Enriching biologically relevant chemical space around 2-aminothiazole template for anticancer drug development

Article abstract of DOI:10.1007/s00044-017-2039-y

Combinatorial library based on a biologically relevant core template, 2-aminothiazole, with immense scope of diversity multiplication was designed for anticancer therapeutics. The diversity elements were incorporated through azomethine linkage on C4 hydrazine terminus in 5-benzoyl-2-arylamino-1,3-thiazole using isopropyl, isobutyl, cyclohexyl, and benzyl fragments and enrichment of chemical space therein was evaluated. Molecular docking of an in-house 200-member virtual library in anticancer target proteins- estrogen receptor (3ERT), cyclin dependent kinase (3FDN), and Aurora kinase (3LAU), identified selective binding of the compounds as ATP competitive inhibitors of 3LAU. The synthetic access to the compounds was realized through a facile and economically viable [4 + 1] ring synthesis strategy employing commercially available reagents. The in vitro cytotoxicity of selected members against human cancer cell lines indicated the potential of the designed scaffold in anticancer drug discovery, where compounds 2b, 3b, and 4b were found to be active against MCF-7 and A549 cell lines in less than ten micro molar concentrations. Moreover the predicted physicochemical properties pointed to the drug appropriateness for most of these molecules, that they obey the rule of five (RO5). Thus we present 2-alkyl/arylamino-4-alkylidene/arylidenehydrazino-5-benzoyl-1,3-thiazoles as a prospective and expandable skeleton for diversity oriented synthesis and in the discovery of selective Aurora kinase inhibitors.

Full text of DOI:10.1007/s00044-017-2039-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2018) 27:23–36
DOI 10.1007/s00044-017-2039-y
ORIGINAL RESEARCH
Enriching biologically relevant chemical space around 2-
aminothiazole template for anticancer drug development
Sarah Titus1 Kumaran G. Sreejalekshmi1
●
Received: 28 February 2017 / Accepted: 16 August 2017 / Published online: 28 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Combinatorial library based on a biologically
relevant core template, 2-aminothiazole, with immense
scope of diversity multiplication was designed for antic-
ancer therapeutics. The diversity elements were incorpo-
rated through azomethine linkage on C4 hydrazine terminus
in 5-benzoyl-2-arylamino-1,3-thiazole using isopropyl,
isobutyl, cyclohexyl, and benzyl fragments and enrichment
of chemical space therein was evaluated. Molecular docking
of an in-house 200-member virtual library in anticancer
target proteins- estrogen receptor (3ERT), cyclin dependent
kinase (3FDN), and Aurora kinase (3LAU), identified
selective binding of the compounds as ATP competitive
inhibitors of 3LAU. The synthetic access to the compounds
was realized through a facile and economically viable
[4 + 1] ring synthesis strategy employing commercially
available reagents. The in vitro cytotoxicity of selected
members against human cancer cell lines indicated the
potential of the designed scaffold in anticancer drug dis-
covery, where compounds 2b, 3b, and 4b were found to be
active against MCF-7 and A549 cell lines in less than ten
micro molar concentrations. Moreover the predicted
physicochemical properties pointed to the drug appro-
priateness for most of these molecules, that they obey the
rule of five (RO5). Thus we present 2-alkyl/arylamino-4-
alkylidene/arylidenehydrazino-5-benzoyl-1,3-thiazoles as a
prospective and expandable skeleton for diversity oriented
synthesis and in the discovery of selective Aurora kinase
inhibitors.
●
●
●
Keywords Chemical space 2-Aminothiazole HAT
●
●
VCHATL Anticancer activity Molecular docking
Introduction
The chemical space (Dobson 2004; Gorse 2006; Lipinski
and Hopkins 2004) comprising small organic molecules is
vast but largely unexplored whereas access to medicinally
relevant chemical space with appropriate design and syn-
thetic techniques is limited (Deng et al. 2013). Hence, the
identification of novel small molecules within biological
activity span is of utmost importance to chemical biology
and drug discovery (Bennani 2012). Consequently,
computer-based de novo drug design (Schneider and
Schneider 2016; Rodrigues et al. 2015; Schneider and
Fechner 2005) is currently re-emerging with a focus on
generating innovative molecular scaffolds by accessing
virtually infinite chemical space. Here, identification of
novel drug-like small molecules with high structural
diversity and quality are the prerequisite for new break-
throughs. The design of novel molecular libraries (Hajduk
et al. 2011) can focus on (i) a relevant therapeutic target
(John Harris et al. 2011; Sheppard and MacRitchie 2013;
Prien 2005) or disease and (ii) the chemistry (Dimova and
Bajorath 2016; Chen et al. 2005) or a desired molecular
function. Despite the presence of numerous recurring
molecular frameworks in bioactive molecules, the major
challenge that still remains is the design of novel scaffolds
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2039-y) contains supplementary material,
which is available to authorized users.
* Kumaran G. Sreejalekshmi
sreeja@iist.ac.in
1
Department of Chemistry, Indian Institute of Space Science and
Technology, Valiamala Post-695 547, Thiruvananthapuram, India

