Discovery and synthesis of 2-amino-1-methyl-1H-imidazol-4(5H)-ones as GPCR ligands; an approach to develop breast cancer drugs via GPCR associated PAR1 and PI3Kinase inhibition mechanism

Article abstract of DOI:10.1016/j.bioorg.2019.02.048

Efforts were taken to synthesis and characterize 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives (4a-u) through a four-step reaction. The achieved compounds in remarkable yield have characterized through standard analytical techniques such as FTIR, LC

Full text of DOI:10.1016/j.bioorg.2019.02.048

Accepted Manuscript
Discovery and synthesis of 2-amino-1-methyl-1H-imidazol-4(5H)-ones as
GPCR ligands; an approach to develop breast cancer drugs via GPCR associated
PAR1 and PI3Kinase inhibition mechanism
S.R. Ashok, M.K. Shivananda, A. Manikandan, R. Chandrasekaran
PII:
S0045-2068(18)31568-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.048
YBIOO 2823
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 December 2018
16 February 2019
21 February 2019
Please cite this article as: S.R. Ashok, M.K. Shivananda, A. Manikandan, R. Chandrasekaran, Discovery and
synthesis of 2-amino-1-methyl-1H-imidazol-4(5H)-ones as GPCR ligands; an approach to develop breast cancer
drugs via GPCR associated PAR1 and PI3Kinase inhibition mechanism, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.02.048
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Discovery and synthesis of 2-amino-1-methyl-1H-imidazol-4(5H)-ones as
GPCR ligands; an approach to develop breast cancer drugs via GPCR
associated PAR1 and PI3Kinase inhibition mechanism
Ashok S.R1, Shivananda M.K1,*, Manikandan A2,** and Chandrasekaran R3
1Dept. Of Studies & Research in Chemistry, Tumkur University, Tumkur-572102, Karnataka, India.
2Dept. of Biotech, Vellore Institute of Technology (VIT), Vellore-632014, Tamil Nadu, India.
3Sai Supreme Chemicals, Gummidipoondi, Chennai-601201, Tamil Nadu, India.
* Corresponding author email: shivanmk@yahoo.com
** Corresponding author email: mailtomicromani@gmail.com
Abstract
Efforts were taken to synthesis and characterize 2-amino-1-methyl-1H-imidazole-4(5H)-one
derivatives (4a-u) through a four-step reaction. The achieved compounds in remarkable yield
have characterized through standard analytical techniques such as FTIR, LC-MS, NMR,
HRMS, and elemental analysis. Present study mainly aimed to evaluate 4a-u as G protein-
coupled receptors (GPCR). In the mechanism, stimulation of phosphoinositide 3-
kinase (PI3K) and Akt (protein kinase B) is a general reaction activated by a series of
membrane-bound receptors such as GPCR. Protease-activated receptor-1 (PAR1) is a
subfamily of related GPCR, which triggered by the division of fragment of its extracellular
domain. Therefore, molecular docking is done to ensure the inhibition of PAR1 and
PI3Kinase. PI3Kinase is a chief enzyme in the development of breast cancer via the
Akt/mTOR pathway. Thus, in vitro PI3Kinase inhibition and anti-breast cancer studies has
also done to screen medicinally important compounds among (4a-u). Based on the best
binding affinity, in vitro relative % activity and IC50 values, compounds 4a, 4g, 4i, 4n, and 4u
were screened for further preclinical studies in animal model evaluations.
Keywords: 2-amino-1-methyl-1H-imidazol-4(5H)-one; Anticancer; GPCR; Molecular
docking; PAR1; PI3Kinase

Introduction
Small molecule drugs are discoverable through conventional and modern drug discovery
techniques [1-3]. Imidazole-based drugs are ubiquitous since they are most effective against a
wide range of drug targets [4]. They are efficacious towards various cancers [4] and active
inhibitors against protein kinases [5]. Imidazole-based antifungal and antimycobacterial
agents have shown remarkable activities in the recent past decade [6]. In this study, we have
approached a four-step synthesis of 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives
(4a-u). The final confirmed structures of these compounds were subjected to a series of in
silico preliminary bioactivity screening in order to adjudge their most suitable and efficacious
bioactivity (Fig. 1) to be executed. Free online tools such as Molinspiration
(www.molinspiration.com), PASSonline (www.pharmaexpert.ru/passonline/) and MedChem
designer (www.simulations-plus.com). All results obtained from these tools were centralized
with correlations between the most favorably predicted bioactivity. From the four repeated
prediction results, the compounds have shown favorable prediction values against G protein-
coupled receptors (GPCRs) ligand, Enzyme inhibition, and Kinase inhibition.
Figure 1. Bioactivity prediction results
GPCRs establish receptors from enormous protein families that are recognized from
the external molecules of the corresponding cell environment and induces internal signal

transduction pathways and simultaneously also stimulating the other accompanying cellular
processes [7,8]. GPCRs also recognized as the heptahelical receptors, G protein-linked
receptors (GPLR), and Serpentine receptors. GPCRs also was known as the seven-(pass)-
transmembrane domain receptors (since they are Coupling with G proteins), and 7TM
receptors (because they pass through the cell membrane seven times) [8]. GPCRs are highly
associated and intricate in several diseases, in fact, GPCRs being the drug target of about
34% of all recent drug discovery researches [9-11]. GPCRs are involved majorly in the
cAMP and PI3Kinase signal pathways [12]. Protease-Activated Receptors (PARs), a sub-
family of GPCRs, often extremely expressed in platelets (thrombocytes), myocytes (muscle
cell), endothelial cells, and neurons (nerve cells) [13]. PAR1-4 are the members of the seven-
transmembrane GPCR superfamily, and are almost expressed all over the body and
responsible for various diseases including inflammation and cancers [14,15]. Therefore, the
present study targeted PAR1 and PI3Kinase inhibition studies along with anti-breast cancer
investigations for 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives (4a-u) (Fig. 2).
Figure 2. Proposed final structures of 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives

Materials and methods
All organic chemicals and solvents have procured from Sigma-Aldrich, Merck, Spectrochem,
and AVRA. Solvents and common reagents used as without doing any further purification.
For thin-layer chromatography (TLC) analysis, Merck pre-coated plates (silica gel 60 F254)
used and the 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 were performed using OptiMelt automated melting point
1
system and are uncorrected. H NMR (300 MHz) and 13C NMR (300 MHz) spectra were
recorded on Bruker-300 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.
General procedure for the synthesis of title compounds, 2-amino-1-methyl-1H-
imidazole-4(5H)-one derivatives (4a-u).
General procedure for the synthesis of ethyl 1H-indole-5-carboxylate (1): To a 1000 mL
three neck RB flask indole-4-carboxylic acid (1eq, 1mol), ethanol (10V) were added. The
reaction mass was stirred for 10 mins at 20-30°C. Thionyl chloride (2.0eq, 2mol) added
slowly on stirring condition and heated the reaction mass to reflux. The obtained clear
solution stirred and refluxed further for 2 hours. The reaction mass cooled to 20-30°C and
stirred for another 10 mins. 10V of MTBE (Methyl tert-butyl ether) added as anti-solvent and
stirred for 30 mins. The off-white solid material filtered and washed with 2V of MTBE. The
material was suction dried till no MLR and the obtained mass was dried further under
vacuum at 50-60°C for 6 hours to get ethyl 1H-indole-5-carboxylate (1) with ≈90 % yield. (-
1H NMR (300MHz, DMSO-d6) δ: 11.49 (S, 1H, -NH), 8.29 (S, 1H, Ar-H), 7.76-7.73 (d,

