Sn(II)-Mediated facile approach for the synthesis of 2-aryl-2: H -indazole-3-phosphonates and their anticancer activities

Article abstract of DOI:10.1039/c7nj00843k

A convenient three-component method was developed for the synthesis of various 2-aryl-2H-indazole-3-phosphonates using SnCl₂·2H₂O under conventional heating techniques. The transition metal free reaction, mild reaction conditions, one-pot operation, open flask conditions, wide substrate scope and good yields are a few important features of this methodology. This protocol proceeds with high atom economy via the formation of α-aminophosphonates followed by the generation of an indazole ring through N-N bond formation, eliminating water as a by-product. The cytotoxic properties of the 2-aryl-2H-indazole-3-phosphonate derivatives were investigated against A549 and HepG-2 cells. Interestingly, compounds 4d and 4g showed potent cytotoxic properties against HepG-2 cells and compound 4p showed significant cytotoxic properties against A549 cells. Intracellular visualization was done using a laser scanning confocal microscope. DNA fragmentation, colony formation, and apoptosis studies, flow cytometry and western blot analysis for 4p against A549 cells have also been reported.

Full text of DOI:10.1039/c7nj00843k

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Sn(II)-mediated facile approach for the synthesis of 2-aryl-2H-
indazole-3-phosphonates and their anticancer activity
N. Sudhapriya,ac C. Balachandran,b S. Awaleb and P. T. Perumal*a
Received 00th January 20xx,
Accepted 00th January 20xx
A convenient three-component method was developed for the synthesis of various 2-aryl-2H-indazole-3-phosphonates
using SnCl2.2H2O under conventional heating techniques. Transition metal free reaction, mild reaction conditions, one-pot
operation, open flask condition, wide substrate scope and good yields are few important features of this methodology.
This protocol proceeds with high atom economy via the formation of α-aminophosphonates followed by the generation of
indazole ring through N-N bond formation eliminating water as a by-product. Cytotoxic properties of 2-aryl-2H-indazole-3-
phosphonate derivatives were investigated against A549 and HepG-2 cells. Interestingly, compound 4d and 4g showed
potent cytotoxic properties against HepG-2 cells and compound 4p showed significant cytotoxic properties against A549
cells. Intracellular visualization was done by using a laser scanning confocal microscope. DNA fragmentation, colony
formation, apoptosis studies, flow cytometry and western blot analysis of 4p against A549 cells have also been reported.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
H3CHN
HO
O
O
Me
From an ecological perspective, the multicomponent reactions
(MCRs) are found as a powerful tool that allow multiple bond
formation events taking place in a single vessel, and exhibit
economy of steps and most often atom economy, selectivity
and low levels of by-product generation.1 Nitrogen-containing
heterocycles are an important class of organic compounds and
in fact, they are widely distributed in nature and are
ubiquitous structural motifs as they occur in numerous natural
substances, compounds of pharmaceutical interest and
commodity chemicals.2 Therefore the need of green and
sustainable methods for the highly efficient synthesis of these
molecules with important biological activities has become a
topic of interest for organic chemists.3
N
F
N
N
N
Me
Me
HN
S
N
Me
N
N
N
O
H3C
Cl
O
N
Cl
Viral polymerase inhibitor
NH2
N
S
anti-cancer agent (Ionidamine)
O
H
O
VEGF inhibitor (pazopanib)
O
P
O
P
R1O
R1O
OR1
OR1
NEt
N
N
R2
N
N
Our designed molecules
Me
OH
R
OHC
HO
R
N
H
Indazole ring system display a broad range of biological
properties, such as anticancer,4 anti-inflammatory,5
antipyretic,6 anti-HIV7 and contraceptive8 activities. For
example, drugs like pazopanib (VEGF inhibitor),9 lonidamine
(anti-cancer agent)10 and viral polymerase inhibitor11
N
H
glybomine B R = OH
glybomine C R = OMe
heptaphylline R = H
7-methoxyheptaphylline
Fig. 1 Selected examples of bio-active scaffolds and our designed molecules
incorporate this basic scaffold. Owing to its biological method for synthesis of 2H-indazole via halogen-bond-assisted
importance, synthesis of 2H-indazoles through efficient benzyl C–H functionalization using I2.13a Lee and co-workers
strategies remains the most challenging task for the organic developed the one-pot method for the synthesis of 2H-
chemist in recent years (Fig. 1).12 Xi et al reported an efficient
indazoles via palladium-catalyzed deacylative cross-coupling
and denitrogenative cyclization of 2-iodoazoarenes and 2-
iodoaryltriazenes with acyldiazoacetates.13b Yang et al
a.Organic Chemistry Division, CSIR-Central Leather Research Institute, Adyar,
described an efficient protocol for the synthesis of
Chennai 600 020, India.
b.Division of Natural Drug Discovery, Department of Translational Research,
Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-
0194, Japan.
indazolo[2,3-a]quinoline derivatives via ruthenium catalyzed
and viscible-light-mediated photocatalysis (Fig. 2).13c
c. Department of Chemistry, Sri Shakthi Institute of Engineering & Technology,
Coimbatore, Tamil Nadu 641062.
Furthermore, organophosphorous compounds are found in an
array of clinically useful drugs14 hence they have become the
subject of immense interest in recent years.15 Especially,
†Electronic Supplementary Informaꢀon (ESI) available:1H, 13C and 31
NMR spectra of compounds.CCDC 1444407 (4f).For ESI and crystallographic data in
CIF or other electronic format see DOI:
P
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aminophosphonate subunit incorporated molecules could then purified by column chromatography and then subjected
effectively improve the anticancer activity.16
to reductive cyclization using an eco-friendly SnCl2.2H2O as a
reducing agent and catalyst under conventional heating
techniques in EtOH.27 To our delight it afforded the cyclic
product 4f in 91% yield. It was isolated as a white solid on
completion of the reaction as indicated by TLC, followed by
work-up and purification by column chromatography. The
1
product was characterized by H and 13C NMR spectroscopy
and HRMS analysis. Then we have checked feasibility of the
Kabachnik-Fields reaction and reductive cyclization in one-pot
fashion. To the merit of our significantly developed method
the desired product (89%) was obtained without any
considerable change in the yield.
Table 1.Optimization of reaction conditions for the preparation of 4f
O
NH2
MeO
i) 80 °C, 3 h
ii) Conditions
P
OMe
O
CHO
NO2
P
H
N
OMe
MeO
OMe
N
Fig.2 Some typical methods for the synthesis of indazoles
OMe
4f
1a
3a
2c
Transition-metal catalyzed reactions have become an essential
tool for the formation of indazole scaffolds,17 however a main
weakness of this strategy is associated with removal of the
metal residue.18 Hence, reaction protocols that enable the
formation of these compounds in the absence of transition
metals are fascinating and considered sustainable.19 Based on
state-of-the-art techniques, we are currently interested in
developing greener approach for the synthesis of nitrogen-
containing heterocycles.20 Nowadays, N-N bond forming
reactions has become an intriguing task for the researchers.21
Shi et al described an efficient synthesis of 2H-indazoles via
reductive cyclization of 2-nitrobenzylamines using SnCl2•2H2O
and they have also used low-valent titanium reagent (TiCl4/Zn)
for their further studies.22-23 In this context, we previously
reported the synthesis of 2H-indazole derivatives through N-N
bond formation using SnCl2•2H2O as eco-friendly catalyst.24
Considering biological importance of indazole moiety and in
connection with our efforts to develop new methods for the
synthesis of phosphorus-containing compounds,25 we herein
reveal the first example of a one-pot preparation of diverse 2-
aryl-2H-indazole-3-phosphonates and their preliminary
cytotoxicity studies. To the best of author’s knowledge, there
are no reports on the one-pot synthesis of novel 2-aryl-2H-
indazole-3-phosphonates through SnCl2•2H2O mediated
methodology and their anticancer activity.
Feed
ratioa
1:2
1:3
1:3
1:4
1:5
1:4
1:4
1:4
1:4
1:4
1:4
1:2
1:4
Entry
Reducing agent
Time (h)
Yieldb(%)
1
2
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, EtOH
SnCl2.2H2O, MeOH
SnCl2.2H2O, HCl-EtOH
SnCl2.2H2O, HCl-EtOH
2
2
4
2
2
3
4
1
2
2
2
2
2
60
72
76
89
81
87
85
87c
85c
88d
84
59
62
3
4
5
6
7
8
9
10
11
12
13
aRatio between the reactant and reducing agent.b Isolated yield after column
c
chromatography and reactions carried out using 3 mL of EtOH and at 70 °C.