24
Med Chem Res (2018) 27:23–36
capable to direct functional groups in a well-defined three
dimensional (3D) space. The structural modification of
compounds with established physiological or pharmacolo-
gical effects for the production of novel and high quality
lead molecules is an important aspect in medicinal chem-
istry and hence a key factor in drug development. In this
context, heterocycles which can orient functional groups
favorably are considered to be privileged structures and
their presence in overall bioactive compound population
gained them a significant position in modern drug devel-
opment (Welsch et al. 2010; Dua et al. 2011; Song et al.
2014). Among heterocyclic ring systems, 1,3-thiazole has
been the topic of interest for many researchers across
the world because of its presence in many natural products
and biomolecules and its wide spectrum of pharmaco-
logical activities. Biological importance of thiazoles
varies from pharmacophoric and bioisosteric elements to
spacers that made them suitable core in design and devel-
opment of novel therapeutic agents (Ayati et al. 2015;
Dilek Altıntop et al. 2016; Kashyap et al. 2012). Various
structural modifications around thiazole core identified
its role as an important pharmacophore in many clinically
used drugs. Among different synthetic derivatives of
1,3-thiazoles, 2-amino-1,3-thiazoles are an attractive
moiety in medicinal chemistry with a wide spectrum of
biological activities (Das et al. 2016; Cheng et al. 2016;
Gallardo-Godoy et al. 2011; Gorczynski et al. 2004). Our
research group has been interested in the design, virtual
screening, synthesis, and biological evaluation of novel
heterocyclic tumor growth inhibitors and apoptosis inducers
as potential new anticancer agents. The recurrence of 2-
aminothiazole motif in a number of compounds with
reported anticancer activities such as DAT1 and related
compounds (Sengupta et al. 2005; Romagnoli et al. 2009;
Paula et al. 2013) motivated us to select 2-aminothiazole as
a template for the design of a novel scaffold for anticancer
drug design.
For enriching biologically relevant chemical space
(BRCS) around the 2-aminothiazole core, we were
encouraged by the growing interest in hydrazones
with varied physical, chemical, and biological activities
(Rollas and Küçükgüzel 2007; Tian et al. 2010) along
with expanding knowledge in their potential anticancer
activities, (Green et al. 2001; Kalinowski et al. 2007;
Lovejoy and Richardson 2002; Dandawate et al. 2014; Nasr
et al. 2014; Kaplánek et al. 2015a, b; Yu et al. 2015),
especially thiazolyl hydrazones (Novinson et al. 1976;
Holla et al. 2003; Savini et al. 2004; Altıntop et al. 2014;
Dilek Altıntop et al. 2014). Our continued interest in the
synthesis and biological evaluations of thiazole com-
pounds, especially 2,4-diamino-5-ketothiazoles, and inten-
sifying our research further, we designed the next higher
homolog of DAT1 by providing an azomethine linkage at
C4 which resulted in 2-amino-4-alkylidene/arylidenehy-
drazino-5-benzoyl-1,3-thiazoles (HAT) and accomplished a
one pot synthetic protocol (Titus and Sreejalekshmi 2014)
to access these compounds. The broad structural diversity
encompassed by these novel entities store immense poten-
tial for lead generation in chemical genetics and drug
discovery.
The present article describes the synthesis of a series of
HAT and their in vitro anticancer activity against
six NCI approved human cancer cell lines. Expansion of the
chemical space around the scaffold was carried out
by the design of a 200-member virtual combinatorial
HAT library (VCHATL) and in silico screening
in well-established anticancer target proteins viz; estrogen
receptor (3ERT), cyclin dependent kinase (CDK1), and
aurora kinase (3LAU). It is worth noting that the predicted
ADME properties further highlight the potential
of the designed scaffold in anticancer drug design and
development.
Materials and methods
Experimental
Aminoguanidine nitrate (AGN) isothiocyanates, α-
bromoketones and CDCl3 were purchased from Aldrich
chemical company and were used as such. Ketones/alde-
hydes,
DMF, DMSO, Et3N, and NaOH were purchased from
Merck chemicals and the solvents were purified using
general procedure. Silica-coated plates for thin layer
chromatography (TLC) were obtained from Merck, India,
and spots were visualized under ultraviolet (UV) light
or in iodine chamber. All the synthesized compounds were
characterized by spectroscopic techniques (1H nuclear
magnetic resonance (NMR), 13C NMR, mass spectrometry
(MS), Fourier transform infrared (FT-IR)), melting
point (mp) determination and elemental analysis. Mp
determination was carried out using ThermoScientific mp
apparatus. NMR spectra were recorded using Bruker
AV III 500 MHz FT-NMR spectrometer using CDCl3 as
solvent. Liquid chromatography/mass spectrometry (LC/
MS) spectra were recorded on Varian Inc LC/MS spectro-
meter. HR-MS was obtained using Waters Micromass
Q-TofmicroTM (YA105) spectrometer. Electrospray
ionization-mass spectrometry (ESI-MS) was recorded using
ThermoScientific ExactiveOrbitrap mass spectrometer.
Infrared (IR) spectra were obtained using Perkin Elmer
Spectrum 100 FT-IR spectrometer. Elemental analysis was
carried out with Perkin Elmer 2400 Series CHNS/O
Analyzer.

