Synthesis of phthalazine derivative based organic nanoflakes in aqueous solvent as a potential nano-anticancer agent: A new approach in medical field

Article abstract of DOI:10.1016/j.molstruc.2019.127156

A simple phthalazine derivative 13-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-1H-indazolo[1,2-b] phthalazine-1,6,11(2H,13H)-trione (IDP) was synthesized by Knoevenagel condensation with Michael addition reaction. The present articles deals with preparation

Full text of DOI:10.1016/j.molstruc.2019.127156

Journal Pre-proof
Synthesis of phthalazine derivative based organic nanoflakes in aqueous solvent as a
potential nano-anticancer agent: A new approach in medical field
Saubai B. Wakshe, Shilpa R. Patil, Audumbar D. Patil, Prafulla B. Choudhari,
Sandeep B. Patil, Prashant V. Anbhule, Daewon Sohn, Govind B. Kolekar
PII:
S0022-2860(19)31265-7
DOI:
https://doi.org/10.1016/j.molstruc.2019.127156
MOLSTR 127156
Reference:
To appear in: Journal of Molecular Structure
Received Date: 10 July 2019
Revised Date: 5 September 2019
Accepted Date: 28 September 2019
Please cite this article as: S.B. Wakshe, S.R. Patil, A.D. Patil, P.B. Choudhari, S.B. Patil, P.V. Anbhule,
D. Sohn, G.B. Kolekar, Synthesis of phthalazine derivative based organic nanoflakes in aqueous solvent
as a potential nano-anticancer agent: A new approach in medical field, Journal of Molecular Structure
(2019), doi: https://doi.org/10.1016/j.molstruc.2019.127156.
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Synthesis of phthalazine derivative based organic nanoflakes in aqueous solvent as a
potential nano-anticancer agent: A new approach in medical field
1
1
1
2
Saubai B.Wakshe , Shilpa R. Patil , Audumbar D.Patil , Prafulla B. Choudhari , Sandeep B.
1
1
3
1,3
Patil , Prashant V.Anbhule , Daewon Sohn *, Govind B. Kolekar *
1
Fluorescence Spectroscopy Research Laboratory, Department of Chemistry, Shivaji University,
Kolhapur, 416004, MS, India
2
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Near
Chitranageri, Kolhapur 416013, MS, India
3
Department of Chemistry, Hanyang University, Seoul 04763, Republic of Korea
*
Corresponding author gbkolekar@yahoo.co.in , dsohn@hanyang.ac.kr
1

Abstract:
A simple phthalazine derivative 13-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-1H-
indazolo[1,2-b]phthalazine-1,6,11(2H,13H)-trione (IDP) was synthesized by Knoevenagel
condensation with Michael addition reaction. The present articles deals with preparation of
highly fluorescent nanoparticles as prepared drug (IDP) by simple reprecipitation method using
single aqueous solvent. The DLS measurement predicted the 150 nm size of a synthesized IDP
NPs. Furthermore UV-Visible and fluorescence spectroscopy measurement showed a distinct
peak for IDP NPs from their parent molecule. Scanning Electron Microscopy (SEM)
microphotograph of air dried film of nanoparticles exhibited nanoflakes type and feathery
morphology. The highly fluorescent nature of IDP NPs ultimately revealed in high quantum
yield than IDP parent molecule. These fluorescent organic nanoflakes then further applied as
anticancer agent. Interestingly it showed better activity than IDP molecules. Thus this is the first
report to use organic nanoflakes as an anticancer agent, which may open new avenues in field’s
medical and related sciences.
Keywords: IDP Synthesis, Fluorescent Organic nanoparticle, Photophysical properties,
anticancer activity, Molecular docking
2

