Design, synthesis and biological evaluation of novel Schiff base-bridged tetrahydroprotoberberine triazoles as a new type of potential antimicrobial agents

Article abstract of DOI:10.1039/c6md00688d

A series of novel Schiff base-bridged tetrahydroprotoberberine (THPB) triazoles were designed, synthesized and characterized for the first time. Antimicrobial assay showed that some of the prepared compounds exerted stronger antibacterial and antifungal activities than the reference drugs. Especially, THPB triazole 7a gave low MIC values of 0.5, 1 and 2 μg mL^-1 against B. yeast, M. luteus and MRSA, respectively. Further experiments indicated that the highly active molecule 7a was able to rapidly kill the MRSA strain and did not trigger the development of bacterial resistance even after 14 passages. The preliminary exploration for the antimicrobial mechanism revealed that compound 7a could effectively intercalate into calf thymus DNA to form a 7a-DNA supramolecular complex, and its Zn²⁺ complex had the ability to directly cleave pUC19 DNA, which suggested that compound 7a might be a potentially dual-targeting antibacterial molecule. It was also found that compound 7a could be efficiently stored and carried by human serum albumin (HSA), and the hydrophobic interactions and hydrogen bonds played important roles in the transportation of HSA to the active molecule 7a.

Full text of DOI:10.1039/c6md00688d

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ARTICLE
Design, synthesis and biological evaluation of novel Schiff base-
bridged tetrahydroproberberine triazoles as new type of potential
antimicrobial agents†‡
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
b
DOI: 10.1039/x0xx00000x
Jun-Rong Duan, Han-Bo Liu, Ponmani Jeyakkumar,§ Lavanya Gopala,# Shuo Li,* Rong-Xia
a a
Geng and Cheng-He Zhou*
www.rsc.org/medchemcomm
A series of novel Schiff base-bridged tetrahydroprotoberberine (THPB) triazoles were designed, synthesized and
characterized for the first time. Antimircobial assay showed that some of the prepared compounds exerted stronger
antibacterial and antifungal activities than the reference drugs. Especially, THPB triazole 7a gave low MIC values of 0.5, 1
and 2 μg/mL against B. yeast, M. luteus and MRSA, respectively. Further experiments indicated that the highly active
molecule 7a was able to rapidly kill the MRSA strain and did not trigger the development of bacterial resistance even after
4 passages. The preliminary exploration for antimicrobial mechanism revealed that compound 7a could effectively
1
2+
intercalate into calf thymus DNA to form 7a−DNA supramolecular complex, and its Zn complex had ability to directly
cleave pUC19 DNA, which suggested that compound 7a might be a potentially dual-targeting antibacterial molecule. It was
also found that compound 7a could be efficiently stored and carried by human serum albumin (HSA), and the hydrophobic
interactions and hydrogen bonds played important roles in the transportation of HSA to the active molecule 7a.
electronic interactions, thus exerting potent biological activities.
Notably, despite of its long term clinical use, the incidence of
resistance of berberine is less reported. This attracts special
interest in investigating berberines for possible further
antimicrobial use and other medicinal potentiality like
1
. Introduction
4
The increasing resistance of clinical drugs especially currently
antimicrobial agents is triggering high morbidity and mortality,
which has become one of the most critical health concerns
1
anticancer, antiviral, antiinflammatory, antiparasitic activities
worldwide. Currently, an urgent global task is to combat drug
5
etc
.
However, berberine exhibits poor solubility, low
resistance, and it has been universally recognized that no action
today means no cure tomorrow. The developments of
structurally novel molecules with distinct action mechanisms
from the wellꢀknown class of antimicrobial agents are therefore
bioavailability in vivo and some of the severe side effects such
as anaphylactic shock and drug eruption, and these have
seriously limited its useful profile. Therefore much effort has
been devoting towards the structural modification of berberine.
Literature revealed that structural changes such as disruption of
2
of great importance to medical community.
Berberine has been commonly used in clinic with long
history of more than 2000 years in traditional Chinese herbs to
treat intestinal infections such as acute gastroenteritis, bacillary
dysentery and cholera. The unique structure of berberine with
a
backbone enables berberineꢀbased derivatives to readily bind
with biomolecules like deoxyribonucleic acid (DNA) or
enzyme via noncovalent forces such as
symmetry or molecular planarity were proved to be an effective
6
way to improve
the
intrinsic
water
solubility.
3
Tetrahydroprotoberberine (THPB) was found to be good
7
structural alternative with large medicinal potentiality. An
quaternary nitrogen and large desirable πꢀconjugated
increasing exploitation is directing towards THPBs for their
8
medicinal value, especially in antiꢀinfective aspects.
Azole compounds are well known types of heterocycles with
various bioactivities, have been extensively designed and
π–π stacking and
9
synthesized as antimicrobial drugs. Particularly triazole
nucleus is a polyꢀnitrogen electronꢀrich heterocycle present in
many biologically active compounds. It is generally considered
a
Institute of Bioorganic
& Medicinal Chemistry, Key Laboratory of Applied
Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, PR China. E-mail: that triazole fragment is stable to metabolic degradation and
zhouch@swu.edu.cn; Fax: +86 23 68254967; Tel: +86 23 68254967
b
capable of forming hydrogen bonds, thus the introduction of
triazole nucleus is beneficial to improve the binding with
School of Chemical Engineering, Chongqing University of Technology, Chongqing
00054, PR China. E-mail: lishuo@cqut.edu.cn
The authors declare no competing interests.