J=9Hz 1H, Ar-H), 7.49-7.47 (m, 2H, Ar-H), 6.61 (S, 1H, Ar-H), 4.32-4.30 (quat, J=6Hz,
2H, -OCH2) and 1.35-1.32 (t, J=3Hz and J=6Hz, 3H, CH3))
General procedure for the synthesis of N-substituted-ethyl-indole-5-carboxylate (2a-o):
To a 100 ml single neck, RB flask, ethyl 1H-indole-5-carboxylate (1) (1eq, 1mol), DCM
(20V), tetra butyl ammonium hydrogen sulfate (0.010eq) was added and the reaction mass
was stirred for 5mins at 20-30°C. Then, Potassium hydroxide powder (3.0eq) added to the
clear solution and stirred at 0-5°C for 10mins. After that, the respective alkyl halides/sulfonyl
halides or carbamates (1.5-2.0eq) were added and the reaction mass stirred for reaction
completion at 20-30°C. The reaction completion was confirmed over TLC/LCMS. After the
completion of the reaction, the mass was filtered out using a hyflo bed and washed with 2V
of DCM. Further, the filtered content dried over anhydrous sodium sulfate and the volatiles
distilled off under reduced pressure to get the crude gummy/semi-solid material. These crude
materials were purified using column chromatography with Silica as the stationary phase and
EtOAc: n-Heptane as the mobile phase to get pure products (2a-o). The pure material thus
obtained in a range of 60-90 % yield of the target product.
General procedure for the synthesis of N-substituted-ethyl-indole-5-carboxylic acid (3a-
o): To a 100 ml single neck RB flask, N-substituted-ethyl-indole-5-carboxylate (1eq), THF:
MeOH: H2O (1:1:0.5) (20V) were added and the reaction mass stirred for 5mins at 20-30°C.
Lithium hydroxide (5.0eq) added and stirred for further 4-6 hours at 20-30°C until the
reaction completion confirmed by TLC/LCMS. Upon the reaction completion, the reaction
mass was concentrated under vacuum and the residual material was diluted with 20V of
water. The pH was adjusted to 3-4 using 3N HCI and the product was extracted using ethyl
acetate (2x15 V). The separated ethyl acetate layer washed with water (10 V), dried over
anhydrous sodium sulfate, and concentrated under vacuum. The obtained products (3a-o)
dried thoroughly under vacuum and taken for coupling reaction without further purification.

Synthesis of 2-amino-1-methyl-1H-imidazole-4(5H)-one (creatinine) derivatives (4a-u):
Compounds (4a-u) synthesized from N-substituted-4-indole carboxylic acids (3a-o) and
derivatives (4p-u) from heterocyclic carboxylic acids using standard amidation reaction. To a
100 mL single neck RB flask, (N-substituted-4-indole carboxylic acids (3a-u)) (1eq/1mol),
creatinine (1.5eq/1.5mol), HATU (1 eq/1 mol) and DMF (8-12 V) were added under nitrogen
atmosphere and stirred for 5mins at 20-30 °C. Upon a clear solution formed, DIPEA (2eq or
2mol) was added and the reaction mass stirred again for 12-16 hours at 20-30 °C. After 12-16
h, TLC/LCMS confirmed the completion of the reaction. Ice-cold water (20 volumes) was
added slowly to the reaction mixture under stirring condition. The product was extracted with
dichloromethane (2x10 volumes). The combined dichloromethane extract washed with (10
volumes) water and the organic layer separated then dried over anhydrous sodium sulfate.
Further, the volatiles was distilled off under reduced pressure to get the crude product. The
crude material further purified using column chromatography with silica as the stationary
phase and MDC: MeOH as the mobile phase to get pure products (4a-u) with 60-88% yield.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1-(methylsulfonyl)-1H-indole-5-
carboxamide (4a): 1-(methylsulfonyl)-1H-indole-5-carboxylic acid was used to prepare 4a.
Melting point: 128-130 °C. IR (KBr): 3360 (amide N-H stretch), 3205 (aromatic C-H
stretch), 2903 (-CH stretch), 1624 (amide –C=O stretch), 1671 (aromatic C=C stretch), 1268
(-C-N stretch), 1242 (–S=O stretch) and 721 (S-C stretch) Cm-1; 1H NMR (300 MHz,
DMSO-d ) δ: 11.31 (s, 1H), 8.58 (s, 1H), 8.24-8.21 (d, J=9, 1H), 7.92-7.89 (d, J=9, 1H),
6
7.68-7.67 (d, J= 3, 1H), 6.99-6.98 (d, J=3, 1H, 4.16 (s, 2H, 3.15 (s, 3H), 2.91 (s, 3H, CH3).
13C-NMR δ: 174.78 (-CH2CONH2), 172.34 (-CCONH); 158.72, 137.12, 130.29, 128.73,
126.08, 123.79, 112.78 and 108.93 (Aromatic C), 51.78 (-NCH2), 48.86 (-NCH3) and 32.35 (-
SCH3) ppm. LC-MS: 335.05 (M+1). Yield: 88%; Anal calcd for: C14H14N4O4S; Elemental

Analysis calculated: C, 50.29; H, 4.22; N, 16.76; S, 9.59; found: C, 50.30; H, 4.23; N, 16.77;
S, 9.59; HRMS Calculated 334.3500 [M+] m/z, Found 334.3504.
1-(cyclopropylsulfonyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4b): 1-(cyclopropylsulfonyl)-1H-indole-5-carboxylic acid was used to prepare
4b. Melting point: 124-126 °C. IR (KBr): 3352 (amide N-H stretch), 3195 (aromatic C-H
stretch), 2909 (-CH stretch), 1616 (amide –C=O stretch), 1656 (aromatic C=C stretch), 1234
1
(-C-N stretch), 1208 (–S=O stretch) and 717 (S-C stretch) Cm-1; H NMR ( 300 MHz,
DMSO-d ) δ: 11.34 (s, 1H), 8.59 (s, 1H), 8.26-8.23 (d, J=9, 1H), 8.0-7.97 (d, J=9, 1H), 7.74-
6
7.73 (d, J=3, 1H), 7.03-7.02 (d, J=3, 1H), 4.16 (s, 2H), 3.20 (m, 4H), 1.33-1.32 (d, J=3,
2H), 1.15-1.13 (d, J=3, 2H). 13C-NMR δ: 174.78 (-CH2CONH2), 172.33 (-CCONH); 158.69,
137.02, 132.73, 130.38, 128.45, 123.72, 121.18, 112.94 and 109.21 (Aromatic C), 51.74 (-
CH3NCH2CO), 31.54 (-NHCH3), 30.98 (-SCHCH2CH2), 6.30 (-CCH2CH2), ppm. LC-MS:
361.05 (M+1). Yield: 72%; Anal calcd for: C16H16N4O4S; Elemental Analysis calculated: C,
53.32; H, 4.48; N, 15.55; S, 8.90; found: C, 53.32; H, 4.48; N, 15.56; S, 8.90; HRMS
Calculated 360.3880 [M+] m/z, Found 360.3882.
1-(isobutylsulfonyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4c): 1-(Isobutylsulfonyl)-1H-indole-5-carboxylic acid was used to prepare 4c.
Melting point: 118-120 °C. IR (KBr): 3346 (amide N-H stretch), 3210 (aromatic C-H
stretch), 2923 (-CH stretch), 1642 (amide –C=O stretch), 1668 (aromatic C=C stretch), 1244
(-C-N stretch), 1230 (–S=O stretch) and 740 (S-C stretch) Cm-1; 1H NMR (300 MHz, DMSO-
d ) δ: 11.34 (s, 1H), 8.59 (s, 1H), 8.26-8.23 (d, J=9, 1H,), 7.96-7.93 (d, J=9, 1H), 7.74-7.73
6
(d, J=3, 1H), 7.03-7.02 (d, J=3, 1H), 4.16 (s, 2H), 3.59-3.57 (d, J=6, 2H), 3.20 (s, 3H),
2.08-1.98 (t, J=3, 1H), 0.99-0.96 (d, J=9, 6H). 13C-NMR δ: 174.77 (-CH2CONH2), 172.30 (-
CCONH); 158.69, 136.86, 132.75, 130.27, 128.23, 126.08, 123.77, 112.69 and 109.08

