Design and synthesis of conjugated azo-hydrazone analogues using nano BF3·SiO2 targeting ROS homeostasis in oncogenic and vascular progression

Article abstract of DOI:10.1016/j.biopha.2017.08.076

Disrupted redox balance is implicated in multiple pathologies including malignant progression and tumor angiogenesis. In this investigation, we report the design and development of novel and effective ROS detoxifying azo-hydrazone molecules targeting mali

Full text of DOI:10.1016/j.biopha.2017.08.076

Biomedicine&Pharmacotherapy95(2017)419–428
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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Original article
Design and synthesis of conjugated azo-hydrazone analogues using nano
BF3·SiO2 targeting ROS homeostasis in oncogenic and vascular progression
Zabiullaa, V. Vigneshwaranb, A. Begum Bushraa, G.S. Pavankumarb, B.T. Prabhakarb,
Shaukath Ara Khanuma,⁎
a
Department of Chemistry, Yuvaraja’s College (Autonomous), University of Mysore, Mysore, Karnataka, India
Molecular Oncomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College (Autonomous), Kuvempu University,
b
Karnataka, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Azo-hydrazone
ROS
Malignancy
Angiogenesis
Disrupted redox balance is implicated in multiple pathologies including malignant progression and tumor an-
giogenesis. In this investigation, we report the design and development of novel and effective ROS detoxifying
azo-hydrazone molecules targeting malignant pathologies and neoangiogenesis. A series of azo-derivatives
conjugated to hydrazones moieties (9a–j) were synthesized using Nano BF3·SiO2. The compounds (9a–j) were
screened for in-vitro antioxidant and lipid peroxidation inhibitory activity. Among the series 9a–j, compound 9f
potently quenched biologically relevant radicals such as superoxide and hydrogen peroxide which emerged as
the lead ROS detoxifying molecules. Compound 9f potently inhibited the proliferative capability of Daltons
Lymphoma Ascites (DLA) tumor cells in-vivo in dose dependent manner. Regressed tumor progression was
correlated with pronounced endogenous antioxidant enzyme superoxide dismutase and catalase in-vivo. Also,
ROS levels were severely suppressed in 9f treated mice as assessed by lapsed lipid peroxidation. Altered enzymic
and ROS levels in-vivo by 9f were implicated in suppressed VEGF secretion leading to regressed tumor neo-
vasculature and tumor growth. Considering together, it is evident that the synthetic azo-hydrazone analogue 9f
with potent ROS scavenging efficacy inhibits tumor progression and neo-angiogenesis.
Dalton’s lymphoma ascites tumor
1. Introduction
is a critical process in the growth, invasion, and metastases of trans-
formed cells. Several lines of recent evidences suggest a pivotal role for
Redox homeostasis is dependent on the delicate balance between
the rate and the magnitude of oxidant production and their elimination
over time [1]. Oxidative stress generally describes a condition in which
cellular antioxidant defence mechanisms are insufficient to inactivate
ROS, or excessive ROS are produced, or both. Upregulated ROS levels
lead to “non-specific” damage of macromolecules such as DNA, proteins
and lipids [2]. This subsequently leads to generation of a new array of
oxidation mediated metabolic agents through lipid and protein perox-
idation, which are highly destructive than ROS themselves [3].
Growing body of evidence indicates that persistently high ROS levels
have been detected in almost all cancers, as a consequence of genetic,
metabolic and microenvironment-associated alterations [1]. As a con-
sequence, deregulated redox balance is strongly implicated in multiple
aspects of malignant progression and resistance to therapeutic inter-
ventions [4]. Tumor growth relies on formation of angiogenesis which
ROS signalling in augmenting tumor angiogenesis aiding cancer pro-
gression. The reciprocity between oxidative stress and angiogenesis has
been centered specifically on the VEGF signalling pathway, engaged in
promoting neovasculature [3]. Therefore the phenomenon of ROS-
driven cancer progression and angiogenesis has substantially fueled a
long-standing interest in the development of effective antioxidant mo-
lecules to attenuate neoplastic proliferation through ROS detoxification
[4]. Nitrogen-containing compounds are one of the most fruitful and
extensively developing fields in chemistry. These compounds display
various kinds of biological activities [5–7]. Hydrazones moiety plays an
important key role in heterocyclic chemistry [8–13]. It is a class of
organic compounds with structure R1R2C = NNH2. Hydrazones nucleus
exhibited immense pharmacological activities. They are present in
many of the bioactive heterocyclic compounds that are of very im-
portant use because of their various biological and clinical applications.
Abbreviations: DLA, Daltons lymphoma ascites; VEGF, vascular endothelial growth factor; MVD, microvessel density; TBARS, thiorbarbituric acid reactive substances; DPPH,2, 2
diphenyl-1-picryl-hydrazyl-hydrate; ROS, reactive oxygen species; ELISA, Enzyme linked immunosorbent assay; MDA, malondialdehyde; SOD, superoxide dismutase; H & E, hematoxylin
and eosin
⁎
Corresponding author.
E-mail address: shaukathara@yahoo.co.in (S.A. Khanum).
http://dx.doi.org/10.1016/j.biopha.2017.08.076
Received 11 April 2017; Received in revised form 3 August 2017; Accepted 19 August 2017
0753-3322/©2017ElsevierMassonSAS.Allrightsreserved.

Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
Scheme 1. Synthesis of heteroaryl hydrazide.
(Hetero) aroyl hydrazones demonstrate a wide range of biological ac-
tivities, including anti-microbial, anti-bacterial, anti-mycobacterial,
antifungal, anti-viral, anti-tuberculosis, anti-malarial and anticancer
activities [14–21]. On the other hand azo compounds have shown di-
verse biological activities including antimicrobial antioxidant and an-
titumor attributes [22–28]. Intrigued to develop a biologically active
and potent pharmacophore for oxidant stress and tumor angiogenic
pathologies, we in this investigation performed chemical combination
of hydrazone and azo compounds to synthesize novel azo-hydrazaide
analogous (9a–j) using Nano BF3·SiO2. The main objective of this in-
vestigation was to select the potent radical quenching azo-hydrazone
molecules and analyze its inhibitory effect on in-vivo tumor progression
and neo-angiogenesis using Dalton’s lymphoma murine tumor model.
using nano BF3·SiO2 as a catalyst) using sodium hydroxide and ethanol
as solvent delivered the expected final products 9a–j in a good yield.
(Scheme 3). All the structures of newly synthesized compounds were
assigned on the basis of their spectroscopic data; IR, NMR, LC–MS and
C, H, N analysis. The spectra of compounds 4a was confirmed with the
appearance of NHeNH2 stretching bands at 3100–3205 in IR spectra. In
proton NMR, the appearance of two singlet of NH2 and OCH2 at δ 4.21
and δ 4.83 respectively and also revealed multiplet signal in the range δ
6.5–7.7 for aromatic protons as well as singlet at 9.5 for NH proton. The
mass spectra of compound 4a gave significant stable M + 1 peak at m/z
168 evident the formation of 4a. Further, a spectrum of the title com-
pound 6a was considered as a representative example of the series 6a–l.
