Unveiling novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinolines as GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation valuations; development of a new class of anticancer drugs with thrombolytic effects

Article abstract of DOI:10.1016/j.bioorg.2018.09.011

In the present study, novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinolines (4a-l) were recognized and evaluated as G-Protein Coupled Receptor (GPCR) ligands through molecular evaluations. Thrombin mediates adhesion of mast cell, a type of cell abundantly found in connective tissue and releasing histamine and other substances during inflammatory and allergic reactions, through phosphoinositol 3-kinase pathway. With this background, as preliminary, 4a-l are resolute to be potential leads, designated from their effective phosphoinositol 3-kinase (PI3-Kinase) inhibition potentials, best-docked scores, comparative ligand efficiency, and significant structural attributes evaluated by ab initio simulations. Since thrombin is one of the main reason for various cancer invasion in association with PI3Kinase, a thrombolytic potential of the compounds also analyzed. The experimental in vitro studies confirmed the significant enhancement as PI3Kinase inhibitors and appreciable enhancement in MTT assay of breast and skin cancer cell lines. Significantly, acetophenone substituent in the quinoline scaffold could be coherent to note the significant binding affinity to all the evaluated drug targets.

Full text of DOI:10.1016/j.bioorg.2018.09.011

Accepted Manuscript
Unveiling novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinolines as
GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation val-
uations; Development of a new class of anticancer drugs with thrombolytic
effects
P. Thangarasu, S. Thamarai Selvi, A. Manikandan
PII:
S0045-2068(18)30688-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.011
YBIOO 2508
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 July 2018
26 August 2018
7 September 2018
Please cite this article as: P. Thangarasu, S. Thamarai Selvi, A. Manikandan, Unveiling novel 2-cyclopropyl-3-
ethynyl-4-(4-fluorophenyl)quinolines as GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation
valuations; Development of a new class of anticancer drugs with thrombolytic effects, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.09.011
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Unveiling novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinolines as
GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation
valuations; Development of a new class of anticancer drugs with thrombolytic
effects
Thangarasu Pa, Thamarai Selvi Sc,* and Manikandan Ab,**
aResearch and Development Centre, Bharathiar University, Coimbatore - 641046, India.
bPG and Research Department of Chemistry, Government Arts College, Coimbatore- 641018, Tamil Nadu, India.
cDepartment of Biotechnology, School of Bio-Sciences and Technology, VIT University, Vellore-632014, India.
Corresponding authors:* thamaraimohan@gmail.com; ** mailtomicromani@gmail.com
Abstract
In the present study, novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinolines (4a-l) were
recognized and evaluated as G-Protein Coupled Receptor (GPCR) ligands through molecular
evaluations. Thrombin mediates adhesion of mast cell, a type of cell abundantly found in
connective tissue and releasing histamine and other substances during inflammatory and allergic
reactions, through phosphoinositol 3-kinase pathway. With this background, as preliminary, 4a-l
are resolute to be potential leads, designated from their effective phosphoinositol 3-kinase (PI3-
Kinase) inhibition potentials, best-docked scores, comparative ligand efficiency, and significant
structural attributes evaluated by ab initio simulations. Since thrombin is one of the main reason
for various cancer invasion in association with PI3Kinase, a thrombolytic potential of the
compounds also analyzed. The experimental in vitro studies confirmed the significant
enhancement as PI3Kinase inhibitors and appreciable enhancement in MTT assay of breast and
skin cancer cell lines. Significantly, acetophenone substituent in the quinoline scaffold could be
coherent to note the significant binding affinity to all the evaluated drug targets.
Keywords: Anti-inflammatory; Anticancer; GPCR Ligands; Molecular docking; MTT assay;
PAR1; PI3Kinase; Quinolines; XRD.

Introduction:
Due to the presence of highly expressible affinity to various proteins, which are involving in a
disease’s instigation and development, makes quinoline and their derivatives are acting as an
important class of compounds in new drug design and development. Quinoline and its fused
heterocyclic compounds are biologically as well as medicinally significant functional entities
[1,2]. So far, a wide range of quinoline-based derivatives were targeted as anticancer [3],
antibiotic [4], anti HIV [5], anti-mycobacterial [6], antimalarial [7], anti-inflammatory [8],
antihypertensive [9], antibacterial [10] and cardiovascular [11] agents. Present work deals about
the preparation of novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinoline and its related
compounds to evaluate them as effective G-Protein Coupled Receptor (GPCR) ligands through
Protease-Activated Receptor-1 (PAR-1) expressions. To achieve the targeted quinoline
derivatives, we have approached two methods namely Ohira-Bestmann synthesis [12, 13] and
Sonogashira coupling methods [14, 15].
Comparatively, according to our experimental experience, Ohira-Bestmann synthesis was
reliable. For this reason, we opt Ohira-Bestmann synthesis and followed by Sonogashira
Coupling methods to achieve novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinoline
derivatives (Fig. 1) 4a-l and the method was most reliable and economical. A prediction based
preliminary screening against the most potent bioactivity possessed by 4a-l executed. Open
bioactivity prediction online-sites like Molinspiration and PASSonline used for the prediction.
Molinspiration, a proficient system for the calculation of drug-likeness score, allows predicting
GPCR ligands, ion channel modulators, kinase inhibitors, nuclear receptor ligands, protease
inhibitors and other enzyme targets (http://www.molinspiration.com/cgi-bin/properties).

PASSonline selecting the prospective compounds for High-throughput screening
(http://www.pharmaexpert.ru/ PASSonline/predict.php).
Figure 1. 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinoline derivatives
Figure
2.
Bioactivity
prediction
data
of
2-cyclopropyl-3-ethynyl-4-(4-
fluorophenyl)quinolines (4a-l)
Fig. 2 depicts the vicissitudes of 4a-l towards all the above-mentioned categories. In the
first look, GPCR ligand potency (data set in blue) of all compounds (4a-l) and impotency of 4f
towards all activity was recognized. The dataset (Fig. 2) in black are the obtained possible

enzyme inhibition potential values of 4a-l. This reveals the possible antagonism effects of 4a-l
against any enzyme that are highly responsible for numerous diseases and their developments.
The derived kinase inhibition values of 4a-l prescribed to execute a selective kinase mechanism
based biological activity where the GPCR playing a vital role. Protease-activated receptor 1
(PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (Gq/11, Gi/o, and
G12/13) and their linked signaling pathways are regulating a broad range of pathophysiological
processes [16]. Most importantly, the coagulation and fibrinolytic systems contribute to
malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis
[17]. Tissue factor (TF) is the cell surface receptor for coagulation factor VII/VIIa (FVII/VIIa).
TF is expressed on various cell types and its expression is up-regulated by oncogenic
transformation, conferring a procoagulant phenotype to cancer cells [18,19].
Methods and Materials
All organic chemicals and solvents procured from Sigma-Aldrich, Merck, and Spectrochem.
Solvents (commercial) and chemicals used as received without doing any further purification.
For thin-layer chromatography (TLC) analysis, Merck pre-coated plates (silica gel 60 F254) used
and spots visualized under UV light. Merck silica gel 60 (230-400 mesh) was used for flash
column chromatography and the eluting solvents are indicated in the procedures. Melting point
(mp) determinations performed using an OptiMelt automated melting point system and are
1
13
uncorrected. H NMR (400 MHz) and C NMR (100 MHz) spectra were recorded on Bruker-
400 MHz instrument. Mass spectra recorded on Agilent ion trap MS and Infrared (IR) spectra
recorded on Perkin Elmer FT-IR spectrometer. HPLC analysis of all the compounds carried out
on an Agilent 1260 series HPLC with an Agilent Extend-C18 column.

