Design, synthesis, biological and in silico evaluation of coumarin-hydrazone derivatives as tubulin targeted antiproliferative agents

Article abstract of DOI:10.1016/j.bioorg.2019.103143

Coumarin-based different series of hydrazone derivatives were synthesized and evaluated for anticancer activity against four different human cancer cell lines. The activity of the compounds were compared with doxorubicin as a standard drug and all the compounds exhibited good to moderate cytotoxicity with IC₅₀ values ranging from 6.07 to 60.45 μM against all the examined cancer cell lines. Based on the screening results, it was concluded that the compounds 12a and 18a were the most promising medicinal entities. In vitro tubulin polymerisation inhibition assay was performed for the compounds 12a and 18a and these two compounds displayed good potency when compared with colchicine as the standard drug. The interaction of these compounds with tubulin protein was also studied with the help of molecular docking technique using Discovery studio software. Furthermore, the molecular and ADMET properties of the compounds were computed with Osiris property software and PreADMET server. The compounds exhibited exciting in vitro and in silico results. Hence we propose that the compounds 12a and 18a could be developed as tubulin targeted potential antiproliferative agents.

Full text of DOI:10.1016/j.bioorg.2019.103143

Accepted Manuscript
Design, Synthesis, Biological and In silico evaluation of Coumarin-hydrazone
Derivatives as Tubulin targeted Antiproliferative agents
Pilli Govindaiah, Naresh Dumala, Irshad Mattan, Paramjit Grover, M. Jaya
Prakash
PII:
S0045-2068(19)30352-9
https://doi.org/10.1016/j.bioorg.2019.103143
103143
DOI:
Article Number:
Reference:
YBIOO 103143
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2019
18 July 2019
19 July 2019
Please cite this article as: P. Govindaiah, N. Dumala, I. Mattan, P. Grover, M.J. Prakash, Design, Synthesis,
Biological and In silico evaluation of Coumarin-hydrazone Derivatives as Tubulin targeted Antiproliferative agents,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103143
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Design, Synthesis, Biological and In silico evaluation of Coumarin-hydrazone
Derivatives as Tubulin targeted Antiproliferative agents
Pilli Govindaiaha*, Naresh Dumalab, Irshad Mattana, Paramjit Groverb, Jaya Prakash. Ma
aDepartment of Chemistry,National Institute of Technology, Rourkela-769008,Odisha, India.
bToxicology Unit, Applied Biology Division,CSIR- Indian Institute Of Chemical Technology,
Hyderabad-500007,Telangana, India.
*Corresponding author.
Email address:govipharma@gmail.com (Pilli Govindaiah)
Abstract:
Coumarin-based different series of hydrazone derivatives were synthesized and evaluated for
anticancer activity against four different human cancer cell lines. The activity of the
compounds were compared with doxorubicin as a standard drug and all the compounds
exhibited good to moderate cytotoxicity with IC50 values ranging from 6.07 to 60.45 µM
against all the examined cancer cell lines. Based on the screening results, it was concluded
that the compounds 12a and 18a were the most promising medicinal entities. In vitro tubulin
polymerisation inhibition assay was performed for the compounds 12a and 18a and these two
compounds displayed good potency when compared with colchicine as the standard drug.
The interaction of these compounds with tubulin protein was also studied with the help of
molecular docking technique using Discovery studio software. Furthermore, the molecular
and ADMET properties of the compounds were computed with Osiris property software and
PreADMET server. The compounds exhibited exciting in vitro and in silico results. Hence we
propose that the compounds 12a and 18a could be developed as tubulin targeted potential
antiproliferative agents.
Key Words: Coumarin, Hydrazone, ADMET, Anticancer, Lipinski’s rule, Molecular
docking, Tubulin.
1

1. Introduction:
The design and optimization of anticancer agents with the acceptable
pharmacodynamics, toxicological and pharmacokinetic [1, 2] are the fundamental focuses of
medicine development. Using in silico methods for prediction of these characteristics in the
initial evolution stages is extremely beneficial for the drug innovation process. Identification
of the right target and a strong mechanism of action would effectively reduce the time span in
the drug discovery process [3]. The effective molecules can be thoroughly examined in
computational models even before molecular synthesis stage. Furthermore, pharmacokinetic
studies can provide useful insights into the behaviour of absorption, distribution and pathway
of drug metabolism with biological sites (e.g. cytochrome P450 isoforms) [4]. Lipinski’s rule
may be used as decision gate to facilitate the development of orally bioavailable drugs [5].
Molecular docking is one of the highly used computational techniques to have an idea of the
suitable target(s) and active sites of the potential drug molecules.
Cancer is one of the life-threatening diseases which affects almost 60 human organs
[6]. It is a highly heterogeneous and complex disease caused by some etiological agents,
many of which are unknown. No single chemotherapeutic agent has been 100% successful in
the treatment of cancer. Moreover, existing chemotherapeutic agents are progressively
becoming less effective due to development of drug-resistance by the cancer cells [7], along
with the side effects such as hepatotoxicity, myelotoxicity, neurotoxicity, pulmonary toxicity,
urinary toxicity and cardiac toxicity upon their long-term use. Various research groups
around the globe are working to produce efficient anticancer drugs with high effectiveness
and low side effects [8].
Coumarins are a category of naturally occurring organic compounds (mainly found in
plants) with several medicinal properties [9]. These compounds belong to the benzopyrones
class and are one of the significant sources for the new anticancer agents [10]. Coumarins
have been reported to have a wide variety of pharmacological activities such as antibacterial
[11], antialzheimer [12], antidiabetic [13], antioxidant [13], anticoagulant [14], anti-
inflammatory, analgesic [15] etc. Various research groups around the world have reported
many anticancer agents with coumarin moeity. Nasr et al. in 2014 reported 6-brominated
coumarin derivatives that exhibited potential anticancer activity against Hep-G2 and CCRF
cell lines [16]. Further in 2017 Batran et al. reported 4-hydroxy coumarins and their
anticancer behaviour against breast cancer (MCF-7) and cervical cancer (HeLa) [17].
2

