Design, synthesis and biological evaluation of berberine-benzimidazole hybrids as new type of potentially DNA-targeting antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2016.06.031

A series of novel berberine-benzimidazole derivatives were conveniently and efficiently synthesized and characterized by NMR, IR, MS and HRMS spectra. Most of the prepared compounds showed effective antimicrobial activities in contrast with clinical norfloxacin, chloromycin and fluconazole. Especially, compound 5d exhibited good anti-MRSA, anti-Escherichia coli, and anti-Salmonella typhi activity with low MIC values of 2–8?μg/mL, which were comparable or even superior to reference drugs. The preliminarily interactive investigation revealed that the most active compound 5d could effectively intercalate into DNA to form 5d-DNA complex and cleavage DNA by agarose gel electrophoresis experiments. It was also found that compound 5d was able to efficiently permeabilize the membranes of both Gram-positive (MRSA) and Gram-negative (E. coli DH52) bacteria. Experiments and molecular docking both showed that human serum albumin (HSA) could effectively transport compound 5d and hydrophobic interactions and hydrogen bonds play important roles in the association of compound 5d with HSA.

Full text of DOI:10.1016/j.ejmech.2016.06.031

Accepted Manuscript
Design, synthesis and biological evaluation of berberine-benzimidazole hybrids as
new type of potentially DNA-targeting antimicrobial agents
Ponmani Jeyakkumar, Ling Zhang, Srinivasa Rao Avula, Cheng-He Zhou
PII:
S0223-5234(16)30510-4
10.1016/j.ejmech.2016.06.031
EJMECH 8692
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2016
Revised Date: 16 June 2016
Accepted Date: 17 June 2016
Please cite this article as: P. Jeyakkumar, L. Zhang, S.R. Avula, C.-H. Zhou, Design, synthesis
and biological evaluation of berberine-benzimidazole hybrids as new type of potentially DNA-
targeting antimicrobial agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.06.031.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of berberine-benzimidazole hybrids as new type of
potentially DNA-targeting antimicrobial agents
Ponmani Jeyakkumar† , Ling Zhang, Srinivasa Rao Avula‡, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of
Chongqing Municipality, School of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, People’s Republic of China
A series of berberine−benzimidazole hybrids were synthesized and screened for their
antimicrobial activities, further studies with pET-28a (E. coli DNA), calf thymus DNA, human
serum albumin and molecular docking simulation were investigated.
i
1.8
i
0.4
1.6
a
1.4
0.2
a
1.2
a
1.0
i
DNA+NR
0.0
0.8
400
450
500
550
600
Wavelength (nm)
0.6
0.4
0.2
0.0
DNA+NR
250 300
350
400
450
500
550
600
Wavelength (nm)
†Ph.D Candidate from India
‡ Postdoctoral fellow from CSIR-Central Drug Research Institute, Lucknow 226031, India.

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Title page
Title:
Design, synthesis and biological evaluation of berberine-benzimidazole hybrids as new type of potentially
DNA-targeting antimicrobial agents
Author names and affiliations:
Ponmani Jeyakkumar† , Ling Zhang, Srinivasa Rao Avula‡, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,
People’s Republic of China
* Corresponding Address:
Prof. Cheng-He Zhou, School of Chemistry and Chemical Engineering, Southwest University, Chongqing
400715, People’s Republic of China
Tel.: +86-23-68254967; Fax: +86-23-68254967
E-mail: zhouch@swu.edu.cn (Cheng-He Zhou)
Abstract
A series of novel berberine-benzimidazole derivatives were conveniently and efficiently synthesized and
characterized by NMR, IR, MS and HRMS spectra. Most of the prepared compounds showed effective
antimicrobial activities in contrast with clinical norfloxacin, chloromycin and fluconazole. Especially,
compound 5d exhibited good anti-MRSA, anti-E. coli, and anti-S. typhi activity with low MIC values of 2-
8 µg/mL, which were comparable or even superior to reference drugs. The preliminarily interactive
investigation revealed that the most active compound 5d could effectively intercalate into DNA to form 5d-
DNA complex and cleavage DNA by agarose gel electrophoresis experiments. It was also found that
compound 5d was able to efficiently permeabilize the membranes of both Gram-positive (MRSA) and
Gram-negative (E. coli DH52) bacteria. Experiments and molecular docking both showed that human
†Ph.D Candidate from India
‡ Postdoctoral fellow from CSIR-Central Drug Research Institute, Lucknow 226031, India.
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serum albumin (HSA) could effectively transport compound 5d and hydrophobic interactions and hydrogen
bonds play important roles in the association of compound 5d with HSA.
Keywords: Berberine, Benzimidazole, Antimicrobial, DNA, HSA
1. Introduction
Berberine is well-known natural isoquinoline alkaloid with a quaternary nitrogen and desirable large π-
conjugated backbone. It has drawn much attention in medicinal chemistry especially antimicrobial aspect in
very recent years. Berberine has been playing a quite important role in the treatment of intestinal infections
such as acute gastroenteritis, cholera and bacillary dysentery. Much effort has been directing towards the
development of novel berberine derivatives with medicinal applications [1]. Literature revealed that
lipophilic substituents at C-9 position of berberine could helpfully enhance the solubility, antimicrobial
activities and broaden antibacterial spectrum [2] which overwhelmingly compel us with great interest to
further structurally modify berberine in order to discover new class of antimicrobial agents. Butyl berberine
A1 exhibited better antifungal activities (MIC = 0.25–1 µg/mL) against Cryptococcus neoformans,
Candida species than the Amphotericin B [3]. Substitution of the butyl group by methyl fragment as shown
in berberine A2 resulted in decreased bioactivities (MIC = 1–8 µg/mL) [4]. Ethyl berberine A3 was an
effective anti-tubercular agent (MIC = 0.125 mg/mL) and exhibited more potent effect against rifampicin
(RIF)- and isoniazid (INH)-resistant mycobacterium tuberculosis strains than both RIF and INH, suggesting
a new mechanism of action [5]. In recent years, the introduction of heterocycles into berberine as
antimicrobial agents has also attracted extensive interest [6–8]. Berberine-triazole A4 showed potent
antibacterial activity especially against Escherichia coli DH52, Shigella dysenteriae, Pseudomonas
aeruginosa and Proteus vulgaris with low MIC values ranging from 2 to 64 µg/mL, which were
comparable to or even better than the reference drugs berberine, chloromycin, norfloxacin and fluconazole
[6, 7]. Berberine-metronidazole A5 exhibited effective antimicrobial activities and particularly gave
comparable or even better anti-Gram-negative (MIC = 4–8 µg/mL) and anti-Candida mycoderma (MIC = 1
µg/mL) efficacies in comparison to reference drugs chloromycin, norfloxacin, berberine, fluconazole [8].
The above observations showed that the introduction of heterocycles into berberine skeleton is of great
development value as potential antimicrobial agents, which has provoked considerable interest to develop
azoles-based berberines as new antimicrobial drugs (Figure 1) [8].
Azoles are currently the most popular class of antimicrobial agents used in medicine. The development
of azole based drugs has achieved much advance in medicinal chemistry [9–11]. Benzimidazole derivatives
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are structural isosteres of naturally occurring purine. They can easily interact with biomolecules of the
living systems exhibiting diverse biological activity such as antihelmintic [12], antihistaminic [13],
anticancer [14], antiviral [15], anti-inflammatory [16], antiproliferative [17], antioxidant [18], anticoagulant
properties [19]. In recent years, much literature has revealed that benzimidazole derivatives could
effectively prevent the synthesis of microbial nucleic acids and proteins [20], thus inhibiting the growth of
various microorganisms [21], which suggests that benzimidazole moiety should have potential
antimicrobial activities. Much work has been directing towards the development of structurally novel
benzimidazole compounds as antimicrobial agents [22]. Especially the appearance of drug-resistant strains
among community acquired infections and the rapid global spread of resistant clinical isolates make
infective diseases become one of the most challenges to the public health and scientific communities
worldwide [23]. Therefore, there is an emergent need to develop new antimicrobial drugs with novel
chemical structures and mechanism of action.
Considering the above aspects, it was of great interest for us to prepare berberine-benzimidazole
derivatives, which were expected to overcome drug-resistance and increase antimicrobial bioactivities.
Literature revealed that substituents could modulate molecular flexibility and regulate the lipid-water
partition coefficient [24–27]. Rationally, various aliphatic chains with different lengths and substituted
phenyl moieties including chloro, fluoro and nitro groups were further modified based on the preliminary
active screening (Scheme 1).