Reactions carried out at 80°C. d 5 mL of EtOH was used
To optimize the reaction conditions, further reactions were
carried out by varying amount of reagent, solvent,
temperature and time. In order to make our protocol greener,
we have used EtOH as the reaction medium. First, we have
optimized the feed ratio of reagent to substrate using EtOH
(Table 1). The use of 2 equiv. of SnCl2.2H2O produced the
cyclized product 4f in 60% yield (Table 1, entry 1). The yield of
the product 4f increased as the ratio was increased from 1:2 to
1:4 (Table 1, entries 1-4). However, further increase of ratio to
1:5 failed to improve the yield of the product 4f (Table 1, entry
5). Moreover, we also examined the time factor which
influences the yield of the product. Best results were obtained
by the use of 4 equiv. of SnCl2.2H2O resulted in the formation
of product 4f in 89 % yield in 2 h (Table 1, entry 4) (monitored
by TLC). Further increment of time did not improve the yield
(Table 1, entries 6-7). To further optimize reaction conditions,
a similar test was carried out with different temperatures
(Table 1, entries 8-9), the reaction rate was increased at an
Result and Discussion
Chemistry
In our continuing effort to exploit the formation of 3-
substituted-2H-indazole, we envisioned the development of an
efficient protocol for the synthesis of 2-aryl-2H-indazole-3-
phosphonates. At the outset, a model reaction was performed
for the synthesis of α-aminophosphonate from 2-
nitrobenzaldehyde 1a (1.0 mmol), p-anisidine 2c (1.0 mmol)
and dimethylphosphite 3a (1.5 mmol) by following the
reported literature procedure.26 The obtained product was
2 | J. Name., 2012, 00, 1-3
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elevated temperature (80 °C) without significantly improving On studying the reaction scope we next devoted our efforts by
the yield. Change of solvent quantity neither decreased the varying the 2-nitrobenzaldehydes to synthesize 2-aryl-2H-
reaction time nor improved the yield of the product (Table 1, indazole-3-phosphonates analogs (Table 3). A reaction of 1b
entry 10). The reaction was also carried out using MeOH as a bearing a two methoxy group as the electron-donating group
reaction medium but the yield was not improved remarkably on the 2-nitrobenzaldehyde with 2c or 2e and 3a at 70 °C for
(Table 1, entry 11). Finally, to improve solubility the reaction 2h provided the desired product 4n and 4o in 84% and 86%
was conducted using SnCl2.2H2O, HCl-EtOH. The system yield respectively. Similarly, treatment of 1c bearing O(-CH2)2
afforded the cyclized product 4f in lower yields (Table 1, with 2c or 2e and 3a afforded the corresponding products 4p
entries 12-13).
and 4q in 80 and 82% yield respectively. A combination of 1d
or 1e bearing a chloro or bromo group with 2e and 3a resulted
in the corresponding products 4r and 4s in 77 and 79% yield
respectively.
Table 2.Exploring the scope of aromatic amines for the synthesis of 2-aryl-2H-indazole-
3-phosphonates (4a-m)a
O
i) 80 °C, 3 h
ii) SnCl2.2H2O
O
NH2
R3
O
P
O
Table 3. The scope of 2-nitrobenzaldehyde for the synthesis of compounds (4n-s)a
OR3
R2
P
H
H
EtOH, 70 °C, 2 h
R3O
OR3
N
O
NO2
N
i) 80 °C, 3 h
R2
2a-l
MeO
O
NH2
P
ii) SnCl2.2H2O
EtOH, 70 °C, 2 h
4a-m
OMe
1a
3a,b
O
R3
H
P
H
N
MeO
OMe
N
NO2
R3
R2
1b-e
2c,e
3a
4n-s
[a] Isolated yield after column chromatography
Considering the general applicability of this reaction using
various substituted arylamines, dialkyphosphites and 2-
nitrobenzaldehydes, the potential use of carbazolamine to
afford the complex and diverse 2-aryl-2H-indazole-3-
phosphonates derivatives was next examined (Table 4). A
[a] Isolated yield after column chromatography
With the reductive cyclization conditions optimized, a series of
2-aryl-2H-indazole-3-phosphonates were prepared to examine
the scope of the methodology. The reaction showed a broad
substrate scope and it allowed the employment of a variety of
substituents in aromatic amines (o-, m- and p-substituted)
bearing electron donating and electron withdrawing groups
(Table 2). Generally, substrates with an electron rich group,
such as -Me and -OMe on the benzene rings, provided the
combination of 1a with 3a or
afforded the corresponding
phosphonates 4t and 4u in 85 and 87% yield respectively. The
reaction of 1c 2m with 3a provided the desired products 4v
in 76-82% yield. The results are depicted in the Table 4. All
b
and 2m at 70 °C for 2 h
2-aryl-2H-indazole-3-
-e
,
-
x
1
the prepared compounds (4a-x) were well characterized by H
and 13C NMR and mass techniques. The structure of
compounds 4a-x is in accord with elemental analysis, IR, NMR
(1H, 13C and 31P) and mass spectral data as illustrated for a
representative example 4f. The H NMR spectrum of product
corresponding products in high yield (Table 2, 4b
-f
) when
1
compared to the unsubstituted aromatic amine (Table 2, 4a).
Conversely, using substrate with a bulk group (tBu) on the
phenyl ring (2,5-di-tert-butylaniline) was tolerated rather well,
providing 4g in 78% yield. The decrease in the yield of the
product 4g might be due to the steric effect (Table 2, 4g).
4f showed a characteristic doublet absorption peak at the
chemical shift 8.05 ppm, which was assigned to the hydrogen
adjacent to the N-atom in the indazole ring which confirmed
the formation of cyclized product 4f (Fig.
on S6†). In 13C NMR
spectrum of 4f, the doublets due to C-P coupling at the
chemical shift values δ (ppm) 122.6 (d, J = 220.0 Hz), 128.3 (d, J
= 18.6 Hz), 148.6 (d, J = 16.3 Hz) confirmed the formation of
dimethyl(2-(4-methoxyphenyl)-2H-indazol-3-yl)phosphonate 4f
Notably, the product yields (4h
decreased when halogen substituents (F, Cl or Br) were
introduced in the substrates (Table 2, 4h ). The electron
–l) of the reactions were slightly
-l
(Fig
confirms the formation of the product 4f (Fig
.
on S6†). The 31P NMR of the purified compound further
withdrawing (-CF3) substituent provided the desired product in
68% yield (Table 2, 4m). It is evidenced by the fact that
electron-rich arylamines afforded relatively higher yields than
that of electron deficient analogs.
.
on S25†). High
resolution mass spectrum of compound 4f also showed peak
at m/z 355.0820 which corresponded to [M+Na]+(Fig. on S29†).
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The structural evidence could be secured from X-ray analysis On the basis of the above control experiments and reported
performed for a representative compound 4f (Fig. 3).28
literature,22,29 a tentative mechanistic proposal for the
formation of representative indazole phosphonate 4f is
described in Scheme 1. Presumably, this transformation is
triggered by the less basic anisidine 2c which manifest proton-
Table 4 .Synthesis of 2-aryl-2H-indazole-3-phosphonates (4t-x)a from carbazolamine
donor property and forms a pre-reaction complex
phosphate 3b. Intermediate presumably adds with 2-
nitrobenzaldehyde 1a results in an imine
A with the
A
B
hydrophosphorylation of which gives the α-aminophosphonate
5f. The nitro group of α-aminophosphonate was reduced to
form nitroso compound C in the presence of SnCl2.2H2O. Then
the nucleophilic attack of amine to the reduced nitroso group
followed by elimination of water produced the desired product
4f.
O
O
MeO
O
P
EtO
OMe
P
MeO
OEt
P
O
O
OMe
NEt
N
NEt
N
NEt
N
N
N
N
Pharmacology
4v
, 82%
4u
4t
, 85%
, 87%
Cytotoxic properties
O
The synthesis of a phosphonate core containing a heterocyclic
scaffold is a very attractive field of research due to its
pharmacological importance.30 The synthesized compounds
were tested against A549 and HepG-2 cells at 24 h using MTT
assay and they showed moderate to good cytotoxic
properties.The cytotoxic activity of the compounds against
A549 and HepG-2 cells were observed at 100 to 12.5 μg/mL
concentration. In general compounds with electron donating
substituents show better activity than the other compounds.