4
†
‡
biomolecular targets and increase water solubility of target
Electronic supplementary information (ESI) available. See DOI: 10.1039/
10
compounds.
Therefore triazole moiety is prevalently
x0xx00000x
§
Ph.D candidate from India.
Postdoctoral fellow from Sri Venkateswara University, Tirupati 517502, India.
employed in drug design, so far a large number of predominant
triazoleꢀbased medicinal drugs have been successfully
#
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1
1
developed and prevalently used in clinic, especially in etc
.
Their importantly clinical uses have been motivating
antimicrobial aspect, such as fluconazole, itraconazole, extensive efforts to construct new bioactive molecules based on
voriconazole, posaconazole, efinaconazole and terconazole triazole fragment.
Fig. 1 Design of novel Schiff base-bridged tetrahydroproberberine triazoles.
1
4
Our previous work showed that the introduction of azoles and regulating the lipidꢀwater partition coefficient. Rationally,
such as imidazole, triazole and benzimidazole moieties into the various aliphatic chains with different lengths and substituted
Cꢀ9 or Cꢀ12 position of berberine backbone was not only benzyl moieties including chloro, fluoro and nitro groups were
beneficial to enhance the antimicrobial activities but also widen incorporated into Cꢀ9 position of THPB to investigate their
the antimicrobial spectrum, including against methicillinꢀ effects on antimicrobial activities. The synthetic route of target
1
2
resistant Staphylococcus aureus (MRSA). However, to our THPB triazoles was shown in Scheme 1. All the newly
best knowledge, the combination of azoles and THPB backbone synthesized compounds were confirmed by spectral analysis
has been rarely reported. In view of above consideration and as and screened against Gramꢀpositive bacteria including MRSA,
an extension of our ongoing research on bioactive heterocycles, Gramꢀnegative bacteria and fungi. Aqueous solubility,
it is of great interest for us to combine THPB with triazole bactericidal kinetic assay and antibacterial resistance to the
nucleus to generate a new structural type of potentially highly active molecule were also investigated. In order to
antimicrobial agents via Schiff base bridge which is a validated explore the preliminary antimicrobial action mechanism, the
moiety to beneficially improve the water solubility and the interaction of the most active triazole derivative with DNA was
1
3
antimicrobial efficacy (Fig. 1). It is expected that these further carried out by fluorescence and UVꢀvis absorption
hybrids of THPB with triazole moiety could not only spectroscopy as well as agarose gel electrophoresis.
beneficially enhance the antimicrobial activities and broaden Additionally, the binding behavior of the highly active
the antimicrobial spectrum, but also helpfully improve water molecule to human serum albumin (HSA) was investigated to
solubility. Literature revealed that lipophilic substituents on preliminarily study the absorption, distribution, and metabolism.
berberine especially at Cꢀ9 position could significantly
influence the bioactivities by modifying molecular flexibility
Scheme 1. Reagents and conditions: (i) berberine, 20 mm Hg, 190 °C, 15 min; CH
3
CH
2
OH-HCl; (ii) CH
3
OH, NaBH
4
, rt 4 h; (iii) (a) HMTA/TFA, 120 °C, 3 h; (b) 10%
H
2
SO
4
, 90–100 °C, 2 h; (iv) CH
3
CH
2
OH, CH
3
COOH, reflux, 5 h; (v) alkyl bromides and halobenzyl halides, K CO , DMF, 80 °C.
2 3
2
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1
13
base. All the new compounds were confirmed by IR, H NMR,
NMR and HRMS spectra (ESI‡).
C
2
. Chemistry
All the target compounds were conveniently prepared from
commercial berberine chloride, 4Hꢀ1,2,4ꢀtriazolꢀ4ꢀamine, alkyl
3
. Results and discussion
bromides and halobenzyl halides. As depicted in Scheme 1, 3.1 Antimicrobial activities
berberrubine 2 was easily obtained in 84.6% yield by the 9ꢀ
The obtained results in Table 1 showed that almost all the
synthesized THPBs displayed better in vitro biological activity
than precursor berberine against most of the tested strains.
1
5
demethylation of berberine chloride 1 at 190 °C under vacuum, and
was further reduced by NaBH to give THPB 3 at 0 °C in 63.1%
yield. The formylation of compound 3 by hexamethylenetetramine
HMTA) in trifluoroacetic acid under reflux produced the important
4
Towards M. luteus and B. typhi, the intermediate
4 with a formyl
(
group at the 12ꢀposition of THPB gave comparable or superior
antibacterial efficacy to chloromycin and norfloxacin.
Generally, the target THPB triazoles gave better inhibitory
potency than precursor berberine and the corresponding
16
intermediate 4 in high yield of 76.5%, which proceeded more
1
7
facilely and efficiently in comparison to the literature method. The
latter was condensed with 4Hꢀ1,2,4ꢀtriazolꢀ4ꢀamine in ethanol under
reflux using a catalytic amount of glacial acid to afford the target
Schiff base 5 in 88.3% yield. The further structural modification of
compound 5 by alkyl or substituted aromatic halides generated the
alkyl derivatives 6a−f and aralkyl ones 7a−h with the yields from
intermediate 4, and some of them were much more active than
reference drugs. It revealed that the triazole fragment at the 12ꢀ
position of THPB should play an important role in exerting
antibacterial potency.