(Aromatic C), 60.98 (-CH3NCH2CO), 51.74 (-NHCH3), 30.97 (-SCH2CH), 24.78 (-
SCH2CHCH3) and 22.10 (-CCH3CH3), ppm. LC-MS: 377.1 (M+1). Yield: 68 %; Anal calcd
for: C17H20N4O4S; Elemental Analysis: calculated: C, 54.24; H, 5.36; N, 14.88; S, 8.52;
found: C, 54.24; H, 5.36; N, 14.89; S, 8.52; HRMS Calculated 376.4310 [M+] m/z, Found
376.4310.
1-(phenylsulfonyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4d): 1-(Phenylsulfonyl)-1H-indole-5-carboxylic acid was used to prepare 4d.
Melting point: 130-132 °C. IR (KBr): 3332 (amide N-H stretch), 3245 (aromatic C-H
stretch), 2939 (-CH stretch), 1663 (amide –C=O stretch), 1646 (aromatic C=C stretch), 1280
(-C-N stretch), 1265 (–S=O stretch) and 786 (S-C stretch) Cm-1; 1H NMR (300 MHz, DMSO-
d ) δ: 11.32 (s, 1H), 8.49 (s, 1H), 8.24-8.21 (d, J=9, 1H), 8.08-8.06 (d, J=6, 3H), 7.95-7.93
6
(d, J=6, 1H), 7.77-7.72 (m, 1H), 7.67-7.62 (m, 2H), 7.03-7.02 (d, J= 3, 1H), 4.14 (s, 2H)
and 3.20 (s, 3H). 13C-NMR δ: 174.54 (-CH2CONH2), 172.29 (-CCONH); 158.70, 137.40,
136.56, 135.27, 133.16, 130.73, 130.38, 128.30, 127.20, 126.36, 123.73, 112.96 and 110.68
(Aromatic C), 51.74 (-NCH2) and 30.95 (-NCH3) ppm. LC-MS: 397.1 (M+1). Yield: 74.1 %;
Anal calcd for: C19H16N4O4S; Elemental Analysis calculated: C, 57.57; H, 4.07; N, 14.13; S,
8.09; found: C, 57.57; H, 4.08; N, 14.14; S, 8.09; HRMS Calculated 396.4210 [M+] m/z,
Found 396.4213.
1-(ethanesulfonyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4e): 1-(ethanesulfonyl)-1H-indole-5-carboxylic acid was used to prepare 4e.
Melting point: 125-127 °C. IR (KBr): 3390 (amide N-H stretch), 3223(aromatic C-H stretch),
2946 (-CH stretch), 1645 (amide –C=O stretch), 1665 (aromatic C=C stretch), 1266 (-C-N
stretch), 1253 (–S=O stretch) and 768 (S-C stretch) Cm-1; 1H NMR (300 MHz, DMSO-d ) δ:
6
11.29 (s, 1H), 8.55 (s, 1H), 8.21-8.18 (d, J=9, 1H), 7.91-7.88 (d, J=9, 1H), 7.68-7.67 (d, J=

3, 1H), 6.98-6.97 (d, J=3, 1H), 4.14 (s, 2H), 3.70-3.63 (d, J=6, 2H), 3.15 (s, 3H), 1.09-1.04
(d, J= 9, 3H). 13C-NMR δ: 174.75 (-CH2CONH2), 172.30 (-CCONH); 158.69, 137.07,
130.25, 128.66, 126.03, 123.75, 112.75 and 108.89 (Aromatic C), 51.75 (-NCH2), 48.84 (-
NCH3). 30.98 (-SCH2CH3) and 8.24 (-SCH2CH3) ppm. LC-MS: 371.1 (M+Na). Yield: 77.6
%; Anal calcd for: C15H16N4O4S; Elemental Analysis calculated: C, 51.72; H, 4.63; N, 16.08;
S, 9.20; found: C, 51.72; H, 4.64; N, 16.09; S, 9.21; HRMS Calculated 348.3770 [M+] m/z,
Found 348.3774.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1-tosyl-1H-indole-5-carboxamide (4f):
1-tosyl-1H-indole-5-carboxylic acid was used to prepare 4f. Melting point: 119-121 °C. IR
(KBr): 3380 (amide N-H stretch), 3228(aromatic C-H stretch), 2931 (-CH stretch), 1652
(amide –C=O stretch), 1675 (aromatic C=C stretch), 1252 (-C-N stretch), 1270 (–S=O
stretch) and 791 (S-C stretch) Cm-1; 1H NMR (300 MHz, DMSO-d ) δ: 11.26 (s, 1H), 8.44 (s,
6
1H), 8.18-8.15 (d, J=9, 1H), 8.00-7.88 (m, 4H), 7.40-7.36 (m, 2H), 6.96 (s, 1H), 4.10 (s,
2H), 3.12 (s, 3H) and 2.31 (s, 3H). 13C-NMR δ: 174.56 (-CH2CON), 172.28 (-CCONH);
158.69, 146.17, 136.54, 134.51, 133.07, 130.78, 130.71, 128.31, 127.25, 126.27, 123.68,
112.97 and 110.52 (Aromatic C), 51.74 (-NCH2) 30.96 (-NCH3) and 21.49 (-CCH3) ppm.
LC-MS: 411.3 (M+1). Yield: 80.8 %; Anal calcd for: C20H18N4O4S; Elemental Analysis
calculated: C, 58.53; H, 4.42; N, 13.65; S, 7.81; found: C, 58.53; H, 4.43; N, 13.64; S, 7.82;
HRMS Calculated 410.1049 [M+] m/z, Found 410.1048.
1-acetyl-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carboxamide
(4g): 1-acetyl-1H-indole-5-carboxylic acid was used to prepare 4g. Melting point: 112-114
°C. IR (KBr): 3412 (amide N-H stretch), 3201(aromatic C-H stretch), 2920 (-CH stretch),
1624 (amide –C=O stretch), 1634 (aromatic C=C stretch) and 1256 (-C-N stretch) Cm-1; 1H
NMR (300MHz, DMSO-d ) δ: 11.27 (S, 1H, -NH), 8.47 (S, 1H, Ar-H), 8.38-8.35 (d, J=9Hz,
6

1H, Ar-H), 8.17-8.14 (d, J=9Hz, 1H, Ar-H), 7.93 (S, 1H, Ar-H), 6.88 (S, 1H, Ar-H), 4.11
13
(S, 2H, CH2), 3.14 (S, 3H, CH3) and 2.51 (S, 3H, CH3). C-NMR δ: 174.93 (-CH2CON),
172.31 (-CH3CON), 170.23(-CCONH), 158.62, 137.36, 130.50, 128.73, 126.32, 123.03,
115.59 and 109.23 (Aromatic C), 51.73 (-NCH2), 30.98 (-NCH3) and 24.35 (-NCOCH3) ppm.
LC-MS: 321.05 (M+Na). Yield: 68.2 %; Anal calcd for: C15H14N4O3; Elemental Analysis
calculated: C, 60.40; H, 4.73; N, 18.78; O, 16.09; found: C, 60.40; H, 4.74; N, 18.78; O,
16.09; HRMS Calculated 298.3020 [M+] m/z, Found 298.3022.
1-benzoyl-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carboxamide
(4h): 1-benzoyl-1H-indole-5-carboxylic acid was used to prepare 4h. Melting point: 120-122
°C. IR (KBr): 3419 (amide N-H stretch), 3224(aromatic C-H stretch), 2916 (-CH stretch),
1645 (amide –C=O stretch), 1618 (aromatic C=C stretch) and 1241 (-C-N stretch) Cm-1; 1H
NMR (300 MHz, DMSO-d ) δ: 11.30 (s, 1H), 8.53 (s, 1H), 8.33-8.30 (d, J=9, 1H), 8.23-8.20
6
(d, J=9, 1H), 7.81-7.60 (m, 5H), 7.48-7.47 (d, J= 3, 1H), 6.90-6.89 (d, J=3, 1H), 4.12 (s, 2H)
and 3.16 (s, 3H). 13C-NMR δ: 174.83 (-CH2CON), 172.31 (-CHCON), 168.81(-CCONH),
158.68, 137.94, 134.22, 133.29, 132.73, 130.81, 129.60, 129.23, 126.35, 123.22, 115.55 and
109.50 (Aromatic C), 51.75 (-NCH2) and 31.01 (-NCH3) ppm. LC-MS: 361.1 (M+1). Yield:
78.4 %; Anal calcd for: C20H16N4O3; Elemental Analysis calculated: C, 66.66; H, 4.48; N,
15.55; O, 13.32; found: C, 66.66; H, 4.48; N, 15.55; O, 13.33; HRMS Calculated 360.3730
[M+] m/z, Found 360.3732.
Methyl-5-((1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)carbamoyl)-1H-indole-1-
carboxylate (4i): 1-(methoxycarbonyl)-1H-indole-5-carboxylic acid was used to prepare 4i.
Melting point: 119-120 °C. IR (KBr): 3442 (amide N-H stretch), 3220(aromatic C-H stretch),
2911 (-CH stretch), 1702 (ester –C=O stretch), 1631 (amide –C=O stretch), 1611 (aromatic
C=C stretch), 1241 (-C-N stretch) and 1109 (C-O stretch) Cm-1; 1H NMR (300 MHz, DMSO-

