Novel berberine triazoles: Synthesis, antimicrobial evaluation and competitive interactions with metal ions to Human Serum Albumin

Article abstract of DOI:10.1016/j.bmcl.2012.12.036

A series of novel berberine triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All target compounds and their precursors were screened for antimicrobial activities in vitro against four Gram-positive bacteria, four Gram-negative bacteria and two fungal strains. Bioactive assay indicated that most of the prepared compounds exhibited good antibacterial and antifungal activities 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. The competitive interactions between compound 5a and metal ions to Human Serum Albumin (HSA) revealed that the participation of Mg²⁺ and Fe³⁺ ions in compound 5a-HSA association could result in the concentration increase of free compound 5a, shorten the storage time and half-life of compound 5a in the blood, thus improving its antimicrobial efficacy.

Full text of DOI:10.1016/j.bmcl.2012.12.036

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1008–1012
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel berberine triazoles: Synthesis, antimicrobial evaluation and
competitive interactions with metal ions to Human Serum Albumin
Shao-Lin Zhang a, Juan-Juan Chang a, Guri L.V. Damu a, , Bo Fang a, Xiang-Dong Zhou b, Rong-Xia Geng a,
,
⇑
Cheng-He Zhou a,
⇑
a Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
b School of Pharmaceutical Sciences, Third Military Medical University, Chongqing 400038, 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 berberine triazoles were synthesized and characterized by IR, NMR, MS and HRMS spec-
tra. All target compounds and their precursors were screened for antimicrobial activities in vitro against
four Gram-positive bacteria, four Gram-negative bacteria and two fungal strains. Bioactive assay indi-
cated that most of the prepared compounds exhibited good antibacterial and antifungal activities with
Received 29 September 2012
Revised 26 November 2012
Accepted 12 December 2012
Available online 20 December 2012
low MIC values ranging from 2 to 64 lg/mL, which were comparable to or even better than the reference
drugs Berberine, Chloromycin, Norfloxacin and Fluconazole. The competitive interactions between com-
pound 5a and metal ions to Human Serum Albumin (HSA) revealed that the participation of Mg2+ and Fe3+
ions in compound 5a–HSA association could result in the concentration increase of free compound 5a,
shorten the storage time and half-life of compound 5a in the blood, thus improving its antimicrobial
efficacy.
Keywords:
Berberine
1,2,4-Triazole
Antibacterial
Antifungal
Ó 2012 Elsevier Ltd. All rights reserved.
Human Serum Albumin
Berberine is a well-known natural isoquinoline alkaloid which
is mainly found in roots and rhizomes of several plants such as
berberidaceae, ranunculaceae and papaveraceae.1 In recent years,
it has been demonstrated to possess a variety of pharmacological
activities like antimicrobial,2 antineoplastic,3 antiviral,4 antiinflam-
matory,5 antiprotozoal,6 antidiarrheal,7 antileishmanial8 ones, etc.
Especially as antimicrobial agent, berberine has been playing a
quite important role in the treatment of intestinal infections such
as acute gastroenteritis, cholera and bacillary dysentery. This has
attracted numerous efforts towards the development of novel ber-
berine derivatives with medicinal applications. So far a large
amount of work has focused on the C-8, C-9 and C-13 positions
of berberine by incorporating various types of substituents includ-
ing aliphatic chains and heterocycles such as thiophene, piperidine
and so on.9 Noticeably, some lipophilic substituents at C-9 position
could helpfully enhance the antimicrobial activities and broaden
antibacterial spectrum,10 which overwhelmingly compels us with
great interest to further structurally modify berberine in order to
discover new antimicrobial agents with more efficient and broad
spectrum. In spite that various heterocyclic rings like thiophene,
pyrrole, piperidine, carbazole, etc. have been extensively intro-
duced into berberine, however, to our best knowledge, the combi-
nation of berberine with 1,2,4-triazole has been seldom studied. In
view of this, herein we combined berberine and 1,2,4-triazole to af-
ford a series of hybrids as a new type of Fluconazole analogues.