< Figure 1 >
2. Results and discussion
2.1. Chemistry
The preparation of the target compounds was outlined in Scheme 1. Intermediate 2 could be efficiently
prepared in yield of 79% by selective demethylation of berberine chloride 1 at 9-position heated at 190 °C
under vacuum (20 mm Hg) for 45 minutes [28]. Berberrubine 2 was further reacted with chloroacetic acid
by nucleophilic substitution in dry DMF at 60 °C to produce compound 3 in yield of 50% [29,30], and then
cyclized with substituted benzene-1,2-diamines in 4 mol/L hydrochloric acid solution at 110 °C in good
yields to afford compounds 4a−c. Alkyl derivatives 5a−i and aralkyl ones 6a−g were obtained in yields of
28–53% by the reaction of compound 4a with different aliphatic or substituted aromatic halides
respectively in dry DMF at 60 °C using potassium carbonate as base.
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< Scheme 1 >
2.2. Biological activity
All the newly synthesized compounds were evaluated for their in vitro antimicrobial activities against
four Gram-positive bacteria Staphylococcus aureus ATCC 6538, Methicillin-resistant Staphylococcus
aureus N315 (MRSA), Bacillus subtilis ATCC 21216, and Micrococcus luteus ATCC4698; four Gram-
negative bacteria Bacillus proteus ATCC 13315, Escherichia coli JM 109, Pseudomonas aeruginosa and
Bacillus typhi and four fungi Candida albicans ATCC 76615, Candida utilis, Saccharomyces cerevisia and
Aspergillus flavus using two fold serial dilution technique (Supporting information) in 96-well micro-test
plates recommended by National Committee for Clinical Laboratory Standards (NCCLS) with the positive
control of clinically antimicrobial drugs chloromycin, norfloxacin and fluconazole [31]. pET-28a E. coli
DNA plasmid was kindly provided by Dr. Jianping Xie (State Key Laboratory Breeding Base of Eco-
Environment and Bio-Resource of the Three Gorges Area, School of Life Sciences, Southwest University,
Chongqing, China).
2.2.1. Antibacterial activity
The obtained results (depicted in Table 1) revealed that most of the prepared compounds could
effectively inhibit the growth of all the tested strains in vitro. Table 1 showed valuable effects of aliphatic
substituents on biological activities.
< Table 1 >
< Figure 2 >
Generally, intermediates showed moderate activity against all the tested bacteria. Particularly, compound
4b with fluoro moiety gave better inhibitory activity against S. aureus (MIC = 8 µg/mL), B. proteus (MIC
= 16 µg/mL) and S. typhi (MIC = 16 µg/mL) than chloromycin (MIC = 16 µg/mL, 16 µg/mL, 32 µg/mL
respectively). The further structure–activity relationship revealed that the obvious effect of the substituents
at the N-position of benzimidazoles on biological activity. In comparison with the halobenzyl derivatives
6a–g, most of alkyl ones 5a–i exerted relatively better activities in inhibiting the growth of the tested
strains. Notably, compound 5d with heptyl group exhibited broad-spectrum and good antibacterial
efficacies with MIC values between 2 to 16 µg/mL, and it gave better biological activity against S. typhi
(MIC = 2 µg/mL), E. coli (MIC = 4 µg/mL), S. aureus (MIC = 4 µg/mL), P. aeruginosa (MIC = 16 µg/mL)
in comparison with chloromycin (MIC = 32 µg/mL, 32 µg/mL, 16 µg/mL, 32 µg/mL respectively).
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Especially, its anti-MRSA (MIC = 8 µg/mL) activity was better than the chloromycin (MIC = 16 µg/mL)
and even equal to norfloxacin (MIC = 8 µg/mL). The above results indicated that compound 5d should
have large possibility as antimicrobial agent. Besides, compound 5b with the propyl group gave stronger
inhibition against MRSA (MIC = 4 µg/mL) than norfloxacin (MIC = 8 µg/mL), and displayed better anti-S.
typhi activity (MIC = 4 µg/mL) than chloromycin (MIC = 32 µg/mL), even equal to norfloxacin (MIC = 4
µg/mL). Compound 5e with the octyl group was also able to inhibit the growth of P. aeruginosa (MIC = 8
µg/mL) actively superior to norfloxacin (MIC = 16 µg/mL). Decyl derivative 5f gave stronger inhibition
against S. typhi (MIC = 2 µg/mL) than norfloxacin (MIC = 4 µg/mL). This might be resulted from the
moderately better water solubility. However, longer aliphatic chain attached benzimidazole-berberine
derivatives showed relatively poor antibacterial activities. Compounds 5h and 5i with longer aliphatic
chains gave poor biological activities.
Most of the substituted benzyl group attached in berberine-benzimidazole derivatives gave moderate
potent in the tested bacteria. Introducing ortho- and para-substituted halide derivatives were more active in
the tried bacteria than the meta-substituted halide derivatives. Particularly, 2,4-dichlorobenzyl one 6g gave
better inhibition against MRSA (MIC = 4 µg/mL) than chloromycin (MIC = 16 µg/mL) and norfloxacin
(MIC = 8 µg/mL). However, when the electron withdrawing group like NO2 (compound 6f) was introduced,
there is no enhancement of biological activity.
2.2.2. Antifungal activity
< Figure 3 >
The in vitro antifungal data (Table 1) indicated that target compounds displayed moderate to good
inhibitory potencies against tested fungi. Moreover, the fungus A. flavus was more sensitive than other
fungi. Most of the aromatic derivatives were more active against A. flavus and C. utilis than aliphatic-
substituted ones. In particularly, compound 6d also gave excellent inhibitory potencies against A. flavus
(MIC = 4 µg/mL) in comparison to fluconazole (256 µg/mL). Compounds 6g (2 µg/mL), 6b (4 µg/mL) and
6d (4 µg/mL) showed the stronger inhibition against C. utilis (2 µg/mL) than fluconazole (8 µg/mL).
2.2.3. Compound 5d interaction with pET-28a DNA
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The DNA-cleaving activity of compound 5d towards pET-28a DNA was studied under physiological
conditions (5 h, 37 °C). The ability of binding to supercoiled pET-28a DNA was determined by use of
agarose gel electrophoresis (GE) (Figure S1). It can be seen that compound 5d could capable of converting
pET-28a DNA into open circular form, and that the cleaving activity showed a strong dependence on the
concentration of compound 5d. When the concentration of compound 5d was about 0.1 mM, almost all the
CCC form was converted to the OC form. Such dependence has lent strong support that compound 5d
catalyzed the cleaving.
2.2.4. Bacterial membrane permeabilization
< Figure 4 >
< Figure 5 >
Natural and synthetic membrane active antibacterial agents offer a hope to solve the problem of bacterial
resistance as the membrane-active nature imparts low propensity for the development of resistance. The
ability of compound 5d to permeabilize the bacterial cell membrane was studied using propidium iodide
(PI). This dye can pass through the membrane of compromised bacterial cells and fluoresces upon binding
to the DNA. As can be seen from Figure 4 and Figure 5, the tested compound was efficient in
permeabilizing the membranes of both Gram-positive (MRSA) and Gram-negative (E. coli DH52) bacteria.
2.3. Interactions with calf thymus DNA
DNA is one of the targets for the studies of biologically important small molecules such as antimicrobial
drugs. There is growing interest in investigating the interaction of small molecules with DNA for the
rational design and construction of new and efficient drugs targeted to DNA. To explore the possible
antimicrobial action mechanism, the binding behavior of compound 5d with calf thymus DNA (DNA
model with medical importance, low-cost and ready availability properties) was studied on a molecular
level in vitro using neutral red (NR) dye as a spectral probe by UV−visible spectroscopic methods.
2.3.1 Absorption spectra of DNA in the presence of compound 5d
< Figure 6 >
The application of absorption spectroscopy is one of the most useful techniques in DNA-binding studies.
In absorption spectroscopy, hypochromism and hyperchromism are very important spectral features to
distinguish the change of DNA double-helical structure. Due to the strong interaction between the
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electronic states of intercalating chromophore and that of the DNA base, the observed large hypochromism
strongly suggests a close proximity of the aromatic chromophore to the DNA bases. With a fixed
concentration of DNA, UV-vis absorption spectrum was recorded with the increasing amount of compound
5d. As shown in Figure 6, UV-vis spectra displayed that the maximum absorption peak of DNA at 260 nm
exhibited the proportional increase with the increasing concentration of compound 5d. Meanwhile the
absorption value of simply sum of free DNA and free compound 5d was a little greater than the measured
value of 5d−DNA complex. These meant a weak hypochromic effect existed between DNA and compound
5d. Furthermore, the intercalation of the aromatic chromophore of compound 5d into the helix and the
strong overlap of π-π* states in the large π-conjugated system with the electronic states of DNA bases were
consistent with the observed spectral changes [32, 33]. Based on variations in the absorption spectra of
DNA upon binding to 5d, eq 1 can be utilized to calculate the binding constant (K).
