RSC Advances

View Article Online

PAPER View Journal | View Issue

An expedient route to highly diversified [1,2,3]-

Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

triazolo[1,5-a][1,4]benzodiazepines and their

evaluation for antimicrobial, antiproliferative and in
Cite this: RSC Adv., 2015, 5, 66260

silico studies†
N. Sudhapriya,a A. Nandakumar,a Y. Arun,a P. T. Perumal,*a C. Balachandranb
and Nobuhiko Emib

An efficient diversity oriented synthesis of [1,2,3]triazolo[1,5-a][1,4]benzodiazepines has been developed by

sequential diazotization, azidation and cycloaddition reactions in a one-pot fashion. This strategy allows an
easy accessibility of triazole fused [1,4]benzodiazepines in good yields. The main objective of this
methodology is to introduce various substituents at all possible positions under mild reaction conditions.
All the synthesized compounds were evaluated for their antimicrobial, anticancer and in silico activity.
Among the tested compounds (2a–n), the derivatives 2a, 2b, 2d, 2k, 2g, 2j, 2m and 2l have displayed a
broad spectrum of antibacterial activity. Anticancer activity results revealed that compounds 2a, 2g and
Received 28th June 2015
Accepted 27th July 2015
2m exhibited potent in vitro anticancer activity against A549 lung adenocarcinoma cancer cell line.
Further, molecular docking studies of all the synthesized compounds were performed to gain a
DOI: 10.1039/c5ra12497b
comprehensive understanding of the plausible binding modes and also to compare the theoretical and
www.rsc.org/advances experimental results of these compounds.

known as antidepressants11 and compound (5), derived from

Introduction the further elaborated [1,2,3]triazolo[1,4]benzodiazepine skel-
Benzodiazepines are privileged heterocyclic structures and their eton, displays activity as protease inhibitors (Fig. 1).12
synthesis has been receiving much attention in the eld of Careful examination of the literature indicates that the
medicinal and pharmaceutical chemistry owing to their appli- majority of syntheses have been developed through inter/intra
cations as anticonvulsants,1 anti-inammatories,2 HIV inhibi- molecular cycloaddition to generate the triazole ring.13 The
tors,3 farnesyl-transferase inhibitors4 and receptor ligands in efficiency of the cycloaddition has been successfully demon-
neurodegenerative diseases.5 Benzodiazepine derivatives have strated in both aqueous14 and organic solvents involving metal
also been reported to possess antibacterial, antifungal and catalyzed15/metal-free reactions16 and various thermal condi-
antitumor activities.6 Fused heterocyclic benzodiazepines have tions. Ideally an intramolecular 1,3-dipolar cycloaddition reac-
attracted considerable attention due to their highly potent tion can be expected to furnish two annulated cyclic rings in
anxiolytic7 or anti-depressant activity.8 1,2,3-Triazole derived comparison to the intermolecular format. Several groups have
molecules were found to have antiprotozoal, antiviral, anti-
leishmanial and anticancer properties.9
Furthermore, fused-ring systems consisting of multifarious
heterocycles such as 1,4-benzodiazepine and triazole substruc-
tures have attracted considerable attention due to their highly
potent biological activities. Some of the examples for triazolo
benzodiazepines are alprazolam (1), estazolam (2) are used as
anxiolytic agents10 whereas triazolam (3), adinazolam (4) are

a
Organic & Bio-Organic Chemistry Division, CSIR – Central Leather Research Institute,
Adyar, Chennai-600020, India. E-mail: [email protected]
b
Department of Hematology, Fujita Health University, 1-98 Dengakugakubo,
Kutsukake-cho, Toyoake, Aichi 470-1192, Japan
† Electronic supplementary information (ESI) available. CCDC 1036732. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c5ra12497b Fig. 1 Selected examples of triazolo-[1,4]benzodiazepines.

66260 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Paper RSC Advances

multistep format to promote 1,3-dipolar cycloaddition with

limited applications.18
Numerous reports demonstrate the formation of triazole
fused benzodiazepines. Though the efficient methodologies to
prepare the diversely functionalized fused 1,4-benzodiazepines
remains a challenge to modern synthetic organic chemists.19
Therefore, new and efficient methods that could be carried out
under milder and eco-friendly condition always demand special
importance to the eld of synthetic organic chemistry. Martin
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

et al. have synthesized 1,4-benzodiazepines fused with a 1,2,3-

triazole ring and diversied them via a variety of refunctional-
izations.20 Copper catalyzed tandem Ullmann C–N coupling
followed by azide–alkyne cycloaddition approach has been
described by Majumdar and co-worker.21 Eycken group have
reported Cu-catalyzed azidation–cyclization reaction for the
synthesis of fused triazoles.22 Although the above mentioned
method is efficient, it is limited to the aliphatic substituents at
C3-position and it requires metal-catalyst. Our rationale is
based on the idea of assembling an aromatic substituent at C3-
position of triazolo-1,4-benzodiazepines involving in situ
generated aryl azide under mild reaction condition (Scheme 1).
Scheme 1 Methods for the preparation of triazolo-1,4- Prompted by the synthetic interest and inevitable medicinal
benzodiazepines.
properties of triazole fused benzodiazepines we report herein a
facile route to highly substituted [1,2,3]triazolo[1,5-a][1,4]
benzodiazepines under metal-free conditions and the evalua-
Table 1 Optimization of reaction conditions
tion of the biological activity of resulting compounds.

Results and discussion

Chemistry
We have developed many general approaches for preparing the
various heterocyclic scaffolds of possible medical relevance by a
strategy that involves sequencing multicomponent assembly
processes (MCAPs) with subsequent cyclizations.23 As a contin-
Isolated yield uation of these investigations, we found an expedient route to a
Entry Solvent Temp (
C) Time (h) (%) compound bearing the triazolo-1,4-benzodiazepine scaffold.
Our synthetic approach started with 2-amino-N-benzylpro-
1 Diethyl ether 0 5 Tracea pargylamine 1a as a model substrate to synthesize fused tri-
2 Diethyl ether 0 12 Tracea
3 Benzene 60 12 20b
azoles 2a mild reaction condition. This precursor 1a can be
4 Toluene 80 12 30b obtained using the sequential A3-coupling reaction of N-benzyl-
5 Toluene 100 24 45b 1-(2-nitrophenyl)methanamine, aromatic aldehyde, alkyne fol-
5 DMSO 120 24 48c lowed by the reduction of –NO2 group (as mentioned in the
6 EtOH 60 12 55c ESI†). In an exploratory experiment the reaction conditions for
7 H2O/DMSO 100 12 68c
8 H2O/EtOH 100 12 75c
the one-pot diazotization, azidation and cycloaddition were
9 H2O rt 12 50c investigated.
10 H2O rt 24 62c The investigation on the optimization of reaction parameters
11 H2O 80 12 78c included temperature, solvent and reaction time (Table 1). A
12 H2O 100 12 86c variety of solvents were explored which included diethyl ether,
13 H2O 100 24 85c
benzene, toluene, EtOH, DMSO and H2O. The reaction was
a
NaNO2 (2 eq.), 1 N HCl, glacial acetic acid (7 mL), NaN3 (6 eq.). found to proceed well using polar protic solvents (Table 1
b
NaNO2 (2 eq.), 1 N HCl, NaN3 (2 eq.). c NaNO2 (1.4 eq.), 2 N HCl,
NaN3 (1.4 eq.).
entries 6–13) when compared to other solvents (Table 1, entries
1–5). Interestingly H2O was found to be the best solvent for the
formation of fused triazoles (Table 1, entries 7–13). Subse-
quently the effect of reaction temperature was studied (Table 1,
reported intramolecular azide–alkyne 1,3-dipolar cycloaddition
entries 9–12). At room temperature the reaction proceeded
strategy for the synthesis of triazole linked polyhetrocycles.17
slowly and resulted in a lower yield of the product. The increase
However most of these strategies predominantly follows
in temperature increased the rate of the reaction. The scope of