Interestingly, among all the tested compounds the -Me and -
tBu substituted compounds 4d and 4g showed very high
activity 52.9 % and 58.1 % with IC50 value at 12.5 μg/mL. In
particular, among the tested compounds 4a and 4o showed
MeO
O
P
OMe
MeO
P
Br
OMe
Cl
NEt
N
NEt
N
N
N
4x
4w
, 79%
, 76%
[a] Isolated yield after column chromatography
very good activity 57.6
%
(for each) at 25 μg/mL
4e 4f
concentrations against HepG-2 cell line. Compounds 4c
,
,
and 4p appeared to be next active series with IC50 value at 50
μg/mL (Fig. 4).
100
12.5 µg/mL
25 µg/mL
50 µg/mL
100 µg/mL
80
60
40
20
0
Fig.3 ORTEP diagram of compound 4f
DMSO 4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
Fig. 4 Anticancer activity of synthesized compounds (4a
Positive control Cisplatin 21.5±0.032 µM (IC50
-t) against HepG-2 cells.
)
Analysis of the screening data (Fig. 5) revealed that compound
4p possessing -OMe and -Me groups, exhibited maximum
cytotoxicity 51.2 % at 12.5 μg/mL concentration against A549
lung adenocarcinoma cancer cell line compared to other
analogues. Compounds 4d and 4g appeared to be the second
most active series with IC50 value 25 μg/mL. A moderate
inhibitory potency was observed in compound 4o at 44.40
μg/mL (IC50) in A549 cells. However, compounds 4a
, 4c, 4f, 4j,
4m, 4n, 4q and 4s showed least activity against A549 exhibiting
IC50 values at 97.46 μg/mL, 89.72 μg/mL, 82.82 μg/mL, 96.52
μg/mL, 88.18 μg/mL, 99.40 μg/mL, 92.92 μg/mL and 85.32
μg/mL respectively. The concentrations that are used in the
Scheme 1 Mechanism for the formation of 4f
4 | J. Name., 2012, 00, 1-3
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experiment decreased the cell viability significantly (P<0.05) in and nuclear condensation were found after treatment with 4p
concentration dependent manner. The synthesized against A549 cells, 4d and 4g treatment against HepG-2 cells
compound 4p showed no toxicity against Vero cells up to 2 under confocal microscopic image respectively (Fig. 6 & 7).
a
mg/mL.
FITC stain showed membrane level changes, DAPI and PI stain
showed fragmentation and condensation of nuclei.
100
12.5 µg/mL
25 µg/mL
50 µg/mL
100 µg/mL
80
60
40
20
0
DMSO 4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
Fig.5 Anticancer activity of synthesized compounds (4a-t) against A549 cells.
Positive control Cisplatin 18±0.059 µM (IC50)
Fig. 7 Apoptosis analysis was confirmed by fluorescence staining method in A549
cells using different probes such as FITC (Fluorescein Isothiocyanate), PI
(Propidium Iodide) and DAPI (4',6-diamidino-2-phenylindole). DAPI (Blue color),
FITC (Green color), PI (Red color) and merged image. A549 control cells (a1-DAPI,
a2-FITC, a3-PI & a4-merged) and 4p (12.5 µg/mL: b1-DAPI, b2-FITC, b3-PI & b4-
merged). Notably, our results indicated that after treatment with 4p exhibited
significant membrane level changes, fragmentation and condensation of nuclei
when compared to the control cells.The fluorescent signals of DAPI, FITC and PI
were examined under confocal laser scanning microscope, Zeiss, 2009.Arrows
indicate apoptotic cells.
Confocal microscopy studies
DNA fragmentation
DNA fragmentation was demonstrated by incubating A549
cells with 12.5 µg/mL concentration of 4p for 24 h. DNA
fragmentation became apparent with 12.5 µg/mL compound
treatment and this response was dose dependent. When cells
were treated with 12.5 µg/mL DNA ladders were visible from
24 h after treatment (Fig. 8).
Fig. 6 Apoptosis analysis was confirmed by fluorescence staining method in
HepG-2 cells using different probes such as FITC (Fluorescein Isothiocyanate), PI
(Propidium Iodide) and DAPI (4',6-diamidino-2-phenylindole). DAPI (Blue color),
FITC (Green color), PI (Red color) and merged image. HepG-2 control cells (a1-
DAPI, a2-FITC, a3-PI & a4-merged), 4d (12.5 µg/mL: b1-DAPI, b2-FITC, b3-PI & b4-
merged) and 4g (12.5 µg/mL: c1-DAPI, c2-FITC, c3-PI and c4-merged).Notably,
our results indicated that after treatment with 4d and 4g exhibited significant
membrane level changes, fragmentation and condensation of nuclei when
compared to the control cells. The fluorescent signals of DAPI, FITC and PI were
examined under confocal laser scanning microscope, Zeiss, 2009.Arrows indicate
apoptotic cells.
We used fixation technique for confocal images to
identification of apoptotic cells. A549 (4p-12.5 µg/mL) and
HepG-2 (4d and 4g - 12.5 µg/mL) cells were treated and
subjected to confocal microscopic studies.In this study, we
used DAPI, FITC and PI fluorescence probes. FITC acts as a
phosphatidyl serine tracer and suggests the presence of
apoptosis. DAPI binds strongly to A-T rich regions in DNA and
pass through an intact cell membrane. PI can penetrate cells Fig. 8. Detection of DNA fragmentation by agarose gel electrophoresis (2%). The
apoptotic DNA fragmentation was detected by agarose gel electrophoresis in
where the cell membrane has been compromised. Our results A549 cancer cells treated with compound 4p for 24 h. lane 1-1kb DNA ladder,
lane2-control, lane 3-DMSO (0.5%) and 12.5 µg/mL.
showed that significant morphological changes, fragmentation
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Colony formation assays
24 h there was dramatic increase in the apoptosis effect on
A549 cells when compared to the control cells (Fig. 10). The
12.5 µg/mL showed 31.7% of apoptosis; 25 µg/mL showed
44.8 % of apoptosis and 50 µg/mL showed 64.4 % of apoptosis
in A459 cells for 24 h. As the concentration of 4p increased
early, late and dead apoptotic cells were gradually increased
when compared to the control cells.
To determine the potential anticancer effect of 4p in A549
cells, in vitro colony formation assays were undertaken. A549
cells were treated with 4p at concentrations of 12.5 and 25
µg/mL. As shown in Fig. 9, 12.5 and 25 µg/mL concentrations
in 10 days incubation, significantly increasing the colony
forming ability when compared to the control.
Western blotting
Mostly p53 is a tumor suppressor protein in all cancers. This
protein does not directly take part in the apoptosis pathway;
however, it regulates a host of other genes that lead to cell
arrest or apoptosis. Because the apoptotic function of p53 is
critical for tumor suppression, reconstitution of inactive p53
dependent pathways is an appealing approach currently being
explored for anticancer treatment.31 Several apoptotic stimuli
provoke a fast translocation of p53 to the outer mitochondrial
membrane.32 In the present study pro-apoptotic pathway
associated with compound 4p treatment, namely the
expression of p53 in A549 cells treated with 12.5 and 25 μg/mL
concentration for 24 h, was tested by western blotting
analysis. Compared to the control, cells treated with
compound 4p were strongly up-regulated at protein level (Fig.
11).
Fig. 9 Effect of colony formation assay with the synthesized compound 4p
against A549 cells. A549 cells were treated with or without compound 4p and
number of colonies were counted 2 weeks later after cells being stained with
crystal violet. Data shown are representatives of three separate experiments.
Apoptosis analysis using flow cytometry
Fig. 10 A549 cells were treated with 12.5, 25 and 50 µg/mL of 4p for 24 h and
apoptosis was confirmed by flow cytometry analysis (annexin V-FITC/PI kit). The
results showed that compound 4p dramatically increased the apoptosis when
compared to the control cells. Annexin V-positive cells were considered as
apoptotic cells. Data are mean ± SD of three independent experiments with each
experiment conducted in triplicate. R2 = live cells; R3 = Early apoptotic cells; R4 =
Late apoptotic cells; R5 = dead cells.
Fig. 11 A549 cells were treated with 4p (12.5 and 25 µg/mL) and proteins were
extracted with using RIPA buffer. The extracted proteins were subjected to
western blot analysis to identification of apoptosis involving genes. We used
following genes for protein expression study such as p53, Bax, cytochrome-c,
caspase-3 and Bcl-2. Our results indicated that p53, Bax, cytochrome-c, caspase-
3 were increased dramatically and Bcl-2 was down regulated in A549 cells
following treatment of 4p for 24 h. GAPDH is a control.