Coumarin sulphonamides demonstrated their anticancer activity against HeLa cell lines [18].
From the previous reports we observed that toluenesulfonyl, benzoyl and isoniazid hydrazone
derivatives showed significant anticancer activity [19–22].
Tubulin protein is one of the important targets in the treatment of cancer due to its
involvement in wide variety of cellular processes like mitosis, cell division, intracellular
transport, cell shape and migration in eukaryotic cells [23]. Tubulin targeted drugs e.g. vinca
alkaloids, colchicine derivatives and podophyllotoxins are having prominent use in the
treatment of cancer [24,25].
The potential significance of coumarins as well as hydrazone derivatives encouraged
us to design, synthesize new coumarin based hydrazone derivatives and to investigate their
potential for in vitro antiproliferative activity and in vitro tubulin polymerisation inhibition
assay. In this present work, we have also performed molecular docking studies, in silico
molecular properties, ADMET evaluation studies for all synthesized compounds.
2. Materials and Methods:
All reactions were carried out in 100 mL and 250 mL RBFs (round bottom flask). All
the starting materials were purchased from Sigma and Himedia and used without further
purification. Dulbecco’s modified Eagle medium (DMEM), MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide], trypsin-EDTA, antibiotic-antimycotic solution,
phosphate buffered saline (Ca2+, Mg2+ free; PBS), dimethyl sulfoxide (DMSO) were
purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Foetal bovine serum
(FBS) was purchased from Gibco, USA. Cell culture 96 well plates and plastic wareswere
obtained from Techno Plastic Products (TPP) (CH-8219, Trasadingen, Switzerland). All
other chemicals were obtained locally and were of analytical grade.
The progress of the reactions was monitored by thin layer chromatography (TLC)
with ethyl acetate-hexane (1:1) mixture as eluent. Melting points of the compounds were
recorded on a Digital Auto Melting/ Boiling point apparatus (LABARD LIM-252) and are
uncorrected. FT-IR spectra were recorded on RX-I Perkin Elmer Instrument using KBr pellet.
1
All NMR spectra were recorded on (400 MHz H NMR, 100 MHz 13C NMR) Bruker FT-
NMR spectrometer and chemical shifts were expressed in δ units relevant to Tetra methyl
silane(TMS) signal as an internal source in DMSO-d6. Signals were indicated as s (singlet), d
(doublet), t (triplet) m (multiplet, when multiplicity is complex) for 1H NMR. Coupling
3

constants, J was recorded in Hz. Mass of the synthesized compounds were recorded by
Liquid chromatography-Mass spectrometry (LC-MS; Perkin Elmer, Flexar SQ 300 ms).
2.1 Chemistry
2.1.1 Synthesis of compound 3:
A mixture of R-Ph-OH (10 mmol), Malonic acid (10 mmol), ZnCl2 (30 mmol) and
POCl3 (30 mmol) was stirred at 70 ºC for 24 hrs. Then it was allowed to cool and poured into
ice-cold water. The precipitate was collected, washed several times with water, dissolved in 1
N NaOH, and the insoluble suspension was removed by filtration. The filtrate was acidified
with concentrated HCl, and the precipitate was collected. The crude compound was
crystallized from methanol solvent.
2.1.2 Synthesis of compound 4:
In 250 mL RB flask 3 g of compound 3 was taken, and to this 15 mL of acetic acid
and 5 mL of POCl3 were added; the mixture was refluxed for 3 hrs. Then the solution was
poured into ice-cold water. The precipitate was collected and washed several times with
distilled water. Then the product was purified by chromatography (1:1, ethyl acetate: hexane)
to have white colour solid with 75 % yield [26].
2.1.3 General procedure for the synthesis of compounds (6a-c, 12a-c and 18a-c)
To the 1 mmol of compound 4 in a 100 mL RB flask, 1 mmol of compound 5 was
added and dissolved in ethanol. To the reaction mixture added few drops of acetic acid, the
contents of flask was allowed to stirr for 2 hrs, at 60 ºC. After completion of the reaction, the
mixture was poured into ice-cold water; the residue was collected by filtration, and then
recrystallized from ethanol/DMF (4:1) solvent mixture to afford the final desired compounds
[26].
2.1.3.1 N′-(1-(4-hydroxy,7-methyl-2-oxo-2H-chromen-3-yl)ethylidene)benzohydrazide (6a):
-1
Light yellow colour solid, yield (58 %) M.P: 210-212 ºC, FT-IR (KBr) 휈
̅
/cm : 3502, 3116,
1670, 1607; 1H NMR (400 MHz, DMSO-d6, δ ppm) 15.71 (s, 1H), 11.81 (s, 1H), 7.85 (d, J =
7.2 Hz, 2H),7.78 (d, J = 8Hz, 2H), 7.61 (t, J=7.2Hz, 1H), 7.55 (t, J = 7.6Hz, 2H),7.27 (d, J =
2 Hz, 1H) 7.10 (m, 2H), 2.51 (s, 3H), 2.43 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm):
4

