2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile as novel inhibitor of receptor tyrosine kinase and PI3K/AKT/mTOR signaling pathway in glioblastoma

Article abstract of DOI:10.1016/j.ejmech.2019.01.021

Nerve growth factor receptor (NGFR), a member of kinase protein, is emerging as an important target for Glioblastoma (GBM) treatment. Overexpression of NGFR is observed in many metastatic cancers including GBM, promoting tumor migration and invasion. Hydrazones have been reported to effectively interact with receptor tyrosine kinases (RTKs). We report herein the synthesis of 23 arylhydrazones of active methylene compounds (AHAMCs) compounds and their anti-proliferative activity against GBM cell lines, LN229 and U87. Compound R234, 2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile, was identified as the most active anti-neoplastic compound, with the IC₅₀ value ranging 87 μM - 107 μM. Molecular docking simulations of the synthesized compounds into the active site of tyrosine receptor kinase A (TrkA), demonstrated a strong binding affinity with R234 and concurs well with the obtained biological results. R234 was found to be a negative regulator of PI3K/Akt/mTOR pathway and an enhancer of p53 expression. In addition, R234 treated GBM cells exhibited the downregulation of cyclins, cyclin-dependent kinases and other key molecules involved in cell cycle such as CCNE, E2F, CCND, CDK6, indicating that R234 induces cell cycle arrest at G1/S. R234 also exerted its apoptotic effects independent of caspase3/7 activity, in both cell lines. In U87 cells, R234 induced oxidative effects whereas LN229 cells annulled oxidative stress. The study thus concludes that R234, being a negative modulator of RTKs and cell cycle inhibitor, may represent a novel class of anti-GBM drugs.

Full text of DOI:10.1016/j.ejmech.2019.01.021

Accepted Manuscript
2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile as novel inhibitor of receptor
tyrosine kinase and PI3K/AKT/mTOR signaling pathway in glioblastoma
Anisha Viswanathan, Dinesh Kute, Aliyu Musa, Saravanan Konda Mani, Vili
Sipilä, Frank Emmert-Streib, Fedor I. Zubkov, Atash V. Gurbanov, Olli Yli-Harja,
Meenakshisundaram Kandhavelu
PII:
S0223-5234(19)30031-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.021
EJMECH 11028
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 November 2018
Revised Date: 9 January 2019
Accepted Date: 9 January 2019
Please cite this article as: A. Viswanathan, D. Kute, A. Musa, S.K. Mani, V. Sipilä, F. Emmert-
Streib, F.I. Zubkov, A.V. Gurbanov, O. Yli-Harja, M. Kandhavelu, 2-(2-(2,4-dioxopentan-3-
ylidene)hydrazineyl)benzonitrile as novel inhibitor of receptor tyrosine kinase and PI3K/AKT/mTOR
signaling pathway in glioblastoma, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.021.
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ACCEPTED MANUSCRIPT
2
-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile as novel inhibitor of receptor
tyrosine kinase and PI3K/AKT/mTOR signaling pathway in glioblastoma
1
1
2
3
1
Anisha Viswanathan , Dinesh Kute , Aliyu Musa , Saravanan Konda Mani , Vili Sipilä , Frank
2
4
5,6
7,8
Emmert-Streib , Fedor I. Zubkov , Atash V. Gurbanov , Olli Yli-Harja , and
1
,
Meenakshisundaram Kandhavelu *
1
Molecular Signaling Lab, Faculty of Medicine and Health Technology, Tampere University and
BioMediTech, P.O. Box 553, 33101 Tampere, Finland.
2
Predictive Medicine and Data Analytics Lab, Faculty of Medicine and Health Technology,
Tampere University and BioMediTech, P.O. Box 553, 33101 Tampere, Finland.
3
Centre of Advanced Study in Crystallography & Biophysics, University of Madras, Chennai –
6
00 025
4
Organic Chemistry Department, Faculty of Science, Peoples’ Friendship University of
Russia (RUDN University), 6 Miklukho-Maklaya St., Moscow, 117198, Russian Federation
5
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco
Pais, 1049–001 Lisbon, Portugal
6
Department of Chemistry, Baku State University, Z. Xalilov Str. 23, Az 1148 Baku, Azerbaijan
7
Computaional Systems Biology Group, Faculty of Medicine and Health Technology, Tampere
University and BioMediTech, P.O. Box 553, 33101 Tampere, Finland.
8
th
Institute for Systems Biology, 1441N 34 Street, Seattle, WA 98103-8904, USA

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Abstract
Nerve growth factor receptor (NGFR), a member of kinase protein, is emerging as an important
target for Glioblastoma (GBM) treatment. Overexpression of NGFR is observed in many
metastatic cancers including GBM, promoting tumor migration and invasion. Hydrazones have
been reported to effectively interact with receptor tyrosine kinases (RTKs). We report herein the
synthesis of 23 arylhydrazones of active methylene compounds (AHAMCs) compounds and their
anti-proliferative activity against GBM cell lines, LN229 and U87. Compound R234, 2-(2-(2,4-
dioxopentan-3-ylidene)hydrazineyl)benzonitrile, was identified as the most active anti-neoplastic
compound, with the IC50 value ranging 87 µM - 107µM. Molecular docking simulations of the
synthesized compounds into the active site of tyrosine receptor kinase A (TrkA), demonstrated a
strong binding affinity with R234 and concurs well with the obtained biological results. R234
was found to be a negative regulator of PI3K/Akt/mTOR pathway and an enhancer of p53
expression. In addition, R234 treated GBM cells exhibited the downregulation of cyclins, cyclin-
dependent kinases and other key molecules involved in cell cycle such as CCNE, E2F, CCND,
CDK6, indicating that R234 induces cell cycle arrest at G1/S. R234 also exerted its apoptotic
effects independent of caspase3/7 activity, in both cell lines. In U87 cells, R234 induced
oxidative effects whereas LN229 cells annulled oxidative stress. The study thus concludes that
R234, being a negative modulator of RTKs and cell cycle inhibitor, may represent a novel class
of anti-GBM drugs.
INTRODUCTION:
Glioblastoma Multiforme (GBM) is WHO grade IV astrocytoma, representing a highly
heterogeneous group of neoplasms that are among the most aggressive and challenging cancers
to treat. Though highly heterogeneous, GBM shares common essential characteristics with other