A0
ξC
ξC
1
(1)
=
+
×
A− A0 ξD−C − ξC ξD−C − ξC K[Q]
A0 and A represent the absorbance of DNA in the absence and presence of compound 5d at 260 nm, ξC
and ξD-C were absorption coefficients of compound 5d and DNA-compound 5d complex respectively. The
plot of A0/(A-A0) versus 1/[compound 5d] was constructed by using the absorption titration data and linear
fitting (Figure 7), yielding the binding constant, K = 4.12×104 L/mol, R = 0.9945, SD = 0.0937 (R is the
correlation coefficient. SD is standard deviation).
< Figure 7 >
2.3.2 Absorption spectra of NR interaction with DNA
Neutral Red (NR) is a planar phenazine dye and is structurally similar to other planar dyes, for example,
acridine, thiazine, and xanthene. It has been demonstrated that the binding of NR with DNA is an
intercalation binding in recent years. Therefore, NR was employed as a spectral probe to investigate the
binding mode of 5d with DNA in the present work. The absorption spectra of the NR dye upon the addition
of DNA are shown in Figure 8. It is apparent that the absorption peak of the NR at around 460 nm showed
the 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 DNA-NR complex formation.
< Figure 8 >
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2.3.3 Absorption spectra of competitive interaction of compound 5d and NR with DNA
Figure 10 displayed the absorption spectra of a competitive binding between NR and 5d with DNA. As
shown, with the increasing concentration of 5d, the maximum absorption, around 525 nm of the DNA-NR
complex decreased. However, a slight intensity increase was observed in the developing band around 475
nm. Compared with the absorption band at around 450 nm of the free NR in the presence of the increasing
concentrations of DNA (Figure 9), the spectra in Figure 9 (inset) exhibited the reverse process. The results
suggested that compound 5d intercalated into the double helix of DNA by substituting for NR in the DNA-
NR complex.
< Figure 9 >
2.4. Interactions of compound 5d with HSA
HSA is the principal extracellular protein of the circulatory system, and accounts for about 60% of the
total plasma proteins corresponding to a concentration of 42 mg/mL and provides about 80% of the colloid
osmotic pressure of blood. It is well accepted that the overall distribution, metabolism, and efficacy of
drugs can be changed by their affinity to HSA, and many promising new drugs have been rendered
ineffective because of their unusually high affinity for this protein. So the investigations of interactions
between drugs or bioactive small molecules and HSA are not only beneficial to provide a proper
understanding of the absorption, transportation, distribution, metabolism and excretion properties of drugs,
but also significant to design, modify and screen drug molecules.
2.4.1 UV- visible absorption spectral study
UV-vis absorption measurement is a very easy operational method applicable to explore the structural
change of protein and to identify the complex formation. In the binding experiment, UV−vis absorption
spectroscopic method was adopted to evaluate the binding behavior between compound 5d and HSA. As
shown in Figure 10, the absorption peak observed at 278 nm was attributed to the aromatic rings in
Tryptophan (Trp-214), Tyrosine (Tyr-411) and Phenylalanine (Phe) residues in HSA. With the addition of
compound 5d, the peak intensity increased, indicating that compound 5d could interact with HSA and the
peptide strands of HSA were extended. However, the maximum absorption wavelength remained
unchanged, implying that the interactions of compound 5d and HSA was a non covalent interaction, which
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occurred via the π-π stacking between aromatic rings of compound 5d and Trp, Tyr and Phe residues
possessed conjugated π-electrons and located in the binding cavity of HSA [34].
< Figure 10 >
2.5. Molecular docking studies with HSA
HSA contains three homologous α-helical domains (I−III), each of which is composed of subdomains A
and B. The principal regions of ligand binding sites in HSA are located in hydrophobic cavities in the
subdomains IIA and IIIA [35, 36]. In this study molecular docking simulation has been used to improve the
understanding of the interaction of drug to HSA [37–39]. As shown in the Figure 11 binding mode of
compound 5d with HSA with high docking score of 8.31. As clearly evidenced, compound 5d surrounded
by LYS-212, ALA-215, ALA-213, TRP-214, ALA-210 and PHE-211. Berberine methoxy group (-OCH3)
and benzimidazole-3-N bind with LYS-212 residue. Hydrophobic interaction exists between the aromatic
rings of compound 5d and HSA. Through the interaction are dominated by hydrophobic, specific electronic
interaction and hydrogen bonds between compound 5d and LYS-212 are involved in the binding process.
< Figure 11 >
3. Conclusion
In conclusion, a series of berberine-benzimidazoles were successfully synthesized by a convenient and
efficient procedure. Their structures were confirmed by 1H NMR, 13C NMR, IR and HRMS spectra. The in
vitro antimicrobial evaluation revealed that most of the synthesized compounds exhibited good bioactivities
against the tested bacterial strains. The prepared compound 5d showed low MIC value of 2−8 ꢀg/mL
against S. aureus, E. coli, and S. typhi which was comparable or even superior to reference drugs. The
preliminarily interactive investigation revealed that the most active compound 5d could effectively
intercalate into DNA to form 5d-DNA complex. Furthermore, it was also found that compound 5d could
cleavage DNA by agarose gel electrophoresis experiments. Molecular docking showed that human serum
albumin (HSA) could effectively transport compound 5d and hydrophobic interactions and hydrogen bonds
play important roles in the association of compound 5d with HSA.
4. Experimental Methods
4.1. General information
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All reagents were commercial and used without further purification. All reactions were monitored by
TLC, silica gel plates with fluorescence F254 was used. Melting points were recorded in the X-6 melting
point apparatus and uncorrected. Column chromatography carried out by Aluminium oxide and silica gel
(80-120). FT-IR spectrum was carried on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge, MA,
USA) using KBr pellets in the 400 - 4000 cm-1 range. 1H NMR and 13C NMR spectrum was recorded on a
Bruker AV 600 spectrometer using TMS as an internal standard. The following abbreviations were used to
designate aryl groups: Bim = benzimidazoyl, Ph = phenyl. The 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 spectrum was recorded on LCMS-
2010A and HRMS. UV spectra was recorded at room temperature on a TU-2450 spectrophotometer (Puxi
Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells. HSA, Calf thymus DNA and
NR were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris, NaCl, HCl was analytical purity.
Sample masses were weighed on a microbalance with a resolution of 0.1 mg.
4.1.1. Synthesis of berberrubine (2)
Berberine hydrochloride (1, 2.5 g, 7 mmol) was placed in the round bottom flask and in a vacuum,
stirred at 190 °C for 20 min to give the crude product, which was recrystallized by ethanol (30 mL) and
concentrated HCl (5 mL) to give compound 2 (1.8 g) as yellow solid. Yield: 80%.
4.1.2. Synthesis of 6H-benzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium,9-carboxymethoxy)-5,6-dihydro-10-
methoxy-chloride (3)
Berberrubine 2 (1.0 g, 2.8 mmol) and KOH (110 mg, 2.8 mmol) in DMF (6 mL) were stirred at 60 °C for
45 min, then the reaction mixture was cooled room temperature. Chloroacitic acid (390 mg, 4.2 mmol) and
TBAI (200 mg, 0.05 mmol) were added then slowly raised temperature up to 60 °C and maintained for 5 h.
After the reaction was completed (monitored by TLC, eluent, chloroform/methanol, 2/1, V/V), the reaction
mass was cooled to room temperature and diethyl ether (15 mL) was added then filtered. The isolated solid
was washed with chloroform (10 mL) and diethyl ether (10 mL) and dried. The crude compound was
purified by column chromatography (chloroform/methanol, 10/1−2/1, V/V) to afford compound 3 (640 mg)
as yellow solid. Yield: 50%.
4.1.3. Synthesis of 2-berberinoxymethoxy-1H-benzo[d]imidazole (4a)
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Benzene-1,2-diamine (1.0 g, 9.3 mmol) and compound 3 (3.8 g, 9.3 mmol) in HCl (4N, 30 mL) were
stirred at 110 °C for 5 h, then cooled to room temperature and neutralized (pH = 5 to 7) with ammonia
solution (25%). Yellow solid was filtered and washed with minimum quantity of water (10 mL). The crude
product was purified by alumina column chromatography (chloroform/methanol, 10/1, V/V) to afford the
target compound 4 (704 mg) as yellow solid. Yield: 45%. mp: 178–180 ºC. IR (KBr, cm-1) ν: 3436 (NH),
2917, 2846 (CH2, CH3), 1631, 1601, 1505, 1422 (aryl skeleton, C=C), 1394, 1348, 1272, 1227, 1142,
1102, 1061, 1038, 936, 889, 749 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 12.74 (s, 1H, Bim -NH), 9.91 (s,
1H, 8-H), 8.94 (s, 1H, 13-H), 8.25 (d, J = 9.2 Hz, 1H, 12-H), 8.04 (d, J = 9.1 Hz, 1H, 11-H), 7.78 (s, 1H, 1-
H), 7.62 (d, J = 7.9 Hz, 1H, Bim -H), 7.55 (d, J = 7.9 Hz, 1H, Bim -H), 7.23 (t, J = 7.5 Hz, 1H, Bim -H),
7.18 (t, J = 7.5 Hz, 1H, Bim -H), 7.10 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.59 (s, 2H, 9-OCH2-), 4.87 (t,
J = 6.2 Hz, 2H, 6-H), 4.09 (s, 3H, -OCH3), 3.20–3.14 (m, 2H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6)
δ: 151.2, 150.4, 150.1, 148.2, 145.9, 143.3, 142.1, 137.9, 134.7, 133.4, 131.0, 127.1, 124.7, 123.1, 122.1,
122.0, 120.8, 120.6, 119.5, 112.0, 108.9, 105.9, 102.6, 69.3, 57.7, 55.9, 26.8 ppm; HRMS (TOF): calcd for
C27H22ClN3O4 [M-Cl]+, 452.1605, found, 452.1604.
