Synthesis of novel 1,4-disubstituted 1,2,3-triazolo-bosentan derivatives - evaluation of antimicrobial and anticancer activities and molecular docking

Article abstract of DOI:10.1039/c5ra18618h

Novel 1,4-disubstituted 1,2,3-triazolo bosentan derivatives 1a-n from bosentan 2 were synthesized in good yields by sequential chlorination, azidation followed by Cu(i) catalyzed 1,3-dipolar cycloaddition. All obtained compounds 1a-n were evaluated for their antimicrobial and in vitro anticancer activities and by in silico docking studies. Among all tested compounds 1e,f and 1h-j show better antimicrobial activities against the tested bacteria and fungi. When subjected to anticancer testing, compounds 1g-j and 1n show significant activities against both A549 and SKOV-3 cell lines with IC₅₀ values at 7.81 μg mL^-1 and among them compound 1i exhibited very potent activity. In addition, no toxicity was calculated up to 2 mg mL^-1 in Vero cells. In silico studies were conducted to investigate the possible bonding modes of 1a-n with target receptors namely DNA topoisomerase IV (4 EMV) and anaplastic lymphoma kinase (2XP2). Among them, compounds 1e and 1h show maximum binding energies with 4EMV and 2XP2 receptors, respectively which also exhibited good antimicrobial and potent anticancer activities.

Full text of DOI:10.1039/c5ra18618h

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ARTICLE
Synthesis of Novel 1,4-Disubstituted 1,2,3-Triazolo-Bosentan
Derivatives – Evaluation of Antimicrobial, Anticancer activities
and Molecular Docking
Received 00th January 20xx,
Accepted 00th January 20xx
K. Easwaramoorthi,a,c A. Jeya Rajendran,*,a K. Chennakesava Rao,b,c Y. Arun,b C. Balachandran,d
P.T. Perumal,b Nobuhiko Emi,d S.M. Mahalingame V.Duraipandiyanf,g N.A. Al-Dhabig
DOI: 10.1039/x0xx00000x
www.rsc.org/
Novel 1,4-disubstituted 1,2,3-triazolo bosentan derivatives 1a-n from bosentan 2 were accomplished in good yields by
sequential chlorination, azidation followed by Cu(I) catalyzed 1,3-dipolar cycloaddition. All obtained compounds 1a-n were
evaluated for their antimicrobial, in vitro anticancer activities and in silico docking studies. Among all tested compounds
1e-f and 1h-j have shown better antimicrobial activities against tested bacteria and fungi. When subjected to anticancer
activity, compounds 1g-j and 1n have shown significant activities against both A549 and SKOV-3 cell lines with IC50 values
at 7.81 µg/mL and among them compound 1i has exhibited very potent activity. Beside no toxicity was calculated up to 2
mg/mL in Vero cells. In silico studies were conducted to know the possible bonding modes of 1a-n with target receptors
namely DNA Topoisomerase IV (4 EMV) and Anaplastic Lymphoma Kinase (2XP2). Among them, compounds 1e and 1h
have shown maximum binding energies with 4EMV and 2XP2 receptors, respectively which also exhibited good
antimicrobial and potent anticancer activities.
Endothelin A (ETA) and B (ETB) receptors, have been implicated
in the development of several cancers through activation of
Introduction
As per WHO, multidrug-resistant organisms (MDRO) are an
increasing threat to the human life as well as the animal
kingdom.1-5 Microorganisms gain resistance by mutation of
their DNA and tolerate the effect of standard antimicrobial
drugs resulting in uncured diseases/infections. Cancer is
another major threat to human life. When in the early years of
a person’s life, normal body cells divide faster to allow him/her
to grow well. The division of cell rate decreases when the
person becomes an adult and it happens only to replace
exhausted cells or to heal injuries. Abnormal growth of cells
results to form cancers which are very difficult to cure and end
with death in most cases. Most of the anticancer drugs have
side effects on normal cells and result in some abnormalities in
health condition. Hence, this situation stresses the recurrent
development of new drugs that are more efficient and less
expensive6 than the existing drugs.
pathways involved in cell proliferation, migration, invasion,
osteogenesis and angiogenesis.7 Targeting ET receptor and its
antagonism constitute as an attractive and challenging
approach for cancer therapy.8-9
A large number of ET
antagonists have been developed and studied in clinical trials
for chronic heart failure, hypertension, cancer and fibrosis.10-13
Bosentan, the dual ETA/ETB receptor antagonist is approved for
treatments of pulmonary arterial hypertention14-16 and used in
combination chemotherapy with cisplatin17 and paclitaxel for
ovarian cancer.18 The development of potent bosentan
derivative as specific, selective and dual ET receptor antagonist
represents an effective treatment for cancer as a mono drug.
Structural modification of bosentan may lead to increase in the
selective dual activity with ET receptor and may cause less or
no hepatotoxic (damage to liver – side effect of Bosentan) and
anemia.19-20 The current research activity focuses on the
preparation of various 1,4-disubstituted 1,2,3-triazoles linked
with the pyrimidine ring of bosentan to give triazolo-bosentan
derivatives in order to test their anticancer, antimicrobial
activities and carry out molecular docking studies.
a.
Department of Chemistry, Loyola College, Chennai-600034, India, Phone: +91
944-411-6528, Email: jeyaadmaterial@gmail.com
b.Organic Chemistry Division, CSIR-CLRI, Chennai-600020, India
c. R&D Centre, Malladi Drugs & Pharmaceuticals Ltd., Chennai-600124, India.
d.Department of Hematology, Fujita Health University,
1-98, Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan.
e. Centre for Drug Discovery and Department of Chemistry, Purdue University,
Indiana
Generally, having two is better than one and the same concept
is applicable to hybrid drugs which can have enhanced
biological activity and can address the issues on drug
resistance.21-23 Many hybrid drugs are known with more
advantages, as they can potentially overcome the
pharmacokinetic drawbacks than their precursors and
conventional drugs.24-26 These can be prepared by adjoining to
f. Division of Ethnopharmacology, Entomology Research Institute, Loyola College,
Chennai-600034, India
g.Department of Botany and Microbiology, Addiriyah Chair for Environmental
Studies, College of Science, King Saudi University,P.O.Box.2455, Riyadh-11451,
Saudi Arabia.
†Electronic Supplementary Informaꢀon (ESI) available: IR, NMR, Mass and Docking.
This journal is © The Royal Society of Chemistry 20xx
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two different drugs by a covalent bond or by an adequate optimize the method (Table 1). Terminal alkynes 5a′-f′ were
fusion.27 Hybrid drugs such as NOSH-Aspirin28, Artemisinin- obtained in situ by heating their corresponding amines 5a-f
quinine29, reversed Chloroquine30 are well known (Figure 1).
with propargyl chloride in the presence of a base (Scheme 2).
Terminal alkynes 5g -k were also made in situ by converting
′
′
their corresponding acids 5g-k to acid chlorides followed by
treating with propargyl alcohol (Scheme 2). Terminal alkynes
5l-n were procured commercially and used as such. About 78-
95% of overall yields (Scheme 4 and Table 2) were obtained
when the optimized reaction conditions mentioned in this
article were followed. The compounds 1a-n were obtained as
pale brown to brown amorphous solids and further
purification also resulted in amorphous solids since single
crystal XRD studies were not performed.
Optimization of reaction conditions
Various solvents, bases, catalysts, reaction temperature and
time have been explored to optimize the reaction conditions
during this research work (Table 1) using Scheme 3 as a model
experiment. During the study, it was noticed that better
results were obtained when CuI (10 mol%), DIPEA and n-
BuOH/Water mixture were used at room temperature for
about 10 h (Table 1, entry 6). Usage of excess CuI did not
improve the yield (Table 1, entry 5, 14 & 15) and usage of
other copper salts was resulted in very poor conversions
(Table 1, entry 2-3). It was understood that reaction can
proceed in other polar solvents but had not gone to
completion (Table 1, entries 6-11 & 12). The reaction did not
proceed when other bases such as triethylamine and
potassium hydroxide were used instead of DIPEA (Table 1,
entries 12-15).
Fig. 1: Hybrid drugs
Hybrid compounds of pyrimidines which are associated with
other heterocycles exhibited good biological activities.31
Presently, the interest in developing new hybrid molecules
from pyrimidine derivatives is increasing with vast applications
in medicinal chemistry.32-37 Click chemistry is a brilliant concept
in organic chemistry which involves synthesizing complex
molecules in high yields with simple isolation techniques.38-39
Various 1,4-disubstituted 1,2,3-triazole derivatives have been
reported in literature using this well known click chemistry
approach by Cu(I) catalyzed azide-alkyne 1,3-dipolar
cycloaddition (CuAAC)40-45 which have broad applications in
medicinal chemistry.46-49
The structures of all novel 1,2,3-triazole 1a-n compounds were
elucidated with the help of IR, 1H-NMR, 13C-NMR and Mass
spectra as exemplified for compound 1e. Characteristic IR
bands of 1e: The broad band at 3422 cm-1 represents the –NH
stretching. The medium band at 2963 cm-1 indicates the
aliphatic stretching. The sharp bands that appear at 1674,
1605 & 1564 cm-1 show the presence of benzenoid rings. The
band present at 1393 cm-1 confirms the presence of S=O
stretching. The bands at 1252, 1219 & 1173 cm-1 support the
presence of –C–N and –C–O stretching. The characteristic
bands at 864, 762 & 694 cm-1 represent the existence of para-
& ortho- disubstituted and monosubstituted aromatic rings
respectively. The signals of 1H & 13C -NMR of compound 1e are
explained in Figure 1. A distinguishing peak observed at m/z:
748 in the mass spectrum of 1e corresponds to the protonated
molecular ion [M+H]+.