d ) δ: 11.28 (s, 1H), 8.49 (s, 1H), 8.20-8.13 (t, J=9, 2H), 7.79-7.78 (d, J=3, 1H), 6.89-6.88 (d,
6
J=3, 1H), 4.11 (s, 2H), 4.02 (s, 3H), 3.15 (s, 3H). 13C-NMR δ: 174.89 (-CH2CON), 172.30(-
CCONH), 158.62, 151.28, 137.18, 132.54, 130.33, 127.33, 126.12, 123.37, 114.42 and
109.23 (Aromatic C), 54.77 (-NCH2), 51.73 (OCH3) and 30.98 (-NCH3) ppm. LC-MS:
337.05 (M+Na). Yield: 61.1 %; Anal calcd for: C15H14N4O4; Elemental Analysis calculated:
C, 57.32; H, 4.49; N, 17.83; O, 20.36; found: C, 57.32; H, 4.49; N, 17.84; O, 20.37; HRMS
Calculated 314.3010 [M+] m/z, Found 314.3012.
Ethyl-5-((1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)carbamoyl)-1H-indole-1-
carboxylate (4j): 1-(ethoxycarbonyl)-1H-indole-5-carboxylic acid was used to prepare 4j.
Melting point: 115-117 °C. IR (KBr): 3440 (amide N-H stretch), 3224(aromatic C-H stretch),
2921 (-CH stretch), 1743 (ester –C=O stretch), 1632 (amide –C=O stretch), 1619(aromatic
C=C stretch), 1245 (-C-N stretch) and 1145 (C-O stretch) Cm-1; 1H NMR (300 MHz, DMSO-
d ) δ: 11.28 (s, 1H), 8.49 (s, 1H), 8.20-8.12 (t, J=9, 2H), 7.79-7.77 (d, J=3, 1H), 6.88-6.87 (d,
6
J=3, 1H), 4.51-4.43 (t, J=9, 2H), 4.11 (s, 2H), 3.15 (s, 3H) and 1.43-1.39 (t, J=6, 3H). 13C-
NMR δ: 174.92 (-CH2CON), 172.29(-CCONH), 158.62 (-NCOO), 150.74, 137.16, 132.50,
127.32, 126.11, 123.36, 114.46 and 109.12 (Aromatic C), 63.98 (-OCH2CH3) 51.72 (-NCH2),
30.97 (-NCH3) and 14.59 (OCH2CH3) ppm. LC-MS: 351.05 (M+Na). Yield: 74.4 %; Anal
calcd for: C16H16N4O4; Elemental Analysis calculated: C, 58.53; H, 4.91; N, 17.06; O, 19.49;
found: C, 58.53; H, 4.91; N, 17.07; O, 19.48; HRMS Calculated 328.3280 [M+] m/z, Found
328.3284.
1-methyl-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carboxamide
(4k): 1-methyl-1H-indole-5-carboxylic acid was used to prepare compound 4k. Melting
point: 110-112 °C. IR (KBr): 3329 (amide N-H stretch), 3201(aromatic C-H stretch), 2864 (-
CH stretch), 1601 (amide –C=O stretch), 1629 (aromatic C=C stretch) and 1271 (-C-N

1
stretch) Cm-1; H NMR (300MHz, DMSO-d ) δ: 11.24 (s, 1H), 8.49 (s, 1H), 8.03-8.00 (d,
6
J=9, 1H), 7.47-7.40 (m, 2H), 6.57 (s, 1H), 4.09 (s, 2H), 3.82 (s, 3H), 3.14 (s, 3H). 13C-NMR
δ: 176.05 (-CH2CON), 172.24 (-CCONH), 158.18, 131.31, 128.65, 127.91, 123.48, 123.01,
109.40 and 102.38 (Aromatic C), 51.61 (-NCH2), 33.10 (-CH2NCH3) and 30.92 (-CHNCH3)
ppm. LC-MS: 271.2 (M+1). Yield: 69.2 %; Anal calcd for: C14H14N4O2; Elemental Analysis
calculated: C, 62.21; H, 5.22; N, 20.73; O, 11.84; found: C, 62.21; H, 5.22; N, 20.74; O,
11.84; HRMS Calculated 270.2920 [M+] m/z, Found 270.2973.
1-(ethoxymethyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carbox
-amide (4l): 1-(ethoxymethyl)-1H-indole-5-carboxylic acid was used to prepare 4l. Melting
point: 119-121 °C. IR (KBr): 3346 (amide N-H stretch), 3254(aromatic C-H stretch), 2871 (-
CH stretch), 1623 (amide –C=O stretch), 1651 (aromatic C=C stretch), 1280 (-C-N stretch)
1
and 1154 (-C-O-C stretch) Cm-1; H NMR (300 MHz, DMSO-d ) δ: 11.27 (s, 1H), 8.51 (s,
6
1H), 8.06-8.03 (d, J=9, 1H), 7.61-7.55 (m, 2H), 6.64 (s, 1H), 5.59 (s, 2H), 4.09 (s, 2H,),
3.44-3.37 (m, 2H), 3.14 (S, 3H), 1.07-1.03 (d, J=6, 3H). 13C-NMR δ: 175.83 (-CH2CON),
172.26 (-CCONH); 158.28, 138.62, 130.77, 129.60, 128.57, 123.55, 123.47, 110.05 and
103.57 (Aromatic C), 75.66 (-NCH2O), 63.72 (-CH3NCH2CO), 51.62 (-OCH2CH3), 30.91 (-
NHCH3) and 15.27 (-OCH2CH3) ppm. LC-MS: 315.10 (M+1). Yield: 73.4 %; Anal calcd for:
C16H18N4O3; Elemental Analysis calculated: C, 61.14; H, 5.77; N, 17.82; O, 15.27; found: C,
61.14; H, 5.77; N, 17.83; O, 15.26; HRMS Calculated 314.3450 [M+] m/z, Found 314.3454.
1-(cyclopropylmethyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4m): 1-(cyclopropylmethyl)-1H-indole-5-carboxylic acid was used to prepare
4m. Melting point: 124-126 °C. IR (KBr): 3382 (amide N-H stretch), 3221(aromatic C-H
stretch), 2869 (-CH stretch), 1622 (amide –C=O stretch), 1667 (aromatic C=C stretch) and
1276 (-C-N stretch) Cm-1; 1H NMR (300MHz, DMSO-d ) δ: 11.24 (s, 1H), 8.49 (s, 1H), 8.01-
6