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 66260–66270 | 66261
 View Article Online

RSC Advances Paper

reaction time was also studied (Table 1 entries 12 & 13). Thus
the best result was obtained when 1.4 eq. of NaNO2, 2 N of HCl,
1.4 eq. of NaN3 and 2 mL of H2O were used for the in situ
generation of azide and followed by the cycloaddition reaction
to obtain the desired product in a single step.
With these optimal conditions in hand, we set out to explore
the scope of our method for the preparation and diversication
Scheme 2 Preparation of diversified triazolo-1,4-benzodiazepines.
of 2-amino-N-benzylpropargylamines (Scheme 2).
We can diversify the nal products by varying N-benzyl/alkyl-
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

1-(2-nitrophenyl)methanamine, aromatic aldehyde, alkyne used

for the A3-coupling reaction (see ESI†). We utilized a variety of
commercial aldehydes such as paraformaldehyde, 1-naph-
thaldehyde, furan-3-carbaldehyde and other aromatic alde-
hydes (R1–CHO) (R1 ¼ –Ph, p-MeC6H4, o-FC6H4, p-ClC6H4, p-
BrC6H4) (Scheme 3). Aliphatic aldehyde provides higher yield
when compared to aromatic and hetero aromatic aldehydes.
The halo substituents gave lower yield when compared to the
electron donating methyl substituent on the phenyl ring of
aldehydes. Next, we examined the substrate scope by varying the
alkynes substituents. The reaction is tolerant for both alkyl and
aryl substituted terminal alkynes (R2 ¼ n-butyl, p-MeC6H4, p-
FC6H4). Methyl substituted aromatic alkynes gives higher yield
than the other alkynes. The desired compounds are obtained as
a single diastereoisomer. At the amine position we have intro-
duced alkyl (n-butyl, n-hexyl) and benzyl substituents. The main
advantage of this method is that we can introduce the substit-
uents even at the R3 position (Scheme 4).
Scheme 3 Scope of aldehydes. A plausible reaction mechanism for the formation of triazole
fused [1,4]benzodiazepines is depicted in Scheme 5. 2-Amino-N-
benzylpropargylamine 1 was subjected to diazotization reaction
using sodium nitrite in dilute HCl to generate diazonium salt A.
Subsequent treatment of in situ generated diazonium salt A with
sodium azide produced the corresponding azido compound B.
Concurrent intramolecular azide–alkyne cycloaddition of B
under thermal conditions would lead to the formation of
annulated 7- and 5-membered heterocyclic rings as a desired
product 2.
The structure of all prepared compounds were elucidated by
the 1H, 13C NMR experiments and further proved by the mass data.
The disappearance of characteristic peak corresponds to
–NH2 protons in 1H NMR spectra and the disappearance of
peaks corresponds to acetylenic carbons at the d value 84.9, 88.8
ppm indicates the formation of cyclized product. The stereo-
chemistry of the compounds is clearly identied from the single
crystal X-ray analysis of compound 2e (Fig. 2)24 which further
supports the NMR spectroscopy.

Pharmacology
Antimicrobial activity. All the synthesized compounds were
screened for the antimicrobial activities against eleven bacteria
and two fungi using well method. The results revealed that most
of the synthesized compounds exhibited good antimicrobial
activities against S. paratyphi B, E. aerogens, S. epidermidis, S.
Scheme 4 Exploring the scope of alkynes (2i–k) and various substit- typhimurium, K. pneumonia, P. aeruginosa and S. aureus. The
uents at R3 and R4 (2l–n). results are summarized in Table 2 and Fig. 3. Compounds 2a,
2b, 2c, 2d, 2g, 2j and 2m have shown excellent activities nearly

66262 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Paper RSC Advances

Anticancer activity. Anti-cancer activity studies have been

performed for the synthesized compounds 2a, 2b, 2g, 2j, 2l and
2m (Table 4 and Fig. 5). All the tested compounds showed good
cytotoxicity activity against cell line, however some of the
synthesized compounds showed prominent cytotoxic activity in
vitro against A549 adenocarcinoma lung cancer cell line. The
anticancer activity against A549 cell line was observed at 200 to
50 mg mL1 concentration. In general alkyl substituents at –C3
and –N4 position shows better activity than the other
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

compounds. Also, naphthyl substituent at –C3 position

increases the activity of the product. Interestingly, among all the
tested compounds 2a, 2m and 2g showed very good activity with
IC50 value at 50 mg mL1. In particular, among the tested
compounds 2a, 2m and 2g showed very high activity 64.3%,
61.9% and 75.3% at 200 mg mL1 concentrations against A549
lung adenocarcinoma cancer cell line. All concentrations used
in the experiment decreased the cell viability signicantly (P <
Scheme 5 Proposed mechanism for the formation of 2.
0.05) in a concentration-dependent manner.
Molecular docking studies. To rationalize the pharmaco-
logical results, molecular docking studies were performed using
the AutoDock Tools (ADT) version 1.5.6 and AutoDock version
4.2.5.1 docking program25 on a DNA gyrase (PDB ID: 2XCS) and
Anaplastic Lymphoma Kinase (ALK) receptor (PDB ID: 2XP2)26
to simulate the interaction of the synthesized compounds with
the receptor binding site.
The co-crystallized ligand was extracted from the complex
and submitted for one-ligand run calculation in order to verify
the reproducibility of the docking calculation. This reproduced
top scoring conformation falling within root-mean-square
deviation (RMSD) value of 1.63 Å with bound X-ray conforma-
tion for 2XP2, suggesting this method is valid enough to be used
for docking studies of other compounds.
Fig. 2 ORTEP of compound 2e. The same protocol was applied to all the synthesized
compounds and docking simulation was performed in the same
active site using AutoDock aer the validation study. All dock-
equal to the standard and 2k is showing better activity than the ings were taken into 2.5 million energy evaluations were per-
standard drug against S. aureus at 1 mg per well. Moreover the formed for each of the test molecules. Docked ligand
compounds 2a, 2b, 2d, 2k, 2g, 2j, 2m and 2l showed good conformations were analyzed in terms of energy, hydrogen
antibacterial activity over the others. All tested compounds bonding, hydrophobic and p–p interaction between ligand and
showed moderate antifungal activity against C. albican and M. DNA topoisomerase IV receptor. The ligand–receptor interac-
pachydermatis. tions were clearly analyzed, and nal coordinates of the ligand
The Minimum Inhibitory Concentration (MIC) values of and receptor were saved. Aer docking, output was exported to
active compounds against bacteria are given in Table 3 and PyMOL soware for display of the ligand with the receptor
Fig. 4. Signicant MIC values were observed against gram binding site.27 The free energy of binding (FEB) of all
positive and gram negative bacteria. The results revealed that compounds were calculated from the docking scores (Table 5).
the fused triazoles 2a, 2b, 2d, 2j, 2l and 2m have shown good Docking studies of synthesized compounds with 2XCS
antibacterial activity against tested organisms. Among all tested receptor show that all the docked compounds bind efficiently
compounds phenyl ring containing compound 2b has shown with the receptor and exhibits free energy of binding value from
signicant MIC values against P. vulgaris, S. exneri, 4-methyl 9.13 to 11.07 kcal mol1. Interestingly, among all the
substituted aromatic rings containing compound 2j is potent compounds docked, compound 2g exhibits very high binding
against S. aureus (MRSA), P. vulgaris and S. exneri. The naph- with 2XCS receptor and forms two polar interactions with three
thyl substituted compound 2g is active against S. exneri and P. amino acid namely MET-1121, resulted in the binding energy of
vulgaris. The N-butyl substituted fused triazole compound 2m 11.07 kcal mol1. As shown in Fig. 6, in the compound 2g, two
showed signicant MIC values against P. vulgaris and S. aureus nitrogens of triazole ring interact with N–H of MET-1121, forms
(MRSA). two polar interactions with the distance of 2.1 and 2.5 Å. In