Med Chem Res (2018) 27:23–36
25
Synthesis
4-methoxyphenylisothiocyanate (9 mmol, 1.48 g). The iso-
lated product after purification by recrystallization (EtOH/
Water-1:1) was then reacted with phenacylbromide (10
mmol, 1.99 g) following the general procedure and pure 1b
was obtained after recrystallization from ethanol–acetone
(1:1) mixture as yellow solid). Yield 92%; mp 238–240 °C;
IR (UATR)υmax: 3268, 3076, 2996, 1695, 1231, and 1030
General procedure for the synthesis of 2-amino-4-
alkylidene/arylidenehydrazino-5-benzoyl-1,3-thiazoles
(HAT)
The HATs were synthesized by [4 + 1] ring closure
between a C–N–C–S fragment aminoamidinothiourea
(AATU) and α-halo ketone. AATUs were synthesized by
treating a suspension of aminoguanidine nitrate (10 mmol,
1.37 g) and NaOH (10 mmol, 0.40 g) in DMF for 10 min at
room temperature followed by the addition of 10 mmol of
the ketone/aldehyde and further stirring for 45–50 min.
Further, isothiocyanate (9 mmol) in DMF was added drop
wise and stirred for 1 h followed by addition of the mixture
to crushed ice for the precipitation of the solid product.
AATU was purified and isolated by recrystallization from
appropriate solvents. In the ring closure step, AATU (10
mmol) in DMSO and α-bromoketone (10 mmol) were
reacted in presence of 1.2 molar excess of Et3N for 10 min
at room temperature and then was added to crushed ice to
obtain the solid HAT which was collected by filtration, air-
dried and purified by recrystallization from suitable
solvents.
cm−1 1H NMR (CDCl3, 500 MHz, ppm): δ = 12.08 (s,
;
1NH), 8.95 (s, 1ArNH), 7.74–7.76 (m, 2ArH), 7.41–7.46
(m, 3ArH), 7.25–7.28 (m, 1ArH), 6.86–6.88 (m, 1ArH),
6.82–6.83 (m, 1ArH), 6.72–6.76 (m, 1ArH), 3.79 (s, 3H,
OCH3), 2.03 (s, 3H, CH3), 1.97 (s, 3H, CH3); 13C NMR
(CDCl3, 125 MHz, ppm): δ = 183.8 (C, C-10), 171.9 (C, C-
9), 162.0 (C, C-2), 152.2 (C, C-4), 141.2 (C, C-17), 135.8
(C, C-14), 135.6 (C, C-6), 130.5 (C, C-5), 130.2 (C, C-12),
128.2 (C, C-13), 127.1 (C, C-7), 121.5 (C, C-8), 94.6 (C, C-
11), 55.4 (C, C-18), 25.0 (C, C-16), 17.1 (C, C-15); EIMS
m/z 364[M]+ (100), 308 (20), 259 (23), 133 (48), 132 (34),
105 (63), 91 (16), 77 (37), 56 (48); anal. calcd. for
C20H20N4O2S: C, 65.90; H, 5.53; N, 15.37. Found: C,
65.77; H, 5.46; N, 15.29.
5-(4-Flurobenzoyl)-4-isopropylidenehydrazino-2-phenyla-
minothiazole (1c) Yellow solid (EtOH–CH3COCH3, 1:1).
(The corresponding AATU was obtained using acetone (10
mmol, 0.580 g), AGN (10 mmol, 1.37 g) and phenyli-
sothiocyanate (9 mmol, 1.21 mg) and following the general
procedure. It was purified by recrystallization using EtOH/
Water (1:1) and then treated with 4-fluorophenacylbromide
(10 mmol, 2.77 g). It was purified by recrystallization using
ethanol–acetone (1:1) mixture to obtain pure yellow solid).
Yield 90 %; mp 224–225 °C; IR (UATR) υmax: 3340, 3279,
5-Benzoyl-4-isopropylidenehydrazino-2-phenylaminothia-
zole (1a) Yellow solid (EtOH–CH3COCH3,1:1). (The
required AATU was synthesized using acetone (10 mmol,
0.580 g), AGN (10 mmol, 1.37 g) and phenylisothiocyanate
(9 mmol, 1.21 g) following the general procedure. The
isolated product after recrystallization from EtOH/Water
(1:1) was treated with phenacylbromide (10 mmol, 1.99 g)
in presence of Et3N following the general procedure. The
crude product was purified by recrystallization using
ethanol–acetone (1:1) mixture to obtain the pure product as
1
3062, 2945, 1670, 847, and 755 cm−1; H NMR (CDCl3,
500 MHz, ppm): δ = 12.08 (s, 1NH), 9.10 (s, 1ArNH),
7.76–7.80 (m, 2ArH), 7.38–7.42 (m, 2ArH), 7.28–7.30 (m,
2ArH), 7.21–7.24 (m, 1ArH), 7.12 (t, J = 8.5 Hz, 2ArH),
2.03 (s, 3H, CH3), 1.96 (s, 3H, CH3); 13C NMR
(CDCl3,125 MHz, ppm): δ = 182.6 (C, C-10), 171.4 (C, C-
14), 165.0 (C, C-13), 163.0 (C, C-5), 162.1 (C, C-2), 152.4
(C, C-4), 138.4 (C, C-17), 137.4 (C, C-6), 129.5 (C, C-8),
125.8 (C, C-9), 121.4 (C, C-12), 115.5 (C, C-7), 95.0 (C, C-
11), 24.9 (C, C-16), 17.1 (C, C-15); anal. calcd. for
C19H17FN4OS: C, 61.94; H, 4.65; N, 15.22. Found: C,
59.37; H, 3.88; N, 14.45.
yellow solid). Yield 97%; mp 206–208 °C; IR (UATR)υmax
:
3350, 3220, 3060, 1676, 749, and 690 cm−1 1H NMR
;
(CDCl3, 500 MHz, ppm): δ = 12.09 (s, 1NH), 8.45 (s,
1ArNH), 7.76–7.77 (m, 1ArH), 7.40–7.48 (m, 5ArH),
7.28–7.29 (m, 3ArH), 7.21–7.24 (m, 1ArH), 2.10 (s, 6H,
CH3); 13C NMR (CDCl3, 67.9 MHz, ppm): δ = 184.2 (C, C-
10), 161.8 (C,C-2), 152.3 (C, C-4), 141.3 (C, C-17), 138.2
(C, C-6), 130.7 (C, C-5), 129.7 (C, C-12), 128.4 (C, C-14),
127.2 (C, C-8, C-13), 125.6 (C, C-9), 120.9 (C,C-7), 94.4
(C, C-11), 25.2 (C, C-16), 17.2 (C, C-15); EIMS m/z 350
[M]+ (40), 245 (6), 119 (21), 105 (37), 77 (100), 56 (19);
anal. calcd. for C19H18N4O2S: C, 65.12; H, 5.18; N, 15.99.
Found: C, 65.08; H, 5.14; N, 15.83.
4-Isopropylidenehydrazino-2-(4-methoxyphenylamino)-5-
(naphth-2-oyl)thiazole (1e) Yellow solid (EtOH–CH3
COCH3,1:1). (The desired AATU was synthesized using
acetone (10 mmol, 0.580 g), AGN (10 mmol, 1.37 g) and 4-
methoxyphenylisothiocyanate (9 mmol, 1.48 g) following
the general procedure. The purified product obtained by
recrystallization (EtOH/Water-1:1) was then treated with 2-
bromoacetylnaphthalene (10 mmol, 2.49 g) following the
5-Benzoyl-4-isopropylidenehydrazino-2-(4-methoxypheny-
lamino)thiazole (1b) Yellow solid (EtOH–CH3CO
CH3,1:1). (The desired AATU was synthesized using
acetone (10 mmol, 0.580 g,), AGN (10 mmol, 1.37 g) and