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tumors, such as, uncontrolled proliferation, evading of apoptosis, invasiveness, avoidance of
immune surveillance, resistance to chemotherapy and Reactive Oxygen Species (ROS)
generation and angiogenesis [1].
Receptor tyrosine kinases (RTKs) are important regulators of intracellular signal-
transduction pathways mediating cellular processes, including metabolism, cell-cycle control,
survival, proliferation, motility and differentiation, and are often deregulated in cancer[2,3].
RTKs consist of families of growth factor receptors such as the platelet-derived growth factor
receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor
receptor (VEGFR), and epidermal growth factor receptor (EGFR), nerve growth factor receptor
(NGFR) and 15 other subfamilies. Most of the members of RTK family are strongly associated
with oncological diseases through gain-of-function mutations or through receptor/ligand
overexpression [4]. GBM being high in intratumoral heterogeneity exhibits amplification of RTK
expression on tumor cells [5], affecting the sensitivity to various chemo treatments [6]. The main
signalling pathways activated by RTKs are PI3K/Akt, Ras/Raf/ERK1/2 and signal transduction
and activator of transcription (STAT) pathways [7].
Among several RTKs linked with cancer, the NGFR is found to be positively correlated
with astrocytic gliomas and several reports suggests the role of NGFs in GBM growth [8–10].
NGFRs includes two distinct family of receptor Trks and p75NTR and are found to be
differentially expressed in glioblastoma and are identified as effective drugable targets for GBM
[8,11–15]. NGFR is a transmembrane receptor for the neurotrophin family. Neurotrophins
regulate the proliferation and differentiation of neurons in the central nervous system via NGFRs
by activating specific tyrosine kinase receptors which include TrkA, TrkB, and TrkC [12].
Recent reports suggest that inhibition of NGFR could be a therapeutic target considering its role

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in cell proliferation and survival [16], differentiation, cell cycle progression [17], apoptosis
[11,18], neurite outgrowth and retraction [19] myelination [20] cell migration and invasion. The
NGF-NGFR cascade is reported to activate nuclear factor-κB (NF-κB), leading to inhibition of
apoptosis [21]. NGFR is also involved in feedback regulation of P53 and it has been reported
that down regulation of NGFR, induced p53-dependent apoptosis and cell growth arrest, as well
as suppressed tumor growth [22]. Neurotrophin binding to NGFR activates the c-Jun N-terminal
kinase (JNK) signalling cascade, resulting in the activation of p53 and expression of pro-
apoptotic genes such as Bcl-2 [23]. Alternatively, combined activation of NGFR and tumor
necrosis factor receptor member TRAF6 has been shown to promote downstream activation of
NF-κB signalling, which primarily promotes cell survival [24]. Blockade of NGF/Trk signalling
via anti-NGF antibodies or Trk inhibitors reduced cell proliferation and tumorigenesis in a
muscarinic acetylcholine receptor-3 dependent manner, suggesting NGF as an potential target in
tumor treatment and prevention [25,26].
Currently the most targeted molecules in GBM pathway are Protein kinase inhibitors
such as Lonarfanib [27], Sorafenib [28], Topoisomerase targeting Irinotecan [29], PKC inhibitors
such as Enzastaurin [30] and Integrin inhibitors such as Cilengitide [31] and RTK inhibitors. The
use of small-molecule inhibitors targeted against EGFR (Cetuximab and Panitututmab) is a
common anti-tumor therapeutic strategy [32–35]. Anti- PDGFR agent Imatinib has been shown
to inhibit GBM cell proliferation and induces cell cycle arrest in the G1 phase of the cell cycle
[
36]. Similarly, inhibitors of IGFR can modify sensitivity to several chemotherapeutic agents
37–40]. There is still considerable need of more effective chemotherapeutics targeting RTK
[
signaling pathways in GBM disease.

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Hydrazones are a potential class of compounds for new drug development, considering
its established properties as anticonvulsant [41–43], anti-depressant [44,45], analgesic [46,47],
anti-inflammatory [46,48,49], anti-microbial [45], anti-mycobacterial [50], anti-tumoral [51,52]
activities. Among several potential arylhydrazones, Di- and tri-organotin(IV) complexes of
arylhydrazones of methylene active compounds have shown better anti-proliferative effect
against the growth of HCT116 and HEPG2 cancer cells[53]. In addition, hydrazone derivatives
were also identified as potential anti-prostate cancer compounds [54]. These compounds induces
the apoptosis by caspase 9 and 3 activation [55,56]. Lanthanide chelation with hydrazone
derivatives have also expressed higher cytotoxic effect on colon cancer [57]. More recently, we
also studied effect of the panel of many hydrazone derivatives on multiple human brain
astrocytoma cells and the results showed a better anti-proliferation effect at micro molar
level[58]. Considering the prior studies on hydrazone derivatives as a potential anticancer
compound and tyrosine kinase inhibitors[59,60] the current study intend to correlate the
pharmacodynamic behavior of the top hydrazone derivative under evaluation, with the genomic
data, to identify the key pathways affected in the anti-neoplastic behavior, specifically via
tyrosine kinase inhibition.
METHODOLOGY
2
.1 . Chemistry
A series of new 3-(2-(1-(dimethylamino)-1,3-dioxobutan-2-ylidene)hydrazineyl)-2-hydroxy-5-
nitrobenzenesulfonic acid (3, R212), N,N-dimethyl-2-(2-(4-nitrophenyl)hydrazineylidene)-3-
oxobutanamide (16, R221), 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)hydrazineyl)benzoic acid (19, R156), sodium 2-(2-(1,3-dioxo-1,3-diphenylpropan-2-