1
5.2 to 35.4% in DMF at 80 °C using potassium carbonate as the
a,b
Table 1 Antibacterial data as MIC (ꢁg/mL) for compounds 4ꢀ7
.
Gramꢀpositive bacteria
Gramꢀnegative bacteria
Compds
S.
aureus
128
64
256
16
8
8
32
256
8
256
32
16
256
8
16
512
512
16
B.
subtilis
32
512
128
16
8
16
32
256
32
512
32
32
512
16
32
256
>512
32
M.
luteus
2
1
16
1
16
16
64
512
1
E. coli
JM109
256
128
256
16
E. coli
DH52 dysenteriae
256
128
512
32
32
16
64
256
16
256
16
64
512
32
256
512
>512
32
S.
P.
B.
proteus
64
64
32
16
16
16
64
256
16
64
128
512
512
8
32
512
–
32
8
B.
typhi
2
128
256
16
2
16
16
256
4
512
4
1
256
8
32
4
–
MRSA
aeruginosa
4
5
256
16
8
512
256
512
8
8
64
128
512
32
256
16
16
64
64
512
64
256
32
4
16
128
256
16
32
512
128
512
8
16
8
2
512
4
6
6
a
b
8
6c
32
16
32
512
2
512
8
16
256
32
8
4
32
6
d
6e
128
512
32
>512
32
4
0.5
8
16
128
–
32
1
6
f
7
7
a
b
32
2
7c
7
d
256
512
16
128
2
>512
8
2
7e
7
f
7
7
g
h
32
64
256
32
16
16
128
16
8
Berberine
Chloromycin
Norfloxacin
32
4
0.5
4
1
a
Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
S. aureus, Staphylococcus aureus ATCC25923; MRSA, MethicillinꢀResistant Staphylococcus aureus N315; B. subtilis, Bacillus subtilis ATCC6633; M.
b
luteus, Micrococcus luteus ATCC4698; E. coli JM109, Escherichia coli JM109; E. coli DH52, Escherichia coli DH52; S. dysenteriae, Shigella dysenteriae; P.
aeruginosa, Pseudomonas aeruginosa ATCC27853; B. proteus, Bacillus proteus ATCC13315; B. typhi, Bacillus typhi.
The preliminary structure−activity relationships (SARs) Noticeably, compound 6b exhibited the best antiꢀM. luteus
demonstrated the significant effect of the substituents at the 9ꢀ activity with a MIC value of 1 ꢁg/mL, which was superior to
position of THPB on biological activities. In contrast with the standard drugs chloromycin (MIC = 8 ꢁg/mL) and norfloxacin
clinical drugs chloromycin and norfloxacin, the unsubstituted (MIC = 2 ꢁg/mL). Towards E. coli JM109 and B. typhi strains,
compound
5 at the 9ꢀposition of THPB nucleus exhibited compound 6c displayed 8ꢀ and 16ꢀfold more potent inhibitory
weaker activity against all the tested strains except for M. luteus activity (MIC = 4 and 2 ꢁg/mL, respectively) than chloromycin.
which was the most sensitive to compound
value of 1 ꢁg/mL.
5
with low MIC It also demonstrated effective antiꢀS. aureus, antiꢀB. subtilis
and antiꢀS. dysenteriae activities with MIC values of 8 ꢁg/mL,
In general, most of the alkylꢀsubstituted compounds could being more potent than chloromycin. Moreover, when the alkyl
effectively inhibit the growth of all the tested bacterial strains in substituents were extended to decyl and dodecyl groups,
vitro. Among the alkyl derivatives 6a
and the hexyl analogue 6c gave broader antibacterial most of the tested bacterial strains. However, the ethyl
spectrum and better activities with MIC values of 1–32 ꢁg/mL. derivative 6a was also less beneficial for the antibacterial
−f, the butyl compound compounds 6e and 6f showed poor or even no activity against
6b
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efficiency even at high concentration. These results revealed towards C. albicans and 7f towards C. utilis, and this suggested
that either decrease or increase of the alkyl chain length was that these compounds should be much more active than
unfavorable for the bioactivity, and only proper length chain in reference drug fluconazole, and they might have large
THPBs exerted an important influence in enhancing bioactivity. possibility as new antifungal agents and should be worthy to be
The short alkyl chains with poor lipophilicity and long alkyl investigated deeply (ESI‡ Table S1).
chains with good lipophilicity in these THPBs might make
3
.2 Effect of clogP values and aqueous solubility of selected
them unfavorable for being delivered to the binding sites.
In comparison with the alkyl derivatives, most of the aralkylꢀ
substituted ones 7a−h exerted relatively better activities in
inhibiting the growth of the tested strains. Notably, compound
compounds
THPB triazoles 5, 6b, 6c, 6f, 7a, 7c and 7f were further selected for
soluble profile. The water solubility (mg/mL) was evaluated at
physiological pH and the obtained results were depicted in Table 2.
In general, the THPB triazoles were more soluble in water than
berberine. Particularly, the THPB triazole 7f bearing 4ꢀchlorobenzyl
substituent showed the largest water solubility in this series, 7 times
higher than berberine. The highly bioactive derivative 7a possessed
equivalent solubility to its fluorinated counterpart 7c with 2ꢀfold
enhanced soluble potency in contrast with berberine. Remarkably,
the alkyl derivatives were less soluble in water than benzyl ones.
