Topo IA
by hydrogen bonds, but also had the ability to intercalate into calf
thymus DNA and cleave pBR322 DNA, which might be
responsible for its powerful antimicrobial activities (Figure 6).
Therefore, compound 5b was potential to be developed as novel
antimicrobial agent. Some properties including other
antimicrobial mechanism explorations such as antibiofilm and
bacterial membrane permeabilization activity as well as in vivo
antibacterial and antifungal evaluation are currently underway in
our laboratory.
To further explore the binding mode of compound 5b with
DNA, negatively charged I− ion was employed due to the fact
that negatively charged phosphate backbone of DNA is expected
to repel a highly negatively charged quencher. Therefore, when a
small molecule is protected from being quenched by anionic
quencher, the binding mode is intercalation, whereas groove
binding agents should be quenched readily.25
The KSV values of compound 5b quenched by I− ion in the
absence and presence of DNA were calculated to be 104.52 (R =
0.996) and 87.74 (R = 0.994) L mol-1, respectively. It was
apparent that when compound 5b interacted with DNA, the
quenching effect was decreased (Figure S7), which suggested the
binding mode of compound 5b to DNA was intercalation.
Acknowledgments
This work was partially supported by the National Natural
Science Foundation of China (NSFC) (21372186, 21672173) and the
Research Fund for International Young Scientists from International
(Regional) Cooperation and Exchange Program of NSFC (No.
81650110529).
Figure 5. Cleavage of pBR322 DNA by various concentrations of compound
5b. Lane 1 DNA control, Lane 2 DNA + 25 µM compound 5b, Lane 3 DNA
+ 50 µM compound 5b, Lane 4 DNA + 75 µM compound 5b.
References and notes
The DNA-cleaving activity of compound 5b towards pBR322
DNA was studied under physiological conditions (37 °C and pH
= 7.0). Figure 5 showed the GE patterns for the cleavage of
pBR322 DNA by compound 5b of varying concentrations. It can
be seen that compound 5b was capable of converting pBR322
DNA into Form II and that the cleaving activity showed a strong
dependence on its concentration (Lane 4). When the
concentration of compound 5b was about 75 µM, almost all the
Form I was converted to the Form II and Form III (a linear form,
generated from cleavage of both Form I and Form II strand),
which lent strong support that compound 5b catalyzed the
cleavage of pBR322 DNA plasmid by concentration dependence.
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26. Experimental: Melting points are determined on X-6 melting point
apparatus and uncorrected. IR spectra were determined on a Bio-
Rad FTS-185 spectrophotometer in the range of 400-4000 cm-1.
1H NMR and 13C NMR spectra were recorded on a Bruker AV
600 or Varian 300 spectrometer using TMS as an internal standard.
Chemical shifts were reported in parts per million (ppm), the
coupling constants (J) were expressed in hertz (Hz) and signals
were described as singlet (s), doublet (d), triplet (t), as well as
multiplet (m). The mass spectra (MS) were recorded on LCMS-
2010A and the high-resolution mass spectra (HRMS) were
recorded on an IonSpec FT-ICR mass spectrometer with ESI
resource. The following abbreviations were used HMTA:
hexamethelene tetramine; TFA: trifluoroacetic acid; MIC:
minimal inhibitory concentration; IC50: 50% inhibiting
concentration of drug; NR: Neutral Red. All chemicals and
solvents were commercially available, and used without further
purification. The concentration of DNA in stock solution was
determined by UV absorption at 260 nm using a molar absorption
-1
coefficient ξ260 = 6600 L mol-1 cm (expressed as molarity of