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ylidene)hydrazineyl)benzenesulfonate (20, R246), ethyl 2-(2-(4-chlorophenyl)hydrazineylidene)-
3
-oxobutanoate (21, R40), 4-(2-(1-cyano-2-methoxy-2-oxoethylidene) hydrazineyl)benzoic acid
(22, R313), 5-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)isophthalic acid (23, R283) and
known, 3-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)-2-hydroxy-5-nitrobenzenesulfonic acid (1,
1
R2), triaqua[2-(hydroxy-κO)-5-nitro-3-{2-[2-oxo-4-(oxo-κO)pentan-3-ylidene]hydrazinyl-κN }
benzenesulfonato(2-)]iron trihydrate (2, FeR2), (2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)
phenyl)arsonic acid (4, R10), 5-chloro-3-(2-(1-ethoxy-1,3-dioxobutan-2-ylidene)hydrazineyl)-2-
hydroxybenzenesulfonic acid (5, R31), 2-hydroxy-5-nitro-3-(2-(2,4,6-trioxotetrahydropyrimidin-
5
(2H)-ylidene)hydrazineyl)benzenesulfonic acid (6, R142), 5-(2-(1,3-dioxo-1-phenylbutan-2-
ylidene)hydrazineyl)-4-hydroxybenzene-1,3-disulfonic acid (7, R46), 2-(2-(2,4-dioxopentan-3-
ylidene)hydrazineyl) benzenesulfonic acid (8, R237), 4-(2-(2,4-dioxopentan-3-
ylidene)hydrazineyl)benzonitrile (9, R241), 2-(2-(2,4-dioxopentan-3-
ylidene)hydrazineyl)benzonitrile (10, R234), 2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)
benzoic acid (11, R236), 3-(2-(2-nitrophenyl) hydrazineylidene)pentane-2,4-dione (12, R235), 3-
(2-(4-chlorophenyl)hydrazineylidene)pentane-2,4-dione (13, R8), 3-(2-(4-
bromophenyl)hydrazineylidene) pentane-2,4-dione (14, R9), ethyl 2-(2-(4-cyanophenyl)
hydrazineylidene)-3-oxobutanoate (15, R243), 2-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)
hydrazineyl)benzoic acid (17, R244), 2-(2-(2-hydroxy-4-nitrophenyl) hydrazineylidene)-1-
phenylbutane-1,3-dione (18, R48) arylhydrazones of active methylene compounds (AHAMCs)
have been prepared. Synthesis scheme of know compound is give in the supplemental file 1
while novel compounds synthesis scheme is given below.
2
.2. Synthesis of novel arylhydrazone derivatives
The aryl hydrazones R212, R221, R156, R246, R40, R313 and R283 were synthesized via

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Japp−Klingemann reaction[61–67][68–74] between the substituted aryldiazonium chloride and
active methylene compounds in water solution containing sodium hydroxide (Table 1).
2
.2.1. Diazotization
0
.025 mol of substituted aniline was dissolved in 50 mL of water and then 0.025 mol of
crystalline NaOH was added. The solution was cooled in an ice bath to 273 K and then 0.025
mol of NaNO was added; after that 5.00 mL (33 %) HCl was added in portions for 1 h. The
2
temperature of the mixture should not exceed 278 K. The resulting diazonium solution was used
directly in the following coupling procedure.
2
.2.2. Coupling
.025 mol of NaOH was added to a mixture of 0.025 mol of active methylene
0
compound with 50 mL of water. The solution was cooled in an ice bath to ca. 273 K, and a
suspension of aryldiazonium chloride (see above) was added in three portions under vigorous
stirring for 1 h
3
, R212: yield, 77 % (based on N,N-dimethyl-3-oxobutanamide), yellow powder, soluble in
water, methanol, ethanol, and insoluble in chloroform. Anal. Calcd for C12 S (M =
74.3): C, 38.50; H, 3.77; N, 14.97. Found: C, 38.42; H, 3.64; N, 14.84 %. IR, cm : 3459
14 4 8
H N O
–
1
3
1
ν(OH), 2944 ν(NH), 1681 ν(C=O), 1628 ν(C=O···H), 1535 ν(C=N), 742 δ(C−H, Ar). H NMR of
a mixture of enol-azo and hydrazo tautomers, (300.13 MHz, D O). Enol-azo, δ: 2.55 CH , 2.97
2
3
and 3.08 N(CH ) , 7.98−8.11 (2H, C H ). Hydrazo, δ: 2.32 CH , 3.13 and 3.18 N(CH ) ,
3
2
6
2
3
3 2
1
7
.98−8.11 (2H, C H ). H NMR of a mixture of enol-azo and hydrazo tautomers, (300.13 MHz,
6 2
DMSO-d ). Enol-azo, δ: 2.47 CH , 2.77 and 2.96 N(CH ) , 7.98−8.18 (2H, C H ), 9.94 (s, 1H,
6
3
3 2
6
2
HO–Ar), 11.96 (s, 1H, HO-enol). Hydrazo, δ: 2.28 CH
3
, 3.02 and 3.10 N(CH
3
)
2
, 7.98−8.18 (2H,

