Design, synthesis, and biological evaluation of novel carbazole aminothiazoles as potential DNA-targeting antimicrobial agents

Article abstract of DOI:10.1039/c6md00357e

A series of novel carbazole aminothiazoles as a new type of antimicrobial agents were designed, synthesized and characterized by ¹H NMR, ¹³C NMR, IR, MS and HRMS spectra. Some of the carbazole aminothiazoles exhibited good antimicrobial activities; particularly, heptyl-derived carbazole aminothiazole 4f could effectively inhibit the growth of MRSA with an MIC value of 4 μg mL^-1, which was superior to the reference drugs Chloromycin and Norfloxacin. Moreover, cytotoxicity investigation indicated that bioactive compound 4f did not exhibit cytotoxicity to Hep-2 cells within its MIC against MRSA. Preliminary interactive investigation revealed that compound 4f could effectively intercalate into calf thymus DNA to form 4f-DNA complexes which might block DNA replication and thus exert antimicrobial activities. In addition, the binding behavior of compound 4f to DNA revealed that compound 4f could interact with DNA by hydrogen bonds and electrostatic interactions.

Full text of DOI:10.1039/c6md00357e

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: D. Addla, S. Wen,
W. Gao, S. K. Maddili, L. Zhang and C. Zhou, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00357E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 7
View Article Online
DOI: 10.1039/C6MD00357E
MedChemComm
ARTICLE
Design, synthesis, biological evaluation of novel carbazole
aminothiazoles as potential DNA-targeting antimicrobial
agents†‡
Received 00th January 20xx,
Accepted 00th January 20xx
1
,#,†
1,#
1
1,‡
2,*
Dinesh Addla , SiꢀQi Wen , WeiꢀWei Gao , Swetha Kameswari Maddili , Ling Zhang and
DOI: 10.1039/x0xx00000x
1
,*
ChengꢀHe Zhou
www.rsc.org/
A series of novel carbazole aminothiazoles as new type of antimicrobial agents were designed, synthesized and
1
13
characterized by H NMR, C NMR, IR, MS and HRMS spectra. Some of the carbazole aminothiazoles exhibited good
antimicrobial activities, particularly heptyl derived carbazole aminothiazole 4f could effectively inhibit the growth of
MRSA with MIC value of 4 ꢁg/mL, which was superior to the reference drugs Chloromycin and Norfloxacin. Moreover, the
cytotoxicity investigation indicated that bioactive compound 4f did not exhibit cytotoxicity to Hepꢀ2 cells within its MIC
against MRSA. The preliminarily interactive investigation revealed that compound 4f could effectively intercalate into calf
thymus DNA to form 4f–DNA complexes which might block DNA replication and thus exert antimicrobial activities. In
addition, the binding behavior of compound 4f to DNA revealed that compound 4f could interact with DNA by hydrogen
bonds and electrostatic interactions.
active substances including antimicrobial agents such as
5
1
. Introduction
Carbazomycins and Murrayafoline A.
Moreover, recent
research showed that carbazoles could nonꢀcovalently interact
with DNA through intercalation, the minor groove binding or
electrostatic interactions. This encourages great interest in
Infectious diseases caused by bacteria and fungi have been
causing serious health problems and high mortality especially
in portions of South America, Indian subcontinent and tropical
6
1
investigating carbazoles as
a
novel type of potential
antimicrobial agents. Our previous work showed that the
introduction of fiveꢀmembered azoles into the ꢀposition of
fraction of Africa. A large number of antibiotics and synthetic
7
drugs available for clinic play important roles in treatment of
2
N
microbial infections. However, many bacterial and fungal
carbazole could effectively inhibit the growth of the tested
bacteria and fungi which were even superior to the reference
strains have become resistant to one or more drugs along with
the abuse of antibiotics, among them the most challenging one
is methicillinꢀresistant Staphylococcus aureus (MRSA), which
make a number of firstꢀline drugs decrease their effects or
8
drugs Chloromycin and Norfloxacin. In recent years, the
modification of 3ꢀ and 6ꢀpositions of carbazole ring has become
one of hot topics in the exploitation of novel antibacterial
3
totally lose activities. Thus, it is an urgent task to develop
9
agents.
novel antimicrobial drugs with better treatment effectiveness.
Carbazole is an important type of naturally occurring and
synthetic nitrogenꢀcontaining aromatic heterocycle. This special
type of structure of carbazole enables its derivatives readily
interact with various biological molecules such as enzymes and
receptors, and thus display a broad spectrum of biological
4
activities. Carbazole ring is present in a variety of medicinally
1
Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, PR China.
School of Pharmacy, Yancheng Teachers University, Yancheng 224051, PR China.
The authors declare no competing interests.
These two authors contributed equally to this work.
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India.
Ph.D candidate from India.
Corresponding author; zhouch@swu.edu.cn (C. H. Zhou); Tel.: +86ꢀ23ꢀ68254967;
2
†
#
Fig. 1 Design of novel carbazole aminothiazoles.
†
‡
Aminothiazole as an important pharmacological fragment is
prevalently present in quantities of clinical antimicrobial drugs,
Electronic Supplementary Information (ESI) available: [details of any such as Cephalosporins, Tigemonam, Aztreonam, Carumonam,
*
Fax: +86ꢀ23ꢀ68254967.