1
. Introduction:
The stream of phthalazine nitrogen containing heterocycles has been attracted a terrific
interest due to its interesting pharmacological properties and clinical applications. Present
hydrazine moiety in phthalazine is ‘fusion site’ and its backbone for drug design. Its more
importance as building block for key intermediates in the synthesis of many natural products
with a broad range of pharmacological and biological activities including anti-inflammatory [1-
2
], antimicrobial [3], anticancer agents and antitumor compounds [4], antimalerial [5],
anticonvulsant [6], cardiotonic [7], together with unique electrical and optical properties[8-9].
Indazolo phthalazine derivatives can be prepared by one pot, three component condensations of
aldehydes, pthalalhydrazide and dimedone using various catalysts such as Ce(SO4)2·4H2O, p-
TSA[10-11]. In all the reported methods contains harsh reaction conditions and volatile
solvents are used. To overcome such drawbacks, herein we have used 50% aq. hydrotropic
solution for the synthesis of 13-(4-bromo-phenyl)-3,3 dimethyl 2,3,4,13 tetrahydro indazolo(1,2-
b) phthalazine -1,6,11-trione (IDP).The spectroscopic methods such as UV-Visible, FT-IR
and NMR were utilized for the structure elucidation of IDP. By considering fluorescence
properties of organic molecules, detailed spectral analysis was also possible to study with
the aid of modern spectroscopic tools such as single molecule spectroscopy, single molecule
imaging and fluorescence correlation single molecule spectroscopy [12-13]. In continuation
with our previous efforts to introduce fascinating aspects, we have developed an efficient,
inexpensive method for the synthesis of Indazolo phthalazine (IDP) derivatives.
Nowadays nanoparticles has attracted great attention because of potential applications and has
high biological activity, biodegradability [14], good accessibility, larger surface area, excellent
stability and cost-effectiveness [15-16]. Recently nanoparticles have been focused prevailing
3

challenge in medical field. The sensitivity of organic drug nanoparticles has huge interest in life
sciences, chemical sciences and biotechnology [17-18] Nanoparticles pulled in extraordinary
consideration as a result of their potential applications in biomolecules, pharmacology and
furthermore it has optical and electronic properties [19]. Fluorescent Organic Nanoparticles
(
FONS) have most of the wide applications in vitro and in vivo imaging [20] because they allow
much more variability and flexibility in material synthesis, emissive properties, very good water
solubility for nanoparticles preparation [21-22]. Water amongst many other solvents is the
most suitable for biological molecules for example proteins and amino acids [23] .There are
several methods used for the preparation of FONS include emulsification [24], evaporation,
diffusion, and laser ablation [25] .
Parent IDP material has poor stability in deionized water and its spectrally red shifted, less
intense fluorescence emission could be enhanced by using water soluble polymers. Our intention
is to make surface modification of bulk bioactive organic compound via nanoparticles
preparation. Hence we have synthesized IDP NPs through simple reprecipitation method in
aqueous media. It is very popular and strong influence for the preparation of organic drug
nanoparticles because it’s simple and versatile operation [26] .
The synthesized nanoparticles exhibited characteristic properties different than that of
organic bulk compound. The prepared IDP NPs were characterized for its spectroscopic and
surface morphology. The enhanced effect of IDP NPs compared to bulk molecules investigated
for anticancer activity. The possible positions of IDP binding with tubulin were also investigated
by molecular modeling study. Molecular docking and spectroscopic analysis were used to
estimate the relation between the biological activities and chemical structure [27].
4

