Synthesis of novel 1,2,3-triazole substituted-N-alkyl/aryl nitrone derivatives, their anti-inflammatory and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2014.04.052

A series of novel 1,2,3-triazole substituted N-phenyl nitrone derivatives 5a-e were prepared in three steps starting from 1-substituted-1,2,3-triazole-4- carbaldehydes 2 via Schiff's base formation, reduction followed by oxidation. Similarly, 1,2,3-triazo

Full text of DOI:10.1016/j.ejmech.2014.04.052

European Journal of Medicinal Chemistry 80 (2014) 184e191
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 1,2,3-triazole substituted-N-alkyl/aryl nitrone
derivatives, their anti-inflammatory and anticancer activity
P. Sambasiva Rao a, C. Kurumurthy a, B. Veeraswamy a, G. Santhosh Kumar a,
Y. Poornachandra b, C. Ganesh Kumar b, Sathish Babu Vasamsetti c, Srigiridhar Kotamraju c,
,
B. Narsaiah a
*
a Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
c Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,2,3-triazole substituted N-phenyl nitrone derivatives 5aee were prepared in three
steps starting from 1-substituted-1,2,3-triazole-4-carbaldehydes 2 via Schiff’s base formation, reduction
followed by oxidation. Similarly, 1,2,3-triazole substituted N-alkyl nitrone derivatives 6aep were pre-
pared in single step starting from compound 2 on reaction with N-alkyl hydroxylamine hydrochlorides.
All the final compounds were screened for anti-inflammatory and anticancer activity against various
cancer cell lines. Among the compounds tested, the compounds 5a, 5d, 6a, 6b, 6m and 6o exhibited
Received 8 August 2013
Received in revised form
14 April 2014
Accepted 15 April 2014
Available online 18 April 2014
significant inhibition of IL-1
6i and 6o exhibited significant activity against all the cell lines (A549, COLO 205, MDA-MB 231 and PC-3)
at IC50 values of <15 M.
b secretion as a measure of anti-inflammatory activity. Compound 5b, 5c, 6h,
Keywords:
1,2,3-Triazole
Schiff’s base
Nitrone
m
Ó 2014 Elsevier Masson SAS. All rights reserved.
Antiinflammatory
Anticancer activity
1. Introduction
anticancer agents [19e22]. Based on the importance of 1,2,3-
triazole, nitrone function and in continuation of our efforts on the
synthesis of potential molecules [23e26], we have synthesized a
series of novel 1,2,3-triazole substituted-N-alkyl/aryl nitrone de-
rivatives and are presented herein for the first time. In this
communication, N-((1-substituted-1H-1,2,3-triazol-4-yl)methyl)
anilines 4 are oxidized into the corresponding nitrones 5 upon
treatment with 2e3 molar equivalents of hydrogen peroxide in the
presence of 4e5 mol % of selenium dioxide at room temperature in
a single step. The synthesis of 1,2,3-triazole substituted-N-alkyl
nitrone derivatives 6 was accomplished by condensation of 1-
substituted-(1,2,3)triazole-4-carbaldehydes 2 [27] with N-mono
substituted hydroxylamine hydrochlorides.
Nitrones are valuable synthons in the realm of organic synthesis
which are extensively used in the synthesis of biologically active
compounds [1], as spin trap reagents [2], as therapeutic agents [3]
and also behave like 1,3-dipoles in cycloaddition reactions [4]. Two
main synthetic methods commonly used for the preparation of
nitrones are: i) condensation of carbonyl compounds with N-mono
substituted hydroxylamines [5], ii) oxidation of secondary amines
or hydroxylamines [6]. In particular, the oxidative approach pro-
vides the most direct and general method for preparing nitrones.
Stoichiometric oxidants used in these reactions include oxaziridine
[7] or dioxirane [8]. Alternatively, hydrogen peroxide is used as
primary oxidant [9] in the presence of a catalyst such as Na2MoO4
or Na2WO4 [10,11] or MeReO3 [12] or SeO2 [13]. The 1,2,3-triazoles
due to their unique chemical and structural properties have
received much attention over the past few decades and found wide
application in medicinal chemistry [14e18] and especially as
2. Chemistry
The 1-substituted-1,2,3-triazol-4-yl-methanol 1 was oxidized to
an aldehyde 2 using Jones reagent followed by the reaction of an-
iline in acetonitrile at room temperature furnished Schiff’s base 3.
The N-((1-alkyl-1H-1,2,3-triazol-4-yl) methylene) aniline 3 were
reduced to amine 4 using NaBH4 in methanol and the resulting
reduced product further oxidized with 2e3 molar equivalents of
hydrogen peroxide and 4e5 mol% of selenium dioxide to obtain
* Corresponding author.
E-mail
addresses:
narsaiahbanda84@gmail.com,
narsaiah@iict.res.in
(B. Narsaiah).
http://dx.doi.org/10.1016/j.ejmech.2014.04.052
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
185
1,2,3-triazole substituted-N-phenyl nitrone derivatives 5aee. The
activity of various oxidants such as t-BuOOH, m-ClC6H4CO3H and
H2O2 have been examined and amongst them hydrogen peroxide
was identified as the best oxidant. The reaction could proceed
efficiently with H2O2 and SeO2 system in DCM medium. The details
of the reactions are outlined in Scheme 1. Similarly, 1-phenyl-1,2,3-
triazole-4-carbaldehyde 2a was reacted with N-benzyl hydroxyl-
amine hydrochloride using different bases such as Et3N, piperidine,
K2CO3 and NaHCO3 in ethanol and obtained 1,2,3-triazole
Scheme 2. Synthesis of 1,2,3-triazole substituted-N-alkyl nitrone derivatives 6aep.
substituted N-benzyl nitrone derivative 6a in
a single step.
Among all the bases, NaHCO3 is considered as best which gave the
highest yield. The protocol was extended with diverse substituted
1,2,3-triazole-4-carbaldehydes with N-benzyl/t-butyl substituted
hydroxylamine hydrochlorides and obtained respective products in
high yield. The reactions are outlined in Scheme 2 and products
were tabulated in Table 1.
Table 1
Physical properties of compounds 5aee and 6aep.