179.54, 172.78, 153.43, 145.88, 140.50, 133.21, 131.52, 129.18, 128.39, 127.54, 125.93,
125.59, 116.49, 115.11, 95.48, 21.62, 18.15; ESI-MS [M+Na]: 359.79 Da.
2.1.3.2
N′-(1-(4-hydroxy,7-methyl-2-oxo-2H-chromen-3-yl)ethylidene)methylbenzene
-
sulfonohydrazide (6b): White colour solid, yield (55%), M.P: 220-222 ºC, FT-IR (KBr) 휈
̅
/cm
1: 3419, 3206, 1732, 1625, 1569;1H NMR (400 MHz, DMSO-d6, δ ppm) 14.85 (s, 1H), 9.59
(s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.27 (d, J =
2 Hz ), 7.20 (m, 1H), 2.50 (s, 3H), 2.44 (s, 3H) , 2.30 (s, 3H) ; 13C NMR (100 MHz, DMSO-
d6, δ ppm): 169.22, 160.02, 153.90, 153.35, 153.28, 143.84, 135.86, 129.83, 128.19, 126.80,
118.83, 117.89, 114.12, 110.51, 21.42, 21.27, 18.56; ESI-MS [M+Na]: 409.56 Da.
2.1.3.3
N′-(1-(4-methyl,7-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)isonicotinohydrazide
-1
(6c): Orange colour solid, yield (62%), M.P: 236-238 ºC, FT-IR (KBr) 휈
̅
/cm : 3485, 3065,
2678, 1677, 1559; 1H NMR (400 MHz, DMSO-d6, δ ppm) 14.85 (s, 1H), 10.81 (s, 1H), 7.95
(d, J = 7.2 Hz, 2H), 7.88(d, J = 8 Hz, 2H), 7.66 (t, J =7.2 Hz, 1H), 7.57 (t, J=7.6Hz, 2H),
7.28 (d, 1H), 2.38 (s, 3H) , 2.14 (s, 3H) ;13C NMR (100 MHz, DMSO-d6, δ ppm): 177.35,
152.21, 145.82, 143.98, 135.20, 132.52, 129.35, 128.93, 127.26,125.34, 123.62, 116.98,
115.21, 98.31, 21.65, 18.95; ESI-MS [M+Na]: 360.7
2.1.3.4 N′-(1-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)ethylidene)benzohydrazide (12a):
-1
White colour solid, yield (76%), M.P: 230-232 ºC, FT-IR (KBr) 휈
̅
/cm : 3454, 3256, 3084,
1692, 1611; 1H NMR (400 MHz, DMSO-d6, δ ppm) 15.69 (s, 1H), 11.85 (s, 1H), 8.02 (d, J =
12 Hz, 1H), 7.95 ( t, J = 7.2 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 8 Hz, 2H), 7.14 (m,
2H), 2.75 (s, 3H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 179.09, 172.59,
169.11, 165.28, 155.15, 154.13, 133.13, 131.54, 129.12, 128.35, 127.40, 118.48, 117.88,
110.23, 95.37, 21.31; ESI-MS [M+Na]: 361.58 Da.
2.1.3.5
N′-(1-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)ethylidene)4-methylbenzenesulphonyl
-1
hydrazide (12b): White colour solid, yield (70%), M.P: 248-250 ºC, FT-IR (KBr) 휈
̅
/cm :
1
3436, 3076, 2826, 1769, 1616, 1563; H NMR (400 MHz, DMSO-d6, δ ppm) 15.64 (s, 1H),
11.83 (s, 1H), 8.01 (d, J = 8.4Hz, 1H),7.93 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 7.6 Hz,1H), 7.56
(t, J = 7.6 Hz, 2H), 7.12 (m, 2H), 2.73 (s, 3H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6,
5