The IR spectra of the compound 6a was confirmed by the disappearance
of the NH2 stretching and the appearance 2930 (NeH), 1720 (C]O),
1499 (C]N), 3120–3220 (amide, COeNH) absorption peaks (cm−1). In
addition, 1H NMR spectra showed disappearance of NH2 proton and
appearance of N]CH protons at δ 8.5, beside increase in aromatic
proton with a range of δ 6.5–7.8 and also mass spectra gave significant
stable M + 1 peak at m/z 270 which clearly affirmed the formation of
compound 6a. The appearance N]N and disappearance NH2 IR
starching of compound 8a and 7a respectively, also disappearance of
NH2 proton in NMR its evident for formation of compound 8a. Finally,
in title compounds 9a–l, compound 9a taken as a representative ex-
ample to explain characterizations. The IR spectra was supported by the
appearance of N]N stretching band in the IR spectra. Also, the dis-
appearance of N]CH proton, besides, an increase in aromatic protons
in the NMR spectra with M + 1 peak at m/z at 374 confirmed the
product as 9a.
2. Result and discussion
2.1. Chemistry
The synthesis of the title compounds 9a–j was accomplished by a
synthetic procedure as shown in (Scheme 1–3). All the synthesized
compounds were established by IR, proton NMR and mass spectral data.
First, the heterocyclic acetic acid ethyl ester 3a–c were synthesized by
the etherification of heterocyclic hydroxyl compounds 1a–c with chloro
ethyl acetate 2 using dry acetone as a solvent to give compounds 3a–c
[7]. The compounds 3a–c on treatment with 99% hydrazine hydrate
yield heteroaryl hydrazide 4a–c (Scheme 1). The compounds 6a–g were
obtained by refluxing compounds 4a–c with substituted aromatic al-
dehydes 5a–d using ethanol as a solvent. (Scheme 2). Finally, all the
substituted benzylidine hydrazone analogous 6a–g, on treatment with
aryl diazonium salt 8a–e (diazotization of substituted aniline 7a–e
Scheme 2. Synthesis of benzylidene hydrazone analogous.
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Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
Scheme 3. Synthesis of azo-hydrazone analogues.
2.2. Pharmacology
heteroaryl and aryl rings A and B of these conjugates. Among azo–hy-
drazone analogues (9a–j), compound 9f displayed potent inhibitory
effect on ROS and lipid peroxidation. It is evident from this report that
analogue 9f with both electron withdrawing and electron donating
substitution was found to be potent ROS detoxifying and lipid perox-
idation inhibitory molecule. Compound 9f contains 2-nitro and 5-
chloro groups in aryl ring A, 2-chloro group in aryl ring B and one
methyl group in hetroaryl ring which demonstrated potent ROS
quenching attribute and lipid peroxidation inhibition with minimal
inhibitory concentration (IC50) as shown in Fig. 1 as verified by DPPH,
superoxide anion and H2O2 radical scavenging assay. Other tested
compounds (9a–j) with chloro- and nitro- substitution showed marked
radical quenching activity (Fig. 1). Hence this paper provides a novel
insight that multi structured azo-hydrazone pharmacophore with elec-
tron withdrawing and donating substitution could scavenge biologically
relevant radicals involved in malignant pathologies. Finally, con-
sidering the IC50 values and significant SAR, compound 9f was selected
as the lead molecule for further in-vivo antioxidant and tumor angio-
genic studies.
2.2.1. Compound 9f with inhibitory effect on in-vitro ROS and lipid
peroxidation is the lead molecule
Azo and hydrazone compounds were reported to exhibit varied
pharmacological activities. The present investigation sought to develop
series of novel antioxidant molecules (9a–j) by conjugating azo and
hydrazine moieties. Preliminarily, the synthesized compounds 9a–j was
subjected to various independent in-vitro antioxidant assays. Among the
9a–j series, compound 9f potently scavenged the DPPH radical with
IC50 9.6
1.15. Then the compounds were investigated for super-
oxide (O2−) and hydrogen peroxide (H2O2) scavenging activity, as
these reactive oxygen species were associated with various tumorigenic
and tumor angiogenesis process [29]. Results demonstrated that similar
to DPPH assay, compound 9f quenched superoxide anion and H2O2
radicals with high efficiency with IC50 values at 8.56
7.54 1.65 respectively. With respect to the effect on lipid perox-
idation, compound 9f potently inhibited the lipid peroxidation in-vitro
with IC50 15.6 1.74. Taking together the results elucidate that
1.34 and
among the synthesized azo-hydrazone series 9a–j, compound 9f dis-
played the potent ROS scavenging attribute. As ROS is strongly in-
volved in tumor progression and tumor neovasculature, compound 9f
with potent ROS detoxifying potential is opted as the lead molecule for
the further anticancer investigation.
2.2.3. Compound 9f regresses DLA tumor progression in-vivo with minimal
side effects
The in-vivo tumor models are critical for the evaluation of anti-
proliferative efficacy. The in-vivo anti-proliferative potential of com-
pound 9f was investigated using a reliable murine Dalton’s Lymphoma
Ascites (DLA) tumor model. DLA is a widely used cell line to study the
in-vivo anti-proliferative activity which is known for secretion of ascites
fluid containing various growth factors and cytokines for tumor pro-
gression [30,31]. Results revealed that administration of compound 9f
at 20 mg/kg b.w. concentration exhibited a dose dependent decrease in
the tumor development with 83.03% inhibition at the final dose as
assessed by the physical morphology (Data not shown) and body weight
index (Table 1). The reduced tumorigenic index in 9f was reflected in
the decreased cell density as compared to untreated. As a consequence
2.2.2. Structure activity relationship (SAR) of compound 9f
The azo and hydrazone derivatives are known to be pharmacolo-
gically active molecules against various pathological conditions in-
cluding oxidative stress and malignancy. The current investigation re-
ports the multistep synthesis of conjugated azo-hydrazaide moieties.
Structurally, the title compounds are having a basic backbone of one
heteroaryl and two aryl rings A and B with azo and imine link and
terminal amide group. In order to get the insight into the structure-
activity relationship (SAR), we varied the substitutions on both the
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Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
Fig. 1. Screening of potent free radical quenching Azo-Hydrazone analogue from 9a-l series. The IC50 of the antioxidant molecules 9a-l were determined by employing various in-vitro
radical scavenging and liver lipid peroxidation assays. (A) IC50values of 9a-l series compounds against DPPH, superoxide and hydrogen peroxide radical. (B) Lipid peroxidation inhibitory
effect of 9a-l series in-vitro. Peroxidized lipids in liver tissues were estimated by TBARS methods in-vitro. By taking IC50 values of ROS scavenging potential and lipid peroxidation
inhibitory effect, compound 9f was chosen as the lead molecule. Results are the means of three independent determinations, conducted in triplicate. Statistically significant values are
represented as*p < 0.05; **p < 0.01.
abdominal ascites secretion in the 9f treated was considerably re-
strained whereas the control animal depicted abundant secretion
(Table 1). As a toxicological assessment, the gross and histopathological
appearance of liver and spleen in the 9f treated DLA animals were
analyzed which appeared without any abnormalities as contrary to
untreated (Data not shown). Serological and hematological examina-
tions after 9f treatment in non tumor bearing mice indicated neglible
secondary complications (Table 2).
Table 2
Heamatological and serum parameters of non-tumor normal mice following treatment
with compound 9f at day 10.
