Synthesis and bioactive evaluation of novel hybrids of metronidazole and berberine as new type of antimicrobial agents and their transportation behavior by human serum albumin

Article abstract of DOI:10.1016/j.bmc.2013.05.007

A series of novel hybrids of metronidazole and berberine as new type of antimicrobial agents were synthesized and characterized by ¹H NMR, ¹³C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that most of the prepared compounds exhibited effective antibacterial and antifungal activities and some showed comparable or superior potency against Methicillin-resistant Staphylococcus aureus to reference drugs Norfloxacin, Chloromycin and Berberine. The transportation behavior of human serum albumin (HSA) to the highly active compound 5g was evaluated and revealed that the association of imidazole derivative 5g with HSA was spontaneous and the electrostatic interactions played important roles in the transportation of HSA to 5g. The calculated parameters indicated that compound 5g could be effectively stored and carried by HSA.

Full text of DOI:10.1016/j.bmc.2013.05.007

Bioorganic & Medicinal Chemistry 21 (2013) 4158–4169
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioactive evaluation of novel hybrids of metronidazole
and berberine as new type of antimicrobial agents and their
transportation behavior by human serum albumin
⇑
⇑
Ling Zhang, Juan-Juan Chang, Shao-Lin Zhang, Guri L. V. Damu , Rong-Xia Geng , Cheng-He Zhou
Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel hybrids of metronidazole and berberine as new type of antimicrobial agents were syn-
thesized and characterized by 1H NMR, 13C NMR, IR, MS and HRMS spectra. Bioactive assay manifested
that most of the prepared compounds exhibited effective antibacterial and antifungal activities and some
showed comparable or superior potency against Methicillin-resistant Staphylococcus aureus to reference
drugs Norfloxacin, Chloromycin and Berberine. The transportation behavior of human serum albumin
(HSA) to the highly active compound 5g was evaluated and revealed that the association of imidazole
derivative 5g with HSA was spontaneous and the electrostatic interactions played important roles in
the transportation of HSA to 5g. The calculated parameters indicated that compound 5g could be effec-
tively stored and carried by HSA.
Received 30 March 2013
Revised 6 May 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
Imidazole
Metronidazole
Berberine
Antibacterial
Antifungal
Ó 2013 Elsevier Ltd. All rights reserved.
Human serum albumin
1. Introduction
tozoa such as Giardia, Lamblia, and Entomoeba histolytic.15 How-
ever, with the extensive investigations towards nitroimidazoles,
Imidazole derivatives have been attracting increasing interest
due to their large potentiality in medicinal chemistry.1–6 Nitro
modified imidazoles are a unique type of imidazole derivatives in
which nitro fragment could not only enhance lipophilicity of the
target compounds which is favorable for tissue penetration, but
also induce bioactivities through the metabolic activation of nitro
group.7 Nitroimidazole-based compounds exhibited various bioac-
tivities like antibacterial,8,9 anti-tubercular,10 antiparasitic11 and
anticancer12,13 ones. Especially, as antimicrobial agents, many
nitroimidazoles such as metronidazole, benznidazole, ornidazole,
secnidazole, nimorazole and tinidazole have been in widespread
clinical use to treat diseases caused by anaerobic bacteria.4,14 Nota-
bly, despite of their long term clinical use, the incidence of resis-
tance in anaerobic bacteria is still very low. This encourages
continuous researches to focus on the development of such nitro-
imidazoles with potential medicinal application. Particularly,
structurally simple metronidazole as an effective synthetic drug
introduced in 1960 possesses strong inhibitory efficacies against
Gram-negative anaerobic bacteria like Helicobacter pylori and pro-
researches disclosed that the reactive intermediates formed in
microorganisms by the reduction of nitro group in nitroimidazoles
could covalently bind with DNA and trigger the adverse effect. The
sterical protection of nitro group in metronidazole was proved to
be an effective way to improve the metabolism and physicochem-
ical property of such compounds.16 Furthermore, some frequently
used clinical antimicrobial nitroimidazole drugs like nitroimida-
zooxazine PA-824 were reported to be subjected to poor aqueous
solubility and unsuitable binding propensity to proteins in human
plasma (Fig. 1).17–19 Therefore, the development of new types of
nitroimidazoles with broad antimicrobial spectrum, good metabo-
lism and physicochemical property, and suitable binding affinity to
serum albumins has been recently attracting special attention.
Much effort has been oriented towards novel nitroimidazole-based
antimicrobial agents with high efficiency.
Berberine has been commonly used in the clinic as therapeutic
agent to treat infectious diseases such as acute gastroenteritis,
cholera and bacillary dysentery for many years. Its importantly
clinical uses stimulate the continuing researches in antimicrobial
field20,21 and other related investigations such as anticancer,22
antiviral,23 antiinflammatory,24 antiparasitic25,26 activities. The
special structure of berberine with a quaternary nitrogen and large
⇑
Corresponding authors. Tel./fax: +86 23 68254967.
E-mail addresses: geng0712@swu.edu.cn (R.-X. Geng), zhouch@swu.edu.cn
(C.-H. Zhou).
desirable
like
could improve the physicochemical properties of its derivatives,
p
-conjugated backbone could exert noncovalent forces
p–p stacking and electronic interactions. Thereby, it not only
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT),
Hyderabad 500 607, India.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.007

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4159
O
attention for that they could deliver drugs or other bioactive small
molecules to the binding sites.30,31 A thorough binding analysis be-
tween drugs or bioactive small molecules and human serum albu-
min (HSA) may beneficially provide useful information for the
absorption, transportation, distribution, metabolism and excretion
properties of drugs. It might also be significant to the design, mod-
ification and screening of drug molecules. In view of above obser-
vations, it is considerably reasonable for us to further investigate
the transportation behavior by HSA to the highly active prepared
compounds in order to preliminarily evaluate their transportation
and pharmacokinetic properties by fluorescence and UV–vis
absorption spectroscopy on molecular level.
N
O
N
NO2
F3CO
Nitroimidazooxazine (PA-824)
Figure 1. Structure of nitroimidazooxazine (PA-824).
their affinity to transport proteins, and thus enhance their antimi-
crobial activities, but also easily interact with diverse enzymes and
receptors in biological system resulting in broad bioactive spec-
trum. In our previous work,27 a type of amine-derived bis-azoles
has been successfully developed as newly structural Fluconazole
analogues. The tertiary alcohol moiety in Fluconazole was replaced
by a tertiary amino group as bioisoster, and the methylene bridge
between the tertiary alcohol and the triazolyl moieties was substi-
tuted by an ethylene chain (Fig. 2). Bioactive study manifested that
this type of compounds exhibited good or even better antimicro-
bial activities in comparison to reference drugs Chloromycin, Nor-
floxacin and Fluconazole. Here, we would like to replace the azolyl
ethylamino moiety with the clinical metronidazole as bioisoster
fragment and further substitute the other azolyl ring by the berber-
ine skeleton to generate a series of novel hybrids of berberine and
nitroimidazoles as potential antimicrobial agents. This special type
of hybrids might not only be helpful to spatially protect the nitro
group with the potential to improve the metabolism and physico-
chemical property, but also improve the water solubility and bind-
ing affinity by the introduction of tertiary amine moiety thereby
effectively increase their biological activities and broaden active
spectrum. In addition, various halobenzyl moieties were intro-
duced into target compounds to investigate the effect of haloben-
zyl moiety on biological activities, since many halobenzyl
incorporated molecules gave good antimicrobial activities.28,29
The designed structures of this series of novel hybrids of berberine
and nitroimidazole moiety including 4-nitroimidazole, 5-nitroimi-
dazole and 2-methyl-5-nitroimidazole derivatives are shown in
Scheme 1.
2. Results and discussion
2.1. Chemistry
The target hybrids of berberine nitroimidazoles were prepared
from commercial halobenzyl chlorides, diethanolamine and berb-
errubine (Scheme 1). Diethanolamine was N-alkylated with differ-
ent halobenzyl chlorides 1a–e to produce intermediates 2a–e in
excellent yields (92.2–97.8%). The resulting diols 2a–e were
treated in chloroform with phosphorus tribromide to afford
dibromides 3a–e in good yields ranging from 88.3% to 97.4%.27
Further reactions of compounds 3a–e with 4-nitroimidazole
afforded the corresponding mono-azole bromides 4b–k in 18.5–
80.2% yields. Possible reactions of 4-nitroimidazole in the pres-
ence of potassium carbonate are shown in Figure 3. It has been
documented that tautomeric interconversion of the 5-nitro and
4-nitro imidazoles takes place under either acidic or basic condi-
tions. During the N-alkylations of 4-nitroimidazole with alkyl
halides, the acidic conditions favored the 5-nitro orientation
while basic conditions favored the 4-nitro orientation.32 In this
work, potassium carbonate was selected to produce our target
compounds, since the yields were generally poor under acidic
conditions. In the presence of potassium carbonate, 4-nitroimi-
dazole displays four resonance forms (A–D) (Fig. 3). Since
patterns A and C are much more stable than patterns B and D,
the N-alkylation of 4-nitroimidazole could yield N-1 and N-3
alkylated products, and the former is the predominant one due
Serum albumins as the most important and abundant macro-
molecule proteins in the circulatory system have received much
Z
N
NO2
OH
O
O
Z
N
N
N
N
Cl
N
N
H3C
H3CO
H3CO
N
X3
Metronidazole
Z = CH, N
X1, X2, X3 = H, F, Cl
Berberine
X1
X2
The reported tertiary amine bis-azoles
R2
R3
N
N
Target compounds
R1
N
NO2
O
H3C
N
O
N
O
O
O
N
X3
N
O
N
X3
Cl
X2
X1
Cl
X2
X1
H3CO
R1, R2, R3 = H, NO2, CH3
H3CO
Figure 2. Design of novel hybrids of berberine and nitroimidazoles.

