Identification of morpholine based hydroxylamine analogues: Selective inhibitors of MARK4/Par-1d causing cancer cell death through apoptosis

Article abstract of DOI:10.1039/d0nj03474f

Microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulates microtubule dynamics and abets tumor progression by participating in oncogenic signaling pathways. It is overexpressed in multiple human malignancies and no drug is available for this potential therapeutic target at present. Therefore, using the structure based drug design strategy, a library of hydroxylamine derivatives of morpholine were designed and synthesized as small molecule inhibitors of MARK4. Compound 32 having the CF3 group at the ortho position of the phenyl ring tethered with the >CNOH core and the hinge binder morpholine component was found to be a potent and selective inhibitor of MARK4 over thirty other serine-threonine kinases. Study of cell viability and compound induced morphological changes in MCF-7 cancer cells discovered that molecule 32 caused death of cancerous cells through the mechanism of apoptosis. Compound 32 may be transported and delivered to the target site through the blood stream, and has promising antioxidant potential. Such bio-active molecules could serve as optimized lead candidates in drug discovery for cancer treatment through MARK4 inhibition.

Full text of DOI:10.1039/d0nj03474f

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Page 1 of 36
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Identification of morpholine based hydroxylamine analogues: Selective
inhibitors of MARK4/Par-1d causing cancer cell death through
apoptosis
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Mudasir Nabi Peerzada1, Parvez Khan2, Nashrah Sharif Khan2,3 Aysha Gaur1, Fernando
,
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Avecilla4, Md. Imtaiyaz Hassan2, Amir Azam1,*
1Medicinal Chemistry Research Laboratory, Department of Chemistry, Jamia Millia Islamia,
Jamia Nagar, New Delhi-110025, India.
2Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar,
New Delhi-110025, India.
3Department of Biotechnology, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India.
4Grupo Xenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de
Química, Facultade de Ciencias, Universidade da Coruña, Campus A Coruña, 15071, A Coruña,
Spain.
* Corresponding author: E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
Tel.: +91-11-26981717/3254; fax: +91 11 26980229
1

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Abstract
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Microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the
phosphorylation of MAP proteins that regulate microtubule dynamics and abets tumor
progression by participating in oncogenic signaling pathways. It is overexpressed in multiple
human malignancies and no drug is available for this potential therapeutic target at present.
Therefore, using the structure based drug design strategy, a library of hydroxylamine derivatives
of morpholine were designed and synthesized as small molecule inhibitors of MARK4.
Compound 32 having CF3 group at the ortho position of phenyl ring tethered with >C=NOH core
and the hinge binder morpholine component, was found as potent and selective inhibitor of
MARK4 over other thirty serine-threonine kinases. Study of cell viability and compound induced
morphological changes in MCF-7 cancer cells discovered that molecule 32 caused death of
cancerous cells through mechanism of apoptosis. Compound 32 may be transported and
delivered to the target site through blood stream, and has promising antioxidant potential. Such
bio-active molecules could serve as optimized lead candidates in drug discovery for cancer
treatment through MARK4 inhibition.
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Keywords: Hydroxylamine, Morpholine, Small-molecule inhibitors, Crystal structures, MARK4
inhibitors, Apoptosis
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1. Introduction
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Microtubule Affinity-Regulatory Kinase 4 (MARK4), also termed as Par-1d, is a member of
Ser/Thr kinase family that has surfaced as an important therapeutic target for drug development
against cancer. It belongs to the calcium/calmodulin-dependent protein kinases and is involved in
catalyzing the transfer of the γ-phosphate group of ATP to the microtubule-associated proteins
(MAPs).1 The process of phosphorylation maintains the cell polarity, microtubules stability,
protein stability, intracellular signaling, cell cycle control, cell division specifically in the G1/S
checkpoint and many other complex cellular processes due the detachment of MAPs from
microtubule assembly.2,3 Phosphorylation by MARK4 at Thr214 residue activates the
microtubule dynamics and at Ser218 residue of Lys-Xaa-Gly-Ser (KXGS) motifs inhibits its
function. The active site residue Asp181 gets activated through the phosphorylation of Thr214.4,5
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MARK4 gets expressed almost in every organ of human body and its up-regulation
facilities the development of glial tumors, lung carcinoma and leukemia.5 MARK4 plays a
significant role in Wnt signaling pathway and is associated with β-catenin/TCF-dependent
transcription activity that leads to prostate cancer development.6 The overexpression of MARK4
is also allied with nuclear accumulation of β-catenin in hepatic cells that promotes hepatocellular
carcinogenesis.7 Recent reports have established its involvement in breast cancer progression,
migration and metastasis development. MARK4 up-regulation in breast cancer cells excels the
proliferation by inhibiting Hippo signaling pathway.8 The obstruction of phosphorylation process
carried out by MARK4 seems to be one of the promising chemotherapeutic management
strategies of breast cancer.9 The conclusive findings related to the up-regulation of MARK4 in
multiple human malignancies, interference with signal transductions and its occurrence as a
negative regulator of mTORC16, marks it as a potent target for cancer treatment.
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Mostly the kinase inhibiting agents are reversible, ATP-competitive and bind in the ATP-
binding pocket. It has been documented that morpholine based drugs pictilisib (GDC-0941) (1)
and gilteritinib (2) are the FDA approved kinase inhibitors that function by interacting with the
ATP-binding domain of kinases (Figure 1).10,11 Some morpholine derivatives that have been
reported as selective ATP-competitive inhibitors are proposed to act as the hinge-region binders
in PI3K/mTOR inhibitors.12,13 Moreover, the morpholine scaffold is introduced in the
compounds of therapeutic potential for conferring the aqua solubility to the compounds and
increasing the bioavailability.12,14 Several Ser/Thr kinase inhibitors such as LY 294002 (3)15,
copanlisib (4)16, buparlisib (5)17 and GSK-263671 (6)18 containing the morpholine scaffold have
been described in literature.