‡
supplementary information available should be included here]. See DOI:
Sulfathiazole and Abafungin. Its special property motivates the
10.1039/x0xx00000x.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2016, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 7
View Article Online
DOI: 10.1039/C6MD00357E
ARTICLE
Journal Name
extensive studies to construct antimicrobial agents based on 2ꢀ mechanisms, the interaction of bioactive compound 4f with calf
1
0
aminothiazole. Currently, structural modification of bioactive thymus DNA was also carried out by UV–vis absorption
compounds by introducing 2ꢀaminothiazole is one of the most spectroscopy and fluorescence spectra on molecular level.
convenient and rewarding methods to exploit new antimicrobial
agents.
2
. Chemistry
In view of the above considerations and our continuing
efforts to develop carbazoleꢀbased antimicrobial agents, 2ꢀ
aminothiazole was for the first time introduced into the 3ꢀ and
The target
N
ꢀalkylcarbazole aminothiazoles 4a
−
1
j
were
synthesized via multiꢀstep reactions from carbazole
and the
6
ꢀpositions of carbazole backbone to investigate the
contribution to antimicrobial activity. In addition, different
alkyl groups were also introduced into the ꢀposition of
synthetic routine was outlined in Scheme 1. Intermediates 2a
–j
were prepared in yields of 96 98% by the ꢀalkylation of
−
N
N
commercially available carbazole with various alkyl bromides
carbazole with the aim to study biological properties and
understand structureꢀactivity relationships. The design idea of
novel carbazole aminothiazoles was shown in Fig. 1. Their
antibacterial and antifungal activities were evaluated, and the
preliminary antimicrobial mechanism was also investigated.
Moreover, the highly bioactive carbazole aminothiazole derivatives
were further examined for their cytotoxic properties on Human
epidermoid cancer cells (Hepꢀ2) by means of CKKꢀ8 Kit.
Additionally, in order to investigate the antimicrobial action
under a nitrogen atmosphere, and then Friedel–Crafts reactions
of
dichloromethane at room temperature produced carbazoles 3a
with yields of 30
with thiourea in refluxing absolute ethanol afforded the
alkylcarbazole aminothiazoles 4a in yields of 94
Nꢀalkylcarbazoles 2a–j with chloroacetyl chloride in
−j
−
36%. The further treatment of compounds
3
a
−
j
N
ꢀ
−
j
−
96%. All
1
13
new compounds were confirmed by H NMR, C NMR, IR,
MS and HRMS spectra (Supplementary Information).
Scheme 1 Synthetic route of
Nꢀalkylcarbazole aminothiazoles. Conditions and reagents: i) corresponding alkyl bromide, NaH, dry DMF, r.t., 6 h;
ii) ClCH COCl, AlCl , dry dichloromethane, r.t., 24 h; iii) thiourea, absolute ethanol, reflux, 1 h.
2
3
compounds gave weak to moderate antimicrobial activity
against the tested strains in vitro. Preliminary active screening
showed that carbazole aminothiazoles 4a possessed better
antibacterial efficacies than their precursors 3a , which
The newly synthesized compounds were evaluated for their revealed that the 2ꢀaminothiazole fragment was important in
antimicrobial activities in vitro against four Gramꢀpositive exerting antimicrobial activities.
3
. Results and discussion
−
j
3.1. Antimicrobial activities
−
j
bacteria (S. aureus ATCC25923, S. aureus N315, B. subtilis
As shown in Table 1, most of the target compounds were
ATCC6633 and M. luteus ATCC4698), four Gramꢀnegative weak or inactive to the tested fungal strains. Interestingly, some
bacteria (E. coli JM109, P. aeruginosa ATCC27853, E. typhosa prepared compounds exhibited good activities against
ATCC14028 and B. proteus ATCC13315) as well as five fungi Fluconazoleꢀinsensitive
Aspergillus
flavus
.
Specially,
(
C. utilis ATCC9950, A. flavus ATCC204304, B. yeast, compounds 4h and 4i showed comparable efficacies (MIC =
ATCC9763, C. albicans ATCC10231, C. mycoderma 128
ꢀ
g/mL) against A. flavus to Fluconazole (MIC = 256
g/mL). For antibacterial activity, the majority of target
well microtest plates according to the National compounds also exhibited weak or moderate inhibition.
Committee for Clinical Laboratory Standards (NCCLS) However, to our surprise, some compounds with moderate
Supplementary Information). The biological tests were carried length alkyl groups showed good activities against some tested
out in triplicate. Minimal inhibitory concentration (MIC, bacteria. Specially,
ꢀpentylcarbazole aminothiazole 4d
g/mL) was defined as the lowest concentration of new exhibited potent inhibition of P. aeruginosa with MIC value of
compounds that completely inhibited the growth of microbes.
g/mL, which was 8ꢀfold more active than reference drug
Currently available antimicrobial drugs Chloromycin, Chloromycin (MIC = 16
g/mL). Among all the target
Norfloxacin and Fluconazole were used as the positive control. compounds, the heptyl substituted compound 4f with MIC
The values of ClogP were calculated using ChemDraw Ultra value of 4
g/mL gave 2ꢀ16 fold more potent antibacterial
0.0 software. The antibacterial and antifungal data as well as efficacy than Chloromycin and Norfloxacin against MRSA.