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 66260–66270 | 66263
 View Article Online

RSC Advances Paper

Table 2 In vitro antimicrobial activity of synthesized compoundsa

Zone of inhibition in mm

Gram positive bacteria Gram negative bacteria Fungi

S.
S. S. aureus M. E. S. K. P. S. S. P. C. M.
Compounds epidermidis aureus (MRSA) luteus aerogenes typhimurium pneumoniae vulgaris exneri Paratyphi B aeruginosa albicans pachydermatis
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

2a 17 15 15 18 18 16 14 22 21 15 16 NI NI
2b 15 12 13 19 16 14 16 23 25 14 15 10 12
2c 12 13 NI 10 10 11 13 10 NI 12 13 NI NI
2d 12 12 10 14 13 NI 12 10 16 12 14 10 10
2e NI NI NI NI NI NI NI NI NI NI 16 12 12
2f NI NI NI NI NI NI NI NI NI NI NI NI NI
2g 14 13 18 22 20 17 18 23 26 15 NI NI NI
2h 14 12 10 10 11 11 10 12 10 10 12 NI 13
2i 12 10 13 15 12 11 14 11 10 13 14 10 10
2j 15 12 23 18 12 22 17 24 23 14 15 NI 13
2k 13 15 12 16 16 13 14 15 12 14 10 12 10
2l 15 13 10 18 20 20 14 17 19 16 12 NI 13
2m 10 12 23 20 18 22 18 25 21 17 18 10 13
2n NI NI 13 NI 14 12 NI NI NI 10 13 NI NI
Streptomycin 26 14 30 26 22 24 20 30 30 18 30 NA NA
Ketoconazole NA NA NA NA NA NA NA NA NA NA NA 28 26
a
NA – not applicable NI – no inhibition.

Fig. 3 Comparison of antimicrobial activity of synthesized compounds and standard drugs.

addition, naphthyl moiety exhibits hydrophobic interaction of 10.07 kcal mol1. As shown in Fig. 7, in the compound 2g,
with GLY-1117, ALA-1118, ALA-1119 and ALA-1120 amino acids. two nitrogens of triazole ring interact with C]O of ASP-1203,
Synthesized compounds efficiently bind with the active site forms two polar interactions with the distance of 2.7 and 2.9
of 2XP2 receptor and exhibits free energy of binding value from Å. Further, naphthyl and phenyl moieties exhibit hydrophobic
7.70 to 10.07 kcal mol1. All the synthesized compounds interaction with LEU-1122, VAL-1130, ALA-1148, LEU-1198 and
interact in the 24 active site amino acids namely ARG-1120, ALA-1200 amino acids.
LEU-1122, GLY-1123, VAL-1130, GLU-1132, ALA-1148, LYS-
1150, LEU-1196, GLU-1197, LEU-1198, MET-1199, ALA-1200,
GLY-1201, GLY-1202, ASP-1203, SER-1206, PHE-1207, GLU- Conclusions
1210, ARG-1253, ASN-1254, CYS-1255, LEU-1256, GLY-1269
and ASP-1270. Among all the compounds docked, compound In conclusion, an efficient method for the synthesis of highly
2g exhibits very high binding with 2XP2 receptor and forms two diversied [1,2,3]triazolo[1,5-a][1,4]benzodiazepine derivatives
polar interactions with ASP-1203, resulted in the binding energy (2a–n) under mild reaction condition were reported in good
yields via MCR approach and evaluated for their in vitro

66264 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Paper RSC Advances

Table 3 MIC (mg mL1) of compounds tested against bacteria

Gram positive bacteria Gram negative bacteria

S. S. S. aureus M. E. S. K. P. S. S. P.
Compounds epidermidis aureus (MRSA) luteus aerogenes typhimurium pneumoniae vulgaris exneri paratyphi B aeruginosa

2a 125 125 125 125 125 125 250 62.5 62.5 125 125
2b 125 250 250 62.5 125 250 125 31.25 31.25 250 125
2d 250 250 500 250 250 NI 250 500 125 250 250
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

2g 250 250 125 62.5 250 125 125 31.25 31.25 125 125
2j 125 250 31.25 125 250 62.5 125 31.25 31.25 250 31.25
2k 250 125 250 125 125 250 250 125 250 250 500
2l 250 250 250 125 62.5 62.5 250 125 62.5 125 250
2m 500 250 31.25 62.5 125 62.5 125 31.25 62.5 125 62.5
Streptomycin 25 6.25 6.25 6.25 25 6.25 6.25 6.25 6.25 30 25
Ciprooxacin 6.25 <0.78 <0.78 >100 <0.78 >100 6.25 6.25 <0.78 6.25 6.25

Fig. 4 Comparison of MIC (mg mL1) values of synthesized compounds and standard drugs.

Table 4 Anticancer activity of synthesized compounds against A549 cancer cell line

2a 2b 2g 2j 2l 2m

Conc. (mg mL1) % Mean  S.D % Mean  S.D % Mean  S.D % Mean  S.D % Mean  S.D % Mean  S.D

50 21.1 1.134  0.004 21.7 1.124  0.004 55.9 0.632  0.004 4.8 1.367  0.005 4.4 1.373  0.006 35.9 0.921  0.0062
100 34.9 0.935  0.002 34.4 0.942  0.003 64.3 0.512  0.007 28.6 1.025  0.008 26.5 1.056  0.006 52.6 0.681  0.0039
200 64.3 0.512  0.006 51.4 0.698  0.009 75.3 0.354  0.008 41.2 0.845  0.004 35.9 0.921  0.004 61.9 0.546  0.0043

antimicrobial and anticancer studies. Among the series, benzodiazepine can be promising therapeutic agents for A549
compounds 2a, 2b, 2d, 2k, 2g, 2j, 2m and 2l were found to be lung adenocarcinoma cancer cell line.
potent with respect to standard drugs streptomycin and cipro-
oxacin. Anticancer studies revealed that, the compound 2g
possessing naphthyl group showed potent anticancer activity Experimental
against A549 lung adenocarcinoma cancer cell line. In order to
Chemistry
support the in vitro antibacterial and anticancer results, the
synthesized compounds were docked in to the plausible target Analytical TLC was performed on precoated aluminium sheets
enzymes. The binding energies and H-bond interactions with of silica gel 60F254 of 0.2 mm thickness (Merck, Germany).
amino acids in active site of target enzyme well supported the in Melting points were determined on Gallenkamp melting point
vitro results. These compounds [1,2,3]triazolo[1,5-a][1,4] apparatus and are uncorrected. 1H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were recorded in CDCl3 solutions with

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 66260–66270 | 66265
 View Article Online

RSC Advances Paper

Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

Fig. 5 Comparison of anticancer activity of synthesized compounds

against A549 cancer cell line.