S.No.
Hematological and serum parameters
Normal mice
9f treated mice
1.
2.
3.
4.
5.
Alkaline Phosphatase (IU/L)
Creatinine (mg/dL)
Urea (mg/dL)
127.63
0.62
46
2.54
1.02
2.1
129.62
0.67
44
1.13
1.53
1.3
3.6
2.7
RBC (106/μL)
6.86
4.24
2.4
2.1
6.11
4.7
WBC (106/μL)
2.2.4. Compound 9f inhibits ROS generation by pronouncing ROS
neutralizing antioxidant enzymes in-vivo
Results are the means of three independent determinations, each conducted in triplicate.
ROS poses a significant threat to nucleic acids, proteins, and lipids
and, as a result, can lead to a favourable scenario in which oncogenic
transformation takes place [32]. Therefore, in order to improve the
efficacy of therapeutic agents, strategies which reduce and/or eliminate
cellular sources of ROS are needed. So, the anti-proliferative activity of
compound 9f was validated for its implications on ROS and ROS
scavenging antioxidant enzyme levels in-vivo. ROS levels in-vivo was
estimated indirectly by measuring the ROS generated secondary oxi-
dative byproducts. ROS reacts with polyunsaturated or poly desaturated
fatty acids to initiate lipid peroxidation which generates numerous
genotoxic molecules including malondialdehyde (MDA) [2]. Our ex-
perimental observation firmly demonstrates that compound 9f sig-
nificantly decreased ROS in-vivo as evidenced by severely lapsed MDA
(Fig. 2A). Furthermore, the cellular redox balance is maintained by
powerful antioxidant enzyme system that neutralizes ROS [33]. Among
those, the enzymeSuperoxide Dismutases (SODs) that catalyzes the
dismutation ofsuperoxide anion radical to oxygen & hydrogen peroxide,
and catalase that facilitates the decomposition of hydrogen peroxide to
water and oxygen, are pivotal for proper homeostasis [2]. Investigation
on the serum enzyme parameters of 9f treated animal deduced a sig-
nificantly upregulated SOD and catalase levels in-vivo (Fig. 2B & C), in
contrast to the untreated mice which paralleled with tumor growth
index (Table 1). Altogether, it is clear from this observation that the
tumor inhibitory activity of compound 9f is strongly attributed to its
ROS detoxifying behavior.
2.2.5. Compound 9f attenuates VEGF and tumor neovasculature by
modulating ROS
Angiogenesis, the process wherein new blood vessels are formed
from pre existing blood vessels, is a critical determinant of cancer
growth [32]. DLA tumor growth is related to the progression of
Table 1
Tumor inhibitory effect of azo-hydrazone analogue 9f in Dalton’s lymphoma ascites (DLA) tumor model. DLA transplanted mice was administered intraperitoneally with 9f at 20 mg/kg
b.w. concentration for three doses and tumor inhibition of the drug was analyzed. Tumor volume was determined by assessing the body weight of 9f treated mice as compared to control.
S.No
Tumor Parameters
I DOSE
Control
II DOSE
Control
III DOSE
Control
9f
9f
9f
1
2
3
4
Tumor volume (g)
6.4
0
6.7
0.6
3.1
51.6
2.3
5
0.4
0.3
9.2
0
11.5
86
0.6
2.5
72.83
3.4
0.3*
12.4
0
15.2
0.5
0.6
2.1
83.07
3.2
0.2**
Tumor inhibtion (%)
Ascites Secretion (mL)
Cell Count (×106 mL)
0.5
0.5
0.5
0.5
0.2**
0.5
0.2**
0.3**
25
0.5
10
125
0.5
12
Values are indicated in mean
SEM, n = 6, one way ANOVA followed by Tukey’s multiple comparison test. Statistical significant values were expressed as *p < 0.05 and **p < 0.01.
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Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
Fig. 2. Compound 9f potentially decelerates tumor induced lipid peroxidation and increases enzymic antioxidants. The elevated levels of lipid peroxidation and its breakdown product
MDA is highly linked in tumorigenesis and neovasculature. The effect of compound 9f in inhibiting the lipid peroxidation was determined by estimating MDA using TBARS assay. (A)
Compound 9f potently decreases the lipid peroxidation in dose dependant manner. Malignancy is associated with significantly altered ROS scavenging enzymes. Treatment with 9f
potentially elevates the hepatic catalase (B) and superoxide dismutase (C) in murine system. Results are the means of three independent determinations, conducted in triplicate.
Statistically significant values are represented as*p < 0.05; **p < 0.01.
angiogenic complications and enlargement of peritoneal microvessels
[34]. Our results inferred that compound 9f significantly inhibited the
peritoneal microvessels in-vivo as observed visibly in dose dependent
manner (Fig. 3A). The angioinhibitory activity of compound 9f was
further validated by H & E stained peritoneal sections. Results showed
that compound 9f displayed a drastic reduction in microvessel density
(MVD) in contrast to untreated animal confirming its angioinhibitory
activity (Fig. 3B & C). Compelling evidences indicate that ROS promotes
angiogenic signalling cascade governed by cytokines and transcription
factors influencing tumor neovascularization [35,29]. ROS such as Su-
peroxide anion (O2%−) and Hydrogen peroxide (H2O2) promote an-
giogenesis by inducing proangiogenic factors VEGF expression. Com-
pound 9f detoxified ROS in-vivo by up regulating O2% and
H2O2scavengers such as SOD and catalase respectively in 9f treated
mice, as published reports have corroborated that over expression of
those enzymes inhibited tumor neovascularization in mice [29].
Moreover by taking in-vitro ROS scavenging potential of compound 9f
into account (Fig. 1A), it is very evident that the compound attenuated
angiogenesis most possibly through detoxifying the ROS radicals in-
vivo. As indicated above, it has been postulated that main mechanism of
oxidative stress induced angiogenesis involves peroxidized lipids and
vascular endothelial growth factor (VEGF) signalling [3,35]. In-vivo
observation infer that compound 9f has potently decreased the VEGF
levels in tumor bearing mice compared to that of control (Fig. 3D). Also,
there was drastic reduction in the lipid peroxidation in 9f treated ani-
mals as assessed by measuring thiobarbituric acid reactive substances
(TBARS) in-vivo (Fig. 2A).The reduction of lipid peroxidation and
peritoneal MVD with upregulated antioxidant enzymes has con-
siderably influenced the survivability of 9f treated animals (Fig. 3E).
Henceforth these experimental observations provide a clear perspective
that compound 9f inhibits vascularization by quenching ROS and in-
ducing antioxidant enzymes SOD and catalase, thereby leading to re-
gressed VEGF levels and tumor cell proliferation.
3. Conclusion
The present study elucidated the synthesis of azo-hydrazone ana-
logues as potent antioxidant molecules targeting disrupted ROS
homeostasis in malignant and vascular progression. Among the syn-
thetic series 9a–j, compound 9f emerged as the lead molecule with
potent ROS scavenging and lipid peroxidation inhibitory activity.