Results and discussion
Chemistry
The key intermediate azide
bosentan with thionyl chloride in chloroform followed by
treating with sodium azide in N,N-dimethylacetamide (DMA)
(Scheme 1 . The targeted new bis-pyrimidinyl linked 1,2,3-
triazolo-bosentan derivatives 1a-n were prepared via one pot
synthesis by reacting azide with various terminal alkynes 5a
and 5l-n in the presence of CuI catalyst and N, N-
4 was prepared by heating
2
)
4
′-
k′
diisopropylethylamine (DIPEA) in aqueous n-butanol medium
at ambient temperature using a regular click chemistry
approach (Scheme 2). Various conditions have been tried to
2 | J. Name., 2012, 00, 1-3
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Scheme 1: Synthesis of azide 4 from Bosentan 2
Scheme 2: Overview of synthesis of novel 1,4-disubstituted 1,2,3-triazoles 1a-n
DIPEA–diisopropylethylamine, TEA–triethylamine, aisolated yields
δH: 4.73, t,2H, J=12Hz
δC: 56.14, 1C
δH: 4.25, t, 2H, J=8Hz
N
δC: 36.61, 1C
δ
H:4.22, s,2H
δC: 45.35, 1C
δH: 6.67-8.24, m, 16H,
aromatic protons
N
O
N
N
N
N
: 9.10,bs
δH
δC: 97.74-159.24,
N
26 aromatic carbons
O
1e
O
δH: 3.14, m, 1H
δC: 35.21, 1C
NH
OCH3
S
NH
Scheme 3: Synthesis of 1l – Reaction optimization
Table 1: Optimization of reaction conditions
O
H 3
C
H3C
: 9.75 bs
H3C
δH
δC:31.28, 1C
δH: 1.25, s, 9H
δ :28.80,3C
δH: 2.87, m, 2H
δC:30.28, 1C
δH: 3.82, s,3H δH: 2.24& 2.41,m, 2H
δC: 61.13 δC:28.90, 1C
Entry
Base
Catalyst
(mol%)
Solvent
Temp Time Yield
(%)a
C
(°C)
(h)
1
2
3
4
5
6
7
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
CuI (10)
CuBr (10)
CuCl (10)
CuI (5)
n-BuOH
n-BuOH
n-BuOH
n-BuOH
n-BuOH
H2O
25-30
25-30
25-30
25-30
25-30
25-30
25-30
10
10
10
10
10
10
10
75
15
Fig. 2: Characterization of 1e using NMR spectroscopy
Pharmacology
trace
46
CuI (20)
CuI (10)
CuI (10)
93
Antimicrobial activity
48
n-BuOH/
H2O(1:1)
EtOH
95
The synthesized novel bis-pyrimidinyl linked 1,2,3-triazolo-
bosentan derivatives 1a-n were screened for antimicrobial
activity50 using the well method51 at 1000 µg/well against
eleven bacteria and two fungi (Table 3). Minimum inhibitory
8
9
DIPEA
DIPEA
DIPEA
CuI (10)
CuI (10)
CuI (10)
25-30
45-50
45-50
10
20
20
trace
15
EtOH
10
EtOH/
27
concentration (MIC) studies of the synthesized compounds 1a
-
H2O(1:1)
THF
n
were performed according to the standard reference
11
12
13
14
15
DIPEA
TEA
CuI (10)
CuI (10)
CuI (10)
CuI (20)
CuI (20)
25-30
25-30
25-30
25-30
50-55
20
10
10
10
20
60
31
method for bacteria, filamentous fungi52 and yeasts53
(NCCLS/CLSI, 2002). The MIC values of the synthesized
n-BuOH
THF
TEA
46
compounds 1a-n were calculated (Table 3). Standard
KOH
KOH
n-BuOH
n-BuOH
trace
trace
antimicrobial drugs, streptomycin, ciprofloxacin and
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ketoconazole were used as positive controls against bacteria
and fungi respectively in both antimicrobial and MIC study.
Scheme 4: One pot synthesis of 1,4-disubstituted 1,2,3-triazoles 1a-n from azide
Table 2: Novel 1,4-disubstituted 1,2,3-triazoles from bosentan
4 using click chemistry
Compound Terminyl alkynes prepared from
R
m.p (°C)* Yield (%)#
1a
1b
1c
1d
1e
1f
Ephedrine
[N-methyl-(1-hydroxy-1-phenyl)-2-propylamino]
[(1-hydroxy-1-phenyl)-2-propylamino]
[1-phenyl-2-propylamino]
122-125
140-143
99-102
88
78
90
90
88
91
92
84
88
90
87
95
89
87
Norephedrine
Selegiline
Phenylephrine
[N-methyl-2-(3-hydroxyphenyl)-2-hydroxyethylamino]
[2,3-dihydro-1H-inden-1-amino]
148-150
102-105
1-Aminoindane
Noralfentanil
[4-(methoxymethyl)-4-(N-phenyl-N-propionamide)piperidino] 180-181
1g
1h
1i
Benzoic acid
[benzoyloxy]
95-98
2-Bromo-5-methoxybenzoic acid
4-Nitrobenzoic acid
Veratroic acid
[2-bromo-5-methoxybenzoyloxy]
[4-nitrobenzoyloxy]
[3,4-dimethoxybenzoyloxy]
[4-methylbenzoyloxy]
[hydroxymethyl]
160-164
168-171
128-132
140-144
168-170
101-103
105-107
1j
1k
1l
3-Methylbenzoic acid
Propargyl alcohol€
1-Hexyne€
1m
[n-butyl]
1n
1-Ethynyl-1-cyclohexanol€
[(1-hydroxy)cyclohexyl]
* uncorrected melting points, # obtained yields without any purification, €obtained from commercial sources and used as such
The incurred results disclosed good antimicrobial activity of 1e
the synthesized compounds 1a when compared with the others against tested bacteria and fungi. Best MIC values have
standard antimicrobial drugs used in this study. P. aeruginosa been observed for compounds 1h-j amongst others The
-f & h-j have shown better activity when compared with
-n
.
has emerged as one of the most problematic Gram-negative ground for this better activity in 1h and 1j may be due to the
pathogen, with an alarmingly high antibiotic resistance rate. presence of methoxy substitutions of benzoic acid which can
Even with the most effective antibiotics against this pathogen, increase the in vitro activity against gram positive when
namely carbapenems (imipenem and meropenem), the compared to other compounds.55-56 The reason for the best
resistance rate was found to be 15-20.4% amongst aeruginosa activity of 1i may be due to the presence of para-nitro
strains.54 Our current study showed that the synthesized substitution in benzoic acid.
compounds were active against P. aeruginosa. Compounds,
Table 3: Antimicrobial activity of synthesized compounds 1a-n using the well method (zone of inhibition in mm) (1000 µg/well).