It is well known that Fluconazole as the first-line triazole anti-
fungal drug has been attracting special attention for its exceptional
therapeutic records against fungal infections.11 However, the fre-
quent emergence of increasingly serious Fluconazole-resistant
pathogens and its narrow antifungal spectrum12 decreased its
therapeutic efficacy, thus numerous researches were directed to-
wards the further investigations of Fluconazole in order to increase
its therapeutic indexes and broaden antimicrobial spectrum.13–15
Recently, we synthesized a series of novel halobenzyl amine bis-
azoles as new structural analogues of Fluconazole, in which ter-
tiary alcohol was replaced by tertiary amino moiety, the methylene
bridge between tertiary alcohol and triazolyl moieties was substi-
tuted by ethylene chain. Bioactive evaluation showed that this kind
of Fluconazole analogues exhibited good antibacterial and anti-
fungal activities which were comparable to or even better than
the reference drugs Chloromycin, Norfloxacin and Fluconazole.16
This strongly encourages us with special interest to further explore
this type of new skeleton compounds as potential antimicrobial
agents.
⇑
Corresponding authors. Tel./fax: +86 23 68254967 (C.-H.Z.).
E-mail addresses: geng0712@swu.edu.cn (R.-X. Geng), zhouch6848@sina.com
(C.-H. Zhou).
Based on above observations, our target compounds were de-
signed from the following considerations:
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.036

S.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1008–1012
1009
(1) Our previous work revealed that halobenzyl amine bis-
azoles as new structural analogues of Fluconazole possessed
possibility as antimicrobial drugs, thus benzyl amine ethyl
triazole skeleton in the target molecules should be beneficial
to antimicrobial potency.
(2) Berberine as an important antibacterial drug has been play-
ing significant roles in clinic for the treatment of infectious
diseases, while triazole ring possesses unique merits in anti-
microbial especially antifungal aspect. Rationally, it was of
great interest for us to combine berberine fragment and tri-
azole moiety to yield a series of hybrids, which were
expected to increase bioactivities and extend antimicrobial
spectrum.
protein, has many important physiological and pharmacological
functions.19 When drugs bind reversibly to HSA, it could be deliv-
ered to the binding sites. However, some metal ions in plasma
could affect the binding affinity of drugs with HSA, thus causing
the changes of the binding constant of the drug–HSA complex. In
view of this, investigating the interactions of metal ions with
drug–protein association may provide useful information for
transportation, distribution, metabolism of drugs.20 Therefore, it
was necessary for us to study the competitive interactions between
compound 5a and metal ions to HSA.
The synthetic route of berberine triazoles was outlined in
Scheme 1. The desired target compounds were synthesized via
multistep reactions from commercially available halobenzyl chlo-
rides, diethanolamine, triazole and berberine. Intermediates 2a–h
could be efficiently prepared by N-alkylation of different haloben-
zyl chlorides 1a–h with diethanolamine in acetonitrile, then diol
compounds 2a–h were brominated under reflux in chloroform by
phosphorus tribromide to produce the bromides 3a–h according
to our previous procedures.16 Compounds 3a–h were treated with
1,2,4-triazole in the presence of potassium carbonate at 30 °C to af-
ford their corresponding triazoles 4a–h. The berberine chloride
was demethylated at 190 °C under reduced pressure (20 mm Hg)
for 2 h to produce berberrubine in 88.2% yield which was in agree-
ment with literature.4 The latter was reacted with compounds
4a–h in DMF at 110 °C for 20 h approximately to afford the target
(3) Berberine with a quaternary nitrogen and desirable large
p
-conjugated backbone might easily interact with various
active targets in biological system by non-covalent forces
such as stacking and electronic interactions. These were
p–p
helpful to improve physicochemical properties of target
molecules and their affinity to transport proteins, thus
enhancing their antimicrobial activities.
(4) It was disclosed that halobenzyl moiety could greatly influ-
ence the pharmacological properties of drugs by affecting
the rate of absorption and transport in vivo.17 Reasonably,
various halobenzyl moieties were incorporated into the tar-
get molecules to survey their contributions to antimicrobial
efficacy.
compounds 5a–h. Subsequently, hydrochloride
6 was easily
(5) Literature evidenced that the transformation of azoles into
their corresponding salts could enhance antimicrobial effi-
cacy due to the improvement of water solubility.18 Thus,
the selected berberine triazole was converted into hydro-
chloride to test its antibacterial and antifungal activities.
prepared by the reaction of compound 5a with 4 M hydrochloric
acid. All the new compounds were characterized by 1H NMR, 13C
NMR, IR, MS and HRMS spectra.21
All the newly synthesized compounds were evaluated for their
in vitro antimicrobial activities against eight bacteria and two fun-
gi using the standard two folds serial dilution method in 96-well
microtest plates according to the National Committee for Clinical
Laboratory Standards (NCCLS).22 Minimal inhibitory concentration
In continuation of our ongoing interest in the development of
new antimicrobial agents, herein the berberine triazoles 5 and 6
were designed and synthesized. Their antimicrobial activities were
also evaluated in vitro. Further studies about the competitive inter-
actions between compound 5a and metal ions to Human Serum
Albumin (HSA) were explored. HSA, as an important transport
(MIC,
lg/mL) was defined as the lowest concentration of new
compounds that completely inhibited the growth of bacteria.