7.98 (d, J=9, 1H), 7.55-7.51 (m, 2H), 6.59 (s, 1H), 4.08 (s, 4H), 3.14 (s, 3H), 1.25 (s, 1H)
and 0.53-0.40 (m, 4H). 13C-NMR δ: 176.03 (-CH2CON), 172.25 (-CCONH); 158.18, 138.31,
130.20, 128.62, 127.99, 123.52, 123.97, 109.61 and 102.54 (Aromatic C), 51.60 (-
NCH2CH3), 50.31 (-CH3NCH2), 30.91 (-NCH2) 12.03 (-CCH2CH2) and 4.16 (-CCH2CH2)
ppm. LC-MS: 311.10 (M+1). Yield: 76.4 %; Anal calcd for: C16H18N4O3; Elemental Analysis
calculated: C, 61.14; H, 5.77; N, 17.82; O, 15.27; found: C, 61.15; H, 5.82; N, 17.83; O,
15.28; HRMS Calculated 310.3520 [M+] m/z, Found 310.3524.
1-(2-methoxyethyl)-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-
carboxamide (4n): 1-(2-methoxyethyl)-1H-indole-5-carboxylic acid was used to prepare 4n.
Melting point: 117-119 °C. IR (KBr): 3392 (amide N-H stretch), 3230(aromatic C-H stretch),
2891 (-CH stretch), 1642 (amide –C=O stretch), 1654 (aromatic C=C stretch), 1267 (-C-N
1
stretch) and 1104 (-C-O-C stretch) Cm-1; H NMR (300 MHz, DMSO-d ) δ: 11.30 (s, 1H),
6
8.54 (s, 1H), 8.06-8.03 (d, J=9, 1H), 7.58-7.48 (m, 2H), 6.63 (s, 1H), 4.41 (s, 2H), 4.12 (s,
13
2H), 3.72 (s, 2H), 3.27 (s, 3H), 3.19 (s, 3H). C-NMR δ: 176.02 (-CH2CON), 172.25 (-
CCONH); 158.19, 138.50, 130.27, 128.71, 127.98, 123.47, 122.96, 109.66 and 102.60
(Aromatic C), 71.47 (-NCH2CH2), 58.54 (-CH3NCH2CO), 51.60 (-CH2OCH3), 45.98 (-
NHCH3) and 30.91 (-CH2OCH3) ppm. LC-MS: 315.10 (M+1). Yield: 76.9 %; Anal calcd for:
C16H18N4O3; Elemental Analysis calculated: C, 50.29; H, 4.22; N, 16.76; O, 19.14; S, 9.59;
found: C, 50.29; H, 4.23; N, 16.76; O, 19.13; S, 9.59; HRMS Calculated 314.3450 [M+] m/z,
Found 314.3453.
1-benzyl-N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carboxamide
(4o): 1-benzyl-1H-indole-5-carboxylic acid was used to prepare compound 4o. Melting point:
126-128 °C. IR (KBr): 3429 (amide N-H stretch), 3285(aromatic C-H stretch), 2897 (-CH
stretch), 1622 (amide –C=O stretch), 1682 (aromatic C=C stretch) and 1321 (-C-N stretch)

Cm-1; 1H NMR (300MHz, DMSO-d ) δ: 11.25 (s, 1H), 8.50 (s, 1H), 7.99-7.96 (d, J=9, 1H),
6
7.59-7.48 (m, 2H), 7.31-7.23 (m, 5H), 6.64 (S, 1H), 5.46 (s, 2H), 4.08 (s, 2H) and 3.13 (s,
3H). 13C-NMR δ: 175.93 (-CH2CON), 172.26 (-CCONH); 158.21, 138.48, 138.30, 130.82,
129.04, 128.94, 128.23, 127.89, 127.53, 123.58, 123.19, 109.89 and 103.08 (Aromatic C),
51.60 (-NCH2CO), 49.50 (-NCH2Ph) and 30.91 (-NCH3) ppm. LC-MS: 347.10 (M+1). Yield:
80.0 %; Anal calcd for: C20H18N4O2; Elemental Analysis calculated: C, 69.35; H, 5.24; N,
16.17; O, 9.24; found: C, 69.35; H, 5.25; N, 16.17; O, 9.25; HRMS Calculated 346.3900
[M+] m/z, Found 346.3901.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-4-carboxamide (4p): 1H-
indole-4-carboxylic acid was used to prepare 4p; Melting point: 109-111 °C. IR (KBr): 3351
(N-H stretch), 3236 (amide N-H stretch), 3259 (aromatic C-H stretch), 2881 (-CH stretch),
1601 (amide –C=O stretch), 1632 (aromatic C=C stretch) and 1165 (-C-N stretch) Cm-1; 1H
NMR (300 MHz, DMSO-d ) δ: 11.30 (s, 2H), 8.03-8.01 (d, J=9, 1H), 7.61-7.58 (d, J=9,
6
1H), 7.46 (s, 1H), 7.26 (s, 1H), 7.18-7.14 (t, J=6, 1H), 4.09 (s, 2H) 3.14 (s, 2H). 13C-NMR
δ: 176.81 (-CH2CON), 172.31 (-CCONH); 158.17, 137.36, 128.64, 127.66, 127.16, 123.09,
120.41, 115.85 and 103.67 (Aromatic C), 51.61 (-NCH2CO) and 30.99 (-NCH3)ppm. LC-
MS: 257.05 (M+1). Yield: 82.0 %; Anal calcd for: C13H12N4O2; Elemental Analysis
calculated: C, 60.93; H, 4.72; N, 21.86; O, 12.49; found: C, 60.93; H, 4.72; N, 21.86; O,
12.49; HRMS Calculated 256.2650 [M+] m/z, Found 256.2652.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-6-carboxamide(4q):
1H-
indole-6-carboxylic acid was used to prepare compound 4q. Melting point: 111-113 °C. IR
(KBr): 3362 (N-H stretch), 3231 (amide N-H stretch), 3253(aromatic C-H stretch), 2889 (-
CH stretch), 1612 (amide –C=O stretch), 1641 (aromatic C=C stretch) and 1175 (-C-N
stretch) Cm-1; 1H NMR (DMSO-d ) δ: 11.38 (s, 1H), 11.26 (s, 1H), 8.32 (s, 1H), 7.88-7.85 (d,
6

J=9, 1H), 7.58-7.54 (m, 2H), 6.49 (s, 1H), 4.09 (s, 2H), 3.13 (s, 2H). 13C-NMR δ: 176.03 (-
CH2CON), 172.25 (-CCONH); 158.24, 135.82, 131.01, 130.44, 128.88, 120.63, 119.54,
113.95 and 101.79 (Aromatic C), 51.63 (-NCH2CO) and 30.89 (-NCH3) ppm. LC-MS:
257.10 (M+1). Yield: 82.4 %; Anal calcd for: C13H12N4O2; Elemental Analysis calculated:
C, 60.93; H, 4.72; N, 21.86; O, 12.49; found: C, 60.93; H, 4.72; N, 21.87; O, 12.49; HRMS
Calculated 256.2650 [M+] m/z, Found 256.2654.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-7-carboxamide (4r): 1H-
indole-7-carboxylic acid was used to prepare compound 4r. Melting point: 112-114 °C. IR
(KBr): 3359 (N-H stretch), 3224 (amide N-H stretch), 3243 (aromatic C-H stretch), 2884 (-
CH stretch), 1609 (amide –C=O stretch), 1635 (aromatic C=C stretch) and 1169 (-C-N
stretch) Cm-1; 1H NMR (300MHz, DMSO-d ) δ: 11.27 (s, 1H), 10.99 (s, 1H), 8.04-8.02 (d,
6
J=6, 1H), 7.79-7.77 (d, J=6, 1H), 7.42 (s, 1H), 7.12-7.08 (t, J=6, 1H), 6.53 (s, 1H), 4.14 (s,
2H), 3.17 (s, 2H). 13C-NMR δ: 176.09 (-CH2CON), 172.24 (-CCONH); 158.57, 135.32,
129.50, 126.80, 125.28, 124.61, 120.35, 118.80 and 101.76 (Aromatic C), 51.90 (-NCH2CO)
and 31.22 (-NCH3) ppm. LC-MS: 257.10 (M+1). Yield: 83.6 %; Anal calcd for: C13H12N4O2;
Elemental Analysis calculated: C, 60.93; H, 4.72; N, 21.86; O, 12.49; found: C, 60.93; H,
4.72; N, 21.86; O, 12.49; HRMS Calculated 256.2650 [M+] m/z, Found 256.2650.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-indole-5-carboxamide(4s):
1H-
indole-5-carboxylic acid was used to prepare 4s. Melting point: 116-118 °C. IR (KBr): 3356
(N-H stretch), 3213 (amide N-H stretch), 3261(aromatic C-H stretch), 2872 (-CH stretch),
1621 (amide –C=O stretch), 1626 (aromatic C=C stretch) and 1151 (-C-N stretch) Cm-1; 1H
NMR (300 MHz, DMSO-d ) δ: 11.35 (s, 1H), 11.25 (s, 1H, -NH), 8.50 (s, 1H), 7.99-7.96 (d,
6
J=9, 1H), 7.43-7.40 (m, 2H), 6.57 (s, 1H), 4.08 (s, 2H), 3.13 (s, 2H). 13C-NMR δ: 176.15 (-
CH2CON), 172.25 (-CCONH); 158.13, 138.55, 128.60, 127.58, 126.96, 123.26, 123.05,