Compound 9f significantly regressed the proliferation of Dalton’s lym-
phoma ascites tumor cells and tumor angiogenesis by inhibiting VEGF
levels in-vivo. Tumor growth index was paralleled with decreased ROS
and lipid peroxide levels in-vivo and increased ROS detoxifying en-
zymes. Taking together, this study provides clear insight that com-
pound 9f by altering redox signalling components potently inhibited
the tumor growth and tumor neovasculature. Hence azo-hydrazone
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Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
Fig. 3. Compound 9f regresses tumor neovascularization and vascular endothelial growth factor secretion in mice. To validate the angiopreventive effect of compound 9f, DLA induced
peritoneal angiogenesis was induced in mice by culturing DLA cells and administered with compound 9f at 20 mg/kg b.w. (i.p.) for three consecutive doses on alternative day. (A)
Compound 9f antagonized the tumor induced peritoneal angiogenesis in dose dependant manner. (B) The angiogenesis inhibition was revalidated by H & E staining of the peritoneum. (C)
The chart depicting the reduction of micro vascular density (MVD/HPF) in the peritoneum and H & E stained sections. (D) Compound 9f inhibits VEGF-A secretion in tumor mice in dose
dependant manner. (E) Kaplan–Meier graph showing the prolonged life span of 9f treated mice.Statistically significant values are represented as*p < 0.05; **p < 0.01.
analogue 9f could be developed as potential therapeutic molecule for
4.2. Chemistry
malignant neoplasia and tumor angiogenesis targeting oxidant stress.
4.2.1. General procedure for the synthesis of heteraryloxy-acetic acid ethyl
ester 3a-c
4. Materials and methods
A mixture of heteroaryl hydroxy compounds (1a–c, 0.025 mol) and
ethyl cholro acetate (2, 0.037 mol) in dry acetone (40 mL) with anhy-
drous potassium carbonate (0.037 mol) was refluxed for 10 h. The re-
action mixture was cooled and the solvent was removed by distillation.
The residual mass was triturated with cold water to remove potassium
carbonate and extracted with ether (3 × 30 mL). The ether layer was
washed with 10% sodium hydroxide solution (3 × 30 mL) followed by
water (3 × 30 mL) and then dried over anhydrous sodium sulphate and
4.2.1.1. (Pyridin-2-yloxy)-acetic acid ethyl ester 3a. Yield: 90%. M.P.:
158−160°C; IR (KBr) νmax (cm−1): 1680 (C]O), 1150 (CeO),
2900–3100 (CeH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 1.43 (s, 3H,
CH3of ester), 4.32 (d, 2H, CH2of ester), 4.91 (s, 2H, OCH2), 6.5–7.6 (m,
4H, Ar-H).LC–MS m/z 182 (M + 1). Anal. Cal. for C9H11NO3 (181): C,
59.66; H, 6.12; N, 7.73. Found: C, 59.62; H, 6.07; N, 7.69%.
4.1. Experimental section
All solvents and reagents, 2-hydroxypyridine, 8-hydroxyquinoline,
2-methyl-8-quinolinol, ethyl chloro acetate, hydrazine hydrate, ben-
zaldehyde, o-tolualdehyde, 2-hydroxy-4-methoxybenzaldehyde, 5-
chloro-2-nitrobenzaldehyde, aniline, 2-fluoroaniline, 2-bromoaniline,
2-chloroaniline, p-anisidine, commercial nano silica gel and BF3·Et2O,
DPPH, TBARS, TBA and MDA were purchased from Sigma Aldrich
Chemicals Pvt Ltd. with a purity 90–99%. H2O2 was purchased from
Merck Millipore, Massachusetts, United States. NBT and all other che-
micals required for the experimental analysis were obtained from the
Himedia, Mumbai, India. Recombinant VEGF was produced in
Molecular Oncomedicine Laboratory, Sahyadri Science College,
Shivamogga, Karnataka, India. TLC was performed on aluminum-
backed silica plates and visualized by UV-light. Melting points (M.P)
were determined on an electrically heated VMP-III melting point ap-
paratus. The elemental analysis of the compounds was performed on a
Perkin Elmer 2400 elemental analyzer. The results of elemental ana-
lyses were within 0.4% of the theoretical values. The FT-IR spectra
were recorded using KBr discs and Nujol on FT-IR Jasco 4100 infrared
4.2.2. (Quinolin-8-yloxy)-acetic acid ethyl ester 3b
Yield: 79%. M.P.: 116–118 °C; IR (KBr) νmax (cm−1): 1660 (C]O),
1130 (CeO), 2800–3000 (CeH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
1.35 (t, 3H, CH3of ester), 4.25 (d, 2H, CH2of ester), 4.83 (s, 2H, OCH2),
6.9–8.4 (m, 6H, Ar-H).LC–MS m/z 232 (M + 1). Anal. Cal. for
C
13H13NO3 (231): C, 67.52; H, 5.67; N, 6.06. Found: C, 67.48; H, 5.63;
N, 6.01%.
spectrophotometer. 1H NMR spectra were recorded on
a Bruker
400 MHz NMR spectrometer in CDCl3 or DMSO and the chemical shifts
were recorded in parts per million downfield from tetramethylsilane.
Mass spectra were recorded on LC–MS (API-4000) mass spectrometer.
4.2.3. (2-Methyl-quinolin-8-yloxy)-acetic acid ethyl ester 3c
Yield: 84%. M.P.: 120–122 °C; IR (KBr) νmax (cm−1): 1635 (C]O),
1100 (CeO), 2850–3000 (CeH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
1.39 (t, 3H, CH3of ester), 2.32 (s, 3H, Ar-CH3), 4.25 (s, 2H, CH2of ester),
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4.93 (s, 2H, OCH2), 7.0–8.4 (m, 5H, Ar-H).LC–MS m/z 246 (M + 1).
Anal. Cal. for C14H15NO3 (245): C, 68.56; H, 6.16; N, 5.71. Found: C,
68.52; H, 6.11; N, 5.64%.
into ice cold water and the precipitate obtained was filtered and dried
in oven at low temperature. The product was recrystallised from ab-
solute ethanol to afford compounds 6a–g in a good yield.
4.2.6.1. (Pyridin-2-yloxy)-acetic acid (2-methyl-benzylidene)-hydrazone
6a. Yield: 90%. M.P.: 138–140 °C; IR (KBr) νmax (cm−1): 2930
(NeH), 1720 (C]O), 1499 (C]N), 1460 (C]C), 3120–3220 (amide,
COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.35 (s, 3H, CH3), 4.72
(s, 2H, OCH2), 6.5–7.8 (m, 8H, Ar-H), 8 0.5 (s, 1H, CH]N), 9.7 (s, 1H,
NH). LC–MS m/z 270 (M + 1). Anal. Cal. for C15H15N3O2 (269): C,
66.90; H, 5.61; N, 15.60. Found: C, 66.86; H, 5.75; N, 15.57%.
4.2.4. General procedure for the synthesis of heteroaryl hydrazide 4a–c
To compounds (3a–c, 0.01 mol) in ethanol (10 mL), 90% hydrazine
hydrate (0.01 mol) was added in drops and stirred for 3 h at room
temperature. A white solid was obtained. It wasfiltered and washed
with water which on recrystalization with methanol afforded com-
pounds 4a–c.