Synthesis of title compounds, quinoline derivatives (4a-l)
In the present study, the final quinoline derivatives (4a-l) were achieved from 2-cyclopropyl-3-
ethynyl-4-(4-fluorophenyl)quinoline (3). To achieve compound 3, we have used Ohira-
Bestmann synthesis, a single step process. Dimethyl-2-oxopropylphosphonate 6.84 g (0.12 mol),
was added to the mixture of potassium carbonate 21.31 g ( 0.45 mol) and 1H-imidazole-1-
sulfonyl azide hydrochloride 9.35 g (0.13 mol) in acetonitrile (20 ml). The mass was stirred for 2
hr at 25 °C. Then added 10 g of 2-cyclopropyl-4-(4-fluorophenyl)quinoline-3-carbaldehyde (0.10
mol) in methanol (10 ml) to the above suspension, the mass was stirred for 15 hours at 25 °C.
The reaction was completed as indicated by TLC and the mixture was filtered, washed the bed
with ethyl acetate 50 ml and concentrated the filtrate at 50-55 °C under vacuum. 50 ml of water
was added, the product was extracted in ethyl acetate (4* 50 ml), and the combined organic
layers were washed with brine (2* 50 ml), dried with sodium sulfate, filtered and concentrated at
50-55 °C under vacuum. Later stripped up with 10 ml of isopropyl alcohol. Then IPA 30.0 ml
added and the temperature raised to 50-55 °C. The mass stirred at 55-60 °C for 30 mins, the
temperature reduced to 25-30 °C. The mass was stirred again for another 2-3 hours at 25-30 °C.
The mass was filtered and washed the wet cake with 5 ml of IPA, then dried to get pure
solid (5.91 g, 60 %), 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinoline (3). Melting point:
1
115-117 °C. IR (KBr): 3306, 3066, 3009, 2082, 1445, 1224 cm-1. H NMR: NMR (400 MHz,
DMSO-d6) δ ppm: 1.13-1.15 (d, 2H, J=8.0), 1.20 (s, 2H), 2.86 (m, 1H), 4.49 (s, 1H), 7.36-
13
7.51(m, 6H), 7.70-7.74 (t, 1H, J= 8), 7.89-7.91 (d, 1H, J=8). C NMR (100 MHz, DMSO-d6) δ
ppm: 11.10, 15.28, 79.99, 89.78, 115.04, 115.29, 115.54, 124.46, 125.66, 126.31, 130.33,
131.75, 132.35, 146.13, 149.77, 160.88, 162.46, 163.32.MS (EI, m/z): 288.1 (M+1). HPLC

purity: 96.35 %. Anal calcd for C20H14FN: C,83.60; H, 4.91; F 6.61; N, 4.87 %; Found: C,
83.61; H, 4.90; F, 6.60; N, 4.88 %.
General procedure for the preparation of derivatives of 2-cyclopropyl-3-ethynyl-4-(4-
fluorophenyl) quinoline derivatives (4a-l) using Sonogashira Coupling: To a solution of 0.5 g
compound 3 (0.10 mol) in 3 ml DMF, 3.3 mg (0.01 mol) of copper iodide, 0.57 g cesium
carbonate (0.1 mol), 20 mg (0.01 mol) Tetrakis (triphenylphosphine) palladium(0) was added
under nitrogen atomophere. Then aryl halide (4-12) 0.11 mol was added under nitrogen atm, the
mass was stirred for 8 hrs at 75 °C under nitrogen atm. Reaction was completed as indicated by
TLC and the reaction mass was filtered through cellite bed, washed the bed with ethyl acetate
(10 ml). The product layer (Ethyl acetate layer)was then washed with water 10.0 ml, sodium bi
sulphite 10 ml followed by saturated sodium chloride solution (5.0 ml) and dried over (Na2SO4).
The solvent was distilled off and the resulting product was purified by column chromatography
using ethyl acetate and n-hexane to get the pure product (4a-l). Yield observed was around 85-90
% for all the derivatives.
1-(3-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)phenyl)ethanone (4a): The aryl
halide 3- Bromo aceto phenone was used to prepare 4a. Melting point: 143.4-145.2 °C. IR
(KBr): 3063, 2997, 2212, 1427, 1384, 1681, 1512, 1222, 812 cm-1. 1H NMR: NMR (400 MHz,
DMSO-d6) δ ppm: 1.17-1.19 (d, 2H, J= 7.2), 1.24 (s, 2H), 2.56 (s, 3H), 2.94-2.95 (m, 1H), 7.44-
7.52 (m, 6H), 7.54-7.60 (m, 2H), 7.69-7.72 (m, 2H), 7.89-7.93 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ ppm: 11.19, 15.59, 26.60, 54.83, 87.27, 97.03, 115.15, 115.36, 122.44, 124.42,
125.62, 126.30, 128.43, 128.58, 129.21, 130.29, 130.48, 131.94, 132.02, 132.42, 135.04, 137.03,
146.33, 149.42, 160.94, 161.93, 163.38, 196.98. MS (EI, m/z): 406.3 (M+1). HPLC purity:
99.80 %. Anal calcd for C28H20FNO: C,82.94; H, 4.97; F,4.89; N, 3.45, O, 3.95 %; Found:

C,82.95; H, 4.99; F, 4.87; N, 3.47, O, 3.96 %; HRMS Calculated [M+] m/z 405.4724, Found
405.4724.
1-(4-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)phenyl)ethanone (4b): The
aryl halide 4-Bromo aceto phenone was used for the preparation of 4b. Melting point: 165.1-
1
166.5 °C. IR (KBr): 3068, 3001, 2210, 1677, 1438, 1356, 1222, 834 cm-1. H NMR: NMR (400
MHz, DMSO-d6) δ ppm: 1.18-1.20 (d, 2H, J= 8), 1.25 (s, 2H), 2.57 (s, 3H), 2.93-2.94 (m,1H) ,
7.37-7.39 (d, 2H, J=8), 7.42-7.49 (m, 4H), 7.51-7.61 (m, 2H), 7.72-7.75 (m, 1H), 7.88-7.98 (m,
13
3H). C NMR (100 MHz, DMSO-d6) δppm: 11.20, 15.65, 26.72, 30.65, 89.43, 97.13, 115.21,
115.43, 124.44, 125.75, 126.44, 128.47, 128.63, 130.51, 131.18, 131.95, 132.04, 146.45, 149.63,
162.04, 197.17. MS (EI, m/z): 406.5 (M+1). HPLC purity: 99.66 %. Anal calcd for C28H20FNO:
C,82.94; H, 4.97; F, 4.89; N, 3.45, O, 3.95 %; Found: C,82.92; H, 4.95; F, 4.89; N, 3.47, O, 3.97
%; HRMS Calculated [M+] m/z 405.4724, Found 405.4725.
3-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)benzaldehyde (4c): Aryl halide 3-
Bromo benzaldehyde was used for the preparation of 4c. Melting point: 117.6-119.6 °C. IR
1
(KBr): 3057, 3003, 2215, 1697, 1435, 1383, 1223 cm-1. H NMR: NMR (400 MHz, DMSO-d6)
δ ppm: 1.17-1.19 (d, 2H, J= 8), 1.24 (s, 2H), 2.95 (s, 3H), 7.36-7.45 (m, 4H), 7.51-7.53 (d, 1H,
13
J=8.0), 7.56-7.62 (m, 3H), 7.70-7.73 (m, 1H), 7.87-7.91 (m, 2H), 9.97 (s, 1H). C NMR (100
MHz, DMSO-d6) δ ppm: 11.15, 15.60, 87.61, 96.57, 115.17, 115.25, 115.39, 122.89, 124.40,
125.65, 126.32, 128.59, 129.57, 129.65, 130.33, 130.71, 131.61, 131.91, 131.99, 132.34, 136.35,
146.33, 146.36, 149.36, 160.95, 162.00, 163.31, 192.32. MS (EI, m/z): 392.2 (M+1). HPLC
purity: 90.80 %. Anal calcd for C27H18FNO: C,82.85; H, 4.63; F, 4.85; N, 3.58; O, 4.09 %;
Found: : C,82.87; H, 4.65; F, 4.82; N, 3.60, O, 4.07 %; HRMS Calculated [M+] m/z 391.4454,
Found 391.4456.