111.08 and 103.00 (Aromatic C), 51.59 (-NCH2CO) and 30.89 (-NCH3)ppm. LC-MS: 257.10
(M+1). Yield: 80.5 %; Anal calcd for: C13H12N4O2; Elemental Analysis calculated: C, 60.93;
H, 4.72; N, 21.86; O, 12.49; found: C, 60.93; H, 4.72; N, 21.86; O, 12.49; HRMS Calculated
256.2650 [M+] m/z, Found 256.2653.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)quinoline-6-carboxamide
(4t):
quinoline-6-carboxylic acid was used to prepare 4t. Melting point: 117-119 °C. IR (KBr):
3382 (N-H stretch), 3254 (amide N-H stretch), 3251(aromatic C-H stretch), 2859 (-CH
stretch), 1613 (amide –C=O stretch), 1601 (aromatic C=C stretch) and 1163 (-C-N stretch)
Cm-1; 1H NMR (300 MHz, DMSO-d ) δ: 11.35 (s, 1H), 8.99 (s, 1H), 8.84 (s, 1H), 8.58-8.55
6
(d, J=9, 1H), 8.48-8.45 (d, J=9, 1H), 8.09-8.06 (d, J=9, 1H), 7.62-7.59 (m, 1H), 4.14 (s, 2H),
13
3.19 (s, 2H). C-NMR δ: 174.07 (-CH2CON), 172.35 (-CCONH); 159.01, 152.54, 149.64,
137.95, 135.33, 129.69, 129.18, 127.68, and 122.40 (Aromatic C), 51.58 (-NCH2CO) and
31.10 (-NCH3) ppm. LC-MS: 269.05 (M+1). Yield: 83.9 %; Anal calcd for: C14H12N4O2;
Elemental Analysis calculated: C, 62.68; H, 4.51; N, 20.88; O, 11.93; found: C, 62.68; H,
4.51; N, 20.89; O, 11.94; HRMS Calculated 268.2760 [M+] m/z, Found 268.2761.
N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole-5-carboxamide
(4u): 1H-benzo[d]imidazole-5-carboxylic acid was used to prepare 4u. Melting point: 113-
115 °C. IR (KBr): 3367 (N-H stretch), 3242 (amide N-H stretch), 3267(aromatic C-H
stretch), 2871 (-CH stretch), 1629 (amide –C=O stretch), 1613 (aromatic C=C stretch) and
1124 (-C-N stretch) Cm-1; 1H NMR (300MHz, DMSO-d ) δ: 11.30 (s, 1H), 8.48 (s, 1H), 8.37
6
13
(s, 1H), 8.11-8.08 (d, J=9, 1H), 7.65-7.62 (d, J=9, 1H), 4.10 (s, 2H), 3.13 (s, 2H). C-NMR
δ: 175.39 (-CH2CON), 172.31 (-CCONH); 158.46, 144.40, 141.50, 138.1, 131.57, 123.86,
117.83 and 114.78 (Aromatic C), 51.67 (-NCH2CO) and 30.91 (-NCH3) ppm. LC-MS:
258.10 (M+1). Yield: 86.7%; Anal calcd for: C12H11N5O2; Elemental Analysis calculated: C,

56.03; H, 4.31; N, 27.22; O, 12.44; found: C, 56.04; H, 4.32; N, 27.22; O, 12.44; HRMS
Calculated 257.2530 [M+] m/z, Found 257.2534.
Connotation among PARs and PI3Kinase – and the assay protocol PI3Kinase inhibition
activity of 4a-u
Protease-Activated Receptors (PARs) mostly expressed on the exterior region of almost all
cell types and intracellular signal transducer enzyme, PI3Kinase, play an important role in
regulating the functional reactions in haemopoietic cells such as neutrophils, eosinophils,
mast cells, monocytes, and B- and T-cells that are highly involving in the cancer development
[16]. Accordingly, a chemical entity that inhibiting PI3 Kinases would be the ideal drug
target against cancers, especially breast cancer due to the high involvement of PI3Kinase in
it. Therefore, the inhibitory potentials of 2-amino-1-methyl-1H-imidazole-4(5H)-one
derivatives (4a-u) has evaluated by means of Glutathione-coated 96 well plate assay [17-20].
Pictilisib (GDC-0941) was commercially obtained and served as the standard drug. The
kinase and 2-amino-1-methyl-1H-imidazole-4(5H)-ones 4a-u were pre-incubated about 10-
15 minutes before adding the substrate (PIP2 (Phosphatidylinositol biphosphate)). 5X kinase
reaction buffer (5 µL) was added after adding 5 µL/well of PIP2 substrate to corresponding
wells. Millipore water was added to each well to bring the final volume to 25 µL/well. Then
the reaction mix was incubated at temperature for 1 hour. The absorbance read at 450 nm. As
the result different, the relative % activity to b-PIP3 was calculated by means of the following
formula,
OD of samples (buffer, kinase & inhibitors)
-----------------------------------------------------
OD of B-PIP3 average
x 100
% inhibition =
Molecular docking evaluations
Molecular docking study was carried out using Autodock version 4.2.6 (MGL Tools 1.5.6
was used as the Graphical User Interface) [21,22]. Argus lab 2.0 was used to cleaning the

receptor protein and the ligands 4a-u. Based on the predicted biological activity values (Fig.
1), medicinal value assessments of 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives
(4a-u) towards GPCR ligand (through PAR1 inhibition) and PI3Kinase inhibition was
measured via molecular docking studies since they are mainly involved in cancer
development. PAR1 is the characteristic adherent component of the family of GPCRs that
ease cellular events to thrombin and associated kinases and proteases. To get more insight
into the mode of binding interactions to GPCR and PI3Kinase, the ligands structures (4a-u)
was docked into the active site of GPCR and PI3Kinase. The PDB X-ray crystallography
structures of PAR1, PDB ID: 2GNJ and PI3Kinase, PDB ID: 3HHM.
Anti-breast cancer studies - Michigan Cancer Foundation-7 (MCF-7) cell lines
Anti-breast cancer experimentations by means of Michigan Cancer Foundation-7 (MCF-7)
cell culture widely used for emerging novel diagnostic examinations and new treatments for
breast cancer [23]. The early passage MCF-7 cell lines used in this study were established
and cultivated as described earlier [17-20]. Cells were cultured in Dulbecco's Modified
Eagle's Medium (DMEM) which was supplemented with 10% Fetal Bovine Serum (FBS),
100 μg/ml streptomycin and (100U) 20 μg/ml penicillin. Cells were incubated at 37 °C in 5%
CO2 atmosphere. For the anti-proliferative activity comparison to normal and cancerous cells,
the normal breast epithelial cells (MCF-10) in addition to MCF-7 cells. These cells were
cultured in 1:1 combination of DMEM and Ham's F12 medium with 20 mg/ml of epidermal
growth factor (EGF), 0.01 mg/ml insulin, 100 μg/ml cholera toxins, 500 μg/ml
hydrocortisone, and 5% chelex treated horse serum were added. For the biological activity
assessments, 1 ml of homogenized cell suspension of both normal and cancerous cells
dispensed in each well of a separate microtiter plate and kept in a desiccator underneath 5%
CO2 atmosphere. Further, the cells observed under the inverted microscope after 48 hours of

incubation and assessed for their physiological and morphological changes if any. 0.05 ml of
the compounds 4a-u along with the standard (Doxorubicin) dissolved in 4.95 ml of DMSO to
get a working concentration of 1 mg/ml. This working concentration was set freshly and
filtered over a 0.45-micron filter prior to bioassay.
MTT assay for anticancer evaluations
To execute the MTT assay, about 5000 cells were seeded in a Glutathione-coated 96-well
flat-bottom titer plate and incubated for 24, 48, and 72 hours at 37 ºC in 5 % CO2 atmosphere.
Different aliquots of compounds (4a-u) (25 – 250 µg/mL) were added and incubated at room
temperature. After the incubation, the medium was removed from corresponding wells of the
working plate. The wells were washed several times with Phosphate Buffer Solution; Further,
100 µL of the working MTT dye in DMEM media was added and incubated for 120 minutes.
With this, the MTT lysis buffer (100 µL) was included and incubation was continued for 4
hrs more. The absorbance was measured at 570 nm and the cell viability was calculated using
the following formula,
Cell viability (%) = Mean OD/Control OD x 100 %
Hydrogen peroxide (H2O2) radical scavenging evaluation of 4a-u
The connection between free radicals and breast cancer is well known. Recent researches
suggest that free radicals are playing a vital role in breast cancer development [24,25].
Antioxidant properties of 4a-u assessed by H2O2 scavenging assay [26]. The assay performed
in triplicate with Ascorbic acid as standard. Compounds 4a-u and Ascorbic acid assessed
with a range of concentration from 25- 250 g/mL and incubated by adding 0.6 mL of fresh
H2O2 (40 mM) at 37 °C for 60 minutes. Ascorbic acid used as positive control. Absorbance at
230 nm was recorded to determine the relative H2O2 % scavenging rate of compounds 4a-u.