Table 5 Binding energy and the interaction of ligands with the DNA
gyrase and ALK receptor

Binding energya (kcal mol1)

Compound DNA gyrase (2XCS) ALK (2XP2)

2a 10.24 7.70
2b 10.66 8.58
2c 9.79 8.89
2d 10.60 8.65
2e 9.93 8.95
2f 10.24 9.07
2g 11.07 10.07
2h 9.84 8.24
2i 9.32 8.17
2j 10.24 8.37
2k 9.75 8.42
2l 10.48 8.94
2m 9.13 7.77
2n 9.48 8.24
Crystallized ligand 13.81 8.40 Fig. 6 Docking simulation in the active site of 2XCS receptor.
Standard drug 7.94b, 5.85c
a
Calculated by AutoDock. b Streptomycin. c Ciprooxacin.
NaNO2 (1.26 mmol) in 2 mL H2O dropwise during 35 min and
the mixture was allowed to stir for another 30 min at the same
TMS as an internal standard on a Brucker Avance DPX-400 MHz temperature. A solution of NaN3 (1.26 mmol) in 2 mL H2O was
instrument. Proton chemical shis (d) are relative to tetrame- added dropwise during 35 min under ice-cooled condition and
thylsilane (TMS, d ¼ 0.00) as internal standard and expressed in the stirring was continued for another 15 min. The reaction
parts per million. The number of protons (n) for a given reso- mixture was allowed to come to room temperature during about
nance was indicated as nH. Coupling constants (J) are given in 45 min. It was then heated at 100
C for 12 h to obtain a desired
hertz. Spin multiplicities are given as s (singlet), d (doublet), t fused triazole.
(triplet) and m (multiplet). Mass spectra were recorded under 5-Benzyl-3-phenyl-5,6-dihydro-4H-benzo[f][1,2,3]triazolo[1,5-
Mass spectra were recorded using ESI/HRMS at 60 000 resolu- a][1,4]diazepine (2a). White solid. Yield: 88%. mp: 154–156
C.
tion in Thermoscientic Exactive mass spectrometer and ESI/
1
H NMR (400 MHz, CDCl3) d 7.96 (d, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼
MS using a Thermo Finnigan LCQ Advantage MAX 6000 ESI 7.6 Hz, 2H), 7.59 (t, J ¼ 7.6 Hz, 1H), 7.51–7.37 (m, 5H), 7.35 (d, J
mass spectrometer. Elemental analyses were recorded using a ¼ 4.4 Hz, 4H), 7.33–7.27 (m, 1H), 3.74 (d, J ¼ 6.4 Hz, 4H), 3.65 (s,
Thermo Finnigan FLASH EA1112 CHN analyzer. 2H). 13C NMR (100 MHz, CDCl3) d 145.4, 137.9, 136.9, 131.1,
130.7, 130.2, 129.6, 129.1, 128.9, 128.88, 128.6, 128.3, 127.6,
127.5, 122.8, 60.3, 54.5, 45.1. HRMS (ESI): mass calculated for
Experimental procedure for the synthesis of (2a–n) C23H21N4 [M + H]+ 353.1761, found, [M + H]+ 353.1772.
To a stirred and cooled (0–3
C) solution of 2-amino-N-benzyl- 5-Benzyl-3,4-diphenyl-5,6-dihydro-4H-benzo[f][1,2,3]triazolo
propargylamine (0.90 mmol) in 2 N HCl (8.0 mL) was added [1,5-a][1,4]diazepine (2b). White solid. Yield: 86%. mp: 197–199

66266 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Paper RSC Advances

calculated for C30H27N4 [M + H]+ 443.2230, found, [M + H]+

443.2223.
5-Benzyl-4-(2-uorophenyl)-3-phenyl-5,6-dihydro-4H-benzo-
[f][1,2,3]triazolo[1,5-a][1,4]diazepine (2d). White solid. Yield:
76%. mp: 151–153
C. 1H NMR (400 MHz, CDCl3) d 7.93 (d, J ¼
7.6 Hz, 1H), 7.48–7.40 (m, 4H), 7.37 (t, J ¼ 7.4 Hz, 1H), 7.25–7.30
(m, 9H), 6.98 (dd, J ¼ 13.2, 7.2 Hz, 1H), 6.80 (t, J ¼ 7.6 Hz, 1H),
6.70–6.65 (m, 1H), 5.40 (s, 1H), 3.95 (d, J ¼ 13.6 Hz, 1H), 3.78 (q,
J ¼ 13.6 Hz, 2H), 3.64 (d, J ¼ 13.6 Hz, 1H). 13C NMR (100 MHz,
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

CDCl3) d 161.1, 158.6, 146.6, 137.9, 136.9, 132.5, 130.7, 130.5,

130.0 (d, J ¼ 3.3 Hz), 129.6, 129.2, 129.1 (d, J ¼ 8.3 Hz), 128.8,
128.7, 128.6 (d, J ¼ 4.4 Hz), 128.3, 128.0, 127.6, 126.2 (d, J ¼ 11.7
Hz), 123.3 (d, J ¼ 3.4 Hz), 122.6, 115.2, 115.0, 59.0, 54.1, 53.3. MS
(ESI) for C29H23FN4 m/z ¼ 447 [M + H]+. Elemental analysis calc
for C29H23FN4: C, 78.01; H, 5.19; F, 4.25; N, 12.55 found, C 78.04,
H, 5.16; F, 4.23; N, 12.58.
5-Benzyl-4-(4-chlorophenyl)-3-phenyl-5,6-dihydro-4H-benzo-
[f][1,2,3]triazolo[1,5-a][1,4]diazepine (2e). White solid. Yield:
80%. mp: 181–183
C. 1H NMR (400 MHz, CDCl3) d 7.66 (d, J ¼
8.0 Hz, 1H), 7.61–7.59 (m, 2H), 7.42–7.41 (m, 3H), 7.32–7.28 (m,
1H), 7.27–7.23 (m, 5H), 7.17–7.19 (m, 2H), 6.98–6.93 (m, 4H),
5.38 (s, 1H), 3.94 (dd, J ¼ 26.4, 12.8 Hz, 2H), 3.70 (d, J ¼ 12.8 Hz,
1H), 3.51 (d, J ¼ 12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) d
147.5, 139.0, 137.6, 137.1, 132.6, 132.4, 130.4, 130.3, 129.9,
129.2, 129.0, 128.9, 128.8, 128.5, 128.4, 128.1, 128.0, 127.9,
127.7, 122.3, 60.7, 56.6, 54.8. HRMS (ESI): mass calculated for
C29H24ClN4 [M + H]+ 463.1684, found, [M + H]+ 463.1676.
5-Benzyl-4-(4-bromophenyl)-3-phenyl-5,6-dihydro-4H-benzo-
[f][1,2,3]triazolo[1,5-a][1,4]diazepine (2f). White solid. Yield:
79%. mp: 178–180
C. 1H NMR (400 MHz, CDCl3) d 7.66 (d, J ¼
7.6 Hz, 1H), 7.60–7.59 (m, 2H), 7.42–7.41 (m, 3H), 7.33–7.29 (m,
1H), 7.27–7.23 (m, 5H), 7.17–7.16 (m, 2H), 7.10 (d, J ¼ 8.0 Hz,
2H), 6.90 (d, J ¼ 8.0 Hz, 2H), 5.35 (s, 1H), 3.94 (dd, J ¼ 27.0, 12.8
Hz, 2H), 3.69 (d, J ¼ 12.8 Hz, 1H), 3.51 (d, J ¼ 12.8 Hz, 1H). 13C
NMR (100 MHz, CDCl3) d 147.5, 139.5, 137.5, 137.1, 132.3, 130.9,
Fig. 7 Docking simulation in the active site of 2XP2 receptor.
130.4, 130.3, 129.8, 129.3, 129.0, 128.9, 128.8, 128.5, 128.4,
128.3, 128.1, 127.7, 122.3, 120.8, 60.7, 56.6, 54.8. MS (ESI) for
C29H23BrN4 m/z ¼ 507 [M + H]+. Elemental analysis calc for