ClogP values were depicted in Table 1, most of the prepared The preliminary structure activity relationship demonstrated
ATCC9888) using the standard two folds serial dilution method
in 96
ꢀ
-
(
N
ꢁ
2
ꢀ
ꢀ
ꢀ
1
2
| J. Name., 2016, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 3 of 7
View Article Online
DOI: 10.1039/C6MD00357E
Journal Name ARTICLE
that the substituent on
Nꢀposition of carbazole exerted great activities than those of shorter ones, because the longer
influence on the antibacterial efficacies. It could be observed aliphatic group might improve the physiochemical properties
that the prepared compounds with long hydrophobic alkyl chain which could beneficially enhance the absorption rate and
such as pentyl and heptyl groups showed better antimicrobial transport of bioactive compound in vivo
.
a, b, c, d
Table 1 In vitro antimicrobial data as MIC values (µg/mL)
for compounds 3a−j and 4a−j.
Gram-negative bacteria
Gramꢀpositive bacteria
Fungi
Compds ClogP
E.
B.
P.
E.
S.
B.
M.
C.
A.
B.
C.
C.
MRSA
coli
512
256
512
128
512
512
512
512
256
256
128
256
128
256
128
128
512
256
128
128
16
proteus aeruginosa typhosa
aureus subtilis luteus utilis flavus yeast albicans mycoderma
3
3
3
a
b
c
3.80
4.33
4.86
5.39
5.29
6.44
6.98
8.04
10.15
11.21
4.88
5.41
5.94
6.47
6.70
7.53
8.06
9.11
11.23
12.29
ꢀ1.09
ꢀ0.78
ꢀ
512
128
256
512
16
256
256
512
128
16
512
256
128
256
256
256
128
64
512
256
512
256
256
512
512
256
512
256
256
256
128
128
256
128
512
512
512
256
32
512
512
256
512
512
512
512
512
256
256
512
256
256
128
64
256
512
256
256
512
512
512
256
128
128
256
128
128
64
32
16
64
128
256
256
8
2
ꢀ
512
512
256
512
512
512
512
256
256
512
256
128
256
256
512
256
512
512
512
256
32
256
512
256
512
256
512
512
128
256
512
128
128
128
64
256
256
128
128
256
512
512
256
256
512
128
128
256
128
256
256
512
256
128
256
ꢀ
256
512
256
128
512
512
512
512
256
256
256
256
256
256
512
256
256
128
128
256
ꢀ
128
256
512
128
512
512
256
256
512
256
256
256
128
128
256
128
512
256
256
256
ꢀ
512
256
512
256
512
512
512
256
512
512
64
128
512
128
256
256
256
512
256
256
512
64
256
64
64
128
64
3
d
3
3
e
f
g
512
512
256
256
512
256
256
128
256
256
256
128
128
128
256
32
3
3
h
3
3
4
i
j
a
4
b
64
4
c
128
128
256
128
512
128
512
256
ꢀ
4
d
2
32
4
4
e
f
g
256
64
128
512
512
512
512
16
4
32
128
256
512
64
4
4
512
256
256
512
64
512
128
512
512
ꢀ
h
4
4
A
B
C
i
j
16
ꢀ
4
ꢀ
2
ꢀ
4
ꢀ
8
ꢀ
1
ꢀ
2
ꢀ
ꢀ
8
ꢀ
ꢀ
16
ꢀ
32
ꢀ
32
256
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b
MRSA, MethicillinꢀResistant Staphylococcus aureus N315; S. aureus, Staphylococcus aureus ATCC25923; B. subtilis, Bacillus subtilis
Pseudomonas aeruginosa ATCC27853; B.
Bacillus proteus ATCC13315; E. typhosa, Eberthella typhosa ATCC14028; C. utilis, Candida utilis ATCC9950; A. flavus, Aspergillus
flavus ATCC204304; S. cervisea, Sacchromycese cervisea ATCC9763; C. albicans, Candida albicans ATCC10231; C. mycoderma Candida
ATCC6633; M. luteus, Micrococcus luteus ATCC4698; E. coli, Escherichia coli JM109; P. aeruginosa
proteus
,
,
,
mycoderma ATCC9888
.
c
A
= Chloromycin, B = Norfloxacin, C = Fluconazole.
d
ClogP values were calculated by ChemDraw Ultra 10.0
The lipid/water partition of drugs plays an important role in 100 and 125
ꢀg/mL. Cells were cultured for 24 h in these
exerting bioactivities by influencing the transportation, solutions. The cell viability was examined by microplate reader.
1
1
distribution, metabolism and excretion in organisms.
The Cytotoxicity results (Fig. 2) showed that the cell viability of the
ClogP values have been widely employed to predict tested compounds was at least 85% within concentration of 125
transportation and bioactivities of drugs and it is well known ꢁg/mL. It indicated that the tested compounds 4d and 4f did not
that, for ideally pharmacokinetic and pharmacodynamic exhibit cytotoxicity to Hepꢀ2 cells within their MIC against P.
properties in a drug, the distribution coefficient is a moderate aeruginosa and MRSA.