4.2.4.1. (Pyridin-2-yloxy)-acetic acid hydrazide 4a. Yield: 76%. M.P.:
170–172 °C; IR (KBr) νmax (cm−1): 1645 (amide, C]O), 3205 (-NH-),
−NH2 show two bands one at 3200–3350 (-NH sym), 3300–3450 (eNH
anti). 1H MR (400 MHz) (CDCl3) δ (ppm): 4.21 (s, 2H, NH2), 4.83 (s,
2H, OCH2), 6.5-7.7 (m, 4H, Ar-H), 9.5 (s, 1H, NH),.LC–MS m/z 168 (M
+ 1). Anal. Cal. for C7H9N3O2 (167): C, 50.29; H, 5.43; N, 25.14.
Found: C, 50.25; H, 5.39; N, 25.08%.
4.2.6.2. (Pyridin-2-yloxy)-acetic acid (2-hydroxy-4-methoxy-benzylidene)
-hydrazone 6b. Yield: 84%. M.P.: 116–118 °C; IR (KBr) νmax (cm−1):
2926 (NeH), 1715 (C]O), 1599 (C]N), 1472 (C]C), 3130–3230
(amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 3.53 (s, 3H,
OCH3), 4.8 (s, 2H, OCH2), 6.2-7.7 (m, 7H, Ar-H), 8.7 (s, 1H, CH]N),
9.5 (s, 1H, NH), 10.3 (s, 1H, OH). LC–MS m/z 302 (M + 1). Anal. Cal.
for C15H15N3O4 (301): C, 59.79; H, 5.02; N, 13.95. Found: C, 59.72; H,
4.95; N, 13.89%.
4.2.4.2. (Quinolin-8-yloxy)-acetic acid hydrazide 4b. Yield: 80%. M.P.:
163–165 °C; IR (KBr) νmax (cm−1): 1655 (amide, C]O), 3200 (eNHe),
eNH2 show two bands one at 3230–3350 (eNH sym) and 3300–3450
(eNH anti). 1H NMR (400 MHz) (CDCl3) δ (ppm): 4.32 (s, 2H, NH2),
4.84 (s, 2H, OCH2), 7.1-8.7 (m, 6H, Ar-H), 9.3 (s, 1H, NH). LC–MS m/z
218 (M + 1). Anal. Cal. for C11H11N3O2 (217): C, 60.82; H, 5.10; N,
19.34. Found: C, 60.78; H, 5.06; N, 19.31%.
4.2.6.3. (2-Methyl-quinolin-5-yloxy)-acetic acid (2-methyl-benzylidene)-
hydrazone 6c. Yield: 90%. M.P.: 112–114 °C; IR (KBr) νmax (cm−1):
2874 (NeH), 1690 (C]O), 1489 (C]N), 1535 (C]C), 3120–3220
(amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.33 (s, 3H,
CH3), 2.51 (s, 3H, CH3), 4.67 (s, 2H, OCH2), 7.4–8.1 (m, 9H, Ar-H), 8
0.6 (s, 1H, CH]N), 9.7 (s, 1H, NH). LC–MS m/z 334 (M + 1). Anal. Cal.
for C20H19N3O2 (333): C, 72.05; H, 5.74; N, 12.60. Found: C, 71.95; H,
5.68; N, 12.57%.
4.2.4.3. (2-Methyl-quinolin-8-yloxy)-acetic acid hydrazide 4c. Yield:
75%. M.P.: 159–161°C; IR (KBr) νmax (cm−1): 1643 (amide, C]O),
3180 (eNHe), −NH2 show two bands one at 3200–3320 (eNH sym)
and 3290–3350 (–NH anti). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.42
(s, 2H, CH3), 4.43 (s, 2H, NH2), 4.87 (s, 2H, OCH2), 6.9–8.5 (m, 5H, Ar-
H), 9.7 (s, 1H, NH). LC–MS m/z 232 (M + 1). Anal. Cal. for C12H31N3O2
(231): C, 62.33; H, 5.67; N, 18.17. Found: C, 62.29; H, 5.63; N, 18.12%.
4.2.6.4. (Quinolin-8-yloxy)-acetic acid (2-methyl-benzylidene)-hydrazone
6d. Yield: 92%. M.P.: 125–127 °C; IR (KBr) νmax (cm−1): 2956 (NeH),
1730 (C]O), 1553 (C]N), 1465(C]C), 3220–3310 (amide, COeNH).
1H NMR (400 MHz) (CDCl3) δ (ppm): 2.39 (s, 3H, CH3), 4.68 (s, 2H,
OCH2), 6.7–8.2 (m, 10H, Ar-H), 8.4 (s, 1H, CH]N), 9.9 (s, 1H, NH).
LC–MS m/z 320 (M + 1). Anal. Cal. for C15H15N3O2 (319): C, 71.46; H,
5.37; N, 13.16. Found: C, 71.42; H, 5.31; N, 13.09%.
4.2.5. Drying of ethanol (Super dry ethanol)
Ethyl alcohol of a high degree of purity is frequently required in
preparative organic analysis. Ethyl alcohol was dry using quick lime
(lumps of clean marble) was heated for 3–6 h in an oven at 200 °C.
Rectified sprit (ethyl alcohol) was poured into R.B flasks containing
freshly burned quick lime and the flask fitted with a doubled surface
condenser carrying a dry CaCl2 guard tube on top. The mixture was
gently refluxed on an oil bath for six hours and allowed to stand
overnight. It was then distilled it to have absolute ethyl alcohol. Super
dry alcohol can be prepared from the product of dehydration of recti-
fied sprit using activated magnesium. A clean dry magnesium (5 g)
turnings and iodine (0.5 g) were placed in flask fallowed by 50–70 mL
alcohol. The mixture was warmed until the iodine has disappeared,
heating was continued until all the magnesium was converted into
ethylate. Then, the absolute alcohol was added and the mixture was
refluxed for 30 min, then alcohol was distilled off directly into the
vessel in which it was stored. The reactions involved during the process
are
4.2.6.5. (2-Methyl-quinolin-8-yloxy)-acetic
acid
(5-chloro-2-nitro-
benzylidene)-hydrazone 6e. Yield: 85%. M.P.: 137–139 °C; IR (KBr)
νmax (cm−1): 2945 (NeH), 1740 (C]O), 1523 (C]N), 1520 (C]C),
3130–3230 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 3H, CH3), 4.80 (s, 2H, OCH2), 6.5–7.8 (m, 8H, Ar-H), 8 0.5 (s,
1H, CH]N), 9.7 (s, 1H, NH). LC–MS m/z 399 (M + 1). Anal. Cal. for
C
19H15ClN4O4 (398): C, 57.22; H, 3.79; N, 14.05. Found: C, 57.17; H,
3.73; N, 13.96%.
4.2.6.6. (2-Methyl-quinolin-8-yloxy)-acetic acid (2-hydroxy-4-methoxy-
benzylidene)-hydrazone 6f. Yield: 87%. M.P.: 110–113 °C; IR (KBr)
νmax (cm−1): 2935 (NeH), 1690 (C]O), 1512 (C]N), 1454 (C]C),
3100–3215 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
2.36 (s, 3H, CH3), 3.52 (s, 3H, OCH3), 4.69 (s, 2H, OCH2), 6.3–8.2 (m,
8H, Ar-H), 8.1 (s, 1H, CH]N), 10.2 (s, 1H, NH), 10.5 (s, 1H, OH).