S. No
Compound no
R
R1
Yield (%)a
m.p (ꢀC)
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
C6H5
4-FC6H4
4-ClC6H4
C8H17CH2CH2
C8F17CH2CH2
C6H5
C6H5
C6H5
C6H5
C6H5
64
62
61
58
54
88
85
84
88
79
82
74
77
89
85
84
88
82
85
71
74
126e128
132e134
135e136
124e125
142e144
150e152
160e162
134e136
174e176
142e144
148e150
180e182
194e196
144e146
145e146
108e110
156e158
76e78
3. Results and discussion
C6H5
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
3.1. Anti-inflammatory activity
4-FC6H4
4-MeOC6H4
4-CF3C6H4
C6H13CH2CH2
C8H17CH2CH2
C6F13CH2CH2
C8F17CH2CH2
C6H5
9
All the final compounds 5aee and 6aep were screened for anti-
inflammatory activity in the presence of PMA (phorbol 13-
myristate 12-acetate)-induced inflammation using THP-1 mono-
cyte cell system [31]. Among the compounds tested, the com-
pounds 5a, 5d, 6a, 6b, 6m and 6o exhibited significant inhibition of
10
11
12
13
14
15
16
17
18
19
20
21
4-FC6H4
IL-1
7.9e9.9
a well known COX-2 inhibitor showed an IC50 of 18
the other hand, none of the compounds even at 20
b
secretion as a measure of anti-inflammatory activity (IC50
M, Pl. see Table 2). Under the same conditions, piroxicam,
M (Table 2). On
M concen-
4-MeOC6H4
4-CF3C6H4
C6H13CH2CH2
C8H17CH2CH2
C6F13CH2CH2
C8F17CH2CH2
m
m
6m
6n
6o
6p
m
82e84
140e142
164e166
tration had any effect on cell viability (data not shown). This clearly
indicates that the anti-inflammatory activities of these compounds
are not due to the induction of cytotoxicity in these cells. The
structureeactivity relationship (SAR) revealed that the activity is
independent of structure and there was no clear cut relationship.
Further studies are underway to optimize the lead molecule.
a
Isolated yields.
the compounds showed significant reduction in cell viability in all
the tested cell lines in a concentration-dependent manner. Among
the derivatives, compound 5b, 5c, 6h, 6i and 6o exhibited signifi-
cant activity against all the cell lines at micro molar concentration
3.2. In vitro cytotoxicity
with IC50 values of <15
exhibited moderate activity against all the cell lines at IC50 values of
<50 M. Similarly, the compound 6j showed moderate activity on
all the cell lines at IC50 value of <90 M and compound 5e showed
cytotoxicity against A549 cell line only at IC50 value of <86.8 M.
mM. Compounds 6k, 6l, 6m, 6n and 6p
The compounds 5aee and 6aep were also screened against four
human cancer cell lines such as A549, COLO 205, MDA-MB 231 and
PC-3 by MTT assay method [32]. IC50 values of the test compounds
on different cancer cell lines were calculated and the results are
presented in Table 3. Based on the results, it is evident that most of
m
m
m
Scheme 1. Synthesis of 1,2,3-triazole substituted-N-phenyl nitrone derivatives 5aee.

186
P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
Table 2
4.1. Preparation of alkyl/aryl azides [23]
IL-1b
secretion inhibition efficacy of the synthesized compounds 5aee and 6aep.a
IL-1b (IC50 m
M)b
4.1.1. General procedure
S. No
Compounds
Iodo alkane (10.0 mmol) and sodium azide (30.0 mmol) were
taken in 40 mL acetone: water (3:1) and heated to 60 ꢀC for 8 h.
After completion of reaction, cooled to room temperature and
excess solvent was removed under vacuum. The aqueous residue
was extracted with hexane (3ꢁ, 30 mL each time) and the com-
bined extracts were washed with water, then dried over anhydrous
sodium sulfate. The solvent was removed under vacuum to obtain
alkyl azide in quantitative yield and used in the next reaction
without further purification.
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
8.8 ꢂ 1.6
NA
NA
9.9 ꢂ 2.3
11.7 ꢂ 1.6
8 ꢂ 2.6
9.7 ꢂ 3.5
17.3 ꢂ 1.6
15.4 ꢂ 3.2
13.1 ꢂ 2.6
15.6 ꢂ 0.8
12.1 ꢂ 3.1
17.0 ꢂ 2.6
NA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4.2. Preparation of 1-substituted-1,2,3-triazol-4-yl-methanol (1)
14 ꢂ 1.1
13.5 ꢂ 2.8
14.4 ꢂ 1.0
7.9 ꢂ 1.36
4.2.1. General procedure
In an argon atmosphere, alkyl/aryl azide (5.4 mmol), propargyl
alcohol (8 mmol) were taken in t-BuOH (5 mL) followed by
addition of an aqueous Cu(OAc)2 solution (5 mol%, 1 mL). The
reaction was stirred for 18 h at room temperature and monitored
by TLC. The resulting mixture was diluted with dichloromethane;
the organic layer was separated and washed with water. Dried
over sodium sulfate and purified by passing through a column
packed with silica gel using n-hexane: ethyl acetate (6:4) as
solvents.
20.0 ꢂ 1.67
8.1 ꢂ 2.01
15.0 ꢂ 3.7
18.0 ꢂ 2.7
6p
Piroxicamc
a
THP1 monocytes were pre-treated with 5, 10 and 20 mM concentrations of the
above mentioned 1,2,3-triazole substituted nitrone derivatives 5aee and 6aep for
2 h before simulation with 100 nM Phorbol 13-myristate 12-acetate (PMA) to induce
inflammation for a period of 48 h. At the end of the treatment, conditioned media
was collected and the level of IL-1b was measured by ELISA as described in the
experimental section.
(1-Phenyl-1H-(1,2,3)triazol-4-yl)-methanol (1a) [28]
(1-(4-Fluoro-phenyl)-1H-(1,2,3)triazol-4-yl)-methanol (1b) [23]
(1-(4-Chlorophenyl)-1H-(1,2,3)triazol-4-yl)-methanol (1c) [29]
(1-decyl-1H-(1,2,3)triazol-4-yl)-methanol(1d) [23]
(1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluoro-decyl)-1H-
(1,2,3)triazol-4-yl)-methanol (1e) [19].
b
IC50 values are mean ꢂ SD of three independent experiments, NA: indicates IC50
value >20
m
M.
c
Piroxicam, a known anti-inflammatory agent was used as a positive control.
The structureeactivity relationship (SAR) studies revealed that 4-
fluorophenyl or 4-chlorophenyl substitution on the triazole moi-
ety and phenyl, t-butyl on nitrone moiety promoted the cytotox-
icity, whereas the benzyl group substituent on the nitrone moiety
exhibited no additional advantage in improving the cytotoxicity.
Among all the derivatives, compounds 5b, 5c, 6h and 6o showed
potent cytotoxicity against all the tested cell lines at IC50 values of
4.3. Preparation of 1-substituted-1,2,3-triazol-4-carbaldehyde (2)
4.3.1. General procedure
The 1-substituted-1,2,3-triazol-4-yl-methanol 1 (4 mmol) was
taken in dry acetone (10 mL), cooled to 0 ꢀC and the Jones reagent
(CrO3 þ H2SO4 þ Acetone) (4 mmol) was added slowly over a
period of 15 min. The reaction mixture was stirred for 20 min at
0 ꢀC. After completion of reaction, filtered through the short pad of
celite and the filtrate was collected and concentrated under vac-
uum. The residue was purified by passing through a column
packed with silica gel using petroleum ether/EtOAc (8:2) as
eluents.
<12 mM. However, compounds 6aeg did not show any cytotoxicity.
Further, it was confirmed that the promising compound 6h
exhibited apoptotic cell death based on the caspase-3 assay
(Table 4). Nevertheless, it is contemplated that a slight structural
modification of these active derivatives may yield prospective
anticancer drugs.