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Perusal of literature divulged that hydroxylamines act as key structural motif in several
chemotypes endowed with anticancer activities including kinase inhibition.19–22 Such compounds
have been found to inhibit the kinase function by interacting at the ATP binding pocket and
thereby prevent the phosphorylation process which in turn disrupts the microtubule assembly.24
Designing hybrid molecules in which two or more pharmacophores are covalently linked is a
rationally attractive approach toward the development of effective therapeutic molecules that
have different modes of action and reduced side effects.25 Further, the small molecule inhibitors
play the pivotal role in the inhibition of various kinases pertaining to the human kinome and are
being utilized extensively in the drug development. The small molecule ATP competitors have
the major access to interact with the active cleft residues of the kinases and hence show
maximum inhibition potential.26,27
In addition, as the MARK4 expression plays crucial role in the induction of oxidative
stress in adipocytes and therefore, the inhibition of MARK4 may result into the reduction of
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reactive oxygen species (ROS) generation which thereby relieves the oxidative stress in cells and
prevents oncogenesis.28,29 The hydroxylamines are reported to be the potent radical scavengers
and hence possess the significant antioxidant potential.30,31 Considering the important
pharmacological properties such as ease in diffusion, membrane permeability, transport,
bioavailability, binding affinity, potency associated with small ring heterocyclic motifs32–36 and
our earlier reports on microtubule affinity-regulating kinase 437–39, we herein report the design
and synthesis of novel hydroxylamine derivatives of morpholine as a new class of potent
MARK4 inhibitors with substantial antioxidant potential.
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O
N
N
N
O
O
O
N
N
S
N
N
O
N
N
O
N
N
O
N
O S O
NH
N
NH2
Pictilisib (1)
LY 294002 (3)
Gilteritinib (2)
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O
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N
N
NH2
HO
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O
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NH2
N
F
N
O
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HN
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O
O
Copanlisib (4)
Buparlisib (5)
GSK-263671 (6)
Figure 1. Morpholine heterocyclic scaffold in some potent Ser/Thr kinase inhibitors.
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2. Results and Discussion
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2.1. Chemistry
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In the context of our ongoing endeavor for MARK4 inhibitor development, a series of
morpholin-4-yl-methylidene hydroxylamines (Scheme 1) were synthesized from substituted
aldehydes (1-8) which were converted into their corresponding arylaldoximes (9-16) in presence
of hydroxylamine hydrochloride under low temperature. The arylaldoximes (9-16) were then
chlorinated in presence of N-Chlorosuccinimide (NCS) and dimethylformamide (DMF) to get
carboximidoyl chlorides (17-24). These intermediates were reacted with morpholine in presence
of hindered base triethylamine (TEA) to form arylhydroxylamine derivatives of morpholine (25-
32). All the synthesized compounds were characterized by using various analytical techniques
1
such as H NMR, 13C NMR, ESI-MS and their purity was established by elemental analysis
(CHNS). All compounds have a purity grade of ≥ 95%.
OH
H
OH
Cl
R2
O
R2
N
R2
N
a
b
R1
R1
R1
(1-8)
(9-16)
(17-24)
c
25; R1=H, R2= H
29; R1=C2H5, R2= H
30; R1=Cl, R2= H
26; R1=OCH3, R2= H
27; R1=CH3, R2= H
OH
N
R2
N
31; R1=NO2, R2= H
O
R1
28; R1=OC2H5, R2= H 32; R1=H, R2= CF3
(25-32)
Scheme 1. Reagents and conditions: (a) NH4OH.HCl, 3N NaOH, EtOH, reflux, 14-20 h; (b)
NCS, DMF 60 °C 8-12 h; (c) morpholine, DCM, TEA, 0 °C, 15-20 h.
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2.2. X-ray Crystallography
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Crystals suitable for measuring by X-ray crystallography were found for the compounds 25, 28
and 32. Compound 25 crystallizes in orthorhombic crystal system and compounds 28 and 32 in
triclinic crystal system (Table 1). The compound 25 contains two molecules in the asymmetric
unit, which correspond with two atropoisomers (Figure S1). These atropoisomers are present in
all the structures. In 25, a large Flack parameter, 0.6(14), was observed and Friedel pairs were
determined and their number was 25280. Figure 2 shows a drawing of probability ellipsoids of
the compounds 25, 28 and 32. Bond lengths and angles are similar to other morpholine
derivatives with hydroxilamines (Table S1). Intermolecular hydrogen bonds appear in the
structures and they are summarized in Table S2.
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Non covalent interactions are important in the crystal packing (Figure 3). In compound
25, the principal interaction is between non-binding pair of the oxygen atoms of hydroxylamine
groups and -CH2- groups of morpholine rings (~ 2.6 Å). In compound 28, C-H···π (~ 2.8 Å)
interactions, between -CH2- groups and π clouds of the phenyl rings and interactions between the
non-binding pair of the oxygen atoms of substituents with -CH2- of morpholine groups (~ 2.6 Å)
are the more intense. In compound 32, fluorine atoms interact with hydrogen atoms of sp3
carbons of morpholine rings of the next molecules (~ 2.6 Å) and the morpholine groups form an
antiparallel sandwich structures by -CH2- groups and non-binding pair of the oxygen atoms of
morpholine groups interactions (~ 2.66 Å). Differences in the predominant interactions may
explain the different behavior of these compounds when interacting with MARK4 target.