LC–MS m/z 366 (M + 1). Anal. Cal. for C20H19N3O4 (365): C, 65.74; H,
5.24; N, 11.50. Found: C, 65.71; H, 5.19; N, 11.44%.
CaCO3 + H2O → Ca(OH)2 + CO2
Mg + 2C2H5OH → H2 + Mg(OC2H5)
Mg(OC2H5) + H2O → Mg(OH)2 + 2C2H5OH
4.2.6.7. (Quinolin-8-yloxy)-acetic acid benzylidene-hydrazone 6g. Yield:
78%. M.P.: 135–137 °C; IR (KBr) νmax (cm−1): 2894 (NeH), 1715 (C]
O), 1567 (C]N), 1493 (C]C), 3110–3210 (amide, COeNH). 1H NMR
(400 MHz) (CDCl3) δ (ppm): 4.73 (s, 2H, OCH2), 7.1-8.3 (m, 11H, Ar-
H), 8 0.6 (s, 1H, CH]N), 10.5 (s, 1H, NH). LC–MS m/z 306 (M + 1).
Anal. Cal. for C18H15N3O2 (305): C, 70.81; H, 4.95; N, 13.76. Found: C,
70.78; H, 4.89; N, 13.71%.
4.2.6. General procedure for the synthesis of benzylidene hydrazones
analogous 6a-g
A mixture substituted aromatic aldehyde (5a-d, 0.01 mol) and
heteroaryl hydrazide (4a-c, 1.37 g, 0.01 mol) in 15 mL of super dry
ethanol was refluxed for 7 h with few drops of acid. The completion of
reaction was confirmed by TLC. The reaction mixture was then poured
4.2.7. Preparation of 15 mol% nano BF3·SiO2
Nano BF3·SiO2 was easily prepared from nano silica gel and BF3·Et2O
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Biomedicine&Pharmacotherapy95(2017)419–428
according to the literature. In short, BF3·Et2O (2.65 mL) was added
drop-wise to a slurry containing nano silica gel (7 g) and ethanol
(30 mL, instead of chloroform). The mixture was stirred for 1 h at room
temperature. The resulted suspension was filtered to obtain the white
solid named nano BF3·SiO2 (15 mol%). The acidic capacity of nano
BF3·SiO2 was 2.07 mmolg−1 and determined via titration of 0.2 g of
solid acid with standard solution of NaOH [36].
4.2.8.4. (2-Methyl-quinolin-8-yloxy)-acetic acid [(2-bromo-phenylazo)-o-
tolyl-methylene]-hydrazone 9d. Yield: 78%. M.P.: 118–120 °C; IR (KBr)
νmax (cm−1): 2930 (NeH), 1499 (C]N), 1530 (C]C), 568 (C-Br), 1460
(N]N), 3120–3220 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ
(ppm): 2.24 (s, 3H, CH3), 2.29 (s, 3H, CH3), 4.81 (s, 2H, OCH2), 6.7-8.5
(m, 13H, Ar-H), 9.7 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 174.2, 159.5,
155.9, 155.3, 139.9, 138.2, 134.2, 132.9, 132.5, 131.5, 130.7, 129.3,
128.9, 128, 127.8 126.6, 125.3, 123.4, 123, 119.1, 106.3, 78.3, 21.3,
4.2.8. General procedure for the synthesis of (hetero-aryloxy)-acetic acid
(phenylazo-methylene)-hydrazone (Azo-hydrazone analogues)
16.2. LC–MS m/z 516.15 (M + 1). Anal. Cal. for C26H22BrN5O2
(515.10): C, 60.47; H, 4.29; N, 13.56. Found: C, 60.43; H, 4.22; N,
13.53%.
Benzylidine hydrazone analogous 6a–g were dissolved in ethanolic
solution of sodium hydroxide (15 mL). The mixture was cooled down to
0 °C and kept ready for the coupling reaction (stock solution). At the
other side, substituted aryl diazonium salt 8a–e were prepared with
subsituted aniline 7a–e by diazotization using nano BF3·SiO2 as a cat-
alyst.
4.2.8.5. (Quinolin-8-yloxy)-acetic acid (phenylazo-o-tolyl-methylene)-
hydrazone 9e. Yield: 75%. M.P.: 135–37 °C; IR (KBr) νmax (cm−1):
2956 (NeH), 1512 (C]N), 1496 (C]C), 1453 (N]N), 3140–3250
(amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.27 (s, 3H,
CH3), 4.68 (s, 2H, OCH2), 6.9–8.8 (m, 15H, Ar-H), 9.6 (s, 1H, NH). 13C
NMR (DMSO-d6) δ: 175.6, 159.6, 157.5, 148.2, 139.8, 137.2, 135.8,
130.9, 129.7, 127.3, 126.8, 126.1, 125.4, 123.8, 122.6, 120.1, 118.7,
105.3, 80.4, 15.3. LC–MS m/z 424.20 (M + 1). Anal. Cal. for
diazotization (To synthesize aryldiazonium salts 8a–e, aromatic
amines 7a–e (4 mmol) was stirred with NaNO2 (5 mmol) using nano
BF3·SiO2 as a catalyst at −5–0 °C). This diazonium solution was added
in drop wise manner to basic buffer solutions as prepared above. The
residual acidic sites on nano BF3·SiO2 in diazotization step can partially
decompose benzylidine hydrazone analogous to the starting materials
in non-basic medium. So, NaOH amount should be sufficient before
adding of aryl diazonium salts (Medium pH of 10–12 is suitable).
Reaction mixture cooled down to 0 °C, in ice bath with constant stirring
for the coupling reaction. Care was taken for to not let temperature
exceed −5–0 °C. The mixture was stirred for 3–4 h at the same tem-
perature. After completion of the reaction (TLC), the mixture was wa-
shed with distilled water (50 mL) for naturalization of additional NaOH
and then by acetone (30 mL) for separation of nano BF3·SiO2.
Evaporation of the solvent followed by column chromatography af-
forded compounds 9a–j in good yields. The compound was re-
crystallized from methanol to obtained pure product 9a–j.
C25H21N5O2 (423.17): C, 70.91; H, 5.00; N, 16.54. Found: C, 70.85;
H, 4.96; N, 16.48%.
4.2.8.6. (2-Methyl-quinolin-8-yloxy)-acetic
acid
[(5-chloro-2-nitro-
phenyl)-(2-chloro-phenylazo)-methylene]-hydrazone
9f. Yield: 68%.
M.P.: 146–148 °C; IR (KBr) νmax (cm−1): 2930 (NeH), 1595 (C]N),
1543 (C]C), 724 (CeCl), 1354 (NO2), 1470 (N]N), 3120–3220
(amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.27 (s, 3H,
CH3), 4.71 (s, 2H, OCH2), 6.7–8.4 (m, 12H, Ar-H), 9.7 (s, 1H, NH). 13C
NMR (DMSO-d6) δ: 174.6, 160.3, 157.9, 156.2, 149.0, 143.3, 140.8,
137.1, 135.3, 133.7, 131.3, 130, 129.5, 128.7, 127.8, 127, 125.1,
125.3, 122.1, 119.2, 108.7, 76.4, 19.3. LC–MS m/z 537.11 (M + 1).