Currently available antimicrobial drugs Berberine, Chloromycin,
Norfloxacin and Fluconazole were used as the positive control.
Cl
N
N
N
OH
OH
Br
Br
R1
R2
Br
R1
R2
R1
R2
R1
R2
a
b
c
N
N
N
R3
R3
R3
1
2
R3
3
4
O
O
Cl
Cl
N
N
d
O
O
e
OCH3
OCH3
OH
OCH3
N
N
N
N
F
O
O
O
N
N
•2HCl
Cl
N
Cl
N
f
N
R1
O
N
O
R2
R3
O
5
6
OCH3
F
OCH3
1-5: a, R1 = F, R2 = H, R3 = F, b, R1 = F, R2 = H, R3 = H
c, R1 = H, R2 = F, R3 = H, d, R1 = Cl, R2 = H, R3 = Cl
e, R1 = H, R2 = Cl, R3 = Cl f, R1 = Cl, R2 = H, R3 = H
g, R1 = H, R2 = Cl, R3 = H h, R1 = H, R2 = H, R3 = Cl
Scheme 1. Reagents and conditions: (a) CH3CN, diethanolamine, 50 °C, 10–12 h; yields 92–98%; (b) CHCl3, PBr3, refluxed, 2 h; yields 88–98%; (c) 1,2,4-triazole, K2CO3, CH3CN,
rt to 50 °C, 12 h; yields 40–63%; (d) berberine, 20 mm Hg, 190 °C, 15 min; (e) DMF, 110 °C, 20 h; (f) 4 M HCl, diethyl ether/chloroform, 30 °C, 0.5 h.

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S.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1008–1012
Table 1
In vitro antibacterial activity for compounds 4–6 expressed as MIC (lg/mL)
Compds
Gram-positive bacteria
Gram-negative bacteria
S. dysenteriae P. aeruginosa
MRSA
S. aureus
B. subtilis
M. luteus
E. coli
P. vulgaris
4a
4b
4c
4d
4e
4f
4g
4h
128
128
128
256
256
256
256
256
8
64
64
64
64
64
128
128
128
4
256
256
256
256
256
256
256
256
8
32
64
64
32
32
64
64
512
8
32
32
64
32
32
64
128
128
4
64
64
64
128
128
256
256
256
4
128
128
128
128
128
128
256
256
8
128
256
256
128
256
512
>512
512
2
5a
5b
8
4
8
8
4
4
8
4
5c
5d
5e
5f
5g
5h
6
8
8
8
8
8
8
8
16
2
8
8
8
8
16
8
4
16
16
16
32
32
16
8
8
4
8
8
8
16
2
4
8
16
32
32
8
8
16
16
32
32
16
4
4
8
8
16
32
32
2
16
16
32
16
4
2
Chloromycin
Norfloxacin
Berberine
16
8
128
16
0.5
512
32
1
>512
8
2
>512
32
16
>512
32
4
256
32
16
256
32
8
128
MRSA, 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.
Table 2
32
Effect of metal ions on the binding constants of compound 5a–HSA complex at 298 K
a
Systems
105 Kb (L/mol)
Kb/K0
Rb
S.D.c
28
24
20
16
12
8
HSA–compound 5a
1.079
1.032
1.077
1.049
1.017
0.953
0.905
1.039
0.940
1.204
1.148
1.00
0.990
0.994
0.993
0.994
0.991
0.994
0.998
0.992
0.992
0.993
0.996
0.146
0.116
0.122
0.123
0.129
0.093
0.036
0.106
0.089
0.139
0.100
C. albicans
HSA–compound 5a–Ca2+
HSA–compound 5a–Ce3+
HSA–compound 5a–Cu2+
HSA–compound 5a–K+
HSA–compound 5a–La3+
HSA–compound 5a–Ni2+
HSA–compound 5a–Pb2+
HSA–compound 5a–Zn2+
HSA–compound 5a–Fe3+
HSA–compound 5a–Mg2+
0.956
0.998
0.972
0.943
0.883
0.839
0.963
0.871
1.116
1.064
C. mycoderma
4
a
K0 is the binding constant of compound 5a–HSA complex in the absence of
metal ions; Kb is the binding constants of compound 5a–HSA complex with metal
0
ions.