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1
3
1
C
6
H
2
), 9.94 (s, 1H, HO–Ar), 13.60 (s, 1H, NH). C{ H} NMR (100.61 MHz, DMSO-d
6
). Enol-
azo, δ: 24.7, 34.0 and 34.8 (CH ), 109.1 (Ar−N=N), 116.2 (C–N), 125.6 and 131.6 (2Ar−H),
3
1
36.4 (Ar−SO H), 139.7 (Ar−NO ), 146.8 (Ar−OH), 162.0 (CH C−O), 194.5 (N(CH ) C=O).
3 2 3 3 2
Hydrazo, δ: 28.8, 37.3 and 38.1 (CH ), 116.9 and 123.5 (2Ar−H), 123.5 (Ar−NH−N), 130.7
3
(Ar−SO H), 132.2 (C=N), 139.6 (Ar−NO ), 145.4 (Ar−OH), 194.5 (N(CH ) C=O), 196.0
3 2 3 2
1
(
CH C=O). H NMR of a mixture of enol-azo and hydrazo tautomers, (300.13 MHz, CD OD).
3
3
Enol-azo, 2.54 CH
3
, 2.92 N(CH
, 8.23−8.36 (2H, C
9.8 and 34.7 (CH ), 107.5 (Ar−N=N), 109.2 (C–N), 117.9 and 121.0 (2Ar−H), 133.4
3
)
2
, δ: 8.23−8.36 (2H, C
6
H
2
). Hydrazo, δ: 2.34 CH
3
, 3.18 and
13
1
3
2
.18 N(CH
3
)
2
6
H
2
). C{ H} NMR (100.61 MHz, CD OD). Enol-azo, δ: 24.8,
3
3
(
Ar−SO H), 140.5 (Ar−NO ), 148.0 (Ar−OH), 164.5 (CH C−O), 196.7 (N(CH ) C=O).
3 2 3 3 2
Hydrazo, δ: 28.8, 38.2 and 38.8 (CH ), 110.7 and 111.1 (2Ar−H), 112.3 (Ar−NH−N), 118.4
3
(
Ar−SO H), 133.2 (C=N), 134.8 (Ar−NO ), 141.8 (Ar−OH), 196.7 (N(CH ) C=O), 200.4
3
2
3 2
(
CH C=O).
3
1
6, R221: yield 72 % (based on N,N-dimethyl-3-oxobutanamide), orange powder soluble in
DMSO, methanol, ethanol and acetone, and insoluble in water. Elemental analysis: C H N O
4
1
2
14
4
(M = 278.27); C 51.75 (calc. 51.80); H 5.02 (5.07); N 20.08 (20.13) %. IR (KBr): 2930 ν(NH),
−
1 1
1
663 ν(C=O), 1634 ν(C=O···H), 1595 ν(C=N) cm . H-NMR in DMSO-d
), 2.78 (3H, CH ), 2.99 (6H, CH ), 7.38−7.49 (4H, Ar−H), 12.68 (1H, N−H). C-{ H} NMR
in DMSO-d , δ (ppm): 24.4 (CH ), 33.7 (CH ), 36.4 (CH ), 116.1 (2Ar−H), 126.0 (2Ar−H),
6
, δ (ppm): 2.42 (3H,
13
1
CH
3
3
3
6
3
3
3
1
29.1 (C=N), 138.4 (Ar−NO ), 142.4 (Ar−NH−N), 163.3 and 194.3 (C=O).
2
1
9, R156: yield, 67 % (based on barbituric acid), yellow powder, soluble in water, methanol,
ethanol, and insoluble in chloroform. Anal. Calcd for C H N O (M = 276.20): C, 47.83; H,
1
1
8
4
5
–
1
2
.92; N, 20.28. Found: C, 47.65; H, 2.90; N, 20.11 %. IR, cm : 3365 ν(OH), 3214, 3019, 2855

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1
ν(NH), 1715 ν(C=O), 1657 ν(C=O), 1592 ν(C=O···H), 1563 ν(C=N). H NMR (300.13 MHz,
DMSO-d ) δ: 7.94−8.21 (4H, C H ), 11.35 (s, 1H, NH), 11.54 (s, 1H, NH) and 14.31 (s, 1H,
6
6
4
1
3
1
NH). C{ H} NMR (100.61 MHz, DMSO-d ) δ: 111.3 and 117.9 (4Ar−H), 125.4 (Ar−COOH),
6
1
36.2 (C=N), 146.8 (Ar−NH−N=), 151.8, 157.6, 159.1 and 163.9 (C=O).
2
0, R246: yield 64 % (based on 1,3-diphenylpropane-1,3-dione), grey powder is soluble in
DMSO, methanol, ethanol and acetone, and insoluble in hexane. Elemental analysis:
C H N NaO S (M = 43041; C 58.45 (calc. 58.60); H 3.43 (3.51); N 6.34 (6.51) %. IR (KBr):
21
15
2
5
−
1
1
3
450 ν(OH), 3045 ν(NH), 1681 ν(C=O), 1607 ν(C=O···H), 1587 ν(C=N) cm . H-NMR in
1
3
DMSO, internal TMS, δ (ppm): 7.12−7.98 (14H, Ar−H), 13.42 (s, 1H, N−H). C-NMR in
DMSO, internal TMS, δ (ppm): 125.42 (Ar−H), 127.13 (Ar−H), 128.25 (Ar−H), 128.36 (Ar−H),
1
1
28.43 (Ar−H), 128.56 (Ar−H), 128.90 (Ar−H), 129.49 (Ar−H), 130.38 (Ar−H), 132.43 (Ar−H),
32.85 (Ar−H), 133.08 (Ar−H), 133.729 (Ar−H), 134.05 (Ar−H), 134.67 (C=N), 138.19
(
Ar−CO), 137.21 (Ar−CO), 138.16 (Ar−NH−N), 146.45 (Ar−SO Na), 191.44 (C=O), 192.52
3
(C=O).
2
1, R40: yield 96% (based on ethyl 3-oxobutanoate), yellow powder soluble in DMSO,
methanol, ethanol, chloroform and acetone, and insoluble in water. Elemental analysis:
C H ClN O (M = 268.70); C 53.55 (calc. 53.64); H 4.74 (4.88); N 10.37 (10.43)%. IR (KBr):
12
13
2
3
-
1 1
3
1
453 (NH), 1676 (C=O), 1643 (C=O···H), 1610 (C=N) cm . H NMR in DMSO-d
6
, δ (ppm):
), 7.23–7.76 (4H, Ar–H), 13.23 (s, 1H, N–H).
), 32.33 (CH ), 60.48 (CH ), 116.48 (2Ar–H),
.31 (s, 3H, CH
3
), 2.40 (s, 3H, CH
3 2
), 4.04 (2H, CH
1
3
1
C–{ H} NMR in DMSO-d
6
, d (ppm): 13.29 (CH
3
3
2
1
19.24 (Ar–Cl), 134.20 (2Ar–H), 135.44 (C=N), 144.28 (Ar–NH–N), 196.12 (C=O), 197.66
(C=O).