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L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
X2
X1
X2
X1
X2
X1
X2
X1
Br
Br
OH
X3
X3
X3
X3
I
III
II
N
N
N
Cl
R2
1a-e
2a-e
3a-e
Br
N
N
OH
R3
4a-k
R1
1
1
1-3 a
b
:
, X = F, X2 = H, X3 = F; , X = H, X2 = Cl, X3 = Cl
IV
c, X1 = Cl, X2 = H, X3 = H; d, X1 = H, X2 = Cl, X3 = H
e, X1 = H, X2 = H, X3 = Cl
X3
R2
N
R1
X2
X1
OCH3
OCH3
OCH3
OH
N
O
O
V
R3
N
N
O
O
Cl
O
O
Cl
N
Cl
N
O
6
7
5a-k
OCH3
4-5
:
a, X1 = F, X2 = H, X3 = F, R1 = H,
R2 = NO2, R3 = CH3; b, X1 = F, X2 = H, X3 = F, R1 = NO2, R2 = H, R3 = H
, X1 = H, X2 = Cl, X3 = Cl, R1 = NO2, R2 = H, R3 = H;
, X1 = H, X2 = Cl, X3 = H, R1 = NO2, R2 = H, R3 = H;
, X1 = Cl, X2 = H, X3 = H, R1 = NO2, R2 = H, R3 = H
c
e
d
f
1
, X = H, X2 = H, X3 = Cl, R1 = NO2, R2 = H, R3 = H
g, X1 = F, X2 = H, X3 = F, R1 = H,
R2 = NO2, R3 = H; h, X1 = H, X2 = Cl, X3 = Cl, R1 = H, R2 = NO2, R3 = H
, X1 = Cl, X2 = H, X3 = H, R1 = H,
R2 = NO2, R3 = H;
R2 = NO2, R3 = H
, X1 = H, X2 = Cl, X3 = H, R1 = H, R2 = NO2, R3 = H
j
i
, X1 = H, X2 = H, X3 = Cl, R1 = H,
k
Scheme 1. Synthetic route of berberine nitroimidazoles. Reagents and conditions: (I) CH3CN, diethanolamine, 50 °C, 10–12 h, yields 92.3–98.6%; (II) CHCl3, PBr3, refluxed, 2 h,
yields 88.1–98.5%; (III) 4-nitroimidazole or 2-methyl-5-nitroimidazole, K2CO3, CH3CN, 60 °C, 10–12 h, yields 18.4–80.2%; (IV) berberrubine, DMF, 110 °C, 20 h, yields 23.0–
38.7%; (V) berberine, 20 mm Hg, 190 °C, 15 min, yield 88.4%.
NO2
NO2
NO2
NO2
NO2
K2CO3
HN
N
HN
N
N
HN
N
C
N
N
N
A
D
B
4-nitroimidazole
RX
RX
R
N
N R
NO2
N
N
NO2
main product
minor product
Figure 3. Possible reactions of 4-nitroimidazole in the presence of potassium carbonate.
to the relative low acidity of –NH– proton compared to
corresponding compounds 4a–k in DMF at 110 °C for 20 h in prac-
5-nitroimidazole. Actually, synthesis of 5-nitroimidazole deriva-
tives has rarely been reported,33,34 which is probably due to the
nonavailability of 5-nitroimidazole. In the preparations of 4-nitro-
imidazoles 4b–f, 5-nitroimidazole derivatives 4g–k were obtained
as by-products. As for 2-methyl-5-nitroimidazole, the presence of
the methyl substituent at C-2 possibly reduces the acidity of the –
NH– protons induced by the nitro group at the C-5 position.
Therefore, the reaction of compound 3a with 2-methyl-5-nitroim-
idazole afforded a single product 4a.
tical yields (23.0–38.7%).
2.2. Spectral analysis
All newly synthesized compounds were characterized by 1H
NMR, 13C NMR, IR, MS and HRMS spectra. The spectral analyses
were in accordance with the assigned structures, and listed in
the experimental section.
Generally, there are two kinds of reported methods for demeth-
ylation of berberine chloride including fusion with pyridine hydro-
chloride and thermal decomposition. The first method was lack of
selectivity, while the second one could selectively and conve-
niently produce demethylation at 9-position. Possibly, under the
reduced pressure (20 mm Hg) at 190 °C, berberine could generate
the ketoform intermediate, which could be easily converted into
berberrubine in the presence of hydrochloric acid.35–37 In this
work, berberrubine 7 was prepared in 88.4% yield by demethyla-
tion of berberine chloride 6 at 190 °C under reduced pressure
(20 mm Hg) for 15 min.
2.2.1. IR spectra
In IR spectra, compounds 4a–k gave moderate absorption bands
at 3085–3014 cmꢀ1 and 2971–2821 cmꢀ1 attributed to the stretch-
ing vibration of aromatic and aliphatic C–H, respectively. While the
aromatic frame exhibited characteristic stretching frequencies in
the region between 1603 and 1482 cmꢀ1. All target compounds
5a–k gave moderate absorption at 3152–3011 cmꢀ1 due to the
stretching vibration of aromatic C–H. The absorption bands as-
cribed to aliphatic C–H stretching vibrations were seen at two re-
gions of 2972–2921 and 2857–2816 cmꢀ1, while the aromatic
frame exhibited characteristic stretching frequencies in the region
of 1601–1474 cmꢀ1. Furthermore, two strong and sharp peaks at
1570–1528 and 1350–1341 cmꢀ1 in compounds 5a–k were
Finally, target nitroimidazoles 5a–k were conveniently
obtained by the alkylation of berberrubine 7 with a series of

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4161
attributed to the vibration of N@O bond. All the other absorption
bands were also observed at expected regions.
the lowest concentration of new compounds that completely
inhibited the growth of bacteria. Currently available antimicrobial
drugs such as Berberine, Chloromycin, Norfloxacin and Fluconazole
were used as standard drugs.
2.2.2. 1H NMR spectra
In 1H NMR spectra, for compounds 4b–k, it is expected that
chemical shift values of Hc proton in 5-nitroimidazole derivatives
4g–k (Table 1) should be more downfield, due to the adjacence of
strong electron-withdrawing nitro group, than the 4-nitroimidaz-
oles 4b–f. In this case, protons at 4.39–4.31 ppm were attributed
to the Hc protons of 5-nitroimidazoles 4g–k, while compounds with
similar peak at 4.06–3.98 ppm were identified as the 4-nitroimidaz-
ole isomers 4b–f. Additional evidence supporting the present struc-
tural assignment can be obtained from the chemical shift values of
Ha and Hd protons. The Hd protons between the nitro group and a sp2
nitrogen in 5-nitro isomers 4g–k (7.92–7.84 ppm) were expected to
be more downfield than the Ha protons (7.75–7.67 ppm) placed be-
tween the nitro group and a sp3 nitrogen in the 4-nitro isomers 4b–f.
In the 1H NMR spectra of compounds 5b–k, 4-nitroimidazoles 5b–f
(8.19–7.83 ppm) gave higher shifts for Ha when compared with pre-
cursors 4b–f (7.75–7.67 ppm), while 5-nitroimidazoles 5g–k (7.89–
7.79 ppm) displayed relatively lower shifts for Hd than correspond-
ing 4g–k (7.92–7.84 ppm).
2.3.1. Antimicrobial activity
The antimicrobial results as shown in Table 2 revealed that
most of the prepared compounds 4 and 5 could effectively inhibit
the growth of all tested strains in vitro. For the prepared nitroim-
idazoles 4a–k, the tested strains were quite sensitive to com-
pounds 4g and 4i with MIC values of 1–32
difluorobenzyl derivative 4g gave low inhibitory concentration
(MIC = 8 g/mL) towards P. aeruginosa, which was two times more
active than clinical drug Clinafloxacin (MIC = 16 g/mL). The anti-
C. mycoderma activity (MIC = 4 g/mL) also was comparable to ref-
erence drug Fluconazole (MIC = 4 g/mL).
lg/mL. Especially, 2,4-
l
l
l
l
In comparison to their precursors 4a–k, compounds 5a–k dis-
played stronger antimicrobial efficacy and broader antimicrobial
spectrum. Notably, some synthesized nitroimidazoles showed
superior potency against MRSA to reference drugs Norfloxacin,
Chloromycin and Berberine. Especially, 2,4-difluorobenzyl deriva-
tive 5g and 2-chlorophenyl compound 5i exhibited noticeable
and broad spectrum antimicrobial activities against all the tested
2.2.3. 13C NMR spectra
bacteria and fungi with MIC values of 1–32
teria MRSA was sensitive to the target compounds 5g and 5i with
MIC values of 16 and 8 g/mL, respectively, which were compara-
ble to reference drugs Chloromycin (MIC = 16 g/mL) and Norflox-
acin (MIC = 8 g/mL). Particularly, compound 5g gave low
inhibitory concentration towards S. dysenteriae and P. vulgaris with
MIC values of 4 g/mL, which were comparable to or even better
than the reference drugs Chloromycin, Berberine and Norfloxacin.
The anti-C. mycoderma activity (MIC = 1 g/mL) also was fourfold
more potent than the reference drug Fluconazole (MIC = 4 g/
mL). In addition, compound 5i also exhibited good activity against
S. aureus with MIC value of 2 g/mL, which was eightfold and 256-
lg/mL. Excitedly, bac-
The 13C NMR spectral analyses were in accordance with the as-
signed structures. No large differences were found in 13C chemical
shifts after the conversions of compounds 4a–k into nitroimidaz-
oles 5a–k. The signals at 162.7–157.9 and 163.8–150.5 ppm in
compounds 4a–k and 5a–k were assigned to the halo attached car-
bons. The carbons connected with nitro group in compounds 4 and
5 gave relative high signals at d 139.1–135.5 ppm and the 13C
chemical shifts of other carbons in nitroimidazole ring were ob-
served at d 136.0–131.0 ppm, while all the other carbons gave
13C peaks at the expected regions.
l
l
l
l
l
l
l
2.3. Biological activity
fold more potent than Chloromycin and Berberine, respectively. It
could also remarkably inhibit the growth of B. subtilis and E. coli
with MIC values of 4 lg/mL, which were fourfold more potent than
All newly synthesized compounds 4a–k and 5a–k and their pre-
cursors were evaluated for in vitro antibacterial and antifungal
activities against four Gram-positive bacteria (Staphylococcus aur-
eus ATCC25923, Methicillin-resistant S. aureus N315, Bacillus subtilis
ATCC6633 and Micrococcus luteus ATCC4698), four Gram-negative
bacteria (Escherichia coli DH52, Proteus vulgaris ATCC6896, Pseudo-
monas aeruginosa and Shigella dysenteriae) and two fungal strains
(Candida albicans ATCC76615 and Candida mycoderma) using two-
fold broth dilution method in 96-well microtest plates recom-
mended by National Committee for Clinical Laboratory Standards
(NCCLS).38 The biological tests were carried out in triplicate.
Chloromycin. These antimicrobial results manifested that the
incorporation of berberine moiety into target compounds should
be beneficial for the antibacterial and antifungal potency. To our
surprise, generally all 4-nitroimidazoles 5b–f displayed lower anti-
microbial activities than that of 5-nitroimidazoles 5g–k.
In view of above discussion, the antimicrobial efficacies should
be closely related to nitroimidazole ring and halobenzyl group to
some extent. For this serial compounds, nitroimidazoly moieties
contributed to the antibacterial activities in the order of 5-niotro-
imidazoly >4-nitroimidazoly. Difluorobenzyl group was more
helpful for increasing antibacterial and antifungal efficacy in
comparison to other halobenzyl ones.39
Minimal inhibitory concentration (MIC,
lg/mL) was defined as
Table 1
Some 1H NMR data (d ppm) of 4-nitroimidazole and 5-nitroimidazole derivatives 4b–
k
2.3.2. Analysis of LogP values
Lipophilicity/hydrophilicity governs various biological pro-
cesses such as the transportation, distribution, metabolism and
secretion of biological molecules. A good knowledge of the lipo-
philicity/hydrophilicity is essential to predict the transportation
and activity of drugs. Therefore, the lipophilicity/hydrophilicity ex-
pressed as octanol/water partition coefficient (LogP) was deter-
mined experimentally by traditional saturation shake flask
method combining with UV–vis spectrophotometric approach
(Supplementary data). According to the Lipinski’s rule-of-five, all
the synthesized compounds 5a–k had desirable LogP values (no
more than five) as given in Table 3. They had suitable lipophilicity
that was favorable for them to permeate through biological mem-
brane and to be delivered to the binding sites.
Br
Hc
Br
Hc
Hb
Hb
N
N
N
N
X1
X2
X1
X2
N
N
Ha
O2N
NO2
Hd
X3
4g-k
X3
4b-f
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
Ha
7.71 7.75 7.73 7.69 7.67 ––
––
––
––
––
Hb 7.48 7.47 7.52 7.43 7.45 7.69 7.67 7.66 7.65 7.66
Hc
Hd ––
4.06 4.03 3.99 3.98 3.99 4.36 4.33 4.39 4.31 4.32
–– –– –– –– 7.86 7.91 7.84 7.90 7.92