4. Experimental section
1-Phenyl-1H-(1,2,3)triazol-4-carbaldehyde (2a) [30]
1-(4-Fluorophenyl)-1H-(1,2,3)triazol-4-carbaldehyde (2b) [23]
1-(4-Chlorophenyl)-1H-(1,2,3)triazol-4-carbaldehyde (2c) [29]
1-Decyl-1H-(1,2,3)triazol-4-carbaldehyde (2d) [23]
1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluoro-decyl)-1H-
(1,2,3)triazole-4-carbaldehyde (2e) [19].
Melting points of all the compounds was recorded on Casia-
Siamia (VMP-AM) melting point apparatus and are uncorrected.
IR spectra were recorded on a PerkineElmer FT-IR 240-C spectro-
photometer using KBr optics. 1H NMR spectra were recorded on
Bruker AV 300 MHz and INOVA 500 MHz in CDCl3, 13C NMR spectra
were recorded on Bruker AV 75 MHz in CDCl3 & DMSO-d6 using
TMS as an internal standard. CHN analysis was recorded on a Vario
EL analyser. All high-resolution spectra were recorded on QSTARXL
hybrid MS/MS system (Applied Bio systems, USA) under electro
spray ionization. All the reactions were monitored by thin layer
chromatography (TLC) on pre-coated silica gel 60 F254 (mesh); spots
were visualized with UV light. Merck silica gel (60e120 mesh) was
used for column chromatography. All the final compounds were
analyzed by HPLC to evaluate purity using SHIMADZU LC-20 in-
strument. Separation was carried out by using a mobile phase of
ammonium acetate: acetonitrile (40:60) on Zodiac C18 Column
4.4. Preparation of N-((1-alkyl-1H-1,2,3-triazol-4-yl) methylene)
aniline (3)
4.4.1. General procedure
The 1-substituted-1,2,3-triazol-4-carbaldehyde 2 (4 mmol) and
aniline (4 mmol) were taken in acetonitrile (10 mL). The reaction
mixture was stirred at room temperature for 6 h and after
completion of reaction, as monitored by TLC, acetonitrile was
removed under vacuum. The residue was purified by passing
through a column packed with silica gel using petroleum ether/
EtOAc (8:2) as eluents.
(150 mm ꢁ 4.6 mm, 5
mM) in an isocratic mode at a flow rate of
1.0 ml/min with Photo-diode Array (PDA) detection at 254 nm.

P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
187
Table 3
C
15H11ClN4: C, 63.72; H, 3.92; N, 19.82%: Found: C, 63.74; H, 3.91; N,
Anticancer activity of compounds 5aee and 6aep.a IC50b values in
mM.
19.85%.
S. No Compound
A549
COLO 205
MDA-MB 231 PC-3
N-((1-Decyl-1H-1,2,3-triazol-4-yl)methylene)aniline (3d) [23]
1
5a
e
e
e
e
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl)-
1H-1,2,3-triazol-4-yl)methylene)aniline (3e) [19]
2
3
4
5b
5c
5d
12.5 ꢂ 0.22 10.2 ꢂ 0.27
9.8 ꢂ 0.09
8.9 ꢂ 0.43
11.4 ꢂ 0.32
11.1 ꢂ 0.36
e
10.5 ꢂ 0.24
9.7 ꢂ 0.42
e
e
e
5
6
7
8
5e
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
86.8 ꢂ 0.42
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4.5. Preparation of N-((1-substituted-1H-1,2,3-triazol-4-yl)methyl)
aniline (4)
e
e
e
e
9
e
e
4.5.1. General procedure
10
11
12
13
14
15
16
17
18
19
20
21
22
e
e
A solution of N-((1-alkyl-1H-1,2,3-triazol-4-yl)methylene)ani-
line 3 (4.7 mmol) in MeOH (10 mL) was cooled at 0 ꢀC. Then NaBH4
(9.4 mmol) was added slowly and the final mixture was stirred
during 2 h at room temperature. After completion of the reaction,
solvent was evaporated in vacuo and the residue was treated with
H2O (50.0 mL). Extracted with EtOAc (3 ꢁ 10.0 mL), combined ethyl
acetate layer was dried over anhydrous NaSO4 and evaporated in
vacuo. The residue was purified by column chromatography [SiO2,
petroleum ether/EtOAc (7:3)].
e
e
e
e
8.8 ꢂ 0.19
9.4 ꢂ 0.18
8.4 ꢂ 0.33
8.1 ꢂ 0.32
15.4 ꢂ 0.34
72.1 ꢂ 0.52
23.8 ꢂ 0.28
42.0 ꢂ 0.18
24.6 ꢂ 0.11
19.8 ꢂ 0.08
11.2 ꢂ 0.22
25.7 ꢂ 0.18
0.6 ꢂ 0.28
17.4 ꢂ 0.17 15.2 ꢂ 0.15 14.8 ꢂ 0.28
87.0 ꢂ 0.32 58.9 ꢂ 0.48 66.4 ꢂ 0.22
23.7 ꢂ 0.44 19.8 ꢂ 0.27 23.1 ꢂ 0.09
43.6 ꢂ 0.35 44.7 ꢂ 0.38 48.2 ꢂ 0.15
31.3 ꢂ 0.25 30.8 ꢂ 0.52 29.7 ꢂ 0.26
24.8 ꢂ 0.42 22.1 ꢂ 0.25 20.8 ꢂ 0.22
11.5 ꢂ 0.41
30.5 ꢂ 0.15 28.8 ꢂ 0.28 29.7 ꢂ 0.38
0.8 ꢂ 0.22 0.7 ꢂ 0.42 0.8 ꢂ 0.44
9.5 ꢂ 0.34
9.8 ꢂ 0.32
6p
Doxorubicinc
N-((1-Phenyl-1H-1,2,3-triazol-4-yl)methyl)aniline (4a)
a
Exponentially growing cells were treated with different concentrations of 1,2,3-
triazole substituted-N-phenyl nitrone derivatives 5aee and 1,2,3-triazole
substituted-N-alkyl nitrone derivatives 6aep for 24 h and cell growth inhibition
was analyzed through MTT assay.
White solid, Yield: 76%, m.p.: 102e104 ꢀC, IR (KBr, cmꢃ1): 3340
(eNH), 2988 (HC]C), 1521 (eC]C); 1H NMR (DMSO-d6, 300 MHz):
b
IC50 is defined as the concentration, which results in a 50% decrease in cell
d
4.51 (s, 2H, eCH2eN), 4.62 (br.s, 1H, NH), 6.63e6.74 (m, 3H, Ar-H),
7.12 (d, J ¼ 7.2 Hz, 2H, Ar-H), 7.19 (m, 3H, Ar-H), 7.59 (d, J ¼ 8.3 Hz,
2H, Ar-H), 7.85 (s, 1H, Ar-H); 13C NMR (DMSO-d6, 75 MHz):
149.1,
136.8, 132.2, 129.7, 128.5, 120.8, 120.1, 119.2, 112.7, 42.5; MS (ESI,
70 eV): 251 m/z (M þ H); Anal. Calcd. for C15H14N4: C, 71.98; H, 5.64;
N, 22.38%. Found: C, 71.99; H, 5.66; N, 22.42%.
number as compared with that of the control cultures in the absence of an inhibitor
and were calculated from the plotted absorbance data for the doseeresponse
d
curves. The values (in
m
M) represent the mean
ꢂ
SE of three independent
experiments.
c
Doxorubicin was employed as positive control, e indicates IC50 value >100 mM.