Among the alkylated THPBs 6b, 6c and 6f, the hexyl derivative 6c
was endowed with more potent aqueous solubility, more than five
times as soluble as berberine. Unfortunately, the replacement of
hexyl group by dodecyl moiety dramatically led to a decrease in
solubility. These results indicated that the insertion of triazole moiety
in THPB backbone should be beneficial for the improvement of
aqueous solubility.
7a
without substitution on phenyl ring gave the best
antibacterial efficiencies with MIC values of 1–32 ꢁg/mL
towards the corresponding bacteria. It was found that M. luteus
,
MRSA and P. aeruginosa were sensitive to THPB triazole 7a
with MIC values of 1, 2 and 8 ꢁg/mL, respectively, which were
superior to those of chloromycin and norfloxacin. Excitedly, the
target compounds 6a, 6b, 7a, 7c and 7g exerted good biological
activities against MRSA with MIC values of 2–8 ꢁg/mL, which
were more active than chloromycin (MIC = 16 ꢁg/mL).
Especially compound 7a exhibited 8ꢀ and 4ꢀfold more activity
towards MRSA with low concentration (MIC = 2 ꢁg/mL) than
chloromycin and norfloxacin, respectively. These indicated that
compound 7a had the potency to be a lead molecule in the
development of more effective antimicrobial agents with board
spectrum. Continual study revealed that the orthoꢀ and para
ꢀ
Hydrophobic/lipophilic property played an important role in
predicting pharmacokinetic properties of a drug and its interaction
with macromolecular targets. The calculated lipid/water partition
coefficients (clogP) of some target compounds and berberine were
substituted benzyl derivatives 7c 7d and 7f were more effective
,
against most of the tested bacteria than the metaꢀsubstituted
ones 7b and 7e. Particularly, 2ꢀchlorobenzyl derivative 7d gave
potent inhibition against P. aeruginosa (MIC = 2 ꢁg/mL) and B.
typhi (MIC = 1 ꢁg/mL), which was more active than
chloromycin and norfloxacin. Unfortunately, 4ꢀnitrobenzyl
compound 7h displayed relatively poor activity against most of
the tested bacteria, which suggested that the introduction of
shown in Table 2.
The synthesized compounds 6b−c, 7a, 7c and 7f
with moderate clogP values exhibited better antimicrobial activities,
indicating that compounds with suitable lipophilicity were favorable
to permeate through biological membrane and to be delivered to the
binding sites.
electron withdrawing group like NO
antibacterial activity. Among all the prepared compounds, the
ꢀchlorobenzyl derivative 7e gave the strongest activity against
2
might be not helpful for
Table 2 ClogP values and evaluation of solubility of selected compounds
3
a,b
(
Mean ± SD, n = 3) .
E. coli JM109 with a MIC value of 0.5 ꢁg/mL, it showed that this
compound might be hopeful to be developed as special antiꢀE.
coli JM109 agent.
Compds
ClogP
2.99
0.58
2.65
3.70
6.88
2.83
2.97
3.54
Aqueous solubility
4
5
0.079
0.392
0.121
0.442
0.097
0.178
0.185
0.613
0.085
±
±
±
±
±
±
±
±
±
0.002
0.024
0.016
0.028
0.006
0.027
0.013
0.034
0.005
The antifungal evaluation in vitro showed no remarkable
6
b
activities for 12ꢀformyl THPB
triazole against the tested fungi except for B. yeast which was
highly sensitive to compound (MIC = 1 ꢁg/mL). However,
4 and 9ꢀunsubstituted THPB
6
c
5
6
f
4
7
a
c
the structural modification at the 9ꢀposition of THPB nucleus
generated THPB triazole derivatives which displayed potent
antifungal activities towards most of the tested fungal strains. It
was noticed that the antifungal abilities of the alkyl compounds
displayed similarity to their antibacterial efficiencies. The
suitable length of alkyl chain seemed to be hexyl chain, the
hexyl derivative 6c exerted the best antifungal efficacy with
MIC values ranging from 2 to 32 ꢁg/mL against all the tested
fungi, better than other alkyl derivatives with shorter or longer
7
7
f
Berberine
−
a
b
ClogP values were calculated by ChemDraw Ultra 14.0. In mg/mL,
determined in pH 7.4 phosphate buffer, at 30 °C.
3
.3 Bactericidal kinetic assay
The ability of an antibacterial agent to rapidly eradicate MRSA
would decrease when this agent emerged resistance towards
1
8
chain length. Among the aralkyl THPB triazoles 7a−h, the 4ꢀ
bacteria. To examine the antibacterial potency of the promising
compound, a timeꢀkill assay was performed by checking the viability
of exponentially growing MRSA against the highly active compound
chlorobenzyl derivative 7a gave more effective antifungal potency
with MIC values of 0.5–32 ꢁg/mL than other aralkylꢀsubstituted
compounds. Particularly, the same low MIC values of 0.5 ꢁg/mL
7
a. As shown in Fig. 2, compound 7a showed more than 3 log
were observed for THPB triazole 7a towards B. yeast, 7b
(
CFU/mL) reduction in the number of viable bacterial within an hour
4
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at a concentration of 4 × MIC. Fig. 2 indicated a rapidly killing difficult to develop resistance against compound 7a than the clinical
effect for compound 7a against MRSA.
drug norfloxacin.
1
1
1
4
2
0
8
6
4
2
300
250
Compound 7a
Norfloxacin
Control
Compound 7a
200
150
100
50
0
0
1
2
3
4
5
6
7
0
2
4
6
8
10
12
14
16
Time (h)
Passages
Fig. 2 Time−kill kineꢀcs of compound 7a (4 × MIC) against MRSA.