The cell populations are analyzed by different phases such as
R2 (living cells), R3 (early apoptosis cells), R4 (late apoptosis
cells) and R5 (dead cells). In this study, we used different
concentrations of 4p such as 12.5, 25 and 50 µg/mL for 24 h to
analyze the apoptosis effects. Apoptotic cells were measured
by annexin V-FITC/PI staining assay as per manufacturer’s
guidelines. The results showed that after treatment with 4pfor
Bcl-2 is mainly localized in the outer mitochondrial membrane.
Bcl-2 also plays a vital role in regulating response to
chemotherapy, hormonal therapy and irradiation. In addition,
over expression of Bcl-2 may cause the accumulation of cells in
6 | J. Name., 2012, 00, 1-3
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the G0 phase of cell cycle division and contribute to
chemoresistance. Moreover, Bcl-2 members act as repressors
of apoptosis by blocking the release of cytochrome c, while
proapoptotic members act as promoters. These effects are
more dependent on the balance between Bcl-2 and Bax than
on Bcl-2 quantity alone.33 BH3 molecules need multi domain
BH3 proteins such as Bax and Bak to exert their intrinsic
proapototic activity. In the present study, we determine the
signal pathway of A549 cell apoptosis by synthesized
compound 4p, following the incubation of compound 4p with
the 12.5 and 25 μg/mL concentrations for 24 h, protein
expression of Bcl-2, Bax, cytochrome c and caspase-3
Experimental section
Chemistry
All reagents and solvents that are used were purchased from
commercial suppliers (Aldrich and Merck) and used without
further purification. All reactions were performed in reaction
vessels with magnetic stirring in open flask and no special
precautions were taken to exclude air from the reaction
vessels. The reactions were monitored by thin layer
chromatography using Merck silica gel 60 F254 and visualized
by UV light detection. Analytical TLC was performed on pre-
activation were confirmed by western blotting analysis. Bcl-2 coated aluminium sheets of silica gel 60F254 of 0.2 mm
protein was significantly down regulated after treatment with
compound 4p at the concentrations of 12.5 and 25 μg/mL for
24 h. The expressions of Bax pro-apoptotic gene was gradually
increased after treatment with compound 4p when compared
to the control (Fig. 11). Upon the induction of apoptosis, Bax
experiences a conformational change and translocates to the
mitochondrial membranes where it incorporates and mediates
the release of cytochrome c from the intermembrane space
into the cytosol.Cytochrome c released in the cytosol, interacts
with Apaf-1, leading to the instigation of caspase-9. Activated
caspase-9 then activates caspase-3, which subsequently
thickness (Merck, Germany). Column chromatography was
carried out on silica gel (100-200mesh), SRL. India. Melting
points were determined on Gallenkamp melting point
apparatus in capillaries and are uncorrected. Infrared (IR)
spectra were recorded on a Perkin Elmer FT-IR spectrometer
as KBr pellets and neat sample for liquids.1H NMR (400 MHz),
13C NMR (100 MHz) and 31P NMR (163 MHz) spectra were
recorded in CDCl3/TMS solution with TMS as internal standard
on a Bruker spectrometer. Mass spectra were recorded using
ESI/HRMS at 60000 resolutions in Thermo scientific Exactive
activates the rest of the caspase cascade and leads to mass spectrometer. Elemental analyses were recorded using a
apoptosis. Activated caspases cause the cleavage of nuclear
lamin and breakdown of the nucleus through caspase-
3.Moreover, our results revealed that the expression level of
cytochrome c increased after treatment with compound 4p in
A549 cells. The release of cytochrome c causes cleavage of
caspase-9 which contributes to the activation of caspase-3.
Compound 4p significantly increased cleavage of caspase-3 at
24 h. The increase of cleaved caspase-3 was linked with the
decrease of pro-caspase-3 (Fig. 11).
ThermoFinnigan FLASH EA1112 CHN analyzer.
General procedure for the preparation of indazole phosphonates
(4a
A mixture of 2-nitrobenzaldehyde 1a
aromatic amine 2a (1 mmol), dialkylphosphite 3a-b
-x)
-
e
(1 mmol), substituted
(1.5
-m
mmol), under a neat condition, without any catalyst were
heated at 80 °C for 3 h to yield α-aminophosphonates. To the
reaction mixture was added 3 ml of EtOH and 4 equiv. of
SnCl2.2H2O and heated at 70 °C for 2 h. After the completion of
reaction it was filtered through celite by washing with
ethylacetate and added water (50 mL) extracted with same
solvent (2x20 mL). The combined organic layer washed with
saturated NaOH, brine solution, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude material
was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate (5-20%) to afford the desired 2-
Conclusions
In conclusion, we have demonstrated an efficient and greener
one-pot SnCl2.2H2O mediated reductive cyclization protocol for
the synthesis of 2-aryl-2H-indazole-3-phosphonates. This
method is economical, practical, reliable and more
advantageous as it involves milder reaction conditions, broad
substrate scope, shorter reaction times, high yields and usage
of green solvent. This protocol allowed the high atom
economy and the formation of N-N bond in an open flask
operation affording two molecules of water as the side
product. Evaluation of in vitro anticancer activities towards
HepG-2 and A549 cell lines revealed that the compounds 4d
and 4g exhibited good anticancer properties against HepG-2
cell line and very promising anticancer activity was shown by
compound 4p against A549 lung cancer cell line. We further
aryl-2H-indazole-3-phosphonate 4a-x in good yield.
Diethyl(2-phenyl-2H-indazol-3-yl)phosphonate
(4a):Yellow
oily liquid; IR (Neat): 762, 816, 981, 1012, 1220, 1271, 1497,
1536, 2975, 3087 cm-1;1H NMR (400 MHz, CDCl3) δ 1.19 (t, J =
7.0 Hz, 6H), 3.94-4.04 (m, 2H), 4.05-4.14 (m, 2H), 7.28-7.33 (m,
1H), 7.37-7.43 (m, 1H), 7.48-7.56 (m, 3H), 7.65-7.72 (m, 2H),
7.83-7.88 (m, 1H), 8.13 (d, J = 8.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 16.1 (d, J= 6.8 Hz), 62.8 (d, J = 5.6 Hz), 118.2, 121.3,
123.7 (d, J = 217.8 Hz), 124.7, 126.4, 127.0, 128.4 (d, J = 18.5
Hz), 128.7, 129.5, 140.9, 148.7 (d, J = 16.1 Hz); HRMS (ESI): calc
for C17H19N2NaO3P [M+ Na]+ 353.1026, found 353.1040.
demonstrated
confocal
microscopic
studies,
DNA
fragmentation, western blot and flow cytometry analysis for
the potential compound 4p. The above results suggested that
4p exhibited significant anticancer activity against A549 cells
and it could be a potent candidate for the treatment of lung
cancer.
Diethyl(2-(m-tolyl)-2H-indazol-3-yl)phosphonate (4b): White
solid; mp: 80-82 °C; IR (KBr): 742, 829, 964, 1026, 1238, 1259,
1378, 1456, 1525, 2985, 3045 cm-1;1H NMR (400 MHz, CDCl3) δ
1.17-1.24 (m, 6H), 2.45 (s, 3H), 3.95-4.05 (m, 2H), 4.05-4.15 (m,
2H), 7.27-7.35 (m, 2H), 7.35-7.43 (m, 2H), 7.46-7.53 (m, 2H),
This journal is © The Royal Society of Chemistry 20xx
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7.82-7.88 (m, 1H), 8.12 (dt, J = 8.8, 1.0 Hz, 1H); 13C NMR (100 3H), 1.26 - 1.25 (2s, 6H), 3.56 (s, 3H), 3.59 (s, 3H), 7.29 (d, J =
MHz, CDCl3) δ 16.1 (d, J = 6.8 Hz), 21.3, 62.8 (d, J = 5.5 Hz), 8.0 Hz, 2H), 7.30-7.35 (m, 1H), 7.38-7.43 (m, 1H), 7.48 (t, J =
118.2, 121.3, 123.4, 123.6 (d, J = 218.2 Hz), 124.6, 126.9 (d, J= 7.8 Hz, 1H), 7.85-7.91 (m, 1H), 8.06 (d, J = 8.8 Hz, 1H);13C NMR
1.7 Hz), 128.3(d, J = 18.6 Hz), 128.5, 130.2, 138.8, 140.8, 148.6 (100 MHz, CDCl3) δ 22.3, 25.7, 28.7, 29.7, 52.9 (d, J = 6.1 Hz),
(d, J= 16.2 Hz); HRMS (ESI): calc for C18H21N2NaO3P [M+ Na]+ 114.8, 118.7, 120.9, 123.3, 123.7 (d, J = 192.2 Hz), 127.2 (d, J =
367.1182, found 367.1186.