2
. Experimental
2
.1 Preparation of IDP drug
The required IDP compound for the study was synthesized by following previous reports
[10-11]. The three-component condensation reaction consist of 4-bromobenzaldehyde (1 mmol),
phthalahydrazide (1 mmol) and dimedone (1 mmol) (Scheme1) added in 50% aq. hydrotropic
solution. A reaction mixture was stirred for 2 hours at room temperature. After completion of the
reaction, yellow product was filtered by using Whatman filter paper. The crude product was then
washed with chloroform and mother liquor was collected in watch glass. The obtained final
product was recrystallised by using ethanol, chloroform as solvent. Whole reaction was
accompanied by establishment of drop on TLC plate by using ethyl acetate as mobile phase.
1
The structure of the product was confirmed by their spectral properties like IR, H NMR,
1
3
C NMR etc and melting point was observed at 266°C determined by using open capillary.
Br
O
CHO
O
O
O
O
O
NH
NH
5
0% Aq. hydrotrope
+
+
N
N
O
Br
1
2
3
4
Scheme 1: Synthesis of 13-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-1H-indazolo[1,2-b]
phthalazine-1,6,11(2H,13H)-trione
2
.2 Synthesis of IDP Nanoparticles
Rapid, easy and proficient method carried out in laboratory to synthesize IDP
-4
nanoflakes. A 5 mL solution of drug IDP in acetonitrile (1×10 M) was quickly injected into the
2
0 mL water using micro injector. Complete solution constantly thrilled using magnetic stirrer
5

for 24 hrs and for 1 hr it was sonicated at 30°C. The aqueous suspension of IDP nanoparticles
was used for further characterization and morphology studies.
2
.3 Material Characterization
The NMR (Brucker 300 MHz) and IR (Shimadzu IR-450 FT-IR spectrophotometer)
spectra of prepared compounds was recorded by using tetra methyl silane as an internal standard
and KBr pallets respectively. Fluorescence spectra of IDP NPs were monitored at its excitation
wavelength and recorded on Spectrofluorometer (JASCO, Japan, and model FP-8300) by using
double distilled water. Absorption spectra were measured using UV-3600 UV-Visible
spectrophotometer (Analytic jena). SEM (JEON-6360 Japan) was carried to examine the
morphology and size of nanoparticles. DLS histogram was recorded by using Malvern Zetasizer
(nano ZS-90).
2
.4 Procedure for determination of quantum yield
The more commonly used method for the determination of quantum yield of the
fluorescence involves comparison with a standard material reflects without loss of exciting
wavelength. Quantum yield depends on degree on particle synthesis and surface
modification. Fluorescence spectra were measured on a spectrofluorimeter and absorption
spectra were recorded on a dual beam UV-Visible spectrophotometer. Quantum yield were
recorded at room temperature. The above model gives correct excitation spectra through
the use of quinine sulphate in 0.1M H SO as reference. The excitation source in this set up
2
4
xenon lamp was used. The excited sample makes fluorescence emission in all directions. A
mirror placed at the sample is used to reflect light into the emission monochromator. A 10
6

mm pathlength cuvette was placed in the reflected beam and synchronously scanning the
excitation and emission at the same wavelength. The ultraviolet absorption was measured
using the method described under experimental section. The three different solutions i.e.
Quinine sulphate, IDP in acetonitrile and aqueous suspension of IDP NPs were prepared
and diluted to give absorbance in the range 0.09-0.1 absorbance unit. The absorption
spectra of quinine sulphate were observed at 350 nm while bulk IDP and nanoflakes shows
3
88 nm and 281nm respectively. Emission spectra of bulk IDP and IDP NPs were observed
at 470 and 453, 480 respectively. The fluorescence intensity of bulk IDP was observed 995
while nanoflakes showed 8205.
2
.5 MTT Assay
5
Cells were incubated at a concentration of 1 × 10 cells/ml in culture medium for 24 h at
3
7°C and 5% CO . Cells were seeded at a concentration (70ꢀl) 105 cells /well in 100 ꢀl culture
2
medium and 100ꢀl isolated compounds and 0.1ml of 5-FU into micro plates respectively (tissue
culture grade and 96 wells). Control wells were incubated with DMSO (0.2% in PBS) and cell
line. All samples were incubated in triplicate. Controls were maintained to determine the control
cell survival and the percentage of live cells after culture. Cell cultures were incubated for 24 hr
at 37°C and 5% CO in CO incubator. After incubation, the medium was completely removed
2
2
and added 20 ꢀl of MTT reagent (5mg/ml in PBS). After addition of MTT, cells incubated for 4
o
hrs at 37 C in CO incubator. The wells for formazan crystal formation observed under
2
microscope. The yellowish MTT was reduced to dark coloured formazan by viable cells only.
After removing the medium completely, 20ꢀl of DMSO Added (kept for 10 min) and incubate at
7