26
Med Chem Res (2018) 27:23–36
general procedure. The crude product was purified by
recrystallization using ethanol–acetone (1:1) mixture to
obtain the product as yellow solid). Yield 90 %; mp
223–224 °C. IR (UATR) υmax: 3395, 3269, 3054, 2996,
217–218 °C; IR (UATR) υmax: 3268, 3201, 2870, 1698,
836, and 729 cm−1; 1H NMR (CDCl3, 500 MHz, ppm): δ =
12.12 (s, 1NH), 8.32 (s, 1ArNH), 7.71 (d, J = 7.0 Hz,
2ArH), 7.36–7.45 (m, 3ArH), 7.20–7.22 (m, 2ArH),
6.89–6.93 (m, 2ArH), 3.81 (s, 3H), 2.44–2.46 (m, 1H),
2.33–2.36 (m, 1H), 2.06 (s, 3H), 2.01–2.05 (m, 1H),
1.09–1.23 (m, 2H); 13C NMR (CDCl3,125 MHz, ppm): δ =
183.7 (C, C-10), 173.0 (C, C-9), 162.5 (C, C-2), 156.7 (C,
C-18), 152.1 (C, C-4), 141.4 (C, C-6), 131.1 (C, C-5), 130.5
(C, C-13), 128.4 (C, C-12), 127.2 (C, C-14), 124.7 (C, C-7),
114.7 (C, C-8), 94.6 (C, C-11), 55.5 (C, C-19), 31.9 (C, C-
15), 24.1 (C, C-17), 17.2 (C, C-16); LCMS m/z: 393.30
[M − 1]+.
1
1636, 1231, 1030, 823, and 776 cm−1; H NMR (CDCl3,
500 MHz, ppm): δ = 12.19 (s, 1NH), 8.29 (s, 1ArNH), 8.20
(s, 1ArH), 7.87–7.91 (m, 1ArH), 7.84–7.86 (m, 2ArH),
7.80–7.82 (m, 1ArH), 7.49–7.55 (m, 2ArH), 7.21–7.22 (m,
2ArH), 6.89–6.90 (m, 2ArH), 3.80 (s, 3H, OCH3), 2.08 (s,
6H, CH3); 13C NMR (CDCl3, 125 MHz, ppm): δ = 183.8
(C, C-10), 173.4 (C, C-9), 162.5 (C, C-2), 158.2 (C, C-23),
152.3 (C, C-4), 138.8 (C, C-6), 134.3 (C, C-5), 132.6 (C, C-
12), 131.2 (C, C-13, C-18), 128.9 (C, C-14), 128.3 (C, C-
15), 127.7 (C, C-16), 127.3 (C, C-17), 126.5 (C, C-19),
124.7 (C, C-7, C-20), 114.9 (C, C-8), 95.0 (C, C-11), 55.5
(C, C-24), 25.2 (C, C-21), 17.3 (C, C-22); LCMS m/z:
430.15 [M]+.
4-Isobutylidenehydrazino-5-(naphth-2-oyl)-2-phenylami-
nothiazole (2d) Yellow solid (EtOH–CH3COCH3,1:1).
(The AATU for 2d was synthesized using 2-butanone (10
mmol, 0.721 g), AGN (10 mmol, 1.37 g) and phenyli-
sothiocyanate (9 mmol, 1.21 g). The product was recrys-
tallized using EtOH/Water (1:1) and then treated with 2-
bromoacetylnaphthalene (10 mmol, 2.49 g) following the
general procedure. The crude product was purified by
recrystallization using ethanol–acetone (1:1) mixture to
obtain the product as yellow solid). Yield 90%; mp
237–238 °C; IR (UATR) υmax: 3282, 3098, 3052, 1629,
832, and 759 cm−1; 1H NMR (CDCl3, 500 MHz, ppm): δ =
12.13 (s, 1NH), 8.24 (s, 1ArH), 8.53 (s, 1ArNH), 7.89–7.92
(m, 3ArH), 7.83–7.88 (m, 3ArH), 7.51–7.57 (m, 3ArH),
7.36–7.39 (m, 2ArH), 2.39 (dd, J = 7.5 Hz, 2H), 2.08 (s,
3H), 1.13 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 125 MHz,
ppm): δ = 184.1 (C, C-10), 171.2 (C, C-9), 161.9 (C, C-2),
156.8 (C, C-4), 138.7 (C, C-23), 138.3 (C, C-12), 134.4 (C,
C-5), 132.6 (C, C-6), 129.7 (C, C-18), 128.9 (C, C-14),
128.4 (C, C-20), 127.8 (C, C-15), 127.4 (C, C-16), 126.6
(C, C-17), 125.6 (C, C-19), 124.3 (C, C-8), 120.9 (C, C-7),
95.1 (C, C-11), 32.1 (C, C-15), 15.2 (C, C-17), 11.4 (C, C-
16); LCMS m/z: 414.10 [M]+.
4-isopropylidenehydrazino-5-(naphth-2-oyl)-2-phenylami-
nothiazole (1f) Yellow solid (EtOH–CH3COCH3,1:1).
(The corresponding AATU was obtained by the reaction
between acetone (10 mmol, 0.580 g), AGN (10 mmol, 1.37
g) and phenylisothiocyanate (9 mmol, 1.21 g). The recrys-
tallized product (EtOH/Water-1:1) was then treated with 2-
bromoacetylnaphthalene (10 mmol, 2.49 g) following the
general procedure. The crude product was purified by
recrystallization using ethanol–acetone (1:1) mixture to
obtain the product as yellow solid). Yield 90%;mp
258–259 °C; IR (UATR) υmax: 3282, 3098, 3052, 1629,
832, and 759 cm−1; 1H NMR (CDCl3, 500 MHz, ppm): δ =
12.15 (s, 1NH), 8.24 (s, 1ArH), 8.19 (s, 1ArNH), 7.90–7.93
(m, 2ArH), 7.83–7.88 (m, 2ArH), 7.51–7.57 (m, 2ArH),
7.37–7.41 (m, 2ArH), 7.25–7.27 (m, 2ArH), 7.19–7.22 (m,
1ArH), 2.16 (s, 3H, CH3), 2.12 (s, 3H, CH3); 13C NMR
(CDCl3,125 MHz, ppm): δ = 184.1 (C, C-10), 171.2 (C, C-
9), 161.9 (C, C-2), 152.4 (C, C-4), 138.7 (C, C-23), 138.2
(C, C-12), 134.4 (C, C-5), 132.6 (C, C-6), 129.7 (C, C-18),
128.9 (C, C-14), 128.4 (C, C-20), 127.8 (C, C-15), 127.4
(C, C-16), 126.6 (C, C-17), 125.6 (C, C-19), 124.3 (C, C-8),
120.9 (C, C-7), 95.0 (C, C-11), 25.2 (C, C-21), 17.3 (C, C-
22); LCMS m/z: 400.12 [M]+.
5-Benzoyl-4-cyclohexylidenehydrazino-2-(4-methoxyphe-
nylamino)thiazole (3b) Yellow solid (EtOH–CH3CO
CH3,1:1). (The desired AATU was synthesized using
cyclohexanone (10 mmol, 0.981 g), AGN (10 mmol, 1.37 g)
and 4-methoxyphenylisothiocyanate (9 mmol, 1.48 g)
following the general procedure. The product after recrys-
tallization (EtOH/Water-1:1) was reacted with phenacyl-
bromide (10 mmol, 1.99 g) following the general procedure.
The crude product was purified by recrystallization using
ethanol–acetone (1:1) mixture to obtain the product as
yellow solid). Yield 80%; mp. 246–247 °C; IR (UATR)
υmax: 3276, 3198, 2938, 2858, 1678, 831, and 731 cm−1; 1H
NMR (CDCl3, 500 MHz, ppm): δ = 12.34 (s, 1NH), 8.36 (s,
1ArNH), 7.70–7.72 (m, 2ArH), 7.38–7.47 (m, 3ArH),
7.20–7.22 (m, 2ArH), 6.89–6.92 (m, 2ArH), 3.81 (s, 3H),
5-Benzoyl-4-isobutylidenehydrazino-2-(4-methoxyphenyla-
mino)thiazole (2b) Yellow solid (EtOH–CH3COCH3,1:1).
(The AATU for the synthesis of 2b was obtained by
reacting 2-butanone (10 mmol, 0.721 g), AGN (10 mmol,
1.37 g) and 4-methoxyphenylisothiocyanate (9 mmol, 1.48
g) following the general reaction procedure. The purified
AATU (recrystallization using EtOH/Water-1:1) was then
treated with phenacylbromide (10 mmol, 1.99 g) and the
crude product obtained on work up was purified by
recrystallization using ethanol-acetone (1:1) mixture to
obtain the product as yellow solid). Yield 80%; mp