Compounds
Organism
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
C
Gram positive bacteria
Bacillus subtilis
Micrococcus luteus
Staphylococcus aureus
Staphylococcus epidermidis
Gram negative bacteria
Klebsiellapneumoniae
Salmonella typhimurium
Proteus vulgaris
16
15
13
14
18
12
-
-
-
-
-
14
14
12
10
12
20
13
14
14
12
13
14
18
17
12
22
20
15
15
21
19
21
11
24
19
16
14
19
14
12
10
14
-
-
-
-
14
14
17
15
18
18
16
20
24
26
14
26
16
13
12
13
15
16
14
15
14
13
16
-
15
18
12
-
-
-
-
-
-
-
15
-
12
16
18
19
15
16
18
17
16
13
12
16
13
10
21
14
17
18
20
20
23
18
-
19
-
18
-
10
-
-
-
-
-
-
-
-
-
-
-
13
-
16
14
18
19
17
18
16
20
24
30
30
22
30
30
14
15
14
15
13
20
25
18
22
22
15
21
18
21
19
20
20
17
18
17
10
12
10
13
16
16
18
16
16
14
Shigella flexneri
Enterobacter aerogenes
Pseudomonas aeruginosa
Staphylococcus aureus MRSA
Fungi
Candida albicans
Malassesia pachydermatis
16
-
15
13
-
-
-
-
-
-
-
-
15
-
-
-
14
-
16
12
-
-
10
13
14
-
28
26
4 | J. Name., 2012, 00, 1-3
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C: Streptomycin - standard antibacterial agent; C: Ketoconazole - standard antifungal agent
Table 4: Minimum inhibitory concentration (MIC) of synthesized compounds 1a-n
Compounds
Organism
1a
1b
1d
1e
1f
1g
1h
1i
1j
1k
1m
1n
C-1
C-2
Gram positive bacteria
Bacillus subtilis
Micrococcus luteus
Staphylococcus aureus
Staphylococcus epidermidis
Gram negative bacteria
Klebsiella pneumonia
Salmonella typhimurium
Proteus vulgaris
62.5
250
250
250
62.5
500
-
125
250
500
500
62.5
62.5
250
250
125
125
250
62.5
62.5
500
250
250
31.25
62.5
250
31.25
62.5
125
62.5
125
250
62.5
125
500
500
250
250
250
125
250
125
125
125
62.5
25
<0.78
>100
<0.78
6.25
6.25
6.25
25
125
62.5
31.2
250
500
250
250
125
125
250
250
250
125
-
250
62.5
500
250
-
125
125
125
125
250
62.5
125
62.5
250
125
125
125
125
31.2
125
125
250
500
125
31.25
500
62.5
125
125
125
-
62.5
-
500
-
250
-
125
250
125
125
125
125
125
6.25
30
6.25
6.25
25
6.25
>100
6.25
<0.78
<0.78
6.25
250
250
250
125
500
250
62.5
62.5
31.25
125
62.5
62.5
125
62.5
125
500
500
500
250
250
125
125
125
125
250
Shigella flexneri
125
Enterobacter aerogenes
Pseudomonas aeruginosa
Staphylococcus aureus MRSA
62.5
31.25
31.2
25
6.25
<0.78
C-1: Streptomycin and C-2: Ciprofloxacin - standard antibacterial agents
Anticancer activity
other compounds. Among the tested compounds 1g,i-j
& 1n
showed potent cytotoxicity activity against SKVO-3 cell line
Based on antimicrobial results, compounds 1b and 1e-n were with IC50 values at 7.81 µg/mL. All concentrations used in the
taken selectively and screened for anticancer activity against experiment decreased the cell viability significantly (P<0.05) in
A549 and SKOV-3 cancer cell lines.57 All the tested compounds a concentration-dependent manner. Interestingly, significant
showed good cytotoxicity activity against A549 and SKOV-3 cell anticancer results were seen in triazolo-bosentan derivatives
lines, however, some of the synthesized compounds showed prepared by using aromatic carboxylic acids 5g-j except 1k.
prominent cytotoxicity activity in vitro against A549 and SKOV- Compound 1b, prepared from an amine 5b was found to be
3 cell lines. The anticancer activity against A549 and SKOV-3 least active when compared to others. Compound 1i has
cell lines were observed in 250 to 7.81 µg/mL concentrations shown best activity for both cell lines and the reason might be
(Table 5,6 and Figures 3,4). The results showed that the presence of an electron withdrawing group (-NO2) at the
compounds 1e-j
& 1l have shown significant activity against para- position of benzoic acid 5i. Triazolo-bosentans obtained
A549 cell line when compared to other compounds. In from aliphatic alkynes 1l-n also showed good activity. Toxicity
particular, Ig,i-j & 1l have exhibited very potent cytotoxicity study was tested against Vero cells. Interestingly triazolo-
activity against A549 cell line with IC50 values at 7.81 µg/mL. bosentan derivatives of 1e-j, 1l, and 1n showed no toxicity up
Interestingly significant activity against SKOV-3 cell lines was to 2 mg/mL.
also noticed for compounds 1e-j
& 1l-n when compared with
Table 5. Anticancer activity of synthesized compounds against A549 cancer cell line
Concentration
(µg/mL)
Compound
1b
1e
89.2
0.211
±0.00839 ±0.00712
84.9
1f
1g
96.3
0.072
±0.00337
95.8
1h
94.7
0.103±
0.00441
87
1i
1j
1k
47.2
1.033
±0.00881
35.6
1l
1m
19
1.586
±0.00557
16.7
1n
93.7
0.123
±0.00997
80.9
250
%
31.3
1.345
91.5
0.166
96.2
94.2
95.6
Mean
±S.D.
%
0.074
±0.00118
95.5
0.114±
0.00890
90.9
0.087
±0.00879
90.8
0.18
±0.00325
27
1.429
±0.00412
24.9
1.47
±0.00522
16.4
1.636
±0.00632
13.1
1.701
125
62.5
0.735
Mean
±S.D.
%
Mean
±S.D.
%
Mean
±S.D.
%
Mean
±S.D.
%
0.295
0.082
±0.00559 ±0.00660 ±0.00559
0.255
0.088
0.179±
±0.00578 ±0.00229 0.00773
1.261
1.631
0.123
±0.00836 ±0.00706 ±0.00967 ±0.00997
25.6
1.456
62.5
31.25
15.63
7.81
51.2
18.2
1.602
±0.00578 ±0.00631 ±0.00669 ±0.00397 0.00559
13.5
1.694
82.7
0.338
81.5
0.363
95.3
0.091
86.3
0.268±
80.6
0.38
14.9
1.666
56.1
0.859
0.955±
0.00436
42.9
1.118±
0.00947
30.9
1.352±
0.00871
26.7
1.436±
0.00856
±0.00771 ±0.00756 ±0.00805 ±0.00361
71.9
0.55
60.6
0.771
79.9
0.393
75
0.489±
14.5
1.675
77.9
0.432
10.2
1.759
18.6
1.594
±0.00743 ±0.00801 ±0.00598 ±0.00772
±0.00309 ±0.00779 ±0.00330 ±0.00433 0.00397
71.6
0.556±
11.9
1.725
68.1
0.625
51.2
0.956
75
0.489
11.1
1.741
±0.00491 ±0.00699 ±0.00671 ±0.00589
8.1
1.799
66.1
0.663
8.2
1.797
15.5
1.654
±0.00203
5.2
1.856
±0.00399 ±0.00597 ±0.00449 ±0.00507 0.00227
8.6
1.789
±0.00580
62 57.8
0.745 0.826±
±0.00888 ±0.00379 ±0.00409 0.01025
30.5
1.361
68.2
0.623
54.1
0.899
6.7
1.826
9.6
1.77
Mean
±S.D.
±0.00654
±0.00513 ±0.00712 ±0.00334 ±0.00665
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Fig. 3: Comparison of anticancer activity of synthesized compounds against A 549 cancer cell line
(Note: No toxicity was observed up to 2mg/mL in Vero cells)
Table 6: Anticancer activity of synthesized compounds against SKOV-3 cancer cell line
Concentration Compound
(µg/mL)
1b
48.9
Mean 0.653
1e
71.2
0.368
±0.00443
54
1f
1g
94.3
0.073
±0.00702
87.2
1h
94
0.077
±0.00507
92.2
1i
1j
1lk
39.9
0.768
±0.00338
34.9
1l
1m
84.7
0.195
±0.00337
77.7
1n
91.6
0.107
±0.00447
81
250
%
81.2
0.24
94.7
0.06
92.6
0.095
95.3
0.06
±0.00447
93.9
0.078
S.D.
%
±0.00338
40.5
±0.00556
64.9
0.448
±0.00773 ±0.00564
40.4
0.761
8±0.00560 ±0.00363
93.7
0.08
±0.00609
91.6
0.107
±0.00544
85.2
0.189
±0.00224
81.9
0.231
±0.00697
76.4
125
83.6
0.21
Mean 0.76
0.588
±0.00561 ±0.00881
0.164
0.1
0.831
0.285
0.242
S.D.
%
±0.00812
89.8
0.13
±0.00569 ±0.00501 ±0.00510 ±0.00429 ±0.00366
62.5
34.1
42
0.741
80.3
0.252
81.8
0.232
23.4
0.978
±0.00438 ±0.00588 ±0.00553 ±0.00577 ±0.00228
63.8
0.462
65.3
0.443
86.3
0.175
Mean 0.841
±S.D.
%
±0.00201 ±0.00779 ±0.00501 ±0.00328 ±0.00328
31.6
Mean 0.874
31.25
15.63
7.81
40.1
0.765
30.1
0.893
69.5
0.389
65.8
0.437
77.6
0.286
16.2
1.07
54.9
0.576
50.1
0.637
80.7
0.246
S.D.
%
±0.00198 ±0.00278 ±0.00337 ±0.00209 ±0.00449
24.6
±0.00371 ±0.00579 ±0.00438 ±0.00804 ±0.00509
28.6
0.912
22
0.996
63.6
0.465
±0.00854 ±0.00900 ±0.00886 ±0.00667 ±0.00579
57.1
0.548
72.1
0.356
9.2
1.159
51.1
0.625
31.2
0.878
75.3
0.315
±0.00399 ±0.00416 ±0.00688 ±0.00331 ±0.00708
Mean 0.963
±S.D.