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L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
Table 2
In vitro antimicrobial activities for compounds 4–5 expressed as MIC (lg/mL)
Compds
Gram-positive bacteria
Gram-negative bacteria
Fungi
C. mycoderma
MRSA
S. aureus
B. subtilis
M. luteus
E. coli
S. dysenteriae
P. aeruginosa
P. vulgaris
C. albicans
4a
4b
4c
4d
4e
4f
4g
4h
4i
16
64
64
64
128
128
32
32
16
64
128
16
32
64
16
32
64
16
16
8
64
32
32
16
64
64
32
32
8
16
64
16
16
16
4
256
64
32
32
64
64
64
32
8
64
64
64
32
32
4
32
64
32
16
4
32
128
32
1
128
16
64
64
64
128
16
128
64
64
128
128
16
16
16
64
32
8
32
32
32
64
8
2
>512
—
32
32
32
32
128
128
32
16
8
64
128
128
16
16
16
32
64
8
32
16
16
32
256
128
8
64
16
32
32
64
256
8
16
16
32
64
128
16
16
16
64
64
8
128
32
64
64
512
128
16
32
16
256
256
64
16
16
64
64
64
4
128
32
32
16
128
64
16
16
8
64
64
64
16
16
8
16
64
8
8
4
32
32
—
—
>512
1
128
8
64
128
128
128
4
128
32
64
128
128
8
64
128
32
16
1
64
32
32
64
—
8
16
256
256
256
4
4j
4k
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
A
B
C
4
16
256
128
4
2
8
256
32
32
4
256
—
8
32
8
8
2
8
4
8
8
16
16
64
32
32
8
16
32
16
8
128
—
4
32
32
32
16
>512
—
16
16
32
16
256
—
16
16
0.5
512
—
—
128
4
>512
—
128
—
D
aMinimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
bA = Chloromycin, B = Norfloxacin, C = Berberine, D = Fluconazole.
cMRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus, Staphylococcus aureus ATCC25923; B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus ATCC 4698;
E. Coli, Escherichia coli DH52; S. dysenteriae, Shigella dysenteriae; P. aeruginosa, Pseudomonas aeruginosa; P. vulgaris, Proteus vulgaris; C. albicans, Candida albicans; C. mycoderma,
Candida mycoderma.
Table 3
LogP values of compounds 5a–k
Compds
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5 k
LogP
ꢀ0.34 0.01
0.50 0.03
0.53 0.01
0.51 0.03
0.57 0.02
0.48 0.04
0.52 0.02
0.55 0.04
0.56 0.01
0.54 0.03
0.53 0.02
2.5
2.0
1.5
1.0
0.5
0.0
1.10
0.88
0.66
0.44
0.22
2.3.3. Solubility and stability
278 (nm) R = 0.999
340 (nm) R = 0.999
The solubility of the target compounds 5a–k was tested. Phos-
phate buffer (pH 7.5) was added to the tested samples, and the
resulting suspension was equilibrated by sonication (10 min) and
shaking at room temperature for 20 h, followed by centrifugation.
The supernatant was analyzed by UV spectrometry to determine
the concentration of samples in solution using a regression curve
(Supplementary data).
The stability of the prepared compounds 5a–k was determined
by UV spectrometry (Supplementary data). The experimental re-
sults (Table 4) indicated that all the tested compounds 5a–k were
stable at the pH values (6.5–8.5) and temperatures (30–80 °C).
0.00
0.0
0.5
1.0
1.5
2.0
2.5
Concentration (10-5 mol/L)
HSA
2.4. Binding discussion
Compound 5g only
250
300
350
400
2.4.1. UV–vis absorption spectral study
Wavelength (nm)
UV–vis absorption spectroscopic method is a convenient tech-
nique for measuring the structural change of protein and the forma-
tion of complex. The UV–vis absorption measurement to study the
interaction of compound 5g with HSA has been carried out and
the results are shown in Figure 4. Two absorption peaks at 278
and 340 nm were observed, and the peak intensity increased with
the addition of compound 5g. The enhancement of absorbance
intensity evidences the binding interaction of compound 5g to
HSA. This phenomenon might result from the slightly increased
hydrophobicity of the microenvironment of Tryptophan (Trp-214)
residue.40
Figure 4. Effect of compound 5g on the UV–vis absorption of HSA,
c(HSA) = 1.0 ꢁ 10ꢀ5 mol/L; c(compound 5g)/(10ꢀ5 mol/L): 0, 0.2, 0.4, 0.6, 0.8, 1,
1.2, 1.4, 1.6, 1.8, 2.0, 2.2 (T = 286 K, pH 7.40). The inset corresponds to the
absorbance at 278 and 340 nm with different concentrations of compound 5g.
2.4.2. Fluorescence quenching mechanism
Fluorescence quenching is considered as an effective approach
to investigate the transportation ability of HSA to small molecules.
Trp-214 is the only one tryptophan residue in HSA as fluorophore

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4163
2700
2400
2100
1800
1500
1200
900
3.30
0.5
0.4
0.3
0.2
0.1
HSA
C
D
a
l
2.75
2.20
B
C
1.65
250 260 270 280 290 300
Wavelength (nm)
A: Compound 5g
B: Compound 5g-HSA
C: HSA
1.10
600
D: Difference
0.55
A
300
Compound5g only
D
0
0.00
320
340
360
380
400
420
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Figure 5. Emission spectra of HSA in the presence of various concentrations of
compound 5g. c(HSA) = 1.0 ꢁ 10ꢀ5 mol/L; c(compound 5g)/(10ꢀ5 mol/L), a–l: from
0.0–2.2 at increments of 0.20; dash line shows the emission spectrum of compound
5g only; T = 286 K, kex = 295 nm.
Figure 7. UV–vis spectra of HSA in the presence of compound 5g: A, absorption
spectrum of compound 5g only; B, absorption spectrum of compound 5g/HSA 1:1
complex; C, absorption spectrum of HSA only; D, difference between absorption
spectrum of compound 5g/HSA 1:1 complex and compound 5g, c(HSA) = c(com-
pound 5g) = 1.0 ꢁ 10ꢀ5 M. The curves C and D for the wavelength ranging from
250–300 nm were depicted in the inset.
1.8
1.6
T = 286 K
12
1.4
T = 286 K
T = 298 K
T = 298 K
1.2
1.0
0.8
0.6
0.4
0.2
0.0
T = 310 K
10
T = 310 K
8
6
4
2
0.0
0.5
1.0
1.5
2.0
5
0
1
2
3
4
5
10 [Q] (mol/L)
10-5 [Q]-1 (L/mol)
Figure 6. Stern–Volmer plots of compound 5g-HSA system at three different
temperatures.
Figure 8. Modified Stern–Volmer plots.
capable of fluorescence quenching. The changed fluorescence
intensity of Trp-214 resulted from the interaction of HSA with
other small molecules could be reflected in the fluorescence spec-
tra of HSA in the UV region.
The effect of compound 5g on HSA fluorescence intensity at
286 K was shown in Figure 5. Evidently, a progressive decrease in
the fluorescence intensity was caused by quenching, accompanied
with the blue shift wavelength (from 342.8–339.8 nm) in the albu-
min spectrum. This suggested an increased hydrophobicity of the
region surrounding the single Try-214 residue.31
Table 5
Stern–Volmer quenching constants for the interaction of compound 5g with HSA at
various temperatures
pH
7.4
T (K)
10ꢀ4KSV (L/mol)
Ra
SDb
295
304
310
3.325
3.160
3.080
0.999
0.999
0.998
0.024
0.030
0.031
a
R is the correlation coefficient.
SD is standard deviation.
b
The fluorescence quenching data can be analyzed by the well-
known Stern–Volmer equation41
:
where F0 and F represent fluorescence intensities in the absence and
presence of compound 5g, respectively. KSV is the Stern–Volmer
quenching constant, and [Q] is the concentration of compound 5g.
F0
¼ 1 þ Ksv½Qꢂ
ð1Þ
F
Table 4
Solubility of compounds 5a–k
Compds
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
Solubility (mg/L)
60.6
94.0
88.5
95.1
50.8
64.8
98.1
80.8
86.4
71.3
68.1