H2O2 dissolved in phosphate buffer read at 230 nm taken as control OD. The relative %
scavenging of H2O2 and Ascorbic acid was determined by the following formula:
% H2O2 reducing activity = [OD0−OD1/OD0] × 100
Where OD0 is the optical density (OD) of the control and OD1 is the OD of 4a-u.
In-vitro thrombolytic valuations to exemplify PAR1 inhibitory potentials of 4b and 4g
In order to evaluate the PAR1 inhibition potentials possessed by compounds 4b and 4g,
thrombolytic activity assessment was executed [26]. Protease-activated receptors (PARs)
belong to the GPCR family that proteolytically activated by numerous proteases. Stimulus of
fibrinolysis through plasmin (infusion of tissue plasminogen) activator enabling
thrombolysis. The coagulation and fibrinolytic systems contribute to malignancy by
increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis [27]. PAR
intermediated crosstalk between coagulation and fibrinolysis has been studied broadly and
well-known [27]. Coagulation proteases utilize their tumor-triggering activities by initiating
PAR-1 and PAR-2 [28, 29].
For in vitro thrombolysis assessment, 100 μL (30,000 I.U) of suspension of streptokinase
prepared and used as stock solution. Streptokinase and Millipore water were used as
thrombolytic standard and non-thrombolytic control, correspondingly. Each healthy
volunteers from various age groups donated 5 ml of blood (human, n = 4). The collected
blood was transferred to each of the Eppendorf tubes (750 μL) to obtain clots at room
temperature. Serum was articulated by not upsetting the clot formed. The most active
compounds 4b and 4g were administered at different concentrations and tubes were incubated
at room temperature for 2 hours and the fluid development was perceived. To measure the
serum amount produced, the weight of blood comprising tubes were weighed before and after

the removal of serum. The assessment was repeated up to four times. The differences in
weight measured before and after clot lysis specified as a percentage.
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. A difference was considered statistically significant if
p≤0.05. The 50 % inhibitory concentration (IC50) was calculated from the dose-response
curve obtained by plotting percentage inhibition versus concentration.
Results and discussions
In this study, initially, we have tried for the –NH substitution of creatinine coupled indole
derivative
(N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazole-2-yl)-1H-indole-5-carboxamide
(4s) but due to poor solubility of the compound in solvents reaction did not complete and
percentage of product formation was less. In order to get the good quality and yield of the
desired compound, a four-step reaction was explored at normal room temperature without
using any external energy for reaction completion and achieved the high pure 2-amino-1-
methyl-1H-imidazole-4(5H)-one derivatives (4a-u) with remarkable yields according to
scheme 1 and scheme 2. The first step is esterification of indole carboxylic acid by using
standard esterification reaction with thionyl chloride in ethanol as a solvent to get
corresponding acid ester. After esterification, the product was easily isolated with full yield
by adding MTBE as anti-solvent and after drying, the product was completely free from
excess thionyl chloride used in the reaction.

Scheme 1. The synthetic route for the preparation of title compounds (4a-o).
The second step is the N-substitution of indole carboxylic acid ester with substituting
reagents in presence of a base and this was a very challenging step in the concern of isolation
of product due to the sensitivity of N-substituted indole ester to aqueous media in presence of
a base. However the same issue was successfully addressed by opting MDC as solvent and
NaOH powder as a base, this combination along with catalyst TBAF resulted in very ease of
isolation method due to immiscibility of NaOH powder in MDC. After reaction completion,
NaOH powder removed by celite filtration and product contained MDC layer washed with
water in the absence of a base.
Table 1. Optimization of reaction conditions for the synthesis of intermediates (2a-o).
*
Entry
1
Base
TEA
TEA
TEA
TEA
TEA
TEA
TEA
TEA
Catalyst
----
Solvent
THF
THF
THF
THF
T °C
5-10
Time
% of Yield
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
2.00 hr
40
45
35
50
40
48
65
68
70
85
95
93
95
89
76
2
3
4
5
6
7
8
9
10
11
12
13 a
14
15
----
----
25-30
25-30
25-30
25-30
25-30
25-30
10-15
10-15
10-15
10-15
10-15
20-30
20-30
20-30
TBAF(a)
TBAB(b)
TBAF
TBAF
TBAF
TBAF
TBAF
TBAF
TBAF
TBAF
TBAF
TBAF
THF
Methyl THF
MDC
MDC
MDC
MDC
MDC
MDC
MDC
THF
DIPEA
NaOH
NaOH powder
KOH powder
NaOH powder
NaOH powder
NaOH powder
Toluene
13a The optimized reaction conditions for indole N-H substitution reaction; * The yields are mentioned for the
isolated products; (a) Tetra-n-butylammonium fluoride. (b) Tetra-n-butylammonium bromide

Scheme 2. The synthetic route for the preparation of title compounds (4p-u).
Finally resulted in the high recovery of product with remarkable yield. The third step is
the hydrolysis of N-substituted indole carboxylic acid ester without disturbing the N-
substitution of indole by using the mild base as LiOH in presence protic solvent mixture
MeOH:THF: water (1:1:0.5). The fourth step is acid amine coupling by using standard
coupling agent HATU with the well-known base as diisopropylethylamine (DIPEA) in best
protic solvent DMF to get the title compounds. The achieved compounds in remarkable yield
have characterized through standard analytical techniques such as LC-MS, NMR, HRMS,
and elemental analysis. Table 1-3 are displaying the details of reaction optimization of
compounds 4a-u. Indole N-H substitution reaction using different substituting reagents was
optimized by using methylsulfonyl chloride as substituting reagent with different bases,
solvents, catalysts and different temperature ranges to get intermediate compounds 2a with
high yield (Table 1).
Table 2. Optimization of reaction conditions for the preparation of intermediates 3a-o.
Solvent
Composition
% of Yield
Entry
Base
Solvent
T °C
Time
*
1
2
3
4
5
6
7
8
9
10
11
12
13
14 a
15
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
KOH
MeOH
MeOH
MeOH
THF
THF
1
1
1
1
1
1
1:1
1:1
1:1:1
1:1:1
1:1:1
1:1:1
1:1:1
1:1:0.5
1:1:0.5
5-10
25-35
Reflux
5-10
25-35
Reflux
25-35
25-35
25-35
5-10
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
4.00 hr
1.00 hr
2.00 hr
10.0 hr
NA
NA
NA
NA
NA
NA
NA
NA
30
NA
NA
25
65
96
THF
Water:MeOH
THF:MeOH
THF:MeOH:water
THF:MeOH:water
THF:MeOH:water
THF:MeOH:water
THF:MeOH:water
THF:MeOH:water
THF:MeOH:water
5-10
KOH
25-35
25-35
25-35
5-10
LiOH
LiOH
LiOH
NA
14a The optimized reaction conditions for Ethyl ester hydrolysis reaction of N-substituted indole carboxylate
compounds; * The yields are mentioned for the isolated products; NA – incomplete reaction.