δ ppm): 179.10, 172.68, 169.10, 155.18, 154.15, 133.12, 131.57, 129.12, 128.34, 127.42,
118.50, 117.90, 110.24, 95.39, 21.31, 18.09; ESI-MS [M+Na]: 411.58 Da.
2.1.3.6 N′-(1-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)ethylidene)isonicotinohydrazide (12c):
-1
Orange colour solid, yield (85%), M.P: 242-244 ºC, FT-IR (KBr) 휈
̅
/cm : 3195, 3067, 2287,
1663, 1596; 1H NMR (400 MHz, DMSO-d6, δ ppm) 15.75 (s, 1H), 10.85 (s, 1H), 8.83 (t, J =
1.2 Hz, 2H), 8.02 (d, J = 8.4 Hz 1H), 7.95 (s, 2H), 7.13 (m, 2H), 2.74 (s, 3H); 13C NMR (100
MHz, DMSO-d6, δ ppm): 176.32, 167.25, 155.21, 154.35, 145.28,136.73, 132.55, 131.80,
127.28, 126.95, 119.34, 115.41, 107.29, 96.42, 26.01.
ESI-MS [M+Na]: 362.68 Da.
2.1.3.7 N′-(1-(4-hydroxy-2-oxo-2H-Chromen-3-yl)ethylidene)benzohydrazide (18a):
ο
-1
White colour solid, yield (82%), M.P: 222-224 C, FT-IR (KBr) 휈̅/cm : 3408, 3217, 1706,
1
1650; H NMR (400 MHz, DMSO-d6, δ ppm) 15.72 (s, 1H), 11.83 (s, 1H), 7.99 (d, J = 1.6
Hz, 1H), 7.95 (m, 2H), 7.65 (m, 2H), 7.56 (t, J = 8 Hz, 2H), 7.31 (m, 2H), 2.74 (s, 3H); 13C
NMR (100 MHz, DMSO-d6, δ ppm): 179.71, 172.68, 165.30, 153.54, 134.81, 133.14, 131.56,
129.14, 128.35, 126.12, 124.32, 120.05, 116.78, 95.72, 18.10; ESI-MS [M+Na]: 345.53 Da.
2.1.3.8
N′-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)4-methylbenzenesulphonyl
-1
hydrazide (18b): White colour solid, yield (80%), M.P: 212-214 ºC, FT-IR (KBr) 휈
̅
/cm :
1
3443, 3065, 2826, 1677, 1566; H NMR (400 MHz, DMSO-d6, δ ppm) 14.82 (s, 1H), 7.84
(m, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.63 (m, 1H), 7.48 (d, J = 8 Hz, 2H), 7.26 (m, 2H), 2.60 (s,
3H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 179.28, 177.55, 166.10, 153.44,
145.47, 135.22, 134.01, 130.66, 128.13, 126.10, 124.44, 119.22, 116.79, 97.30, 21.54, 18.02;
ESI-MS [M+Na]: 395.66 Da.
2.1.3.9 N′-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)isonicotinohydrazide (18c):
-1
Orange colour solid, yield (85%), M.P: 244-246 ºC, FT-IR (KBr) 휈
̅
/cm : 3417, 3084, 2659,
1
1677, 1559; H NMR (400 MHz, DMSO-d6, δ ppm) 15.74 (s, 1H), 8.82 (d, J = 5.6 Hz,
2H),7.95 (m, 3H), 7.54 (t, J = 8.4 Hz, 1H), 7.30 (m, 2H), 2.75 (s, 3H); 13C NMR (100 MHz,
DMSO-d6, δ ppm): 178.32, 173.16, 169.23, 155.46, 154.29, 133.72, 131.53, 129.96, 128.74,
127.45, 118.51, 117.56, 110.68, 96.35,18.29; ESI-MS [M+Na]: 346.81 Da.
6

2.1.4 Synthesis of (7-hydoxy-4-methyl)-2H-Chromen-2-one (21):
A mixture of m-cresol (10 mmol), ethyl acetoacetate (10 mmol), and added H2SO4
(5mmol) reacted at 10 ºC for 2.5 hrs. The precipitate was collected, washed several times
with water, dissolved in 1 N NaOH, and the insoluble suspension was removed by filtration.
The filtrate was acidified with concentrated HCl, and the precipitate was collected. Crude
1
compound was recrystallized using methanol (yield 85%). H NMR (400 MHz, DMSO-d6, δ
ppm) 11.62 (s, 1H, OH), 8.02 (d, J = 8.2 Hz, 1H), 6.88 (m, 1H), 6.76 (d, J = 8Hz, 1H), 6.42
(s, 1H), 3.38 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 161.96, 161.05,. 156.29,
151.26, 125.96, 115.31, 110.55, 108.23, 102.71, 18.98.
2.1.5 Synthesis of (3-acetyl-7-hydroxy-4-methyl)-2H-Chromen-2-one (22):
To the 5 g of compound 21, 25 mL of acetic acid and 8 mL of POCl3 was added in
250 mL RBF. Refluxed the mixture for 3 hrs and poured the hot reaction mixture into ice-
cold water then collected the precipitate and washed with water for 3 times. Light yellow
colour solid, yield (72%): 1H NMR (400 MHz, DMSO-d6, δ ppm) 10.43 (s, 1H,), 7.96 (d, J =
7.2 Hz, 1H),7.83 (d, J = 8 Hz, 2H), 5.85 (s, 1H), 2.70 (s, 3H), 2.40 (s, 3H); 13C NMR (100
MHz, DMSO-d6, δ ppm): 179.72, 167.53, 153.30, 138.12, 125.30, 123.26, 116.67, 116.42,
113.53, 96.25, 23.05, 21.75.
2.1.6 General method for the synthesis of compounds (24a-c)
To the solution of 22 (1 mmol) in ethanol (20 mL) in 100 mL RB (round bottom) flask,
added 1 mmol of compound 5 and few drops of acetic acid, stirred the mixture for 2 hrs at 60
ºC. After completion of the reaction, the mixture was poured into ice-cold water then filtered
and the precipitate was collected by vacuum filtration. Further the precipitate was
recrystallized from ethanol/DMF (4:1) solvent mixture to afford final desired compounds.
2.1.6.1 N'-(1-(7-hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)ethylidene)benzohydrazide (24a):
-1
White colour solid, yield (65%), M.P: 218-220 ºC, FT-IR (KBr) 휈
̅
/cm : 3452, 3047, 1668,
1
1556; H NMR (400 MHz, DMSO-d6, δ ppm) 15.71 (s, 1H), 11.79 (s, 1H), 7.95 (d, J = 7.2
Hz, 2H), 7.88 (d, J = 8 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.13 (m,
2H), 2.49 (s, 3H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm) 179.74, 172.69,
7