Fig. 3 Evaluation of resistance development against compound 7a in
3.4 Resistance study
bacterial strain MRSA.
The emergence of bacterial resistance towards most of the clinical
drugs currently is a serious problem, especially MRSA strain
3
.5 Molecular modeling
1
9
To rationalize the observed antibacterial activity and investigate the
antiꢀMRSA action mechanism of THPB triazole 7a, molecular
docking study was performed between compound 7a and MRSA
towards norfloxacin. Hence, it would be important to evaluate the
propensity of THPB triazole 7a to induce bacterial resistance. This
work investigated the ability of susceptible pathogen MRSA to
develop resistance against compound 7a, and norfloxacin was used
as a positive control. The standard strain of MRSA was exposed to
the increasing concentrations of compound 7a from MIC for the
sustained passages, and the new MIC values were determined
against each passage of MRSA. Fig. 3 exhibited no obvious change
in the MIC for compound 7a even after 14 passages, whereas
norfloxacin gave significant increase in the MIC after 5 passages
against MRSA. The obtained results suggested that MRSA was more
2
DNA (PDB ID: 2XCS). As shown in Fig. 4, the OCH O group of the
THPB 7a was adjacent to the ARGꢀ1122 and METꢀ1121 residues,
forming three hydrogen bonds with a distance of 2.1, 2.4 and 2.9 Å,
respectively. Besides, the ARGꢀ1122 and GLYꢀ1072 residues could
also form hydrogen bonds with methoxy group of THPB fragment
and N atom of triazole ring. The hydrogen bonds might be favorable
to stabilize the 7a−DNA complex, which might be responsible for
the good inhibitory efficacy of compound 7a against MRSA.
Fig. 4 Molecular modeling of compound 7a and MRSA DNA.
indicate a close proximity of the aromatic chromophore to the DNA
bases due to the interaction between the electronic states of
intercalating chromophore and that of the DNA base.
With a fixed concentration of DNA, UVꢀvis absorption spectra
were recorded with sequentially increasing amount of compound 7a.
As shown in Fig. 5, UVꢀvis spectra gave a proportional increase at
3
.6 Interactions with calf thymus DNA
DNA is the main cellular target for studies with small molecules of
biological importance, and as a therapeutic target it has been
extensively employed in rational design and construction of new and
effective drugs. Calf thymus DNA was commonly selected as DNA
model due to its medically important, lowꢀcost and ready available
properties. To explore the possible mechanism of antimicrobial
action, the interaction of the highly active compound 7a with DNA
at molecular level was investigated by UVꢀvis spectroscopy.
21
2
0
2
60 nm for the maximum absorption peak of DNA and a slightly red
shift with the increasing concentration of compound 7a. Meanwhile,
the absorption value of simply sum of free DNA and free compound
7
a was a little greater than the measured value of 7a−DNA complex
3.6.1 Absorption spectra of DNA in the presence of compound 7a (inset of Fig. 5). This suggested a hypochromic effect between DNA
and compound 7a. Furthermore, the intercalation of the
chromophore fragment of compound 7a into the DNA helix and the
strong overlap of πꢀπ* states of the large πꢀconjugated system with
In absorption spectroscopy, hyperchromism and hypochromism are
perceived as important spectral features to distinguish change of the
DNA doubleꢀhelix structure. As literature reported, hypochromism
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the electronic states of DNA bases were in accordance with the the presence of the increasing concentrations of DNA (ESI
‡
Fig. S2), the absorbance at the same wavelength exhibited the
reverse process (inset of Fig. 6). The results suggested that
compound 7a could intercalate into the double helix of DNA by
observed spectral changes.
On the basis of variations in the absorption spectra of DNA
upon binding to compound 7a, the binding constant
K can be
substituting NR in the DNA–NR complex.
calculated by the following equation:
0.6
0
A
ξC
ξC
1
(1)
1.5
i
=
+
×
i
0
0.4
A− A ξD−C −ξC ξD−C −ξC K[Q]
and A represent the absorbance of DNA in the absence and
presence of compound 7a at 260 nm, ξ and ξD–C are the absorption
coefficients of compound 7a and compound 7a−DNA complex,
0
A
0
.2
1
.0
.5
a
C
DNA+NR
480
0.0
a
420
450
510
540
0
0
Wavelength (nm)
respectively. The plot of A /(A–A ) versus 1/[compound 7a] is
0
constructed by using the absorption titration data and linear fitting
4
(
(
ESI‡, Fig. S1), yielding the binding constant, K = 2.27 × 10 L/mol,
R = 0.999, SD = 0.110). The experimental results revealed that
0.0 DNA+NR
00
THPB triazole 7a possessed a good DNAꢀbinding ability with calf
thymus DNA, which was also consistent with the above mentioned
hypochromic effect.
3
400
500
600
700
Wavelength (nm)
Fig.