4.1.4. Synthesis of 2-berberinoxymethoxy-5-fluro-1H-benzo[d]imidazole (4b)
4-Fluorobenzene-1,2-diamine (1. 0 g, 9.3 mmol) and compound 3 (3.8 g, 9.3 mmol) in HCl (4N, 30 mL)
were stirred at 110 °C for 5 h, then cooled to room temperature and neutralized (pH = 6-7) with ammonia
solution (25%). Yellow solid was filtered and washed with minimum quantity of water (10 mL). The crude
compound was purified by alumina column chromatography (chloroform/methanol, 10/1, V/V) to afford
the target compound 4b (704 mg) as yellow solid. Yield: 42%. mp: 185–187 ºC. IR (KBr) ν: 3440 (NH),
3052 (Ar-CH), 2946, 2909, 2843 (CH2, CH3), 1631 (aryl skeleton, C=C), 1602, 1504, 1481, 1454, 1393,
1360, 1299, 1270, 1226, 1189, 1138, 1106, 1061, 1036, 996, 969, 934, 913, 864, 802, 762, 656, 459, 427
1
cm-1; H NMR (600 MHz, DMSO-d6) δ: 12.93 (s, 1H, Bim -NH), 9.96 (s, 1H, 8-H), 8.95 (s, 1H, 13-H),
8.24 (d, J = 9.2 Hz, 1H, 12-H), 8.05 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.59 (d, J = 32.0 Hz, 1H,
Bim -H), 7.40 (d, J = 32.1 Hz, 1H, Bim -H), 7.10 (s, 1H, 4-H), 7.06 (s, 1H, Bim -H), 6.18 (s, 2H, -OCH2O-
), 5.58 (s, 2H, 9-OCH2-), 4.91 (t, J = 6.2 Hz, 2H, 6-H), 4.08 (s, 3H, -OCH3), 3.19 (t, J = 6.2 Hz, 2H, 5-H)
ppm; 13C NMR (151 MHz, DMSO-d6) δ: 151.1, 150.4, 148.2, 145.9, 142.1, 138.1, 133.4, 131.1, 127.2,
124.8, 122.1, 120.8, 120.7, 108.9, 105.9, 102.6, 69.2, 57.7, 55.9, 26.8 ppm; HRMS (TOF): calcd. for
C27H21ClFN3O4 [M-Cl]+ , 470.1511, found, 470.1512.
11

ACCEPTED MANUSCRIPT
4.1.5. Synthesis of 2-berberinoxymethoxy-5-chloro-1H-benzo[d]imidazole (4c)
4-Chlorobenzene-1,2-diamine (1. 0 g, 9.3 mmol) and compound 3 (3.8 g, 9.3 mmol) in HCl (4N, 30 mL)
were stirred at 110 °C for 5 h, then cooled to room temperature and neutralized (pH = 6 to 7) with ammonia
solution (25%). Yellow solid was filtered and washed with minimum quantity of water (10 ml). The crude
compound was purified by alumina column chromatography (chloroform/methanol, 10/1, V/V) to afford
the target compound 4c (704 mg) as yellow solid. Yield: 49%. mp: 193–195 ºC. IR (KBr) ν: 3435 (NH),
3203 (Ar-CH), 3052 (Ar-CH), 2947, 2909 (CH2, CH3), 1630 (aryl skeleton, C=C), 1604, 1569, 1503,
1479, 1448, 1424, 1393, 1364, 1298, 1271, 1225, 1189, 1143, 1102, 1062, 1036, 997, 970, 934, 914, 863,
808, 650, 426 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 12.95 (d, J = 25.5 Hz, 1H, Bim -NH), 9.96 (d, J =
11.1 Hz, 1H, 8-H), 8.95 (s, 1H, 13-H), 8.24 (d, J = 9.2 Hz, 1H, 12-H), 8.05 (d, J = 9.1 Hz, 1H, 11-H), 7.79
(s, 1H, 1-H), 7.72–7.63 (m, 1H, Bim -H), 7.62 – 7.55 (m, 1H, Bim -H), 7.29–7.18 (m, 1H, Bim -H), 7.10 (s,
1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.59 (s, 2H, 9-OCH2-), 4.98–4.86 (m, 2H, 6-H), 4.08 (s, 3H, -OCH3),
3.25–3.15 (m, 2H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 151.1, 150.4, 148.2, 145.9, 142.1, 138.1,
133.5, 131.1, 127.2, 124.8, 122.0, 120.8, 120.7, 108.9, 105.9, 102.6, 69.2, 57.7, 56.0, 26.9 ppm; HRMS
(TOF): calcd. for C27H21Cl2N3O4 [M-Cl]+ , 486.1215, found, 486.1215.
4.1.6. Synthesis of 2-berberinoxymethoxy-1-ethyl-1H-benzo[d]imidazole (5a)
Compound 4a (500 mg. 1.0 mmol) and potassium carbonate (110 mg, 0.8 mmol) in DMF (2.5 mL) were
stirred at 0 °C for 1 h, then 1-bromoethane (140 mg, 1.3 mmol) was added and slowly raised to room
temperature and 60 °C for 7 h maintained. After the reaction was completed (monitored by TLC, eluent,
chloroform/methanol, 99/1, V/V), the mixture was cooled to room temperature and diethylether (10 mL)
was added and filtered. The solid was further washed with diethylether (20 mL) and dried over under
vacuum. The crude product was purified by alumina column chromatography (chloroform/methanol, 99/1)
to afford the compound 5a (270 mg) as yellow solid. Yield: 52%. mp: 168–170 ºC. IR (KBr) ν: 2809, 2725,
2657 (CH2, CH3), 1630 (aryl skeleton, C=C), 1593, 1508, 1397, 1348, 1273, 1229, 1148, 1104, 1039, 877,
760 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 9.78 (s, 1H, 8-H), 8.96 (s, 1H, 13-H), 8.24 (d, J = 9.2 Hz, 1H,
12-H), 8.06 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.70–7.62 (m, 2H, Bim -H), 7.32 (t, J = 7.4 Hz,
1H, Bim -H), 7.24 (t, J = 7.4 Hz, 1H, Bim -H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.63 (s, 2H, 9-
OCH2-), 4.87 (t, J = 6.2 Hz, 2H, 6-H), 4.55 (q, J = 7.1 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.20–3.13
(m, 2H, 5-H), 1.45 (t, J = 7.2 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 151.4, 150.4, 149.4,
12

ACCEPTED MANUSCRIPT
148.2, 145.8, 142.6, 142.0, 138.0, 135.3, 133.6, 131.1, 127.0, 124.9, 123.4, 122.4, 122.2, 120.8, 120.8,
119.9, 111.1, 108.9, 105.9, 102.6, 68.1, 57.6, 56.0, 39.0, 26.8, 15.7 ppm; HRMS (TOF): calcd for
C29H26ClN3O4 [M-Cl]+, 480.1918, found, 480.1916.
4.1.7. Synthesis of 2-berberinoxymethoxy-1-propyl-1H-benzo[d]imidazole (5b)
Compound 5b (271 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromopropane (15 mg, 1.3 mmol). Yield: 50%. mp: 158–160 ºC; IR (KBr) ν: 2966, 2924 (CH2,
CH3), 1630, 1603, 1506, 1458 (aryl skeleton, C=C), 1396, 1347, 1271, 1228, 1141, 1104, 1038, 962, 751
cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 9.78 (s, 1H, 8-H), 8.96 (s, 1H, 13-H), 8.24 (d, J = 9.2 Hz, 1H, 12-
H), 8.06 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.70–7.63 (m, 2H, Bim -H), 7.31 (t, J = 7.6 Hz, 1H,
Bim -H), 7.23 (t, J = 7.5 Hz, 1H, Bim -H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.63 (s, 2H, 9-OCH2-
), 4.86 (t, J = 6.2 Hz, 2H, 6-H), 4.45 (t, J = 7.3 Hz, 2H, -NCH2-), 4.04 (s, 3H, -OCH3), 3.19–3.13 (m, 2H, 5-
H), 1.92–1.84 (m, 2H, -N(CH2)CH2-), 0.94 (t, J = 7.4 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-d6)
δ: 151.4, 150.4, 149.6, 148.2, 145.8, 142.3, 141.9, 138.0, 135.7, 133.5, 131.1, 126.9, 124.9, 123.3, 122.3,
122.1, 120.8, 120.7, 119.9, 111.3, 108.9, 105.9, 102.6, 67.9, 57.6, 56.0, 45.4, 26.8, 23.4, 11.6 ppm; HRMS
(TOF): calcd for C30H28ClN3O4 [M-Cl]+, 494.2074, found, 494.2072.