3-((4-chlorophenyl)ethynyl)-2-cyclopropyl-4-(4-fluorophenyl)quinoline (4d):Aryl halide 4-
Bromo chloro benzene was used for the preparation of 4d. Melting point: 151.3-153.2 °C. IR
1
(KBr): 3042, 3008, 2210, 1439, 1416, 1225, 1013 cm-1. H NMR: NMR (400 MHz, CDCl3) δ
ppm: 1.17-1.19 (d, 2H, J=8), 1.23-1.24(d, 2H, J=4), 2.91-2.95 (m, 1H), 7.27-7.29 (d,2H, J=8),
7.43-7.50 (m, 6H), 7.56-7.60 (m, 2H), 7.71-7.75 (m, 1H), 7.90-7.92 (d, 1H, J=8). 13C NMR (100
MHz, CDCl3) δ ppm: 10.80, 15.97, 87.77, 97.07, 115.09, 115.31, 116.29, 121.50, 125.13,
125.82, 125.87, 128.73, 129.11, 129.74, 131.78, 131.86, 132.45, 133.0, 134.54, 147.01, 149.25,
162.59, 163.95. MS (EI, m/z): 398.3 (M+2). HPLC purity: 94.52 %. Anal calcd for C26H17ClFN:
C,78.49; H, 4.31; Cl 8.91; F, 4.78; N, 3.52 %; Found: C,78.48; H, 4.32; Cl 8.93; F, 4.77; N, 3.51
%; HRMS Calculated [M+] m/z 397.8774, Found 397.8774.
2-cyclopropyl-4-(4-fluoro phenyl)-3-(phenyl ethynyl) quinoline (4e): The aryl halide, Iodo
benzene was used for the preparation of 4e. Melting point: 101.2-103.0 °C. IR (KBr): 3059,
1
3005, 2205, 1600, 1487, 1442, 1218, 1093, 1024 cm-1. H NMR: NMR (400 MHz, CDCl3) δ
ppm: 1.16-1.18 (d, 2H, J= 8), 1.24 (s, 2H), 2.94 (m, 1H),7.25-7.26 (d, 2H, J=4) , 7.36-7.44 (m,
3H), 7.54-7.57 (m, 3H), 7.63-7.82 (m, 2H), 7.85-7.90 (m, 1H), 8.69-8.72(d, 2H, J=12). 13C NMR
(100 MHz, CDCl3) δ ppm: 11.00, 15.52, 86.35, 97.92, 115.06, 115.27, 115.64, 121.96, 124.43,
125.53, 126.18, 128.53, 128.63, 128.92, 129.13, 130.05, 130.62, 130.92, 131.85, 131.93, 132.06,
132.44, 146.19, 148.84, 161.94, 163.30. MS (EI, m/z): 364.4 (M+1). HPLC purity: 94.85 %.
Anal calcd for C26H18FN:C,85.93; H, 4.99; F, 5.23; N, 3.85 %; Found: C,85.95; H, 4.98; F, 5.24;
N, 3.86 %; HRMS Calculated [M+] m/z 363.4354, Found 363.4353.
3-((6,7-bis(2-methoxyethoxy)quinolin-2-yl)ethynyl)-2-cyclopropyl-4-(4-fluorophenyl)
quinoline (4f): The aryl halide 2-chloro-6,7-bis(2-methoxyethoxy)quinoline was used for the
preparation of 4f: Melting point: 133.8-135.6 °C. IR (KBr): 3059, 2983, 2200, 1443, 1180 cm-1.

1H NMR: NMR (400 MHz, CDCl3) δ ppm: 1.19-1.22 (m, 2H), 1.43-1.44(m, 2H), 3.04-3.08 (m,
1H), 3.51 (s, 6H), 3.86-3.90 (m, 4H), 4.06-4.08 (t, 2H, J=8), 4.31-4.33 (t, 2H, J=8), 7.02 (s, 1H),
7.23-7.28 (m, 3H), 7.39-7.43 (m, 1H), 7.53-7.58 (m, 3H), 7.68-7.72 (m, 1H), 8.00-8.02 (d, 1H,
J=8), 9.11 (s, 1H). 13C NMR (100 MHz, CDCl3) δ ppm: 11.29, 16.07, 59.32, 68.62, 68.84,
70.33, 70.53, 93.73, 94.29, 104.86, 107.57, 114.75, 115.44, 115.65, 121.25, 124.86, 126.05,
130.47, 131.75, 131.83, 132.47, 147.53, 148.34, 150.23, 150.59 , 153.75, 156.10, 161.56, 163.25,
164.03. MS (EI, m/z): 564.3 (M+1). HPLC purity: 97.37 %. Anal calcd for C35H31FN2O4:
C,74.72; H, 5.55; F, 3.38; N, 4.98; O, 11.37 %; Found: C,74.74; H, 5.55; F, 3.6; N, 4.99; O,
11.39 %; HRMS Calculated [M+] m/z 562.6414, Found 562.6414.
2-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)-4-fluorobenzoic acid (4g): Aryl
halide used for the preparation of compound 3g is 2-Bromo 4-fluoro benzoic acid. Melting point:
181.2-182.7 °C. IR (KBr): 3435, 3017, 2853, 1728, 1654, 1515, 1246cm-1. 1H1 NMR: NMR
(400 MHz, CDCl3) δ ppm: 1.18-1.19 (m, 2H), 1.20 (s, 2H), 2.95-3.01 (m, 1H), 7.45-7.51 (m,
4H) , 7.58-7.62 (m, 2H), 7.62-7.70 (m, 2H), 7.73-7.76 (m, 1H), 7.91-7.93 (d, 1H, J=8), 8.00 (s,
13
1H), 13.59 (s, 1H, D2O exchangable). C NMR (100 MHz, CDCl3) δ ppm: 11.31, 15.62, 88.73,
95.42, 115.02, 115.17, 115.39, 121.81, 124.37, 125.70, 126.38, 128.61, 130.47, 131.97, 132.31,
133.38, 136.91, 146.48, 149.87, 162.06, 165.07. MS (EI, m/z): 426.40 (M+1). HPLC purity:
95.57 %. Anal calcd for C27H17F2NO2: C, 76.23; H, 4.03; F, 8.93; N, 3.29; O, 7.52 %; Found: C,
76.24; H, 4.02; F, 8.93; N, 3.30; O, 7.53 %; HRMS Calculated [M+] m/z 425.4348, Found
425.4349.
4-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)-3-methylbenzoic acid (4h): The
ayl halide 4-Bromo 3-methyl benzoic acid wasused for the preparation of 4h. Melting point:
202.1-204.5 °C. IR (KBr): 3433, 3070, 3006, 2201, 1692, 1653, 1488, 1263, 1224 cm-1.1H NMR:

NMR (400 MHz, DMSO-d6) δ ppm: 1.17-1.19 (d, 2H, J=8), 1.25 (s, 2H), 2.07 (S, 3H), 2.94-2.95
(m, 1H), 7.39-7.48 (m, 5H) , 7.53-7.56 (m, 2H), 7.61-7.64 (m, 2H), 7.66-7.79 (m, 1H), 7.90-7.92
(d, 1H, J=8), 13.01 (s, 1H, D2O exchangable). 13C NMR (100 MHz, DMSO-d6) δ
ppm:10.90,15.66, 19.51, 92.60, 96.13, 115.35, 115.57, 124.60, 125.67, 126.11, 126.35, 126.63,
128.56, 130.19, 130.35, 130.66, 131.65, 132.61, 139.34, 146.26, 149.23, 161.00, 161.99, 166.67.
HPLC purity: 90.74 %. MS (EI, m/z): 564.3 (M+1). Anal calcd for C28H20FNO2: C,79.79; H,
4.78; F, 4.51; N, 3.32; O, 7.59 %; Found: C,79.80; H, 4.77; F, 4.53; N, 3.30; O, 7.58 %; HRMS
Calculated [M+] m/z 421.4714, Found 421.4716.
4-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)benzoic acid (4i):Aryl halide 4-
Bromo benzoic acid was used for the preparation of compound 4i. Melting point: 196.2-198.1
1
°C. IR (KBr): 3448, 3056, 3004, 2204, 1680, 1420, 1224 cm-1. H NMR: NMR (400 MHz,
DMSO-d6) δ ppm: 1.18-1.24 (d, 4H), 2.3 (m, 1H), 7.37-7.46 (m, 8H), 7.57-7.71 (m, 1H), 7.91
(m, 3H), 13.13 (s, 1H, D2O exchangable). 13C NMR (100 MHz, DMSO-d6) δ ppm: 11.05, 15.59,
89.0, 97.07, 115.14, 115.23, 115.35, 124.39, 124.65, 126.17, 126.31, 128.58, 129.47, 130.34,
130.73, 131.03, 131.87, 131.95, 132.27, 146.40, 149.39, 160.93, 162.01,166.51. MS (EI, m/z):
408.5 (M+1). HPLC purity: 94.93 %. Anal calcd for C27H17FNO2: C,79.59; H, 4.45; F, 4.66; N,
3.44; O, 7.85 %; Found: C,79.58; H, 4.46; F, 4.68; N, 3.45; O, 7.86 %; HRMS Calculated [M+]
m/z 407.4444, Found 407.4445.
2-bromo-6-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)benzoic acid (4j): Aryl
halide 2-Bromo 5-iodo benzoic acid was used for the preparation of 4j. Melting point: 210.2-
211.8 °C. IR (KBr): 3449, 3061, 3004, 2204, 1676, 1420, 1227 cm-1. 1H NMR: NMR (400 MHz,
DMSO-d6) δ ppm: 1.16-1.20 (m, 2H), 1.24 (s, 2H), 2.95-3.01 (m, 1H), 7.45-7.51 (m, 4H) , 7.58-
7.62 (m, 2H), 7.67-7.70 (m, 2H),7.73-7.76(t, 1H, J=6.4), 7.91-7.93 (d, 1H, J=8.4), 8.00 (s, 1H),

13.18 (s, 1H, D2O exchangable).13C NMR (100 MHz, DMSO-d6) δ ppm: 11.31,15.62, 88.73,
95.42, 115.02, 115.17, 115.39, 121.81, 124.37, 125.76, 128.61, 130.47, 132.97, 132.31, 133.38,
136.91, 146.84, 149.87, 162.06, 165.07. MS (EI, m/z): 486.6 (M+2). HPLC purity: 94.90 %.
Anal calcd for C27H17BrFNO2: C,66.68; H, 3.52; Br, 16.43; F, 3.91; N 2.88; O, 6.58 %; Found:
C, 66.70; H, 3.54; Br, 16.44; F, 3.90; N, 2.89; O, 6.59 %; HRMS Calculated [M+] m/z
486.3404, Found 486.3405.
2-chloro-5-((2-cyclopropyl-4-(4-fluorophenyl) quinolin-3-yl)ethynyl) benzoic acid (4k): The
ayl halide 5-Bromo 2-chloro benzoic acid was used for the preparation of 4k. Melting point:
181.2-182.7 °C. IR (KBr): 3431, 3075, 3012, 1725, 1556, 1512, 1419, 1193, 1171, 1041cm-1. 1H
NMR: NMR (400 MHz, CDCl3) δ ppm: 1.18-1.20 (d, 2H, J=8), 1.24 (s, 2H), 2.89-2.95 (m, 1H),
7.36-7.38 (d, 2H, J=8), 7.43- 7.51 (m, 4H), 7.57 -7.61 (m, 4H), 7.73-7.76 (m, 1H), 7.91-7.93 (d,
13
1H, J=8), 13.65 (s, 1H, D2O exchangable). C NMR (100 MHz, CDCl3) δ ppm: 11.19, 15.60,
59.68, 88.21, 95.89, 115.17, 115.37, 121.02, 124.40, 125.67, 126.37, 128.59, 130.41, 131.25,
131.93, 132.01, 132.30, 132.91, 134.27, 146.39, 149.53, 160.95, 161.99, 163.40, 165.72. MS (EI,
m/z): 442.3 (M+1). HPLC purity: 97.41 %. Anal calcd for C35H31FN2O4: C,74.72; H, 5.55; F,
3.38; N, 4.98; O, 11.37 %; Found: C,74.74; H, 5.56; F, 3.39; N, 4.97; O, 11.35 %; HRMS
Calculated [M+] m/z 441.8864, Found 441.8866.
1-(2-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)ethynyl)phenyl)ethanone (4l): Aryl
halide 2-Bromo aceto phenone was used for the preparation of 4l. Melting point: 177.5-178.5 °C.
IR (KBr): 3067, 3004, 2204, 1767, 1438, 1384, 1221 cm-1. 1H NMR: NMR (400 MHz, DMSO-
d6) δ ppm: 1.21-1.29 (m, 4H), 3.21 (m, 1H), 7.22-7.24 (d, 1H, J=8), 7.47-7.54 (m, 5H), 7.56-
13
7.60 (m, 3H), 7.76-7.77 (m, 1H), 7.85-7.87 (d, 1H, J=8), 7.94-7.96 (d,1H,J=8). C NMR (100
MHz, DMSO-d6) δ ppm: 10.90, 11.09, 14.71, 15.43, 28.82, 90.72, 96.93, 115.17, 115.39, 115.57,