153.55, 145.82, 140.60, 133.11, 131.58, 129.14, 128.29, 127.59, 125.96, 125.49, 116.65,
115.08, 95.44, 21.64, 18.11; ESI-MS [M+Na]: 359.68 Da.
2.1.6.2N'-(1-(7-hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)ethylidene)4-
methylbenzenesulfonohydrazide (24b): White colour solid, yield (72%), M.P: 224-226 ºC,
-1
1
FT-IR (KBr) 휈̅/cm : 3417, 3074, 1668, 1663, 1559; H NMR (400 MHz, DMSO-d6, δ ppm)
14.93 (s, 1H), 10.81 (s, 1H), 7.31 (t, J = 8.4 Hz, 3H), 7.48 (d, J = 8Hz, 2H), 7.08 (t, J = 10.8
Hz, 2H), 2.48 (s, 3H), 2.41 (s, 3H) , 2.35 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm)
179.27, 177.42, 153.50, 146.28, 145.41, 134.08, 130.65, 128.31, 128.12, 127.65, 125.94,
125.57, 116.68, 115.07, 97.00, 23.07, 21.65, 17.97; ESI-MS [M+Na]: 409.58 Da.
2.1.6.3
N'-(1-(7-hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)ethylidene)isonicotinohydrazide
-1
(24c): Orange colour solid, yield (78%), M.P: 212-214 ºC, FT-IR (KBr) 휈
̅
/cm : 3444, 3056,
2641, 1695, 1559; 1H NMR (400 MHz, DMSO-d6, δ ppm): 15.15 (s, 1H), 10.41 (s, 1H), 8.80
(d, J = 8, 2H), 7.85 (d, J = 7.2 Hz, 2H), 7.89 (t, J = 8 Hz, 1H), 7.47 (t, J = 8.8 Hz, 2H), 7.11
(m, 2H), 2.48 (s, 3H), 2.73 (s, 3H) , 2.48 (s, 3H); 13C NMR (100 MHz, DMSO-d6, δ ppm):
176.58, 154.55, 146.53, 144.48, 135.28, 131.62, 129.35, 128.92, 126.95, 126.35, 124.27,
117.48, 114.15, 98.09, 22.68, 18.91; ESI-MS [M+Na]: 360.72 Da.
2.2 Anticancer Evaluation:
2.2.1 Cell culture:
A549 derived from human lung adenocarcinoma cells, HeLa derived from human
cervical cancer cells, SK-N-SH derived from human neuroblastoma cells, MCF7 derived
from human mammary gland adenocarcinoma cells and NRK-49F derived from normal rat
kidney cells were procured from National Centre for Cell Science (Pune, India), maintained
in DMEM media supplemented with 10% FBS, 0.1mM non-essential aminoacids, 1mM
sodium pyruvate, 2mM L-glutamine and antibiotics (60 μg /mL penicillin, 100 μg/mL
streptomycin) at 37 °C in a 5% CO2 humidified incubator with a subculture period of 2–3
days.
2.2.2 Preparation of samples for MTT assay:
All the test compounds were taken in DMSO and further dilutions were made with
sterile PBS (1×) to get desired concentrations of 10 mg/mL. All formulations were filtered
8

through 0.22 µm sterile filter and 20 minutes of UV radiation before adding to the 96 well
plates containing cells [27].
2.2.3 Cytotoxicity evaluation (MTT assay):
All the synthesized compounds were screened for in vitro cytotoxic activity against a
panel of human tumour cell lines along with normal rat kidney cells (NRK 49F).The assay is
based on the reduction of MTT by the mitochondrial dehydrogenase of viable cells into
purple formazan crystals which gets dissolved in DMSO and measured at 570 nm. Briefly,
5x103 exponentially growing cells were seeded into each 96 well plate (counted by Trypan
blue exclusion dye method) allowed to grow till 60-70% , then compounds were added to the
culture medium with the final concentrations ranging from 0.1, 1, 5 and 10 μM/mL and
along with controls {negative (without compound) and positive (Doxorubicin)} incubated
for 24 hoursin CO2 incubator at 37 °C with a 90% humidified atmosphere and 5% CO2. Then
the media of the wells were replaced with 90 µl of fresh serum-free media and 10 µl of MTT
(5mg/mL of PBS), plates were incubated at 37 °C for 2 hrs, there after the above media was
discarded and dried. 100 µL of DMSO was added in each well at 37 °C for 5 mins continuous
added. The purple formazan crystals were dissolved and absorbance was immediately
measured at 570 nm using Spectra Max plus 384 UV-Visible plate reader (Molecular
Devices, Sunnyvale, CA, USA). IC50 values were determined by regression analysis software
package of MS-excel.
2.3 In vitro Tubulin Polymerization Inhibition Assay:
Tubulin was purified from goat brain by repeated cycles of polymerization and
depolymerisation initially in PEM buffer [100mM PIPES, 1 mM MgSO4, 2 mM EGTA and 1
mM GTP] containing 50% glycerol and later in GEM [1M sodium glutamate with pH 6.9,
1mM EGTA and 1mM MgSO4] buffer. The crude protein homogenate was obtained from
minced and homogenised goat brain tissue. The homogenate was polymerised at 37 °C, pH =
6.9 with 1 mM GTP. The polymerised microtubules were sedimented with 151240 g at 35
°C. The sedimented microtubules were redissolved in ice cold PEM. The process was
repeated four times to obtain the purified tubulin. Further, the tubulin protein was mixed with
different concentrations of compounds (12a and 18a) in PEM buffer (pH= 6.9), DMSO (8%),
GTP (1mM). Colchicine that inhibits microtubule polymerisation was used as positive control
at different concentrations for the inhibition assay. Microtubule polymerisation was
performed at 37 °C. The polymerised microtubules were sedimented with 25200 g at 37 °C
9