4.1.8. Synthesis of 2-berberinoxymethoxy-1-pentyl-1H-benzo[d]imidazole (5c)
Compound 5c (290 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromopentane (185 mg, 1.3 mmol). Yield: 53%; mp: 153–155 ºC; IR (KBr) ν: 2959, 2927, 2853,
2658 (CH2, CH3), 1631, 1598, 1506, 1458 (aryl skeleton, C=C), 1397, 1348, 1275, 1229, 1142, 1105,
1
1036, 963, 932, 873, 747 cm-1; H NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.96 (s, 1H, 13-H),
8.24 (d, J = 9.2 Hz, 1H, 12-H), 8.07 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.2 Hz, 2H,
Bim -H), 7.31 (t, J = 7.5 Hz, 1H, Bim -H), 7.23 (t, J = 7.6 Hz, 1H, Bim -H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H,
-OCH2O-), 5.63 (s, 2H, 9-OCH2-), 4.90–4.83 (m, 2H, 6-H), 4.47 (t, J = 7.4 Hz, 2H, -NCH2-), 4.02 (s, 3H, -
OCH3), 3.21–3.14 (m, 2H, 5-H), 1.90–1.78 (m, 2H, -N(CH2)CH2-), 1.39–1.33 (m, 4H, -N(CH2)2(CH2)2-),
0.85 (t, J = 6.8 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9,
142.4, 141.9, 138.0, 135.7, 133.5, 131.1, 126.9, 124.9, 123.4, 122.3, 122.2, 120.8, 120.7, 119.9, 111.2,
13

ACCEPTED MANUSCRIPT
108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 44.0, 29.8, 28.9, 26.8, 22.3, 14.3 ppm; HRMS (TOF): calcd for
C32H32ClN3O4 [M-Cl]+, 522.2387, found, 522.2383.
4.1.9. Synthesis of 2-berberinoxymethoxy-1-heptyl-1H-benzo[d]imidazole (5d)
Compound 5d (300 mg) was prepared as yellowish brown solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromoheptane (220 mg, 1.3 mmol). Yield: 51%; mp: 141–143 ºC; IR (KBr) ν: 2964, 2920, 2656,
2390 (CH2, CH3), 1631, 1600, 1507, 1412 (aryl skeleton, C=C), 1347, 1273, 1228, 1104, 1037, 757 cm-1;
1H NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.96 (s, 1H, 13-H), 8.24 (d, J = 9.1 Hz, 1H, 12-H),
8.07 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.1 Hz, 2H, Bim-H), 7.31 (t, J = 7.6 Hz, 1H,
Bim-H), 7.23 (t, J = 7.6 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.63 (s, 2H, 9-OCH2-),
4.91–4.81 (m, 2H, 6-H), 4.46 (t, J = 7.3 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.20–3.12 (m, 2H, 5-H),
1.87–1.78 (m, 2H, -N(CH2)CH2-), 1.39–1.31 (m, 2H, -N(CH2)2CH2-), 1.31–1.26 (m, 2H, -N(CH2)3CH2-),
1.25–1.16 (m, 4H, -N(CH2)4(CH2)2-), 0.80 (t, J = 6.6 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-d6)
δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7, 133.6, 131.1, 127.0, 125.0, 123.3, 122.4,
122.2, 120.8, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 44.1, 31.6, 30.1, 28.8, 26.9, 26.7,
22.4, 14.3 ppm; HRMS (TOF): calcd for C34H36ClN3O4 [M-Cl]+, 550.2708, found, 550.2706.
4.1.10. Synthesis of 2-berberinoxymethoxy-1-octyl-1H-benzo[d]imidazole (5e)
Compound 5e (310 mg) was prepared as yellowish brown solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromooctane (237 mg, 1.3 mmol). Yield: 51%; mp: 139–141 ºC; IR (KBr) ν: 2923, 2851, 2663, 2361
(CH2, CH3), 1631, 1595, 1506, 1457 (aryl skeleton, C=C), 1396, 1348, 1271, 1227, 1104, 1039, 744 cm-1;
1H NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.96 (s, 1H, 13-H), 8.24 (d, J = 9.2 Hz, 1H, 12-H),
8.06 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.7 Hz, 2H, Bim-H), 7.31 (t, J = 8.0 Hz, 1H,
Bim-H), 7.23 (t, J = 7.9 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.63 (s, 2H, 9-OCH2-),
4.86 (t, J = 6.2 Hz, 2H, 6-H), 4.46 (t, J = 7.4 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.19–3.12 (m, 2H, 5-
H), 1.86–1.79 (m, 2H, -N(CH2)CH2-), 1.38–1.32 (m, 2H, -N(CH2)2CH2-), 1.30–1.25 (m, 2H, -N(CH2)3CH2-
), 1.24–1.17 (m, 6H, -N(CH2)4(CH2)3-), 0.80 (t, J = 6.9 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-
d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7, 133.6, 131.1, 127.0, 125.0, 123.3,
14

ACCEPTED MANUSCRIPT
122.3, 122.2, 120.2, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 44.1, 31.6, 30.1, 29.1, 29.0,
26.9, 26.7, 22.5, 14.3 ppm; HRMS (TOF): calcd for C35H38ClN3O4 [M-Cl]+, 564.2857; found, 564.2856.
4.1.11. Synthesis of 2-berberinoxymethoxy-1-decyl-1H-benzo[d]imidazole (5f)
Compound 5f (260 mg) was prepared as yellowish brown solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromodecane (272 mg, 1.3 mmol). Yield: 40%; mp: 127–129 ºC; IR (KBr) ν: 2923, 2858 (CH2,
CH3), 1631, 1606, 1559, 1506, 1476, 1422 (aryl skeleton, C=C), 1399, 1360, 1336, 1247, 1227, 1144,
1104, 1036, 962, 935, 742 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.97 (s, 1H, 13-H),
8.25 (d, J = 9.2 Hz, 1H, 12-H), 8.07 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.4 Hz, 2H,
Bim-H), 7.31 (t, J = 7.8 Hz, 1H, Bim-H), 7.23 (t, J = 7.6 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -
OCH2O-), 5.63 (s, 2H, 9-OCH2-), 4.87 (t, J = 6.2 Hz, 2H, 6-H), 4.46 (t, J = 7.4 Hz, 2H, -NCH2-), 4.03 (s,
3H, -OCH3), 3.21–3.12 (m, 2H, 5-H), 1.86–1.78 (m, 2H, -N(CH2)CH2-), 1.36–1.31 (m , J = 14.9, 7.5 Hz,
2H, -N(CH2)2CH2-), 1.29–1.24 (m, J = 13.0, 7.7 Hz, 2H, -N(CH2)3CH2-), 1.22–1.19 (m, 2H, -N(CH2)4CH2-
), 1.18–1.10 (m, 8H, -N(CH2)5(CH2)4-), 0.82 (t, J = 7.1 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-
d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7, 133.6, 131.1, 127.0, 124.9, 123.3,
122.3, 122.2, 120.8, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.1, 57.6, 56.1, 49.1, 44.1, 31.7, 30.1, 29.3,
29.3, 29.1, 26.9, 26.7, 22.5, 14.4 ppm; HRMS (TOF): calcd for C37H42ClN3O4 [M-Cl]+, 592.317; found,
592.3174.
4.1.12. Synthesis of 2-berberinoxymethoxy-1-dodecyl-1H-benzo[d]imidazole (5g)
Compound 5g (270 mg) was prepared as brown solid according to the procedure described for compound
5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol) and 1-
bromododecane (306 mg, 1.3 mmol). Yield: 41%; mp: 127–129 ºC; IR (KBr) ν: 2924, 2852 (CH2, CH3),
1
1631, 1604, 1562, 1507, 1458 (aryl skeleton, C=C), 1399, 1345, 1272, 1227, 1105, 1038, 751 cm-1; H
NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.97 (s, 1H, 13-H), 8.25 (d, J = 9.2 Hz, 1H, 12-H), 8.07
(d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.3 Hz, 2H, Bim-H), 7.31 (t, J = 7.7 Hz, 1H, Bim-
H), 7.23 (t, J = 7.6 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.63 (s, 2H, 9-OCH2-), 4.87
(t, J = 6.1 Hz, 2H, 6-H), 4.46 (t, J = 7.4 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.21–3.11 (m, 2H, 5-H),
1.86–1.78 (m, 2H, -N(CH2)CH2-), 1.35–1.31 (m, 2H, -N(CH2)2CH2-), 1.27–1.25 (m, 2H, -N(CH2)3CH2-),
1.24–1.22 (m, 2H, -N(CH2)4CH2-), 1.16–1.07 (m, 12H, -N(CH2)5(CH2)6-), 0.83 (t, J = 7.1 Hz, 3H, -CH3)
15

ACCEPTED MANUSCRIPT
ppm; 13C NMR (151 MHz, DMSO-d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7,
133.6, 131.1, 127.0, 125.0, 123.3, 122.3, 122.2, 120.8, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.0, 57.6,
56.0, 49.1, 44.1, 31.7, 30.1, 29.4, 29.4, 29.3, 29.1, 29.1, 26.9, 26.7, 22.5, 14.4 ppm; HRMS (TOF): calcd
for C39H46ClN3O4 [M-Cl]+, 620.3483; found, 620.3484.