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Table 1. Crystal Data and Structure Refinement for the Compounds 25, 28 and 32
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Compound
Formula
25
28
32
C11 H14 N2 O2
206.24
C13 H18 N2 O3
250.29
C12 H13 F3 N2 O2
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Formula weight
T, K
274.24
100(2)
100(2)
100(2)
Wavelength, Å
Crystal system
Space group
0.71073
0.71073
Triclinic
P1
0.71073
Triclinic
P1
Orthorhombic
Pna21
a/Å
b/Å
c/Å
19.6320(10)
5.7579(3)
18.7135(11)
90
6.8280(3)
8.4251(4)
12.0384(5)
101.2290(10)
95.1410(10)
109.126(2)
632.92(5)
2
7.2135(9)
9.5319(10)
9.6366(11)
84.670(4)
68.742(4)
82.721(4)
611.74(12)
2
/º
β/º
90
γ/º
V/Å3
90
2115.4(2)
8
Z
F000
880
268
284
Dcalc/g cm-3
/mm-1
1.295
0.091
1.313
1.489
0.094
0.132
3.01 to 28.31
0.1073
2.63 to 28.32
0.0579
3.05 to 28.34
0.0482
/ (º)
Rint
Crystal size/ mm3
Goodness-of-fit on F2
0.08 x 0.20 x 0.22 0.11 x 0.11 x 0.18
0.05 x 0.17 x 0.20
1.038
1.041
1.036
R1[I>2(I)] a
wR2 (all data) b
0.0472
0.0402
0.0435
0.1079
0.1027
0.0976
Largest differences peak
and hole (eÅ-3)
0.168 and -0.224
0.421 and -0.211
0.352 and -0.241
aR1 = Fo - Fc/Fo. bwR2 = [w(Fo2 -Fc2)2]/[w(Fo ) ]1/2
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(25)
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Figure 2. Molecular structures for morpholine derivatives 25, 28 and 32. All the non-hydrogen
atoms are presented by their 50% probability ellipsoids. ORTEP drawing were done
with SHELXL package.
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(25)
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Figure 3. Non covalent interactions present in compounds 25, 28 and 32. Heteroatoms of
different substituent’s can determine the predominant interaction with the MARK4
target. Drawings were made with mercury 3.7 program in balls and sticks. Carbon
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atoms in gray, hydrogen atoms in white, nitrogen atoms in blue, oxygen atoms in red
and fluorine atoms in yellow.
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2.3. Molecular Docking
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The binding mode of synthesized molecules (25-32) with amino acid residues of MARK4 was
studied using molecular docking. Autodock VinaTM in combination with PyRxTM was employed
to get a molecular insight of each MARK4-ligand complex.40,41 Initially, the designed un-
substituted molecule (25) of the series was subjected to docking for finding out the mode of
interaction with MARK4. It was found that designed starting compound 25 showed efficient
binding and interaction with the various active site residues of MARK4. The six membered
morpholine ring of compound 25 showed π-alkyl interactions with Ala195 and Val70 whereas
the hydrophobic phenyl substituent interacts with Ala83, Ala195, Val116, Met111, Val70 and
Lys85 via π-alkyl interaction. The nitrogen atom of the -NOH group shows polar interaction with
Asp196 whereas its hydrogen atom forms the hydrogen bond with Lys85 residue (Figure S2).
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The binding energy and MARK4 residues interacting with each of the synthesized ligand
is provided in supporting information (Table S3, Figures S2-S5). All synthesized compounds
showed significant binding energy in the range of –6.2 kcal/mol to –7.1 kcal/mol. It was found
that the complex of MARK4 with synthesized ligands 25-32 was stabilized by substantial
number of non-covalent interactions such as hydrogen bonding, van der Waals forces that are
formed with the active site residues of MARK4 (Figures S2-S5). The consistency of docking
protocol was examined by re-docking the known inhibitor of MARK4 (pyrazolopyrimidine
inhibitor 5RC, Figure S5D-E) into the active site of the MARK452.
2.4. MARK4 Inhibition and Structure Activity Relationship (SAR)
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After the in silico evaluation of binding affinities of these compounds 25-32, MARK4 enzyme
inhibition studies was performed using ATPase assay. For conducting this study, we have
expressed and purified recombinant MARK4 as described earlier.42 For initial screenings, we
performed single dose (20 μM) enzyme inhibition studies and found that compound 32 showed
maximum inhibition potential at this dose. These studies suggested compound 32 as most active
molecule, thus it was further evaluated for enzyme inhibition studies in the concentrations range
of 0-20 μM (Figure 4). The cumulative results of docking and enzyme inhibition studies showed
that compound 32 bind to the amino acid residues of MARK4 active site and inhibited its activity
significantly (Table 2).
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Figure 4. Enzyme inhibition studies of MARK4 with compound 32: (A) Shows the
hydrolysis of 32Pi from [γ-32P] ATP, position of 32Pi and ATP spots are indicated. Lane
1, negative control (no protein); lane 2, 100 nM MARK4 (positive control); and lanes
labeled as 0.5, 1, 2, 5, 10 and 20 shows the concentration of compound 32; (B)
ATPase inhibition (% hydrolysis of Pi) with increasing concentrations of compound
32 is shown as a function of concentration quantified by comparing with positive
control.
The scaffold variation was performed on the either side of the >C=NOH core for finding
out the impact of the varied electronic arrangement on the MARK4 enzymatic activity by the
target molecules 25-32. All the evaluated compounds of the Scheme 1 share the common
structural features of central >C=NOH core and hydrophobic substituent. The hydroxylamine
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group was coupled with morpholine ring which acts as the hinge region binder in most of the
kinase inhibitors. The initial compound 25 bearing unsubstituted phenyl ring was observed to be
less potent when evaluated for MARK4 inhibitory activity. However, the introduction of electron
releasing groups (ERGs) such as OCH3 (26), CH3 (27), C2H5 (28) and Cl (30) at C-4 position in
phenyl ring as R1 substituents still lead to the minimal MARK4 inhibition except that of OC2H5
(29) which resulted in improved enzymatic activity (Figure 5). The replacement of these ERGs
with strongly electron withdrawing NO2 group (31) at the same position resulted into the slight
enhancement in the potency. Surprisingly, multifold MARK4 inhibition was noticed when CF3
group was incorporated as R2 substituent in the phenyl ring and keeping the R1 as H atom (Table
2).