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2
2, R313: yield 87% (based on methyl 2-cyanoacetate), yellow powder soluble in DMSO,
methanol, ethanol, chloroform and acetone, and insoluble in water. Elemental analysis:
C H N O (M = 247.21); C 53.38 (calc. 53.44); H 3.54 (3.67); N 16.95 (17.00)%. IR (KBr):
11
9
3
4
-1 1
3
470 (OH), 3349 (NH), 1670 (C=O), 1646 (C=O···H), 1614 (C=N) cm . H NMR in DMSO-d ,
6
δ (ppm): 3.56 (s, 3H, OCH ), 7.28–7.86 (4H, Ar–H), 11.70 (s, 1H, O–H). 13.08 (s, 1H, N–H).
3
1
3
1
C–{ H} NMR in DMSO-d , d (ppm): 58.20 (OCH ), 116.40 (2Ar–H), 124.58 (Ar–COOH),
6
3
1
33.72 (2Ar–H), 135.13 (C=N), 145.04 (Ar–NH–N), 162.06 (C=O), 196.29 (C=O), 197.80
C=O).
3, R283: yield 94% (based on pentane-2,4-dione), yellow powder soluble in DMSO, methanol,
ethanol, chloroform and acetone, and insoluble in water. Elemental analysis: C13 (M =
92.25); C 53.37 (calc. 53.43); H 4.08 (4.14); N 9.46 (9.59)%. IR (KBr): 3378 (OH), 3290 (NH),
(
2
12 2 6
H N O
2
1
-1 1
668 (C=O), 1626 (C=O···H), 1607 (C=N) cm . H NMR in DMSO-d , δ (ppm): 2.43 (s, 3H,
6
1
3
1
CH ), 2.46 (s, 3H, CH ), 8.13–8.31 (3H, Ar–H), 13.99 (s, 1H, N–H). C–{ H} NMR in DMSO-
3
3
d , δ (ppm): 26.27 (CH ), 31.29 (CH ), 119.34 (2Ar–H), 126.76 (Ar–H), 134.02 (Ar–COOH),
6
3
3
1
35.45 (Ar–COOH), 136.18 (C=N), 141.80 (Ar–NH–N), 167.71 (C=O), 167.94 (C=O), 196.32
(C=O), 197.01 (C=O).
2
.3. Computational assessment of Ligand-Receptor interaction
The structures of 23 ligands were drawn in SDF format using the tool Marvin Sketch
[Marvin Draw 5.1.5, 2008, Chemaxon Ltd., Budapest]. The ligands were prepared for docking
simulations using the LigPrep module of the Schrödinger suite of tools. Most probable tautomers
and all possible stereo isomers were generated to study the activity of individual stereotypes of
each ligand. In the final stage of LigPrep, compounds were minimized with OPLS-2001 Force

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field [75]. The Human nerve growth receptor structure with PDBID 1HE7 (Resolution- 2 Å) is
downloaded from Protein Data Bank [76]. The protein structure was prepared using the ‘Protein
Preparation Wizard’ of the Schrödinger suite (Schrödinger, LLC, New York, NY, 2009). The
docking grid on the receptor has been generated based on the co-crystallized ligand. Docking
was performed on prepared receptor and 23 ligands by using the Glide docking program [Glide,
version 5.5, Schrödinger, LLC, New York, NY, 2009].
2
.4.Cell Culture
Human GBM cell lines, U87 cells were grown in Minimum Essential Medium (MEM,
Product# 51416C, Sigma-Aldrich, St. Louis, MO) with 10% FBS 2mM sodium pyruvate
Product# S8636, Sigma-Aldrich, St. Louis, MO), 1% Penicillin-Streptomycin and 0.025mg/ml
(
Amphotericin B. LN229 cells were cultured in Dulbecco’s Modified Eagle Medium - high
glucose (DMEM, Catalog# L0102, Biowest) containing 5% FBS (Product # F1051, Sigma-
Aldrich, St. Louis, MO), 1% Penicillin-Streptomycin (Product # P4333, Sigma-Aldrich, St.
Louis, MO) and 0.025mg/ml Amphotericin B (Sigma-Aldrich, St. Louis, MO). Cells were
maintained at 37°C in a humidified incubator supplemented with 5% CO . Biological and
2
technical repeats were used for each condition.
2
.5.In vitro cytotoxicity assay
Cytotoxicity assay was performed to determine the cell growth inhibitory effect of the
compounds following treatment for 48hrs on the GBM cell lines, U87 and LN229. This assay
was performed in two stages. First, a high concentration, 100µM, was used for all the
compounds as well as for the positive control Cisplatin (Sigma-Aldrich, USA). Following which,
in the second stage, the compounds, which exhibited better activity in the previous step, were

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selected, and different concentrations (100µM, 75µM, 50µM, 25µM, and 10µM) were used to
determine the IC50 of each compound. Treated cells were harvested by centrifugation at 1200
rpm for 10min. Cell viability was determined using trypan blue staining, using a Countess II
Automated Cell Counter (Thermo Fisher Scientific) to count the number of live and dead cells.
The inhibition percentage of each sample was determined using the following formula to
determine the dose-response curve. From the dose-response curve, IC value of each compound
5
0
was calculated.
Mortality % was calculated using the following formula:
Mortalityꢀꢁ%ꢂ
ꢃꢄꢅꢆꢀꢇꢈ. ꢈꢉꢀꢊꢆꢋꢌꢄꢅꢋꢄꢍꢀꢎꢄꢏꢏꢐꢀꢁꢑꢃꢒꢓꢀꢎꢈꢆꢋꢌꢈꢏꢂ − ꢃꢄꢅꢆꢀꢇꢈ. ꢈꢉꢀꢋꢌꢄꢅꢋꢄꢍꢀꢎꢄꢏꢏꢐꢀ × 100
ꢃꢄꢅꢆꢀꢇꢈ. ꢈꢉꢀꢊꢆꢋꢌꢄꢅꢋꢄꢍꢀꢎꢄꢏꢏꢐꢀꢁꢑꢃꢒꢓꢀꢎꢈꢆꢋꢌꢈꢏꢂ
=
Double staining assay
U87 and LN229 cell lines were grown as described previously, for 24hrs, followed by
treatment with IC50 value of R234 and incubated for 48hrs. Negative (untreated) and positive
Cisplatin) controls were maintained. Apoptosis/necrosis detection was carried out using
(
Annexin V FITC and PI (Thermo Fisher Scientific). The apoptosis determination was performed
following the standard protocol from the manufacture. Briefly, the cells were cultured in 6 well-
5
plate with the initial cell density of 7 × 10 cells/well. The cells were incubated for 48hrs with
R234, positive control and untreated cells conditions and then harvested and washed in cold
PBS. The cell pellets were then resuspended in 1X Annexin-binding buffer provided in the kit.
Consequently, 5µL of FITC conjugated Annexin V and 1µL of the 100µg/ml PI working