Med Chem Res (2018) 27:23–36
27
2.49–2.52 (m, 2H), 2.34–2.38 (m, 2H), 1.63–1.72 (m, 6H);
13C NMR (CDCl3, 125 MHz, ppm): δ = 183.7 (C, C-10),
173.4 (C, C-9), 162.8 (C, C-2), 158.3 (C, C-15), 158.2 (C,
C-4) 141.4 (C, C-6), 131.2 (C, C-13), 130.5 (C, C-5), 128.4
(C, C-12), 127.2 (C, C-14), 124.7 (C, C-7), 114.9 (C, C-8),
94.6 (C, C-11), 55.5 (C, C-19), 35.2 (C, C-16), 27.2 (C, C-
16′), 26.9 (C, C-17), 25.6 (C, C-17′), 25.9 (C, C-18); LCMS
m/z: 421.12 [M + 1]+.
128.4 (C, C-13), 128.2 (C, C-14), 128.3 (C, C-16), 127.3
(C, C-16), 127.2 (C, C-7), 124.8 (C, C-17), 114.4 (C, C-18),
94.6 (C, C-11), 55.4 (C, C-20); LCMS m/z: 429.30
[M + 1]+.
In vitro anticancer screening
The compounds were screened for their anticancer activity
using the colorimetric sulforhodamine B (SRB) assay fol-
lowing the reported protocol (Skehan et al. 1990; Vichai
and Kirtikara 2006). Six human cancer cell lines namely
MCF-7, SW-620, HL-60, A549, OVCAR-3, and SK-MEL-
2 were selected from the NCI’s (National Cancer Institute)
cell line panel. Three consecutive screening experiments
were carried out for each compound in a 96-well plate (5 ×
103 cells/well). The dose-response in each of the experi-
ments was measured at four different concentrations (10,
20, 40, and 80 μg/mL) of the compounds in DMSO or
alcohol and in each experiment Adriamycin (ADR) was
used as a positive control at the same concentration levels.
The percentage growth was evaluated spectro-
photometrically against controls that were not treated with
test compounds.
4-Cyclohexylidenehydrazino-5-(4-methoxybenzoyl)-2-phenyl-
aminothiazole (3c) Yellow solid (EtOH–CH3COCH3,1:1).
(The corresponding AATU was synthesized using cyclohex-
anone (10 mmol, 0.981 g), AGN (10 mmol, 1.37 g) and phe-
nylisothiocyanate (9 mmol, 1.21 g). The recrystallized product
(EtOH/Water-1:1) and 4-methoxyphenacylbromide (10 mmol,
2.29 g) was reacted following the general procedure. The
crude product was purified by recrystallization using
ethanol–acetone (1:1) mixture to obtain the product as yellow
solid). Yield 63%; mp 224–225 °C; IR (UATR) υmax: 3296,
1
1629, 1545, 1244, and 1058 cm−1; H NMR (CDCl3, 500
MHz, ppm): δ = 12.29 (s, 1NH), 8.38 (s, 1ArNH), 7.75–7.77
(m, 2ArH), 7.38–7.42 (m, 2ArH), 7.27–7.28 (m, 2ArH),
7.19–7.22 (m, 1ArH), 6.92–6.94 (m, 2ArH), 3.85 (s, 3H),
2.51–2.53 (m, 2H), 2.40–2.43 (m, 2H), 0.69–1.75 (m, 4H);
13C NMR (CDCl3,125 MHz, ppm): δ = 183.4 (C, C-10),
170.9 (C, C-14), 161.9 (C, C-2), 161.6 (C, C-14), 156.2 (C, C-
15), 151.9 (C, C-4), 138.5 (C, C-17), 133.8 (C, C-5), 133.7 (C,
C-13), 129.6 (C, C-8), 129.2 (C, C-6), 125.5 (C, C-9), 121.1
(C, C-12), 113.7 (C, C-7), 98.0 (C, C-11), 55.4 (C, C-19), 31.9
(C, C-16), 24.1 (C, C-16′), 17.2 (C, C-17), 15.2 (C, C-17′),
11.3 (C, C-18); ESIMS m/z: 421.16 [M + H]+; anal. calcd. for
C23H24N4O2S: C, 65.69; H, 5.76; N, 13.33. Found: C, 66.18;
H, 5.91; N, 13.55.
Virtual screening
All the computational studies were carried out using
Schrodinger software suite 2014 (Schrodinger LLC 2014).
The ligand structures used in this study were constructed
using the graphical tool, Maestro 9.6 and geometry opti-
mization and partial atomic charge assignment of ligands
were done with the help of Ligprep using the optimized
potentials for liquid simulations-all atom (OPLS-AA) force
field (Jorgensen et al. 1996). The X-ray crystal structures of
proteins were retrieved from RCSB Protein Data Bank
(PDB ID codes 3ERT, 3LAU, and 3FDN). Prior to calcu-
lations, the raw crystal structures were prepared and the
water molecules were removed using protein preparation
tool (PrepWizard) in the software package. All docking
calculations were performed using the Extra Precision (XP)
mode of a grid-based ligand docking method, GLIDE 6.1
(Friesner et al. 2006). The grid was generated in such a way
that grid center was located at the mid-point of the longest
atom–atom distance in the respective co-crystallized ligand
in the protein. The native ligand was used as the reference
for the grid generation. Inside the grid, at most 800 poses
per ligands were generated keeping the default variables as
such. The free energy of binding for each ligand was cal-
culated by the sum of all the interactions like hydrogen
bonding, hydrophobic, lipophilic etc. The lowest estimated
free energy from various binding conformations of each
ligand was calculated, ranked by dock score functions and
5-Benzoyl-4-benzylidenehydrazino-2-(4-methoxyphenyla-
mino)thiazole (4b) Yellow solid (EtOH-CH3COCH3,1:1).
(The required AATU was synthesized using benzaldehyde
(10 mmol, 1.06 g), AGN (10 mmol, 1.37 g) and 4-
methoxyphenyl isothiocyanate (9 mmol, 1.48 g) following
the general procedure. Then the recrystallized product
(EtOH/Water-1:1) was treated with phenacylbromide (10
mmol, 1.99 g) following the general procedure. The crude
product was purified by recrystallization using
ethanol–acetone (1:1) mixture to obtain 4b as yellow solid).
Yield 85%; mp 193–194 °C; IR (UATR) υmax: 3220, 3040,
1718, 826, and 725 cm−1 1HNMR (CDCl3, 500 MHz,
;
ppm): δ = 12.39 (s, 1NH), 8.05 (s, 1ArNH), 7.98 (s, 1H),
7.64–7.67 (m, 3ArH), 7.51–7.54 (m, 3ArH), 7.46–7.48 (m,
2ArH), 7.35–7.42 (m, 3ArH), 7.23–7.31 (m, 1ArH), 6.94
(d, J = 9 Hz, 2ArH), 3.83 (s, 3H); 13CNMR (CDCl3, 125
MHz, ppm): δ = 184.2 (C, C-10), 173.6 (C, C-9), 161.8 (C,
C-2), 158.0 (C, C-19), 155.4 (C, C-4), 141.3 (C, C-6), 133.8
(C, C-15), 131.3 (C, C-5), 130.7 (C, C-8), 129.6 (C, C-12),