Anal. Cal. for C25H18Cl2N6O4 (536.08): C, 55.88; H, 3.38; N, 15.64.
Found: C, 55.82; H, 3.33; N, 15.58%.
4.2.8.1. (Pyridin-2-yloxy)-acetic
acid (phenylazo-o-tolyl-methylene)-
hydrazone 9a. Yield: 90%. M.P.: 114–116 °C; IR (KBr) νmax (cm−1):
2930 (NeH), 1563 (C]N), 1459 (C]C), 1454 (N]N), 3120–3220
(amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.28 (s, 3H,
CH3), 4.68 (s, 2H, OCH2), 6.6-7.8 (m, 13H, Ar-H), 9.8 (s, 1H, NH). 13C
NMR (DMSO-d6) δ: 175.8, 166.2, 160.7, 149.5, 140.2, 138.4, 137.5,
135.7, 133.8, 131.3, 128.4, 125.3 120.6, 109.3, 80.1, 16.2. LC–MS m/z
374.17 (M + 1). Anal. Cal. for C21H19N5O2 (373.15): C, 67.55; H, 5.13;
N, 18.76. Found: C, 67.55; H, 5.13; N, 18.76%.
4.2.8.7. (2-Methyl-quinolin-8-yloxy)-acetic acid [(2-hydroxy-4-methoxy-
phenyl)-(4-methoxy-phenylazo)-methylene]-hydrazone 9g. Yield: 90%.
M.P.: 128–130 °C; IR (KBr) νmax (cm−1): 2960 (NeH), 1512 (C]N),
1613 (C]C), 1476 (N]N), 3220–3320 (amide, COeNH). 1H NMR
(400 MHz) (CDCl3) δ (ppm): 2.26 (s, 3H, CH3), 3.8 (s, 6H, OCH3), 4.68
(s, 2H, OCH2), 6.2-8.5 (m, 12H, Ar-H), 9.8 (s, 1H, NH), 10.3 (s, 1H, 0H).
13C NMR (DMSO-d6) δ: 175.2, 166.7, 163.2, 159.6, 158.8, 155.6, 154.4,
148.8, 133.4, 131.2, 130.0, 128.8, 122.9, 121.7, 119.7, 116.9, 113.8,
110.7, 107.8, 102.9, 101.4, 78.7, 56.0, 21.3. LC–MS m/z 500.23 (M
+ 1). Anal. Cal. for C27H25N5O5 (499.19): C, 64.92; H, 5.04; N, 14.0.
Found: C, 64.88; H, 4.98; N, 13.96%.
4.2.8.2. (Pyridin-2-yloxy)-acetic acid [(4-methoxy-phenylazo)-o-tolyl-
methylene]-hydrazone 9b. Yield: 85%. M.P.: 110–112 °C; IR (KBr) νmax
(cm−1): 2920 (NeH), 1480 (C]N), 1531 (C]C), 1460 (N]N),
3120–3220 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
2.26 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 4.68 (s, 2H, OCH2), 6.8–7.7 (m,
12H, Ar-H), 9.7 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 174.3, 165.1,
161.7, 154.6, 148.5, 140.2, 139.7, 138.4, 133.8, 130.5, 128.3, 127.6,
125.3, 120.6, 119.8, 117.1, 111.8, 80.8, 57.5, 17.6. LC–MS m/z 404.18
(M + 1). Anal. Cal. for C22H21N5O3 (403.16): C, 65.50; H, 5.25; N,
17.36. Found: C, 65.43; H, 5.21; N, 17.29%.
4.2.8.8. (Pyridin-2-yloxy)-acetic acid [(2-chloro-phenylazo)-(2-hydroxy-
4-methoxy-phenyl)-methylene]-hydrazone
9h. Yield:
75%.
M.P.:
154–156 °C; IR (KBr) νmax (cm−1): 2924 (NeH), 1562 (C]N), 1512
(C]C), 673 (CeCl), 1499 (N]N), 3120–3220 (amide, COeNH). 1H
NMR (400 MHz) (CDCl3) δ (ppm): 3.68 (s, 3H, OCH3), 4.72 (s, 2H,
OCH2), 6.4–7.8 (m, 11H, Ar-H), 9.4 (s, 1H, NH). 13C NMR (DMSO-d6) δ:
173.9, 166.5, 164.1, 159.8, 155.3, 149.6, 138.8, 134.5, 131.4, 130.4,
128.7, 127.4, 115.1, 110.3, 109.7, 106.3, 102.4, 78.5, 53.9. LC–MS m/z
440.14 (M + 1). Anal. Cal. for C21H18ClN5O4 (439.10): C, 57.34; H,
4.12; N, 15.92. Found: C, 57.31; H, 4.06; N, 15.89%.
4.2.8.3. (Pyridin-2-yloxy)-acetic acid [(2-fluoro-phenylazo)-(2-hydroxy-
4-methoxy-phenyl)-methylene]-hydrazone
9c. Yield:
88%.
M.P.:
121–123 °C; IR (KBr) νmax (cm−1): 2915 (NeH), 1489 (C]N), 1568
(C]C), 1115 (C-F), 1475 (N]N), 3110–3210 (amide, COeNH). 1H
NMR (400 MHz) (CDCl3) δ (ppm): 3.7 (s, 3H, OCH3), 4.72 (s, 2H,
OCH2), 6.5–7.7 (m, 11H, Ar-H), 9.5 (s, 1H, NH), 10.3 (s, 1H, OH). 13C
NMR (DMSO-d6) δ: 173.8, 168.73, 166.1, 162.8, 158.7, 156.3, 148.6,
139.3, 130.4, 130.8, 123.7, 117.1, 115.6, 109.7, 108.5, 104.8, 99.6,
83.2, 53.4. LC–MS m/z 424.13 (M + 1). Anal. Cal. for C21H18FN5O4
(423.13): C, 59.57; H, 4.29; N, 16.54. Found: C, 59.49; H, 4.25; N,
16.51%.
4.2.8.9. (Pyridin-2-yloxy)-acetic
acid
[(2-bromo-phenylazo)-o-tolyl-
methylene]-hydrazone 9i. Yield: 90%. M.P.: 136–137 °C; IR (KBr) νmax
(cm−1): 2930 (NeH), 1515 (C]N), 1457 (C]C), 551 (C-Br), 1456
(N]N), 3140–3230 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ
(ppm): 2.32 (s, 3H, CH3), 4.68 (s, 2H, OCH2), 6.4–7.7 (m, 12H, Ar-H),
9.9 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 176.5, 168.2, 159.8, 150.6,
141.3, 137.8, 135.9, 132.4, 131.8, 130.4, 129.2, 128.9, 127.5, 124.6,
122.5, 116.3, 115.5, 80.3, 15.5. LC–MS m/z 452.11 (M + 1). Anal. Cal.
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Zabiulla et al.
Biomedicine&Pharmacotherapy95(2017)419–428
for C21H18BrN5O2 (451.06): C, 55.76; H, 4.01; N, 15.48. Found: C,
55.72; H, 3.95; N, 15.41%.
on Animals (CPCSEA) guidelines for laboratory animal facility (NCP/
IAEC/CL/101/05/2012-13).