N-((1-Phenyl-1H-1,2,3-triazol-4-yl)methylene)aniline (3a)
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)aniline (4b)
White solid, Yield: 91%, m.p.: 110e112 ꢀC, IR (KBr, cmꢃ1): 3018
(HC]C), 1532 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
7.16e7.24
White solid, Yield: 69%, m.p.: 128e129 ꢀC, IR (KBr, cmꢃ1): 3328
(eNH), 2972 (HC]C), 1531 (eC]C); 1H NMR (DMSO-d6, 300 MHz):
d
(m, 5H, Ar-H), 7.32e7.36 (m, 3H, Ar-H), 7.80 (d, 2H, J ¼ 7.8 Hz, Ar-H),
d
4.55 (s, 2H, eCH2eN), 4.32 (br.s, 1H, NH), 6.69e6.79 (m, 3H, Ar-H),
8.42 (s, 1H, Ar-H), 8.71 (s, 1H, eCH]N); 13C NMR (DMSO-d6,
7.18 (d, J ¼ 7.3 Hz, 2H, Ar-H), 7.22 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.68 (d,
J ¼ 8.3 Hz, 2H, Ar-H), 7.83 (s, 1H, Ar-H); 13C NMR (DMSO-d6,
75 MHz):
d 151.8, 151.1, 147.2, 136.7, 130.5, 128.3, 127.2, 122.7, 121.6,
120.4; MS (ESI, 70 eV): 249 m/z (M þ H); Anal. Calcd. for C15H12N4:
75 MHz):
d 160.2, 147.1, 133.8, 132.2, 129.4, 127.5, 121.8, 120.4, 119.1,
C, 72.56; H, 4.87; N, 22.57%: Found: C, 72.54; H, 4.89; N, 22.61%.
112.7, 42.3; MS (ESI, 70 eV): 269 m/z (M þ H); Anal. Calcd. for
15H13FN4: C, 67.15; H, 4.88; N, 20.88%. Found: C, 67.18; H, 4.86; N,
20.89%.
C
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)aniline
(3b) [23]
N-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)aniline
N-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)aniline (4c)
(3c)
White solid, Yield: 72%, m.p.: 126e128 ꢀC, I.R. (KBr, cmꢃ1): 3309
(eNH), 2978 (HC]C), 1511 (eC]C); 1H NMR (DMSO-d6, 300 MHz):
White solid, Yield: 94%, m.p.: 132e134 ꢀC, IR (KBr, cmꢃ1): 3012
(HC]C), 1554 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d 7.12e7.21
d
4.61 (s, 2H, eCH2eN), 4.54 (br.s, 1H, NH), 6.71e6.80 (m, 3H, Ar-H),
(m, 5H, Ar-H), 7.26 (d, 2H, J ¼ 8.1 Hz, Ar-H), 7.68 (d, 2H, J ¼ 8.1 Hz,
7.21 (d, J ¼ 7.24 Hz, 2H, Ar-H), 7.28 (d, J ¼ 8.2 Hz, 2H, Ar-H), 7.81 (d,
Ar-H), 8.43 (s, 1H, Ar-H), 8.73 (s, 1H, eCH]N); 13C NMR (DMSO-d6,
J ¼ 8.2 Hz, 2H, Ar-H), 7.86 (s, 1H, Ar-H); 13C NMR (DMSO-d6,
75 MHz): d 151.7, 151.2, 147.0, 134.9, 134.3, 130.0, 128.8, 127.2, 122.3,
75 MHz): d 154.2, 147.4, 134.8, 132.8, 128.4, 126.5, 120.8, 120.4, 118.1,
121.9, 121.4; MS (ESI, 70 eV): 283 m/z (M þ H); Anal. Calcd. for
112.2, 42.5; MS (ESI, 70 eV): 283 m/z (M þ H); Anal. Calcd. for
Table 4
Caspase-3 activity on compound 6h.
S. No.
Test compound (concentration)
Fluorescence units (FU/mg/min)
12 h
24 h
36 h
48 h
1
2
3
4
5
Control
1055.6 ꢂ 108.2
2473.2 ꢂ 104.1
2541.4 ꢂ 125.1
3033.7 ꢂ 136.8
3331.1 ꢂ 356.8
2115.3 ꢂ 83.2
3148.5 ꢂ 281.9
3802.2 ꢂ 182.9
3791.8 ꢂ 327.0
4007.5 ꢂ 213.5
2192.3 ꢂ 115.9
3396.8 ꢂ 209.1
4082.5 ꢂ 176.6
4794.2 ꢂ 152.1
4994.1 ꢂ 149.2
2642.4 ꢂ 37.5
4280.8 ꢂ 170.8
4700.5 ꢂ 28.9
6690.3 ꢂ 173.7
7240.4 ꢂ 271.9
6h (8
6h (10
m
M)
M)
m
Doxorubicin (1
Doxorubicin (2
m
m
M)
M)

188
P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
N-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)aniline
oxide (5c)
C15H13ClN4: C, 63.27; H, 4.60; N, 19.68%. Found: C, 63.29; H, 4.61; N,
19.62.
White solid, Purity: 98.1%; Yield: 61%, m.p.: 135e136 ꢀC, IR.
(KBr, cmꢃ1): 2911 (HC]C), 1571 (eC]N); 1H NMR (DMSO-d6,
N-((1-Decyl-1H-1,2,3-triazol-4-yl)methyl)aniline (4d)
White solid, Yield: 79%, m.p.: 98e99 ꢀC, IR (KBr, cmꢃ1): 3324
(eNH), 2945 (HC]C), 1518 (eC]C); 1H NMR (DMSO-d6,
300 MHz):
d
7.39 (d, J ¼ 7.49 Hz, 2H, Ar-H), 7.45e7.52 (m, 3H, Ar-
H), 7.52 (d, J ¼ 7.49 Hz, 2H, Ar-H), 7.64 (d, J ¼ 7.54 Hz, 2H, Ar-H),
8.08 (s, 1H, Ar-H), 9.11 (s, 1H, HC]N); 13C NMR (DMSO-d6,
300 MHz):
d
0.88 (t, J ¼ 7.05 Hz, 3H, eCH3), 1.23e1.33 (m, 14H, e
75 MHz):
d 142.2, 134.1, 132.8, 132.2, 128.7, 127.5, 127.03, 125.2,
(CH2)7e), 1.84e1.90 (m, 2H, eCH2), 4.20 (br.s, 1H, eNH), 4.30 (t,
J ¼ 6.9 Hz, 2H, eNCH2), 4.45 (s, 2H, eCH2eNH), 6.67 (d, J ¼ 7.1 Hz,
2H, Ar-H), 6.74 (t, J ¼ 7.1 Hz, 1H, Ar-H), 7.18 (t, J ¼ 8.05 Hz, 2H, Ar-
123.7, 122.4, 115.5; MS (ESI, 70 eV): 299 m/z (M þ H); Anal. Calcd.
for C15H11ClN4O: C, 60.31; H, 3.71; N, 18.76%. Found: C, 60.36; H,
3.72; N, 18.75%.