16.3 Hz), 126.6, 130.4, 136.8, 146.3, 148.2 (d, J = 16.3 Hz); MS
White (ESI) for C23H32N2O3P: m/z 415 [M+H]+; Elemental analysis calc
Diethyl(2-(p-tolyl)-2H-indazol-3-yl)phosphonate(4c):
solid; mp: 83-84 °C; IR (KBr): 756, 826, 960, 1026, 1218, 1254, for C23H31N2O3P: C 66.65; H 7.54, N 6.76, O 11.58, P 7.47,
1362, 1449, 1512, 2926, 2980, 3066 cm-1; 1H NMR (400 MHz, found C 66.61, H, 7.60; N, 6.78; O, 11.55; P, 7.43.
CDCl3) δ 1.21 (td, J = 7.0, 0.4 Hz, 6H), 2.45 (s, 3H), 3.94-4.05 (m, Dimethyl(2-(3,4-difluorophenyl)-2H-indazol-3-
2H), 4.05-4.16 (m, 2H), 7.28-7.34 (m, 3H), 7.36-7.42 (m, 1H), yl)phosphonate(4h):Semi solid; IR (neat): 747, 815, 846, 1151,
1
7.52-7.58 (m, 2H), 7.82-7.88 (m, 1H), 8.11 (dt, J = 8.6, 0.9 Hz, 1228, 1467, 1551, 1638, 3052 cm-1; H NMR (400 MHz, CDCl3)
1H); 13C NMR (100 MHz, CDCl3) δ 16.08 (d, J = 6.8 Hz), 21.3, δ 3.73 (s, 3H), 3.76 (s, 3H), 7.29-7.37 (m, 2H), 7.40-7.44 (m,
62.78 (d, J = 5.5 Hz), 118.2, 121.3, 123. 6 (d, J = 217.8), 124.6, 1H), 7.46-7.52 (m, 1H), 7.55-7.56 (m, 1H), 7.82-7.88 (m, 1H),
126.1, 126.9, 128.3 (d, J = 18.7 Hz), 129.2, 138.5, 139.6, 148.6 8.03 (dt, J = 8.4, 1.0 Hz, 1H);13C NMR (100 MHz, CDCl3) δ 53.23
(d, J = 16.2 Hz); HRMS (ESI): calc for C18H21N2NaO3P [M+ Na]+ (d, J = 5.6 Hz), 103.9, 104.1, 110.2 (dd, J = 5.7, 3.2 Hz), 116.2
367.1182, found 367.1190.
(dd, J = 19.7, 1.8 Hz), 117.3 (dd, J = 18.2, 1.5 Hz), 119.7 (d, J =
255.5 Hz), 122.66 (dd, J = 6.8, 3.9 Hz), 125.4, 127.5, 128.4 (d, J
Dimethyl(2-(2,4-dimethylphenyl)-2H-indazol-3-
yl)phosphonate (4d): White solid; mp: 81-83 °C; IR (KBr): 751, = 18.3 Hz), 148.9 (d, J = 16.1 Hz); MS (ESI) for C15H14F2N2O3P:
832, 968, 1022, 1228, 1254, 1368, 1444, 1517, 2985, 3062 cm- m/z 339 [M+H]+; Elemental analysis calc for C15H13F2N2O3P: C
1; 1H NMR (400 MHz, CDCl3) δ 1.99 (s, 3H), 2.41 (s, 3H), 3.63 (s, 53.26, H 3.87, F 11.23, N 8.28, O 14.19, P 9.16, found C 53.24,
3H), 3.66 (s, 3H), 7.13 (d, J = 8.4 Hz, 1H), 7.17 (s, 1H), 7.21-7.27 H 3.83, F 11.26, N 8.32, O 14.17, P 9.12.
(m, 1H), 7.27-7.35 (m, 1H), 7.38-7.44 (m, 1H), 7.83-7.89 (m, Dimethyl(2-(2-chlorophenyl)-2H-indazol-3-
1H), 8.06 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 17.0, yl)phosphonate(4i): White solid; mp: 88-90 °C; IR (KBr): 678,
21.3, 53.0 (d, J= 5.7 Hz), 115.1, 118.4, 121.0, 123.7(d, J= 221.9 751, 796, 874, 1238, 1254, 1388, 1492, 1521, 1628, 2985, 3137
Hz), 124.7, 126.6, 126.7, 127.2, 127.5(d, J = 18.9 Hz), 131.3, cm-1;1H NMR (400 MHz, CDCl3) δ3.71 (s, 3H), 3.74 (s, 3H), 7.28-
135.3, 137.3, 140.1, 148.5 (d, J = 16.2 Hz); MS (ESI) for 7.35 (m, 1H), 7.38-7.46 (m, 1H), 7.46-7.54 (m, 2H), 7.58-7.64
C17H20N2O3P: m/z 331 [M+H]+; Elemental analysis calc for (m, 1H),7.72 (t, (m, 1H),7.72 (t, J = 2.0 Hz, 1H), 7.83-7.88 (m,
C17H19N2O3P: C 61.81, H 5.80, N 8.48, O 14.53, P 9.38 found C 1H),8.05 (dt, J = 2.0 Hz, 1H), 7.83-7.88 (m, 1H),8.05 (dt, J = 8.8,
61.87, H 5.84, N 8.4, O 14.55, P 9.37.
1.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 53.20 (d, J = 5.7 Hz),
Diethyl(2-(4-methoxyphenyl)-2H-indazol-3-yl)phosphonate
113.2,114.9,118.40,121.0,122.7
(d,
(d,
J
=
218.7
18.2
(4e): White solid; mp: 93-95 °C; IR (KBr): 767, 848, 951, 1034, Hz),124.5,125.2,126.6,127.4,128.5
J
=
1
1226, 1267, 1354, 1445, 1524, 2926, 2980, 3049 cm-1; H NMR Hz),129.8,130.3,134.5,141.7, 148.9 (d, J = 16.3 Hz); MS (ESI) for
(400 MHz, CDCl3) δ 1.23 (t, J = 7.2 Hz, 6H), 3.89 (s, 3H), 3.95- C15H15ClN2O3P: m/z337 [M+H]+; Elemental analysis calc for
4.05 (m, 2H), 4.05-4.15 (m, 2H), 6.99-7.05 (m, 2H), 7.27-7.32 C15H14ClN2O3P:C 53.51, H 4.19, Cl 10.53, N 8.32, O 14.26, P
(m, 1H), 7.36-7.44 (m, 1H), 7.56-7.62 (m, 2H), 7.80-7.88 (m, 9.20, found C 53.55, H 4.16, Cl 10.57, N 8.29, O 14.23, P 9.26.
1H), 8.10 (dt, J = 8.4, 0.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ Diethyl(2-(4-chlorophenyl)-2H-indazol-3-yl)phosphonate (4j):
16.1 (d, J = 6.8 Hz), 55.6, 62.8 (d, J = 5.5 Hz), 113.8, 118.2, White solid; mp: 84-86 °C; IR (KBr): 751, 776, 874, 1148, 1216,
121.3, 123.6 (d, J = 218.0 Hz), 124.5, 124.8, 126.8, 127.5, 128.2 1382, 1495, 1520, 1628, 2985, 3132 cm-1;1H NMR (400 MHz,
(d, J = 18.6 Hz), 134.0, 148.5 (d, J = 16.1 Hz), 160.3; 31P NMR CDCl3) δ 1.24 (t, J = 7.0 Hz, 6H), 3.97-4.08 (m, 2H), 4.08 - 4.18
(163 MHz, CDCl3) δ 6.8; HRMS (ESI): calc for C18H21N2NaO4P (m, 2H), 7.27 - 7.33 (m, 1H), 7.38 - 7.44 (m, 1H), 7.48 - 7.54 (m,
[M+ Na]+ 383.1131, found 383.1128.
2H), 7.62-7.68 (m, 2H), 7.82-7.88 (m, 1H), 8.09 (d, J = 8.4 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 16.1 (d, J = 6.6 Hz), 62.9 (d, J
Dimethyl(2-(4-methoxyphenyl)-2H-indazol-3-yl)phosphonate
(4f): White solid; mp: 110-113 °C; IR (KBr): 759, 848, 955, 1026, = 5.5 Hz), 118.3, 121.3, 123.8 (d, J = 217.2 Hz), 124.9, 127.2,
1
1232, 1254, 1351, 1443, 1522, 2926, 2980, 3052 cm-1; H NMR 127.6, 128.4 (d, J = 18.3 Hz), 128.9, 135.5, 139.4, 148.8 (d, J =
(400 MHz, CDCl3) δ 3.67 (s, 3H), 3.70 (s, 3H), 3.89 (s, 3H), 6.97 - 16.1 Hz); HRMS (ESI): calc for C17H18ClN2NaO3P [M+ Na]+
7.06 (m, 2H), 7.28-7.32 (m, 1H), 7.37-7.44 (m, 1H), 7.81-7.90 387.0636, found 387.0634.