Fluconazole
5a 5b 5c 5d 5e 5f 5g 5h
Compounds
6
b
R is the correlation coefficient.
SD is the standard deviation.
c
Figure 1. In vitro antifungal activity for compounds 5 and 6 expressed as MIC (
mL); C. albicans, Candida albicans; C. mycoderma, Candida mycoderma.
lg/
and distribution in organism. Noticeably, compounds 5a–d
(MIC = 8
ence drugs Chloromycin and Berberine against MRSA, respectively,
and equipotent to Norfloxacin (MIC = 8 g/mL). All these indicated
that compound 5a could be employed to further develop novel po-
tent board-spectrum antimicrobial agents.
Compound 5a was transformed into water soluble hydrochlo-
ride 6, which was expected to improve its antimicrobial efficacy.
The bioactive results manifested that hydrochloride 6 showed
remarkable antibacterial and antifungal activities with MIC values
lg/mL) were 2- and 16-fold more potent than the refer-
l
The antibacterial results were shown in Table 1. It was observed
that all the newly prepared berberine triazoles 5 and 6 could effec-
tively inhibit the growth of the tested strains to some extent.
For the tested berberine triazoles 5a–h, all displayed good
inhibitory efficacy towards Gram-positive, Gram-negative bacteria
and fungi. Compound 5a with 2,4-difluorobenzyl moiety gave
strong antibacterial activity (MIC = 2–8
tested bacteria, which was comparable to or even better than the
reference drugs, especially for Proteus vulgaris (MIC = 2 g/mL), it
was 4-, 16- and 64-fold more potent than the reference drugs Nor-
floxacin (MIC = 8 g/mL), Chloromycin (MIC = 32 g/mL) and Ber-
berine (MIC = 128 g/mL), respectively. Meanwhile, compound 5a
was also sensitive to fungus Candida mycoderma with MIC value
of 2 g/mL, which was twofold more effective than that of Fluco-
lg/mL) against all the
ranging from 2 to 8
for Escherichia coli and Shigella dysenteriae, compound 6 exhibited
low inhibitory concentration of 2 g/mL, which was twofold better
lg/mL against all the tested strains. Especially
l
l
than its precursor 5a, and 2- to 16-fold more efficient than Chloro-
mycin and Norfloxacin. Moreover, compound 6 also exerted anti-
fungal efficacy against Candida albicans and Candida mycoderma,
respectively. The enhanced activities of compound 6 might be
attributed to the improved water solubility in comparison with
its corresponding precursor 5a.
HSA is a major depot and transport protein which could bind,
transport and deliver drugs to their target organs. However, there
are some metal ions in plasma which can form complexes with
l
l
l
l
nazole (Fig. 1). These results demonstrated that the existence of
fluorine atom on the benzyl moieties in this series of berberine tri-
azoles should be of special importance in microbial inhibition
probably due to its easy and efficient formation of non-covalent
forces which could be helpful for the biological transportation

S.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1008–1012
1011
complex, suggesting the presence of these metal ions could
increase the concentration of free compound 5a, shorten the
storage time and half-life of compound 5a in the blood, and
thus improving its antimicrobial efficacy.
In summary, a series of novel berberine triazoles were success-
fully prepared through an easy, convenient and economic synthetic
procedure. Their structures were characterized by IR, NMR, MS and
HRMS spectra. The in vitro antimicrobial evaluation showed that
most synthesized berberine triazoles could effectively inhibit the
growth of all the tested bacteria and fungi strains, some even dis-
played excellent antimicrobial activities in contrast to their posi-
tive control. Particularly, compound 5a with 2,4-difluorobenzyl
group and its corresponding hydrochloride 6 gave the most excel-
1500
1200
900
600
300
0
HSA
2+
HSA+Ca
a
i
Compound 5a
lent antibacterial and antifungal efficacies (MIC = 2–8 lg/mL),
which suggested that further investigations were necessary to
optimize these potential compounds as more efficacious antimi-
crobial agents. These results showed that the combination of ber-
berine and triazole moiety was greatly helpful for the
antimicrobial activities. Competitive interactions indicated that
the participation of Mg2+ and Fe3+ ions in compound 5a–HSA com-
plex could increase the concentration of free compound 5a, short-
en the storage time and half-life of compound 5a in the blood, and
thus improving its antimicrobial efficacy. Other related researches
including the binding behaviors (thermodynamic properties, bind-
ing parameters and interactions mechanism, etc.) and docking
studies between compound 5a and HSA, the effect factors on anti-
microbial activities of the target compounds such as other hetero-
cyclic azoles (benzotriazole, imidazole, benzimidazole and their
derivatives, etc.), toxicity investigation along with in vivo bioactive
evaluation are active in progress in our group. All these will be re-
ported in the future full paper.