C. 1H NMR (400 MHz, CDCl3) d 7.69 (d, J ¼ 7.6 Hz, 1H), 7.63– C29H23BrN4: C, 68.64; H, 4.57; Br, 15.75; N, 11.04 found, C,
7.62 (m, 2H), 7.41–7.40 (m, 3H), 7.26–7.18 (m, 8H), 7.05–6.94 68.67; H, 4.55; Br, 15.71; N, 11.06.
(m, 5H), 5.46 (s, 1H), 3.99 (d, J ¼ 12.8 Hz, 1H), 3.90 (d, J ¼ 13.2 5-Benzyl-4-(naphthalen-1-yl)-3-phenyl-5,6-dihydro-4H-benzo
Hz, 1H), 3.71 (d, J ¼ 13.2 Hz, 1H), 3.52 (d, J ¼ 12.8 Hz, 1H). 13C [f][1,2,3]triazolo[1,5-a][1,4]diazepine (2g). White solid. Yield:
NMR (100 MHz, CDCl3) d 147.4, 140.3, 137.8, 137.2, 133.0, 130.6, 77%. mp: 64–65
C. 1H NMR (400 MHz, CDCl3) d 8.60 (d, J ¼ 8.0
130.3, 130.0, 129.0, 129.0, 128.8, 128.7, 128.5, 128.3, 128.1, Hz, 1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.68 (d,
127.8, 127.6, 126.8, 126.7, 122.3, 60.7, 57.2, 54.7. HRMS (ESI): J ¼ 8.0 Hz, 1H), 7.54–7.41 (m, 4H), 7.36–7.32 (m, 3H), 7.27–7.20
mass calculated for C29H24N4 [M + H]+ 429.2074, found, [M + H]+ (m, 4H), 7.15–7.06 (m, 6H), 5.86 (s, 1H), 4.01 (d, J ¼ 16.8 Hz, 1H),
429.2072. 3.85 (d, J ¼ 16.8 Hz, 1H), 3.79 (d, J ¼ 13.6 Hz, 1H), 3.65 (d, J ¼
5-Benzyl-3-phenyl-4-(p-tolyl)-5,6-dihydro-4H-benzo[f][1,2,3]- 13.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) d 137.6, 137.0, 134.3,
triazolo[1,5-a][1,4]diazepine (2c). White solid. Yield: 87%. mp: 134.1, 131.0, 130.4, 129.1, 129.0, 128.7, 128.5, 128.4, 128.3,
149–150
C. 1H NMR (400 MHz, CDCl3) d 7.78 (d, J ¼ 8.0 Hz, 1H), 128.2, 128.0, 127.9, 127.8, 127.5, 127.3, 126.3, 125.8, 125.0,
7.57–7.59 (m, 2H), 7.39–7.38 (m, 3H), 7.32–7.27 (m, 1H), 7.17– 123.8, 122.6, 58.4, 57.0, 52.6. HRMS (ESI): mass calculated for
7.24 (m, 7H), 6.92 (d, J ¼ 8.0 Hz, 2H), 6.81 (d, J ¼ 8.0 Hz, 2H), C33H27N4 [M + H]+ 479.2230, found, [M + H]+ 479.2226.
5.41 (s, 1H), 3.99 (d, J ¼ 13.2 Hz, 1H), 3.85 (d, J ¼ 13.1 Hz, 1H), 5-Benzyl-4-(furan-3-yl)-3-phenyl-5,6-dihydro-4H-benzo[f]-
3.68 (d, J ¼ 13.2 Hz, 1H), 3.54 (d, J ¼ 13.2 Hz, 1H), 2.15 (s, 3H). [1,2,3]triazolo[1,5-a][1,4]diazepine (2h). White solid. Yield:
13
C NMR (100 MHz, CDCl3) d 137.2, 136.5, 132.5, 130.7, 130.3, 82%. mp: 168–170
C. 1H NMR (400 MHz, CDCl3) d 7.83 (d, J ¼
129.0, 128.8, 128.7, 128.6, 128.4, 128.2, 128.1, 127.5, 126.7, 7.6 Hz, 1H), 7.63–7.61 (m, 2H), 7.43–7.33 (m, 4H), 7.32–7.18 (m,
124.9, 122.3, 121.1, 60.4, 57.4, 54.6, 20.9. HRMS (ESI): mass 7H), 7.02 (s, 1H), 6.90 (s, 1H), 5.81 (s, 1H), 5.32 (s, 1H), 3.98 (d, J

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 66260–66270 | 66267
 View Article Online

RSC Advances Paper

¼ 12.8 Hz, 1H), 3.85 (d, J ¼ 13.2 Hz, 1H), 3.68 (d, J ¼ 13.2 Hz, 120–122
C. 1H NMR (400 MHz, CDCl3) d 7.81–7.79 (m, 1H),
1H), 3.52 (d, J ¼ 12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) d 7.69–7.67 (m, 2H), 7.45–7.37 (m, 3H), 7.31–7.27 (m, 2H), 7.25–
146.5, 142.8, 140.8, 137.8, 137.2, 132.2, 130.5, 130.3, 130.1, 7.22 (m, 1H), 6.84 (q, J ¼ 8.9 Hz, 4H), 5.45 (s, 1H), 3.95 (d, J ¼
129.1, 128.9, 128.8, 128.5, 128.4, 128.1, 127.6, 126.0, 122.2, 13.2 Hz, 1H), 3.56 (d, J ¼ 13.2 Hz, 1H), 2.73–2.69 (m, 2H), 2.16 (s,
109.0, 60.6, 55.0, 51.2. HRMS (ESI): mass calculated for 3H), 1.55–1.48 (m, 2H), 1.40–1.31 (m, 2H), 0.87 (t, J ¼ 7.4 Hz,
C27H23N4O [M + H]+ 419.1866, found, [M + H]+ 419.1858. 3H). 13C NMR (100 MHz, CDCl3) d 147.0, 137.7, 137.1, 136.4,
5-Benzyl-3-butyl-4-(p-tolyl)-5,6-dihydro-4H-benzo[f][1,2,3]tri- 133.7, 130.8, 130.3, 130.1, 128.7, 128.7, 128.6, 128.5, 128.3,
azolo[1,5-a][1,4]diazepine (2i). Yellow oil. Yield: 75%. 1H NMR 128.0, 126.7, 122.1, 58.5, 55.9, 54.5, 30.0, 20.9, 20.3, 13.9. HRMS
(400 MHz, CDCl3) d 7.90 (d, J ¼ 7.6 Hz, 1H), 7.48–7.43 (m, 3H), (ESI): mass calculated for C27H29N4 [M + H]+ 409.2387, found,
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