4.2.8.10. (Quinolin-8-yloxy)-acetic acid [(2-methoxy-phenylazo)-phenyl-
methylene]-hydrazone 9j. Yield: 89%. M.P.: 143–145 °C; IR (KBr) νmax
(cm−1): 2945 (NeH), 1514 (C]N), 1620 (C]C),1480 (N]N),
3120–3220 (amide, COeNH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
3.81 (s, 3H, OCH3), 4.67 (s, 2H, OCH2), 7.1–8.6 (m, 15H, Ar-H), 9.6 (s,
1H, NH). 13C NMR (DMSO-d6) δ: 175.3, 165.8, 159.3, 155.8, 150.4,
142.0, 137.8, 135.3, 131.8, 130.3, 129.3, 128.8, 127.6, 126.1, 123.5,
122.1, 119.3, 114, 108.9, 78.5, 55.3. LC–MS m/z 440.17 (M + 1). Anal.
Cal. for C25H21N5O3 (439.16): C, 68.33; H, 4.82; N, 15.94. Found: C,
68.29; H, 4.77; N, 15.90%.
4.4.2. Animal tumor models and treatment
DLA cells were cultured in-vivo by intra-peritoneal (i.p) transplan-
tation to develop ascites tumor by adopting previous reports [34]. After
the optimal growth of the tumor, three doses of 9f were administered to
test mice at a concentration 20 mg/kg body weight through in-
traperitoneal route. On day 10 after the final treatment, ascitic lym-
phoma growth parameters including percentage of tumor inhibition,
ascites secretion and tumor cell counts were analyzed. The number of
animals survived and the life span duration after the treatment period
were documented separately. To examine the physiological effect of 9f
in normal non-tumor mice, the serum of 9f treated animals were sub-
jected to the estimation of alkaline phosphatase (ALP), creatinine and
urea. Liver and spleen were surgically removed from test and control
animals for the histopathological analysis.
4.3. Pharmacology
4.3.1. DPPH photometric assay
The DPPH radical scavenging activity of the synthesized azo-hy-
drazone analogues was evaluated as described earlier with slight
modification [37]. Briefly, 2.5 mL of 0.2 mM DPPH methanol solution
along with each compound (9a–j series) in different concentration
range (1–200 μg/mL) was mixed well and incubated in dark at room
temperature. After 30 min absorbance was read at 518 nm. Ascorbic
acid was used as a positive control.
4.4.3. Peritoneal microvessel density (MVD) assessement
The angiogenic response of compound 9f in tumor condition was
assessed by peritoneal microvessel density and H & E staining as de-
scribed previously [30]. Shortly, the formaldehyde-fixed peritoneum of
the control and 9ftreated were processed for H & E staining. Then
quantification of MVD was performed using light optical microscopy in
the areas of peritoneum containing the highest number of capillaries.
4.3.2. Hydrogen peroxide scavenging assay
The H2O2 radical scavenging activity of the azo-hydrazone analo-
gues (9a–j) was determined as described previously [38]. Briefly, 2 mM
of H2O2 solution was prepared in 50 mM phosphate buffer pH 7.4. The
synthetic drugs 9a–j series (0.1 mL of 1–200 μg/mL concentration)
were mixed with 0.4 mL of 50 mM phosphate buffer (pH 7.4). Then,
0.6 mL H2O2 solution was added and the absorbance was read at
230 nm.
4.4.4. In-vivo lipid peroxidation assay
In-vivo lipid peroxidation in the compound 9f treated and control
tumor bearing animals were done as per the previously described
methods [41]. Briefly, the whole liver was dissected out and a 10%
tissue homogenate was prepared in ice cold 100 mM PBS pH-7.2. Liver
tissue homogenate of the control and 9f treated were the estimation of
lipid peroxidation.
4.3.3. Superoxide scavenging assay
4.4.5. Assays for in-vivo hepatic antioxidant enzyme
Compound 9a–j series was subjected to scavenging activity for su-
peroxide anion radical (O2.−) generated by NADH/PMS system ac-
cording to previous methods with slight modifications [39]. Shortly, the
reaction mixture consisted of 9a–j series (1–200 μg/mL concentration),
166 μM NADH, 107.5 μM NBT and 2.7 μM PMS dissolved in 19 mM of
potassium phosphate buffer (pH 7.4). After 5 min of incubation at
25 °C, absorbance was measured at 560 nm using eppendorf spectro-
photometer.
Hepatic antioxidant enzyme Superoxide dismutase and catalase
were estimated in the 9f treated and control tumor bearing animals as
per the previous protocols [31]. Liver tissue homogenate of control and
9f treated mice were used for the estimation of enzyme levels.
4.4.6. VEGF-ELISA
The in-vivo secretion of VEGF was quantified in serum of control and
9f treated DLA bearing mice by ELISA as per described protocols
[42,43]. On note, 100 μL of serum from the control and 9f treated were
coated in a coating buffer at 4 °C overnight. Subsequently, wells were
incubated with anti-VEGF-A antibodies (Sigma Aldrich, USA), followed
by re-incubation with secondary antibodies tagged to alkaline phos-
phatase. VEGF-A was measured by reading the absorbance at 405 nm
using PNPP as substrate.
4.3.4. In-vitro lipid peroxidation assay
The effect of compound 9a–j series on in-vitro lipid peroxidation was
performed by the methods as described earlier by measuring thio-
barbituric acid reactive substances (TBARS) with slight modifications
[40]. Analyses were done using total mice liver homogenate in cold
50 mM phosphate buffer (plus KCl 125 mM). The reactions were started
by addition of Fe(II) to solutions (0.5 mL final volume) containing
10 mM phosphate (pH 7.2), KCl 125 mM, 5% v/v liver homogenate,
compound 9a–j and H2O2. This was followed by the addition of 0.2 mL
7% phosphoric acid plus 0.2 mL TBA solution to the reaction mixture.
After 15 min of boiling, solutions were cooled and absorbance was read
at 532 nm.
4.5. Statistical analysis
Values were expressed as mean
standard error (SEM). Statistical
significance was evaluated by one-way analysis of variance (ANOVA)
followed by Student’s t-test (*p < 0.05) and (**p < 0.01).
Acknowledgements
4.4. Anti-tumor activity and angiogenesis studies
Zabiulla gratefully acknowledges the financial support provided by
the Department of Science and Technology, New Delhi, Under INSPIRE-
Fellowship scheme [IF140407]. Shaukath Ara Khanum thankfully ac-
knowledges the financial support provided by VGST, Bangalore, under
CISEE Programme [Project sanction order: No. VGST/CISEE/282]. B.T.
Prabhakar gratefully acknowledges the grant extended by DBT (6242-
P37/RGCB/PMD/DBT/PBKR/2015), UGC(F.No.41-507/2012 (SR) and
VGST (VGST/GRD231/CISEE/2013-14).
4.4.1. Animal and ethical statement
Swiss albino mice aged 5–6 weeks, weighing 25–30 g were housed
under standard laboratory conditions and fed with commercial rodent
meal and water ad libitum. All the animal experimentations were ap-
proved by the Institutional Animal Ethics Committee (IAEC), National
College of Pharmacy, Shimoga, India, in accordance with the
Committee for the Purpose of Control and Supervision of Experiments
427

Zabiulla et al.
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