Table 3. Optimization of reaction conditions for the preparation of final compounds 4a-u.
*
Entry
Base
TEA
TEA
TEA
TEA
DIPEA
DIPEA
DIPEA
DIPEA
DIPEA
Solvent
THF
THF
THF
THF
Coupling reagents
DCC(c)/HOBt(d)
DCC/HOBt
EDC.HCl(e)/HOBt
HATU(f)
T °C
25-35
Reflux 6.00 hr
Reflux 6.00 hr
25-35
25-35
25-35
25-35
25-35
25-35
Time
6.00 hr
% of Yield
NA
NA
NA
NA
NA
88
1
2
3
4
6.00 hr
6.00 hr
6.00 hr
6.00 hr
6.00 hr
6.00 hr
5
THF
HATU
HATU
6 a
7
DMF
DMA
DMF
DMF
HATU
82
80
61
8
9
HBTU(g)
EDC.HCl/HOBt
6a The optimized reaction conditions for the preparation of final compounds.
* The yields are mentioned for the isolated products. NA- Incomplete reactions.
(c) Dicyclohexylcarbodiimide. (d) Hydroxybenzotriazole.
(e) N′-ethyl carbodiimide hydrochloride.
(f) (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluoro
phosphate.
(g) 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate
Ethyl ester hydrolysis reaction of N-substituted indole carboxylate compounds 2a-o was
optimized by using ethyl 1-(methylsulfonyl)-1H-indole-5-carboxylate (2a) compound with
different bases, solvents, solvent mixtures, solvent mixtures with varied composition and
different temperature ranges to get intermediate compounds 3a with high yield (Table 2). The
coupling reaction of N-substituted indole carboxylic acid compounds 3a-o and Creatinine (2-
amino-1-methyl-1H-imidazole-4(5H)-one) was optimized by using 1-(methylsulfonyl)-1H-
indole-5-carboxylic acid (3a) compound with different bases, solvents and different
temperature ranges to get final pure compounds 4a with high yield (Table 3).
Results of molecular docking studies
In molecular docking studies, the results of PAR1 has attained the maximum requirement, but
not up to the level of PI3Kinase results. With a binding affinity range of 0.25 to 1.75 µM
inhibitory constant (ki) values, all the compounds were able to inhibit PAR1. Therefore, we
took only the PI3Kinase inhibition results to discuss elaborately here. The lowest binding
affinity and best-docked poses had taken as the key measurements in order to screen a
compound as efficacious towards the proposed biological activity.

Figure 2. Molecular docking study results. Note: 4A. Binding energy values (kcal/mol) of 4a-u; 4B.
Obtained inhibitory constant (ki) values of 4a-u (best compounds are highlighted oval shape); 4C. Best docked
pose (compound 4g into the binding pocket of PI3Kinase PDB ID: 3HHM); 4D. Molecular interaction of
PI3Kinase and compound 4g (Stick and balls = 4g; sticks only = amino acid residues of PI3Kinase).
The molecular mechanistic values such as least binding energy (kcal/mol), ligand
efficiency, and inhibitory constant (ki) have retrieved and based on those values the
compounds 4a-u were ranked. Top five compounds among 4a-u had screened for further in
vitro studies. In the results, the remarkable binding affinity of all compounds has recognized
through the established binding energy values (-7.20 to -12.17 kcal/mol) and the inhibitory
constant values (1.35 to 0.085 µM) (Fig. 2 & B). Excellent molecular mechanistic values are
shown by the compounds 4b, 4g, 4i, 4n, and 4u enforced us to screen them for further in vitro
investigations to unveil their PI3Kinase inhibitory potentials.
PI3Kinase inhibition and relevant molecular interactions of 4a-u to PI3Kinase
The inhibition potential of present study compounds 4b, 4g, 4i, 4n and 4u that has screened
from molecular docking studies based on molecular mechanistic values assessed through
competitive ELISA method. Four repeated assays were done (n=4). The obtained results
statistically optimized and mentioned with ±SD.

Figure 3. Results of PI3kinase inhibition activity of present study compounds 4b, 4g, 4i, 4n,
and 4q. Note: 3A. Relative percentage activity in comparison to Doxorubicin; 3B. Inhibitory constant values
(IC50 values); 3C. Dose-response curve of compound 4g and Doxorubicin.
In the results, almost all compounds showed equal or more activity than the standard drug
Pictilisib (Fig. 3 A-C). Compounds 4b and 4g dominated for the activity while comparing the
standard Pictilisib (78.14±2.64 relative % activity and IC50 0.78±0.02) by attaining the
highest relative percentage activity (84.16±2.12 and 87.94±1.89 respectively) and lowest
inhibitory constant (IC50 0.75±0.02 and 0.5±0.02). Fig. 3D illustrates the anticipated
interaction to establish the highest relative percentage activity with the lowest inhibitory
constant by the compound 4g. The study results also used to screen these two compounds for
further anticancer studies in breast cancer cell lines and their antioxidant potentials.
Anticancer study results
Compounds 4b and 4g screened based on strong PI3Kinase inhibition used to assess their
anti-cancer potentials against breast-cancer cell lines. We have also assessed the cytotoxicity
of these compounds against normal breast epithelial cells in order to have the activity
variations between these two different but same environmental type cells. Expectedly,

compound 4g showed excellent anti-proliferative effects than the standard doxorubicin. At
the same time 4b also equally active as 4g. Only a negligible variation of result values found
between 4b and 4g. The calculated relative percentage activity was excellent for 4b
(85.25±1.98) and 4g (87.25±1.84) while comparing with Doxorubicin (80.18±2.75).
Figure 4. The appearance of normal breast epithelial cells (MCF-10) (left) and cancerous
breast epithelial cells after 2 hours after treating compound 4g.
The IC50 values were 0.48±0.05, 0.25±0.02 and 1.05±0.15 for 4b, 4g and Doxorubicin
respectively. From these results, the ability or potential to act as an effective PI3Kinase
inhibitor or breast cancer therapeutics by 4b and 4g had recognized. Fig. 4 illustrates the
timely evaluated anticancer study result variations between the cancerous (MCF-7) and
normal breast epithelial cells. Remarkably, there was no necrosis or adverse effects found on
normal epithelial cells (MCF-10) even up to eight-hour observation. However, necrosis was
observed on cancerous cells from 30 minutes of incubation after treating the compounds 4b
and 4g.
Results of radical scavenging effects of compounds 4b, 4g, and Ascorbic acid
Excellent free radical scavenging potential observed for compounds 4b and 4g while
comparing Ascorbic acid. Scavenging potential of 4g was almost 98.42±1.76 %, 4a
96.30±1.85, Ascorbic acid 86.24±2.46 and the IC50 was 54 nM, 62 nM and 1 µM for 4g, 4b
and Ascorbic acid respectively.

Results of PAR1 inhibition through thrombolytic assay
Thrombin is one of the vital factors and playing a major role in the advancement of numerous
diseases. In the current approach, the clot lysis activity of compounds 4b and 4g, assessed for
PAR1 inhibition through thrombolytic effects. Subsequently administering 4b, 4g, and
streptokinase (standard), the dry weight of the Eppendorf tube with clotted blood measured as
the result by removing serum released due to the thrombin lysis activity by both compounds
and standard in every 15 minutes. In the results, compounds, 4b (92.15±1.28 % lysed) and 4g
(96.25±1.42 % lysed) were established most active thrombolytic effects. They were able to
lyse the whole blood-clot within 15-30 minutes after the treatment while streptokinase
(standard) (87.12±1.72 % lysed) utilized more than 1 hour to the whole completion of lysis.
Furthermore, it was observed that there was no variations or differences in clot lysis activity
on the variedly collected blood samples collected from three set of age group (18-24 (n=4);
25-35 (n=4); 36-40 (n=4)) and 41 above (n=4).
Conclusion
In conclusion, 21-member family of, four-step involved 2-amino-1-methyl-1H-imidazole-
4(5H)-one derivatives (4a-u) synthesized in very good yield and characterized through
conventional analytical techniques. As indicated through preliminary bioactivity predictions,
these compounds evaluated as PAR1, PI3Kinase inhibitors, and anticancer agents through
various established techniques. In the end, compounds 4b and 4g found to have remarkable
activities against proposed biological activities and screened for future pre-clinical studies
over animal models.
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