for 30 mins. The concentration of free tubulin in the supernatant was measured by Bradford
assay [28].
2.4 Computational methodology
2.4.1 Molecular Docking Simulations;
The protein for the present investigation is tubulin which was retrieved from the
Protein Data Bank (PDB) with the code 4YJ3 [10]. Protein preparation was performed by
removing all the ligands, heteroatoms and water molecules followed by addition of missing
hydrogens [29,30]. To make the protein ready for docking, the energy minimization was
conducted until satisfied convergence gradient was obtained and then active site was selected
around the substrate. Coumarin derivatives were drawn with the help of Chemdraw 3D
software. Further, energy minimization done with CHARMm force field and was imported
into the DSv16.1.0 [31]. The docking studies were performed with the CDOCKER, available
in the Discovery studio software, for analyzing the binding affinities between the ligand and
the protein. The prepared protein and ligands were imported into the DSv16.1.0 and the
docking protocol was executed [32–34]. The docking results were studied with -CDOCKER
interaction energy, hydrogen bond interaction and the binding mode pattern.
2.4.2 ADMET and Drug-likeness Studies;
The compounds were evaluated for their drug-likeness properties and
pharmacokinetic properties which are retrieved by using the Osiris property explorer [35] and
predicted by using ADMET server [36,37].
3 Results and Discussion:
3.1 Chemistry
The synthetic protocols of the target compounds were summarised in Scheme-I and II.
Scheme-I: (6a-c, 12a-c and 18a-c) compounds were synthesized from phenol and it’s
other derivatives (3) as a precursor, which was prepared by condensation of 1 and malonic
acid. The formed coumarin (3) was further acylated with acetic acid in the presence of
phosphorus oxychloride. Acylatedcoumarin (4) was further reacted with three different
aromatic hydrazides to afford the final targeted compounds (Scheme-I).
10

Sheme-II: 24a-c compounds were synthesized from 7-hydroxy 4-methyl coumarin as
a precursor, which was prepared from m-cresol and ethyl acetoacetate in the presence of
sulphuric acid. Further, the formed coumarin (21) was acylated by using acetic acid in the
presence of phosphorus oxychloride. Acylatedcoumarin (22) was finally reacted with three
different aromatic hydrazides to afford the final compounds (Scheme-II).
3.2 Antiproliferative assay:
We have evaluated all the synthesized coumarin derivatives for the screening of
anticancer activity using MTT assay against four human cancer cell lines A549 derived from
human lung adenocarcinoma cells, HeLa derived from human cervical cancer cells, SK-N-
SH derived from human neuroblastoma cells, MCF-7 derived from mammary gland
adenocarcinoma cells and NRK-49F derived from normal rat kidney cells using doxorubicin
as standard drug. Based on the screening results, we observed that these compounds exhibited
good to moderate cytotoxicity with IC50 values ranging from 6.07 to 60.45 µM against all the
tested cancer cell lines. The results suggested the compounds 12a and 18a are promising
antiproliferative agents due to their functional group diversity. The cytotoxicity results are
shown in Table-1.
3.3 Tubulin Polymerisation inhibition assay:
Many of the chemotherapeutic agents used in cancer today target tubulin. By targeting
tubulin they cause mitotic arrest and thus are termed as antimitotic drugs. These drugs attack
the cancer cells at both primary and metastatic phase. These agents primarily bind to the beta-
tubulin protein in the mitotic spindle. The most potent compounds (12a and 18a) find in
MTT assay were allowed for tubulin polymerisation inhibition assay. The compounds
showed good inhibitory activity compared with standard antitubulin drug colchicine. The IC50
values of compounds 12a and 18a are 14.25 and 16.96 µM respectively.
3.4 Molecular docking simulation:
Molecular docking studies were carried out to identify the interactions between
compounds and target protein. Discovery studio software was used for the docking
simulation where the ligands were docked into the protein to optimize the scoring function.
The crystal structure of the tubulin protein (PDB; 4YJ3) was obtained from the PDB
(www.rcsb.org/pdb). Both the compounds 12a and 18a were docked with the target protein.
11

The docking studies on protein revealed that these ligands showed good covalent cross-
linkage mode of binding with the tubulin. Each compound showed three strong conventional
hydrogen bond interactions. Figure 1 depicted the interactions of 12a ligand with protein
exhibiting hydrogen bonds with Asn-101, Thr-179 and ASN-228 amino acid residues.
Figure 2 depicted the interactions of 18a ligand with protein. The compound exhibited
hydrogen bonds with GLN-11, ALA-12, and ASN-206 amino acid residues.
3.5 Physicochemical properties:
To evaluate the drug-like properties we used Lipinski’s rule of five, according to this
rule the orally bioavailable drug should have molecular weight (MW) should be less than 500
Daltons, partition coefficient (LogP) should not greater than 5, hydrogen bond acceptors
(HBA) are not more than 10 and hydrogen bond donors (HBD) are not more than 5. The
results were predicted by using Osiris property explorer software [35]. The results
demonstrated that all the compounds obey the Lipinski rule and showed good properties
(Table 3).
3.6 Pharmacokinetic properties:
The evaluation of pharmacokinetic properties is probably the most important aspect to
develop a novel drug molecule. The drug molecule requires good pharmacokinetic properties
along with medicinal properties. To determine the pharmacokinetic behavior of our
synthesized compounds PreADMET server was employed. The general evaluation in
ADMET, absorption properties such as human intestinal absorption (HIA), CaCo-2
permeability, MDCK cell permeability, skin permeability (log kp) results indicate that the
compounds are showing good absorption properties to cross the gut wall by passive diffusion
mechanism to reach the target. In a general sense the drugs are majorly distributed through
plasma proteins. However some drugs can distribute through blood brain barrier (BBB). The
distribution properties of our compounds suggest that these are distributed through plasma
proteins only and there is no ability to cross BBB (Table-4). The compounds metabolic
properties were predicted by the behavior of cytochrome P450 and their isoform enzymes.
Other than CYP_2C9, CYP_2C9 isoforms all other isoforms of CYP450 are not showing any
competitive inhibitory or substrate activity with the all synthesized compounds (Table-5).
12