(m, 1H), 8.05 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ Diethyl(2-(4-bromophenyl)-2H-indazol-3-yl)phosphonate (4k):
53.1 (d, J = 5.6 Hz), 55.6, 113.9, 118.3, 121.0, 122.6 (d, J= 220.0 White solid; mp: 87-89 °C; IR (KBr): 772, 759, 825, 1176, 1228,
Hz), 124.8, 126.9, 127.4, 128.3 (d, J = 18.6 Hz), 133.8, 148.6 (d, 1376, 1489, 1565, 1645, 2994, 3098 cm-1;1H NMR (400 MHz,
J = 16.3 Hz), 160.4; 31P NMR (163 MHz, CDCl3) δ 9.8. HRMS CDCl3) δ 1.24 (t, J = 7.0 Hz, 6H), 3.98-4.08 (m, 2H), 4.08-4.18
(ESI): calc for C16H17N2NaO4P [M+ Na]+ 355.0818, found (m, 2H), 7.27-7.33 (m, 1H), 7.38-7.45 (m, 1H), 7.56-7.61 (m,
355.0820.
2H), 7.63-7.70 (m, 2H), 7.78-7.89 (m, 1H), 8.09 (d, J = 8.8 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 16.1 (d, J = 6.7 Hz), 63.0 (d, J
Dimethyl(2-(2,5-di-tert-butylphenyl)-2H-indazol-3-
yl)phosphonate (4g): Semi solid; IR (neat): 735, 756, 785, 820, = 5.6 Hz), 118.3, 121.3, 123.6, 126.1 (d, J = 233.8 Hz), 127.9,
975, 1125, 1217, 1383, 1526, 2979, 3094 cm-1;1H NMR (400 128.4 (d, J = 18.3 Hz), 131.9, 139.9, 148.8 (d, J = 16.0 Hz);
MHz, CDCl3) δ1.06 (s, 3H), 1.08 (s, 3H), 1.20 (s, 3H), 1.21 (s,
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HRMS (ESI): calc for C17H18BrN2NaO3P [M+ Na]+ 431.0131, 2H);13C NMR (100 MHz, CDCl3) δ 21.3, 52.9 (d, J = 5.5
found 431.0136.
Hz),94.4,96.1,101.4,121.5 (d, J = 220.0 Hz),125.4 (d, J = 18.9
Hz),125.8,128.9,129.3,129.7,138.4,139.3,146.1 (d, J = 16.8
Dimethyl(2-(4-iodophenyl)-2H-indazol-3-yl)phosphonate
(4l):Semi solid; IR (neat): 762, 813, 877, 1142, 1217, 1374, Hz),147.8, 149.6; HRMS (ESI): calc for C17H18N2O5P [M+ H]+
1492, 1525, 1623, 2981, 3113, 3410 cm-1;1H NMR (400 MHz, 361.0948, found 361.0951.
CDCl3) δ 3.66 (s, 3H), 3.69 (s, 3H), 7.33-7.29 (m, 1H), 7.39-7.43 Dimethyl(2-(4-methoxyphenyl)-2H-[1,3]dioxolo[4,5-f]indazol-
(m, 1H), 7.52-7.55 (m, 2H), 7.63-7.70 (m, 2H), 7.84-7.90 (m, 3-yl)phosphonate (4q): Semi solid; IR (neat): 723, 817, 931,
1H), 8.07 (dt, J = 8.4, 1.0 Hz, 1H);13C NMR (100 MHz, CDCl3) δ 1018, 1218, 1248, 1359, 1497, 2996, 3061 cm-1; 1H NMR (400
53.1 (d, J = 5.7 Hz), 118.4, 121.1, 122.7 (d, J = 219.3 Hz), 124.9, MHz, CDCl3) δ3.65 (s, 3H), 3.68 (s, 3H), 3.87 (s, 3H), 6.02 (s,
126.2, 127.0, 128.42 (d, J = 18.5 Hz), 128.8, 129.7, 140.8, 148.7 2H), 6.96-7.04 (m, 2H), 7.05-7.11 (m, 1H), 7.30 (d, J = 0.4 Hz,
(d, J = 16.3 Hz); MS (ESI) for C15H15IN2O3P: m/z429 [M+H]+; 1H), 7.49-7.56 (m, 2H);13C NMR (100 MHz, CDCl3) δ 29.7, 52.9
Elemental analysis calc forC15H14IN2O3P:C 42.08, H 3.30, I (d, J = 5.5 Hz), 55.5, 94.3, 96.1, 101.4, 113.3, 113.8, 114.6,
29.64, N 6.54, O 11.21, P 7.23,found C 42.13, H 3.33, I 29.66, N 114.9, 121.7 (d, J = 219.8 Hz), 125.3(d, J = 18.9 Hz), 127.2,
6.50, O 11.26, P 7.26.
133.9, 146.0 (d, J = 16.8 Hz), 147.8, 149.6, 160.1; MS (ESI) for
C17H18N2O6P: m/z377 [M+H]+; Elemental analysis calc
Dimethyl(2-(3-(trifluoromethyl)phenyl)-2H-indazol-3-
yl)phosphonate (4m):Semi solid; IR (neat): 763, 820, 1043, forC17H17N2O6P:C 54.26, H 4.55, N 7.44, O 25.51, P 8.23,found
1104, 1132, 1165, 1219, 1338, 1511, 1596, 3131, cm-1; 1H NMR C 54.20, H 4.58, N 7.49, O 25.46, P 8.27.
(400 MHz, CDCl3) δ 3.71 (s, 3H), 3.73 (s, 3H), 7.36-7.30 (m, 1H), Dimethyl(5-chloro-2-(4-methoxyphenyl)-2H-indazol-3-
7.47-7.40 (m, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.80 (d, J = 7.6 Hz, yl)phosphonate (4r) :White solid; mp: 104-105 °C; IR (KBr):
1H), 7.89-7.84 (m, 1H), 7.92 (d, J = 8.0 Hz, 1H), 8.00 (s, 1H), 685, 732, 758, 809, 929, 1041, 1236, 1377, 1528, 1595, 1626,
8.07 (dt, J = 8.4, 0.8 Hz, 1H);13C NMR (100 MHz, CDCl3) δ 53.13 3082, 3127 cm-1. 1H NMR (400 MHz, CDCl3) δ3.68 (s, 3H), 3.71
(d, J = 5.7 Hz), 118.4,121.1,122.0 (d, J = 30.1 Hz),123.5 (q, J = (s, 3H), 3.89 (s, 3H), 6.99-7.05 (m, 2H),7.34 (dd, J = 9.2, 2.0 Hz,
3.9 Hz),124.4 (d, J = 82.2 Hz),125.4,126.3 (q, J = 3.7 1H), 7.53-7.59 (m, 2H),7.79 (ddd, J = 9.1, 2.2, 0.4 Hz, 1H),8.06
Hz),127.5,128.6 (d, J = 18.2 Hz), 129.5,129.7,131.37 (d, J = 33.1 (dd, J = 1.8, 0.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 53.2 (d, J
Hz),141.1,149.0 (d, J = 16.2 Hz); MS (ESI) for C16H15F3N2O3P: = 5.6 Hz), 55.6,113.9,119.8 (d, J = 12.4 Hz),122.5 (d, J = 220.0
m/z 371 [M+H]+; Elemental analysis calc for C16H14F3N2O3P: C Hz), 127.3,128.5,128.8 (d, J = 18.2 Hz),130.7,133.5,146.9 (d, J =
51.90, H 3.81, F 15.39, N 7.57, O 12.96, P 8.37 found C 51.94, H 15.9 Hz), 160.5; HRMS (ESI): calc for C16H16ClN2NaO4P [M+ Na]+
3.87, F 15.32, N 7.56, O 12.99, P 8.34.
389.0428, found 389.0436.