330
360
390
420
450
Wavelength (nm)
Figure 2. Emission spectra of HSA+Ca2+ in the presence of various concentrations of
compound 5a. c(HSA) 1.0 Â 10À5 mol/L; c(Ca2+)/c(HSA) = 1:1; c(compound 5a)/
(10À5 mol/L), a–i: from 0.0 to 2.0 at increments of 0.25; dash line shows the
emission spectrum of compound 5a only; T = 298 K, kex = 295 nm.
HSA
3+
1500
1200
900
600
300
0
HSA+Fe
a
i
Acknowledgments
This work was partially supported by National Natural Sci-
ence Foundation of China [No. 21172181, 81250110089 (The Re-
search Fellowship for International Young Scientists from
International (Regional) Cooperation and Exchange Program)], the
key program from Natural Science Foundation of Chongqing
(CSTC2012jjB10026), the Specialized Research Fund for the
Doctoral Program of Higher Education of China (SRFDP
20110182110007).
Compound 5a
330
360
390
420
450
Wavelength (nm)
Figure 3. Emission spectra of HSA+Fe3+ in the presence of various concentrations of
compound 5a. c(HSA) 1.0 Â 10À5 mol/L; c(Fe3+)/c(HSA) = 1:1; c(compound 5a)/
(10À5 mol/L), a–i: from 0.0 to 2.0 at increments of 0.25; dash line shows the
emission spectrum of compound 5a only; T = 298 K, kex = 295 nm.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.
036.
HSA, thus influencing the binding affinity between drug and albu-
min. Therefore, the effect of metal ions including major elements
References and notes
like Ca2+, K+, Mg2+ ions and trace elements such as Cu2+, Zn2+
,
Ni2+ and Fe3+ ions on the binding constant of compound 5a–HSA
complex was investigated at 298 K (Supplementary data). As evi-
dent from Table 2, the competition between the metal ions and
compound 5a led to the binding constants of compound 5a–HSA
complex ranging from 83.9% to 111.6% of the values of binding
constant in absence of metal ions. The altered binding constants
might be explained by the conformational changes of HSA struc-
ture caused by binding to metal ions, which in turn affected pro-
tein binding to drugs. Clearly, the presence of metal ions such as
Ca2+, Ce3+, Cu2+, K+, La3+, Ni2+, Pb2+ and Zn2+ ones decreased the
binding constants of compound 5a–HSA complex, thus causing
compound 5a to be quickly cleared from blood,23 which may lead
to more doses of compound 5a to achieve the desired therapeutic
effectiveness (Fig. 2). While the participation of Mg2+ and Fe3+ ions
(Fig. 3) increased the binding constants of compound 5a–HSA
1. Grycová, L.; Dostál, J.; Marek, R. Phytochemistry 2007, 68, 150.
2. (a) Samosorn, S.; Tanwirat, B.; Muhamad, N.; Casadei, G.; Tomkiewicz, D.;
Lewis, K.; Suksamrarn, A.; Prammananan, T.; Gornall, K. C.; Beck, J. L.; Bremner,
J. B. Bioorg. Med. Chem. 2009, 17, 3866; (b) Park, K. D.; Lee, J. H.; Kim, S. H.; Kang,
T. H.; Moon, J. S.; Kim, S. U. Bioorg. Med. Chem. Lett. 2006, 16, 3913; (c) Park, K.
D.; Cho, S. J.; Moon, J. S.; Kim, S. U. Bioorg. Med. Chem. Lett. 2010, 20, 6554.
ˇ
ˇ
3. (a) Letašiová, S.; Jantová, S.; Cipák, L.; Múcková, M. Cancer Lett. 2006, 239, 254;
(b) Kuo, H. P.; Chuang, T. C.; Yeh, M. H.; Hsu, S. C.; Way, T. D.; Chen, P. Y.; Wang,
S. S.; Chang, Y. H.; Kao, M. C.; Liu, J. Y. J. Agric. Food Chem. 2011, 59, 8216; (c) Ma,
Y.; Ou, T. M.; Tan, J. H.; Hou, J. Q.; Huang, S. L.; Gu, L. Q.; Huang, Z. S. Bioorg. Med.