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Binds with MARK4 active residue Asp 196
R2
NOH
2
1
3
4
Hydrophobic
Fragment
N
Hinge binder, interacts with
the allosteric site of MARK4
A
B
O
6
R1
5
Figure 5. Structure of the designed molecules depicting the tactical variation on either side of
hydroxylamine central core.
Since the MARK4 inhibitory activity shown by the electron withdrawing groups was
more effective as compared to the ERGs in Scheme 1. Therefore, in terms of structure activity
relationship, it can be concluded that the MARK4 inhibition of studied molecules was substituent
dependent. The compound 32 that has –CF3 group was most potent among all other chemotypes
which supported the earlier reports that the molecules bearing trifluoromethyl group occupy
prime importance in the medicinal chemistry due to their significant drug like properties such as
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momentous lipophilicity, absorption and metabolic stability.43,44 Moreover, the presence of –CF3
group may have drifted the activity of compound 32 due to its promising physiological
significance mostly in terms of conferring the excellent lipophilicity and absorption to the
molecule across the cellular plasma membrane for enhanced interaction with the MARK4
enzyme.
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Table 2. MARK4 and human cancer cell line proliferation inhibition profile of compounds
25-32
MARK4
Inhibition
IC50 (µM)
MCF-7
IC50 (µM)
HepG2
IC50 (µM)
Compound
R1
R2
H
OCH3
CH3
OC2H5
C2H5
Cl
H
H
25
˃20
˃20
˃20
14.34
˃20
˃20
19.54
3.17
-
35.24
30.43
27.52
22.34
33.24
38.24
24.41
5.27
32.32
29.44
26.33
20.77
30.22
34.72
28.27
9.31
26
H
27
H
28
H
29
30
H
NO2
H
H
31
CF3
-
32
-
paclitaxel
0.05
0.07
2.5. Kinase Selectivity Profiling of Compound 32
Kinase inhibitors usually show off-target activities with same/different kinase families which are
responsible for undesired side effects. Therefore, in order to check out the selectivity, the
identified potent MARK4 inhibitor compound 32 was evaluated against a panel of 30 kinases of
the same family (CAMK family). The results of kinase selectivity profiling showed that
compound 32 inhibited the MARK4 more strappingly (>80%) as compared to other kinases of
same family (Figure 6). It was found that, at the studied dose, out of 30 kinases, 23 kinases
showed <20% inhibition, 6 kinases showed 20-40% inhibition and MARK4 showed >80%,
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indicating the remarkable selectivity of compound 32 towards MARK4 inhibition. The inhibition
pattern indicated that at tested concentration, compound 32 moderately inhibited MARK1,
CAMKIV and MAPKAPK2 and DAPK1 (Table S4). The reason behind these observations
could be the similar architecture of binding pockets of these kinases. Taken together, the results
of kinase selectivity clearly suggested that compound 32 showed high selectivity towards
MARK4 inhibition.
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Compound
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n
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c
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Z
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M
(
e
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l
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S
v
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A
H
M
i
o
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r
M
C
A
t
a
n
C
g
o
e
c
N
e
v
A
i
t
i
s
o
P
<20%
20-40%
>80%
B
Figure 6. Single dose kinase selectivity assay: (A) Kinase inhibition results with a panel of 30
kinases for compound 32, (B) Pie chart presentation for selectivity data. The selected
panel of 30 kinases was incubated with 10µM dose of each compound and analyzed for
percent activity values. The percent kinase activity was calculated and presented as a
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percentage of inhibition (%). In a total of 30 kinases, 23 kinases showed <20%
inhibition, 6 kinases showed 20-40% inhibition and only MARK4 exhibit >80%
inhibition.
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2.6. Binding Affinity towards MARK4 and HSA
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Binding studies of compound 32 with MARK4 and human serum albumin (HSA) was
performed using fluorescence quenching method. Recombinant MARK4 was titrated with
increasing concentrations of compound 32 and fluorescence emission was recorded. Similar
experiment was carried out with HSA. The decrease in the fluorescence emission of
MARK4/HSA was analyzed using modified Stern-Volmer relation (for details see Supporting
Information) and binding constant Ka was estimated (Figure 7). The results showed that
compound 32 possess high bonding affinity towards MARK4 with Ka value of 3.8 × 106 M-1.
However, for HSA the observed value of binding constant for compound 32 was 1.13× 103 M-1.
These results advocated that compound 32 bind strongly with MARK4. The observed moderate
binding affinity for the HSA indicates a desirable transport affinity and drug likeness of the
molecule. HSA is the major carrier protein responsible for the transport of different molecules
across the blood stream and thus the molecules as like compound 32 showing moderate affinities
for HSA will be easy to transport.37
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Figure 7. Fluorescence binding studies of compound 32 with MARK4 and HSA: (A)
Fluorescence emission of MARK4 (10 µM)/HSA (25 µM) was recorded with the
increasing concentrations of 32. Emission spectrum showing quenching of
MARK4/HSA fluorescence with the increasing concentration of compound 32
(excitation is at λ280 nm and emission range is 300-400 nm). (B) Showing the fitted
fluorescence quenching data using Modified Stern-Volmer relation obtained from
MARK4/HSA fluorescence with increasing concentration of 32. This plot is used for
the estimation of binding affinity (Ka).