%
6
22.7
0.987
9.5
60.1
0.51
50.3
0.63
63.4 63.3
0.468 0.469
±0.00608 ±0.00399 ±0.00701 ±0.00209 ±0.00884
3.2
1.236
33.7
0.847
22.2
0.993
Mean 1.201 1.156
S.D. ±0.00337 ±0.00458 ±0.00927
0.301
±0.00345 5±0.00670 ±0.00555
Fig. 4: Comparison of anticancer activity of synthesized compounds against SKOV-3 cancer cell line
In silico studies
using the AutoDock Tools (ADT) version 1.5.6 and AutoDock
version 4.2.5.1 docking program59-60 to investigate the
Molecular docking is a useful instrument to acquire knowledge potential binding mode of inhibitors. The DNA Topoisomerase
on protein-ligand interaction which is very significant in drug IV receptor is required for maintenance of proper DNA
discovery.58 All the synthesized pyrimidinyl linked 1,2,3- topology during transcription and replication in bacteria.61
triazolo-bosentan derivatives 1a
-n
were subjected to Anaplastic Lymphoma Kinase (ALK) is a Tyrosine kinase
molecular docking studies with DNA topoisomerase IV (PDB ID: receptor. Due to its critical role of aberrant signalling in cancer,
4EMV) and Anaplastic Lymphoma Kinase (ALK) (PDB ID: 2XP2) it is an attractive oncology target for therapeutic
6 | J. Name., 2012, 00, 1-3
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intervention.62 The obtained free energy of binding (FEB) was interact with C=O of LEU-1122 and ASP-1203 to form three
computed for all the synthesized compounds 1a-n (Table 7)
.
polar interactions with bond lengths of 2.7, 3.2 and 3.4 Å. In
addition, nitrogen and oxygen of triazole and phenoxy groups
Table 7 Free energy of binding (FEB) of 1a-n
A
B
C
Free energy of binding (kcal/mol)a
DNA Topoisomerase IV Anaplastic Lymphoma
Compound
(4EMV)
-6.95
-5.23
-6.58
-5.53
Kinase (2XP2)
1a
1b
1c
1d
NC
-8.54
NC
NC
1e
1f
-9.29
-7.97
-8.40
-6.71
-7.09
-8.66
-8.01
-8.32
-9.55
-7.94
1g
1h
1i
1j
-8.13
-7.04
-6.08
-7.44
-7.28
-9.80
-6.70
-8.70
-8.42
-8.78
-8.28
-8.42
1k
1l
1m
1n
Inhibitor
aCalculated by Autodock, NC-not calculated, Inhibitor: Co-
crystallized inhibitor with protein
Molecular docking outcomes of synthesized compounds 1a-n
with 4EMV receptor established that all the docked
compounds 1a-n bind efficiently with the receptor and exhibit
free energy of binding value from –5.23 to –9.29 kcal/mol.
Interestingly, among all the compounds docked, compound 1e
exhibits very high binding with 4EMV receptor and forms five
polar interactions with three amino acids, namely ASP-78, GLY-
82 and ARG-140, resulting in the binding energy of –9.29
kcal/mol. In the compound 1e, N-H attached with triazole
interacts with the ASP-78 and forms two polar interactions
with bond lengths of 1.7 and 3.3 Å respectively. Likewise, two
nitrogens of triazole interact with the GLY-82 and form two
Fig. 5: Docking with 4EMV receptor (A) Method validation using
crystallised and docked ligand; (B) Docking mode of all the compounds; (c)
Docking mode of the most binding energy compound 1e.
polar interactions with bond lengths of 3.2 and 3.5
Å
interact with C=O and N-H of MET-1199 and LYS-1150 to form
two polar interactions of 2.0 and 3.4 Å respectively. Also, N-H
and S=O interact with C=O of GLY-1123 to form two polar
interactions in 2.0 and 3.4 Å respectively. Binding interaction
respectively. Aside from this, pyrimidine nitrogen forms a polar
interaction with the ARG-140 with a bond length of 2.9 Å (Fig.
5).
with the 2XP2 receptor is shown in Fig.6
.
Docking of synthesized compounds with 2XP2 revealed that,
compounds efficiently bind with the active site of 2XP2
receptor and exhibits free energies of binding from -6.70 to
-9.55 kcal/mol. Compounds interact with the active site amino
acids of 2XP2 namely ARG-1120, 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-1210, ARG-1253, ASN-1254,
CYS-1255, LEU-1256, GLY-1269 and ASP-1270.Among all the
compounds docked, compound 1h exhibited very high binding
with 2XP2 receptor and formed seven polar interactions with
five amino acids and resulted in a binding energy of -9.55
kcal/mol. In the compound 1h, nitrogens of two pyrimidines
Conclusions
In summary, we have described facile one pot synthesis of
novel bis-pyrimidinyl linked 1,4-disubstituted 1,2,3-triazoles
1a-n (triazolo-bosentans) with good yields from bosentan
2 as
a
potent hybrid drugs with dual biological activities
(antimicrobial and anticancer) and rationalized with respective
proteins using docking study. During this work good
antimicrobial activities were observed for compounds 1e-f
&
1h-j against tested bacteria and fungi, amongst which, 1h and
1j exhibited very good activity. In vitro cytotoxicity (lung and
ovarian) studies against A549 and SKOV-3 cell lines revealed
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prominent cytotoxic activity of compounds 1e-n. Among all Fig. 6: Docking with 2XP2 receptor (A) Method validation using
crystallised and docked ligand; (B) Docking mode of all the compounds; (C)
Docking mode of the most binding energy compound 1h.
triazolo-bosentans screened, (1g,i-j) and (1l,e-j,l-n) showed
very significant in vitro cytotoxicity against A549 and SKOV-3
cell lines respectively with IC50 value at 7.81 μg/mL. Active
compounds 1e-j, 1l and 1n were tested against Vero cells and
Acknowledgements
no toxicity was observed up to 2 mg/mL. When correlated the
Sincere thanks to the management of Malladi Drugs &
Pharmaceuticals Ltd., Chennai, India for their support. This
project was supported by King Saud University, Deanship
Scientific Research, College of Science, Research Center.
structure and activity, it was concluded that antimicrobial and
anticancer activities were enhanced by the methoxy
substitutions of benzoic acid ring. Among the tested
compounds, best anticancer activity was observed for p-nitro
substituted triazolo-bosentan 1i which also exhibited good
antimicrobial activity. Free energies of binding were calculated
for the synthesized compounds against 4EMV and 2XP2
receptors. Among them, 1e has shown a better binding energy
(-9.29 kcal/mol) against 4EMV receptor with five polar
interactions by three amino acids and 1h has shown better
binding energy (-9.55 kcal/mol) against 2PX2 receptor with
seven polar interactions by five amino acids. Based on
significant anticancer results, this class of compounds can be
considered as a good starting point of development of
Experimental
Chemistry
Melting points were measured using a Veego melting point
apparatus model VMP-PM. Thin layer chromatography (TLC)
was carried out using pre-coated Merck TLC Silicagel 60 F254
and spots were detected using Ultra-Violet light. IR spectra
were recorded (KBr pellet) on Shimadzu Prestige 21 FTIR
1
instrument in the range of 4000 to 400 cm-1. H NMR and 13C
bosentan
2 based dual endothelin receptor (ET) antagonists
NMR spectra were recorded using a Bruker-Avance 300 MHz
FT-NMR spectrometer (300 and 75 MHz, respectively) using
DMSO-d6 as solvent and TMS as internal standard. Chemical
shifts (δ) were mentioned in parts per million (ppm). The
following abbreviations are used: s-singlet, d-doublet, t-triplet,
q-quintet and m-multiplet. Low resolution mass spectra were
recorded on Agilent 6110LC/MS mass spectrophotometer
using ESI mode. The elemental analysis was done (sample
thoroughly dried under vacuum) using a Thermo Fischer Flash
1112 Series elemental analyzer. For antimicrobial activity,
the reference cultures were obtained from the Institute of
Microbial Technology (IMTECH), Chandigarh, India-160 036
and the remaining cultures were prevailed from the
Department of Microbiology, Christian Medical College,
Vellore, Tamil Nadu, India.
for efficient mono therapy for cancer.
A
B
Experimental procedure for the synthesis of compound (3):
Thionyl chloride (8 mL, 0.11 mol) was added to bosentan
2
(55g, 0.10 mol) in chloroform (500 mL) and heated to 55 to 60
°C for 3 h under nitrogen. Excess thionyl chloride and
chloroform were completely knocked off under vacuum to get
a yellow solid which was filtered and washed with acetone
(120 mL). Obtained 48g of solid after drying.