4164
L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
In order to verify the presence of static or dynamic quenching in
compound 5g-HSA complex, we have plotted F0/F against Q and
the results are shown in Figure 6.
2.4.4. Binding mode and thermodynamic parameters
There are generally four kinds of noncovalent interactions like
hydrogen bonds, van der Waals forces, electrostatic and hydropho-
bic bonds, which exert an important effect on small molecules
binding to proteins.44 The thermodynamic parameters enthalpy
The mechanisms of fluorescence quenching, often classified as
dynamic quenching or static quenching were depended on temper-
ature and viscosity. As higher temperatures lead to larger diffusion
coefficients, the quenching constants are expected to increase with
a gradually increasing temperature in dynamic quenching. Never-
theless, the increasing of temperature might induce a smaller static
quenching constant due to the dissociation of weakly bound
complexes.
The calculation of constant KSV from Stern–Volmer plots (Ta-
ble 5) demonstrated the effect on fluorescence quenching by com-
pound 5g at each temperature (295, 304, and 310 K) studied. The
result showed that the Stern–Volmer quenching constant KSV was
inversely correlated with temperature, which manifested that the
probable quenching mechanism of compound 5g-HSA binding
reaction was initiated by ground-state complex formation.40
The difference absorption spectroscopy was used to obtain
spectra to reconfirm that the probable fluorescence quenching
mechanism of HSA by compound 5g was mainly initiated by
ground-state complex formation. The UV–vis absorption spectrum
of HSA and the difference absorption spectrum between HSA-com-
pound 5g complex and compound 5g at the same concentration
could not be superposed (Fig. 7), this result suggested that the
probable quenching mechanism of fluorescence of HSA by com-
pound 5g was mainly a static quenching procedure.31
(
D
H) and entropy (
dence for confirming the interactions between small molecules
and protein. If the enthalpy change ( H) does not vary obviously
over the studied temperatures range, then its value and that of en-
tropy change ( S) can be evaluated from the van’t Hoff equation:
DS) change of binding reaction are the main evi-
D
D
D
RT
H
DS
R
lnK ¼ ꢀ
þ
ð4Þ
where K is analogous to the associative binding constants at the cor-
responding temperature and R is the gas constant. In order to ex-
plain the binding model between compound 5g and HSA, the
thermodynamic parameters were calculated from the van’t Hoff
plots. The enthalpy change (DH) was estimated from the slope of
the van’t Hoff relationship (Fig. 9). The free energy change (
DG)
was then calculated from the following equation:
D
G ¼
D
H ꢀ T
D
S
ð5Þ
Table 7 summarized the values of
D
H,
D
G and
D
S. The negative
values of free energy
DG of the interaction between compound 5g
and HSA suggested that the binding process was spontaneous, and
10.9
InK = 3.38 + 2148/T
a
2.4.3. Binding constant and site
R = 0.990, S.D. = 0.058
For a static quenching process, the data could be described by
10.8
10.7
10.6
10.5
10.4
10.3
the modified Stern–Volmer equation42
:
F0
1
1
1
¼
þ
ð2Þ
D
F
faKa ½Qꢂ fa
where
D
F is the difference in fluorescence intensity in the absence
and presence of compound 5g at concentration [Q], fa is the fraction
of accessible fluorescence, and Ka is the effective quenching con-
stant for the accessible fluorophores, which are analogous to asso-
ciative binding constants for the quencher-acceptor system. The
dependence of F0/DF on the reciprocal value of quencher concentra-
tion [Q]ꢀ1 is linear with the slope equaling to the value of (faKa)ꢀ1
.
The value faꢀ1 is fixed on the ordinate. The constant Ka is a quotient
3.20
3.25
3.30
3.35
1000/T (K-1)
3.40
3.45
3.50
ꢀ1
of the ordinate fa and the slope (faKa)ꢀ1. The modified Stern–Vol-
mer plots were Figure 8 and the calculated results were depicted in
Table 6.
Figure 9. Van’t Hoff plots of the compound 5g-HSA system.
The numbers of binding sites and the equilibrium binding con-
stants can also be calculated according to the Scatchard
equation43
:
Table 6
Binding constants and sites of compound 5g-HSA system at pH 7.4
r=D ¼ nKb ꢀ rKb
ð3Þ
T(K) Modified Stern–Volmer
Scatchard Method
Method
where Df is the molar concentration of free small molecules, r is the
moles of small molecules bound per mole of protein, n is binding
sites multiplicity per class of binding sites, and Kb is the equilibrium
binding constant. The Scatchard plots (Supplementary data) and the
Kb and n were listed in Table 6.
10ꢀ4Ka (L/mol)
10ꢀ4Kb (L/
R
SD
n
mol)
286 5.49
298 3.78
310 3.08
5.08
3.80
3.02
0.994 0.009 1.17
0.997 0.005 1.37
0.996 0.005 1.46
The results of the modified Stern–Volmer and Scatchard plots
for the compound 5g-HSA system at different temperatures were
shown in Table 6. The decreasing trend of Ka and Kb with increasing
temperatures was in accordance with KSV’s dependence on temper-
atures. The value of binding sites n was approximately one, which
indicated the interaction of compound 5g with HSA seemed to be
the presence of one high affinity binding site. The results also man-
ifested that the binding constants were moderate and the effect of
temperatures was not obvious, therefore compound 5g might be
stored and carried by HSA.
Table 7
Thermodynamic parameters of compound 5g-HSA system at different temperatures
T(K)
D
H (kJ/mol)
D
G (kJ/mol)
DS (J/mol K)
286
298
310
ꢀ25.89
ꢀ26.23
ꢀ26.58
ꢀ17.86
28.09

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4165
the negative values of enthalpy (
was mainly enthalpy-driven and involved an exothermic reaction,
the entropy ( S) was unfavorable for it. A positive ( S) value is fre-
quently taken as a typical evidence for hydrophobic interaction,
which is consistent with the above discussion. Therefore, enthalpy
D
H) indicated that the binding
4.1.1. General procedures for the HSA binding study
The concentration of HSA was fixed at 1.0 ꢁ 10ꢀ5 mol Lꢀ1 and
that of compound 5g was varied from 0 to 2.2 ꢁ 10ꢀ5 mol Lꢀ1 at
increments of 0.2ꢁ10ꢀ5 mol Lꢀ1 in the fluorescence measure-
ments. In the quenching experiments, the concentrations of HSA
solution were stabilized at 1.0 ꢁ 10ꢀ5 mol Lꢀ1, and solutions of
D
D
change
DH < 0 and entropy change DS > 0 obtained in this case
indicated that the electrostatic interactions played an important
compound 5g with concentrations varied from
0
to
role in the binding of compound 5g to HSA (Table 7).40
2.4 ꢁ 10ꢀ5 mol Lꢀ1 at increments of 0.2 ꢁ 10ꢀ5 mol Lꢀ1 were added
to the HSA solution. Fluorescence spectra (kex = 295 nm) were
measured after the mixtures were incubated at 295, 304 and
310 K for 24 h, respectively.
3. Conclusions
In conclusion, a series of novel berberine nitroimidazoles was
successfully synthesized by a convenient and efficient procedure
starting from commercially available halobenzyl chlorides, dietha-
nolamine and berberine. Their structures were confirmed by 1H
NMR, 13C NMR, MS, IR and HRMS spectra. The in vitro antimicrobial
evaluation revealed that most of the synthesized berberine nitro-
imidazoles could effectively inhibit the growth of the tested strains
and some showed comparable or superior potency against Methi-
cillin-resistant S. aureus to reference drugs Norfloxacin, Chloromy-
cin and Berberine. Especially, nitroimidazole derivative 5g with
2,4-difluorobenzyl moiety exhibited effective antibacterial and
antifungal activities and particularly gave comparable or even bet-
4.1.2. General procedures for the preparation of intermediates
diols (2a–e) and bromides (3a–e)
The intermediates 2a–e and 3a–e were prepared according to
the previously reported methods.27
4.1.3. 2-Bromo-N-(2,4-difluorobenzyl)-N-[2-(2-methyl-5-nitro-
1H-imidazol-1-yl)ethyl]ethanamine (4a)
A mixture of 2-methyl-5-nitroimidazole (1.29 g, 0.01 mol) and
potassium carbonate (2.80 g, 0.02 mol) in acetonitrile (5 mL) was
stirred at 60 °C for 1 h. After the mixture was cooled to room tem-
perature, compound 3a (4.20 g, 0.01 mol) was added and stirred
for 2 h (monitored by TLC, eluent, ethyl acetate/petroleum, 1:2,
v/v). After the solvent was evaporated under reduced pressure,
and the resulting residue was extracted with ethyl acetate
(3 ꢁ 30 mL), the organic layers were combined, dried over anhy-
drous sodium sulfate, and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatogra-
phy eluting with ethyl acetate/petroleum (1:2, v/v) to afford the
target compound 4a (3.03 g) as white solid. Yield: 74.1%; mp:
ter anti-Gram-negative (MIC = 4–8
lg/mL) and anti-C. mycoderma
(MIC = 1 g/mL) efficacies in comparison to reference drugs. Fur-
l
ther transportation behavior of HSA to compound 5g manifested
that nitroimidazole 5g could be effectively stored and carried by
HSA. The experimental parameters indicated that the quenching
mechanism of fluorescence of HSA by compound 5g was a static
quenching procedure. The association of compound 5g with HSA
was spontaneous and the electrostatic interactions played impor-
tant roles in the transportation of HSA to 5g.
107–108 °C; IR (KBr)
2851 (CH2, CH3), 1602, 1535, 1499 (aromatic skeleton), 1420,
1327, 1289, 1234, 1137, 1049, 1031, 975, 853, 826 cmꢀ1 1H
m: 3085, 3067 (Ar–H), 2966, 2954, 2918,
;
4. Experimental
NMR (300 MHz, CDCl3): d 7.75 (s, 1H, Im 4-H), 7.20–6.76 (m, 3H,
Ph 3,5,6-H), 3.93 (t, J = 6.2 Hz, 2H, Im-CH2), 3.68 (s, 2H, PhCH2),
3.38 (t, J = 6.0 Hz, 2H, Br–CH2), 3.00 (t, J = 6.2 Hz, 2H, Im-CH2CH2),
2.89 (t, J = 6.0 Hz, 2H, Br–CH2CH2), 2.33 (s, 3H, Im-CH3) ppm; MS
(m/z): 404 [M+H]+. HRMS (TOF) calcd for C15H17BrF2N4O2:
[M+H]+, 404.2298; found, 404.2290.
4.1. General methods
Melting points were recorded on X-6 melting point apparatus
and uncorrected. TLC analysis was done using pre-coated silica
gel plates. FT-IR spectra were carried out on Bruker RFS100/S
spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pel-
lets in the 400–4000 cmꢀ1 range. NMR spectra were recorded on a
Bruker AV 300 spectrometer using TMS as an internal standard.
The chemical shifts were reported in parts per million (ppm),
the coupling constants (J) were expressed in hertz (Hz) and sig-
nals were described as singlet (s), doublet (d), triplet (t), as well
as multiplet (m). The following abbreviations were used to desig-
nate aryl groups: Im, nitroimidazolyl; Ph, phenyl; Ber, berberinyl.
The mass spectra were recorded on LCMS-2010A and the high-
resolution mass spectra (HRMS) were recorded on an IonSpec
FT–ICR mass spectrometer with ESI resource. All fluorescence
spectra were recorded on F-7000 Spectrofluorimeter (Hitachi, To-
kyo, Japan) equipped with 1.0 cm quartz cells, the widths of both
the excitation and emission slit were set as 2.5 nm, and the exci-
tation wavelength was 295 nm. Fluorescence spectra were re-
corded at 286, 298, 310 K in the range of 300–450 nm. The UV
spectrum was recorded at room temperature on a TU-2450 spec-
trophotometer (Puxi Analytic Instrument Ltd, of Beijing, China)
equipped with 1.0 cm quartz cells. HSA was obtained from Sig-
ma–Aldrich (St. Louis, MO, USA). Tris, NaCl and HCl were analyti-
cal purity. HSA was dissolved in Tris–HCl buffer solution (0.05 M
Tris, 0.15 M NaCl, pH 7.4). Sample masses were weighed on a
microbalance with a resolution of 0.1 mg. All other chemicals
and solvents were commercially available, and were used without
further purification.
4.1.4. 2-Bromo-N-(2,4-difluorobenzyl)-N-[2-(4-nitro-1H-
imidazol-1-yl)ethyl]ethanamine (4b)
Compound 4b was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.70 g,
0.015 mol), potassium carbonate (4.25 g, 0.03 mol) and compound
3a (5.52 g, 0.015 mol). The target compound 4b (2.74 g) was ob-
tained as white solid. Yield: 46.8%; mp: 101–102 °C; IR (KBr) m:
3072, 3014 (Ar–H), 2966, 2925, 2821 (CH2), 1602, 1527, 1490 (aro-
matic skeleton), 1427, 1325, 1288, 1237, 1141, 1049, 1020, 984,
861, 820 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.71 (s, 1H, Im 5-H),
;
7.48 (s, 1H, Im 2-H), 7.16–6.79 (m, 3H, Ph 3,5,6-H), 4.06 (t,
J = 6.2 Hz, 2H, Im-CH2), 3.67 (s, 2H, PhCH2), 3.38 (t, J = 6.0 Hz, 2H,
Br–CH2), 2.99 (t, J = 6.2 Hz, 2H, Im-CH2CH2), 2.90 (t, J = 6.0 Hz, 2H,
Br–CH2CH2) ppm; MS (m/z): 390 [M+H]+. HRMS (TOF) calcd for
C
14H15BrF2N4O2: [M+H]+, 390.2032; found, 390.2040.
4.1.5. 2-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(4-nitro-1H-
imidazol-1-yl)ethyl]ethanamine (4c)
Compound 4c was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.15 g,
0.01 mol), potassium carbonate (2.99 g, 0.02 mol) and compound
3b (3.98 g, 0.01 mol). The target compound 4c (2.37 g) was ob-
tained as white solid. Yield: 55.4%; mp: 115–117 °C; IR (KBr) m:
3078, 3018 (Ar–H), 2971, 2953, 2825 (CH2), 1595, 1523, 1482 (aro-
matic skeleton), 1410, 1327, 1285, 1229, 1124, 1051, 1029, 981,