Dimethyl(5,6-dimethoxy-2-(p-tolyl)-2H-indazol-3-
Dimethyl(5-bromo-2-(4-methoxyphenyl)-2H-indazol-3-
yl)phosphonate (4n):Semi solid; IR (neat): 755, 819, 1026, yl)phosphonate (4s):White solid; mp: 134-136 °C; IR (KBr):
1227, 1284, 1500, 1519, 1594, 2831, 3046 cm-1;1H NMR (400 683, 724, 757, 816, 1047, 1197, 1237, 1369, 1506, 3060 cm-1;
MHz, CDCl3) δ2.45 (s, 3H), 3.64 (s, 3H), 3.67 (s, 3H), 3.97 (s, 1H NMR (400 MHz, CDCl3) δ3.68 (s, 3H), 3.71 (s, 3H), 3.89 (s,
3H), 4.00 (s, 3H), 7.09 (d, J = 2.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 3H),6.99-7.06 (m, 2H),7.46 (dd, J = 9.2, 2.0 Hz, 1H), 7.53-7.59
2H), 7.33 (s, 1H), 7.46-7.54 (m, 2H);13C NMR (100 MHz, CDCl3) (m, 2H),7.73 (ddd, J = 9.1, 2.1, 0.5 Hz, 1H),8.25 (dd, J = 1.8, 0.6
δ 21.3, 52.9 (d, J = 5.4 Hz), 56.1 (d, J = 12.6 Hz), 96.0, 97.9, Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 53.2 (d, J = 5.6 Hz), 55.6,
120.7 (d, J = 156.7 Hz), 124.5 (d, J = 19.1 Hz), 125.8, 128.8, 113.9,118.8,119.9,122.4 (d, J = 220.1 Hz),123.3,127.3,129.5 (d,
129.3, 129.8, 138.4, 139.3, 145.2 (d, J = 16.6 Hz), 150.0, 151.9; J = 18.1 Hz),130.8,133.5,147.0 (d, J = 15.9 Hz),160.6; MS (ESI)
MS (ESI) for C18H22N2O5P: m/z377 [M+H]+; Elemental analysis for C16H17BrN2O4P: m/z 411 [M+H]+; Elemental analysis calc for
calc forC18H22N2O5P:C 57.45, H 5.62, N 7.44, O 21.26, P 8.23, C16H17BrN2O4P: C 46.74, H 3.92, Br 19.43, N 6.81, O 15.56, P
found C 57.48, H 5.67, N 7.49, O 21.21, P 8.20.
7.53, found C 46.71, H 3.96, Br 19.41, N 6.85, O 15.52, P 7.48.
Dimethyl(5,6-dimethoxy-2-(4-methoxyphenyl)-2H-indazol-3-
Dimethyl(2-(9-ethyl-9H-carbazol-3-yl)-2H-indazol-3-
yl)phosphonate (4o): Brown solid; mp: 152-154 °C; IR (KBr): yl)phosphonate (4t):White solid; mp: 112-115 °C; IR (KBr): 772,
737, 812, 953, 1021, 1215, 1248, 1357, 1443, 1502, 2989, 3056 796, 1019, 1231, 1269, 1491, 1597, 2951, 3048, 3452 cm-1; 1H
cm-1; 1H NMR (400 MHz, CDCl3) δ3.65 (s, 3H), 3.68 (s, 3H), 3.89 NMR (400 MHz, CDCl3) δ 1.49 (t, J = 7.2 Hz, 3H), 3.64 (s, 3H),
(s, 3H), 3.97 (s, 3H), 4.00 (s, 3H), 7.03-6.98 (m, 2H), 7.09 (d, J = 3.67 (s, 3H), 4.44 (q, J = 7.2 Hz, 2H), 7.26-7.35 (m, 2H), 7.40-
2.0 Hz, 1H), 7.33 (s, 1H), 7.56-7.49 (m, 2H);13C NMR (100 MHz, 7.46 (m, 1H), 7.48 (s, 1H), 7.49-7.57 (m, 2H), 7.76 (dd, J = 8.6,
CDCl3) δ 52.9 (d, J = 5.4 Hz), 55.6, 56.1 (d, J = 12.5 Hz), 95.9, 2.2 Hz, 1H), 7.87-7.93 (m, 1H), 8.11 (dd, J = 8.2, 1.0 Hz, 2H),
97.9, 113.8, 120.8 (d, J = 219.9 Hz), 124.3, 124.5, 127.3, 134.0, 8.39 (d, J = 2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 13.9, 37.9,
145.2 (d, J = 16.8 Hz),150.0, 151.9, 160.1; MS (ESI) for 53.01 (d, J = 5.6 Hz), 108.1, 108.9, 118.4 (d, J = 10.2 Hz), 119.5,
C18H22N2O6P: m/z393 [M+H]+; Elemental analysis calc 120.8, 121.1, 121.8, 122.8 (d, J = 3.4 Hz), 122.9 (d, J = 220.2
forC18H21N2O6P:C 55.10, H 5.39, N 7.14, O 24.47, P 7.89, found Hz), 123.7, 124.0, 124.7, 126.5, 126.8, 128.5(d, J = 18.8 Hz),
C 55.08, H 5.36, N 7.12, O 24.44, P 7.85.
132.6, 140.1, 140.8, 148.6 (d, J = 16.3 Hz); 31P NMR (163 MHz,
CDCl3) δ 10.1; HRMS (ESI): calc for C23H23N3O3P [M+ H]+
Dimethyl(2-(p-tolyl)-2H-[1,3]dioxolo[4,5-f]indazol-3-
yl)phosphonate(4p):Semi solid; IR (neat): 737, 810, 1038, 420.1472, found 420.1475.
1236, 1276, 1491, 1520, 1597, 2831, 3050 cm-1;1H NMR (400 Diethyl(2-(9-ethyl-9H-carbazol-3-yl)-2H-indazol-3-
MHz, CDCl3) δ 2.44 (s, 3H), 3.64 (s, 3H),3.67 (s, 3H),6.02 (s, 2H), yl)phosphonate (4u):White solid; mp: 98-100 °C; IR (KBr): 772,
7.07 (dd, J = 2.0, 0.4 Hz, 1H), 7.28-7.30 (m, 3H), 7.46-7.51 (m, 798, 1021, 1226, 1271, 1491, 1597, 2951, 3046, 3452 cm-1; 1H
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Journal Name
NMR (400 MHz, CDCl3) δ 1.19 (t, J = 7.1 Hz, 6H), 1.50 (t, J = 7.2 55.44, H 4.25, Br 16.04, N 8.43, O 9.63, P 6.22,found C 55.39, H
Hz, 3H), 3.95-4.05 (m, 2H), 4.05-4.15 (m, 2H), 4.47 (q, J = 7.2 4.27, Br 16.06, N 8.48, O 9.60, P 6.27.
Hz, 2H), 7.28-7.29 (m, 1H), 7.31-7.35 (m, 1H), 7.42-7.49 (m, Biological assays
1H), 7.51 (s, 1H), 7.52-7.58 (m, 2H), 7.79 (dd, J = 8.6, 2.2 Hz,
Cytotoxic properties
1H), 7.88-7.95 (m, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.4
Hz, 1H), 8.42 (d, J = 2.0 Hz, 1H);13C NMR (100 MHz, CDCl3) δ
A549, HepG-2 and Vero cells were obtained from ATCC, USA.
A549, HepG-2 and Vero cell lines were kept in DMEM with 10
13.8, 16.1 (d, J = 6.9 Hz), 37.9, 62.7 (d, J = 5.5 Hz), 108.0, 108.9,
% Fetal Bovine Serum. In this study, we used different
118.2, 118.6, 119.5, 120.8, 121.4, 122.7 (d, J = 15.5 Hz), 123.9,
concentrations of synthesized compounds (100, 50, 25and
124.0 (d, J = 218.7 Hz), 124.5, 126.4, 126.8, 128.4 (d, J = 18.9
12.5 µg/mL) to determine the cytotoxic properties. The cells
Hz), 132.8, 140.0, 140.8, 148.6 (d, J = 16.2 Hz); MS (ESI) for
C25H27N3O3P: m/z448 [M+H]+; Elemental analysis calc
were maintained at 37 °C with relative humidity. After
treatment of synthesized compounds, the media was
forC25H26N3O3P:C 67.10, H 5.86, N 9.39, O 10.73, P 6.92,found
separated from the plate and added 100 µL of medium
C 67.07, H 5.82, N 9.43, O 10.75, P 6.97.
containing 1 mg/mL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
Dimethyl(2-(9-ethyl-9H-carbazol-3-yl)-2H-[1,3]dioxolo[4,5-
diphenyl-tetrazolium bromide (MTT) to the plate. The cells
f]indazol-3-yl)phosphonate(4v):Brown solid; mp: 94-96 °C; IR
were incubated for 4 h and then the solution in the medium
(KBr): 765, 793, 812, 1026, 1215, 1257, 1498, 1597, 3043, 3437
was taken apart. An aliquot of 100µL of DMSO was added to
cm-1; 1H NMR (400 MHz, CDCl3) δ 1.39 (t, J = 7.2 Hz, 3H), 3.53
the plate. The cytotoxicity was determined by measuring the
(s, 3H), 3.56 (s, 3H), 4.33 (q, J = 7.2 Hz, 2H), 5.94 (s, 2H), 7.04
absorbance of the converted dye at 570 nm in an ELISA reader.