1
2
intermediate between hydrophilicity and hydrophobicity. The
calculated lipid/water partition coefficients (ClogP) of all
prepared compounds were shown in Table 1. The ClogP of
Compound 4d
Compound 4f
1
00
80
60
compounds 4a
alkyl chain, and an enhancement of the antimicrobial activities
was observed in compounds 4a , but decrease in compounds
, these might explain the reason that higher lipophilic
compounds were difficult to access the binding sites.
.2. Cytotoxicity investigation
–j increased with the increasing length of the
–
f
4g–j
4
0
3
20
0
The highly bioactive carbazole aminothiazoles 4d and 4f were
further examined for their cytotoxic properties on Human
1
3
5
10
25
50
100
125
epidermoid cancer cells (Hepꢀ2) by means of CKKꢀ8 Kit.
Compounds 4d and 4f were dissolved in DMSO to prepare the
Concentration of compounds (µg/mL)
Fig. 2 Cytotoxic assay of target compounds 4d and 4f on Hep-2 tested by
stock solutions, and then the tested compounds were prepared CCK-8 Kit. Each data bar represents an average of three parallels, and error
bars indicate one standard deviation from the mean.
in medium to obtain the required concentrations of 5, 10, 25, 50,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2016, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 7
View Article Online
DOI: 10.1039/C6MD00357E
ARTICLE
.3. Interactions with calf thymus DNA
Journal Name
0
3
A
ξC
ξC
1
(1)
=
+ ξD−C
×
0
A− A
ξD−C − ξC
− ξC K[Q]
DNA is an informational molecule encoding the genetic
information used in the development and function of almost all
the known living organisms, which has been regarded as an and presence of compound 4f at 260 nm,
important target for exploitation of novel antimicrobial drugs.
Here, the binding behavior of compound 4f with calf thymus 4f
0
A
and
A
represent the absorbance of DNA in the absence
and
absorption coefficients of compound 4f and compound
ξ
ξD–C are the
C
1
4
0
0
−
DNA complexes respectively. The plot of A /(A–A ) versus
DNA, a DNA model that is medical important, low cost and 1/[compound 4f] is constructed by using the absorption titration
readily available, was studied on molecular level in vitro using data and linear fitting (Supplementary Information: Fig. S1),
4
neutral red (NR) dye as a spectral probe by UV–vis absorption yielding the binding constant,
spectroscopy and fluorescence spectra.
K
= 2.71 × 10 L/mol, R =
1
5
0.9992, SD = 0.05248 (R is the correlation coefficient. SD is
standard deviation).
3
4
.3.1. Absorption spectra of DNA in the presence of compound
f
3.3.2 Absorption spectra of NR interactions with DNA
Absorption spectroscopy is one of the most useful techniques It was apparent that the absorption peak of the NR around 460
used in DNA
binding studies. Hypochromism and nm displayed gradual decrease with the increasing
−
hyperchromism are very important spectral features to concentration of DNA, and a new band around 530 nm
distinguish the change of DNA doubleꢀhelical structure in developed. This was attributed to the formation of the new
1
6
absorption spectroscopy.
Due to the strong interaction DNAꢀNR complexes. An isosbestic point at about 504 nm also
between the electronic states of intercalating chromophore and provided evidence of DNAꢀNR complexes formation. To
that of the DNA bases, the observed large hypochromism further understand the interactions between compound 4f and
strongly displays
chromophore to the DNA bases. With a fixed concentration of compound 4f were investigated. NR is a planar phenazine dye,
DNA, the UV
vis absorption spectra were recorded with the which is structurally similar to other planar dyes like thiazines
increasing amount of compound 4f. As shown in Fig. 3, and xanthenes with higher stability, lower toxicity and more
UV
vis spectra suggested that the maximum absorption peak of convenient application. In recent years, it has been
DNA at 260 nm exhibited proportional increase and red shift demonstrated that the binding of NR with DNA is an
a
close proximity of the aromatic DNA, the absorption spectra of competitive interaction of
−
−
1
8
with the increasing concentration of compound 4f. Meanwhile intercalation mode.
Therefore, NR was used as a spectral
the absorption value of simply sum of free DNA and free probe to investigate the binding mode of 4f with DNA in this
1
9
compound 4f was a little greater than the measured value of 4f
–
work.
The absorption spectra of the NR dye upon the
DNA complexes. This meant that a strong hypochromic effect addition of DNA were displayed in Fig. 4. The research results
existed between DNA and compound 4f. Furthermore, the showed that the absorption peak of the NR around 460 nm
intercalation of the aromatic chromophore of compound 4f into exhibited gradual decrease with the increasing concentration of
*
the helix and the strong overlap of π
−
π states in the large DNA, and a new band around 530 nm developed. This was
π
−
conjugated system with the electronic states of DNA bases attributed to the formation of the new DNA–NR complexes.