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L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
865, 822 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 7.75 (s, 1H, Im 5-H),
3.73 (s, 2H, PhCH2), 3.37 (t, J = 6.0 Hz, 2H, Br–CH2), 2.98 (t,
J = 6.2 Hz, 2H, Im-CH2CH2), 2.90 (t, J = 6.0 Hz, 2H, Br–CH2CH2)
ppm; MS (m/z): 390 [M+H]+. HRMS (TOF) calcd for
7.47 (s, 1H, Im 2-H), 7.34–6.98 (m, 3H, Ph 2,5,6-H), 4.03 (t,
J = 6.2 Hz, 2H, Im-CH2), 3.69 (s, 2H, PhCH2), 3.43 (t, J = 6.0 Hz, 2H,
Br–CH2), 3.02 (t, J = 6.2 Hz, 2H, Im-CH2CH2), 2.89 (t, J = 6.0 Hz, 2H,
Br–CH2CH2) ppm; MS (m/z): 423 [M+H]+. HRMS (TOF) calcd for
C
14H15BrF2N4O2: [M+H]+, 390.2032; found, 390.2030.
C
14H15BrCl2N4O2: [M+Na]+, 442.9653; found, 442.9651.
4.1.10. 2-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(5-nitro-1H-
imidazol-1-yl)ethyl]ethanamine (4h)
4.1.6. 2-Bromo-N-(2-chlorobenzyl)-N-[2-(4-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4d)
Compound 4d was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.14 g,
0.01 mol), potassium carbonate (2.93 g, 0.02 mol) and compound
3c (4.54 g, 0.013 mol). The target compound 4d (2.92 g) was ob-
Compound 4h was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.15 g,
0.01 mol), potassium carbonate (2.99 g, 0.02 mol) and compound
3b (3.98 g, 0.01 mol). The target compound 4h (1.13 g) was ob-
tained as white solid. Yield: 26.3%; IR (KBr)
m: 3079, 3015 (Ar–H),
2974, 2950, 2822 (CH2), 1594, 1522, 1487 (aromatic skeleton),
tained as white solid. Yield: 75.0%; mp: 89–90 °C; IR (KBr)
m
:
1415, 1323, 1286, 1227, 1128, 1054, 1025, 986, 869, 826 cmꢀ1
;
3091, 3067 (Ar–H), 2923, 2843 (CH2), 1593, 1519, 1485, 1435 (aro-
1H NMR (300 MHz, CDCl3): d 7.91 (s, 1H, Im 4-H), 7.67 (s, 1H, Im
2-H), 7.33–6.93 (m, 3H, Ph 2,5,6-H), 4.33 (t, J = 6.2 Hz, 2H, Im-
CH2), 3.75 (s, 2H, PhCH2), 3.41 (t, J = 6.0 Hz, 2H, Br–CH2), 3.00 (t,
J = 6.2 Hz, 2H, Im-CH2CH2), 2.88 (t, J = 6.0 Hz, 2H, Br–CH2CH2)
ppm; MS (m/z): 423 [M+H]+. HRMS (TOF) calcd for
matic skeleton), 1371, 1286, 1223, 1126, 1053, 1033, 982, 874,
824 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.73 (s, 1H, Im 5-H), 7.52
;
(s, 1H, Im 2-H), 7.32–7.17 (m, 4H, Ph 3,4,5,6-H), 3.99 (t, J = 6.2 Hz,
2H, Im-CH2), 3.76 (s, 2H, PhCH2), 3.46 (t, J = 6.0 Hz, 2H, Br–CH2),
3.07 (t, J = 6.2 Hz, 2H, Im-CH2CH2), 2.92 (t, J = 6.0 Hz, 2H, Br–
CH2CH2) ppm; MS (m/z): 389 [M+H]+. HRMS (TOF) calcd for
C
14H15BrCl2N4O2: [M+Na]+, 442.9653; found, 442.9650.
C
14H16BrClN4O2: [M+Na]+, 409.0043; found, 409.0040.
4.1.11. 2-Bromo-N-(2-chlorobenzyl)-N-[2-(5-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4i)
4.1.7. 2-Bromo-N-(3-chlorobenzyl)-N-[2-(4-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4e)
Compound 4e was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.12 g,
0.01 mol), potassium carbonate (3.00 g, 0.02 mol) and compound
3d (3.96 g, 0.01 mol). The target compound 4e (2.21 g) was ob-
Compound 4i was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.14 g,
0.01 mol), potassium carbonate (2.93 g, 0.02 mol) and compound
3c (4.54 g, 0.013 mol). The target compound 4i (1.06 g) was ob-
tained as white solid. Yield: 26.9%; IR (KBr)
2926, 2847 (CH2), 1597, 1518, 1483, 1436 (aromatic skeleton),
1376, 1284, 1225, 1125, 1056, 1036, 984, 873, 821 cmꢀ1 1H
m: 3095, 3068 (Ar–H),
tained as white solid. Yield: 57.4%; mp: 68–69 °C; IR (KBr)
m
:
;
3068, 3021 (Ar–H), 2959, 2823 (CH2), 1588, 1524, 1489, 1441 (aro-
NMR (300 MHz, CDCl3): d 7.84 (s, 1H, Im 4-H), 7.66 (s, 1H, Im 2-
H), 7.29–7.14 (m, 4H, Ph 3,4,5,6-H), 4.39 (t, J = 6.2 Hz, 2H, Im-
CH2), 3.73 (s, 2H, PhCH2), 3.41 (t, J = 6.0 Hz, 2H, Br–CH2), 3.05 (t,
J = 6.2 Hz, 2H, Im-CH2CH2), 2.94 (t, J = 6.0 Hz, 2H, Br–CH2CH2)
ppm. MS (m/z): 388 [M+H]+. HRMS (TOF) calcd for C14H16BrClN4O2:
[M+Na]+, 409.0043; found, 409.0041.
matic skeleton), 1325, 1287, 1250, 1146, 1075, 1046, 983, 861,
822 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.69 (s, 1H, Im 5-H), 7.43
;
(s, 1H, Im 2-H), 7.24–7.01 (m, 4H, Ph 2,4,5,6-H), 3.98 (t, J = 6.2 Hz,
2H, Im-CH2), 3.73 (s, 2H, PhCH2), 3.43 (t, J = 6.0 Hz, 2H, Br–CH2),
3.02 (t, J = 6.2 Hz, 2H, Im-CH2CH2), 2.87 (t, J = 6.0 Hz, 2H, Br–
CH2CH2) ppm; MS (m/z): 389 [M+H]+. HRMS (TOF) calcd for
C
14H16BrClN4O2: [M+Na]+, 409.0043; found, 409.0041.
4.1.12. 2-Bromo-N-(3-chlorobenzyl)-N-[2-(5-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4j)
4.1.8. 2-Bromo-N-(4-chlorobenzyl)-N-[2-(4-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4f)
Compound 4f was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.13 g,
0.01 mol), potassium carbonate (2.99 g, 0.02 mol) and compound
3e (4.70 g, 0.013 mol). The target compound 4f (3.09 g) was ob-
Compound 4j was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.12 g,
0.01 mol), potassium carbonate (3.00 g, 0.02 mol) and compound
3d (3.96 g, 0.01 mol). The target compound 4j (0.87 g) was ob-
tained as white solid. Yield: 22.6%; IR (KBr)
2953, 2827 (CH2), 1581, 1525, 1487, 1444 (aromatic skeleton),
1321, 1288, 1252, 1148, 1077, 1047, 988, 868, 823 cmꢀ1 1H
m: 3066, 3020 (Ar-H),
tained as white solid. Yield: 80.0%; mp: 124–125 °C; IR (KBr)
m
:
;
3051 (Ar–H), 2962, 2822 (CH2), 1597, 1516, 1491 (aromatic skele-
NMR (300 MHz, CDCl3): d 7.90 (s, 1H, Im 4-H), 7.65 (s, 1H, Im 2-
H), 7.24–6.97 (m, 4H, Ph 2,4,5,6-H), 4.31 (t, J = 6.2 Hz, 2H, Im-
CH2), 3.71 (s, 2H, PhCH2), 3.40 (t, J = 6.0 Hz, 2H, Br–CH2), 3.00 (t,
J = 6.2 Hz, 2H, Im-CH2CH2), 2.88 (t, J = 6.0 Hz, 2H, Br–CH2CH2)
ppm. MS (m/z): 388 [M+H]+. HRMS (TOF) calcd for C14H16BrClN4O2:
[M+Na]+, 409.0043; found, 409.0040.
ton), 1427, 1371, 1272, 1234, 1144, 1050, 1017, 964, 857,
821 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.67 (s, 1H, Im 5-H), 7.45
;
(s, 1H, Im 2-H), 7.26–7.08 (m, 4H, Ph 2,3,5,6-H), 3.99 (t, J = 6.2 Hz,
2H, Im-CH2), 3.70 (s, 2H, PhCH2), 3.40 (t, J = 6.0 Hz, 2H, Br–CH2),
3.00 (t, J = 6.2 Hz, 2H, Im-CH2CH2), 2.88 (t, J = 6.0 Hz, 2H, Br–
CH2CH2) ppm. MS (m/z): 389 [M+H]+. HRMS (TOF) calcd for
C
14H16BrClN4O2: [M+Na]+, 409.0043; found, 409.0039.
4.1.13. 2-Bromo-N-(4-chlorobenzyl)-N-[2-(5-nitro-1H-imidazol-
1-yl)ethyl]ethanamine (4k)
4.1.9. 2-Bromo-N-(2,4-difluorobenzyl)-N-[2-(5-nitro-1H-
imidazol-1-yl)ethyl]ethanamine (4g)
Compound 4g was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.70 g,
0.015 mol), potassium carbonate (4.25 g, 0.03 mol) and compound
3a (5.52 g, 0.015 mol). The target compound 4g (1.27 g) was ob-
Compound 4k was prepared according to the procedure de-
scribed for compound 4a starting from 4-nitroimidazole (1.13 g,
0.01 mol), potassium carbonate (2.99 g, 0.02 mol) and compound
3e (4.70 g, 0.013 mol). The target compound 4k (0.73 g) was ob-
tained as white solid. Yield: 18.5%; IR (KBr)
2821 (CH2), 1598, 1514, 1492 (aromatic skeleton), 1428, 1373,
1275, 1233, 1142, 1053, 1016, 962, 856, 820 cmꢀ1 1H NMR
m: 3049 (Ar-H), 2960,
tained as white solid. Yield: 21.7%; IR (KBr)
2967, 2923, 2823 (CH2), 1603, 1519, 1497, 1421 (aromatic skele-
ton), 1326, 1280, 1239, 1143, 1051, 1023, 981, 864, 824 cmꢀ1 1H
m
: 3076, 3012 (Ar–H),
;
(300 MHz, CDCl3): d 7.92 (s, 1H, Im 4-H), 7.66 (s, 1H, Im 2-H),
7.35–7.18 (m, 4H, Ph 2,3,5,6-H), 4.32 (t, J = 6.2 Hz, 2H, Im-CH2),
3.73 (s, 2H, PhCH2), 3.32 (t, J = 6.0 Hz, 2H, Br–CH2), 3.02 (t,
J = 6.2 Hz, 2H, Im-CH2CH2), 2.98 (t, J = 6.0 Hz, 2H, Br–CH2CH2)
;
NMR (300 MHz, CDCl3): d 7.86 (s, 1H, Im 4-H), 7.69 (s, 1H, Im 2-
H), 7.10–6.75 (m, 3H, Ph 3,5,6-H), 4.36 (t, J = 6.2 Hz, 2H, Im-CH2),