H), 7.41 (s, 1H, Ar-H); 13C NMR (DMSO-d6, 75 MHz):
d 149.3,
130.2, 129.5, 122.7, 120.8, 112.2, 52.4, 42.5, 31.9, 29.6, 28.4, 27.1,
N-((1-Decyl-1H-1,2,3-triazol-4-yl)methylene)aniline oxide (5d)
22.7, 14.2; MS (ESI, 70 eV): 315 m/z (M þ H); Anal. Calcd. for
C
19H30N4: C, 72.57; H, 9.62; N, 17.82%. Found: C, 72.54; H, 9.63; N,
White solid, Purity: 98.4%; Yield: 58%, m.p.: 124e125 ꢀC, IR
(KBr, cmꢃ1): 2987 (HC]C), 1544 (eC]N); 1H NMR (DMSO-d6,
17.85%.
300 MHz):
d
0.88 (t, J ¼ 6.98 Hz, 3H, CH3), 1.24e1.36 (m, 14H, e
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl)-
1H-1,2,3-triazol-4-yl)methyl)aniline (4e)
(CH2)2e), 1.91e2.01 (m, 2H, CH2), 4.44 (t, J ¼ 6.42 Hz, 2H, CH2),
7.41e7.46 (m, 3H, Ar-H), 7.54 (d, J ¼ 8.1 Hz, 2H, Ar-H), 8.10 (s, 1H,
Ar-H), 9.15 (s, 1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
d 139.3,
White solid, Yield: 62%, m.p.: 122e124 ꢀC, IR (KBr, cmꢃ1): 3314
(eNH), 2945 (HC]C), 1518 (eC]C); 1H NMR (DMSO-d6, 300 MHz):
132.1, 129.3, 128.9, 128.7, 127.5, 125.3, 50.3, 31.6, 29.8, 28.1, 22.4,
13.8; MS (ESI, 70 eV): 329 m/z (M þ H); Anal. Calcd. for
d
2.82e2.91 (m, 2H, eCH2), 4.44 (t, J ¼ 6.94 Hz, 2H, eNCH2), 4.67 (s,
C
19H28N4O: C, 69.48; H, 8.59; N, 17.06%: Found: C, 69.52; H, 8.56;
2H, eCH2eNH), 4.78 (br.s, 1H, NH), 6.80e6.89 (m, 3H, Ar-H), 7.32 (t,
J ¼ 8.15 Hz, 2H, Ar-H), 7.88 (s, 1H, Ar-H); MS (ESI, 70 eV): 621 m/z
(M þ H); Anal. Calcd. for C19H13F17N4: C, 36.79; H, 2.11; N, 9.03%.
Found: C, 36.81; H, 2.14; N, 9.10%.
N, 17.09%.
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl)-
1H-1,2,3-triazol-4-yl)methylene)aniline oxide (5e)
4.6. Preparation of N-((1-substituted-1H-1,2,3-triazol-4-yl)
methylene)aniline oxide (5)
White solid, Purity: 97.6%; Yield: 54%, m.p.: 142e144 ꢀC, IR (KBr,
cmꢃ1): 2921 (HC]C),1593 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
2.84e2.95 (m, 2H, eCH2), 4.72 (t, J ¼ 7.2 Hz, 2H, CH2), 7.24 (t,
J ¼ 7.6 Hz, 3H, Ar-H), 7.45 (d, J ¼ 7.7 Hz, 2H, Ar-H), 8.18 (s, 1H, Ar-H),
9.89 (s, 1H, eCH]N); MS (ESI, 70 eV): 635 m/z (M þ H); Anal. Calcd.
for C19H11F17N4O: C, 35.98; H, 1.75; N, 8.83%: Found: C, 35.99; H,
1.74; N, 8.87%.
4.6.1. General procedure
To a mixture of SeO2 (15 mol%) and N-((1-substituted-1H-1,2,3-
triazol-4-yl)methyl)aniline 4 (2.59 mmol) in DCM (5.0 mL) was
added drop wise an aqueous 30% hydrogen peroxide solution
(7.56 mmol) at 0 ꢀC under argon and stirred at room temperature
for 3 h. After completion of reaction as indicated by TLC, DCM was
removed under reduced pressure and the residue was purified by
passing through a column packed with silica gel using petroleum
ether/EtOAc (7:3) as eluents.
4.7. Synthesis of 1,2,3-triazole substituted-N-alkyl nitrone
derivatives (6aep)
4.7.1. General procedure
N-((1-Phenyl-1H-1,2,3-triazol-4-yl)methylene)aniline oxide (5a)
A mixture of 1-substituted-1H-1,2,3-triazole-4-carbaldehyde 2
(1 mmol), N-benzyl/t-butyl hydroxylamine hydrochloride
(1.5 mmol) and sodium bicarbonate (1.5 mmol) in absolute
ethanol (6 mL) was heated at 60 ꢀC until the carbonyl compound
was disappeared (monitored by TLC). The solvent was removed in
vacuo and the reaction mixture was diluted with H2O and
extracted with EtOAc. Combined organic layers dried over anhy-
drous sodium sulfate and concentrated. The residue was purified
by column chromatography (SiO2, mixtures of petroleum ether/
EtOAc).
White solid, Purity: 97.2%; Yield: 64%, m.p.: 126e128 ꢀC, IR (KBr,
cmꢃ1): 2915 (HC]C), 1565 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d
7.28 (t, J ¼ 8.12 Hz, 2H, Ar-H), 7.49e7.58 (m, 5H, Ar-H), 7.80e7.88
(m, 3H, Ar-H), 8.61 (s, 1H, Ar-H), 9.48 (s, 1H, HC]N); 13C NMR
(DMSO-d6, 75 MHz): 139.9, 136.3, 132.0, 129.5, 129.3, 129.0, 127.0,
123.5, 120.2; MS (ESI, 70 eV): 265 m/z (M þ H); Anal. Calcd. for
15H12N4O: C, 68.17; H, 4.58; N, 21.20%. Found: C, 68.15; H, 4.57; N,
21.24%.