Chem. Lett. 2009, 19, 3414.
4. Bodiwala, H. S.; Sabde, S.; Mitra, D.; Bhutani, K. K.; Singh, I. P. Eur. J. Med. Chem.
2011, 46, 1045.
5. (a) Kuo, C. L.; Chi, C. W.; Liu, T. Y. Cancer Lett. 2004, 203, 127; (b) Gautam, R.;
Jachak, S. M. Med. Res. Rev. 2009, 29, 767.
6. Bahar, M.; Deng, Y.; Zhu, X. H.; He, S. S.; Pandharkar, T.; Drew, M. E.; Navarro-
Vázquez, A.; Anklin, C.; Gil, R. R.; Doskotch, R. W.; Werbovetz, K. A.; Kinghorn,
A. D. Bioorg. Med. Chem. Lett. 2011, 21, 2606.
7. Wongbutdee, J. Thai Pharm. Health Sci. J. 2008, 4, 78.

1012
S.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1008–1012
8. Vennerstrom, J. L.; Lovelace, J. K.; Waits, V. B.; Hanson, W. L.; Klayman, D. L.
Antimicrob. Agents Chemother. 1990, 34, 918.
9. (a) Shan, W. J.; Huang, L.; Zhou, Q.; Meng, F. C.; Li, X. S. Eur. J. Med. Chem. 2011,
46, 5885; (b) Ma, Y.; Ou, T. M.; Hou, J. Q.; Lu, Y. J.; Tan, J. H.; Gu, L. Q.; Huang, Z.
S. Bioorg. Med. Chem. 2008, 16, 7582.
(3 Â 30 mL), then the combined organic extracts were dried over anhydrous
sodium sulfate. Subsequently the crude products were purified by column
chromatography eluting with ethylacetate/petroleum (1/2, v/v) to afford the
target compound 4a as oil (1.15 g). Yield: 44.7%. 1H NMR (400 MHz, CDCl3): d
8.14 (s, 1H, Tri 3-H), 7.86 (s, 1H, Tri 5-H), 7.12–6.75 (m, 3H, Ph 3,5,6-H), 4.17 (br
s, 4H, Tri-CH2), 3.64 (s, 2H, Ph-CH2), 3.20 (br s, 2H, Br-CH2), 2.95 (br s, 2H, Tri-
CH2CH2), 2.87 (br s, 2H, Br-CH2CH2) ppm; 13C NMR (100 MHz, CDCl3): d 162.36,
161.17, 151.85, 143.86, 131.76, 120.69, 111.35, 103.88, 56.19, 53.49, 51.17,
48.36, 29.59 ppm; MS (m/z): 346 [M+H]+.
Synthesis of [[N-(2-(1H-1,2,4-triazol-1-yl)ethyl)-N-(2,4-difluoro benzyl)-2-(2-
berberinoxyethoxy) ethanamine] (5a). To a stirred solution of berberrubine
(700 mg, 1.95 mmol) in DMF (5 mL) at 110 °C was added dropwise compound
4a (661 mg, 1.92 mmol) in DMF (5 mL). The reaction was stirred for 20 h and
monitored by TLC (chloroform/methanol, 20/1, v/v). After the reaction come to
the end, the mixture was cooled to room temperature and extracted with
chloroform (3 Â 30 mL). The organic phase was washed with water and dried
over anhydrous sodium sulfate. After the filtrate was concentrated, the crude
product was purified by column chromatography eluting with chloroform/
methanol (20/1, v/v) to afford the compound 5a (140 mg) as yellow solid.