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4167
ppm; MS (m/z): 388 [M+H]+. HRMS (TOF) calcd for
14H16BrClN4O2: [M+H]+, 387.0223; found, 387.0214.
1601, 1542, 1506, 1483 (aromatic skeleton), 1386, 1346, 1272,
1230, 1138, 1064, 981, 821 cmꢀ1 1H NMR (300 MHz, CD3OD): d
C
;
9.61 (s, 1H, Ber 8-H), 8.75 (s, 1H, Ber 13-H), 7.84 (s, 1H, Im 5-H),
8.10 (d, 1H, J = 9 Hz, Ber 12-H), 7.99 (d, 1H, J = 9 Hz, Ber 11-H),
7.71 (s, 1H, Ber 1-H), 7. 46 (s, 1H, Im 2-H), 7.47–7.14 (m, 3H, Ph
2,5,6-H), 7.01 (s, 1H, Ber 4-H), 6.08 (s, 2H, OCH2O), 4.93 (br, 2H,
Ber 6-H), 4.31 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.08 (t, J = 6.2 Hz, 2H,
Im-CH2), 4.04 (s, 3H, OCH3), 3.77 (s, 2H, Ph–CH2), 3.23 (br, 2H,
Ber 5-H), 3.08 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.91 (t, J = 6.2 Hz, 2H,
Im-CH2CH2) ppm; 13C NMR (75 MHz, CD3OD): d 151.4, 148.7,
146.8, 146.7, 146.1, 144.7, 142.8, 139.4, 136.5, 133.2, 133.1,
132.6, 131.7, 130.2, 128.6, 125.4, 124.1, 123.3, 121.2, 119.5,
118.7, 118.2, 117.5, 108.4, 102.9, 72.5, 57.3, 54.2, 53.0, 51.5, 46.0,
28.0, 26.1 ppm; MS (m/z): 663 [MꢀCl]+; HRMS (TOF) calcd for
4.1.14. N-(2,4-Difluorobenzyl)-N-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5a)
To a stirred solution of berberrubine (1.39 g, 3.89 mmol) in DMF
(10 mL), compound 4a (1.60 g, 3.97 mmol) in DMF (5 mL) was
added dropwise. The reaction mixture was then heated at 110 °C
for 20 h. After the reaction was completed (monitored by TLC, elu-
ent, chloroform/methanol, 20:1, v/v), the mixture was extracted
with chloroform (3 ꢁ 30 mL). The organic layer was washed with
water and dried over anhydrous sodium sulfate. After the filtrate
was concentrated under reduced pressure, the crude product was
purified by silica gel column chromatography eluting with chloro-
form/methanol (20:1, v/v) to afford the target compound 5a
(891 mg) as yellow solid. Yield: 33.7%; mp: 197–199 °C; IR (KBr)
C
33H30Cl3N5O6: [MꢀCl]+, 662.1568; found, 662.1574.
m
: 3039 (Ar–H), 2949, 2846 (CH2, CH3), 1600, 1536, 1504, 1477 (aro-
matic skeleton), 1385, 1335, 1272, 1227, 1116, 1022, 933,
829 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.60 (s, 1H, Ber 8-H),
4.1.17. N-(2-Chlorobenzyl)-N-(2-(4-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5d)
Compound 5d was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.07 g,
2.99 mmol) and compound 4d (1.18 g, 3.04 mmol). The target com-
pound 5d (528 mg) was obtained as yellow solid. Yield: 26.6%; mp:
;
8.75 (s, 1H, Ber 13-H), 8.09 (d, J = 9.0 Hz, 1H, Ber 12-H), 7.72 (s,
1H, Im 4-H), 7.97 (d, J = 9.0 Hz, 1H, Ber 11-H), 7.63 (s, 1H, Ber 1-H),
7.30–7.27 (m, 1H, Ph 3-H), 7.14 (br, 1H, Ph 3-H), 7.04 (s, 1H, Ber 4-
H), 6.94 (br, 1H, Ph 5-H), 6.14 (s, 2H, OCH2O), 4.91 (br, 2H, Ber 6-
H), 4.32 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.10 (t, J = 6.2 Hz, 2H, Im-
CH2), 4.02 (s, 3H, OCH3), 3.80 (s, 2H, Ph–CH2), 3.23 (br, 2H, Ber 5-
H), 3.09 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.91 (t, J = 6.2 Hz, 2H, Im-
CH2CH2), 2.28 (s, 3H, Im-CH3) ppm; 13C NMR (75 MHz, CD3OD): d
163.5, 163.4, 161.2, 161.1, 161.0, 159.1, 158.3, 150.1, 148.5, 146.9,
145.7, 145.2, 142.5, 142.1, 137.5, 135.7, 133.4, 132.9, 132.4, 131.1,
123.0, 121.6, 120.9, 119.6, 119.2, 118.9, 118.6, 118.2, 117.9, 111.9,
111.5, 108.9, 104.3, 104.1, 103.8, 102.5, 72.2, 57.4, 57.0, 53.0, 51.5,
46.0, 28.0, 26.9, 22.2 ppm; MS (m/z): 664 [MꢀCl]+; HRMS (TOF)
calcd for C34H32ClF2N5O6: [MꢀCl]+, 664.2315; found, 664.2319.
169–170 °C. IR (KBr)
1537, 1504, 1479 (aromatic skeleton), 1384, 1344, 1273, 1230,
1102, 1034, 926, 823 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.62
m: 3064 (Ar–H), 2957, 2843 (CH2, CH3), 1599,
;
(s, 1H, Ber 8-H), 8.67 (s, 1H, Ber 13-H), 7.83 (s, 1H, Im 5-H), 8.11
(d, 1H, J = 9 Hz, Ber 12-H), 7.98 (d, 1H, J = 9 Hz, Ber 11-H), 7.72 (s,
1H, Ber 1-H), 7.44 (s, 1H, Im 2-H), 7.11–6.88 (m, 4H, Ph 3,4,5,6-
H), 6.11 (s, 2H, OCH2O), 5.46 (br, 2H, Ber 6-H), 4.05 (t, J = 6.2 Hz,
2H, Im-CH2), 4.31 (t, J = 6.0 Hz, 4H, OCH2CH2N), 4.03 (s, 3H,
OCH3), 3.68 (s, 2H, Ph–CH2), 3.50 (br, 2H, Ber 5-H), 3.05 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 3.09 (t, J = 6.2 Hz, 2H, Im-CH2CH2)
ppm; 13C NMR (75 MHz, CD3OD): d 151.1, 149.3, 147.7, 145.4,
145.3, 144.9, 142.9, 139.3, 133.6, 133.4, 132.2, 131.1, 129.6,
128.8, 127.2, 126.6, 125.4, 123.0, 120.9, 118.9, 118.6, 118.2,
117.9, 108.9, 102.5, 72.2, 57.4, 54.5, 53.8, 51.1, 46.4, 28.2,
26.8 ppm; MS (m/z): 628 [MꢀCl]+. HRMS (TOF) calcd for
4.1.15. N-(2,4-Difluorobenzyl)-N-(2-(4-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5b)
Compound 5b was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.83 g,
5.12 mmol) and compound 4b (661 mg, 1.92 mmol). The target
compound 5b (784 mg) was obtained as yellow solid. Yield:
23.0%; mp: 198–200 °C; IR (KBr)
(CH2, CH3), 1601, 1541, 1504, 1483 (aromatic skeleton), 1383,
1345, 1272, 1224, 1100, 1033, 979, 846 cmꢀ1 1H NMR (300 MHz,
CD3OD): d 9.55 (s, 1H, Ber 8-H), 8.74 (s, 1H, Ber 13-H), 7.84 (s,
1H, Im 5-H), 8.17 (d, J = 9.0 Hz, 1H, Ber 12-H), 7.96 (d, J = 9.0 Hz,
1H, Ber 11-H), 7.62 (s, 1H, Ber 1-H), 7.51 (s, 1H, Im 2-H), 7.30–
6.94 (m, 3H, Ph 3,5,6-H), 6.95 (s, 1H, Ber 4-H), 6.12 (s, 2H, OCH2O),
4.95 (br, 2H, Ber 6-H), 4.38 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.02 (s,
3H, OCH3), 4.01 (t, J = 6.2 Hz, 2H, Im-CH2), 3.80 (s, 2H, Ph–CH2),
3.23 (br, 2H, Ber 5-H), 3.03 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.95 (t,
J = 6.0 Hz, 2H, Im-CH2CH2) ppm; 13C NMR (75 MHz, CD3OD): d
164.1, 163.8, 161.7, 161.6, 161.5, 161.2, 159.0, 158.1, 151.5,
148.2, 146.4, 145.8, 145.1, 143.5, 142.9, 139.4, 135.3, 133.6,
132.3, 132.2, 131.1, 125.4, 123.0, 120.9, 119.4, 119.3, 118.9,
118.6, 118.2, 117.9, 112.0, 111.8, 108.9, 104.6, 104.2, 103.8,
102.5, 72.2, 57.1, 54.5, 52.6, 51.8, 46.7, 27.7, 26.4 ppm; MS (m/z):
630 [MꢀCl]+; HRMS (TOF) calcd for C33H30ClF2N5O6: [MꢀCl]+,
630.2159; found, 630.2154.
C
33H31Cl2N5O6: [M–Cl]+, 629.1957; found, 629.1954.
4.1.18. N-(3-Chlorobenzyl)-N-(2-(4-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5e)
Compound 5e was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.77 g,
4.95 mmol) and compound 4e (1.71 g, 4.98 mmol). The target com-
pound 5e (1.38 g) was obtained as yellow solid. Yield: 34.7%; mp:
m: 3074 (Ar–H), 2923, 2847
;
157–158 °C; IR (KBr)
1534, 1505, 1479 (aromatic skeleton), 1386, 1348, 1274, 1228,
1099, 1040, 941, 871 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.62
m: 3031 (Ar-H), 2968, 2844 (CH2, CH3), 1601,
;
(s, 1H, Ber 8-H), 8.22 (s, 1H, Ber 13-H), 8.19 (s, 1H, Im 5-H),
7.87–7.79 (m, 2H, 11, Ber 12-H), 7.53 (s, 1H, Im 2-H), 7.35 (s, 1H,
Ber 1-H), 7.17–7.11 (m, 3H, Ph 2,5,6-H), 6.85 (s, 1H, Ber 4-H),
6.11 (s, 2H, OCH2O), 4.96 (br, 2H, Ber 6-H), 4.53 (t, J = 6.0 Hz, 2H,
OCH2CH2N), 4.04 (t, J = 6.2 Hz, 2H, Im-CH2), 3.98 (s, 3H, OCH3),
3.72 (s, 2H, Ph–CH2), 3.29 (br, 2H, Ber 5-H), 3.07 (t, J = 6.0 Hz, 2H,
OCH2CH2N), 2.92 (t, J = 6.2 Hz, 2H, Im-CH2CH2) ppm; 13C NMR
(75 MHz, CD3OD): d 150.5, 149.3, 146.5, 146.3, 145.9, 143.5,
141.9, 139.5, 138.1, 134.8, 133.4, 131.1, 130.1, 126.6, 125.4,
124.1, 123.0, 120.9, 119.8, 119.1, 118.6, 117.9, 112.5, 108.9,
102.5, 72.4, 57.7, 54.1, 53.4, 51.6, 45.6, 28.1, 27.4 ppm; MS (m/z):
628 [MꢀCl]+; HRMS (TOF) calcd for C33H31Cl2N5O6: [MꢀCl]+,
629.1957; found, 629.1953.
4.1.16. N-(3,4-Dichlorobenzyl)-N-(2-(4-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5c)
Compound 5c was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.27 g,
3.55 mmol) and compound 4c (1.50 g, 3.55 mmol). The target com-
pound 5c (648 mg) was obtained as yellow solid. Yield: 26.1%; mp:
4.1.19. N-(4-Chlorobenzyl)-N-(2-(4-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5f)
Compound 5f was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.06 g,
194–195 °C; IR (KBr) m: 3075, 3011 (Ar–H), 2923, 2851 (CH2, CH3),