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solutions were added to the 100µL of cell suspension. The cells were incubated in dark for 15
min, at room temperature, after which the stained cells were observed for fluorescence to
distinguish apoptotic cells. The image acquisition was done by using EVOS imaging system
(ThermoFisher Scientific) with 20X objective magnification. Approximately 300 cells were used
for each analysis.
2
.6.Caspase Activity assay
In vitro caspase-3 and caspase-7 activity leading to apoptosis was determined using
Caspase-Glo® 3/7 Assay Systems (Promega Corporation). The reagent was prepared as
mentioned by the manufacturer. The U87and LN229 cells were grown overnight in a 96-well-
plate and were treated with IC of R234. Negative control, positive control and blank (medium+
5
0
◦
DMSO+ dye) were maintained. Cells were incubated at 37 C in a humidified incubator
supplemented with 5% CO for 5hrs and then equilibrated at room temperature for 30 min.
00µL of Caspase-Glo 3/7 reagent was added to 100µL of cells/well and was incubated in a
2
1
dark-chamber. The luminescence signal was quantified (Chameleon Multi-label Detection
Platform) at one hour after treatment. Magnitude of fold change in luminescence between treated
and untreated cells were determined using the following formula:
ꢔꢖꢗꢘꢖ −ꢀꢔꢙꢚꢛꢜꢝ
ꢔꢈꢏꢍꢀꢕꢆꢎꢌꢄꢅꢐꢄ =
ꢔꢞꢟꢜꢖꢠꢟꢚ −ꢀꢔꢙꢚꢛꢜꢝ
2
.7.Intracellular Redox potential test
To evaluate the redox potential of R234, a comparative test, using H O and standard
2
2
drug against untreated cells, was performed using H2DCFDA (Catalog no.# D399 Life
Technologies, USA). The U87 and LN229 cells were grown overnight in a 96-well-plate and

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◦
were treated with IC50 of R234 for 5hrs at 37 C in a humidified incubator supplemented with 5%
CO . Negative control, positive control and blank were maintained. Baseline effect due to
2
solvent was determined as well. After 5hrs of treatment, cells were harvested by centrifugation at
3
000 rpm for 10min and incubated with 200µL of 2µM H2DCFDA for 30min at 37ºC in the CO2
incubator. Cells were then washed with pre-warmed PBS and resuspended in 200µL of pre-
warmed medium.100µL of suspension was then transferred to each well, in a 96-well plate and
incubated at room temperature for 20min. Finally, fluorescence signal was measured using
Chameleon Multi-label Detection Platform (Excitation 485 nm, Emission 535 nm).
2
.8. Cell Cycle intervention test
To elucidate the effect of the newly synthesized compound on the cell cycle progression,
6
R234 was examined against U87 and LN229 cells. A cell density of 1ꢀ×ꢀ10 cells were grown
◦
overnight at 37 C in a humidified incubator supplemented with 5% CO
2
, cells were exposed to
R234 at its IC concentration for 48hrs. The cells then were collected by trypsinization, washed
5
0
with cold phosphate buffered saline (PBS) and pellet was resuspended in 100µL of cold PBS.
◦
Subsequently, the cell suspension was added with 900 µL of 70% ethanol and stored at 4 C for
3
0min. Following that, the cells were harvested by centrifugation at 3000rpm, rinsed again by
centrifugation with 1mL cold PBS. The cells were then stained with 20µL of DNA fluorochrome
◦
PI in a solution containing Triton X-100 and RNase and incubated at 37 C in a humidified
2
incubator supplemented with 5% CO , for 15 min. Finally, the fluorescence was detected by
imaging using an EVOS FL Cell Imaging System.
2
.9.RNA Isolation and Gene Expression Evaluation
◦
Cell were incubated with IC of test compound and standard drug Cisplatin for 48hrs at 37 C
5
0

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in a humidified incubator supplemented with 5% CO2. A negative control was maintained as
well. All conditions were conducted in triplicated RNA from all samples in profiling was
isolated using same protocol. Total RNA of >9.25ng/uL was isolated using GeneJET RNA
purification kit (Catalog no.#K0731), according to the manufacturer’s instructions. The yield was
then measured spectrophotometrically using NanoDrop-1000 (Thermoscientific, USA). After
quantification, the cells were considered for quality assessment by TapeStation and expression
assay by Illumina Next Seq High Output profiling followed by analysis and validation studies.
2
.10. Statistical Analysis
All experiments described in the present study were performed as with three biological
and technical repeats. The data were presented as the mean ± standard error of mean. Statistical
analyses between two groups were performed by Student's t-test. Differences among multiple
groups were tested by one-way analysis of variance following by a Dunnett’s multiple
comparison test (GraphPad Prism 7.04). P<0.05 was considered to indicate a statistically
significant difference.
3
. RESULTS
AHAMCs interaction with receptor tyrosine kinase, TrkA
AHAMCs 1-23 were prepared according to Scheme 1 (Figure 1.a). Purity of all compounds (≥
9
5%) was verified by melting point, NMR, elemental analysis and mass spectrometry
measurements (see Supporting Information). Structures of these compounds are presented in
Table 1. To obtain a deeper insight into the mode of action of all the novel derivatives (1-23)
docking study was carried out against human NGFR protein, receptor tyrosine kinase, TrkA. The
Glide docking results of the TrkA of interest with 23 ligands were presented in the Figure 1.b.

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The ligand R234 shows better binding with the receptor compared with other 22 ligands with a
highest Glide score (-4.12). R234 forms interactions with 11 amino acid residues, which is
shown as two-dimensional interaction diagram (Figure 1.c). There are six hydrophobic amino
acid residues, three polar residues and two charged amino acid residues form interaction with the
receptor. The amino acid residues 313LEU and 314ARG form hydrogen-bonding interactions
with the R234. The hydrogen bonding interaction between the TrkA and R234 is shown with
arrow marks in the two-dimensional ligand interaction diagram.

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Table 1. Arylhydrazones of active methylene compounds (AHAMCs).