1
7
0.30
were consistent with the observed spectral changes.
a
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
.0
4
fꢀDNA complex
0.25
DNA+compound 4f
h
0.9
NR
0
0
0
0
.8
.7
.6
.5
i
0.20
i
0.15
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
1
0ꢀ5 [Q]
0.10
a
a
0.05
DNA
0.00
390
420
450
480
510
540
570
600
225
250
275
300
325
350
Wavelength (nm)
Wavelength (nm)
Fig. 4 UV absorption spectra of NR in the presence of DNA at pH 7.4 and
-5
-5
room temperature. c(NR) = 2 × 10 mol/L, and c(DNA) = 0–3.81 × 10 mol/L
Fig. 3 UV absorption spectra of DNA with different concentrations of
compound 4f (pH = 7.4, T = 290 K). Inset: comparison of absorption at 260
nm between the 4f−DNA complex and the sum values of free DNA and free
-
-5
for curves a–i respectively at increment 0.48 × 10 .
3
.3.3 Absorption spectra of competitive interactions of
-
5
compound 4f. c(DNA)= 5.08 × 10 mol/L, and c(compound 4f) = 0–1.75 × 10
5
-5
compound 4f and NR with DNA
mol/L for curves a−h respectively at increment 0.25 × 10 .
The absorption spectra of a competitive binding between NR
and compound 4f with DNA were showed in Fig. 5, with the
gradually increasing concentration of 4f, an apparent maximum
absorption around 460 nm of the DNAꢀNR complexes
Based on the variations in the absorption spectra of DNA
upon binding to compound 4f, equation (1) can be utilized to
calculate the binding constant ( ).
K
4
| J. Name., 2016, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 5 of 7
View Article Online
DOI: 10.1039/C6MD00357E
Journal Name ARTICLE
increased. Compared with the absorption band around 460 nm distance of 2.27 and 2.32 Å. These results indicated that the
of the free NR in the presence of the increasing concentration importance of the aminothiazole fragment in interaction with DNA.
of DNA (Fig. 4), the absorbance at the same wavelength In addition, electrostatic interactions also existed between compound
exhibited a reverse process (inset of Fig. 5). The results 4f with DA7 and DG10 in DNA. These hydrogen bonds and
suggested that compound 4f intercalated into the double helix electrostatic interactions might be beneficial to stabilize the
of DNA by substituting for NR in the DNAꢀNR complexes. In compound–DNA complexes, which might prevent the formation of
addition, the increase of absorbance at 276 nm and fluorescence hydrogen bonds between DNA base pairs to further influence the
quenching spectra also provided evidence for intercalation of stability of DNA, thus inhibiting its physiological function.
compound 4f into DNA (Supplementary Information: Fig. S2).
0.35
1
1
1
0
0
0
0
0
.4
.2
.0
.8
.6
.4
.2
.0
g
4
. Conclusions
g
a
0
.30
In conclusion, a series of Nꢀalkylcarbazole aminothiazoles were
0.25
designed and synthesized for the first time via an easy,
convenient and efficient synthetic route starting from
commercial carbazole. All the new compounds were confirmed
by NMR, IR and HRMS spectra. The in vitro antimicrobial
evaluation revealed that some target compounds could
effectively inhibit the growth of some tested strains. Structureꢀ
activity relationships suggested that the length of alkyl chain
could significantly affect the antimicrobial efficacies, and the
aminothiazole fragment was important to exert biological
activities. Noticeably, carbazole aminothiazole 4f displayed
a
0.20
0.15
400
425
450
475
500
525
550
Wavelength (nm)
DNA+NR
2
50
300
350
400
450
500
550
600
650
Wavelength (nm)
potent inhibition ability against MRSA
neutral red with DNA. c(DNA) = 4.35 × 10 mol/L, c(NR) = 2 × 10 mol/L, which was better than reference drugs. Further cell toxicity
(MIC = 4 ꢀg/mL),
Fig. 5 UV absorption spectra of the competitive reaction between 4f and
ꢀ5
ꢀ5
ꢀ5
and c(compound 4f) = 0–1.5 × 10 mol/L for curves a–g respectively at
assay indicated that compound 4f did not exhibit cytotoxicity to
Hepꢀ2 cells within concentration of 125 µg/mL, which was far
ꢀ5
increment 0.25 × 10 . Inset: absorption spectra of the system with the
increasing concentration of 4f in the wavelength range of 350–600 nm
absorption spectra of competitive reaction between compound 4f and NR higher than its MIC against MRSA. The interactive
with DNA.
investigations by UV–vis spectroscopic and fluorescence
methods revealed that compound 4f could effectively
intercalate into calf thymus DNA to form 4f−DNA complexes
3
.4 Molecular modeling
To rationalize the observed antibacterial activity and investigate
which might further block DNA replication, and thus exerted
the antimicrobial activities. Molecular docking indicated that
compound 4f could bind with DNA through hydrogen bonds
and electrostatic interactions. All of these indicated that
compound 4f was a promising antimicrobial candidate with
good curative effect and low cell toxicity. Further research,
the action mechanism of carbazole aminothiazole 4f, a flexible
20
ligand receptor docking investigation was undertaken. In this work,
molecular docking study was performed between compound 4f and
bacterial DNA (PDB code: 2XCS) to understand their binding
modes.
including the introduction of aralkyl group (eg
.
pꢀfluorobenzyl,
p
ꢀchlorobenzyl, 2,4ꢀdifluorobenzene benzyl) into
N
ꢀposition of
carbazole and interactions with metal ions as well as in vivo
biological studies are currently in progress, and all of these will
be discussed in the future paper.