6 UV absorption spectra of the competitive reaction between
compound 7a and NR with DNA. c(DNA) = 4.20 × 10 mol/L, c(NR) = 2 × 10
-
5
-
5
1.5
1.0
0.5
0.0
1.0
DNAꢀ7a complex
DNA+ compound 7a
-5
0
0
0
.9
.8
.7
mol/L, and c(compound 7a) = 0–2.0 × 10 mol/L for curves a–i respectively
-5
at increment 0.25 × 10 . (Inset) absorption spectra of the system with the
increasing concentration of 7a in the wavelength range of 400–550 nm
absorption spectra of competitive reaction between compound 7a and NR
with DNA.
i
0.6
0
0
.5
.4
0
.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
10
5
[Q]
3.7 Cleavage ability towards pUC19 DNA
DNA cleavage is controlled by relaxation of supercoiled form DNA
Form I). Plasmid pUC19 DNA, a widely used DNA cleavage
substrate, is acircular double stranded DNA (Form I). The cleavage
agent can convert Form I to nicked form (Form II) or linear form
DNA (Form III). In gel electrophoresis, the migration rate of the
three DNA forms is usually in the order: Form I > Form III > Form
a
(
DNA
50
2
300
350
400
Wavelength (nm)
Fig. 5 UV absorption spectra of DNA with different concentrations of
compound 7a (pH = 7.4, T = 303 K). Inset: comparison of absorption at 260 II. In order to assess the DNA cleavage activities, the cleavage of
nm between the 7a−DNA complex and the sum values of free DNA and free
plasmid pUC19 DNA assays was investigated by agarose gel
electrophoresis. The degradation of plasmid pUC19 DNA from
Form I to Form II and Form III in Fig. S3 (ESI‡) suggested that
-5
-
compound 7a. c(DNA) = 5.20 × 10 mol/L, and c(compound 7a) = 0−2.0 × 10
5
-5
mol/L for curves a−i respectively at increment 0.25 × 10 mol/L.
2
+
3.6.2 Absorption spectra of NR interaction with DNA
compound 7a−Zn complex might cleave DNA effectively at low
ꢀ5
concentration about 1.25 × 10 mol/L, and it must be pointed out
To further understand the interaction between compound 7a and
DNA, the absorption spectra of the competitive interaction of
compound 7a were also investigated. Neutral red (NR) is a planar
phenazine dye, which possesses lower toxicity, higher stability and
convenient application in comparison with other common probes. It
2
+
that in Fig. S3 (ESI‡) berberine, compound 7a and Zn ion could
not cleave DNA totally alone. It could be deduced that compound 7a
2
+
was able to form complex with Zn ion, which might further destroy
2
3
DNA directly and thus exert its antibacterial activity.
has been sufficiently demonstrated that the binding of NR with DNA 3.8 Interactions of compound 7a with HSA
2
2
is an intercalation binding. Therefore, NR was employed as a
spectral probe to investigate the binding mode of compound 7a with
DNA in present work. The absorption spectra of the NR dye upon
the addition of DNA were shown in Fig. S2 (ESI‡). The absorption
peak of NR at around 460 nm gradually decreased with the
increasing concentration of DNA, and a new band at around 530 nm
generated, which could be ascribed to the formation of the new
DNA–NR complex. The isosbestic point at 500 nm also provided
evidence of DNA–NR complex formation.
HSA is an important extracellular protein with extensive distribution
in the circulatory system, transports various exogenous and
endogenous molecules, thus significantly affecting absorption,
24
distribution, and metabolism of numerous therapeutic drugs.
Therefore, the molecular characterization of drugs−HSA interaction
is not only beneficial to understand pharmacokinetic properties, but
also instructive to design new drug molecules.
3
.8.1 UVꢀvis absorption spectral study
UV–vis absorption spectroscopic method is a convenient technique
applicable to measure the structural change of protein and to identify
complex formation. The UV–vis absorption measurement to study
the interaction of compound 7a with HSA was carried out and the
results were shown in Fig. 7. The absorption peak at 278 nm was
attributed to the aromatic rings in tryptophan (Trpꢀ214), tyrosine
3
7
.6.3 Absorption spectra of competitive interaction of compound
a and NR with DNA
Figure 6 displayed the absorption spectra of a competitive
binding between compound 7a and NR with DNA. As shown,
an apparent intensity increase was observed around 460 nm
with the increasing concentration of compound 7a
. In
comparison with the absorption around 460 nm of free NR in (Tyrꢀ411) and phenylalanine (Phe) residues in HSA. With the
6
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1
1
1
400
200
000
800
addition of compound 7a, the peak intensity increased, indicating
that compound 7a could interact with HSA. However, the maximum
absorption wavelength remained unchanged, suggesting the
interactions of compound 7a and HSA were noncovalent interactions.
These occurred via π−π stacking between the aromatic rings of
compound 7a and Trp, Tyr and Phe residues, which possess
conjugated πꢀelectrons and are located in the binding cavity of
a
HSA
6
4
2
00
00
00
h
2
5
HSA.
1
.3
Compound 7a only
1.8
1.5
1.2
0.9
0.6
0.3
0.0
1.2
λ
max = 278 nm
1
1
0
0
.1
.0
.9
.8
0
3
R = 0.999
h
a
00
325
350
375
400
425
450
Wavelength (nm)
0.7
Fig. 8 Emission spectra of HSA with various concentrations of compound 7a.
-5 -5
ꢀ
0.20.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
5
c(HSA) = 1.0 × 10 mol/L; c(compound 7a)/(10 mol/L), a−h: from 0.0 to 1.17
at increments of 0.167; blue and red lines are the emission spectrum of
compound 7a only and HSA only, respectively; T = 298 K, λex = 295 nm.