4-tert-Butyl-N-(6-(2-chloroethoxy)-5-(2-methoxyphenoxy)-
C
2,2'-bipyrimidin-4-yl)benzene- sulfonamide (3): Yellow solid.
o
Yield 85%. Mp: 198-200 C; IR: 3252, 2954, 2904, 2835, 1684,
1618, 1570, 1564, 1554, 1499, 1455, 1438, 1397, 1384, 1284,
1253, 1213, 1173, 1162, 1155, 1111, 1083, 1026, 993, 874,
1
833, 827, 741, 698, 679, 625, 572, and 545 cm-1. H-NMR (400
MHz, DMSO-d6): δH: 1.36 (s, 9H), 3.88 (s, 3H), 4.51 (t, 2H, J=8.0
Hz), 5.18 (t, 2H, J=8.0 Hz), 6.76–10.02 (m, 11H, aromatic) and
11.62 (bs, 1H). 13C-NMR (100 MHz, DMSO-d6): δC: 31.33, 35.41,
37.73, 52.59, 56.27, 113.48, 115.93, 120.76, 123.86, 124.58,
126.04, 127.33, 129.15, 138.24, 141.61, 145.71, 147.01,
149.26, 149.52, 153.73, 155.09, 156.59 and 165.98. ESI-MS:
8 | J. Name., 2012, 00, 1-3
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[M+H]+ at m/z 570 (Cl35) & 572 (Cl37) in 3:1 ratio. Anal. Calcd 4.98 (m, 1H), 5.79 (bs, 1H), 6.78-7.72 (m, 17H, aromatic) and
for C27H28ClN5O5S: C, 56.89; H, 4.95; N, 12.29. Found C, 57.12; 8.97 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): δC 21.59, 26.92,
H, 4.99; N, 12.35.
27.17, 29.75, 30.03, 31.59, 34.73, 48.15, 56.15, 63.48,
113.18, 113.29, 113.55, 113.77, 120.84, 120.86, 124.41,
124.55, 125.18, 125.25, 126.29, 126.32, 126.69, 126.76,
Experimental procedure for the synthesis of azide (4):
N,N-dimethylacetamide (250 ml) was charged to 3 (46g, 0.08 126.79, 127.03, 127.10, 127.89, 128.15, 128.67 and 157.98.
mol), followed by sodium azide (6.5g, 0.1 mol) and stirred at ESI-MS m/z 780 [M+H]+. Anal. Calcd for C40H45N9O6S: C, 61.60;
ambient temperature for 16 to 20 h. Completion of the H, 5.82; N, 16.16.. Found C, 62.20; H, 5.87; N, 16.10.
reaction was confirmed by TLC (10% methanol in ethyl
acetate) after which, 500 mL of water was added to the 4-tert-Butyl-N-(6-(2-(4-(((1S,2R)-1-hydroxy-1-phenyl-2-propyl-
reaction mass and the obtained solid was filtered and dried amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxy-
under vacuum. Obtained azide
refrigerator.
4
(43g) was preserved in a phenoxy)-2,2'-bipyrimidin-4-yl)benzenesulfonamide(1b):
Brown amorphous solid. Yield 6.0g (78%). Mp: 140-143 °C. IR:
3356, 2959, 2924, 2853, 1668, 1603, 1562, 1499, 1456, 1393,
N-(6-(2-Azidoethoxy)-5-(2-methoxyphenoxy)-2,2'-
bipyrimidin-4-yl)-4-tert-butylbenzenesulfonamide – [azide 4]: 750, 702, 627, 575 and 548 cm-1. H NMR (400 MHz, DMSO-
1341, 1252, 1219, 1175, 1140, 1103, 1080, 1022, 908, 852,
1
o
Pale brown solid. Yield 93%. Mp: 172-175 C. IR: 3245, 3066, d6): δH0.94 (d, 3H, J=10.5 Hz), 1.24 (s, 9H), 3.18 (s, 2H), 3.83 (s,
2959, 2903, 2835, 2795, 2090, 1681, 1617, 1563, 1550, 1499, 3H), 4.11 (t, 2H, J=12.2 Hz), 4.59 (t, 2H, J=12.2 Hz), 5.10 (d of d,
1455, 1397, 1386, 1333, 1254, 1215, 1179, 1162, 1111, 1084, 1H, J=56 Hz & J=20.2 Hz), 5.92 (m, 1H), 6.56–7.59 (m, 17H),
1
926, 873, 833, 824, 768, 749, 625, 574 and 545 cm-1. H-NMR 8.94 (bs, 1H) and 9.00 (bs, 1H). 13C-NMR (100 MHz, DMSO-d6):
(400 MHz, DMSO-d6): δH: 1.26 (s, 9H), 3.47 (t, 2H, J=6.2 Hz), δC 14.33, 22.62, 29.46, 29.90, 31.40, 31.76, 32.02, 34.86,
3.83 (s, 3H), 4.12 (t, 2H, J=18.3 Hz), 6.67–9.11 (m, 11H, 56.14, 113.23, 114.51, 115.11, 116.05, 120.35, 120.84, 123.19,
aromatic), 11.30 (bs, 1H). 13C-NMR (100 MHz, DMSO-d6): δC: 123.64, 125.15, 126.31, 127.84, 128.19, 128.21, 128.24,
31.23, 35.24, 44.34, 49.06, 56.18, 113.35, 115.33, 120.85, 128.32, 128.66, 128.99, 129.92, 131.86, 133.48, 139.58, 153.83
122.84, 123.29, 124.33, 125.60, 128.53, 138.48, 146.14, and 158.22. ESI-MS m/z 766 [M+H]+. Anal. Calcd for
146.92, 149.33, 151.31, 156.26, 157.27, 158.38 and 159.58.ESI- C39H43N9O6S: C, 61.16; H, 5.66; N, 16.46. Found C, 61.30; H,
MS: [M+H]+ at m/z 577. Anal. Calcd for C27H28N8O5S: C, 56.24; 5.69; N, 16.50.
H, 4.89; N, 19.43. Found C, 56.55; H, 4.93; N, 19.52.
4-tert-Butyl-N-(5-(2-methoxyphenoxy)-6-(2-(4-((methyl(1-
General experimental procedure for one pot synthesis of (1a- phenylpropan-2-yl)-amino)methyl)-1H-1,2,3-triazol-1-yl)-
f): Amine 5a-f (0.010 mol), n-butanol (10 mL), potassium ethoxy)-2,2'-bipyrimidin-4-yl)benzenesulfonamide(1c):
carbonate (1.7g, 0.012 mol, pre-dried at 100 °C) and propargyl Brown amorphous solid. Yield 6.9g (90%). Mp: 99-102 °C. IR:
chloride (0.75g, 0.010 mol) were mixed and heated at 55 to 60 3447, 3063, 2961, 2924, 2853, 1653, 1595, 1562, 1499, 1458,
°C for 6 h. Cooled the mass to 20 °C and charged with water 1393, 1250, 1221, 1175, 1140, 1107, 1076, 849, 746, 702, 631,
(10 mL), DIPEA (1.3g, 0.02 mol), copper iodide (200mg, 10 577 and 546 cm-1. 1H NMR (400 MHz, DMSO-d6): δH1.04 (s,
mol%) followed by azide
4 (5.8g, 0.01 mol) and agitated at 3H), 1.24 (s, 9H), 2.62 (t, 2H, J=11.3 Hz), 3.14 (t, 2H, J=15.3 Hz),
ambient temperature for 6 to 10 h. Completion of the reaction 3.48 (s, 3H), 3.83 (s, 3H), 4.18 (1H, m), 4.24 (d of d, 2H), 4.65 (t,
was checked by TLC (10% methanol/ethyl acetate). To the 2H), 6.60–7.73 (m, 17H, aromatic) and 9.01 (bs, 1H). 13C NMR
mass, 50 mL of water and 50 mL of ethyl acetate were (100 MHz, DMSO-d6): δC 29.47, 29.90, 30.34, 31.39, 31.76,
charged, stirred and filtered to remove inorganic. The organic 32.09, 34.99, 36.07, 45.11, 56.11, 113.13, 114.37, 120.84,
layer was separated, dried over anhydrous sodium 122.46, 122.54, 122.80, 124.79, 124.95, 126.92, 127.50,
sulphate followed by vacuum distillation using a Rota 127.78, 128.08, 128.17, 128.91, 129.08, 129.72, 130.12,
evaporator at below 60 °C, yielded a semi solid. The title 133.33, 153.45 and 158.15. ESI-MS m/z 764 [M+H]+. Anal.
compounds 1a-f were obtained as pale brown to brown Calcd for C40H45N9O5S: C, 62.89; H, 5.94; N, 16.50. Found C,
amorphous solid in between 78-91% yields, when it was 62.85; H, 5.98; N, 16.55.
triturated with n-heptane, filtered and dried under vacuum at
60 °C.