Acknowledgements
This work was partially supported by National Natural Science
Foundation of China (No. 21372186, 21172181), the Research
Fund for International Young Scientists from International
(
Regional) Cooperation and Exchange Program of NSFC (No.
1450110451), and the Specialized Research Fund for the
Doctoral Program of Higher Education of China (SRFDP
0110182110007) and Fundamental Research Funds for the
Central Universities (No. XDJK2016E059)
8
Fig. 6 Threeꢀdimensional conformation of compound 4f docked in DNA.
The docking mode with the lowest binding free energy (ꢀ10.48
kJ/mol) was shown in Fig. 6 (Supplementary Information: Fig. S3).
The docking result of compound 4f with DNA might rationalize the
possible binding behaviors. The amino group of aminothiazole was
2
.
in close proximity to the residue DG 9 of DNA through a hydrogen Notes and references
bond with distance of 2.14 Å. Moreover, the amino group in 4f could
1
(a) C. T. Ng, J. L. Mendoza, K. C. Garcia and M. B. A.
Oldstone, Cell, 2016, 164, 349ꢀ352; (b) R. Page and W. Peti,
also form two hydrogen bonds with DNA residue DG 8 with
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2016, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 7
View Article Online
DOI: 10.1039/C6MD00357E
ARTICLE
Nat. Chem. Biol., 2016, 12, 208ꢀ214; (c) S. B. Levy and B.
Journal Name
98; (d) L. M. T. Frija, A. J. L. Pombeiro and M. N.
Kopylovich, Coordin. Chem. Rev., 2016, 308, 32ꢀ55; (e) A.
Ayati, S. Emami, A. Asadipour, A. Shafiee and A. Foroumadi,
Eur. J. Med. Chem., 2015, 97, 699ꢀ718; (f) S. F. Cui, D.
Addla and C. H. Zhou, J. Med. Chem., 2016, 59, 4488ꢀ4510.
Marshall, Nat. Med., 2004, 10, S122ꢀS129; (d) H. Nikaido,
Annu. Rev. Biochem., 2009, 78, 119ꢀ146; (e) X. J. Fang, P.
Jeyakkumar, S. R. Avula, Q. Zhou and C. H Zhou, Bioorg.
Med. Chem. Lett., 2016, 26, 2584ꢀ2588.
2
3
4
(a) X. M. Peng, G. X. Cai and C. H. Zhou, Curr. Top. Med. 11 (a) D. Hunziker, P. C. Wyss, P. Angehrn, A. Mueller, H. P.
Chem., 2013, 13, 1963ꢀ2010; (b) C. H. Zhou and Y. Wang,
Curr. Med. Chem., 2012, 19, 239ꢀ280; (c) L. Zhang, X. M.
Peng, G. L. V. Damu, R. X. Geng and C. H. Zhou, Med. Res.
Rev., 2014, 34, 340ꢀ437; (d) Y. Cheng, H. Wang, D. Addla
and C. H. Zhou, Chin. J. Org. Chem., 2015, 36, 1ꢀ42 (in
Chinese); (e) H. H. Gong, D. Addla, J. S. Lv and C. H. Zhou,
Curr. Top. Med. Chem., 2016, doi: 10.2174/1568026616
Marty, R. Halm, L. Kellenberger, V. Bitsch, G. Biringer, W.
Arnold, A. Stämpfli, A. SchmittꢀHoffmannb and D. Cousot,
Bioorg. Med. Chem., 2004, 12, 3503ꢀ3519; (b) A. L. Wolfe,
K. K. Duncan, J. P. Lajiness, K. Zhu, A. S. Duerfeldt and D.
L. Boger, J. Med. Chem., 2013, 56, 6845ꢀ6857; (c) P. O.
Steven, D. H. Matthew and A. P. James, J. Med. Chem., 2007,
50, 5227ꢀ5237.
6
66160506145943.
12 P. F. Salas, C. Herrmann, J. F. Cawthray, C. Nimphius, A.
Kenkel, J. Chen, C. Kock, P. J. Smith, B. O. Patrick, M. J.
Adam, C. Orvig, J. Med. Chem., 2013, 56, 1596ꢀ1613.
(a) X. L. Hu, D. Li, L. Shao, X. Dong, X. P. He, G. R. Chen
and D. Chen, ACS Med. Chem. Lett., 2015, 6, 793ꢀ797; (b) V.
T. Trang, H. Takeuchi, H. Kudo, S. Katsuno, T. Shimamura, 13 (a) Y. L. Luo, B. kishore, K. V. Kumar, C. H. Zhou and G. X.
T. Kashiwagi, V. H. Son, T. Sugiura and H. Ukeda, J. Agric.
Food Chem., 2011, 59, 8953ꢀ8960; (c) S. Q. Wen, P.
Jeyakkumar, S. R. Avula, L. Zhang and C. H. Zhou, Bioorg.
Cai, Sci. China Chem., 2015, 58, 483ꢀ494; (b) L. W. Zhu, M.
X. Gao, T. T. Wang, X. Y. Wan, L. L. Zheng and C. Z.
Huang, Nanoscale, 2014, 6, 3868ꢀ3874.