d
C
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)aniline
1-Phenyl-N-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)methan-
oxide (5b)
amine oxide (6a)
White solid, Purity: 97.8%; Yield: 62%, m.p.: 132e134 ꢀC, IR. (KBr,
cmꢃ1): 2967 (HC]C),1556 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
White solid, Purity: 98.8%; Yield: 88%, m.p.: 150e152 ꢀC, IR (KBr,
cmꢃ1): 2957 (HC]C),1598 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d
7.31 (t, J ¼ 8.8 Hz, 2H, Ar-H), 7.51e7.59 (m, 4H, Ar-H), 7.89 (m, 3H,
Ar-H), 8.62 (s, 1H, Ar-H), 9.50 (s, 1H, HC]N); 13C NMR (DMSO-d6,
75 MHz): 162.0, 141.2, 139.3, 130.9, 130.0, 127, 124.6, 123.5, 122.7,
122.3, 120.3; MS (ESI, 70 eV): 283 m/z (M þ H); Anal. Calcd. for
15H11FN4O: C, 63.83; H, 3.93; N,19.85%. Found: C, 63.86; H, 3.94; N,
19.86%.
d 5.11 (s, 2H, CH2N), 7.42e7.47 (m, 3H, Ar-H), 7.48e7.52 (m, 3H, Ar-
H), 7.54 (t, J ¼ 6.79 Hz, 2H, Ar-H), 7.78 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.92
d
(s, 1H, Ar-H), 8.30 (s, 1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
d
140.1,136.5,132.2,129.7,129.5,129.03,127.2,123.7,120.4, 69.6; MS
C
(ESI, 70 eV): 279 m/z (M þ H); HRMS m/z calcd. for C16H15ON4
[M þ H]: 279.1240. Found 279.1233.

P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
189
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-1-
phenylmethanamine oxide (6b)
d
2.79e2.81 (m, 2H, CH2), 4.72 (t, J ¼ 7.5 Hz, 2H, CH2), 5.08 (s, 2H,
CH2N), 7.40e7.48 (m, 5H, Ar-H), 7.85 (s, 1H, Ar-H), 8.91 (s, 1H, HC]
N); MS (ESI, 70 eV): 549 m/z (M þ H); HRMS m/z calcd. for
White solid, Purity: 98.5%; Yield: 85%, m.p.: 160e162 ꢀC, IR. (KBr,
cmꢃ1): 2987 (HC]C),1541 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
C
18H13ON4F13Na [M þ Na]: 571.0774. Found 571.0755.
d
5.12 (s, 2H, CH2N), 7.25 (t, J ¼ 8.9 Hz, 2H, Ar-H), 7.43 (m, 3H, Ar-H),
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl)-
1H-1,2,3-triazol-4-yl)methylene)-1-phenylmethanamine oxide (6h)
7.50 (d, J ¼ 7.9 Hz, 2H, Ar-H), 7.78 (d, J ¼ 7.9 Hz, 2H, Ar-H), 8.04 (s,
1H, Ar-H), 9.27 (s, 1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
White solid, Purity: 96.2%; Yield: 77%, m.p.: 194e196 ꢀC, IR (KBr,
d
162.2, 141.3, 139.1, 130.9, 130.6, 127.1, 124.5, 123.1, 122.7, 122.3,
cmꢃ1): 2992 (HC]C),1588 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
120.2, 70.3; MS (ESI, 70 eV): 297 m/z (M þ H); HRMS m/z calcd. for
C
16H14ON4F [M þ H]: 297.1146. Found 297.1139.
d
2.72e2.90 (m, 2H, CH2), 4.70 (t, J ¼ 6.7 Hz, 2H, CH2), 5.08 (s, 2H,
CH2N), 7.41e7.49 (m, 5H, Ar-H), 7.85 (s, 1H, Ar-H), 8.91 (s, 1H, HC]
N); MS (ESI, 70 eV): 649 m/z (M þ H); HRMS m/z calcd. for
N-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-1-
phenylmethanamine oxide (6c)
C
20H14F17N4O [M þ H]: 649.0890. Found 649.0896.
White solid, Purity: 98.8%; Yield: 84%, m.p.: 134e136 ꢀC, IR (KBr,
2-Methyl-N-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)propan-
2-amine oxide (6i)
cmꢃ1): 2978 (HC]C), 1576 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d
3.87 (s, 3H, OCH3), 5.10 (s, 2H, CH2N), 7.02 (d, J ¼ 8.8 Hz, 2H, Ar-H),
7.40e7.51 (m, 5H, Ar-H), 7.66 (d, J ¼ 8.8 Hz, 2H, Ar-H), 7.90 (s,1H, Ar-
H), 9.21 (s,1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
159.8,139.8,
White solid, Purity: 98.8%; Yield: 89%, m.p.: 144e146 ꢀC, IR (KBr,
cmꢃ1): 3011 (HC]C), 1541 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d
132.5, 129.9, 129.5, 129.2, 129.0, 127.4, 123.4, 122.0, 114.5, 69.5, 55.5;
MS (ESI, 70 eV): 331 m/z (M þ Na); HRMS m/z calcd. for C17H17O2N4
[M þ H]: 309.1346. Found 309.1336.
d
1.65 (s, 9H, 3CH3), 7.45 (t, J ¼ 7.1 Hz, 1H, Ar-H), 7.54 (t, J ¼ 8.1 Hz,
J¼ 7.1Hz, 2H, Ar-H), 7.81(d,J¼8.1Hz,2H, Ar-H),8.12(s,1H, Ar-H),9.35
(s, 1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
140.9, 136.6, 129.7,
d
128.8,123.4,123,120.3, 70.09, 28.03;MS(ESI, 70eV):245m/z(M þ H);
1-Phenyl-N-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)
methylene)methanamine oxide (6d)
HRMS m/z calcd. for C13H17ON4 [M þ H]: 245.1396. Found 245.1390.
N-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-2-
methylpropan-2-amine oxide (6j)
White solid, Purity: 99.2%; Yield: 88%, m.p.: 174e176 ꢀC, IR (KBr,
cmꢃ1): 2986 (HC]C), 1541 (eC]N), 1292 (CeF); 1H NMR (DMSO-
White solid, Purity: 98.2%; Yield: 85%, m.p.: 145e146 ꢀC, IR (KBr,
cmꢃ1): 2911 (HC]C), 1591 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d6, 300 MHz):
J ¼ 8.3 Hz, 2H, Ar-H), 7.92 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.96 (s, 1H, Ar-H),
9.38 (s, 1H, HC]N); 13C NMR (DMSO-d6, 75 MHz):
140.4, 135.5,
d 5.12 (s, 2H, CH2N), 7.43e7.51 (m, 5H, Ar-H), 7.82 (d,
d
d
1.64 (s, 9H, 3CH3), 7.22 (d, J ¼ 9.06 Hz, 2H, Ar-H), 7.78 (d,
J ¼ 9.06 Hz, 2H, Ar-H), 8.13 (s, 1H, Ar-H), 9.31 (s, 1H, HC]N); 13C
134.2, 132.7, 131.5, 128.3, 127.7, 127.1, 125.4, 124.2, 123.6, 123.1, 65.6;
MS (ESI, 70 eV): 369 m/z (M þ Na); HRMS m/z calcd. for
NMR (DMSO-d6, 75 MHz):
d 163.6, 161.7, 141.3, 133.2, 123.3, 117.2,
C
17H14ON4F3 [M þ H]: 347.1114. Found 347.1107.