28
Med Chem Res (2018) 27:23–36
the best docked structure was chosen. All the pharmaceu-
tically relevant properties and physical descriptors of the
optimized ligands were calculated using Qikprop 3.5 in
Schrodinger LLC. The recommended ranges for the
descriptors were derived by comparing those with 95%
known drugs. The physical descriptor values such as MW,
octanol/water partition coefficient, H-bond donor/acceptor
were compared with classical Lipinski’s RO5 and oral
bioavailability was calculated using Caco-2 and MDCK cell
membrane models.
template was designed by the modification of C4 of the ring
with hydrazone fragment to afford HAT. The designed
tetravariant scaffold provides four sites for chemical space
enrichment around the core. (Fig. 1).
As the synthetic feasibility is a crucial factor in drug
discovery, an easy and economically viable synthetic route
to the novel scaffold, HAT was accomplished following a
[4 + 1] thiazole ring formation (Scheme 1) under mild
conditions employing commercially available building
blocks to facilitate automated synthetic protocols.
Different sites for diversity multiplication (R1, R2, R3,
and R4) would facilitate vast expansion of HAT library and
modification of chemical space around the core. Giving
special emphasis on the hydrazono part, by varying R1 and
R2, a contribution from carbonyl component, we designed
isopropylidene (IPHAT), isobutylidene (IBHAT), cyclo-
hexylidene (CyHAT), and benzylidene (BzHAT) (Fig. 2)
derivatives of HAT.
The generality of the synthetic route was established by
synthesis of compounds (1–4) in moderate to excellent
yields (Table 1), where diversity elements R1 and R2 were
introduced as capping units in aminoguanidine nitrate (b)
by treating with carbonyl compounds (a) and the resultant
Schiff bases (c) were condensed with arylisothiocyanate (d)
under basic conditions to afford aminoamidinothioureas (e)
(Sreejalekshmi 2010) as the C–N–C–S precursors for thia-
zole ring synthesis. In the second step, these C–N–C–S
Results and discussion
Chemistry
Driven by the success stories on the anticancer activity of 2-
aminothiazole derivatives, we felt it worth examining the
possibilities of developing unexplored members from this
family. Importance of biologically relevant hydrazone
libraries, especially hydrazone–heterocycle combinations in
drug discovery (Verma et al. 2014) and reported bioactiv-
ities of different hydrazone containing thiazoles, exclusively
2-hydrazinothiazoles (Bharti et al. 2010; Secci et al. 2012;
Carradori et al. 2013; Yurttaş et al. 2013) inspired us to
select the hydrazone fragment for the modification of 2-
aminothiazole core. In the light of the literature survey, a
novel heterocyclic scaffold based on 2-aminothiazole
precursors underwent
a
base assisted elimination–
cyclization–aromatization with α-bromoketones (f) at room
temperature to afford the desired compounds in excellent
yield. Having representative HAT molecules in our hands,
we next proceeded with the investigation of their potential
as anticancer agents.
In vitro anticancer screening
The cytotoxicity of 12 compounds were evaluated in cancer
cell lines—MCF-7, HL-60, SK-MEL-2, A549, OVCAR-3,
and SW620 using SRB assay with Adriamycin (ADR) as
Fig. 1 Novel drug design scaffold combining aminothiazole and
hydrazone fragment
Scheme 1 Chemical synthesis
of HAT

Med Chem Res (2018) 27:23–36
29
Fig. 2 Four classes of HAT
scaffold by varying carbonyl
component
Table 1 Synthesized HAT molecules
ovarian, and skin melanoma cancer cells were found to be
less affected. Considering these facts, we decided to expand
the library of HAT and to proceed with the in silico binding
studies in anticancer target proteins.
Compound R1
R2
R3
R4
% Yield
1a
1b
1c
1da
1e
1f
–CH3 –CH3 –C6H5
–C6H5
97
92
90
94
90
90
80
–CH3 –CH3 4-OMeC6H4 –C6H5
–CH3 –CH3 –C6H5
–CH3 –CH3 –C6H5
4-FC6H4
In silico studies
4-ClC6H4
–CH3 –CH3 4-OMeC6H4 –C12H11
–CH3 –CH3 –C6H5 –C12H11
–CH3 –C2H5 4-OMeC6H4 –C6H5
ADME property prediction
2b
2ca
2d
3b
3c
4b
a
The bioavailability of the test molecules was assessed by
ADME (absorption, distribution, metabolism, and excre-
tion) prediction using Qikprop which relies mainly on
Lipinski Rule-of-Five and Jorgensen rule-of-three viola-
tions. The pharmaceutically relevant properties—octanol/
water and water/gas log Ps, log S, log BB, overall CNS
activity, Caco-2 and MDCK cell permeabilities, and human
oral absorption were calculated (Table 3) and compared
with those of existing drugs. All the compounds showed
properties within the recommended range for 95% of drugs
which suggested the drug-likeness of the designed mole-
cules with theoretically good passive oral absorption.
–CH3 –C2H5 –C6H5
–CH3 –C2H5 –C6H5
4-OMeC6H4 88
–C12H11
90
80
–C6H10
–C6H10
–C6H5
4-OMeC6H4 –C6H5
–C6H5
4-OMeC6H4 63
H
4-OMeC6H4 –C6H5
85
Reported earlier (Titus and Sreejalekshmi 2014)
the positive control and reference. The screening results
suggested the breast cancer cell line, MCF-7, to be more
fragile towards our tested compounds where GI50 values
were <40 μg/mL. Comparison of cytotoxicity of the
tested compounds is presented in Table 2. Among the
tested compounds, the IBHAT derivative-5-benzoyl-4-iso-
butylidenehydrazino-2-(4-methoxyphenylamino)thiazole
(2b) and the BzHAT derivative-5-benzoyl-4-benzylidene-
hydrazino-2-(4-methoxyphenylamino)thiazole (4b) dis-
played anticancer activity against MCF-7 (Fig. 3a) with
GI50 values of 2.32 and 8.16 µg/mL respectively, whereas,
Molecular docking
With the aim of enriching BRCS, an in-house 200-member
virtual combinatorial HAT library (VCHATL) was
designed by varying R3 and R4 in the respective parent
structures (1a–4a) of IPHAT, IBHAT, CyHAT, and
BzHAT which can be realized through the appropriate
reagents during the synthesis (Table 4).
To further establish the appropriateness of HAT library
in anticancer drug development, molecular docking studies
were performed in three different classes of anticancer tar-
get proteins. Suitable X-ray crystal structures of the selected
proteins—estrogen receptor (PDB ID: 3ERT), Cyclin
dependent kinase (PDB ID: 3FDN) and Aurora kinase
(PDB ID: 3LAU) were obtained from the protein data bank
(PDB) and optimized to get the lowest energy 3D structures.
VCHATL was also optimized and screened in the active
sites of individual proteins in a grid-based ligand docking
protocol using GLIDE and results are presented in Table 5.
The docking studies revealed better affinity of CyHAT, in
the
CyHATcounterpart-5-benzoyl–4–cyclohexylidenehy
drazino-2-(4-methoxyphenylamino) thiazole (3b) was
active against lung cancer cell line A549 (Fig.3b) with GI50
valueof 1.61 µg/mL. Moreover, all the three active mole-
cules were found to have potent anticancer activity against
all the tested cell lines. IPHAT derivative-5-benzoyl-4-iso-
propylidenehydrazino-2-(4-methoxyphenylamino)thiazole
(1b) showed moderate anticancer activity against MCF-7
and HL60 cell lines with GI50 = 35.60 and 33.64 µg/mL
respectively and was inactive against the other cell lines
tested.
It was interesting to note that most of tested compounds
showed selective dose-response towards the breast, leuke-
mia and/or lung cancer cell lines whereas the colon,