Med. Chem. Lett., 2016, 26, 2768ꢀ2773; (d) X. M. Peng, L. P. 14 (a) M. M. Konai, C. Ghosh, V. Yarlagadda, S. Samaddar and
Peng, S. Li, A. S. Rao, K.V. Kumer, S. L. Zhang, K. Y. Tam
and C. H. Zhou, Future Med. Chem., 2016, (in press).
(a) H. J. Knölker and K. R. Reddy, Chem. Rev., 2002, 102,
J. Haldar, J. Med. Chem., 2014, 57, 9409ꢀ9423; (b) S. F. Cui,
Y. Ren, S. L. Zhang, X. M. Peng, G. L. V. Damu, R. X. Geng
and C. H. Zhou, Bioorg. Med. Chem. Lett., 2013, 23, 3267ꢀ
3272; (c) H. Z. Zhang, P. Jeyakkumar, K. V. Kumar and C.
H. Zhou, New J. Chem., 2015, 39, 5776ꢀ5796; (d) L. L. Dai,
H. Z. Zhang, S. Nagarajan, S. Rasheed and C. H. Zhou, Med.
Chem. Comm., 2015, 6, 147ꢀ154; (e) L. Zhang, K. V. Kumar,
R. X. Geng and C. H. Zhou, Bioorg. Med. Chem. Lett., 2015,
25, 3699ꢀ3705; (f) H. H. Gong, K. Baathulaa, J. S. Lv, G. X.
Cai and C. H. Zhou, Med. Chem. Comm., 2016, 7, 924ꢀ931.
15 (a) A. J. Berdis, Biochemistry, 2008, 47, 8253ꢀ8260; (b) L.
Zhang, K. V. Kumar, S. Rasheed, S. L. Zhang, R. X. Geng
and C. H. Zhou, Med. Chem. Comm., 2015, 6, 1303ꢀ1310; (c)
L. P. Peng, S. Nagarajan, S. Rasheed and C. H. Zhou, Med.
Chem. Comm., 2015, 6, 222ꢀ229; (d) G. L. V. Damu, Q. P.
Wang, H. Z. Zhang, Y. Y. Zhang, J. S. Lv and C. H. Zhou,
Sci. China. Chem., 2013, 56, 952ꢀ969; (e) P. Jeyakkumar, L.
4303ꢀ4428; (b) P. Rongved, G. Kirsch, Z. Bouaziz, J. Jose
and B. Le, Eur. J. Med. Chem., 2013, 69, 465ꢀ479; (c) F. F.
Zhang, C. H. Zhou and J. P. Yan, Chin. J. Org. Chem., 2010,
30, 783ꢀ796 (in Chinese); (d) K. Thevissen, A. Marchand, P.
Chaltin, E. M. K. Meert and B. P. A. Cammue, Curr. Med.
Chem., 2009, 16, 2205ꢀ2211; (e) T. C. Chang, J. F. Chu, Y. L.
Tsai and Z. F. Wang, Curr. Pharm. Des., 2012, 18, 2002ꢀ
2
013.
(a) S. B. Markad and N. P. Argade, Org. Lett., 2014, 16,
470ꢀ5473; (b) M. Itoigawa, Y. Kashiwada, C. Ito, H.
5
6
5
Furukawa, Y. Tachibana, K. F. Bastow and K. H. Lee, J. Nat.
Prod., 2000, 63, 893ꢀ897.
(a) A. Gluszyńska, Eur. J. Med. Chem., 2015, 94, 405ꢀ426; (b)
F. A. Tanious, D. Ding, D. A. Patrick, R. R. Tidwell and W.
D. Wilson, Biochemistry, 1997, 36, 15315ꢀ15325; (c) F. A.
Tanious, D. Ding, D. A. Patrick, C. Bailly, R. R. Tidwell and
Zhang, S. R. Avula and C. H. Zhou, Eur. J. Med. Chem.
2016, doi: 10.1016/j.ejmech.2016.06.031
,
.
W. D. Wilson, Biochemistry, 2000, 39, 12091ꢀ12101; (d) B. 16 (a) C. D. Kanakis, S. Nafisi, M. Rajabi, A. Shadaloi, P. A.
Maji, K. Kumar, M. Kaulage, K. Muniyappa and S.
Bhattacharya, J. Med. Chem., 2014, 57, 6973ꢀ6988.
Tarantilis, M. G. Polissiou, J. Bariyanga and H. A. Tajmirꢀ
Riahi, Spectroscopy, 2009, 23, 29ꢀ43; (b) X. M. Peng, K. V.
7
(a) C. Uvarani, M. Sankaran, N. Jaivel, K. Chandraprakash,
A. Ata and P. S. Mohan, J. Nat. Prod., 2013, 76, 993ꢀ1000; (b)
Kumar, G. L. V. Damu and C. H. Zhou, Sci. China Chem.,
2015, doi: 10.1007/s11426ꢀ016ꢀ5557ꢀ4.
A. W. Schmidt, K. R. Reddy and H. J. Knoelker, Chem. Rev.
012, 112, 3193ꢀ3328; (c) M. K. Kathiravan, A. B. Salake, A.
S. Chothe, P. B. Dudhe, R. P. Watode, M. S. Mukta and S.
,
17 (a) G. W. Zhang, P. Fu, L. Wang and M. M. Hu, J. Agric.