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Figure 1: a. Scheme: Japp-Klingemann synthesis of hydrazones [61-74]. b. Docking scores of a
panel of 23 novel aryl hydrazones of active methylene compounds (AHMACs) with tyrosine
kinase TrkA. c. Structure activity relationship of R234 with TrkA receptor. d. Cytotoxicity assay
of 23 AHAMCs in U87 cell line. e. Phase contrast image of U87 cells under apoptosis (non-
apoptotic-left, apoptotic-right).
R234 Effectively reduces cell viability and Proliferation of GBM cells
To evaluate the efficiency of the AHMACs molecules as anti-GBM agents, effect on cell
proliferation and viability was assessed for all synthesized compounds. Test compounds were
initially screened against U87 cell line at 100uM concentration (Figure 1.d). Temolozamide and
Cisplatin were used as positive control drugs and effects were evaluated against untreated cells.
Baseline effect due to solvent was deducted. We have found the compound R46 to have least
cytotoxic activity. R234 and R48 induced higher anti-proliferative effect. However, docking of
R234 and R48 along with other compounds showed that R234 has higher affinity than R48 with
the TrkA receptor (Figure 1.b). Hence, the R234 was further investigated to confirm the does-
and time - dependent cytotoxic effect and found to be cytotoxic in GBM cell lines (Figure 1.e)

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To generalize the potency of R234 activity we used multiple GBM cell lines, LN229 and U87.
The dose-response study was performed as described in the methods section and IC50 was
determined for both cell lines. As expected, there was a gradient increase in mortality with
increasing dosage in both cell lines, and IC value of R234 was calculated as 107 µM in U87
5
0
and 87 µM in LN229, whereas IC value of Cisplatin was found to be 53 µM and 101 µM in
5
0
U87 and LN229, respectively (Figure 2.a and b ). Surprisingly, there was steady increase in
cytotoxicity over first 48hrs whereas a loss in cytotoxic activity was observed after 48 hrs, in
both U87 and LN229 cell lines) (Figure 2. c and d). In addition, R234 was also tested against the
growth of normal brain cells (Mouse Embryonic Fibroblasts, MEF). At 100 µM treatment the
cells expressed less than 20% death suggesting that R234 cytotoxic effect is specific to GBM
cells (data not show).

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Figure 2: R234 inhibits GBM cell proliferation in a time and dose dependent manner. a.
U87 cells were treated for 5hrs with 0-200 µM quantity of R234, after 24hrs of cell growth.
Cellular viability was measured by the Trypan Blue exclusion method. Datapoints and error bars
represent mean ± S.E.M (n = 4 per group). * P < 0.05 per one‐way ANOVA. b. U87 cells were
treated for 5hrs, with 0-200 µM quantity of R234, after 24 hrs of cell growth. Datapoints and
error bars represent mean ± S.E.M (n = 4 per group). * P < 0.05 per one‐way ANOVA. c. U87
cells were harvested after 24 hrs of cell growth and subsequently treated with IC value of R234
5
0
upto 72hrs. Proliferation Inhibition was monitored every 24hrs. Data points and error bars
represent mean ± S.E.M (n = 4 per group). * P < 0.05 per one‐way ANOVA. d. LN229 cells
were harvested after 24hrs of cell growth and subsequently treated with IC50 value of R234 upto
7
2hrs. Proliferation Inhibition was monitored every 24hrs. Datapoints and error bars represent
mean ± S.E.M (n = 4 per group). * P < 0.05 per one‐way ANOVA

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R234 Interrupts of Cell Cycle by inducing G1/S arrest
In order to investigate whether R234 induces cell cycle disturbances in GBM cells, cell cycle
analysis was performed using Propidium iodide staining method as described in method section,
following R234 treatment at IC for 24 h. Cells exhibit fluorescence bright red, proportionate to
5
0
their DNA content in each cell phase (Figure 3.a and b). Significant G1/S transition arrest was
observed in U87 cells treated with R234, with a concomitant reduction in the fraction of cells in
S phase. Fraction of cells in G1 phase was 47.7% in R234 treatment, 44.0% in positive control
Cisplatin and 39.7% in untreated cells. In S phase, the percentage of the population was 2.8% in
R234 treatment, 5.6% in positive control and 19.2% in untreated cells (Figure 3.c). Baseline
effect due to DMSO was negligible. However, in LN229, a similar trend of minimal cell fraction
in S phase cells were observed, in treated condition in comparison to untreated condition.
Particularly, higher fraction of cells at G1 phase in was observed in R234 treatment. Low
fraction of cells in G2/M in R234 treated cells clearly indicates that the drug induces cell cycle
arrest at G1/S (Figure 3.d).
Cell cycle gene expression analysis of U87 cell line revealed differential expression of 77
genes associated with G1-S phase, 82 in S phase and 74 in G2/M phase. Down regulation of
ORCs (ORC1, ORC6) and MCMs (MCM3-7) were observed clearly indicating the blocking of

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DNA elongation. Gene expression analysis highlighted the P53 activation and subsequent
downregulation of cyclins, cyclin-dependent kinases and other key molecules involved in cell
cycle such as CCNE, E2F, CCND, CDK6 etc. (Figure 3.e). The high fraction of cell population
in G1 phase in R234 treated cells could be explained by the Rb mediated downregulation of
Cyclin E (CCNE), which is required for the transition from G1 to S phase of the cell cycle.
Figure 3: R234 induces cell cycle arrest in GBM cells. Cells were treated with DMSO
(negative control), Cisplastin (positive control) and R234 for 48hrs. Microscopic image analysis
was performed for cell-cycle distribution. The DNA content was evaluated with Propidium
iodide (PI) staining and fluorescence measured and analysed. a. Representative fluorescence
microscopy images for each treated or untreated groups in U87, in S phase. b. Representative
fluorescence microscopy images for each treated or untreated groups in LN229 cells. c.
Percentage of U87 cells in various phases is fraction of whole population. d. Percentage of
LN229 cells in various phases is fraction of whole population. Data represented as mean ± S.E.M
of triplicates, *p<0.05 as compared with the control group. e. Fold change for the list of
representative genes in the cell cycle and DNA damage pathways. The blue and red colors
represent those genes that were upregulated and downregulated, respectively.
R234 induces apoptosis by negatively regulating stress protective mechanisms and DNA
Damage Repair
Apoptosis assay was performed using Annexin V/PI test to determine the fraction of
apoptotic cells. Apoptotic cells appeared bright red, necrotic cells as bright green and apo-
nectrotic cells as bright orange (Figure 4.a). In U87 cell line, 68.7% of population under R234
treatment were found to be apoptotic, followed by 41.0% in Cisplatin treatment, and 22.4% in
untreated cells (Figure 4.b). In LN229 cell line, this was 57.3% of the population under R234