3
7 C (wrapped with aluminium foil). Triplicate samples were analyzed by measuring the
absorbance of each sample by a micro plate reader at a wavelength of 450 nm.
2
.4.1. Principle Brine Shrimp lethality bioassay
It is simple, fast, economical and widespread biological assay for biologically active
synthesized and ordinary molecules. This technique employs here in vivo lethality by using
Brine nauplii is suitable observer for selecting in the innovation of biologically active
synthesized and ordinary molecules. The cytotoxicity of Brine nauplii interrelated with 9KB
(p=0.036 and kappa = 0.56). It simply uses a greater number of animals for arithmetical
validation and no necessity of instruments. Moreover, there is no need of animal serum for
cytotoxicities. The solution of sea water prepared by weighing and dissolving 38 gm of marine
salt in 1 L distilled water.
2
.4.2. Hatching of Brine Shrimp
Brine shrimp eggs gathered from pet shops was utilized as the test living being. Sea water
was taken in the little tank and shrimp eggs were added to one side of the little tank and then this
side was enclosed. Two days were permitted to hatch the shrimp and to be full grown as nauplii.
Consistent oxygen supply was carried out through the hatching time. The hatched shrimps are
pulled to the light and thus nauplii free from egg shell were gathered from the illuminated part of
the little tank. The nauplii was taken from the fish tank by a pipette and weakened in clean sea
water to enhance visibility and 10 nauplii were taken cautiously by micropipette.
2
.4.3 Counting of nauplii
8

The test tube was inspected via magnifying glass against black surroundings after 24
hours and the quantity of survived nauplii in every tube was counted. The data indicate that, the
percent of lethality of the brine shrimp nauplii was calculated for every concentration.
The efficiency of mortality relationship of product is frequently expressed as a middle
lethal concentration (LC-50). The concentration of the chemical that produces death in half of
the test subjects after a definite exposure time and calculated by linear regression method from
plotting % mortality against correspondent log of concentration revealed in (fig. 6).
2
.5 Molecular Docking Studies
Molecular docking analysis was performed to analysis binding mode of developed IDP
on the tubulin. Crystal structure of the tubulin (PDB ID 4O2B) was downloaded from the free
protein database www.rcsb.org . Protein structures are prepared prior to docking analysis via
removal of water, soap and addition of hydrogen atoms so that native geometry of the proteins is
retained. IDP structure is developed in 2D molecular builder of V life MDS 4.3. 2D structure of
IDP was converted in to the 3D structures and its energy was minimized via application of
Merck molecular force field (MMFF).
3
. Results and Discussion
3
.1. NMR and IR of IDP
1
3-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-1H-indazolo[1,2-b]phthalazine-
o
1
,6,11(2H,13H)-trione Yellow powder mp 266 C;
-1
IR spectra of IDP (KBr): νmax (cm ) 2955, 1650, 1619, 1464, 1375 (Figure S1).
9