1
0
[Q]
HSA
The fluorescence quenching mechanisms are usually classified as
either dynamic quenching or static quenching, which could be
distinguished by their different dependence on temperature and
viscosity. For dynamic quenching, higher temperatures result in
Compound 7a only
50 275 300
2
325
350
375
400
Wavelength (nm)
Fig. 7 Effect of compound 7a to HSA UV-vis absorption, c(HSA) = 1.0 × 10
-5
-5
mol/L; c(compound 7a)/(10 mol/L): 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 (T = faster diffusion and larger amounts of collisional quenching. As a
98 K, pH = 7.4). The inset corresponds to the absorbance at 278 nm with result, the quenching constants are expected to increase with a
2
different concentrations of compound 7a.
gradually increasing temperature in dynamic quenching, but the
reverse effect would be observed for static quenching. The
3.8.2 Fluorescence quenching mechanism
q
calculated values of KSV and K for the interaction of compound 7a
Fluorescence quenching is considered as an effective approach to
investigate the transportation ability of HSA to small molecules. The
fluorescence intensity of Trpꢀ214 may change when HSA interacts
with other small molecules, which could be reflected in the
fluorescence spectra of HSA in the UV region. With a fixed amount
of HSA, the fluorescence changes of HSA (T = 298 K, λex = 295 nm)
in the presence of different amounts of compound 7a were
determined. The blue line in Fig. 8 was the only emission spectrum
of the active molecule 7a, which implied that its fluorescence
intensity was very week and could be negligible in comparison with
the fluorescence of HSA at the excitation wavelength. The maximum
emission peak of HSA at 352 nm in the fluorescence spectra
exhibited a progressive decrease in the fluorescence intensity.
However, the maximum emission wavelength of HSA remained
unchanged, which revealed that Trpꢀ214 did not undergo any change
in polarity, and hence compound 7a was likely to interact with HSA
via the hydrophobic region located in HSA.
with HSA at different temperatures were listed in Table S3 (ESI‡).
The KSV values were inversely correlated with temperature, which
indicated that the probable fluorescence quenching of HSA was
initiated by the formation of 7a–HSA complex rather than by
dynamic collisions. Furthermore, the obtained larger K
q
values (2.55
at 288 K, 298 K, 310
K, respectively) were far exceeded the diffusion controlled rate
1
3
13
13
−1 −1
×
10 , 2.05 × 10 and 1.70 × 10 L mol s
10
constants of various quenchers with a biopolymer (2.0 × 10
L
−
1
−1
mol s ), which suggested that the quenching was not initiated by
the dynamic diffusion process but occurred in the static formation of
2
6
7
a–HSA complex.
The difference in absorption spectroscopy was used to obtain
spectra to reconfirm that the probable fluorescence quenching
mechanism of HSA by compound 7a was mainly initiated by
groundꢀstate complex formation. The UVꢀvis absorption spectrum of
HSA (ESI‡ Fig. S5, curve C) and the difference spectrum between
HSA−compound 7a 1:1 complex and compound 7a (ESI‡ Fig. S5,
curve D) could not be superposed, this result reconfirmed that the
probable fluorescence quenching mechanism of HSA by compound
The fluorescence quenching data can be elucidated by the wellꢀ
known Stern−Volmer equation:
2
7
F0
F
7a might be a static quenching procedure.
(2)
=
1+ KSV
[Q
]
= 1+ Kqτ0
[Q]
3
.8.3 Binding constant and sites
where F
presence of compound 7a, respectively.
Stern−Volmer quenching constant, [Q] is the concentration of
compound 7a, K is the bimolecular quenching rate constant (L
is the fluorescence lifetime of the fluorophore in the
0
and F are the fluorescence intensities in the absence and
For a static quenching procedure, quenching data were analyzed
according to the modified SternꢀVolmer equation:
K
SV (L/mol) is the
F
1
1
1
(3)
q
0
=
+
−
1
−1
ꢀF
fa K
a
Q
[
]
fa
mol s ). τ
0
−9
absence of quencher, assumed to be 6.4 × 10 s for HSA. Hence, the where ∆F denotes the difference in fluorescence in the absence and
Stern−Volmer plots of HSA in the presence of compound 7a at presence of compound 7a at concentration [Q], f is the fraction of
different concentrations and temperatures could be calculated and accessible fluorescence, and K is the effective quenching constant
a
a
were shown in Fig. S4 (ESI‡).
for the accessible fluorophores, which are analogous to associative
binding constants for the quencherꢀacceptor system. The dependence
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−1
of F
linear with the slope equaling to the value of (f
is fixed on the ordinate. The constant K is a quotient of the ordinate compound 7a and HSA indicated that the binding process was
) . The modified Stern−Volmer plots were spontaneous. The negative values of ꢂH suggested that the binding
shown in Fig. S6 (ESI‡), and the calculated results were listed in was predominately enthalpy driven and involved an exothermic
Table S4 (ESI‡). reaction. The positive ꢂS value is frequently taken as a typical
0
/ꢂF on the reciprocal value of quencher concentration [Q] is Table S5 (ESI‡) summarized the values of ꢂH, ꢂG and ꢂS. The
−1
−1
a
a
K
a
) . The value f
negative values of the free energy ꢂG of the interactions between
a
−1
−1
f
a
a a
and the slope (f K
The Scatchard equation could be used to estimate the equilibrium evidence for hydrophobic interaction, which is consistent with the
binding constant (K ) and the number of binding sites (n); it is
described as:
b
above discussion. The negative ꢂH value (−9.498 kJ/mol) observed
cannot be mainly attributed to electrostatic interactions since the ꢂH
values of electrostatic interactions were very small, almost zero.