(d, J = 1.6 Hz, 1H), 7.14-7.20 (m, 1H), 7.27 (s, 1H), 7.36-7.39 (m,
1H), 7.41 (s, 1H), 7.43-7.45 (m, 1H), 7.62 (dd, J = 8.8, 2.0 Hz,
Confocal microscopy studies
1H), 8.01 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 2.0 Hz, 1H);13C NMR
A549 and HepG-2 cells were cultured in medium
(100 MHz, CDCl3) δ13.9, 37.9, 53.0 (d, J = 5.6 Hz), 94.4, 96.2,
supplemented with or without treatment of synthesized
101.4, 108.1, 108.9, 118.3, 119.4, 120.8, 122.0 (d, J = 221.0
compound (A549-4p and HepG-2 4d and 4g). After 24 h of
Hz), 122.7 (d, J = 7.7 Hz), 123.7, 125.4 (d, J = 19.1 Hz), 126.4,
treatment, cells were washed twice with 0.01 M PBS and
132.7, 139.9, 140.8, 146.0 (d, J = 16.9 Hz), 147.8, 149.6; MS
suspended in binding buffer (Acetone: methanol, 1:1). Cells
(ESI) for C24H23N3O5P: m/z464 [M+H]+; Elemental analysis calc
were incubated with FITC (Fluorescein Isothiocyanate), PI
forC24H22N3O5P:C 62.20, H 4.78, N 9.07, O 17.26, P 6.68,found
(Propidium Iodide) and DAPI (4',6-diamidino-2-phenylindole)
C 62.23, H 4.84, N 9.05, O 17.29, P 6.64.
for 30 min at 4 ºC in the dark. Cells were then centrifuged and
Dimethyl(5-chloro-2-(9-ethyl-9H-carbazol-3-yl)-2H-indazol-3-
pellets were smeared. FITC, DAPI and PI fluorescences were
yl)phosphonate (4w): White solid; mp: 174-176 °C; IR (KBr):
immediately observed under confocal laser scanning
752, 815, 967,1048, 1191, 1225, 1372, 1399, 1498, 1519, 1596,
microscope (ZEISS, LSM710, Germany).
3061 cm-1;1H NMR (400 MHz, CDCl3) δ 1.52 (t, J = 7.2 Hz, 3H),
3.67 (s, 3H), 3.70 (s, 3H), 4.46 (q, J = 7.2 Hz, 2H), 7.28-7.32 (m,
DNA fragmentation
1H), 7.39 (dd, J = 9.2, 2.0 Hz, 1H), 7.48-7.59 (m, 3H), 7.76 (dd, J
A549 cells (1×106 cells) were washed twice with PBS. The cells
= 8.6, 2.2 Hz, 1H), 7.86 (dd, J = 9.2, 2.0 Hz, 1H), 8.05-8.20 (m,
were lysed and kept at 4 °C for 15 min. Then lysed cells were
2H), 8.40 (d, J = 2.0 Hz, 1H);13C NMR (100 MHz, CDCl3) δ 13.9,
centrifuged and supernatants were carefully transferred to
37.9, 53.2 (d, J = 5.6 Hz), 108.2, 109.0, 118.3, 119.7 (d, J = 19.0
new tubes and incubated with proteinase K (0.4 mg/mL;
Hz), 120.0, 120.8, 121.8, 122.7 (d, J = 5.7 Hz), 122.9 (d, J =
Sigma-Aldrich) at 58 °C for 60 min and followed by RNase A
220.6 Hz), 123.5, 126.6, 128.4, 128.9 (d, J = 18.3 Hz), 130.6,
(0.4 mg/mL; Sigma-Aldrich) at 37 °C for 60 min. Furthermore,
132.3, 140.1, 140.8, 146.9 (d, J = 16.1 Hz); MS (ESI) for
the supernatants were mixed with 20 μL of 5 M NaCl and 120
C23H22ClN3O3P: m/z454 [M+H]+; Elemental analysis calc
μL of isopropyl alcohol overnight at -20 °C. The supernatants
forC23H21ClN3O3P:C 60.87, H 4.66, Cl 7.81, N 9.26, O 10.58, P
were then collected by centrifugation at 10,000 rpm for 10
6.82,found C 60.84, H 4.61, Cl 7.87, N 9.24, O 10.62, P 6.86.
min. DNA samples were dissolved in TE buffer and separated
Dimethyl(5-bromo-2-(9-ethyl-9H-carbazol-3-yl)-2H-indazol-3-
by 2% agarose gel electrophoresis.
yl)phosphonate (4x):White solid; mp: 182-183 °C; IR (KBr):
687, 725,752, 809,1047, 1197, 1218, 1369, 1392, 1506, 1592,
Toxicity study
3060cm-1;1H NMR (400 MHz, CDCl3) δ 1.52 (t, J = 7.2 Hz, 3H),
Toxicity study was carried out against Vero cells. The toxicity
was determined according to the reported method.34 The
3.67 (s, 3H), 3.70 (s, 3H), 4.47 (q, J = 7.2 Hz, 2H), 7.29-7.33 (m,
1H), 7.48-7.59 (m, 4H), 7.76 (dd, J = 8.6, 2.2 Hz, 1H), 7.80 (dd, J
different concentrations of synthesized compound were tested
= 9.0, 1.8 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.33 (d, J = 1.2 Hz,
1H), 8.39 (d, J = 2.0 Hz, 1H);13C NMR (100 MHz, CDCl3) δ 13.9,
against Vero cells.
37.9,53.2 (d, J = 5.6 Hz),108.2,109.0,118.3, 118.7, 119.6, 119.9,
Colony formation assay
120.8, 122.7 (d, J = 221.0 Hz), 122.7 (d, J = 6.3 Hz), 123.5 (d, J =
A549 cells (500 cells/well) were seeded in 12 well tissue
12.7 Hz), 126.6, 129.6 (d, J = 18.3 Hz), 130.7, 132.3, 140.1,
culture plate for overnight. Treated and untreated cells were
140.8, 147.0 (d, J = 16.0 Hz); MS (ESI) for C23H22BrN3O3P:
m/z498 [M+H]+; Elemental analysis calc forC23H21BrN3O3P:C
incubated for 10 days in at 37ºC in 5% CO2. Colonies were then
10 | J. Name., 2012, 00, 1-3
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ARTICLE
Rodriguez and T. Constantieux, Chem. Soc. Rev.,2011,
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and S. Perumal, Green Chem.,2014,16,1297; (d) T. Vivekan, P.
washed twice in PBS and stained with crystal violet (Sigma) for
1 h and photos were taken.
Vinoth, B. Agieshkumar,
N Sampath, A. Sudalai, J. C.
Apoptosis analysis using flow cytometry
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of 105 cells were mixed with 5 μL of annexin V-FITC and 10μL
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Western blotting
Treated and untreated A549 cells washed twice with PBS and
lysed in ice-cold RIPA buffer after addition of protease
inhibitors. The samples were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with
10-12.5% resolving gel. After SDS-PAGE it was transferred to
nitrocellulose membrane. Membranes were blocked with
blocking buffer for 1 h at room temperature. Membrane was
incubated overnight at 4 °C with the following specific primary
antibodies: Bcl-2 (C 21, SC783- rabbit polyclonal), p53 (FL393-
rabbit polyclonal), caspase-3 (SC7148- rabbit polyclonal),
cytochrome c (SC7159-rabbit IgG), Bax (SC-493, rabbit IgG) and
GAPDH (FL-335- rabbit polyclonal antibody) (Santa Cruz
Biotechnology, Santa Cruz, USA) with dilution of 1:1000.
Further incubation with appropriate horseradish peroxidise
conjugated secondary antibodies (Sigma), depending on the
primary antibody used, was performed for 1 h at room
temperature. Detection of staining signals was captured by
using chem-documentation system after enhancing them with
chemiluminescence kit (Pierce Biotech).35
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Acknowledgements
The authors N. S. and P. T. P thank the Council of Scientific and
Industrial Research (CSIR) – Emeritus Scientist Scheme, Delhi
for financial support.
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