3. . . . . . . . . . . . . . . . . . . . . . . . . . . Zhang et al. Sci China Chem May (2018) Vol.61 No.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
hertz (Hz). The following abbreviations were used to des-
ignate fragments: Ber=berberine, Im=nitroimidaozle, Ph=
phenyl. The high resolution mass spectra (HRMS) were
achieved by IonSpec FTICR mass spectrometer (Waters
Micromass, USA) using ESI resource. All fluorescence
spectra were recorded on F-7000 spectrofluorimeter (Hi-
tachi, Japan) and UV spectra were obtained through TU-
2450 spectrophotometer (Puxi Analytic Instrument Ltd. of
Beijing, China), both of which were equipped with 1.0 cm
quartz cells. The microbial strains were obtained from Si-
chuan Provincial People’s Hospital (Chengdu, China) and
neutral red (NR) was obtained from Sigma-Aldrich (USA).
The RAW 264.7 cells were obtained from Kunming wildlife
cell bank of the Chinese Academy of Sciences, Kunming,
China. Tris(hydroxymethyl)methylamine, sodium chloride
and hydrochloric acid were analytical purity. The masses of
samples were weighed using microbalance with a resolution
of 0.1 mg. All other chemicals and solvents were commer-
cially available and directly used without further purifica-
tion.
rially diluted. RAW 264.7 cells were grown as monolayer in
medium (90% DMEM medium, 10% fetal bovine serum, 1%
penicillin/streptomycin) and maintained at 37 °C in a hu-
midified atmosphere containing 5% CO2. Cells were de-
tached from culture flasks with 0.25% trypsin and 0.03%
ethylene diamine tetraacetic acid (EDTA) and resuspended
in fresh culture medium at a density of 1.5×105 cells/mL. By
using a Falcon 96-well, flat-bottom plate, 100 μL of the cell
suspension was added to each of the wells and the cells were
incubated for 24 h. The tested cells were treated with com-
pound 8f and berberine in triplicate at concentrations of 8,
16, 32, 64, 128, 256, and 512 μg/mL, respectively. After
incubation with compounds for 72 h, 50 μL of a 2.5 mg/mL
solution of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
tetrazolium bromide (MTT) in phosphate-buffered saline
(PBS) was added to each well and further incubated for
3–4 h. The supernatant was removed, and the cells were
dissolved in 150 μL of DMSO. Absorbance values were
measured at 490 nm by microplate reader.
2.5 Resistance study
2.2 Synthesis
The representative compound 8f was selected to investigate
the developing rate of bacterial resistance according to the
reported method. We exposed a standard strain of resistant E.
coli toward increasing concentrations of compound 8f from
sub-MIC (0.5×MIC, MIC is the minimal inhibitory con-
centration) for sustained passages and determined the new
MIC values of compound 8f for each passage of E. coli. The
initial MIC value of compound 8f and norfloxacin was de-
termined against E. coli as mentioned above in antibacterial
assay. For the next MIC experiment, the bacterial dilution
was prepared by using the bacteria from sub-MIC con-
centration of the compound (0.5×MIC). After a 12 h in-
cubation period, again bacterial dilution was prepared by
using the bacterial suspension from sub-MIC concentration
of the compound (0.5×MIC) and assayed for the next MIC
experiment. The process was repeated for 15 passages. The
MIC values for compound 8f against each passage of E. coli
were determined.
The detailed synthetic procedures and spectral data for
compounds 6–8 were available in the Supporting Informa-
tion online.
2.3 Antibacterial assay
The berberine-derived nitroimidazoles 6–8 were evaluated
for their antibacterial activities against six Gram-positive
bacteria (Methicillin-Resistant Staphylococcus aureus N315,
Enterococcus faecalis, Staphylococcus aureus, Staphylo-
coccus aureus ATCC25923, Bacillus subtilis ATCC6633,
Micrococcus luteus ATCC4698) and six Gram-negative
bacteria (Klebsiella pneumoniae, Escherichia coli, Escher-
ichia coli ATCC25922, Pseudomonas aeruginosa, Pseudo-
monas aeruginosa ATCC27853, Acinetobacter baumannii)
including drug-resistant strains (without ATCC number)
isolated from infected patients. The bacterial suspension was
adjusted with sterile saline to a concentration of 1×105 CFU
measured by nephelometer. Initially the compounds were
dissolved in dimethyl sulfoxide (DMSO) to prepare the stock
solutions, then the tested compounds and reference drugs
were prepared in Mueller-Hinton broth (Guangdong Huaikai
Microbial Sci. & Tech Co., Ltd, Guangzhou, China) to obtain
the required concentrations. These dilutions were inoculated
and incubated at 37 °C for 24 h.
3 Results and discussion
3.1 Chemistry
The berberine-derived nitroimidazoles 6–8 were synthesized
via multi-step reactions from natural berberine as outlined in
Scheme 1. Initially, berberrubine 1 was easily obtained
through selective demethylation of commercial berberine in
high yield of 89.8%, and the latter was further reduced by
excessive sodium borohydride to generate structurally ben-
ded derivative 2 with the yield of 57.0%. Compound 2 was
further formylated by hexamethylenetetramine (HMTA)
2.4 Cytotoxicity assay
The stock solutions of compound 8f and berberine
(1024 μg/mL, in DMSO) were prepared in medium and se-