Therefore, ꢂH < 0 and ꢂS > 0 obtained in this case indicated that
both hydrophobic interactions and hydrogen bonds played a major
role in the binding of compound 7a to HSA, and electrostatic
interactions might also involve in the binding process.
log
Fig. 9 displayed the plots of log (F
interaction between HSA and compound 7a at various temperatures.
and n obtained from the Scatchard plots are listed in Table S4
ESI‡). The decreased trend of and with increased
temperatures was in accordance with the temperature dependence of
SV’s. The value of binding site approximates to 1, indicating that
[
(F / F) −1
]
= log K + n log
[
Q
]
(4)
0
b
0
−F)/F versus log[Q] for the
K
(
b
K
a
K
b
K
only one high affinity binding site was present in the interaction of 4. Conclusion
compound 7a with HSA. The results also showed that the binding
In conclusion, a series of novel Schiffꢀbase bridged THPB triazoles
constants were moderate and the effects of temperatures were not
obvious, thus compound 7a might be stored and carried by this
protein.
as antimicrobial agents have been successfully developed for the first
time by an easy, convenient and economic procedure, and their
structures were characterized by HRMS, NMR and IR spectra. The
in vitro biological evaluation revealed that some of the prepared
compounds exerted good to better antibacterial and antifungal
activities in comparison with the reference drugs. Noticeably, THPB
triazole 7a could effectively inhibit the growth of B. yeast, M. luteus
and MRSA with MIC values of 0.5, 1 and 2 ꢁg/mL, respectively.
Further research suggested that compound 7a could rapidly kill
MRSA and induce bacterial resistance more slowly than norfloxacin.
Molecular docking indicated that target compound 7a could bind
with MRSA DNA through hydrogen bonds. SARs revealed that the
combination of triazole fragment and THPB was beneficial to exert
biological activity, and the substituents on the benzyl moiety as well
length of alkyl chain at 9ꢀposition of THPB influenced the
antibacterial potency. The binding investigation of compound 7a
with HSA revealed that this molecule could be effectively
transported by HSA. The preliminary exploration for antimicrobial
0.4
0.2
0.0
T = 288 K
T = 298 K
T = 310 K
ꢀ0.2
ꢀ0.4
ꢀ0.6
ꢀ0.8
ꢀ
0.8
ꢀ0.6
ꢀ0.4
ꢀ0.2
10 [Q]
Fig. 9 Scatchard plots of 7a−HSA system at different temperatures.
.8.4 Binding mode and thermodynamic parameters
0.0
0.2
5
log
(
)
3
Generally, the major forces involved in small molecules and mechanism disclosed that compound 7a could not only form stable
biomolecules interactions include hydrogen bonds, electrostatic 7a–DNA complex with calf thymus DNA by an intercalating mode,
2
8
interactions, van der Waals forces, and hydrophobic interactions.
but also directly cleave pUC19 DNA by formation of complex with
2
+
Thermodynamic parameters like enthalpy change (ꢂH) and entropy Zn ion, thus exerting antibacterial activity. Therefore, this work
change (ꢂS) of binding reaction are the main evidence for revealed that compound 7a might be a potentially dual DNAꢀ
confirming the interactions between small molecules and protein. If targeting antibacterial molecule with better water solubility than
ꢂH does not vary significantly in the temperature range studied, both berberine, which should be a promising start as novel antibacterial
its value and ꢂS can be estimated from the van’t Hoff equation as agent to overcome drug resistance.
follows:
ꢀ
RT
H
ꢀS
R
(
5)
ln K = −
+
Acknowledgements
where K is analogous to the associative binding constants at the This work was partially supported by National Natural Science
corresponding temperature and R is the gas constant. To elucidate Foundation of China (Nos. 21672173, 21372186), the Research
the binding model between compound 7a and HSA, the Fellowship for International Young Scientists from
thermodynamic parameters were calculated from the van’t Hoff International (Regional) Cooperation and Exchange Program of
plots. The enthalpy change (∆H) was estimated from the slope of the China National Natural Science Foundation (81650110529),
van’t Hoff relationship (ESI‡ Fig. S7). The free energy change (∆G) Chongqing Research Program of Basic Research and Frontier
was then calculated from the following equation:
G = ꢀH −TꢀS
(6)
Technology (No.cstc2013jcyjA50012, cstc2016jcyjA0508),
China Postdoctoral Science Foundation funded project
ꢀ
(
2014M562326, 2016T90851).
8
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2
013, 23, 1008ꢀ1012; (c) S. Q. Wen, P. Jeyakkumar, S. R.
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DOI: 10.1039/C6MD00688D
Design, synthesis and biological evaluation of novel Schiff base-bridged tetrahydroproberberine
triazoles as new type of potential antimicrobial agents
a
a
a
a
b
a
Jun-Rong Duan, Han-Bo Liu, Ponmani Jeyakkumar,§ Lavanya Gopala,# Shuo Li,* Rong-Xia Geng
a
and Cheng-He Zhou*
a
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR
China.
b
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China.
§
Ph.D candidate from India.
#
Postdoctoral fellow from Sri Venkateswara University, Tirupati 517502, India.
Synthesis of a series of Schiff base-bridged tetrahydroprotoberberine triazoles as a new type of
potential antimicrobial agents, and preliminary interactions with DNA indicated the possible interaction
mechanism.
1400
1200
1000
HSA
a
800
600
400
200
h
Compound 7a only
0
3
00
325
350
375
400
425
450
Wavelength (nm)