4-tert-Butyl-N-(6-(2-(4-(((2-hydroxy-2-(3-hydroxyphenyl)-
ethyl)(methyl)amino)-methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-
(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)-benzenesulfon-
4-tert-Butyl-N-(6-(2-(4-((((1S,2R)-1-hydroxy-1-phenyl-2-
propyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-ethoxy)- amide (1d): Brown amorphous solid. Yield 7.1g (90%). Mp 148-
5-(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)-benzenesulfon- 150 °C. IR: 3443, 3067, 2963, 2868, 1653, 1693, 1593, 1564,
amide (1a): Pale brown amorphous solid. Yield 6.7g (88%). Mp: 1499, 1456, 1396, 1252, 1217, 1180, 1138, 1103, 1078, 1047,
122-125 °C. IR: 3373, 2961, 2924, 2853, 1658, 1651, 1587, 906, 837, 752, 702, 630 and 577 cm-1. ESI-MS m/z 782 [M+H]+.
1562, 1520, 1499, 1454, 1395, 1250, 1217, 1177, 1138, 1103, Anal. Calcd for C39H43N9O7S: C, 59.91; H, 5.54; N, 16.12. Found
1078, 851, 750, 702, 629 and 577 cm-1. 1H NMR (400 MHz, C, 60.05; H, 5.57; N, 16.24.
DMSO-d6): δH 0.90 (d, 3H, J=14.4 Hz), 1.01 (s, 9H), 2.71 (t, 2H,
J=12.2 Hz), 3.29 (s, 2H), 3.81 (s, 3H), 4.19 (s, 3H), 4.64 (t, 2H),
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4-tert-butyl-N-(6-(2-(4-((2,3-dihydro-1H-inden-1-ylamino)-
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphen-
methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxyphenoxy)- noxy)-2,2'-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-
2,2'-bipyrimidin-4-yl)benzenesulfonamide(1e): Yellowish methyl benzoate (1g): Yellowish brown solid. Yield 6.8g (92%).
brown solid. Yield 6.6g (88%). Mp: 102-105 °C. IR: 3422, 2963, Mp: 95-98 °C. IR: 3144, 3067, 2963, 2926, 2868, 2853, 1719,
2868, 1674, 1605, 1564, 1499, 1458, 1393, 1340, 1252, 1219, 1676, 1605, 1564, 1499, 1452, 1393, 1340, 1271, 1254, 1219,
1
1173, 1111, 1084, 1020, 864, 837, 762, 625 and 575 cm-1. H 1173, 1111, 1084, 1024, 864, 835, 752, 714, 692, 625, 573 and
NMR (400 MHz, DMSO-d6): δH 1.25 (s, 9H), 2.24 &2.41 (m, 2H, 552 cm-1. 1H NMR (400 MHz, DMSO-d6): δH 1.25 (s, 9H), 3.84 (s,
J=7.5 Hz), 2.87 (m, 2H), 3.14 (m, 2H), 3.82 (s, 3H), 4.22 (s, 2H), 3H), 4.34 (t, 2H, J=9.5 Hz), 4.66 (t, 2H, J=8.0 Hz), 5.32 (s, 2H),
4.25 (t, 2H, J=8.0 Hz), 4.73 (t, 2H, J=12 Hz), 6.67–8.24 (m, 16H, 6.70-9.03 (m, 17H, aromatic) and 11.30 (bs, 1H). 13C NMR (100
aromatic) and 9.75 (bs, 2H). 13C NMR (100 MHz, DMSO-d6): δC MHz, DMSO-d6): δC 31.23, 35.25, 51.34, 56.16, 58.33, 59.58,
28.80, 28.90, 30.28, 31.28, 35.21, 36.61, 45.35, 56.14, 61.13, 113.24 to 165.90. ESI-MS m/z 737 [M+H]+. Anal. Calcd for
97.74, 104.52, 108.11, 113.21, 115.20, 120.80, 125.45, 125.50, C37H36N8O7S: C, 60.31; H, 4.92; N, 15.21. Found C, 60.25; H,
125.53, 125.57, 126.47, 126.71, 127.05, 127.16, 128.45, 4.96; N, 15.20.
129.91, 137.73, 138.20, 138.79, 145.41, 149.17, 157.42, 158.36
and 159.24. ESI-MS m/z 748 [M+H]+. Anal. Calcd for (1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphe-
C39H41N9O5S: C, 62.63; H, 5.53; N, 16.86. Found C, 62.53; H, noxy)-2,2'-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-
5.59; N, 16.94.
methyl 2-bromo-5-methoxybenzoate (1h): Pale brown solid.
Yield 7.2g (84%). Mp: 160-164 °C. IR: 3068, 2965, 2905, 1709,
N-(1-(2-(1-((6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxy- 1674, 1602, 1565, 1515, 1500, 1464, 1456, 1419, 1394, 1343,
phenoxy)-2,2'-bipyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl- 1290, 1271, 1253, 1220, 1176, 1140, 1111, 1084, 1022, 869,
oxy)ethyl)-4-(methoxymethyl)piperidin-4-yl)-N-phenylpropio- 835, 763, 753, 696, 627, 575 and 547 cm-1. 1H NMR (400 MHz,
namide (1f):Pale brown solid. Yield 8.1g (91%). Mp 180-181 °C. DMSO-d6): δH 1.26 (s, 9H), 3.77 (s, 3H), 3.83 (s, 3H), 4.32 (t,
IR: 3474, 2965, 2833, 1651, 1589, 1562, 1518, 1499, 1456, 2H), 4.67 (t, 2H), 5.32 (s, 2H), 6.69-9.03 (m, 15H, aromatic) and
1396, 1377, 1323, 1250, 1229, 1178, 1140, 1107, 1078, 1002, 11.30 (bs, 1H).13C NMR (100 MHz, DMSO-d6): δC 31.24, 35.24,
849, 750, 706, 633, 579 and 552 cm-1. 1H NMR (400 MHz, 45.69, 45.76, 47.91, 56.20, 58.98, 110.74, 113.29, 115.52,
DMSO-d6): δH 0.79 (t 3H, J=7.3 Hz), 1.23 (s, 9H), 1.69,(t, 4H), 116.56, 119.56, 120.85, 122.75, 123.23, 124.44, 125.65,
1.98 (t, 4H), 3.32 (s, 3H), 3.52 (q, 2H), 3.85 (t, 2H), 3.93 (s, 2H), 125.91, 128.52, 133.26, 135.26, 138.38, 146.09, 146.69,
4.12 (s, 2H), 4.56 (t, 2H), 6.51-7.74 (m, 17H) and 8.95 (bs, 1H). 149.27, 151.17, 156.31, 157.38, 158.26, 158.85, 159.10 and
13C NMR (100 MHz, DMSO-d6): δC9.97, 23.30, 28.29, 28.70, 165.60. ESI-MS m/z 847 [M+H]+. Anal. Calcd for C38H37BrN8O8S;
30.47, 31.53, 31.59, 34.80, 59.28, 76.57, 79.46, 89.08, 113.80, C, 59.60; H, 4.87; N, 14.63. Found C, 59.69; H, 4.93; N, 14.70.
120.86, 122.40, 124.47, 129.13, 131.52, 133.47, 134.01,
135.10, 136.29, 139.18, 140.51, 141.35, 146.09, 149.15, (1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphe-
150.32, 152.92 and 168.34. ESI-MS m/z 891 [M+H]+.Anal. Calcd noxy)-2,2'-bipyrimidin-4-yl-oxyethyl)-1H-1,2,3-triazol-4-yl)-
for C46H54N10O7S: C, 62.00; H, 6.11; N, 15.72. Found C, methyl-4-nitrobenzoate (1i): Brown solid. Yield 7.9g (88%).
62.39; H, 6.15; N, 15.89.
Mp: 168-171 °C. IR: 3111, 3078, 2965, 2869, 2838, 1726, 1675,
1607, 1565, 1528, 1499, 1456, 1393, 1342, 1267, 1253, 1219,
General experimental procedure for one pot synthesis of 1g-k 1172, 1112, 1102, 1084, 1014, 872, 858, 762, 752, 720, 625,
In a nitrogen atmosphere, thionyl chloride (0.8 mL, 0.011 mol) 575 and 546 cm-1.1H NMR (400 MHz, DMSO-d6): δH 1.25 (s,
was added to acid 5g-k (0.010 mol) in chloroform (10 mL) at 50 9H), 3.83 (s, 3H), 4.34 (t, 2H, J=9.5 Hz), 4.67 (t, 2H, J=8.0 Hz),
to 55 °C for 3 to 4 h. Then it was cooled to about 10 °C and 5.38 (s, 2H), 6.69-9.03 (m, 16H, aromatic) and 11.31 (bs, 1H).
propargyl alcohol (0.56g, 0.010 mol) was added and stirred for 13C NMR (100 MHz, DMSO-d6): δC 31.58, 35.24, 47.82, 56.13,
4-6 h at ambient temperature. Further it was heated to 50 °C 59.05, 59.26, 124.39, 124.75, 125.46, 125.78, 127.62, 128.51,
and the solvent was distilled off completely under vacuum and 131.22, 131.86, 132.12, 135.23, 142.50, 142.81, 146.92,
the obtained residue was charged with n-butanol (10 mL), 148.26, 150.77, 158.25, 160.23, 161.22, 161.58, 162.12, 164.45
water (10 mL), DIPEA (2.6g, 0.02 mol) and copper iodide and 164.56; ESI-MS m/z 891 [M+H]+. Anal. Calcd for
(200mg, 10 mol%) followed by azide
4 (5.8g, 0.01 mol) and C37H35N9O9S: C, 56.84; H, 4.51; N, 16.12. Found C, 56.98; H,
stirred at ambient temperature for 6 to 10 h. Completion of 4.55; N, 16.19.