115.75, 120.00, 124.48, 125.63, 126.26, 127.24, 128.55, 128.87, 129.07, 130.26, 131.57, 131.92,
132.00, 132.19, 132.41, 133.70, 139.44, 146.31, 149.17, 160.94, 162.50, 163.38, 198.80. MS
(EI, m/z): 406.2 (M+1). HPLC purity: 90.08 %. Anal calcd for C28H20FNO: C,82.94; H, 4.97; F,
4.89; N, 3.45, O, 3.95 %; Found: C,82.93; H, 4.90; F, 4.88; N, 3.46, O, 3.94 %; HRMS
Calculated [M+] m/z 405.4724, Found 405.4726.
Single crystal ORTEP and DFT studies
Single-crystal X-ray diffraction data were collected using Oxford XCalibur, Gemini
diffractometer equipped with EOS CCD detector at 25 ºC with MoKα radiation (λ= 0.71073 Aᵒ).
The data reduced using CrysAlisPro Software and multi-scan absorption correction applied using
multi Ψ-scans. The structural model was refined by full-matrix least squares against F2 using the
SHELXL-97 software. Non-hydrogen atoms were refined with anisotropic thermal parameters.
All hydrogen atoms assigned fixed Uiso values, equal to 1.2Ueq of the parent atom for ring
hydrogen atoms. Crystallographic data for the structure had deposited with the Cambridge
Crystallographic Data Centre (CCDC No.:1542607). Molecular graphics done by ORTEP 3 and
that of molecular packing with MERCURY [20]. The quantum chemical calculations performed
by DFT at B3LYP/LAN l2dz level of basis set, using the Gaussian 09 program package [21].
Optimized parameters such as bond lengths and bond angles measured using a molecular
visualization package CHEMCRAFT [22].
Interrelation of thrombin, PARs and PI3 Kinase and the experiment details of PI3Kinase
inhibition activity assay
Thrombin intercedes most of its cellular activities over the triggering of a series of G protein-
coupled receptors (GPCRs). These GPCRs are nothing but the protease-activated receptors

(PARs). These PARs are mainly expressed in the exterior region of almost all cell types [23,24].
In such a condition, Phosphoinositide 3-kinases (PI3 Kinases), an intracellular signal
transducer enzymes, play a vital role in regulating functional responses in a variety of
hemopoietic cells, including neutrophils, monocytes, mast cells, eosinophils, and B- and T-cells
[25]. In a recent research, thrombin found as the mediator of mast cell adhesion through the
activation of phosphoinositol 3-kinase pathways [26]. Thus, the small molecules that were
inhibiting PI3 Kinases could be the effective anticancer medications. In the present study, the
inhibitory potentials of the quinoline derivatives (4a-l) were assessed using Glutathione-coated
96 well plate assay as we reported earlier [27-30]. The kinase and the quinoline compounds 4a-l
were pre-incubated for 10-15 minutes before adding PIP2 (Phosphatidylinositol biphosphate)
substrate. 5 µL of 5X Kinase reaction buffer was added followed by the addition of 5 µL/well of
PIP2 substrate to each well. Double distilled water was added each well to make up the final
volume as 25 µL/well. Further, the reaction mixture incubated at 37 ºC for 60 minutes. The
absorbance read at 450 nm. The relative % to b-PIP3 was calculated using the following formula,
OD of samples (buffer, kinase & inhibitors)
-----------------------------------------------------
OD of B-PIP3 average
x 100
% inhibition =
Molecular docking studies
Based on the bioactivity predictions (Fig. 2), the efficiency of the compounds towards GPCR
ligand and kinase inhibition was initially assessed through molecular docking. Both are highly
involved in many cancer invasions through thrombin. Protease-activated receptor 1 (PAR1) is
the typical adherent element of a family of G-protein-coupled receptors that facilitate cellular
activities to thrombin and associated proteases. To get insight into the mode of binding to GPCR
and Phosphoinositide Kinase (PI3Kinase), the ligands (4a-l) were docked into the active site of

the X-ray crystallography structures of GPCR and PI3Kinase. The PDB structure of PAR1, PDB
ID: 3vw7 (https://www.rcsb.org/structure/3VW7) and PI3Kinase, PDB ID: 3T8M
(https://www.rcsb.org/structure/3T8M). We achieved the docking process by referring our
previous reports with necessary changes that were needed for the present study [31,32].
In-vitro thrombolytic study
The best PI3Kinase inhibitors of this study compound 4a, 4b 4g, and 4j were analyzed for their
thrombolytic effects. The thrombolytic study carried out as reported earlier with necessary
modifications [32]. The coagulation and fibrinolytic systems contribute to malignancy by
increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis [33]. The
mechanism by which coagulation proteases exert their tumor-enhancing effects is, in part, via
activation of protease-activated receptor PAR-1 and PAR-2 [34, 35]. Protease-activated receptors
(PAR) mediated crosstalk among coagulation and fibrinolysis was studied by Troy et al. 2010
[33]. PARs belong to the GPCR family that proteolytically triggered by various proteases.
Stimulation of fibrinolysis by plasmin through the infusion of tissue plasminogen activator
facilitating the thrombolysis.
A stock suspension of streptokinase was prepared (100 μL (30,000 I.U)) and used for in
vitro thrombolysis. 5 ml blood was collected from each healthy volunteers from different age
groups (human, n = 4). The blood was transferred (750 μL) to each of Eppendorf tubes to form
clots by incubating at room temperature for 1 h. Serum was aspirated by not disturbing the clot.
The weight of blood containing Eppendorf tubes measured before and after the clot formation
even after the removal of serum. After administering the compounds 4a, 4b 4g and 4j in various
concentrations, tubes were incubated at room temperature for 120 minutes and the fluid

formation was observed. Tubes yet again weighed after removing fluid that has formed to
recognize the variation in weight after the clot distraction. The variations in weight measured
before and after clot lysis stated as a percentage. Streptokinase (thrombolytic) and Millipore
water (non-thrombolytic) served as standard and control, respectively.
Platelet aggregation and adhesion assay
Platelet aggregation and adhesion assay evaluated for the best PI3Kinase inhibitor of this study
(i.e. compound 4a). Platelet aggregation was determined by a turbidimetric method with a dual
channel aggregometer (Model 591/592, Chrono-Log Corp., Havertown, PA, USA) [36]. Shortly,
250 µL of platelet-rich plasma (PRP) taken along with various concentrations of quinoline
derivatives (25-250 µg/mL) in glass cuvette and pre-incubated for 3 min at room temperature.
The aggregation was commenced by adding collagen (1.5 µg/mL)/ADP (5 µM)/epinephrine (5
µM) and constantly stirred for 10 min (1200 rpm) [37]. Platelet adhesion assay carried out
according to previous reports [38, 39]. In a 96-well microtiter plate, the collagen immobilized by
adding 25 µg of collagen type I in 250 µL of 10 mM (pH 7.4) phosphate buffered saline (PBS) to
each well and incubated overnight at 4 °C. Further, to block the wells, 250 µL of 1 % (w/v)
Bovine Serum Albumin (BSA) in PBS added and incubated at room temperature for 1 h. The 96
plate wells then washed thrice with PBS.
Initially, quinoline derivatives (25-250 µg/mL) added to the collagen-coated wells and
incubated for 15 min, prior to adding PRP, wells washed thrice with PBS. Then, the PRP pre-
treated quinoline derivatives (25-250 µg/mL) added to the collagen-coated wells. Each well of
reaction mixture volume adjusted to 250 µL by adding PBS and incubated at 37 °C. After 2
hours of incubation, the well washed thrice with PBS. Further, the adherent platelets were lysed