1
H NMR (300 MHz, CDCl
3 2
): δ 1.24 (6H, s, 2Me), 2.35 (2H, s, CH C), 3.38 (2H,dd,
CH CO), 6.41 (1H, s, CHN), 7.28-8.37 (8H, m, Ph) (Figure S2).
2
1
3
C NMR (CDCl , 75 MHz) δ: 191.95, 156.01, 154.41, 151.08, 135.53, 134.57, 133.62,
3
1
31.90, 129.00, 128.81, 128.05, 127.75, 122.74, 118.05, 64.40, 50.94, 38.08, 34.64, 28.66, 28.41.
The above spectral data is shown in Supplementary materials (Figure S3).
3
.2 Particle size and morphology
The aqueous suspension of IDP NPs shows particle size histogram on dynamic light
scattering equipment. Histogram showed in (Fig.1) Indicates narrow particle size distribution
and average size diameter of IDP NPs and is about 150 nm. SEM images clearly indicates that
the surface morphology of IDP NPs (Fig. 2) having nanoflakes type and feathery in nature.
3
3
.3 Photophysical properties
.3.1 Absorption Spectra:
Spectral properties of prepared IDP NPs in aqueous suspension were further confirmed
by UV-absorption in (Fig.3). UV-Visible spectra showed a blue shift in aqueous state in
comparison with bulk materials. Absorption spectrum of dilute sample solution of monomer in
acetonitrile is at 388 nm while that of IDP nanoflakes peaked at 281 nm. Due to the presence of
conjugated phenyl rings and high number of delocalized π electrons revealed that the absorption
transition S →S is π→π*. Nanoparticles suspensions were blue shifted in absorption spectrum
0
1
from absorption spectrum of IDP parent solution .The hypsochromic shift and structured
absorption spectrum indicates IDP molecule aggregates in planar cluster and H-bonded
aggregation by π-stacking effects [28].
10

3
.3.2 Fluorescence spectra
The fluorescence emission and excitation spectra of IDP in diluted acetonitrile and
aqueous suspension of IDP NPs are shown in (Fig. 4) and (Fig. 5) respectively. The strong
upgraded emission 453 nm and 480nm proclaimed the most probable π*-π transition owing face
to face π stacking effects in nanoaggregates [29] . Low emission intensity of bulk IDP compound
was due to expenditure of energy for molecular rotation, while increased intensity of
nanoaggregates suspension is because of the molecular rotations hindered by adjacent molecules
and restricted nonradioactive decay thus allows radiative pathway of excited nanoaggregates.
Excitation spectra showed that IDP NPs suspension (red) and diluted solution of IDP in
acetonitrile (black) at 453, 480 nm respectively.
3
.3.3 Quantum Yield measurements
The fluorescence quantum yield (QY) is the fraction of photons absorbed resulting in
emission of fluorescence
ꢅꢆꢇ
ꢈ
ꢉꢂꢃꢄ
ꢀ
ꢁ = ꢀꢁꢂꢃꢄꢅ
ꢅ×
×
ꢇ
ꢆ
ꢉ
ꢈꢂꢃꢄ
ꢂꢃꢄ
Where
is the quantum yield, A is absorbance (also called optical density) at the
excitation wavelength,ꢅηꢇ andꢅη are the square of ref. index of the solvent used to dissolve
ꢇ
ꢊꢋꢌꢅ
samples (Acetonitrile =1.3404) and standard (0.1M H SO =1.7689) respectively. ꢅꢅAꢍꢎꢏꢅꢅꢅꢅis
2
4
absorbance ofꢅsandard,ꢅI is Fluorescence intensity of standard. By using Quinine sulphate in 0.1M
H SO as standard solution recorded absorption and fluorescence spectra. Quantum yield
2
4
calculated by using above formula. It was observed that IDP NPs suspension showed value 6.271
more fluorescent properties comparatively to bulk material having value 0.760. The obtained
results indicated that IDP NPs has higher quantum yield (6.271) than bulk materials (0.760).
11