the reaction was checked by TLC (10% methanol/ethyl
acetate). To the mass, 50 mL of water and 50 mL of ethyl (1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphen-
acetate were charged, stirred and filtered to remove inorganic. oxy)-2,2'-bipyrimidin-4-yl-oxyethyl)-1H-1,2,3-triazol-4-yl)-
Organic layer was separated, dried over anhydrous sodium methyl 3,4-dimethoxybenzoate (1j): Brown solid. Mp 128-132
sulphate followed by vacuum distillation using
a
rota °C. Yield 7.2g (90%). IR: 3605, 3150, 3076, 2964, 2905, 2837,
evaporator at below 60 °C yielded a semi solid. The title 1707, 1675, 1601, 1565, 1514, 1500, 1457, 1418, 1392, 1343,
compounds 1g-k were obtained as a pale brown to brownish 1289, 1271, 1253, 1219, 1174, 1111, 1022, 868, 834, 763, 694,
1
amorphous solids in between 84-92% yield when it was 626, 575 and 546 cm-1. H NMR (400 MHz, DMSO-d6): δH1.26
triturated with n-heptane, filtered and dried under vacuum at (s, 9H), 3.80 (s, 6H), 4.32 (t, 2H), 4.66 (t, 2H), 5.30 (s, 2H), 6.71-
9.02 (m, 17H, aromatic) and 11.30 (bs, 1H). ESI-MS: [M+H]+ at
60 °C.
10 | J. Name., 2012, 00, 1-3
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m/z 797; Anal. Calcd for C39H40N8O9S: C, 58.78; H, 5.06; N, ESI-MS: [M+H]+ at m/z 659. Anal. Calcd for C33H38N8O5S: C,
14.06. Found C, 58.69; H, 5.12; N, 14.04.
60.17; H, 5.81; N, 17.01. Found C, 60.85; H, 5.86; N, 17.15.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphen-
oxy)-2,2'-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-
4-tert-Butyl-N-(6-(2-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-
1-yl)ethoxy)-5-(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)-
o
methyl 3-methylbenzoate (1k): Brown solid. Mp 140-144 benzenesulfonamide (1n): Brown solid. Mp 105-107 C. Yield
oC.Yield 6.6g (87%). IR: 3455, 3139, 3067, 2964, 2904, 2869, 5.9g (87%). IR: 3418, 3140, 3069, 2936, 2860, 1674, 1607,
1718, 1653, 1562, 1500, 1457, 1397, 1278, 1251, 1228, 1214, 1566, 1499, 1456, 1393, 1340, 1285, 1252, 1219, 1171, 1111,
1202, 1179, 1141, 1105, 1081, 1021, 851, 835, 748, 668, 633, 1084, 1047, 1020, 866, 835, 752, 702, 625, 575 & 546 cm-1. 1H-
580 and 551 cm-1. 1H NMR (400 MHz, DMSO-d6): δH 1.23 (s, NMR (400 MHz, DMSO-d6): δH 1.27 (m, 11H), 1.41 (m, 2H), 1.64
9H), 2.36 (s, 3H), 3.83 (s, 3H), 4.16 (t, 2H), 4.62 (t, 2H), 5.32 (s, (m, 4H, J=5.2 Hz), 1.78 (m, 2H), 3.85 (s, 3H), 4.29 (t, 2H, J=5.6
2H), 6.57-8.07 (m, 15H, aromatic) and 8.95 (bs, 1H). ESI-MS: Hz), 4.60 (m, 2H, J=6.4 Hz), 4.77 (bs, 1H), 6.74-9.06 (m, 12H,
[M+H]+ at m/z 751; Anal. Calcd for C38H38N8O7S: C, 60.79; H, aromatic), 11.27 (bs, 1H); 13C-NMR (100 MHz, DMSO-d6): δC
5.10; N, 14.92. Found C, 60.95; H, 5.14; N, 14.96.
22.06, 25.72, 31.24, 35.24, 38.19, 45.89, 47.42, 56.21, 68.41,
113.30, 115.49, 120.90, 121.89, 122.84, 123.22, 124.42,
General experimental procedure for the synthesis of 1l-n: 125.62, 128.51, 138.43, 146.15, 146.80, 147.52, 149.29,
Aliphatic terminal alkyne 5l-n (0.01 mol), n-butanol (6 mL), 151.25, 156.25, 157.41, 158.32 & 159.15;ESI-MS: [M+H]+ at
water (6 mL), DIPEA (2.6g, 0.02 mol) and copper iodide m/z 683; Anal. Calcd. for C35H40N8O6S: C, 59.98; H, 5.75; N,
(200mg, 10 mol%) were mixed followed by azide
4 (5.8g, 0.01 15.99.Found C, 60.12; H, 5.79; N, 16.11.
mol) and agitated at ambient temperature for 6 to 10 h.
Completion of the reaction was checked by TLC (10% methanol Biological assays
in ethyl acetate). To the mass, 50 mL of water and 50 mL of
ethyl acetate were charged, stirred and filtered to remove Materials and methods for antimicrobial activity:
inorganics. Organic layer was separated, dried over anhydrous Streptomycin and Ciprofloxacin (Sigma) were used as positive
sodium sulphate followed by vacuum distillation using a rota control against bacteria. Ketoconazole (Himedia, Mumbai) was
evaparotor at below 60 °C yielded a semi solid. The title used as a positive control against fungi.
compounds, 1l-n were obtained as a pale brown to brown Microbes: The following bacteria and fungi were used for the
amorphous solids in between 87-95% yield when the semi experiment. Bacteria: Bacillus subtilis MTCC 44.1, Klebsiella
solid was triturated with n-heptane, filtered and dried under pneumoniae MTCC 109, Staphylococcus epidermidis MTCC
vacuum at 60 °C.
3615, Micrococcus luteus MTCC 106, Salmonella typhimurium
MTCC 1251, Proteus vulgaris MTCC 1771, Shigellaflexneri
4-tert-Butyl-N-(6-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)- MTCC 1457, Enterobacter aerogenes MTCC 111,
ethoxy)-5-(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)-benz- Staphylococcus aureus MTCC 96, Pseudomonas aeruginosa
enesulfonamide (1l): Yellowish brown solid. Mp 168-170 oC. MTCC 741 and Staphylococcus aureus (MRSA-methicillin
Yield 6.0g (95%).IR: 3447, 3134, 2965, 2870, 2835, 1678, 1661, resistant). The reference cultures were obtained from the
1609, 1568, 1499, 1427, 1394, 1348, 1250, 1219, 1196, 1167, Institute of Microbial Technology (IMTECH), Chandigarh-
1113, 1084, 1026, 856, 833, 756, 692, 625, 573 and 546 cm- 160036, India; Fungi: Candida albicans MTCC 227 and
1.1H-NMR (400 MHz, DMSO-d6): δH 1.27 (s, 9H), 3.84 (s, 3H), Malassesia-pachydermatis. All the remaining cultures were
4.30 (t, 2H, J=9.5 Hz), 4.45 (t, 2H, J=9.5 Hz), 4.61 (s, 2H), 5.13 prevailed from the Department of Microbiology, Christian
(bs, 1H), 6.70-9.05 (m, 12H, aromatic) and 11.27 (bs, 1H). 13C- Medical College, Vellore, Tamil Nadu, India.
NMR (100 MHz, DMSO-d6): δC 22.41, 28.69, 47.77, 55.46,
69.75, 71.11, 111.40, 113.65, 115.24, 118.88, 123.45, 126.56, In vitro well method
128.54, 128.68, 129.04, 134.68, 136.43, 143.13, 143.91, Petri plates were prepared with 20 mL of sterile Mueller
147.00, 148.41, 150.75, 157.66 and 161.20.ESI-MS: [M+H]+ at Hinton agar (MHA) (Hi-media, Mumbai). The test cultures were
m/z 633; Anal. Calcd for C30H32N8O6S: C, 56.95; H, 5.10; N, swabbed on top of a solidified media and were allowed to dry
17.71. Found C, 57.15; H, 5.14; N, 17.85.
for 10 min and a specific amount of synthesised compound at
1mg/disc was added to each well separately. Negative control
was prepared using respective solvents. Streptomycin was
used as positive control against bacteria. Ketoconazole was
4-tert-Butyl-N-(6-(2-(4-butyl-1H-1,2,3-triazol-1-yl)ethoxy)-5-
(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)benzene-
sulfonamide (1m): Brown solid. Mp 101-103 °C.Yield 5.9g used as positive control for fungi. The plates were incubated
(89%). IR: 3136, 3069, 2959, 2870, 1676, 1605, 1564, 1499, for 24 h at 37°C for bacteria and for 48 h at 28°C for fungi.