7.37 (t, J ¼ 7.2 Hz, 3H), 7.31–7.23 (m, 4H), 7.08 (d, J ¼ 7.6 Hz, [M + H]+ 409.2394.
2H), 4.65 (s, 1H), 3.78–3.71 (m, 2H), 3.57 (d, J ¼ 14.0 Hz, 1H), 5-Hexyl-3-phenyl-4-(p-tolyl)-5,6-dihydro-4H-benzo[f][1,2,3]-
3.51 (d, J ¼ 13.6 Hz, 1H), 2.30 (s, 3H), 2.23–2.16 (m, 1H), 2.00– triazolo[1,5-a][1,4]diazepine (2n). Yellow oil. Yield: 81%. 1H
1.93 (m, 1H), 1.52–1.38 (m, 2H), 1.22–1.15 (m, 2H), 0.82 (t, J ¼ NMR (400 MHz, CDCl3) d 7.81–7.79 (m, 1H), 7.69–7.66 (m, 2H),
7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 146.8, 138.6, 137.6, 7.45–7.36 (m, 3H), 7.31–7.27 (m, 2H), 7.26–7.22 (m, 1H), 6.84 (q,
137.1, 135.8, 132.6, 130.5, 129.1, 128.8, 128.6, 128.5, 128.0, J ¼ 8.9 Hz, 4H), 5.45 (s, 1H), 3.94 (d, J ¼ 13.2 Hz, 1H), 3.56 (d, J ¼
127.4, 122.6, 60.3, 57.3, 51.7, 32.3, 24.8, 22.6, 21.1, 13.8. HRMS 13.2 Hz, 1H), 2.70 (t, J ¼ 7.2 Hz, 2H), 2.16 (s, 3H), 1.55–1.47 (m,
(ESI): mass calculated for C28H31N4 [M + H]+ 425.2543, found, 2H), 1.35–1.28 (m, 2H), 1.26–1.20 (m, 4H), 0.86 (t, J ¼ 6.8 Hz,
[M + H]+ 425.2536. 3H). 13C NMR (100 MHz, CDCl3) d 147.0, 137.7, 137.1, 136.4,
5-Benzyl-3,4-di-p-tolyl-5,6-dihydro-4H-benzo[f][1,2,3]triazolo 133.7, 130.9, 130.8, 130.3, 130.1, 128.9, 128.8, 128.7, 128.6,
[1,5-a][1,4]diazepine (2j). Semi solid. Yield: 83%. 1H NMR (400 128.5, 128.3, 128.0, 126.7, 122.1, 58.4, 56.3, 54.5, 31.6, 27.8, 26.8,
MHz, CDCl3) d 7.76 (d, J ¼ 8.0 Hz, 1H), 7.53 (d, J ¼ 7.6 Hz, 1H), 22.5, 20.9, 19.2, 14.0. HRMS (ESI): mass calculated for C29H33N4
7.48 (d, J ¼ 7.6 Hz, 2H), 7.28–7.19 (m, 9H), 6.91 (d, J ¼ 8.0 Hz, [M + H]+ 437.2700, found, [M + H]+ 437.2702.
2H), 6.81 (d, J ¼ 7.6 Hz, 2H), 5.42 (s, 1H), 3.98 (d, J ¼ 12.8 Hz,
1H), 3.86 (d, J ¼ 13.2 Hz, 1H), 3.70 (d, J ¼ 13.2 Hz, 1H), 3.52 (d, J
¼ 13.2 Hz, 1H), 2.41 (s, 3H), 2.15 (s, 3H). 13C NMR (100 MHz, Biological assays
CDCl3) d 138.1, 138.0, 137.3, 136.4, 133.0, 130.2, 130.0, 129.4, Materials and methods for antimicrobial activity. Strepto-
129.0, 128.8, 128.6, 128.5, 128.4, 128.0, 127.8, 127.5, 126.7, mycin and ciprooxacin (Sigma) were used as positive control
122.2, 60.5, 57.5, 54.6, 21.3, 20.8. MS (ESI) for C31H28N4 m/z ¼ against bacteria. Ketoconazole (Himedia, Mumbai) was used as
457 [M + H]+. Elemental analysis calc for C31H28N4: C, 81.55; H, positive control against fungi.
6.18; N, 12.27 found, C, 81.52; H, 6.21; N, 12.25. Tested microbes. The following bacteria and fungi were used
5-Benzyl-3-(4-uorophenyl)-4-(p-tolyl)-5,6-dihydro-4H-benzo- for the experiment. Bacteria: Salmonella paratyphi-B, Pseudo-
[f][1,2,3]triazolo[1,5-a][1,4]diazepine (2k). White solid. Yield: monas aeruginosa MTCC 741, Klebsiella pneumonia MTCC 109,
74%. mp: 144–145
C. 1H NMR (400 MHz, CDCl3) d 7.83 (d, J ¼ Micrococcus luteus MTCC 106, Salmonella typhimurium MTCC
8.0 Hz, 1H), 7.48 (t, J ¼ 6.4 Hz 2H), 7.33 (t, J ¼ 7.4 Hz, 1H), 7.23– 1251, Proteus vulgaris MTCC 1771, Shigella exneri MTCC 1457,
7.19 (m, 7H), 7.04 (t, J ¼ 8.4 Hz, 2H), 6.93 (d, J ¼ 7.6 Hz, 2H), 6.83 Enterobacter aerogenes MTCC 111, Staphylococcus epidermidis
(d, J ¼ 7.6 Hz, 2H), 5.28 (s, 1H), 3.99 (d, J ¼ 13.6 Hz, 1H), 3.82 (d, MTCC 3615, Staphylococcus aureus MTCC 96 and Staphylococcus
J ¼ 13.2 Hz, 1H), 3.66 (d, J ¼ 13.2 Hz, 1H), 3.57 (d, J ¼ 13.6 Hz, aureus (MRSA-methicillin resistant). The reference cultures
1H), 2.16 (s, 3H). 13C NMR (100 MHz, CDCl3) d 161.6, 146.3, were obtained from Institute of Microbial Technology
137.8, 137.1, 136.8, 133.3, 130.3, 130.0, 129.9, 129.8, 128.9, (IMTECH), Chandigarh, India-160 036; fungi: Candida albicans
128.7, 128.6, 128.5, 127.6, 126.8, 122.3, 115.7, 115.5, 59.9, 57.6, MTCC 227 and Malassezia pachydermatis. All the other cultures
54.3, 20.9. MS (ESI) for C31H28N4 m/z ¼ 461 [M + H]+. Elemental were obtained from the Department of Microbiology, Christian
analysis calc for C30H25FN4: C, 78.24; H, 5.47; F, 4.13; N, 12.17 Medical College, Vellore, Tamil Nadu, India.
found C, 78.21; H, 5.51; F, 4.10; N, 12.19. Preparation of inoculums. Bacterial inoculums were
5-Benzyl-8-bromo-3-phenyl-4-(p-tolyl)-5,6-dihydro-4H-benzo- prepared by growing cells in Mueller Hinton broth (MHB)
[f][1,2,3]triazolo[1,5-a][1,4]diazepine (2l). White solid. Yield: (Himedia) for 24 h at 37
C. The lamentous fungi were grown
72%. mp: 185–186
C. 1H NMR (400 MHz, CDCl3) d 7.80 (d, J ¼ on sabouraud dextrose agar (SDA) slants at 28
C for 10 days and
8.4 Hz, 1H), 7.56–7.52 (m, 2H), 7.44 (dd, J ¼ 8.6, 2.2 Hz, 1H), the spores were collected using sterile doubled distilled water
7.39–7.37 (m, 3H), 7.35 (d, J ¼ 2.0 Hz, 1H), 7.23–7.21 (m, 3H), and homogenized. Yeast was grown on sabouraud dextrose
7.19–7.16 (m, 2H), 6.91 (q, J ¼ 8.4 Hz, 4H), 5.44 (s, 1H), 3.97 (d, J broth (SDB) at 28
C for 48–72 h.
¼ 14.0 Hz, 1H), 3.82 (d, J ¼ 13.2 Hz, 1H), 3.68 (d, J ¼ 13.2 Hz, Disc diffusion assay. Antimicrobial activities were carried
1H), 3.55 (d, J ¼ 13.6 Hz, 1H), 2.20 (s, 3H). 13C NMR (100 MHz, out using well method.28 Petri plates were prepared with 20 mL
CDCl3) d 147.4, 137.5, 137.0, 136.8, 136.2, 133.5, 132.9, 131.8, of sterile Mueller Hinton Agar (MHA) (Hi-media, Mumbai). The
131.0, 130.4, 128.9, 128.7, 128.5, 128.3, 128.0, 127.6, 126.8, test cultures were swabbed on the top of the solidied media
123.8, 122.0, 60.0, 57.7, 53.9, 20.9. HRMS (ESI): mass calculated and allowed to dry for 10 min and a specic amount of
for C30H26BrN4 [M + H]+ 521.1335, found, [M + H]+ 521.1338. synthesized compound at 1 mg per well was added to each well
5-Butyl-3-phenyl-4-(p-tolyl)-5,6-dihydro-4H-benzo[f][1,2,3]tri- separately. Negative control was prepared using respective
azolo[1,5-a][1,4]diazepine (2m). White solid. Yield: 84%. mp: solvents. Streptomycin was used as positive control against