with 200 µL lysis buffer (100 mM citrate buffer (pH 5.4) having 0.1 % Triton X-100 and 5
mM p-nitrophenyl phosphate) at 37 °C for 90 min. The reaction arrested by adding 200 µL of 2N
NaOH (stopping reagent) which is inactivating the platelet membrane acid phosphatase activity
and the resultant color produced measured at 405 λmax. Platelet adhesion was expressed in %
adhesion, by considering the PBS-added platelet suspension as 100 percent.
HRBC based hemolysis assay (Hypotonicity induced hemolysis)
Thrombin playing an important role in acute and chronic inflammation [40-42]. Thrombin can
amplify inflammation induced by other stimuli, either through ischemia. Thrombin, a key factor
in coagulation and inflammation, typically elicits cellular responses via activation of protease-
activated receptors (PARs). Thrombin has a well-characterized pro-inflammatory action that has
recently been suggested to occur via activation of its receptor, the proteinase-activated receptor-1
(PAR1) [43,44]. The anti-inflammatory potential of quinoline derivatives evaluated by means of
hypotonic induced (using distilled water) hemolysis assay [45-48]. The hypotonic solution (5 ml)
having sorted the dosages of the quinoline derivatives (25-250 μg/ml) was used to assess the
hemolysis in triplicates. 5 ml of distilled water (vehicle) and another 5 ml of indomethacin (250
μg/ml) used as the control respectively. 100 µL of Erythrocyte suspension was added to each
tube, mixed thoroughly, and incubated for 60 min at 37 °C. Further, the mixture centrifuged for 5
min at 1200 g. The hemoglobin content of the supernatant measured at the absorbance (OD)
range of 540 nm. The percentage hemolysis calculated by considering the hemolysis formed in
distilled water as 100 %. The percent inhibition of hemolysis by the extract calculated thus:
% Inhibitionꢀofꢀhemolysis = 1− (Ab2−Ab1/Ab3−Ab1) ×100
Where,
Ab1 = absorbance of the test sample in isotonic solution

Ab2 = absorbance of the test sample in hypotonic solution
Ab3 = absorbance of control sample in hypotonic solution.
Cytotoxicity assessment of quinoline derivatives by MTT assay
The cell viability after reacting with the proposed quinoline derivatives was assessed by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]. The current procedure adopted
from our previous reports and modified accordingly [27-30]. In a 96 well microtiter plate, the
Platelet-Rich Plasma (PRP) treated with 10 µM Calcium Ionophore and quinoline derivatives
(25-250 µg/mL). PBS used to make the final volume of the reaction mixture up to 150 µL and
incubated at room temperature for 60 minutes, 150 µM of MTT was added and incubated for
another three hours. MTT separated and the obtained formazan crystals liquefied in DMSO and
absorbance recorded at 570 λmax using Tecan Sapphire multi-fluorescence microplate reader. The
cell viability was calculated using the following formula,
Cell viability (%) = Mean OD/Control OD x 100 %
Statistical analysis
All results were expressed as a percentage increase or decrease with respect to control values. All
results were compared by performing one-way ANOVA with Dunnett’s post-test. GraphPad
Prism version 7.1 for Windows, GraphPad Software, USA (www.graphpad.com) was used for
statistical analysis.
Results and Discussion:
Synthesis of proposed 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinoline (3):

There are two synthetic routes were exploited to access desired compound 3. In scheme-1,
compound 3 prepared by Ohira-Bestmann reaction [49], in this one-pot synthesis, aromatic
aldehyde was converted to alkyne. To achieve this, Ohira-Bestmann reagent was prepared from
dimethyl-2-oxopropylphosphonate and imidazole-1-sulfonyl azide in the presence of K2CO3 in
ACN followed by addition of the aldehyde (1) in methanol. After the reaction completed, the
crude product was crystallized using isopropyl alcohol to get pure compound (3).
F
O
O
P
O
O
O
O
MeOH
rt,15h
N3
+
N2
S
N
N
O
3
MeCN,K2CO3,
rt, 2h
P
O
O
. HCl
O
O
N
Ohira-Bestmann reagent
Scheme 1. Synthesis of proposed 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinoline (3)
using Ohira-Bestmann reaction
Synthesis of substituted 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinoline derivatives
(4a-l) from (3):
The derivatives of 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinoline (3) was achieved as per
the synthetic route displayed in Scheme 3 using Sonogashira coupling method. Table 1 depicts
the substituents that were substituted on the scaffolds of 2-cyclopropyl-3-ethynyl-4-(4-
fluorophenyl)quinoline (3). Compounds obtained in excellent yield up to 90 %. In addition, the
molecular structure of the compound 3 was established by single-crystal X-ray diffraction
studies. Then compound 3 subjected to Sonogashira Cross-Coupling Reaction (Scheme 2) with
different aryl halides to get analogs of biologically active molecules [50-55]. The
characterization data of newly synthesized compounds summarized in the experimental section.
The computational studies carried out to confirm the medical efficacy of the molecule [56, 57].

Scheme 2. Synthesis of title compounds (4a-l) using Sonogashira Coupling
Table 1. Details of a to l of final products of scheme 3 (Sonogashira coupling)
S.No Subtract (ArX)
Product a
b
c
d
1
2
3
4
5
3-Bromo acetophenone
4a
4b
4c
4d
4e
H
CH3CO
H
CHO
H
H
H
H
H
H
H
H
4-Bromo acetophenone
3-Bromo benzaldehyde
4-Bromo chloro benzene
Iodobenzene
H
H
H
H
CH3CO
H
Cl
H
2-chloro-6,7-bis-(2-
6
4f
-
-
-
-
methoxyethoxy)quinoline
2-Bromo 4-fluoro benzoic acid
4-Bromo 3-methyl benzoic acid
4-Bromo benzoic acid
2-Bromo 5-iodo benzoic acid
5-Bromo 2-Chloro benzoic acid
2-Bromo aceto phenone
7
8
9
10
11
12
4g
4h
4i
4j
4k
4l
COOH
CH3
H
H
H
H
H
H
COOH
COOH
H
H
F
COOH
COOH
Br
Cl
H
H
H
H
H
H
CH3CO
The ORTEP representation with 50 % probability shown in Fig. 3, which clearly depicts a stable
crystalline lattice structure of CEFQ. The single crystal X-ray studies (XRD) have shown that the
crystal structure is triclinic with space group compound 3 (Table S1 & S2). The optimized
molecular geometry of the isolated CEFQ molecule calculated using DFT at B3LYP/LAN l2dz
level of theory and shown in Fig.4a&b. The calculated bond length and angles listed in Table S3
and compared with SXRD values. It found that most of the calculated values are higher than the
experimental values because theoretical calculations carried for isolated gaseous state molecule
whereas experimental ones are crystalline nature. Normally the C=N bond length in a
heterocyclic ring is greater than 1.34 Å [58].