2.7. Inhibition of Cancer Cell Proliferation
For the determination of antiproliferative potential of synthesized compounds 25-32 on human
malignant cell lines MCF-7 and HepG2, MTT-based cell viability assay was performed. It was
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observed that compound 32 decreases the viability of MCF-7 and HepG2 cells in a dose
dependent manner (Figure 8A). Interestingly, the toxicity evaluation of compound 32 on non-
cancerous HEK293 cells showed that it did not affect the viability of these cells in the studied
concentration range (Figure 8B). These observations suggested that compound 32 selectively
inhibited the growth of MCF-7 and HepG2 cells as compared to HEK293 cells.
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A
B
MCF-7: Comp 32
HepG2: Comp 32
HEK293
100
75
50
25
0
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0
5
10 20 40 80 160 200
-0.5 0.0
0.5
1.0
1.5
2.0
Concentration(M)
log concentration (M)
Figure 8. Cell viability studies: (A) Represents the effect of compound 32 on the viability of
MCF-7 and HepG2 vs log concentration of compound 32. (B) Graphical representation
for cell viabilities of HEK293 cells. Each of selected cells was treated with increasing
concentrations of compound 32 for 48 h. Cell viabilities were presented as the
percentage of the number of viable cells to that of the control. Each data point shown is
the mean ± SD from n=3. (For anticancer activities paclitaxel has been taken as positive
control).
2.8. Apoptosis Studies
The compound 32 was evaluated for its apoptotic potential to understand the mechanism of
action before its therapeutic implications. The MCF-7 and HepG2 cells were incubated with IC50
dose of compound 32 and apoptosis induction was studied using Annexin-V and PI staining. It
was found that the treatment of compound 32 significantly induces apoptosis in MCF-7 and
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HepG2 cells as compared to vehicle controls (Figure 9A-B). Analysis of apoptosis results
showed that compound 32 induce apoptosis in 26% of MCF-7 and 20.8% of HepG2 cells as
compared to control cells. The results of these studies suggested that, in MCF-7 and HepG2 cells
compound 32 induces cell death through mechanism of apoptosis. Interestingly, present
apoptosis studies are in concurrence with earlier reports which emphasized that the inhibitors or
inhibition of MARK4 induces apoptosis in MCF-7, HepG2 and other cancerous cells.38,45,46
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Figure 9. Compound 32 induces apoptosis and decreases the production of ROS. (A)
Representative contour plots showing the anti-FITC-Annexin-V and PI stained cells
after the treatment of compound 32; name of cell line is indicated on right side of the
plot. The MCF-7 and HepG2 cells were treated with IC50 concentrations of compound
32 for 48 h and processed for apoptosis analysis using Annexin-V/PI apoptosis kit. (B)
Bar graphs represent the percentage of apoptotic cells stained with Annexin-V/PI for
triplicate measurements ± SD. (C) Graphical representation for DCF fluorescence,
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MCF-7 and HepG2 cells were treated with compound 32 and analyzed for the ROS
using H2DCFDA staining. Student t-test was used for statistical analysis, *p< 0.05,
**p< 0.01, compared with the vehicle control.
7
8
9
2.9. Determination of Reactive Oxygen Species (ROS) Level
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Cellular redox status plays an important role in regulation of different signaling pathways and
growth maintenance. Respiration and metabolic pathways are the main sources of free radical
production.47 Contrary to normal cells, cancer cells creates their own redox environment that
plays an important role in cancer cell growth and progression.48 Hydroxylamines are known to
decrease the production of reactive oxygen species (ROS), thus we were interested to investigate
the radical scavenging capability of the compound 32. It was observed that after the treatment of
MCF-7 and HepG2 cells with compound 32, the DCF fluorescence deceases, which suggested a
decrease in the levels of cellular ROS (Figure 9C). It has also been reported previously that
overexpression of MARK4 promotes the oxidative stress and therefore inhibition of MARK4
decreases the ROS levels.29 The results of ROS studies are also in close agreement with earlier
reports that inhibition of MARK4 decreases the ROS generation and thus controls cancer cell
growth and proliferation. In the present study, we consider that the significant antioxidant
potential of compound 32 in MCF-7 and HepG2 cells is most probably due the synergistic effect
of free –NOH group, structural features of molecule and the MARK4 inhibition potential, as both
hydroxylamines and MARK4 inhibition have been independently reported for causing the
reduction of ROS in different cancer cells.30,31,45
In addition, all the compounds were observed of having appreciable water solubility in
the range of 240.01to 851.1 mg/mL indicating their efficient bioavailability (Table S5). Often
the FDA approved kinase inhibitors/drugs are difficult to synthesize and involve harsh chemical
reactions. In compendium, the ease of synthesis, small molecule nature, least off target
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interactions and significant antioxidant potential associated with our synthesized molecules, adds
them as a new class of compounds as potent MARK4 inhibitors. The molecular hybridization of
the morpholine scaffold with the substituted hydroxylamine scaffold led to the emergence and
identification of the novel potent MARK4 inhibitors, envisaged with appreciable bioavailability,
anti-proliferative, and apoptotic activities
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3. Conclusions
Novel water soluble hydroxylamine derivatives of morpholine were designed and synthesized
that emerged as small-molecule potent inhibitors of tumor associated MARK4. The compound
32 inhibited the enzymatic activity of MARK4 specifically and showed moderate effect on thirty
other kinases of Seine/theronine family demonstrating the least off target interactions. In silico
and fluorescence studies validated the strong affinity of compound 32 toward MARK4 enzyme.