3
.4 Anticancer activity against MCF-7 cell line (Human breast cancer)
From table. 1 it is clear that the sample nano IDP in 100 µL has greater quantity on MCF
-7 cell line and also seen by percent cell viability and cell inhibition. Whereas the value of cell
viability was less in nano IDP but percentage of cell inhibition was more compared to bulk IDP.
From the above data it was concluded that the sample nano IDP has greater penetration and
cytotoxicity on cancer cell line as compared to standard. From table 2 it was observed that the
sample nano IDP has high percentage of cytotoxicity because percentage of shrimp survival
number is less as compared to parent IDP but cytotxicity index of nano IDP is nearly equal to
standards. So it was concluded that the nano IDP has somewhat equivalent or slightly more
toxicity on shrimp as compared to IDP [30].
3
.5 Molecular Docking
The molecular docking is not possible for IDP nanoflakes because of their nano structure
nature hence we have employed as synthesized IDP compound for molecular docking to
ascertain the mode of action. (Described in S4 and shown in Figure S5 a,b) [31].
4
. Conclusion
In present study, we have synthesized drug nanoparticles via simple re-precipitation
method in aqueous media. The objective of research work was to synthesize drug nanoparticles
in aqueous media to develop simple, quick, efficient assay. DLS result showed a narrow particle
size distribution with average size 150 nm. SEM histogram of air-dried film of aqueous
suspension of IDP NPs showed that nanoflakes and feathery shaped morphology. The formation
of nanoparticles was also evidenced by absorption and fluorescence spectroscopy. Prepared IDP
NPs emitting blue shifted absorption. The QY of IDP NPs was much greater than the IDP bulk
12

materials. The anticancer activity against MCF-7 cell line reveals the enhancement of anticancer
activity of prepared IDP NPs comparatively with bulk organic compound using MTT assay.
Moreover, molecular docking analysis revealed that the IDP was found to be interacting with
tubulin which can be further explored for applications in cancer diagnostics and biomedical
research.
Acknowledgements
The author SBW thanks to Government of Maharashtra and Director, Directorate of
Technical education for granting permission for Ph.D. research work. This work was also
supported by the Brain Pool Program through the National Research Foundation (NRF) of Korea
(BP Grant No.: 2019H1D3A2A01057526).
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16

Figure Caption
1
) Fig. 1: Particle size distribution histogram of IDP NPs obtained by DLS analysis
2
3
) Fig. 2: SEM microphotograph of air dried film of IDP nanoparticles.
Fig. 3: UV-visible absorption spectra of IDP Nanoparticles (red) and IDP in diluted
acetonitrile (black).
) Fig. 4: Emission Spectra of IDP NPs suspension (red) and diluted solution of IDP in
acetonitrile (black) 453, 480 nm respectively
) Fig. 5: Excitation spectra of IDP NPs suspension (red) and diluted solution of IDP in
acetonitrile (black) at 360 nm and 388nm respectively
) Fig. 6: Photographs of micro plate (a) before Inhibition (b) after Inhibition
4
5
6
Figures
1)
2
)
17

3
)
4
)
18

5
)
6
)
19

Table Captions
1
) Table 1: Effects of isolated compound against MCF-7 cell line (Breast cancer cell
line) by MTT assay
) Table 2: Cytotoxic activity of IDP and Nano IDP against Brine shrimp lethality assay
2
Table 1:
Sr. No.
Concentration (ꢀg)
Optical Density Percentage of
cell viability
Percentage of
cell inhibition
-
1
2
3
Control
0.417
0.343
0.302
-
IDP - 100 µg/ml
Nano IDP - 100 µl
82.46
72.43
17.54
27.57
Table 2:
Drugs Conc. of Total
drugs number
Shrimp survived
Total No.
%
LC-50
of Shrimp inhibition (ꢀg)
of
Survived
shrimp
T1
10
10
T2
10
8
T3
9
IDP -
Nano
IDP -
100 µg/ml 10
100 µl 10
29
27
3.33
6.66
>1000
>1000
9
20

Highlights:
•
•
•
•
A simple, quick and selective method for synthesis of organic IDP NPs.
As synthesized IDP NPs are fluorescent and water soluble.
IDP NPs having particle size 150 nm and nanoflakes shape.
IDP NPs showed enhanced anticancerous activity compared to bulk IDP.