Figure 3.ORTEP structural analysis of 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinoline (3).
Figure 4a&b. optimized geometry of CEFQ for 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)
quinoline (3).
But in CEFQ the C=N (C9-N1) bond length is reduced to 1.336 Å due to the substitution
of cyclopropyl ring at the second position. The C-C bond lengths are in the range of 1.397-1.412
Å and 1.389-1.439 Å in fluoro-substituted benzene ring and quinoline aromatic ring respectively.
In the case of a heterocyclic ring, the length is 1.439 Å for C10-C15, C7-C15 bonds, 1.404Å for
C7-C8 and 1.454 Å for the C8-C9 bond. The weakening of C8-C9 bond length is mainly due to
the substitution of cyclopropyl and ethyne group at respective carbon atoms. The ipso C-C-C

angles of such as C8-C7-C15 (118.9ᵒ), C7-C8-C9 (119.3ᵒ) and C8-C9-N1 (121.7ᵒ) of quinoline
ring show deviation from the normal C-C-C (120ᵒ) value. Among these, the positive deviation
(1.7ᵒ) of C8-C9-N1 is mainly due to the ring strain of cyclopropyl ring and the negative deviation
(1.1ᵒ) of C8-C7-C15 attributed to a fluoro-substituted benzene ring.
3.2 Molecular electrostatic potential (MEP)
Molecular electrostatic potential is one of the useful ways to explore the electrostatic
interaction in hydrogen bonding and to study the biological recognition process [59].
Electrostatic potential (ESP) correlates dipole moment, electronegativity, partial charges at the
site of chemical reactivity of the molecule. In ESP diagram, the negative electrostatic potential
(red color) corresponds to an attraction of the proton by the high electron density in the molecule
and the positive electrostatic potential (blue color) corresponds to the repulsion of the proton by
atomic nuclei in regions where low electron density exists and the nuclear charge is incompletely
shielded. This electrostatic potential surface depicts the size, shape, charge density and
electrophilic and nucleophilic reactivity site of the molecule. The increasing order of electron
potential is blue < green < yellow < orange < red.
It inferred from the ESP diagram of CEFQ shown in Fig. 5 that the maximum negative
potential noted over heterocyclic nitrogen atom N1 and F1 that favors the electrophilic attack.
The proton affinity of these atoms clearly visible from red region and mostly assisted by a lone
pair of electrons. Therefore, these sites route the hydrogen bond interactions. The positive
potential (blue color) shows the acceptor nature. The maximum positive region is located on the
ethyne CH and aromatic CH, this confirms the nucleophilic attacking nature of these atoms.

From MEP, it inferred that N1, F1 atoms would bind with the electrophilic centers in proteins
while the aromatic and ethyne CH groups favorably attacked by the nucleophile of the protein.
Figure 5. ESP diagram of CEFQ
3.3 Mulliken charge analysis
The Mulliken population analysis of atomic charge calculated by the density functional
theory method. Fig. 6 shows the plot of the Mulliken atomic net charges in CEFQ. The negative
charge accumulation over the F1 (-0.2429 e) and N1 (-0.0594 e) atoms show its electron
donating nature. It clearly recognized by MEP shows negative potential over these atoms. All
hydrogen atoms are a positive charge, out of which atom H9 (0.2844 e) possess the highest
value. Usually, the hydrogen atom of the ethyne group is highly polarized one due to its
attachment with high electronegative nature of terminal carbon. The large accumulation of
negative charge on the carbon C17 (-0.4762 e) reveals its electronegative nature. Similarly, atom
C1 (0.3172 e) possess the highest value amongst the positively charged carbon atoms owing to
its association with the electronegative F1 atom. The cyclopropane ring carbons C19, C20 (-
0.4514 e) also possess a high negative charge also reveals its electronegativity.

Figure 6. The plot of the Mulliken atomic net charges in CEFQ
3.4 Frontier molecular orbital analysis
In CEFQ, both HOMO and LUMO are delocalized over the quinoline ring, cyclopropyl
and ethyne fragment but maximum electron density is spread over the aromatic C=C. HOMO-
LUMO energy gap is found to be 4.467 eV (Fig. 7). The HOMO→LUMO transition implies an
electron density transfer occur within quinoline ring and cyclopropyl fragment, especially within
aromatic conjugation of CEFQ. DFT based descriptors help to understand the structure of
molecules and their reactivity. Using HOMO and LUMO orbital energies, the ionization energy
and electron affinity can be expressed as I = -EHOMO, A = -ELUMO [60] and the global chemical
reactivity descriptors of molecules such as hardness (η), chemical potential (μ)[61], softness(S),
electronegativity (χ), electrophilicity index (ω) and additional electronic charge ΔNmax have been
defined [62]. The hardness of the molecule is, η = (I-A)/2. The chemical potential of the
molecule is, μ = - (I+A)/2. The softness of the molecule is, ζ =1/2η; Electronegativity of the
molecule is, χ = (I+A)/2; The electrophilicity index of the molecule is, ω = μ2 /2η; Additional
electronic charge, ΔNmax = - μ/ η; Total energy change, ΔET = η/4.

Figure 7. HOMO-LUMO energy gap of compound 3
Table 3. Frontier molecular orbital analysis of 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)
quinoline (3)
Descriptions
E HOMO
E LUMO
Band gap energy, ΔE
Ionization potential, I
Electron affinity, A
Hardness, η
Values
-6.424 eV
-1.956 eV
4.467 eV
6.424 eV
1.956 eV
2.234
Chemical potential, μ
Softness, ζ
Electrophilicity index, ω
Electronegativity, χ
Additional electronic charge, ΔN max
Total energy change, ΔET
-4.19
0.2238
3.929
4.19
1.875
0.5585
Table 3 presents the energies of frontier molecular orbitals, ionization potential, electron
affinity, hardness, chemical potential, softness, electrophilicity index, electronegativity and
additional electronic charge of CEFQ. The smaller energy gap of CEFQ implies its low stability
and it more polarizable which is associated with a high chemical reactivity. As it has low
stability, CEFQ referred to as a soft molecule. The chemical hardness of the molecule related to
the polarizability because a decrease of the energy gap makes easier polarization of the molecule
[63]. The negative value of chemical potential indicates it is the ability of a system to resist the
deformation of electron cloud under small perturbation during the chemical process. The
hardness and softness of the molecule depending on the molecular orbital energies.