66268 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Paper RSC Advances

bacteria. Ketoconazole was used as positive control for fungi. atoms were added, lower occupancy residue structures were
The plates were incubated for 24 h at 37
C for bacteria and for deleted, and any incomplete side chains were replaced using
48 h at 28
C for fungi. Zones of inhibition were recorded in the ADT. Gasteiger charges were added to each atom, and
millimetres and the experiment was repeated twice. merged the non-polar hydrogen atoms to the protein structure.
Minimum inhibitory concentration (MIC). Minimum The distance between donor and acceptor atoms that form a
inhibitory concentration studies of eight compounds were hydrogen bond was dened as 1.9 Å with a tolerance of 0.5 Å,
performed according to the standard reference methods for and the acceptor–hydrogen–donor angle was not less than 120
.
antibacterial activity.29 The required concentrations (500 mg Then, the structures were saved in PDBQT le format for further
mL1, 250 mg mL1, 125 mg mL1, 62.5 mg mL1, 31.25 mg mL1)
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

studies in ADT. A grid box centred on 6.116, 43.906, 40.763 and

of the compound were dissolved in DMSO (2%), and diluted to 29.697, 47.794, 8.863 with dimension of 50  60  50 Å3 and 40
give serial two-fold dilutions that were added to each medium in  40  40 Å3 with 0.375 Å spacing was created around the
96 well plates. An inoculum of 100 mL from each well was binding site of co-crystallised ligand on 2XCS and 2XP2
inoculated. The antifungal agent ketoconazole for fungi and respectively. The centre of the box was set at co-crystallised
streptomycin and ciprooxacin for bacteria was included in the ligand centre and grid energy calculations were carried out.
assay as positive controls. For fungi, the plates were incubated Default parameters were used for the AutoDock docking
for 48 to 72 hours at 28
C and for bacteria the plates were calculation and 50 docked conformations were generated for
incubated for 24 h at 37
C. The MIC for fungi was dened as the each compound. Genetic algorithms was used to calculate the
lowest extract concentration, showing no visible fungal growth energy of the binding interactions. The outputs were exported to
aer incubation time. 5 mL of tested broth was placed on the PyMOL for visual inspection of the binding modes and inter-
sterile MHA plates for bacteria and incubated at respective actions of the compounds with amino acid residues in the
temperature. The MIC for bacteria was determined as the lowest active sites.
concentration of the compound inhibiting the visual growth of
the test cultures on the agar plate. References
Cytotoxic properties. A549 lung adenocarcinoma cancer cell
line was obtained from National Institute of Cell Sciences, 1 M. Rivas, P. Stables, L. Murphree, R. Edwankar, S. Huang,
Pune. A549 cell line was maintained in complete tissue culture H. D. Jain, H. Zhou, S. Majumder, S. Sankar, L. Roth,
medium Dulbecco's Modied Eagle's Medium with 10% fetal W. Sieghart and M. Cook, J. Med. Chem., 2009, 52, 1795.
bovine serum and 2 mM L-glutamine, along with antibiotics 2 J. de Baun, M. Pallos and R. Baker, Chem. Abstr., 1977, 86,
(about 100 international unit per mL of penicillin, 100 mg mL1 5498.
of streptomycin) with the pH adjusted to 7.2. The cytotoxicity 3 E. Korneki, Y. H. Erlich and R. H. Lenox, Science., 1984, 226,
was determined according to the literature method30 with some 1454.
changes. Cells (5000 cells per well) were seeded in 96 well plates 4 J. Kukla, J. Breslin, J. Diamond, P. Grous, Y. Ho, M. Mirands,
containing medium with different concentrations such as 50, D. Rodgers, G. Sherrill, D. Clercq, R. Pauwels, K. Anderies,
40, 30, 20, 10 and 5 mg mL1. The cells were cultivated at 37
C L. J. Moens, C. Janssen and A. Janssen, J. Med. Chem.,
with 5% CO2 and 95% air in 100% relative humidity. Aer 1991, 34, 3187.
various durations of cultivation, the solution in the medium 5 (a) A. G. Katsis, M. E. McPhee, F. Mattner and D. D. Ridley,
was removed. An aliquot of 100 mL of medium containing 1 mg Aust. J. Chem., 1999, 52, 1061; (b) L. Basolo, E. M. Beccalli,
mL1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium E. Borsini, G. Broggini, M. Khansaa and M. Rigamonti,
bromide (Sigma) was loaded in the plate. The cells were Eur. J. Org. Chem., 2010, 1694.
cultured for 4 h and then the solution in the medium was 6 K. M. El-Shaieb, A. A. Hassan and A. S. Abdel-Aal, J. Chem.
removed. An aliquot of 100 mL of DMSO was added to the plate, Res., 2011, 35, 592.
which was shaken until the crystals were dissolved. The cyto- 7 M. Leuens, A. Vermeeren, E. Smink, P. Ruitenbeek and
toxicity against cancer cells was determined by measuring the G. Ramaekers, Psychopharmacology, 2007, 19, 1951.
absorbance of the converted dye at 540 nm in an enzyme linked 8 D. J. Greenblatt, J. S. Harmatz, L. Shapira, N. Engelhardt,
immune sorbent assay reader. Cytotoxicity of each sample was T. A. Gouthro and N. Shader, N. Engl. J. Med., 1991, 324, 1691.
expressed as the half maximal inhibitory concentration (IC50) 9 (a) R. Raj, P. Singh, N. T. Haberkern, R. M. Faucher, N. Patel,
value. The IC50 value is the concentration of test sample that K. M. Land and V. Kumar, Eur. J. Med. Chem., 2013, 63, 897;
causes 50% inhibition of cell growth, averaged from three (b) H. Cheng, J. Wan, M. I. Lin, Y. Liu, X. Lu, J. Liu, Y. Xu,
replicate experiments. J. Chen, Z. Tu, Y. E. Cheng and K. Ding, J. Med. Chem.,
Molecular docking studies. Molecular docking studies were 2012, 55, 2144; (c) A. Regueiro-Ren, Q. M. Xue,
performed using the AutoDock Tools (ADT) version 1.5.6 and J. J. Swidorski, Y. F. Gong, M. Mathew, D. D. Parker,
AutoDock version 4.2.5.1 docking program. Three dimensional Z. Yang, B. Eggers, C. D'Arienzo, Y. Sun, J. Malinowski,
structure of DNA gyrase (PDB ID: 2XCS) and Anaplastic Q. Gao, D. Wu, D. R. Langley, R. J. Colonno, C. Chien,
Lymphoma Kinase (ALK) receptor (PDB ID: 2XP2) receptor were D. M. Grasela, M. Zheng, P. Lin, N. A. Meanwell and
obtained from the Protein Data Bank. The co-crystallized ligand J. F. Kadow, J. Med. Chem., 2013, 56, 1656; (d) R. Adam,
in the receptor structure was removed. Then, the water mole- P. Bilbao-Ramos, B. Abarca, R. Ballesteros, M. E. González-
cules present with the crystal were deleted, the polar hydrogen Rosende, M. A. Dea-Ayuela, F. Estevan and G. Alzuet-Piña,