4168
L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
2.96 mmol) and compound 4f (1.30 g, 3.35 mmol). The target com-
pound 5f (624 mg) was obtained as yellow solid. Yield: 31.8%; mp:
4.1.22. N-(2-Chlorobenzyl)-N-(2-(5-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5i)
205–206 °C; IR (KBr)
1542, 1522, 1483 (aromatic skeleton), 1384, 1345, 1271, 1223,
1101, 1033, 932, 821 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.61
m
: 3066 (Ar–H), 2972, 2819 (CH2, CH3), 1600,
Compound 5i was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (0.89 g,
2.49 mmol) and compound 4i (0.94 g, 2.43 mmol). The pure com-
pound 5i (639 mg) was obtained as yellow solid. Yield: 38.7%;
;
(s, 1H, Ber 8-H), 8.72 (s, 1H, Ber 13-H), 7.83 (s, 1H, Im 5-H), 8.05
(d, J = 9 Hz, 1H, Ber 12-H), 7.97 (d, J = 9 Hz, 1H, Ber 12-H), 7.64 (s,
1H, Ber 1-H), 7.45 (s, 1H, Im 2-H), 7.21–6.92 (m, 5H, 4-H, Ph
2,3,5,6-H), 6.12 (s, 2H, OCH2O), 4.95 (br, 2H, Ber 6-H), 4.48 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 4.08 (t, J = 6.0 Hz, 2H, Im-CH2), 4.03 (s,
3H, OCH3), 3.73 (s, 2H, Ph-CH2), 3.26 (br, 2H, Ber 5-H), 2.97 (t,
J = 6.0 Hz, 4H, OCH2CH2N, Im-CH2CH2) ppm; 13C NMR (75 MHz,
CD3OD): d 150.4, 148.2, 146.7, 146.1, 145.7, 144.1, 143.3, 139.1,
134.5, 133.6, 133.4, 131.1, 129.1, 127.7, 125.4, 123.0, 120.2,
119.6, 118.7, 118.4, 116.1, 108.9, 102.5, 72.2, 57.4, 55.3, 54.4,
52.3, 47.3, 28.2, 27.6 ppm; MS (m/z): 628 [MꢀCl]+; HRMS (TOF)
calcd for C33H31Cl2N5O6: [MꢀCl]+, 629.1957; found, 629.1948.
mp: 231–233 °C; IR (KBr)
CH3), 1600, 1528, 1507, 1475 (aromatic skeleton), 1383, 1349,
1276, 1235, 1102, 1065, 937, 854 cmꢀ1 1H NMR (300 MHz,
m: 3133, 3078 (Ar–H), 2955, 2824 (CH2,
;
CD3OD): d 9.55 (s, 1H, Ber 8-H), 8.68 (s, 1H, Ber 13-H), 7.88 (s,
1H, Im 4-H), 8. 04 (d, J = 9.0 Hz, 1H, Ber 11-H), 7. 97 (d, J = 9.0 Hz,
2H, Ber 12-H), 7.64 (s, 1H, Im 2-H), 7.17–7.12 (m, 4H, Ph 3,4,5-
H), 7.63 (s, 1H, Ber 1-H), 6.85 (s, 1H, Ph 6-H), 6.11 (s, 2H, OCH2O),
4.98 (br, 2H, Ber 6-H), 4.41 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.32 (t,
J = 6.2 Hz, 2H, Im-CH2), 3.99 (s, 3H, OCH3), 3.75 (s, 2H, Ph–CH2),
3.34 (br, 2H, Ber 5-H), 3.07 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.97 (t,
J = 6.2 Hz, 2H, Im-CH2CH2) ppm; 13C NMR (75 MHz, CD3OD): d
150.4, 148.0, 146.4, 145.7, 144.6, 143.2, 142.1, 136.5, 133.4,
133.0, 132.1, 131.1, 129.4, 128.8, 127.1, 126.6, 125.3, 123.0,
120.9, 119.3, 118.7, 118.1, 116.4, 108.2, 102.4, 72.2, 58.6, 57.3,
53.2, 51.2, 46.6, 29.2, 27.3 ppm; MS (m/z): 628 [MꢀCl]+. HRMS
4.1.20. N-(2,4-Difluorobenzyl)-N-(2-(5-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5g)
Compound 5g was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.92 g,
5.36 mmol) and compound 4g (2.09 g, 5.37 mmol). The target com-
pound 5g (907 mg) was obtained as yellow solid. Yield: 25.4%; mp:
(TOF) calcd for
628.1963.
C
33H31Cl2N5O6: [MꢀCl]+, 628.1957; found,
4.1.23. N-(3-Chlorobenzyl)-N-(2-(5-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5j)
Compound 5j was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (0.89 g,
2.49 mmol) and compound 4j (0.85 g, 2.47 mmol). The pure com-
pound 5j (554 mg) was obtained as yellow solid. Yield: 33.5%;
201–203 °C; IR (KBr)
1568, 1506, 1474 (aromatic skeleton), 1368, 1341, 1270, 1230,
1117, 1035, 931, 824 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.64
m: 3053 (Ar–H), 2922, 2850 (CH2, CH3), 1601,
;
(s, 1H, Ber 8-H), 8.76 (s, 1H, Ber 13-H), 7.79 (s, 1H, Im 4-H), 8.05
(d, J = 9.0 Hz, 1H, Ber 12-H), 7.98 (d, J = 9.0 Hz, 1H, Ber 11-H),
7.64 (s, 1H, Im 2-H), 7.64 (s, 1H, Ber 1-H), 7.23–6.91 (m, 4H, 4-H,
Ph 3,5,6-H), 6.11 (s, 2H, OCH2O), 4.94 (br, 2H, Ber 6-H), 4.37 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 4.32 (t, J = 6.2 Hz, 2H, Im-CH2), 4.04 (s,
3H, OCH3), 3.77 (s, 2H, Ph–CH2), 3.25 (br, 2H, Ber 5-H), 3.09 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 2.91 (t, J = 6.2 Hz, 2H, Im-CH2CH2)
ppm; 13C NMR (75 MHz, CD3OD): d 163.7, 163.3, 162.1, 161.5,
161.0, 160.6, 159.3, 158.4, 152.1, 147.7, 146.2, 145.9, 144.3,
143.0, 142.9, 136.0, 135.2, 133.4, 132.3, 132.1, 131.1, 126.0,
123.0, 120.9, 119.4, 119.2, 118.9, 118.6, 118.2, 117.9, 111.5,
108.9, 104.3, 104.1, 103.8, 102.1, 72.3, 58.4, 57.1, 53.2, 51.4, 46.2,
28.5, 27.3 ppm; MS (m/z): 631 [MꢀCl]+; HRMS (TOF) calcd for
mp: 227–229 °C; IR (KBr)
1600, 1534, 1505, 1477 (aromatic skeleton), 1371, 1350, 1270,
1229, 1101, 1037, 928, 869 cmꢀ1 1H NMR (300 MHz, CD3OD): d
m: 3023 (Ar–H), 2969, 2816 (CH2, CH3),
;
9.59 (s, 1H, Ber 8-H), 8.69 (s, 1H, Ber 13-H), 8. 04 (d, J = 9.0 Hz,
1H, Ber 11-H), 7. 97 (d, J = 9.0 Hz, 2H, Ber 12-H), 7.89 (s, 1H, Im
4-H), 7.65 (s, 1H, Im 2-H), 7.70 (s, 1H, Ber 1-H), 7.16–7.02 (m,
3H, Ph 2,5,6-H), 6.96 (s, 1H, Ber 4-H), 6.12 (s, 2H, OCH2O), 4.91
(br, 2H, Ber 6-H), 4.41 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.37 (t,
J = 6.2 Hz, 2H, Im-CH2), 4.05 (s, 3H, OCH3), 3.76 (s, 2H, Ph–CH2),
3.31 (br, 2H, Ber 5-H), 3.09 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.89 (t,
J = 6.0 Hz, 2H, Im-CH2CH2) ppm; 13C NMR (75 MHz, CD3OD): d
150.6, 148.3, 145.7, 145.3, 145.1, 143.5, 143.2, 138.3, 136.2,
134.0, 133.4, 131.1, 130.4, 126.8, 126.0, 124.1, 123.5, 121.3,
118.9, 118.4, 117.9, 117.3, 112.4, 109.5, 102.6, 72.4, 58.9, 57.8,
52.6, 51.3, 45.7, 27.8, 26.7 ppm; MS (m/z): 628 [MꢀCl]+; HRMS
C
33H30ClF2N5O6: [MꢀCl]+, 630.2159; found, 630.2156.
4.1.21. N-(3,4-Dichlorobenzyl)-N-(2-(5-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5h)
Compound 5h was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (1.90 g,
4.50 mmol) and compound 4h (1.59 g, 4.44 mmol). The target
compound 5h (851 mg) was obtained as yellow solid. Yield:
(TOF) calcd for
628.1954.
C
33H31Cl2N5O6: [MꢀCl]+, 628.1957; found,
4.1.24. N-(4-Chlorobenzyl)-N-(2-(5-nitro-1H-imidazol-1-
yl)ethyl)-2-(2-berberinoxyethoxy)ethanamine (5k)
Compound 5k was prepared according to the procedure de-
scribed for compound 5a starting from berberrubine (0.55 g,
1.54 mmol) and compound 4k (0.60 g, 1.55 mmol). The target com-
pound 5k (310 mg) was obtained as yellow solid. Yield: 30.3%; mp:
27.1%; mp: 185–187 °C; IR (KBr)
(CH2, CH3), 1601, 1570, 1506, 1475 (aromatic skeleton), 1367,
1342, 1273, 1231, 1117, 1102, 1036, 975, 825 cmꢀ1 1H NMR
m: 3018 (Ar–H), 2922, 2850
;
(300 MHz, CD3OD): d 9.59 (s, 1H, Ber 8-H), 8.75 (s, 1H, Ber 13-H),
8.05 (d, J = 9.0 Hz, 1H, Ber 12-H), 7.98 (d, J = 9.0 Hz, 1H, Ber 11-
H), 7.88 (s, 1H, Im 4-H), 7.71 (s, 1H, Im 2-H), 7.67 (s, 1H, Ber 1-
H), 7.45 (br, 1H, Ph 5-H), 7.20–7.11 (m, 2H, Ph 2,6-H), 6.95 (s, 1H,
Ber 4-H), 6.13 (s, 2H, OCH2O), 4.92 (br, 2H, Ber 6-H), 4.41 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 4.35 (t, J = 6.2 Hz, 2H, Im-CH2), 4.03 (s,
3H, OCH3), 3.74 (s, 2H, Ph–CH2), 3.22 (br, 2H, Ber 5-H), 3.10 (t,
J = 6.0 Hz, 2H, OCH2CH2N), 2.90 (t, J = 6.2 Hz, 2H, Im-CH2CH2)
ppm; 13C NMR (75 MHz, CD3OD): d 150.5, 148.4, 145.7, 145.4,
145.2, 143.6, 143.2, 136.4, 136.0, 133.4, 133.1, 132.2, 131.3,
130.8, 128.5, 126.0, 125.3, 123.0, 120.9, 119.4, 118.8, 118.1,
116.5, 108.9, 102.5, 72.2, 58.6, 57.2, 53.3, 51.2, 46.1, 28.3,
27.2 ppm; MS (m/z): 663 [MꢀCl]+. HRMS (TOF) calcd for
173–174 °C; IR (KBr)
1542, 1506, 1479 (aromatic skeleton), 1384, 1347, 1273, 1230,
1101, 1038, 937, 825 cmꢀ1 1H NMR (300 MHz, CD3OD): d 9.57
m: 3044 (Ar–H), 2922, 2845 (CH2, CH3), 1600,
;
(s, 1H, Ber 8-H), 8.73 (s, 1H, Ber 13-H), 7.84 (s, 1H, Im 4-H), 8.06
(d, J = 9.0 Hz, 1H, Ber 12-H), 7.96 (d, J = 9.0 Hz, 1H, Ber 11-H),
7.71 (s, 1H, Im 2-H), 7.67 (s, 1H, Ber 1-H), 7.24–7.17 (m, 4H, Ph
2,3,5,6-H), 6.95 (s, 1H, Ber 4-H), 6.13 (s, 2H, OCH2O), 4.87 (br, 2H,
Ber 6-H), 4.39 (t, J = 6.0 Hz, 2H, OCH2CH2N), 4.34 (t, J = 6.2 Hz, 2H,
Im-CH2), 4.05 (s, 3H, OCH3), 3.68 (s, 2H, Ph–CH2), 3.19 (br, 2H,
Ber 5-H), 3.09 (t, J = 6.0 Hz, 2H, OCH2CH2N), 2.91 (t, J = 6.2 Hz, 2H,
Im-CH2CH2) ppm; 13C NMR (75 MHz, CD3OD): d 150.6, 148.3,
145.7, 145.5, 145.2, 142.8, 142.2, 136.4, 134.5, 133.7, 133.4,
C
33H30Cl3N5O6: [MꢀCl]+, 662.1568; found, 662.1563.