4.1.13. Synthesis of 2-berberinoxymethoxy-1-hexadecyl-1H-benzo[d]imidazole (5h)
Compound 5h (290 mg) was prepared as brown solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 1-bromohexadecane (375 mg, 1.3 mmol). Yield: 40%; mp: 123–125 ºC; IR (KBr) ν: 2923, 2851 (CH2,
CH3), 1631, 1595, 1507, 1456 (aryl skeleton, C=C), 1396, 1348, 1272, 1226, 1104, 1035, 962, 740 cm-1; 1H
NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.97 (s, 1H, 13-H), 8.25 (d, J = 9.2 Hz, 1H, 12-H), 8.07
(d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.3 Hz, 2H, Bim-H), 7.30 (t, J = 7.7 Hz, 1H, Bim-
H), 7.23 (t, J = 7.5 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.62 (s, 2H, 9-OCH2-), 4.87
(t, J = 6.2 Hz, 2H, 6-H), 4.46 (t, J = 7.4 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.19–3.13 (m, 2H, 5-H),
1.86–1.79 (m, 2H, -N(CH2)CH2-), 1.36–1.30 (m, 2H, -N(CH2)2CH2-), 1.28–1.22 (m, 4H, -N(CH2)3(CH2)2-),
1.20–1.10 (m, 20H, -N(CH2)5(CH2)10-), 0.84 (t, J = 7.0 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-
d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7, 133.6, 131.1, 127.0, 125.0, 123.3,
122.3, 122.2, 120.8, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 44.0, 31.7, 30.0, 29.5, 29.5,
29.4, 29.3, 29.3, 29.1, 29.1, 26.8, 26.7, 22.5, 14.4 ppm; HRMS (TOF): calcd for C43H54ClN3O4 [M-Cl]+,
676.4109; found, 676.4109.
4.1.14. Synthesis of 2-berberinoxymethoxy-1-octadecyl-1H-benzo[d]imidazole (5i)
Compound 5i (242 mg) was prepared as brown solid according to the procedure described for compound
5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol) and 1-
bromooctadecane (409 mg, 1.3 mmol). Yield: 32%; mp: 121–123 ºC; IR (KBr) ν: 2922, 2851 (CH2, CH3),
1631, 1604, 1560, 1507, 1458 (aryl skeleton, C=C), 1399, 1340, 1273, 1228, 1105, 1037, 962, 741 cm-1; 1H
NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.97 (s, 1H, 13-H), 8.24 (d, J = 9.2 Hz, 1H, 12-H), 8.07
(d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.65 (d, J = 8.1 Hz, 2H, Bim-H), 7.30 (t, J = 7.7 Hz, 1H, Bim-
H), 7.23 (t, J = 7.5 Hz, 1H, Bim-H), 7.09 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.62 (s, 2H, 9-OCH2-), 4.87
(t, J = 6.2 Hz, 2H, 6-H), 4.46 (t, J = 7.4 Hz, 2H, -NCH2-), 4.03 (s, 3H, -OCH3), 3.19–3.13 (m, 2H, 5-H),
1.86–1.79 (m, 2H, -N(CH2)CH2-), 1.36–1.31 (m, 2H, -N(CH2)2CH2-), 1.29–1.23 (m, 4H, -N(CH2)3(CH2)2-),
16

ACCEPTED MANUSCRIPT
1.21–1.10 (m, 24H, -N(CH2)5(CH2)12-), 0.84 (t, J = 7.0 Hz, 3H, -CH3) ppm; 13C NMR (151 MHz, DMSO-
d6) δ: 151.4, 150.4, 149.6, 148.2, 145.9, 142.5, 142.0, 138.0, 135.7, 133.6, 131.1, 127.0, 125.0, 123.3,
122.3, 122.2, 120.8, 120.8, 119.9, 111.2, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 44.1, 31.7, 30.0, 29.5, 29.4,
29.3, 29.1, 29.1, 26.9, 26.7, 22.5, 14.4 ppm; HRMS (TOF): calcd for C45H58ClN3O4 [M-Cl]+, 704.4422;
found, 704.4419.
4.1.15. Synthesis of N-4-flurobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6a)
Compound 6a (207 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 4-flurobenzyl chloride (178 mg, 1.3 mmol). Yield: 34%; mp: 178–180 ºC; IR (KBr) ν: 2924, 2804
(CH2, CH3), 1630, 1603, 1507, 1400 (aryl skeleton, C=C), 1349, 1274, 1228, 1105, 1036, 875, 755 cm-1;
1H NMR (600 MHz, DMSO-d6) δ: 9.79 (s, 1H, 8-H), 8.94 (s, 1H, 13-H), 8.19 (d, J = 9.2 Hz, 1H, 12-H),
8.03 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.71–7.68 (m, 1H, Bim-H), 7.50 (d, J = 7.2 Hz, 1H, Bim-
H), 7.29–7.23 (m, 4H, Ph-H), 7.13 (t, J = 8.8 Hz, 2H, Bim-H), 7.10 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-),
5.76 (s, 2H, Ph-CH2), 5.69 (s, 2H, 9-OCH2-), 4.86 (t, J = 6.2 Hz, 2H, 6-H), 3.94 (s, 3H, -OCH3), 3.20–3.15
(m, 2H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 162.7, 161.1, 151.1, 150.4, 149.9, 148.2, 145.8,
142.5, 141.9, 138.0, 135.7, 133.5, 133.5, 131.1, 129.4, 129.3, 127.0, 124.8, 123.7, 122.7, 122.0, 120.8,
120.7, 120.1, 116.0, 115.9, 111.5, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 46.6, 26.8 ppm; HRMS (TOF):
calcd for C34H27ClFN3O4 [M-Cl]+, 560.1980, found, 560.1982.
4.1.16. Synthesis of N-2-flurobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6b)
Compound 6b (183 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 2-flurobenzyl chloride (177 mg, 1.3 mmol). Yield: 30%; mp: 183–185 ºC; IR (KBr) ν: 2965, 2924
(CH2, CH3), 1631, 1604, 1507, 1458 (aryl skeleton, C=C), 1399, 1344, 1265, 1231, 1178, 1143, 1105,
1
1035, 964, 876, 800, 756 cm-1; H NMR (600 MHz, DMSO-d6) δ: 9.80 (s, 1H, 8-H), 8.94 (s, 1H, 13-H),
8.18 (d, J = 9.2 Hz, 1H, 12-H), 8.03 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.70 (d, J = 7.2 Hz, 1H,
Bim-H), 7.51 (d, J = 7.2 Hz, 1H, Bim-H), 7.36–7.31 (m, 1H, Ph-H), 7.26 (ddd, J = 21.3, 10.3, 4.4 Hz, 3H,
Ph-H), 7.11–7.07 (m, 2H, 4-H, Bim-H), 6.98 (t, J = 7.4 Hz, 1H, Bim-H), 6.18 (s, 2H, -OCH2O-), 5.82 (s,
2H, Ph-CH2), 5.69 (s, 2H, 9-OCH2-), 4.86 (t, J = 6.2 Hz, 2H, 6-H), 3.93 (s, 3H, -OCH3), 3.20–3.14 (m, 2H,
5-H) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 161.2, 159.5, 151.0, 150.4, 150.1, 148.2, 145.8, 142.5,
17

ACCEPTED MANUSCRIPT
141.9, 138.0, 135.7, 133.5, 131.1, 130.4, 130.3, 129.3, 129.3, 127.0, 125.3, 125.2, 124.8, 124.1, 123.9,
123.7, 122.7, 122.0, 120.8, 120.7, 120.1, 116.1, 116.0, 111.3, 108.9, 105.9, 102.6, 67.9, 57.5, 56.0, 41.8,
41.8, 26.8 ppm; HRMS (TOF): calcd for C34H27ClFN3O4 [M-Cl]+, 560.1980; found, 560.1978.