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 66260–66270 | 66269
 View Article Online

RSC Advances Paper

Org. Biomol. Chem., 2015, 17, 4903; (e) N. Pokhodylo, R. J. Fraser, V. O. Rodionov and V. V. Fokin, Org. Lett.,
O. Shyyka and V. Matiychuk, Med. Chem. Res., 2014, 23, 2426. 2010, 12, 4217; (c) A. Qin, C. K. W. Jim, W. Lu,
10 (a) P. J. Snyder, J. Werth, B. Giordani, A. F. Caveney, J. W. Y. Lam, M. Haeussler, Y. Dong, H. H. Y. Sung,
D. Feltner and P. Maruff, Hum. Psychopharmacol. Clin. Exp., I. D. Williams, G. K. L. Wong and B. Z. Tang,
2005, 20, 263; (b) J. Levine, D. P. Cole, K. N. Roy Macromolecules, 2007, 40, 2308; (d) C. R. Becer,
Chengappa and S. Gerson, Depression Anxiety, 2001, 14, 94; R. Hoogenboom and U. S. Schubert, Angew. Chem., Int. Ed.,
(c) G. L. Post, R. O. Patrick, J. E. Crowder, J. Houston, 2009, 48, 4900; (e) P. Li and L. Wang, Lett. Org. Chem.,
J. M. Ferguson, R. J. Bielski, L. Bailey, H. G. Pearlman, 2007, 4, 23; (f) R. Li, D. J. Jansen and A. Datta, Org. Biomol.
Published on 27 July 2015. Downloaded by National Chung Chen University on 14/08/2015 07:17:45.

V. S. Shu and M. W. Pierce, J. Clin. Psychopharmacol., 1991, Chem., 2009, 7, 1921.

11, 249. 17 (a) F. Couty, F. Durrat and D. Prim, Tetrahedron Lett., 2004,
11 (a) D. J. Greenblatt, J. S. Harmatz, L. Shapiro, N. Engelhardt, 45, 3725; (b) J. R. Donald, R. R. Wood and S. F. Martin,
T. A. Gouthro and T. A. Shader, N. Engl. J. Med., 1991, 324, ACS Comb. Sci., 2012, 14, 135; (c) R. K. Arigela,
1691; (b) R. A. Lahti, V. H. Sethy, C. Barsuhn and A. K. Mandadapu, S. K. Sharma, B. Kumar and B. Kundu,
J. B. Hester, Neuropharmacology, 1983, 22, 1277. Org. Lett., 2012, 14, 1804.
12 (a) K. B. Sharpless and R. Manetsch, Expert Opin. Drug 18 K. C. Majumdar and K. Ray, Synthesis, 2011, 3767.
Discovery, 2006, 1, 525; (b) D. K. Mohapatra, P. K. Maity, 19 (a) D. Antonow and D. E. Thurston, Chem. Rev., 2011, 111,
M. Shabab and M. I. Khan, Bioorg. Med. Chem. Lett., 2009, 2815; (b) P. Thirumurugan, D. Matosiuk and K. Jozwiak,
19, 5241. Chem. Rev., 2013, 113, 4905; (c) M. K. Hussain,
13 (a) G. Broggini, G. Molteni and G. Zecchi, Synthesis, 1995, M. I. Ansari, R. Kant and K. Hajela, Org. Lett., 2014, 16, 560.
647; (b) G. Broggini, G. Molteni, A. Terraneo and G. Zecchi, 20 J. R. Donald and S. F. Martin, Org. Lett., 2011, 13, 852.
Tetrahedron, 1999, 55, 14803; (c) L. Garanti, G. Molteni and 21 K. C. Majumdar and S. Ganai, Tetrahedron Lett., 2013, 54,
G. Broggini, J. Chem. Soc., Perkin Trans. 1, 2001, 1816; (d) 6192.
A. W. Thomas, Bioorg. Med. Chem. Lett., 2002, 12, 1881; (e) 22 G. Hooyberghs, H. D. Coster, D. D. Vachhani,
I. Akritopoulou-Zanze, V. Gracias and S. W. Djuric, D. S. Ermolat'ev and E. V. van der Eycken, Tetrahedron,
Tetrahedron Lett., 2004, 45, 8439; (f) V. Gracias, D. Darczak, 2013, 69, 4331.
A. F. Gasiecki and S. W. Djuric, Tetrahedron Lett., 2005, 46, 23 (a) N. Sudhapriya, A. Nandakumar and P. T. Perumal, RSC
9053; (g) G. Broggini, I. DeMarchi, M. Martinelli, Adv., 2014, 4, 58476; (b) N. Sudhapriya, P. T. Perumal,
G. Paladino, T. Pilati and A. Terraneo, Synthesis, 2005, 2246. C. Balachandran, S. Ignacimuthu, M. Sangeetha and
14 (a) K. R. Reddy, K. Rajgopal and M. L. Kantam, Synlett, 2006, M. Doble, Eur. J. Med. Chem., 2014, 83, 190.
957; (b) P. Li and L. Wang, Lett. Org. Chem., 2007, 4, 23; (c) 24 ESI†
I. Kumar and C. V. Rode, Chem. Lett., 2007, 36, 592; (d) 25 (a) M. F. Sanner, J. Mol. Graphics Modell., 1999, 17, 57; (b)
Z. X. Wang and Z. G. Zhao, J. Heterocycl. Chem., 2007, 44, G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
89; (e) F. Alonso, Y. Moglie, G. Radivoy and M. Yus, Adv. R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Synth. Catal., 2010, 352, 3208; (f) F. Alonso, Y. Moglie, Chem., 2009, 30, 2785.
G. Radivoy and M. Yus, J. Org. Chem., 2011, 76, 8394. 26 Protein Data Bank (http://www.rcsb.org/pdb).
15 (a) L. Zhang, X. Chen, P. Xue, H. H. Y. Sun, I. D. Williams, 27 The PyMOL Molecular Graphics System, Version 1.3,
K. B. Sharpless, V. V. Fokin and G. Jia, J. Am. Chem. Soc., Schrödinger, LLC.
2005, 127, 15998; (b) S. Diez-Gonzalez, A. Correa, L. Cavallo 28 C. Balachandran, V. Duraipandiyan, K. Balakrishna,
and S. P. Nolan, Chem.–Eur. J., 2006, 12, 7558; (c) J. Y. Eun, R. L. Sundaram, A. Vijayakumar, S. Ignacimuthu and
M. Ahlquist, I. Bae, K. B. Sharpless, V. V. Fokin and N. A. Al-Dhabi, Environ. Chem. Lett., 2013, 11.
S. Chang, J. Org. Chem., 2008, 73, 5520; (d) V. Aragão- 29 C. Balachandran, Y. Arun, V. Duraipandiyan,
Leoneti, V. L. Campo, A. S. Gomes, R. A. Field and S. Ignacimuthu, K. Balakrishna and N. A. Al-Dhabi, Appl.
I. Carvalho, Tetrahedron, 2010, 66, 9475. Biochem. Biotechnol., 2014, 172, 3513.
16 (a) C. Chowdhury, A. K. Sasmal and B. Achari, Org. Biomol. 30 C. Balachandran, V. Duraipandiyan and S. Ignacimuthu,
Chem., 2010, 8, 4971; (b) S. W. Kwok, J. R. Fotsing, Asian Pac. J. Trop. Biomed., 2012, 712.

66270 | RSC Adv., 2015, 5, 66260–66270 This journal is © The Royal Society of Chemistry 2015