Mechanistically, the compound 32 inhibited the growth of cancerous cells through induction of
apoptosis as revealed by Annexin-V and PI staining. In addition, the identified compound 32
surfaced as a significant antioxidant agent as it reduced the ROS level in MCF-7 and HepG2
cells. The observed potency of the compound 32 indicates that the presence of trifluromethyl
group owns the promising pharmacological significance for the designing of the potent MARK4
inhibitors. The ascertained effectiveness of the designed compounds signifies the valuable
importance of morpholine incorporating hydroxylamine derived small molecules for selective
inhibition of MARK4, having remarkable antioxidant potential and efficient bioavailability.
Compound 32 may serve as lead candidate for the designing of new inhibitors having enhanced
therapeutic potential for the future clinical development in anticancer drug discovery paradigm
through MARK4 inhibition.
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4. Experimental Section
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7
4.1. Materials and Methods
8
9
Chemicals required were purchased from Sigma Aldrich Chemical Company (USA) and Merck
All the chemicals were used as supplied as they were of analytical grade. Aluminum sheets
(Percolated, Silica gel 60 F254) of Merck Germany were used for thin-layer chromatography
(TLC) purposes. For visualizing the reaction mixture spots on TLC, the ultraviolet light of
wavelength (λ) = 254 nm was used. Melting points of the compounds were taken by the help of
Veego instrument having model specifications of REC-22038 A2 and are uncorrected. 1H NMR
of the target compounds were recorded on Bruker Spectrospin DPX 300 MHz or Jeol-500 MHz
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13
spectrophotometers. C NMR was recorded on a Bruker Spectrospin DPX 75 MHz or Jeol-125
MHz spectrometers and in both the cases CDCl3 or DMSO-d6 was used as a solvent and
trimethylsilane (TMS) was taken as the internal standard. Splitting patterns of the peaks are
designated as follows; s, singlet; d, doublet; t: triplet: m, multiplet; Ar: aromatic and morph:
morpholine. ESI-MS (AB-Sciex 2000, Applied Biosystem) was used for recording the mass of
the compounds. Percentage of elements in the compounds was checked by using CHNS analyzer
elementar. Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD
diffractometer.
Synthesis of Compounds (9-16 and 17-24). The synthesis of intermediates (9-16) and
carboximidoyl chlorides (17-24) is given in Supporting Information.
4.2. Procedure for the synthesis of hydroxylamine derivatives of morpholine (25-32)
TEA (0.75mmol) was added to the morpholine (0.75mmol) taken in DCM followed by the drop
wise addition of synthesized carboximidoyl chlorides (17-24, 0.75mmol). The reaction mixture
was stirred for 15-20 hours at 0 °C and the progress of reaction was monitored by employing thin
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layer chromatography (TLC) visualized under 254nm UV irradiation. After completion, the
reaction mixture was extracted with DCM and water. The DCM layer was washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo under reduced pressure. All the crude
products were purified by column chromatography from hexane/ethyl acetate (1:1) as eluent to
furnish the title compounds (25-32).
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N-[(morpholin-4-yl)(phenyl)methylidene]hydroxylamine (25). Yield: 91%; white solid; m.p:
105 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.46 (s, 1H, N-OH), 7.66-7.37 (m, 5H, Ar-H), 3.62 (t,
4H, J=9.0 Hz, morph-CH2), 2.89 (t, 4H, J= 9.0 Hz, morph-CH2). 13C NMR (75 MHz, DMSO-d6):
158.46, 131.45, 129.46, 129.28, 128.58, 66.27, 48.29. ESI-MS (m/z): [M + H] 207.23. Anal.
Calcd. For C11H14N2O2: C, 64.06; H, 6.84; N, 13.58. Found: C, 64.13; H, 6.73; N, 13.57.
N-[(4-methoxyphenyl)(morpholin-4-yl)methylidene]hydroxylamine (26). Yield: 89%; white
°
solid; m.p: 107-108 C. 1H NMR (300 MHz, DMSO-d6): δ 9.39 (s, 1H, N-OH), 7.45-7.29 (m,
2H, Ar-H), 6.99-6.91 (m, 2H, Ar-H), 3.79 (s, 3H, -OCH3), 3.61 (m, 4H, morph-CH2), 2.51-2.46
(m, 4H, morph-CH2). 13C NMR (75 MHz, DMSO-d6): 159.97, 158.09, 131.06, 123.22, 113.94,
66.33, 55.58, 48.54. ESI-MS (m/z): [M + H] 237.20. Anal. Calcd. For C12H16N2O3: C, 61.00; H,
6.83; N, 11.86. Found: C, 61.13; H, 6.65; N, 11.87.
N-[(4-methylphenyl)(morpholin-4-yl)methylidene]hydroxylamine (27). Yield: 91%; white
solid; m.p: 105 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.41 (s, 1H, N-OH), 7.42-7.17 (m, 4H, Ar-
13
H), 3.61-3.44 (m, 4H, -CH2 morph), 2.88-2.70 (m, 4H, morph-CH2), 2.11 (s, 3H, -CH3). C
NMR (75 MHz, DMSO-d6): 158.41, 138.72, 129.44, 129.12, 128.42, 66.30, 48.38, 21.37. ESI-
MS (m/z): [M + H] 221.04. Anal. Calcd. For C12H16N2O2: C, 65.43; H, 7.11; N, 12.72. Found: C,
65.51; H, 7.22; N, 12.79.
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N-[(4-ethoxyphenyl)(morpholin-4-yl)methylidene]hydroxylamine (28). Yield: 89%; white
solid; m.p: 108 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.40 (s, 1H, N-OH), 7.35 (d, 2H, J=8.4 Hz,
Ar-H), 6.97 (d, 2H, J=8.4 Hz, Ar-H), 4.08-4.01 (q, 2H, -OCH2), 3.60-3.59 (m, 4H, morph-CH2),
2.87-2.84 (m, 4H, morph-CH2), 1.36 (t, 3H, J=13.8 Hz, -CH3). 13C NMR (75 MHz, DMSO-d6):
159.25, 131.06, 130.27, 123.05, 114.50, 67.22, 66.33, 63.51, 49.28, 48.55. ESI-MS (m/z): [M +
H] 251.07. Anal. Calcd. For C13H18N2O3: C, 62.38; H, 7.25; N, 11.19. Found: C, 62.21; H, 7.30;
N, 11.08.