4.1.17. Synthesis of N-3-chlorobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6c)
Compound 6c (214 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 3-chlorobenzyl chloride (198 mg, 1.3 mmol). Yield: 34%; mp: 145–147 ºC; IR (KBr) ν: 2920, 2852
(CH2, CH3), 1631, 1606, 1564, 1504, 1460 (aryl skeleton, C=C), 1397, 1345, 1272, 1227, 1143, 1102,
1
1037, 753 cm-1; H NMR (600 MHz, DMSO-d6) δ: 9.80 (s, 1H, 8-H), 8.97 (s, 1H, 13-H), 8.21 (d, J = 9.1
Hz, 1H, 12-H), 8.06 (d, J = 9.1 Hz, 1H, 11-H), 7.82 (s, 1H, 1-H), 7.74 (d, J = 7.2 Hz, 1H, Bim-H), 7.52 (d,
J = 7.6 Hz, 1H, Bim-H), 7.38 (d, J = 8.2 Hz, 2H, Ph-CH), 7.32–7.26 (m, 2H, Bim-H), 7.24 (d, J = 8.1 Hz,
2H, Ph-CH), 7.13 (s, 1H, 4-H), 6.21 (s, 2H, -OCH2O-), 5.81 (s, 2H, Ph-CH2-), 5.72 (s, 2H, 9-OCH2-), 4.92–
4.85 (m, 2H, 6-H), 3.98 (s, 3H, 10-OCH3), 3.26–3.16 (m, 3H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6)
δ: 151.0, 150.4, 149.9, 148.2, 145.8, 142.4, 141.9, 138.0, 136.3, 135.7, 133.5, 132.6, 131.1, 129.1, 129.1,
127.0, 124.8, 123.7, 122.8, 122.0, 120.8, 120.7, 120.1, 111.5, 108.9, 105.9, 102.6, 67.9, 57.6, 56.0, 46.7,
26.8 ppm; HRMS (TOF): calcd for C34H27Cl2N3O4 [M-Cl]+, 576.1685, found, 576.1684.
4.1.18. Synthesis of N-4-chlorobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6d)
Compound 6d (226 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 4-chlorobenzyl chloride (198 mg, 1.3 mmol). Yield: 36%; mp: 148–150 ºC; IR (KBr) ν: 3031 (Ar-CH),
2925, 2658 (CH2, CH3), 1631, 1604, 1567, 1504, 1480, 1462 (aryl skeleton, C=C), 1395, 1354, 1273,
1
1226, 1143, 1102, 1062, 1037, 963, 938, 879, 809, 752 cm-1; H NMR (600 MHz, DMSO-d6) δ: 9.77 (s,
1H, 8-H), 8.94 (s, 1H, 13-H), 8.18 (d, J = 8.8 Hz, 1H, 12-H), 8.03 (d, J = 8.8 Hz, 1H, 11-H), 7.79 (s, 1H, 1-
H), 7.71 (d, J = 6.5 Hz, 1H, Bim-H), 7.49 (d, J = 6.5 Hz, 1H, Bim-H), 7.35 (d, J = 7.4 Hz, 2H, Ph-CH),
7.30–7.20 (m, 2H, Bim-H), 7.20 (d, J = 7.3 Hz, 2H, Ph-CH), 7.10 (s, 1H, 4-H), 6.18 (s, 2H, -OCH2-), 5.78
(s, 2H, Ph-CH2-), 5.68 (s, 2H, 9-OCH2-), 4.85 (s, 2H, 6-H), 3.94 (s, 3H, -OCH3), 3.18 (s, 2H, 5-H) ppm;
13C NMR (151 MHz, DMSO-d6) δ: 150.9, 150.4, 149.9, 148.2, 145.7, 142.5, 141.8, 138.0, 136.3, 135.7,
133.5, 132.6, 131.0, 129.1, 129.0, 126.9, 124.7, 123.7, 122.7, 121.9, 120.8, 120.7, 120.1, 111.4, 108.9,
18

ACCEPTED MANUSCRIPT
105.9, 102.6, 67.9, 57.6, 55.9, 46.6, 26.8 ppm; HRMS (TOF): calcd for C34H27Cl2N3O4 [M-Cl]+, 576.1685;
found, 576.1687.
4.1.19. Synthesis of N-3-flurobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6e)
Compound 6e (171 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol)
and 3-flurobenzyl chloride (177 mg, 1.3 mmol). Yield: 28%; mp: 176–178 ºC; IR (KBr) ν: 3042 (Ar-CH),
2966, 2657 (CH2, CH3), 1631, 1604, 1566, 1506, 1480, 1454 (aryl skeleton, C=C), 1397, 1356, 1272,
1231, 1140, 1104, 1062, 1035, 962, 935, 911, 877, 798, 752, 681 cm-1; 1H NMR (600 MHz, DMSO-d6) δ:
9.81 (s, 1H, 8-H), 8.94 (s, 1H, 13-H), 8.18 (d, J = 9.1 Hz, 1H, 12-H), 8.03 (d, J = 9.0 Hz, 1H, 11-H), 7.79 (s,
1H, 1-H), 7.71 (d, J = 7.3 Hz, 1H, Bim-H), 7.49 (d, J = 6.8 Hz, 1H, Bim-H), 7.39–7.32 (m, 1H, Ph-CH),
7.26 (s, 2H, Bim-H), 7.12–6.99 (m, 4H, Ph-CH, 4-H), 6.18 (s, 2H, -OCH2O-), 5.80 (s, 2H, Ph-CH2-), 5.69
(s, 2H, 9-O-CH2-), 4.87 (s, 2H, 6-H), 3.93 (s, 3H, -OCH3), 3.18 (s, 2H, 5-H) ppm; 13C NMR (151 MHz,
DMSO-d6) δ: 163.5, 161.9, 151.1, 150.4, 150.0, 148.2, 145.8, 142.6, 141.9, 140.2, 140.2, 138.0, 135.7,
133.5, 131.3, 131.2, 131.1, 127.0, 124.8, 123.7, 123.2, 123.2, 122.7, 122.0, 120.8, 120.7, 120.1, 115.0,
114.8, 114.2, 114.0, 111.4, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 46.8, 26.8 ppm; HRMS (TOF): calcd for
C34H27ClFN3O4 [M-Cl]+, 560.1980; found, 560.1979.
4.1.20. Synthesis of N-4-nitrobenzyl-2-berberinoxymethoxy-1H-benzo[d]imidazole (6f)
Compound 6f (230 mg) was prepared as yellow solid according to the procedure described for compound
5a starting from compound 4a (500 mg, 1.0 mmol), potassium carbonate (110 mg, 0.8 mmol) and 4-
nitrobenzyl bromide (266 mg, 1.3 mmol). Yield: 36%; mp: 145–147 ºC; IR (KBr) ν: 2924, 2853, 2658
(CH2, CH3), 1631, 1605, 1567, 1505, 1462 (aryl skeleton, C=C), 1395, 1346, 1271, 1226, 1142, 1104,
1062, 1036, 964, 754 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 9.76 (s, 1H, 8-H), 8.93 (s, 1H, 13-H), 8.18–
8.11 (m, 8.9 Hz, 3H, Ph-CH, Bim-H), 8.01 (d, J = 9.1 Hz, 1H, Bim-H), 7.78 (s, 1H, 1-H), 7.76–7.71 (m, 1H,
Ph-CH), 7.50–7.45 (m, 1H, Ph-CH), 7.40 (d, J = 8.6 Hz, 2H, Bim-H), 7.30–7.25 (m, 2H, 12-H, 11-H), 7.10
(s, 1H, 4-H), 6.18 (s, 2H, -OCH2O-), 5.96 (s, 2H, Ph-CH2-), 5.69 (s, 2H, 9-OCH2-), 4.91–4.83 (m, 2H, 6-H),
3.93 (s, 3H, -OCH3), 3.22–3.14 (m, 2H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 150.9, 150.4, 150.0,
148.2, 147.3, 145.7, 145.1, 142.6, 141.8, 138.0, 135.7, 133.5, 131.1, 128.3, 127.0, 124.8, 124.2, 123.9,
122.9, 121.9, 120.8, 120.7, 120.2, 111.3, 108.9, 105.9, 102.6, 68.0, 57.6, 56.0, 46.8, 26.8 ppm; HRMS
(TOF): calcd for C34H27ClN4O6 [M-Cl]+, 587.1925; found, 587.1925.