L. Zhang et al. / Bioorg. Med. Chem. 21 (2013) 4158–4169
4169
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(m/z): 628 [MꢀCl]+; HRMS (TOF) calcd for C33H31Cl2N5O6: [MꢀCl]+,
628.1957; found, 628.1949.
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at 190 °C in a vacuum oven under reduced pressure (20 mm Hg) for
15 min to obtain berberrubine 7 (25.51 g) in 88.4% yield in agree-
ment with the literature.23
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Acknowledgments
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This work was partially supported by National Natural Science
Foundation
of
China
[Nos.
21172181,
81250110089,
81250110554 (the Research Fund for International Young Scien-
tists from International (Regional) Cooperation and Exchange Pro-
gram)], 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), the Research Funds for the Central Universities
(Nos. XDJK2011D007, XDJK2012B026).
27. Fang, B.; Zhou, C. H.; Rao, X. C. Eur. J. Med. Chem. 2010, 45, 4388.
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Supplementary data
29. Karegoudar, P.; Prasad, D. J.; Ashok, M.; Mahalinga, M.; Poojary, B.; Holla, B. S.
Eur. J. Med. Chem. 2008, 43, 808.
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2011, 48, 114.
Supplementary data (NMR spectra for all of the synthesized
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.05.007.
32. Mallia, M. B.; Subramanian, S.; Mathur, A.; Sarma, H. D.; Venkatesh, M.;
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