4. Structure activity relationship
From the results of in vitro antiproliferative assay and in silico evaluation studies the
structural activity relationship (SAR) of the compounds was established for 4,7-
dihydroxycoumarin and 4-hydroxyl coumarin derivatives which showed good cytotoxicity
results, good drug-likeness as well as good pharmacokinetic properties. Three different
hydrazine derivatives were substituted to the coumarin ring such as benzoyl,
toulenesulphonyl and pyridinoyl aromatic rings. Among these three, the benzoyl derivatives
showed best results when compared with the other two substitutions.
5. Conclusion:
We have successfully synthesized different series of coumarin based hydrazone
derivatives and these compounds were characterized by FT-IR, NMR and Mass spectroscopic
methods. Furthermore, these compounds were screened for anticancer activity against A-549,
Hela, SKNSH, MCF-7 human cancer cell lines by using MTT assay as well as against normal
rat kidney cell line NRK-49F. All the compounds showed good to moderate cytotoxicity.
Among all compounds, 12a and 18a exhibited potent activity against all tested cancer cell
lines. Tubulin polymerisation inhibition assay was performed for the potent molecules 12a
and 18a which showed good inhibition towards tubulin protein when compared with
colchicine. Docking studies were performed to have an idea of the mechanism and binding
modes of the new active compounds towards tubulin protein. The results revealed good
interactions with target tubulin at the active sites and stable interactions with ligands. We
have studied the drug-like properties and ADMET properties of the all compounds. In
conclusion we propose that the compounds 12a and 18a might be used as lead molecules for
further investigation.
Acknowledgments:
We would like to thank National Institute of Technology, Rourkela for necessary
infrastructural and instrumentation facilities and TEQIP-II for partial funding and research
fellowship. We would like to thank Mr. Tuhin Subhra Dash for his valuable suggestions.
Conflicts of Interest:
The authors declared that they have no conflicts of interest.
13

Supporting Information:
1H, 13C NMR, FT-IR and Mass spectra results of the lead compounds are available in the
supporting information.
References:
[1]
[2]
[3]
M. V Rodi, V.M. Leovac, L.S. Jovanovi, L.S. Vojinovi, M.D. Joksovi, T. Stanojkovi,
European Journal of Medicinal Chemistry Synthesis , characterization , cytotoxicity
and antiangiogenic activity of copper ( II ) complexes with 1-adamantoyl hydrazone
bearing pyridine, 115 (2016) 75–81.
S. Fahad, U. Rashid, F. Latif, Z. Ul-haq, Journal of Molecular Graphics and Modelling
Bioisosteric approach in designing new monastrol derivativesꢀ: An investigation on
their ADMET prediction using in silico derived parameters, Journal of Molecular
Graphics and Modelling. 45 (2013) 202–210.
C.A.S. Bergström, C.J.H. Porter, Understanding the Challenge of Beyond-Rule-of-5
Compounds, Advanced Drug Delivery Reviews. 101 (2016) 1–5.
[4]
[5]
E. Turkheimer, M. Waldron, Fayaz Ali Larik, Psychological Bulletin. 126 (2019) 21.
M.Q. Zhang, B. Wilkinson, Drug discovery beyond the “rule-of-five,” Current Opinion
in Biotechnology. 18 (2007) 478–488.
[6]
Y. Tangella, K.L. Manasa, V. Laxma Nayak, M. Sathish, B. Sridhar, A. Alarifi, N.
Nagesh, A. Kamal, An efficient one-pot approach for the regio- and diastereoselective
synthesis of trans-dihydrofuran derivatives: cytotoxicity and DNA-binding studies,
Org. Biomol. Chem. 15 (2017) 6837–6853.
[7]
J. Mahantesh, Rahul S. Kalhapure, S. Rambharose, Ch.Mocktar, T. Govender.
"Synthesis, characterization and antibacterial activity of novel heterocyclic quaternary
ammonium surfactants." Journal of industrial and engineering chemistry 47 (2017):
405-414.
[8]
[9]
H.A.H. Elshemy, M.A. Zaki, Design and synthesis of new coumarin hybrids and
insight into their mode of antiproliferative action, Bioorganic and Medicinal
Chemistry. 25 (2017) 1066–1075.
U. Rashid, F. Rahim, M. Taha, M. Arshad, H. Ullah, T. Mahmood, M. Ali, Synthesis
14