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ACCEPTED MANUSCRIPT
4.1.21. Synthesis of N-2,4-dichlorobenzyl-2-berberinoxymethoxy-1H-enzo[d]imidazole (6g)
Compound 6g (240 mg) was prepared as yellow solid according to the procedure described for
compound 5a starting from compound 4a (500 mg, 1.0 mmol) , potassium carbonate (110 mg, 0.8 mmol)
and 2,4-dichlorobenzyl chloride (240 mg, 1.3 mmol). Yield: 36%; mp: 165–167 ºC; IR (KBr) ν: 2907, 2847,
2658 (CH2, CH3), 1631, 1602 , 1504, 1476, 1460, 1422 (aryl skeleton, C=C), 1393, 1357, 1270, 1226,
1166, 1142, 1103, 1037, 964, 874, 834, 746, 653 cm-1; 1H NMR (600 MHz, DMSO-d6) δ: 9.75 (s, 1H, 8-H),
8.93 (s, 1H, 13-H), 8.16 (d, J = 9.2 Hz, 1H, 12-H), 8.00 (d, J = 9.1 Hz, 1H, 11-H), 7.79 (s, 1H, 1-H), 7.75–
7.74 (m, 1H, Ph-CH), 7.68 (d, J = 2.0 Hz, 1H, Bim-H), 7.43–7.42 (m, 1H, Bim-H), 7.28–7.21 (m, 2H, Bim-
H), 7.23–7.21 (m, 1H, Ph-CH), 7.10 (s, 1H, 4-H), 6.55 (d, J = 8.5 Hz, 1H, Ph-CH), 6.18 (s, 2H, -OCH2O-),
5.81 (s, 2H, Ph-CH2-), 5.65 (s, 2H, 9-OCH2-), 4.86 (t, J = 6.2 Hz, 2H, 6-H), 3.93 (s, 3H, -OCH3), 3.19–3.16
(m, 2H, 5-H) ppm; 13C NMR (151 MHz, DMSO-d6) δ: 150.8, 150.4, 150.2, 148.2, 145.7, 142.5, 141.7,
138.0, 135.8, 133.7, 133.5, 133.4, 133.0, 131.1, 129.5, 129.3, 128.2, 126.9, 124.7, 123.9, 122.9, 121.9,
120.8, 120.7, 120.2, 111.3, 108.9, 105.9, 102.6, 67.9, 57.6, 55.9, 45.0, 40.6, 40.5, 40.4, 40.2, 40.1, 39.9,
39.8, 39.7, 26.8 ppm; HRMS (TOF): calcd for C34H26Cl3N3O4 [M-Cl]+, 610.1295, found, 610.1294.
Acknowledgement
This work was partially supported by National Natural Science Foundation of China [(No. 21172181,
21372186), the Research Fund for International Young Scientists from International (Regional)
Cooperation and Exchange Program (No. 81350110523, 81450110094)], the Key Program of Natural
Science Foundation of Chongqing (CSTC2012jjB10026), the Specialized Research Fund for the Doctoral
Program of Higher Education of China (SRFDP 20110182110007), and the Chongqing Special Foundation
for Postdoctoral Research Pro-posal (Xm2014127).
Supplementary data
Spectral data of all the compounds associated with this article is available as supplementary data
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for Clinical Laboratory Standards, Wayne, PA, (2002).
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Figure and Table Captions
Figure 1 Structures of some reported antimicrobial compounds.
Scheme 1 Synthesis of berberine-benzimidazole hybrids.
Table 1 Antimicrobial data as MIC (µg/mL) for berberine-benzimidazoles 4a-c, 5a-i and 6a-g
Figure 2 In vitro antibacterial activity for compounds 4b, 5d, 5f, 5g, 6b, 6d and chloromycin expressed as
MIC (µg/mL); S. typhi, Salmonella typhi; S. aureus, Staphylococcus aureus ATCC25923.
Figure 3 In vitro antifungal activity for compounds 5c, 5d, 5f, 6a, 6b, 6d, 6e, 6g and Fluconazole
expressed as MIC (µg/mL); C. utilis, Candida utilis; A. flavus, Aspergillus flavus.
Figure 4 Mechanism of antibacterial action of compound 5d at concentrations of 12 × MIC: membrane
permeabilization of E. coli DH52.
Figure 5 Mechanism of antibacterial action of compound 5d at concentrations of 12 × MIC: membrane
permeabilization of MRSA.
Figure 6 Absorption spectra of DNA with different concentrations of compound 5d (pH = 7.4, T = 298 K).
Inset: comparison of absorption at 260 nm between the compound 5d-DNA complex and the sum values of
free DNA and free compound. c(DNA) = 5.44 × 10-5 mol/L, and c(compound 5d) = 0 – 1.33× 10-5 mol/L
for curves a–i respectively at increment 0.17 × 10-5.
Figure 7 The plot of A0/ (A–A0) versus 1/[compound 5d].
Figure 8 UV absorption spectra of NR in the presence of DNA at pH 7.4 and room temperature. c(NR) = 2
×10-5 mol/L, and c(DNA) = 0–3.81 × 10-5 mol/L for curves a−i respectively at increment 0.48 × 10-5.
Figure 9 Absorption spectra of the competitive reaction between compound 5d and NR with DNA:
c(DNA) = 5.44 × 10-5 mol/L, c(NR) = 2 × 10-5 mol/L, and c(compound) = 0–1.99 × 10-5 mol/L for curves
a–i respectively at increment 0.25 × 10-5. (Inset) Absorption spectra of the system with the increasing
concentration of compound 5d in the wavelength range of 380–600 nm.
Figure 10 Effect of compound 5d to HSA UV-vis absorption, c(HSA) = 1.0 x 10-5 mol/L; c(compound
5d)/(10-5 mol/L): 0−8 × 10-5 mol/L (T = 298 K, pH = 7.4). The inset corresponds to the absorbance at 279
nm with different concentrations of compound 5d.
Figure 11 Binding mode of compound 5d to HSA
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Figure 1 Structures of some reported antimicrobial compounds.
25

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Scheme 1 Synthesis of berberine-benzimidazole hybrids.
Reagents and conditions: (i) 190 °C/vacuum, ethyl alcohol/concentrated hydrochloric acid; (ii) potassium
hydroxide, DMF, TBAI, chloroacetic acid, 60 °C; (iii) 4 mol/L hydrochloric acid, substituted benzene-
1,2-diamines, 110 °C; (iv) potassium carbonate, DMF, alkyl bromide, 60 °C; (v) potassium carbonate,
DMF, halobenzyl halide, 60 °C.
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Table 1 Antimicrobial data as MIC (µg/mL) for berberine-benzimidazoles 4(a-c), 5(a-i) and 6(a-g)a-d
Gram-Positive bacteria
Gram-Negative bacteria
Antifungal
C. S.
P.
Compds
S.
B.
M.
B.
E.
S.
C.
A.
MRSA
aerugino
coli
aureus
subtilis luteus proteus
typhi albicans utilis cerevisiae flavus
sa
4a
4b
4c
5a
5b
5c
5d
5e
5f
512
8
256
512
256
128
4
512
256
512
512
64
32
16
128
64
32
32
64
64
32
128
128
512
512
8
256
16
512
32
256
256
512
512
64
16
16
64
512
4
256
512
512
512
64
64
64
256
128
16
8
512
256
512
512
512
512
512
512
512
512
512
512
256
128
512
512
256
128
512
-
32
32
32
256
16
16
32
256
8
64
512
128
64
4
32
512
64
512
256
128
512
512
512
512
512
64
256
128
4
16
32
64
4
32
8
32
16
2
4
8
8
64
8
512
32
8
32
2
16
32
8
16
8
16
32
16
8
5g
5h
5i
128
256
512
512
512
512
512
512
512
4
512
512
512
512
256
512
512
512
512
512
32
16
512
128
512
512
512
256
512
256
512
32
8
16
16
128
128
8
128
128
512
16
8
256
512
512
2
64
128
256
8
64
512
512
128
512
2
512
512
512
512
512
512
128
512
-
6a
6b
6c
6d
6e
6f
32
128
128
8
4
4
512
32
512
64
64
16
2
16
8
256
2
16
4
32
4
16
32
16
8
128
32
512
32
256
256
16
32
4
8
32
64
16
-
32
2
6g
A
8
16
16
32
32
32
-
B
8
4
2
8
1
16
-
-
-
-
C
-
-
-
-
-
-
-
-
1
8
16
256
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b S. aureus, Staphylococcus aureus ATCC25923; MRSA, Methicillin-Resistant Staphylococcus aureus N315; B. subtilis,
Bacillus subtilis; M. luteus, Micrococcus luteus ATCC4698; B. proteus, Bacillus proteus ATCC13315; E. coli, Escherichia
coli JM109; P. aeruginosa, Pseudomonas aeruginosa; S. typhi, Salmonella typhi.
c C. albicans, Candida albicans ATCC76615; C. utilis, Candida utilis; S. cerevisia, Saccharomyces cerevisia; A. flavus,
Aspergillus flavus.
d A = Chloromycin; B = Norfloxacin; C = Fluconazole
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Figure 2 In vitro antibacterial activity for compounds 4b, 5d, 5f, 5g, 6b, 6d and chloromycin expressed as
MIC (µg/mL); S. typhi, Salmonella typhi; S. aureus, Staphylococcus aureus ATCC25923.
Antibacterial Results
30
25
S.typhi
S.aureus
20
15
10
5
0
4b
5d
5f
5g
6b
6d Chloromycin
Compounds
28