Food Chem., 2011, 59, 8944ꢀ8952; (b) X. L. Li and Y. J. Hu,
Biomacromolecules, 2012, 13, 873ꢀ880.
2
Gadhwe, Bioorg. Med. Chem., 2012, 20, 5678ꢀ5698; (d) L. 18 (a) B. T. Yin, C. Y. Yan, X. M. Peng, S. L. Zhang, S.
Hu, Z. R. Li, Y. Li, J. Qu, Y. H. Ling, J. D. Jiang and D. W.
Boykin, J. Med. Chem., 2006, 49, 6273ꢀ6282.
F. F. Zhang, L. L. Gan and C. H. Zhou, Bioorg. Med. Chem.
Lett., 2010, 20, 1881ꢀ1884.
Rasheed, R. X. Geng and C. H. Zhou, Eur. J. Med. Chem.,
2014, 71, 148ꢀ159; (b) G. L. V. Damu, S. F. Cui, X. M. Peng,
Q. M. Wen, G. X. Cai and C. H. Zhou, Bioorg. Med. Chem.
Lett., 2014, 24, 3605ꢀ3608; (c) J. S. Lv, X. M. Peng, B.
Kishore and C. H. Zhou, Bioorg. Med. Chem. Lett., 2014, 24,
308ꢀ313.
8
9
(a) A. W. Schmidt, K. R. Reddy and H. Knölker, Chem. Rev.
,
2012, 112, 3193ꢀ3328; (b) P. Rajakumar, K. Sekar, V.
Shanmugaiah and N. Mathivanan, Eur. J. Med. Chem., 2009, 19 Y. Ni, S. Dua and S. Kokot, Anal. Chim. Acta, 2007, 584, 19ꢀ
4, 3040ꢀ3045; (c) B. P. Bandgar, L. K. Adsul, H. V. Chavan, 27.
S. S. Jalde, S. N. Shringare, R. Shaikh, R. J. Meshram, R. N. 20 (a) S. Hameed, P. A. Raichurkar, P. Madhavapeddi, S.
4
Gacche and V. Masand, Bioorg. Med. Chem. Lett., 2012, 22,
839ꢀ5844; (d) J. Molette, J. Routier, N. Abla, D. Besson, A.
Bombrun, R. Brun, H. Burt, K. Georgi, M. Kaiser, S. Nwaka,
Menasinakai, S. Sharma, P. Kaur, R. Nandishaiah, V.
Panduga, J. Reddy, V. K. Sambandamurthy and D. Sriram,
ACS Med. Chem. Lett., 2014, 5, 820ꢀ825; (b) M. H. Foss, K.
A. Hurley, N. A. Sorto, L. L. Lackner, K. M. Thornton, J. T.
Shaw and D. B. Weibel, ACS Med. Chem. Lett., 2011, 2, 289ꢀ
292; (c) J. I. Perlmutter, L. T. Forbes, D. J. Krysan, K.
EbsworthꢀMojica, J. M. Colquhoun, J. L. Wang, P. M.
Dunman and D. P. Flaherty, J. Med. Chem., 2014, 57, 8540ꢀ
8562.
5
M. Muzerelle and A. Scheer, ACS Med. Chem. Lett., 2013, 4,
1037ꢀ1041.
1
0 (a) S. F. Cui, Y. Wang, J. S. Lv, G. L. V. Damu and C. H.
Zhou, Sci. Sin.: Chim., 2012, 42, 1105ꢀ1131 (in Chinese); (b)
S. F. Cui, C. H. Zhou, R. X. Geng and Q. G. Ji, Chin. J.
Biochem. Pharm., 2012, 33, 311ꢀ315 (in Chinese); (c) D. Das,
P. Sikdar and M. Bairagi, Eur. J. Med. Chem., 2016, 109, 89ꢀ
6
| J. Name., 2016, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
MedChemComm
View Article Online
DOI: 10.1039/C6MD00357E
Design, synthesis, biological evaluation of novel carbazole aminothiazoles as potential DNA-targeting
antimicrobial agents
1
,#,†
1,#
1
1,‡
2,*
Dinesh Addla , Si-Qi Wen , Wei-Wei Gao , Swetha Kameswari Maddili , Ling Zhang and Cheng-He
1
,*
Zhou
1
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
2
School of Pharmacy, Yancheng Teachers University, Yancheng 224051, PR China
#
These two authors contributed equally to this work.
†
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India.
‡
Ph.D candidate from India.
Synthesis of a series of carbazole aminothiazoles as a new type of potential antimicrobial agents, and
preliminary interactions with DNA indicated a possible intercalation mechanism.
0.35
1
.4
g
g
S
S
1.2
1.0
0.8
0.6
0.4
0.2
0.30
H2N
NH2
0.25
a
MIC = 4 g/mL (MRSA)
N
N
a
0.20
Expriment: intercalate into DNA for blocking DNA
replication
0.15
00
4
425
450
475
500
525
550
N
Wavelength (nm)
Molecule docking: interact with DNA by hydrogen
bonds and electrostatic interactions
H3C
DNA+NR
4
0
.0
2
50
300
350
400
450
500
550
600
650
Wavelength (nm)