116.9, 70.4, 28.03; MS (ESI, 70 eV): 285 m/z (M þ Na); HRMS m/z
calcd. for C13H15FN4ONa [M þ Na]þ: 285.1122. Found 285.1116.
N-((1-Octyl-1H-1,2,3-triazol-4-yl)methylene)-1-
phenylmethanamine oxide (6e)
N-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-2-
methylpropan-2-amine oxide (6k)
White solid, Purity: 99.2%; Yield: 79%, m.p.: 142e144 ꢀC, IR (KBr,
cmꢃ1): 2957 (HC]C), 1588 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
d
White solid, Purity: 98.9%; Yield: 84%, m.p.: 108e110 ꢀC, IR (KBr,
cmꢃ1): 2875 (HC]C), 1566 (eC]N); 1H NMR (DMSO-d6,
0.86 (t, J ¼ 6.7 Hz, 3H, CH3),1.21e1.34 (m,10H, e(CH2)5e),1.85e1.96
(m, 2H, CH2), 4.38 (t, J ¼ 7.5 Hz, 2H, CH2), 5.07 (s, 2H, CH2N), 7.39e7.49
300 MHz):
2H, Ar-H), 7.70 (d, J ¼ 9.1 Hz, 2H, Ar-H), 8.12 (s, 1H, Ar-H), 9.25 (s,1H,
HC]N); 13C NMR (DMSO-d6, 75 MHz):
159.8, 140.7, 130.0, 123.5,
d
1.64 (s, 9H, 3CH3), 3.87 (s, 3H, OCH3), 7.04 (d, J ¼ 9.1 Hz,
(m, 5H, Ar-H), 7.83 (s,1H, Ar-H), 8.81 (s,1H, HC]N); 13C NMR (DMSO-
d6, 75 MHz): d 139.5, 132.3, 129.5, 129.3, 129.0, 127.6, 125.5, 69.4, 50.4,
d
31.6, 30.0, 28.8, 26.3, 22.5, 13.9; MS (ESI, 70 eV): 315 m/z (M þ H);
123.2, 121.9, 114.8, 69.9, 55.5, 28.0; MS (ESI, 70 eV): 297 m/z
(M þ Na); HRMS m/z calcd. for C14H19O2N4 [M þ H]: 275.1502.
Found 275.1494.
HRMS m/z calcd. for C18H27ON4 [M þ H]: 315.2179. Found 315.2171.
N-((1-Decyl-1H-1,2,3-triazol-4-yl)methylene)-1-
phenylmethanamine oxide (6f)
2-Methyl-N-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-
yl)methylene)propan-2-amine oxide (6l)
White solid, Purity: 98.1%; Yield: 82%, m.p.: 148e150 ꢀC, IR (KBr,
cmꢃ1): 2892 (HC]C), 1581 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
White solid, Purity: 97.6%; Yield: 88%, m.p.: 156e158 ꢀC, IR (KBr,
cmꢃ1): 2911 (HC]C), 1591 (eC]N), 1287 (CeF); 1H NMR (DMSO-
d
0.87 (t, J ¼ 6.04 Hz, 3H, CH3), 1.22e1.34 (m, 14H, e(CH2)7e), 1.86e
1.96 (m, 2H, CH2), 4.37 (t, J ¼ 6.8 Hz, 2H, CH2), 5.06 (s, 2H, CH2N), 7.40e
d6, 300 MHz):
7.98 (d, J ¼ 9.06 Hz, 2H, Ar-H), 8.15 (s, 1H, Ar-H), 9.44 (s, 1H, HC]
N); 13C NMR (DMSO-d6, 75 MHz):
141.6, 139.3, 130.1, 127.5, 123.3,
d
1.65 (s, 9H, 3CH3), 7.82 (d, J ¼ 9.06 Hz, 2H, Ar-H),
7.48 (m, 5H, Ar-H), 7.83 (s, 1H, Ar-H), 8.81 (s, 1H, HC]N); 13C NMR
(DMSO-d6, 75 MHz):
d 139.5, 132.3, 129.5, 129.2, 129.0, 127.7, 125.5,
d
69.5, 50.5, 31.8, 30.0, 28.3, 22.6,14.0; MS (ESI, 70 eV): 343 m/z (M þ H);
123.03, 120.5, 70.6, 28.3; MS (ESI, 70 eV): 313 m/z (M þ H); HRMS
HRMS m/z calcd. for C20H31ON4 [M þ H]: 343.2492. Found 343.2483.
m/z calcd. for C14H16ON4F3 [M þ H]: 313.1270. Found 313.1263.
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl)-1H-1,2,3-
triazol-4-yl)methylene)-1-phenylmethanamine oxide (6g)
2-Methyl-N-((1-octyl-1H-1,2,3-triazol-4-yl)methylene)propan-2-
amine oxide (6m)
White solid, Purity: 96.8%; Yield: 74%, m.p.: 180e182 ꢀC, IR (KBr,
cmꢃ1): 2899 (HC]C),1565 (eC]N); 1H NMR (DMSO-d6, 300 MHz):
White solid, Purity: 98.8%; Yield: 82%, m.p.: 76e78 ꢀC, IR (KBr,
cmꢃ1): 2968 (HC]C), 1596 (eC]N); 1H NMR (DMSO-d6,

190
P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
300 MHz):
d
0.87 (t, J ¼ 6.8 Hz, 3H, CH3), 1.23e1.36 (m, 10H, e
they were stimulated with 100 nM PMA. After 48 h, the superna-
tants were harvested and assayed for IL-1 levels using an enzyme-
linked immunosorbent assay (ELISA) kit following the manufac-
turer’s instructions (eBiosciences, San Diego, CA, USA) [31]. The
absorbance in each well was measured on a microplate reader at
450 nm and corrected at 570 nm. IC50 values of test compounds on
(CH2)5e), 1.62 (s, 9H, 3CH3), 1.88e1.96 (m, 2H, CH2), 4.38 (t,
b
J ¼ 7.5 Hz, 2H, CH2), 8.06 (s, 1H, Ar-H), 8.85 (s, 1H, HC]N); 13C NMR
(DMSO-d6, 75 MHz): d 140.4,125.5,123.9, 69.9, 50.6, 31.9, 30.2, 28.9,
28.2, 26.6, 22.8, 14.2; MS (ESI, 70 eV): 281 m/z (M þ H); HRMS m/z
calcd. for C15H28ON4Na [M þ Na]: 303.2155. Found 303.2145.
IL-1b
production were calculated as described in previous paper
2-Methyl-N-((1-decyl-1H-1,2,3-triazol-4-yl)methylene)propan-2-
[31].
amine oxide (6n)
4.11. In vitro cytotoxicity assay
White solid, Purity: 98.9%; Yield: 85%, m.p.: 82e84 ꢀC, IR (KBr,
cmꢃ1): 2956 (HC]C), 1549 (eC]N); 1H NMR (DMSO-d6,
4.11.1. Procedure
300 MHz):
d
0.88 (t, J ¼ 6.7 Hz, 3H, CH3), 1.22e1.34 (m, 14H, e
The cytotoxicity was measured using the MTT [3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] accord-
ing to the method of Mossman [32]. Briefly, the cells (2 ꢁ 104) were
seeded in each well containing 0.1 ml of medium in 96 well plates.