30
Med Chem Res (2018) 27:23–36
Table 2 In vitro screening
results of tested compounds
Compound GI50 (µg/mL)a
MCF-7
SW620
A549
HL60
SK-MEL-2
OVCAR-3
1a
1b
1c
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
35.6 2.73
>80
78.2 15.05 51.7 4.26
33.6 21.39 >80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
1d
1e
>80
>80
34.6 4.04
>80
1f
40.6 5.51
2.32 3.15c 40.1 5.85
>80
2b
2c
12.5 1.33
30.2 17.14 36.1 1.69 55.0 38.96
>80
>80
43.8 12.72 >80
>80
>80
27.9 2.67
2d
3b
3c
58.8 4.03
17.3 3.46
>80
>80
>80
1.61 1.41c
41.2 9.73
>80
30.7 4.81
36.4 7.04
16.1 1.95 22.70 4.72
>80 >80
>80
>80
>80
4b
ADRb
a
8.16 2.69c 44.1 6.23
28.7 2.00
<10
38.3 10.61 31.9 0.31 36.1 2.25
31.3 11.78 <10 <10
<10 <10
The values are the mean standard deviation (SD) of three independent experiments performed
Positive control
b
c
Marked in bold indicated a pronounced anticancer activity
Fig.3 Cell viability of active compounds at different concentrations against a MCF-7, b A549
terms of binding scores, towards estrogen receptor protein
whereas BzHAT class had more affinity to Cyclin depen-
dent kinase protein. But, considering the whole VCHATL,
all the members indicated comparatively good affinity
towards the active site of Aurora kinase (AURK) protein
3LAU.
have selective and potent inhibitors, it is essential to
understand the molecular constraints of ATP binding hinge
region of AURK and the structural basis for its interactions
with inhibitors in detail.
Effect of hydrazone substitution on binding affinity
Encouraged by higher binding energy of VCHATL in
3LAU protein, we carried out detailed binding studies of the
library in AURK proteins. Understanding the role of Aurora
kinases in cell cycle and tumorigenesis (Urich et al. 2013)
and the inhibition of Aurora kinase activity by small
molecule ATP competitors is a widely explored and chal-
lenging regime in target-based anticancer therapy (Fancelli
et al. 2006; Carpinelli et al. 2007; Manfredi et al. 2007;
Mortlock et al. 2007; Wilkinson et al. 2007). In order to
The effect of the hydrazone part in the in silico binding of
compounds was evaluated by the detailed binding study of
core compounds 1a, 2a, 3a, and 4a each representing a
particular class of HAT –IPHAT, IBHAT, CyHAT, and
BzHAT respectively. The predicted binding modes in the
active site of the protein (Fig. 4) were interesting where a
difference in the binding pattern was observed with respect
to variations in R1 and R2.

Med Chem Res (2018) 27:23–36
31
Table 3 Comparison of ADME properties of anticancer active HAT
Ligand MW
Donor HB Accept HB QP Polrz QP logP QP log
QPP
Caco
QP logBB #metab RO5
violations
% Human oral
absorption
o/w
HERG
2b
3b
4b
394.49
1
1
1
5.75
5.75
6.25
41.20
42.85
44.61
9.241
4.756
4.896
−6.150
−5.794
−6.748
1731
1428
1615
−0.734
−0.728
−0.742
3
4
2
0
100
100
100
420.52
428.50
0
0
The molecular weight of the tested ligand, MW (130–725 for 95% drugs); estimated number of H-bonds donated by ligand to water molecules,
DonorHB (0–6.0 for 95% drugs); estimated number of H-bonds accepted by ligand from water molecules, AcceptHB (2–20 for 95% drugs); the
predicted polarizability, QP polrz (13.0–70.0 for 95 % of drugs); the predicted octanol/water partition coefficient, QP logP o/w (−2.0 to 6.5); the
predicted IC50 value for blockage of HERG K+ channels, QP log HERG (concern <−5); the predicted apparent Caco-2 cell permeability, QPP
Caco in nm/s (<25 poor, >500 great); the predicted brain/blood partition coefficient, QP log BB (−3.0 to 1.0 for 95% drugs); the number of likely
metabolic reactions, #metab (1–8 for 95 % of drugs)
Table 4 Designed VCHATL
R1
H
–CH3
R2 –CH3 –C2H5
–C6H5
–C6H10
R3
R4
H
H
4-ClC6H5 4-FC6H5 4-OMeC6H5 4-NO2C6H5 4-MeC6H5
4-ClC6H5 4-FC6H5 4-OMeC6H5 4-NO2C6H5 Naphthalene Coumarin Indol
Table 5 Molecular docking results for synthesized library of HAT along with ADR
Compound 3ERT
3FDN
3LAU
G-score Lipophilic
H bond Electro G-score Lipophilic
H bond Electro G-score Lipophilic
H bond Electro
(kcal/
mol)
EvdW
(kcal/
mol)
EvdW
(kcal/
mol)
EvdW
1a
1b
1c
−4.69
−4.26
−5.23
−2.14
−6.65
−6.28
−2.61
−6.47
−6.17
−1.69
−5.69
−4.95
−4.78
−5.14
−4.77
−4.19
−4.26
−5.89
−5.16
−5.56
−4.93
−5.28
−4.75
−4.26
−4.55
−1.18
0
−0.08 −4.46
−3.78
−3.54
−3.32
−3.81
−4.54
−4.43
−4.01
−4.39
−4.09
−3.56
−4.05
−4.13
−3.31
−0.05 −0.1
−0.7 −0.3
−5.52
−6.93
−3.36
−3.98
−3.73
−4.01
−4.32
−4.23
−4.64
−3.35
−4.23
−4.35
−4.01
−4.33
−4.34
−0.92 −0.21
−0.73 −0.23
−0.77 −0.2
−0.22 −0.13 −4.02
−0.97 −0.33 −4.44
−0.03 −0.21 −4.51
−1.16 −0.36 −5.34
−0.1 −5.37
1d
1e
0
0
−0.09
−0.22 −0.2
−3.31
−1.09 −0.31 −6.61
−0.51 −0.29 −7.77
−0.34 −0.05 −7.58
−0.97 −0.27
−0.93 −0.43
1f
−0.7
−0.24 −5.46
−0.11 −1.73
2b
2c
0
−0.7
−0.17
−0.74 −0.49 −5.29
−0.73 −0.2
−1 −0.44 −6.62
−0.56 −0.08 −7.03
−0.49 0.02 −5.87
−6.29
−1.81 −0.57
−0.97 −0.55
2d
3b
3c
0
0
−0.02 −5.49
−0.07 −0.78
−0.7
−0.7
−0.23
−0.18
−1.03 −0.3
−0.57 −0.25 −0.53
−3.3 −1.33 −4.76
−6.08
4b
ADR
−0.38 −0.25 −7.76
−3.48 −0.95 −9.91
−1.03 −0.3
−3.94 −1.06
Compound 1a (G-score = −5.52 kcal/cal) with 25 heavy
atoms was found to bind to ATP hinge region of the protein
using its NH–C–N–C–NH unit for H-bonding between
Ala213 and Pro214 which are significant residues (Yan
et al. 2011) in AURK inhibition. The azomethine part of the
hydrazone as well as the phenyl ring (R4) were occupied in
the solvent exposure regions of the protein active site
whereas the phenyl ring (R3) was completely embedded in
the hydrophobic domain. When the chain length in the
azomethine part was increased by introducing isobutyl (2a,
G-score = −6.56 kcal/mol) or cyclohexyl (3a, G-score =
−6.05 kcal/mol) units, the hydrazono part moved more
towards the activation loop having the Ala-Asp-Phe triad
commonly refered to as the DFG motif. The ligands occu-
pied the active site such that R3 phenyl ring was enclosed in
a hydrophobic pocket and the C4 and C2 substituents on
thiazole moved out of this pocket and exhibited higher
ligand efficiency and binding energy than that of 1a. In the
BzHAT derivative 4a, H-bonding interaction with Ala 213
was absent and the ligand oriented itself near to the acti-
vation loop with hydrazono moiety as well as R4 proximal
to the DFG motif. The lowest binding energy (G-score =
5.09 kcal/mol) of 4a was attributed to the restricted con-
formational rotation of the azomethine part. The binding
affinity of the molecules in 3LAU followed the order 2a >
3a > 1a > 4a clearly indicating the role of hydrazono