Electrophilicity index measures the stabilization energy when the system acquires an additional
electronic charge from the surrounding and it resists the system to exchange electronic charge
with the surrounding.
Molecular docking studies
Molecular docking study was executed in order to recognize the binding affinity and molecular
interaction between present study quinoline derivatives (4a-l) to PAR1 (PDB ID: 3VW7) and
PI3 Kinase (PDB ID: 3T8M). Least binding energy, binding affinity (ki, the inhibitory constant)
and ligand efficiency considered in narrowing the drug efficacy of 4a-l. Table 4 depicts the
obtained molecular mechanistic values from molecular docking studies. Initially, to scrutinize
the most efficacies compounds and most selectively scored receptors; we have summated and
averaged the molecular mechanistic values. With an average least binding energy (-10.03
kcal/mol), ligand efficiency (-0.35) and binding affinity (0.56 µM), compounds 4a-l showed a
most fortunate activity selectiveness over PI3 Kinase (PDB ID: 3T8M). Meanwhile, we have
also observed noteworthy molecular mechanistic values against PAR1 (PDB ID: 3VW7).
Figure 8a.Molecular interaction of 4a to PI3 Kinase (PDB ID: 3T8M) Figure 8b. Compound 4a
into the binding pocket (active site) of PI3 Kinase (PDB ID: 3T8M)

Except for 4d and 4h, remaining all compounds were showed good binding affinity with
an inhibitory constant (ki) range of 0.056 to 0.813 µM. Based on this molecular docking study
results, we have screened the compounds 4a-c, 4e-g, and 4i-l for further PI3 kinase enzyme
inhibition studies. In addition, we have validated the molecular interactions of best-docked
compounds to corresponding receptors PI3 Kinase (PDB ID: 3T8M). Fig.8a&b is depicted as the
pictorial representation of the molecular interaction between PI3 Kinase (PDB ID: 3T8M) and
most potent PI3 Kinase inhibitor (4a) among 4a-l. The established non-covalent (π-π interaction
between aryl ring of 4a to the Phe113 amino acid residue of PI3 Kinase) and hydrogen bond
(between the oxygen moiety of acetoxy group of compound 4a and PI3 kinase) was the source of
evidence to the drug action of 4a (Fig. 8a). The inhibition, perhaps, facilitated by these non-
covalent and hydrogen bonding. In the acetoxy group, the acetyl end is a weekly deactivating
group, thus, the possibility of further biological reaction also less. One of the –OH[+] group of
Serine being the donor and the acceptor is acetoxy group’s –O[-] of 4a (Fig. 8b).
Table 4. Molecular mechanistic values of molecular docking results
Inhibitory constant (ki)
Binding energy (kcal/mol)
Ligand efficiency
µM)
Entry
3VW7
-6.99
-8.27
-7.94
-5.96
-6.35
-7.11
-8.01
-4.16
-7.66
-9.28
-7.91
-7.71
-87.35
-7.27917
3T8M
-11.73
-11.12
-10.86
-7.21
3VW7
-0.23
-0.27
-0.25
-0.21
-0.23
-0.17
-0.26
-0.25
-0.27
-0.27
-0.21
-0.29
-2.91
-0.2425
3T8M
-0.44
-0.41
-0.40
-0.25
-0.28
-0.37
-0.44
-0.23
-0.31
-0.43
-0.30
-0.32
-4.18
-0.34833
3VW7
7.52
0.868
3.56
42.46
22.25
6.11
1.4
68.45
2.44
0.392
1.024
2.62
159.094
13.25783
3T8M
0.056
0.086
0.094
1.242
0.468
0.174
0.075
2.512
0.357
0.060
0.842
0.813
6.779
0.564917
4a
4b
4c
4d
4e
4f
4g
4h
4i
-9.06
-10.20
-11.33
-7.60
-9.98
-11.63
-9.82
-9.87
-120.41
-10.0342
4j
4k
4l
Sum
Average

Results of PI3 Kinase inhibition studies
In recent days, to prevent arterial ischemic incidence, targeting GPCRs mediated intracellular
signaling downstream with PI3kinase being the novel tactics [64]. In fact, even though the
mechanism is unclear, thrombin proteolytic effect of PAR-1activates PI3K and Akt, known to
play an essential role in proliferation (Fig. 9) [65]. Thus, the inhibition of PI3Kinase is essential
to prevent the downregulation of cellular activity through GPCRs and PAR-1 activities. In this
study, as indicated by molecular docking results, PI3Kinase inhibition activity evaluated for the
compounds 4a-c, 4e-g, and 4i-l. The assay repeated thrice in triplicates and the compounds
diluted at various concentration (25-250 µg/mL). The results were expressed in relative %
activity and the IC50 values in µM. We used Wortmannin as the standard drug to assess the
inhibitor/drug efficacy of these compounds. In a recent study, Wortmannin showed 55.4 %
thrombin-induced basal phosphorylation reduction of Thr308 with 70.1 % thrombin inhibition
[65].
In addition to this, Akt (a serine-threonine kinase) was found as part of a Wortmannin-
sensitive signaling pathway in the downstream activity of PI3kinase [66,67]. To unveil the PI3
kinase inhibition efficacy of compounds 4a-c, 4e-g, and 4i-l, we have analyzed the inhibition
assay results in a various point of views. Fig. 10a-d illustrating the results of relative % activity,
dose-responsive activity, 50 % amount of compound needed to have maximum inhibition
activity (IC50) and the comparison of in silico and in vitro activity result’s resemblances. The
relative percentage activity was measured by testing the inhibitory potentials of compounds and
the standard drug at various concentrations (n=4). As depicted in Fig. 10a compounds 4a, 4b,
and 4j showed slightly increased activity while remaining compounds showed lesser activity
than the standard, Wortmannin. With a maximum of 72.57±5.84 % (mean value) of % inhibition

was the best achieved by 4a than Wortmannin (mean value of 67.25±3.84 %). Compounds 4b
and 4j were also showed higher values as 68.98±4.87 % and 68.52±3.94 % respectively than the
standard. We found a dose depended on increased activity up to a concentration of 10-6 (Fig.
10b).
Figure 9. The role of GPCRs in the activation of PI3Kinases, known to play an essential role in
proliferation. Note: GPCRs involving in the activation of PI3K to the plasma membrane and it catalyzes the formation of
PIP3 from phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). PIP3 acts on a range of downstream effectors to promote cellular
responses including proliferation and migration by involving in the Akt/mTOR activation.
In that concentration, 4a was able to inhibit the PI3Kinase up to 90.85±2.86 % while the
standard showed little lower value (86.12±3.48 %). The calculated IC50 values (mean) displayed
in Fig. 10c. 4a was able to inhibit PI3Kinase with the lowest molar quantity (IC50 = 0.45±0.025
µM). Wortmannin has reached its maximum activity at an IC50 of 0.75±0.05 µM while
compounds 4b and 4j were required almost the same amount of IC50 of the standard. We
displaying Fig. 10d to compare the in silico and in vitro results and to validate the reliability of
the proposed PI3Kinase activity. As showed in Fig. 10d, compounds 4a, 4b, 4g, and 4j were best

compounds to score significant molecular mechanistic values also found best for the PI3Kinase
activity. Thus, the reliability between the in silico and in vitro activity found virtuous.
In vitro thrombolytic activity results of compounds 4a, 4b, 4g, and 4j
Thrombin is one of the main factors in the development of various diseases. Thrombin induces
PAR-1 gene expression in endothelial cells via activation of Gi-linked Ras/Mitogen-activated
protein kinase pathway [68]. In the present study, the clot lysis activity of compounds 4a, 4b, 4g,
and 4j assessed to quantify their thrombolytic effects. After treating the compounds and standard
drug, the dry weight of the vial considered as the result by discarding the serum released due to
the thrombin degradation in every 15 minutes.
Figure 10a-d. Results of PI3Kinase inhibition assay. Figure 10a. Relative percentage activity
results of compounds 4a-c, 4e-g,4i-l and Wortmannin. Figure 10b. Dose-response curve of
compounds 4a and Wortmannin. Figure 10c. Calculated IC50 (µM) values of compounds 4a-c,
4e-g,4i-l, and Wortmannin. Figure 10d. Molecular mechanistic values of docking results in order
to compare in vitro and in silico results reliability.