Yield: 36.2%, mp: 210–212 °C; IR (KBr, cmÀ1): 3121, 3052 (Ar-H), 2931, 2857
(CH2, CH3), 1608, 1581, 1560, 1502, 1481, 1388, 1360, 1301, 1142, 1102, 961,
853; 1H NMR (400 MHz, CD3OD): d 9.57 (s, 1H, 8-H), 8.70 (s, 1H, 13-H), 8.47 (s,
1H, Tri 3-H), 8.08 (d, J = 8.8 Hz, 1H, 11-H), 7.98 (d, J = 8.8 Hz, 1H, 12-H), 7.86 (s,
1H, Tri 5-H), 7.66 (s, 1H, 1-H), 7.22–6.82 (m, 3H, Ph 3,5,6-H), 6.97 (s, 1H, 4-H),
6.11 (s, 2H, OCH2O), 4.92 (t, J = 6.0 Hz, 2H, 6-H), 4.39 (t, J = 6.0 Hz, 4H, OCH2CH2,
Tri-CH2), 4.06 (s, 3H, OCH3), 3.79 (s, 2H, Ph-CH2), 3.27 (t, J = 6.0 Hz, 2H, 5-H),
3.06 (t, J = 6.0 Hz, 4H, OCH2CH2, Tri-CH2CH2) ppm; 13C NMR (100 MHz, CD3OD):
10. (a) Fang, B.; Zhou, C. H.; Zhou, X. D. J. Int. Pharm. Res. 2009, 37, 105 (in Chinese);
(b) Li, B.; Zhu, W. L.; Chen, K. X. Acta Pharmacol. Sin. 2008, 43, 773; (c) Li, Y. H.;
Li, Y.; Yang, P.; Kong, W. J.; You, X. F.; Ren, G.; Deng, H. B.; Wang, Y. M.; Wang, Y.
X.; Jiang, J. D.; Song, D. Q. Bioorg. Med. Chem. 2010, 18, 6422; (d) Huang, L.; Luo,
Z. H.; He, F.; Shi, A. D.; Qin, F. F.; Li, X. S. Bioorg. Med. Chem. Lett. 2010, 20, 6649.
11. (a) Zhou, C. H.; Wang, Y. Curr. Med. Chem. 2012, 19, 239; (b) Wan, K.; Zhang, Y.
Y.; Zhou, C. H.; Zhou, X. D.; Geng, R. X.; Ji, Q. G. Chin. J. Antibiot. 2012, 37, 1 (in
Chinese).
12. Wang, Y.; Zhou, C. H. Sci. Sin. Chim. 2011, 41, 1429 (in Chinese).
13. (a) Zhou, C. H.; Gan, L. L.; Zhang, Y. Y.; Zhang, F. F.; Wang, G. Z.; Jin, L.; Geng, R.
X. Sci. China, Ser. B: Chem. 2009, 52, 415; (b) Zhou, C. H.; Zhang, F. F.; Gan, L. L.;
Zhang, Y. Y.; Geng, R. X. Sci. China, Ser. B: Chem. 2009, 39, 208 (in Chinese); (c)
Chang, J. J.; Wang, Y.; Zhang, H. Z.; Zhou, C. H.; Geng, R. X.; Ji, Q. G. Chem. J. Chin.
U 2011, 32, 1970 (in Chinese).
14. Zhang, Y. Y.; Mi, J. L.; Zhou, C. H.; Zhou, X. D. Eur. J. Med. Chem. 2011, 46, 4391.
15. (a) Sheng, C. Q.; Zhang, W. N.; Ji, H. T.; Zhang, M.; Song, Y. L.; Xu, H.; Zhu, J.;
Miao, Z. Y.; Jiang, Q. F.; Yao, J. Z.; Zhou, Y. J.; Zhu, J.; Lv, J. G. J. Med. Chem. 2006,
49, 2512; (b) Sheng, C. Q.; Zhang, W. N. Curr. Med. Chem. 2011, 18, 733.
16. Fang, B.; Zhou, C. H.; Rao, X. C. Eur. J. Med. Chem. 2010, 45, 4388.
17. (a) Zhang, F. F.; Gan, L. L.; Zhou, C. H. Bioorg. Med. Chem. Lett. 2010, 20, 1881; (b)
Wang, X. L.; Wan, K.; Zhou, C. H. Eur. J. Med. Chem. 2010, 45, 4631; (c) Zhang, Y.
Y.; Zhou, C. H. Bioorg. Med. Chem. Lett. 2011, 21, 4349.
18. (a) Gan, L. L.; Zhou, C. H. Bull. Korean Chem. Soc. 2010, 31, 3684; (b) Shi, Y.;
Zhou, C. H. Bioorg. Med. Chem. Lett. 2011, 21, 956; (c) Luo, Y.; Lu, Y. H.; Gan, L. L.;
Zhou, C. H.; Wu, J.; Geng, R. X.; Zhang, Y. Y. Arch. Pharm. 2009, 342, 386.