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N-[(4-ethylphenyl)(morpholin-4-yl)methylidene]hydroxylamine (29). Yield: 90%; white
solid; m.p: 107 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.38 (s, 1H, N-OH), 7.41-7.11 (m, 2H, Ar-
H), 6.96-6.90 (m, 2H, Ar-H), 4.08-4.01 (m, 4H, morph-CH2), 3.61-3.58 (m, 4H, morph-CH2),
2.87-2.84 (m, 2H, -CH2), 1.36 (t, 3H, J=13.8 Hz, -CH3). 13C NMR (75 MHz, DMSO-d6): 156.39,
147.45, 137.58, 130.52, 123.41, 66.60, 65.66, 48.64, 47.67, 21.27, 15.29. ESI-MS (m/z): [M +
H] 235.19. Anal. Calcd. For C13H18N2O2: C, 66.64; H, 7.74; N, 11.96. Found: C, 66.56; H, 7.82;
N, 11.85.
N-[(4-chlorophenyl)(morpholin-4-yl)methylidene]hydroxylamine (30). Yield: 80%; white
solid; m.p: 106 °C; 1H NMR (500 MHz, CDCl3): δ 8.55 (s, 1H, N-OH), 7.45 (d, 2H, , J=8.9 Hz,
Ar-H), 6.97 (d, 2H, , J=8.9 Hz, Ar-H), 3.68 (t, 4H, J=9.6 Hz, morph-CH2), 2.99 (t, 4H, J=9.6 Hz,
morph-CH2). 13C NMR (125 MHz, CDCl3): 160.33, 159.64, 130.58, 122.00, 113.77, 55.20,
47.70. ESI-MS (m/z): [M + H] 101.25. Anal. Calcd. For C11H13ClN2O2: C, 54.89; H, 5.44; N,
11.64. Found: C, 54.97; H, 5.50; N, 11.53.
N-[(morpholin-4-yl)(4-nitrophenyl)methylidene]hydroxylamine (31). Yield: 84%; yellow
solid; m.p: 250 °C. 1H NMR (500 MHz, CDCl3): δ 9.26 (s, 1H, N-OH), 8.11 (d, 2H, , J=8.9 Hz,
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Ar-H), 7.66 (d, 2H, , J=8.9 Hz, Ar-H), 3.72 (t, 4H, J=9.6 Hz, morph-CH2), 3.01 (t, 4H, J=9.6 Hz,
morph-CH2).13C NMR (75 MHz, DMSO-d6): 158.99, 148.34, 128.40, 110.48, 116.00, 66.60,
65.66, 48.64, 47.67. ESI-MS (m/z): [M + H] 252.12. Anal. Calcd. For C11H13N3O4: C, 52.59; H,
5.22; N, 16.73. Found: C, 52.71; H, 5.13; N, 16.84.
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N-{(morpholin-4-yl)[2-(trifluoromethyl)phenyl]methylidene}hydroxylamine (32). Yield:
91%; white solid; m.p: 258 °C. 1H NMR (500 MHz, CDCl3): δ 8.80 (s, 1H, N-OH), 7.69 (d, 1H, ,
J=7.5 Hz, Ar-H), 7.57-7.48 (m, 2H, Ar-H), 7.44 (d, 1H, , J=7.5 Hz, Ar-H), 3.65 (t, 4H, J=9.6 Hz,
morph-CH2), 3.29 (t, 4H, J=9.6 Hz, morph-CH2). 13C NMR (125 MHz, CDCl3): 150.13, 111.25,
131.75, 129.96, 126.88, 126.67, 110.71, 122.52, 66.95, 66.29, 48.85, 46.54. ESI-MS (m/z): [M +
H] 275.08. Anal. Calcd. For C12H13F3N2O2: C, 52.56; H 4.78; N, 10.21. Found: C, 52.69; H,
4.85; N, 10.19.
4.3. X-Ray Crystal Structure Determination
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at
low temperature for compounds 25, 28 and 32 by the - scan method. Reflections were
measured from a hemisphere of data collected from frames, each of them covering 0.3º in . A
total of 33309 for 25, 26952 for 28 and 19936 for 32 reflections measured were corrected for
Lorentz and polarization effects and for absorption by multi-scan methods based on symmetry-
equivalent and repeated reflections. Of the total, 3860 for 25, 2590 for 28 and 2320 for 32,
independent reflections exceeded the significance level (F/F) > 4.0 > 4.0. After data
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collection, in each case the multi-scan absorption correction (SADABS) was applied, and the
structure was solved by direct methods and refined by full matrix least-squares on F2 data using
SHELX suite of programs.50 Hydrogen atoms were located in difference Fourier map and left to
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refine freely, except for C(13) in 28 which were included in calculation position and refined in
the riding mode. Refinements were done with allowance for thermal anisotropy of all non-
hydrogen atoms. A final difference Fourier map showed no residual density outside: 0.168 and -
0.224 e.Å-3 for 25, 0.421 and -0.211 e.Å-3 for 28 and 0.352 and -0.241 e.Å-3 for 32. w =
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1/[σ2(Fo ) + (0.045900 P)2 + 0.063900 P] for 25, 1/[σ2(Fo ) + (0.042100 P)2 + 0.293300 P] for 28
and 1/[σ2(Fo ) + (0.030900 P)2 + 0.391100 P] for 32, where P = (|Fo|2 + 2|Fc|2)/3, were used in the
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latter stages of refinement. Further details of the crystal structures determination are given in
Table 1. CCDC 1965979-1965981 numbers contain the supplementary crystallographic data for
the structures reported in this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/ retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk.