1456, 1393, 1340, 1252, 1219, 1173, 1111, 1084, 1045, 1022, Zones of inhibition were recorded in mm and the experiment
864, 837, 752, 694, 625, 575 and 546 cm-1.1H-NMR (400 MHz, was repeated twice. Bacterial inoculums were prepared by
DMSO-d6): δH 0.88 (t, 3H, J=6.8 Hz), 1.27 (m, 13H, aromatic), growing cells in Mueller Hinton broth (MHB) (Himedia) for 24 h
1.51 (t, 2H, J=7.5 Hz), 3.83 (s, 3H), 4.31 (t, 2H, J=9.5 Hz), 4.57 (t, at 37°C. The filamentous fungi were grown on Sabouraud
2H, J=9.5 Hz), 6.71-9.04 (m, 12H, aromaitc) and 11.27 (bs, 1H). dextrose agar (SDA) slants at 28°C for 10 days and the spores
were collected using sterile doubled distilled water and were
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homogenized. Yeast was grown on a Sabouraud dextrose lower occupancy residue structures were deleted, and any
broth (SDB) at 28°C for 48 h.
incomplete side chains were replaced using the ADT. Further
ADT was used to remove crystal water, Gasteiger charges were
added to each atom, and merged the non-polar hydrogen
Minimum inhibition concentration study:
The required concentrations (1000 µg/mL, 500 µg/mL, 250 atoms to the protein structures. The distance between the
µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.62 µg/mL and donor and an acceptor atoms that form a hydrogen bond was
7.81 µg/mL) of the compound were dissolved in DMSO (2%), defined as 1.9 Å with a tolerance of 0.5 Å, and the acceptor–
and were diluted to give serial two-fold dilutions that were hydrogen–donor angle was not less than 120˚. The structures
added to each medium in 96 well plates. An inoculum of 100 were then saved in PDBQT file format, for further studies in
µL from each well was inoculated. The antifungal agents, ADT. The grid boxes with dimension of 40 × 40 × 40 Å3and 60 ×
Ketoconazole for fungi and Streptomycin for bacteria were 60 × 60 Å3with 0.375 Å spacing and centred on 14.860, 29.555,
included in the assays as positive controls. For fungi, the plates 6.941 and 29.697, 47.794, 8.863was created around the
were incubated for 48 to 72 h at 28 °C and for bacteria the binding site of co-crystallised ligand on 4EMV and 2XP2
plates were incubated for 24 h at 37 °C. The MIC for fungi was respectively. The centre of the box was set at co-crystallised
defined as the lowest extract concentration, showing no visible ligand centre and grid energy calculations were carried out.In
fungal growth after incubation time. 5 µL of the tested broth order to verify the reproducibility of the docking calculations,
was placed on the sterile MHA plates for bacteria and the bound ligand was extracted from the complexes and
incubated at respective temperature. The MIC for bacteria was submitted for one-ligand run calculation. This reproduced top
determined as the lowest concentration of the compound scoring conformation falling within root-mean-square
inhibiting the visual growth of the test cultures on the agar deviation (RMSD) value of 0.58 to 1.53 Å and 1.63 to 1.95 Å
plate.
with bound X-ray conformation for 4EMV and 2XP2
respectively, suggesting this method is valid enough to be used
for docking studies of other compounds. Docking of different
Study of cytotoxicity
A549 (Lung), SKOV-3 (Ovarian) and Vero cells were obtained ligands to protein was performed using AutoDock, following
from ATCC, USA. A549, SKOV-3 and Vero cells were maintained the same protocol used in as that of validation study. All
in complete tissue culture medium DMEM with 10 % Fetal dockings taken into 2.5 million energy evaluations were
Bovine Serum and 2mM L-Glutamine, along with antibiotics performed for each of the test molecules. For each compound,
(about 100 IU/mL of penicillin, 100 µg/mL of streptomycin) 50 docked conformations were generated. The energy
with the pH adjusted to 7.2. The cytotoxicity was determined calculations were done using genetic algorithms. Docked
according to the method57 with some changes. Cells (5×105) ligand conformations were analyzed in terms of energy,
were seeded in 96 well plates containing medium with hydrogen bonding, and hydrophobic interaction between
different concentrations such as 250, 125, 62.5, 31.25, 15.63 ligand and receptor. Detailed analyses of the ligand-receptor
and 7.81 µg/mL. The cells were cultivated at 37°C with 5% CO2 interactions were carried out, and final coordinates of the
and 95% air in 100% relative humidity. After various durations ligand and receptor were saved. For display of the receptor
of cultivation, the solution in the medium was removed. An with the ligand binding site, PyMOL software63 was used. From
aliquot of 100 µL of medium containing 1 mg/mL of 3-(4, 5- the docking scores, the free energy of binding (FEB) of all
dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) compounds were calculated.
was loaded to the plate. The cells were cultured for 4 h and
then the solution in the medium was removed. An aliquot of
Notes and references
1. WHO, Antimicrobial resistance fact sheet no. 194, World
100 µL of DMSO was added to the plate, which was shaken
until the crystals were dissolved. The cytotoxicity against
Health
Organization,
Geneva,
2014.
cancer cells was determined by measuring the absorbance of
the converted dye at 570 nm in an ELISA reader. Cytotoxicity of
each sample was expressed as IC50 value. The IC50 value is the
concentration of test sample that causes 50% inhibition of cell
growth, averaged from three replicate experiments. The
percentage of growth inhibition was calculated using the
following formula; Inhibition (%) = A-B /A x 100 (A-Control
group and B-Treated group).
http://www.who.int/mediacentre/factsheets/fs194/en/
2. A.P. Magirokaos, A. Srinivasan, R.B. Carey, Y. Carmeli, M.E.
Falagas, C.G. Giske, S. Harbarth, J.F. Hindler, G. Kahlmeter,
B. Olsson-Liljequist, D.L. Paterson, L.B. Rice, J. Stelling, M.J.
Struelenes, A. Vatopoulos, J.T. Weber, and D.L. Monnet,
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277.
Molecular docking study
5. P.M. Bennett, Br. J. Pharmacol., 2008, 153, S347.
Molecular docking studies have been done using the AutoDock
Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1
docking program. The DNA topoisomerase IV and ALK
structures were obtained from the Protein Data Bank (PDB ID:
4EMV and 2XP2). The co-crystallized ligands in the receptors
were removed. Then, the polar hydrogen atoms were added,
6. R. Siegel, D. Naishadham and A. Jemal, CA Cancer J. Clin.,
2013, 63, 11.
7. A. Bagnato, M. Loizidou, B.R. Pflug, J. Curwen and J.
Growcott, Brit. J. Pharmacol., 2011, 163, 220.
8. D. Tamkus, A. Al-Janadi, G. Fink, G. Krishnan and N.V.
Dimitrov, J. Cancer Mol., 2009, 4, 163.
12 | J. Name., 2012, 00, 1-3
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ARTICLE
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DOI: 10.1039/C5RA18618H
Synthesis of Novel 1,4-Disubstituted 1,2,3-Triazolo-Bosentan Derivatives –
Evaluation of Antimicrobial, Anticancer activities and Molecular Docking
K. Easwaramoorthi,a A. Jeya Rajendran,*,a K. ChennakesavaRao,b Y. Arun,b C. Balachandran,c
P. T. Perumal,b Nobuhiko Emi,c S.M. Mahalingam,d V.Duraipandiyan,e,f N.A. Al-Dhabif
aDepartment of Chemistry, Loyola College, Chennai-600034, India, Phone: +91 944-411-6528,
Email: jeyaadmaterial@gmail.com
bOrganic Chemistry Division, CSIR-CLRI, Chennai-600020, India
cDepartment of Hematology, Fujita Health University,
1-98, Dengakugakubo, Kutsukake-cho, Toyoake,Aichi 470-1192, Japan.
dCentre for Drug Discovery and Department of Chemistry, Purdue University, Indiana
efDivision of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai-600034, India
fDepartment of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King
Saud University,P.O.Box.2455, Riyadh-11451, Saudi Arabia.
N
N
N
O
O
N
O
O
N
N
N
N
OH
i. Chlorination
N
N
S
N
S
ii. Azidation
O
O
O
NH
O
NH
O
iii. CuAAC
O
1,3-dipolarcycloaddition
one pot synthesis
O
O
2
O2N
A549 - 68.2% activity at 7.81µg/mL
2XP2 - 76.4% activity at7.81µg/mL
1i
*One pot synthesis with good yeids
*Good antimicrobial activity against 4EMV receptot
*Prominet anticancer activity against A549 and SKOV-3 cellines
Docking mode of most active
*Significant in vitro cytotoxicity at 7.81μg/mL
compound 1h with 2XP2 recptor