19. (a) Ibrahim, N.; Ibrahim, H.; Kim, S.; Nallet, J. P.; Nepveu, F. Biomacromolecules
2010, 11, 3341; (b) Hu, Y. J.; Ou-Yang, Y.; Dai, C. M.; Liu, Y.; Xiao, X. H.
Biomacromolecules 2010, 11, 106.
20. Zhang, S. L.; Guri, L. V. D.; Zhang, L.; Geng, R. X.; Zhou, C. H. Eur. J. Med. Chem.
2012, 55, 164.
21. Experimental: Melting points are determined on X-6 melting point apparatus
d 163.79, 162.76, 152.31, 151.98, 151.93, 150.05, 146.18, 145.75, 144.33,
139.80, 135.30, 133.78, 131.96, 127.90, 124.59, 123.50, 122.98, 121.90, 121.66,
112.14, 109.47, 106.63, 104.50, 103.74, 73.22, 57.67, 57.41, 54.78, 54.56, 52.53,
49.16, 28.27 ppm; MS (m/z): 586 [MÀCl]+; HRMS (Tof) calcd for
C
32H30ClF2N5O4: [MÀCl]+, 586.2260; found 586.2252.
Synthesis of [[N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(2-berberinoxyethoxy)ethanamine] hydrochloride (6). To a solution of compound
5a (62 mg, 0.1 mmol) in ethyl ether (10 mL)/chloroform (10 mL) (1/1, v/v) was
added dropwise diluted hydrochloric acid (4 mol/L) until no more precipitate
formed. The precipitate was filtered, washed with chloroform and then light
petroleum ether, and then dried to afford the desired hydrochloride 6 (64 mg)
as yellow solid. Yield: 87.7%, mp: >250 °C; 1H NMR (400 MHz, D2O): d 9.62 (s,
1H, 8-H), 9.32 (s, 1H, Tri 3-H), 8.73 (s, 1H, 13-H), 8.50 (s, 1H, Tri 5-H), 8.02 (d,
1H, J = 8.8 Hz, 11-H), 7.88 (d, 1H, J = 8.8 Hz, 12-H), 7.66 (s, 1H, 1-H), 7.32–6.97
(m, 3H, Ph 3,5,6-H), 6.82 (s, 1H, 4-H), 6.13 (s, 2H, OCH2O), 4.99 (t, J = 6.2 Hz, 2H,
6-H), 4.87 (t, J = 6.4 Hz, 2H, Tri-CH2), 4.53 (t, J = 6.0 Hz, 2H, OCH2CH2), 4.35 (s,
2H, Ph-CH2), 4.03 (s, 3H, OCH3), 3.92 (t, J = 6.4 Hz, 2H, Tri-CH2CH2), 3.80 (t,
J = 6.0 Hz, 2H, OCH2), 3.77 (t, J = 6.2 Hz, 2H, 5-H), MS (m/z): 586 [MÀClÀ2HCl]+.
22. National Committee for Clinical Laboratory Standards Approved Standard
Document. M27-A2, Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts, National Committee for Clinical Laboratory
Standards, Wayne, PA, 2002.
and uncorrected. IR spectra were determined on
a Bio-Rad FTS-185
spectrophotometer in the range of 400–4000 cmÀ1 1H NMR and 13C NMR
.
spectra were recorded on a Bruker AV 300 or Varian 400 spectrometer using
TMS as an internal standard. Chemical shifts were reported in parts per million
(ppm), the coupling constants (J) were expressed in hertz (Hz) and signals were
described as singlet (s), doublet (d), triplet (t), as well as multiplet (m). The
following abbreviations were used to designate aryl groups: Tri, triazoly; Ph,
phenyl. Mass spectra were confirmed on FINIGAN TRACE GC/MS and HRMS.
Synthesis of [N-(2-(1H-1,2,4-triazol-1-yl)ethyl)-2-bromo-N-(2,4-difluorobenzyl)-
ethanamine] (4a). 1,2,4-Triazole (588 mg, 8.52 mmol) and potassium carbonate
(2.32 g, 16.81 mmol) in acetonitrile (10 mL) were stirred at 50 °C for 1 h, then
the mixture was cooled to room temperature, compound 3a (3.01 g,
8.43 mmol) was added and stirred for 2 h. After the reaction was completed
(monitored by TLC, eluent, ethylacetate/petroleum, 1/2, v/v), solvent was
removed under reduced pressure, the residue was extracted with ethylacetate
23. Hu, Y. J.; Wang, Y.; Ou-Yang, Y.; Zhou, J.; Liu, Y. J. Lumin. 2010, 129, 1394.