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treatment, followed by 32.6% in Cisplatin treatment, and 29.3% in untreated cells (Figure 4.c).
Relevantly, the key differentially expressed genes involved in apoptotic process were identified
as FOS, JUN, ITPR, ACTB and ACTG1(Figure 4.d) Transcriptome analysis identified
upregulation of pro-apoptotic genes such as NOXA, PUMA, BIM, and downregulation of IAPs,
HSPs, (Supplementary file 2) that favours cell survival, suggesting the positive role R234 in
cancer cell apoptosis and chemosensitizing. Also, DNA repair genes such as GADD45s and
SESNs were found to be notably downregulated. Genes such as TUBB3, ACTB, TESK1,
PARVA, and ACTN1 were also found to be downregulated suggesting negatively affecting
cellular integrity and survival. Stress response genes such as NQO1, GSTs and TrxR1 were also
downregulated, which is sensitizes the cell to chemotherapy.

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Figure 4: Annexin V/PI double-staining assay of GBM cells treated with R234 for 48h
indicates that the drug effectively induced apoptosis as compared to control cells. a.
Fluorocytogram of U87 cells exhibited upon Annexin V/PI double-staining (top). Phase contrast
image of the treated cells(bottom). b. The histogram represents the percentage of PI_positive
cells, in the U87 population. c. The percentage of PI positive cells, in the U87 population. d. Key
genes involved in Apoptotic process and their Log2(Fold Change), that are differentially
expressed upon R234 treatment, compared to untreated. *p<0.05 as produced by ANOVA test.
Reactive oxygen species (ROS) mediated caspase activation and subsequent cell death has
been repeatedly reported by various studies [77–79]. Both intracellular ROS and caspase in U87
cells were quantified to assess redox flux as subsequent caspase activation due to the treatment
with candidate molecule. The oxidative potential of R234 was determined by ROS assay using
H2DCFDA indicator as described in methods section. The results shows that oxidative stress
increase of 9.5 % in the R234 treated U87 cells when compared to untreated cells, whereas the
standard drug cisplatin and positive control H O marked 3.7% and 1.6 % respectively.
2
2
However, in LN229 cell line there was no statistically significant difference found between
treated and untreated cell (Figure 5.a). These results suggest that ROS may not played a major
role in R234 induced GBM cell death.

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Figure 5: a. Effect of R234 on intracellular ROS production by GBM cells(U87 cells-left,
LN229 cells-right). R234 treatment marginally increased cellular oxidation in both cell lines. b.
Caspase activity displayed by GBM cells analyzed using caspase3/7 luminescence assay kit.
Decrease in caspase activity of cells was observed after treatment with IC of R234 for 5hrs
5
0
compared to control. The values are expressed as means ± SEM of triplicate measurements of at
least two independent experiments. Significant differences compared between control and treated
cells (*p<0.05).
Considering the role of ROS in caspase-mediated apoptosis, we determined the caspase
activity of both cell lines using caspase 3/7 assay after a treatment period of 5hrs with IC50 of
R234. Interestingly, U87 cells treated with R234 displayed a reduction of caspase 3/7, displaying
a decrease of 60.2% in comparison to untreated cancer cells, whereas positive control displayed

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1
.03% increase in caspase activity. In LN229, both candidate drug as well as positive control
exhibited a reduction in caspase activity with 36.1% and 56.4% respectively (Figure 5.b). The
difference between R234 treated and untreated conditions, was confirmed to be statistically
significant, per ANOVA test (P-value < 0.05). Over all, these observations suggest that ROS-
independent and caspase-depended apoptosis is induced by R234.
Differentially expressed genes between GBM cells treated with R234 and control samples
In the DEGs analysis, 22,260 genes were mapped by at least one read in each sample
profile. In total, 5,619 DEGs with a q-value < 0.05 and fold change > 1.5 were detected over the
2
2
comparisons of R234 with Untreated and R234 with Cisplatin in DESeq2 (Supplementary file
). The numbers of differentially expressed genes with more than 2-fold change were higher in
negative control. Therefore, there were higher number of differentially expressed genes in R234
cell line when analyzed with untreated cell line. We applied the MA plot function in DESeq2 to
visualize the top genes with the smallest q-values (Figure 6.a). We investigated the similarity in
differential gene expression profiles regulated untreated samples and Cisplatin treated samples.
The fold-changes in overlapped genes filtered by the q-value < 0.05 were plotted for U87 cell
line. Venn diagrams indicated overlap in genes whose expression was regulated in the same
direction (Figure 6.b). The total number and up- or down-regulated number of DEGs (>=2-fold
change, p-value 0.05) for untreated, Cisplatin and Both (Figure 6.c). We identified 3256 DEGs
between R234 and Untreated (negative control) samples (q-value < 0.05). We also compared the
R234 and Cisplatin samples as a positive control group, both individually and combined as a
single “affected” group. In these comparisons, 2,442 number of DEGs were identified, with the
largest number of DEGs identified in comparison with the untreated samples, and 1,321 out of
5
,619 DEGs are common in both comparison (Supplementary file 2 and Figure 6.b). The

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complete lists of DEGs from the cell line analysis and all pairs of comparisons are presented in
Supplementary file 2.
R234 disrupt the functions and pathways of GBM

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Pathway effects were elucidated by Gene Ontology analysis to analyze up and down
regulated genes concerning DNA damage, cell cycle and apoptosis for R234 and untreated
sample comparison. GO analysis identified the list of genes that were enriched in DNA
replication, protein folding and regulation of transcription in response to stress. These biological
processes are involved in the DNA replication pathway for Cisplatin (Figure 6.d left).
Enrichment analysis for GO molecular function and pathways clearly demonstrated related
phenotypes associated with GBM (Figure. 6.d right). GO terms cadherin binding, damaged DNA
binding for molecular function appeared to be significantly overrepresented, and none
significantly underrepresented. Cadherin binding, a type I membrane protein involved in cell
adhesion and damaged DNA binding, interacting selectively and non-covalently with damaged
DNA have coordinated effect on regulation and function in DNA damage (will discuss the list of
pathways to add).