32
Med Chem Res (2018) 27:23–36
Fig. 4 3D binding mode of 1a–
4a in the active site of 3LAU
protein
Fig. 5 a Hydrophobic enclosure
of 1a in the active site of 3LAU
b Hydrophobic enclosure of
molecules with substituted C2
phenyl groups
moiety in protein binding. It is assumed that the synergic
nitrogen atoms in the NH–C–N–C–NH pattern of HAT is
stereochemically well suited to form H-bonding interactions
with kinase hinge region of the ATP pocket. Since core
scaffold from all the four families showed selective binding
towards the active site of AURK protein and the scaffold is
endowed with additional positions for increasing diversity,
the influence of R3 and R4 on the binding affinity of
molecules inside the active site of 3LAU was next studied.
C5 towards the solvent exposure region contributed to its
destabilization. But, phenyl ring at C2 was buried inside the
hydrophobic enclosure which favored binding of the
molecule. When C2 phenyl ring in 1a was para substituted
with different functional groups, rotation of molecule along
the C2-NH bond occurred that stabilized orientations of the
molecules within hydrophobic enclosure of R3 with lesser
exposure penality of R4 (Fig. 5). In all the cases, exposure
penality of the hydrophobic groups were reduced sig-
nificantly and H-bonding with Ala213 was in the vicinity of
the hydrophobic residues of the active site and thus the
molecules showed higher binding energies than 1a.
Effect of R3 substitution
A similar trend was observed in the case of IBHATwhere
destabilzation caused by solvent exposure of hydrophobic
methyl groups was compensated by the interaction between
The hydrophobically enclosed H-bonding with Ala213
imparted more stability to the binding pose of 1a whereas
the expose penality of the methyl and phenyl group at the

Med Chem Res (2018) 27:23–36
33
the C5 phenyl ring and hydrophophobic residues of the
protein. The molecules were further stabilized by π-cation
interaction between Arg137 and C2 phenyl ring with a
higher binding energy for molecules with substituted phenyl
rings as R3 (Fig. 6).
In cyclohexylidene derivatives, substitution at C2 phenyl
resulted in partial rotation of the molecules along C2-NH
bond and hence a complete enclosure of hydrophobic
groups in the binding pocket consequent to which all C2
phenyl substituted derivatives had higher binding energy
than the core compound 3a (Fig. 7).
In BzHAT class, core molecule 4a bound to the active
site away from hinge region without any H-bonding inter-
actions with binding pocket. The R3 substitution was
found to have significant effect on locating hydrophobic
rings in protein binding site which increased binding
affinity toward protein through H-bonding with Ala213
(Fig. 8).
It was found that in all four classes, R3 substitution was
desirable for better interaction with hinge region and
thereby increased binding energy than corresponding
unsubstituted analogs.
Fig. 6 a 2D interaction
diagramof 2a in the active site of
3LAU b Hydrophobic enclosure
of 2a (red) and molecules with
substituted C2 phenyl groups
(blue) (color figure online)
Fig. 7 a Hydrophobic enclosure
of 3a in the active site of 3LAU
b Hydrophobic enclosure of
molecules with substituted C2
phenyl groups
Fig. 8 3D binding mode of a 4a
and b molecules with substituted
C2 phenyl groups in the active
site of 3LAU protein

34
Med Chem Res (2018) 27:23–36
Effect of R4 substitution
ing affinity was significantly reduced. This decrease was
attributed to solvent exposure of hydrophobic rings. Fur-
thermore, modification of R4 with biologically relevant
heterocyclic ring systems such as coumarin and indole led
to enhanced binding energies wherein a more pronounced
effect was found for indolyl derivatives (Table 6). The in
silico screening in protein active site clearly indicated a
substantial effect of binding pattern of the molecules with
change in the substitution around the core which can be
utilized for hit to lead identification in a structure-based
drug design (SBDD) protocol.
The docking studies on HAT derivatives with substituted
phenyl rings at R4 revealed that in all classes –NO2/–OMe
substitutions increase binding affinity by reducing exposure
penality effected through an orientation change in the active
site (Fig. 9). However, halogen substitutions were not much
influential in modulating the binding affinity of the mole-
cules in the 3LAU active site.
The replacement of phenyl ring at C5 with a poly aro-
matic ring such as naphthalene increased the binding energy
of IPHAT, IBHAT, and CyHAT whereas in BzHAT bind-
Fig. 9 Binding orientations of a
1a and and F-substituted
molecule in 3LAU active site b
Binding orientations of –OMe
and –NO2 substituted
moleculesin 3LAU active site
Table 6 Binding energy of HAT with poly/heteroaromatic R4
R3
R4
GScore, LiopophilicEvdW, Hbond, Electro
IPHAT
IBHAT
CyHAT
BzHAT
−4.69
−5.14
0
−6.56
−3.87
−0.7
−6.05
−4.19
−0.7
−5.09
−3.9
−0.65
−0.41
−0.08
−0.14
−0.19
−7.80
−5.50
−1.33
−0.39
−6.34
−3.94
−0.7
−6.35
−5.63
−1.18
−0.25
−5.41
−5.07
−0.7
−0.29
−0.25
−8.90
−4.91
−1.83
−0.46
−7.17
−4.86
−0.7
0.02
−6.41
−4.79
−0.7
0.06
−6.82
−4.53
−1.11
−0.34
−6.98
−4.48
−1.01
−0.33
−6.89
−3.93
−1.11
−0.49
−7.27
−4.25
−1.05
−0.37
−6.75
−4.48
−0.7
−0.2

Med Chem Res (2018) 27:23–36
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A novel tetravariant scaffold based on 2-aminothiazole
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