After overnight incubation at 37 ꢀC in 5% CO2, the cells were treated
(CH2)7e), 1.62 (s, 9H, 3CH3), 1.88e1.96 (m, 2H, CH2), 4.38 (t,
J ¼ 7.5 Hz, 2H, CH2), 8.05 (s, 1H, Ar-H), 8.85 (s, 1H, HC]N); 13C NMR
(DMSO-d6, 75 MHz): d 140.2,125.3,123.8, 69.8, 50.4, 31.8, 30.0, 28.9,
28.0, 26.4, 22.6, 14.0; MS (ESI, 70 eV): 331 m/z (M þ Na); HRMS m/z
calcd. for C17H32N4ONa [M þ Na]: 331.2468. Found 331.2461.
with 100
20 g) at identical conditions with three replicates each. The final
test concentrations were equivalent to 10e100 M. The cell
l of MTT (5 mg/ml)
ml of different test concentrations of test compounds (2e
m
2-Methyl-N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-1H-
1,2,3-triazol-4-yl)methylene)propan-2-amine oxide (6o)
m
viability was assessed after 24 h, by adding 10
m
per well. The plates were incubated at 37 ꢀC for additional three
hours. The medium was discarded and the formazan blue formed in
White solid, Purity: 97.4%; Yield: 71%, m.p.: 140e142 ꢀC, IR (KBr,
cmꢃ1): 2896 (HC]C), 1556 (eC]N), 1278 (CeF); 1H NMR (DMSO-
the cells was dissolved with 100
ml of DMSO. The rate of color
d6, 300 MHz):
d
1.62 (s, 9H, 3CH3), 2.73e2.92 (m, 2H, CH2), 4.71 (t,
formation was measured at 570 nm using the TRIAD multimode
reader (Dynex Technologies, Inc., Chantilly, VA). The IC50 values
(50% inhibitory concentration) were calculated from the plotted
absorbance data for the doseeresponse curves. IC50 values (in mM)
are indicated as means ꢂ SD of three independent experiments.
J ¼ 7.5 Hz, 2H, CH2), 8.06 (s, 1H, Ar-H), 8.96 (s, 1H, HC]N); MS (ESI,
70 eV): 537 m/z (M þ Na); HRMS m/z calcd. for C15H16ON4F3
[M þ H]: 515.1111. Found 515.1089.
N-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl)-
1H-1,2,3-triazol-4-yl)methylene)-2-methylpropan-2-amine oxide
(6p)
4.12. Caspase-3 assay
The caspase-3 activity of the active compound (6h) was
measured using AFC conjugated Ac-DEVD as a substrate [33]. The
human prostate cancer cells (PC-3) were seeded in 6 well plates
with the cell confluence of 2.5 ꢁ 105 per well and incubated for
24 h. Cells were treated with the active compound (6 h) at two
White solid, Purity: 97.2%; Yield: 74%, m.p.: 164e166 ꢀC, IR (KBr,
cmꢃ1): 2945 (HC]C), 1578 (eC]N), 1289 (CeF); 1H NMR (DMSO-
d6, 300 MHz):
d 1.62 (s, 9H, 3CH3), 2.73e2.91 (m, 2H, CH2), 4.70 (t,
J ¼ 7.3 Hz, 2H, CH2), 8.06 (s, 1H, Ar-H), 8.94 (s, 1H, HC]N); MS (ESI,
70 eV): 615 m/z (M þ H); HRMS m/z calcd. for C17H16F17N4O
([M þ H]þ): 615.1047. Found 615.1051.
different concentrations, 8
mM (IC50 value) and 10
mM (>IC50 value),
along with the standard Doxorubicin at 1
mM and 2
mM, respec-
tively. After treatment, the cells were trypsinized and centrifuged at
3000 ꢁ g for 10 min at 4 ꢀC. Cells were lysed with lysis buffer
(HEPES 250 mM, CHAPS 25 mM, DTT 25 mM) followed by incu-
bation on ice for 20e30 min. The lysate was centrifuged at
16,000 ꢁ g for 20 min at 4 ꢀC and the obtained supernatant was
used for measurement of caspase activity. In a 96 well black poly-
4.8. THP-1 cell culture
THP-1 monocytes were obtained from ATCC and grown in RPMI
1640 (Sigma) containing 10% FBS (Lonza), non essential amino
acids, penicillin (100 U/mL) and streptomycin (100 mg/mL) in
100 mM dishes (SPL life sciences), incubated at 37 ꢀC in a humid-
ified atmosphere containing 5% CO2 and 95% air.
styrene plate, 2 ml of caspase-3 substrate and 1:1 ratio of super-
natant and 2 ꢁ assay buffer (HEPES 200 mM, CHAPS 1%, EDTA
20 mM, DTT 50 mM) were loaded and the reaction was allowed to
take place for 1 h. The fluorescence generated by the release of the
fluorogenic group AFC on cleavage by caspase-3 was measured by
excitation at 400 nm and emission at 505 nm wavelength for every
5 min over a period of 1 h. The liberated fluorescence was
normalized to cell lysate protein using Bradford’s protein estima-
tion method.
4.9. Measurement of cell viability
The effect of synthesized compounds on cell viability was
determined by Trypan blue dye exclusion assay. THP-1 cells were
seeded at a density of 2 ꢁ 105 cells/mL in 24-well plates in tripli-
cates and were treated with 20 mM concentrations of 5aee and 6ae
p compounds in the absence of PMA. After 48 h time period, cells
were harvested and re-suspended in 0.4% Trypan blue (Invitrogen)
and live/dead cells were counted using cell counting chamber
(Invitrogen).
Acknowledgments
Authors are thankful to the Council of Scientific & Industrial
Research (CSIR), New Delhi, India for providing financial assistance
through XII five year plan projects: DITSF code: CSC-0204 and
“Small Molecules in Lead Exploration” (SMiLE). Authors (P. Sam-
basiva Rao, C. Kurumurthy, B. Veeraswamy, G. Santhosh Kumar, Y.
Poornachandra and SBV) are also thankful to CSIR for providing
financial assistance in the form of Senior Research Fellowship and
contingency grant.
4.10. Enzyme-linked immunosorbent assay for IL-1b inhibition
To check the inhibitory effects of compounds 5aee and 6aep on
PMA-induced inflammation, THP-1 monocytes were seeded at a
density of 2 ꢁ 105 cells/mL in 12 well plates. Cells were pre-treated
with 5, 10 and 20 mM concentrations of compounds for 2 h before

P. Sambasiva Rao et al. / European Journal of Medicinal Chemistry 80 (2014) 184e191
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.052.
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