4.4. Molecular Docking
Molecular docking of the synthesized compounds 25-32 was carried out using Autodock Vina
and AutoDock 4 package.40,51 The structure coordinates of MARK4 was taken from Protein Data
Bank (PDB ID 5ES1).52 The 2D as well as 3D structures of all the synthesized hybrid molecules
was drawn in ChemBio3D Ultra 12.0. Molecular docking was performed as described
previously.53 For visualization and structure analysis of the docked complexes of MARK4 and
also to generate 2D docking for the analysis of hydrogen bonds and hydrophobic interactions,
PyMOL viewer (Schrödinger, LLC) and “Receptor-Ligand Interactions” modules of
BIOVIA/Discovery Studio 2017R2 were used.54
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4.5. Enzyme Inhibition Assay
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our synthesized molecules 25-32 as described earlier.38,45 In brief, we have measured free Pi
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generated from MARK4 arbitrated hydrolysis of [γ-32P] ATP. Firstly, MARK4 was incubated
alongwith ice-cold ATP (1 mM) having radioactive labeled [γ-32P] ATP (specific activity 222
TBq mmol−1) for 2 h at 37 °C and subsequently TLC studies was performed. The separated spots
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of hydrolyzed Pi and ATP were visualized using autoradiography. The inference of MARK4
inhibition in terms of percentage hydrolysis of ATP was quantified using imageJ software
(https://imagej.nih.gov/ij/index.html).
4.6. Single Dose Kinase Inhibition Profiling of Compound 32
In vitro biochemical profiling of compound 32 was evaluated with the twenty six members of
CAMK family (CAMK-1 and CAMK-2) of kinases using kinase screening kit by following the
manufacturer’s protocols (Promega, Madison, USA) as described previously.37,55 Briefly, 10µM
of synthesized compound along with 2µl of kinase solution was added to the corresponding well
of assay plate. Serially, 2µl of kinase solution was added to each wells of the plate. The reaction
mixture was gently mixed, centrifuged, and plate was incubated at room temperature (25 °C) for
10 minutes. Following the incubation period, 2µl of working stocks of ATP/substrate was added
carefully to respective well. Mix the assay plate, centrifuge and incubate at room temperature (25
°C) for 60 minutes. Consequently, 5µl of ADP-Glo™ reagent was added to all reaction wells of
the assay plate. Plate was mixed for 2 minutes, and incubated further at room temperature for 40
minutes. As a final point, 10µl of Kinase Detection Reagent was added to each reaction well and
incubate the plate at room temperature for 30 minutes. After the completion of reaction,
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luminescence was measured using an integration time of 0.5 seconds per well. Using net
luminescence of the no-compound control (negative control) reactions to represent 100% kinase
activity, the percent kinase activity was calculated in the compound-containing reactions and
plotted in terms of percent kinase activity inhibition. Additionally, as our lab is working on
different human kinases such as calcium–calmodulin dependent protein kinase IV (CAMKIV),
Fas-Activated Serine/Threonine Kinase (FASTK), PDK3 and Integrin linked kinase (ILK), so we
have also study the selectivity/inhibition potential of compound 32 by malachite green assay as
per our previously reported protocol.56
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4.7. Fluorescence Measurements
Binding affinities of compound 32 MARK4 and HSA was performed using the fluorescence
spectroscopy as per our published protocol.56 The titration of protein was done in triplicates and
for analysis their average was taken. The decrease in the fluorescence intensity of MARK4/HSA
protein with the increasing concentration of compound 32 was used to calculate the binding
constant (Ka) and the number of binding sites (n) present on the protein molecule by using the
modified Stern-Volmer equation.57
log (Fo-F)/F = log Ka + nlog[L]
(1)
where, Fo = Fluorescence intensity of native protein, F = Fluorescence intensity of protein in the
presence of ligand, Ka = Binding constant, n = number of binding sites, L = concentration of
ligand. The binding constant (Ka) and number of binding sites (n) were obtained from the
intercept and slope, respectively.
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For cell viability studies, standard MTT assay was carried out as describe earlier.45,56 Briefly,
the MCF-7, HepG2 and HEK293 cells were plated at a density of 6000-7000 cells/well of a 96-
well culture plate. After 24 h, cells were incubated with different concentrations (0.1–80 μM) of
compounds 25-32 for 48 h at 37 °C in a humidified CO2 incubator. After 48 h, the mixture of
media and compounds was removed and 20 µl of MTT solution (from 5mg/ml stock solution in
PBS, pH 7.4) along with 100 µl of cell growth medium was added to each well of the plate. The
plates were incubated for 4-5 h at 37 °C in the CO2 incubator and after the removal of
supernatant, formazan crystals were dissolved in 100 µl of DMSO. The plates were agitated to
mix the reaction content and the absorbance (A) of purple dissolved formazan was measured at
570/590 nm using an ELISA reader (Bio-Rad). The absorption was converted into percentage
cell viability and used to estimate the IC50 (50% inhibitory concentration) values for synthesized
compounds. For cell proliferation and anticancer activities paclitaxel was used as positive
control.
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4.9. Apoptosis Assay
Induction of apoptosis was analyzed using Annexin-V/PI staining as described previously.42,56
Briefly, 2.5x 105 cells/well of a six well culture plate were plated and after 24 h growth, cells
were incubated with IC50 concentration of 32 for 48 h at 37 ºC. The control cells were treated
with vehicle control (PBS). After 48 h treatment, the cells were trypsinized and collected. The
collected cells were washed three times with PBS and incubated with FITC- Annexin-V/PI in
binding buffer using FITC-Annexin-V kit as per the manufacturer’s instructions (BD-
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