of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and
molecular docking studies, Bioorganic Chemistry. 66 (2016) 111–116.
[10] D. Cao, Y. Liu, W. Yan, C. Wang, P. Bai, T. Wang, M. Tang, X. Wang, Z. Yang, B.
Ma, L. Ma, L. Lei, F. Wang, B. Xu, Y. Zhou, T. Yang, L. Chen, Design, Synthesis,
and Evaluation of in Vitro and in Vivo Anticancer Activity of 4-Substituted
Coumarins: A Novel Class of Potent Tubulin Polymerization Inhibitors, Journal of
Medicinal Chemistry. 59 (2016) 5721–5739.
[11] D. Patel, P. Kumari, N.B. Patel, Synthesis and biological evaluation of coumarin based
isoxazoles, pyrimidinthiones and pyrimidin-2-ones, Arabian Journal of Chemistry. 10
(2017) S3990–S4001.
[12] F.C. Torres, K.L. Vanzolini, A.A. Merlo, B. Gauer, M. Holzschuh, S. Andrade, M.
Piedade, S.C. Garcia, I. Carvalho, D.F. Kawano, V.L. Eifler-lima, Q.B. Cass, Fernando
C. Torres, 27 (2016) 1541–1550.
[13] H. Niu, W. Wang, J. Li, Y. Lei, Y. Zhao, W. Yang, C. Zhao, B. Lin, S. Song, S. Wang,
A novel structural class of coumarin-chalcone fibrates as PPARalpha/gamma agonists
with potent antioxidant activities: Design, synthesis, biological evaluation and
molecular docking studies., European Journal of Medicinal Chemistry. 138 (2017)
212–220.
[14] A. Montagut-Romans, M. Boulven, M. Jacolot, S. Moebs-Sanchez, C. Hasco�t, A.
Hammed, S. Besse, M. Lemaire, E. Benoit, V. Lattard, F. Popowycz, Synthesis and
biological evaluation of C-3 aliphatic coumarins as vitamin K antagonists, Bioorganic
and Medicinal Chemistry Letters. 27 (2017) 1598–1601.
[15] P. Srivastava, V.K. Vyas, B. Variya, P. Patel, G. Qureshi, M. Ghate, Bioorganic
Chemistry inhibition activities , and molecular docking study of 7-substituted
coumarin derivatives, Bioorganic Chemistry. 67 (2016) 130–138.
[16] T. Nasr, S. Bondock, M. Youns, Anticancer activity of new coumarin substituted
hydrazide e hydrazone derivatives, European Journal of Medicinal Chemistry. 76
(2014) 539–548.
[17] R.Z. Batran, D.H. Dawood, S.A. El-Seginy, M.M. Ali, T.J. Maher, K.S. Gugnani, A.N.
Rondon-Ortiz, New Coumarin Derivatives as Anti-Breast and Anti-Cervical Cancer
15

Agents Targeting VEGFR-2 and p38α MAPK, Archiv Der Pharmazie. 350 (2017) 1–
19.
[18] X. Lu, Z. Wang, S. Ren, F. Shen, R. Man, H. Zhu, Bioorganic & Medicinal Chemistry
Letters Coumarin sulfonamides derivatives as potent and selective COX-2 inhibitors
with efficacy in suppressing cancer proliferation and metastasis, Bioorganic &
Medicinal Chemistry Letters. 26 (2016) 3491–3498.
[19] H. He, X. Wang, L. Shi, W. Yin, Z. Yang, H. He, Y. Liang, Bioorganic & Medicinal
Chemistry Letters Synthesis , antitumor activity and mechanism of action of novel 1 ,
3-thiazole derivatives containing hydrazide – hydrazone and carboxamide moiety, 26
(2016) 3263–3270.
[20] M.S. Alam, S.U. Choi, D.U. Lee, Synthesis, anticancer, and docking studies of salicyl-
hydrazone analogues: A novel series of small potent tropomyosin receptor kinase A
inhibitors, Bioorganic and Medicinal Chemistry. 25 (2017) 389–396.
[21] T. Mukund P, L. Klingler, V. Arun, A. Kumar, R. Sadana, D. Kumar. "Design and
synthesis of bis (indolyl) ketohydrazide-hydrazones: Identification of potent and
selective novel tubulin inhibitors." European Journal of Medicinal Chemistry 136
(2017): 184-194.
[22] V. Eva, S. Polanc, M. Kočevar, J. Košmrlj, K. Horváti, S. Bősze, J. Stolaříková, A.
Imramovský, J. Vinšová. "New series of isoniazid hydrazones linked with electron-
withdrawing substituents." European Journal of Medicinal Chemistry 46, no. 12
(2011): 5902-5909.
[23] G. Wang, J. Qiu, X. Xiao, A. Cao, F. Zhou, Synthesis, biological evaluation and
molecular docking studies of a new series of chalcones containing naphthalene moiety
as anticancer agents, Bioorganic Chemistry. 76 (2018) 249–257.
[24] M.A. Shareef, D. Duscharla, G. Ramasatyaveni, N.R. Dhoke, A. Das, R. Ummanni,
A.K. Srivastava, Investigation of podophyllotoxin esters as potential anticancer agents:
Synthesis, biological studies and tubulin inhibition properties, European Journal of
Medicinal Chemistry. 89 (2014) 128–137.
16

[25] A. Radakovic, D.L. Boger, High expression of class III β-tubulin has no impact on
functional cancer cell growth inhibition of a series of key vinblastine analogs,
Bioorganic and Medicinal Chemistry Letters. 28 (2018) 863–865.
[26] P. Govindaiah, N. Dumala, P. Grover, M.J. Prakash, Synthesis and biological
evaluation of novel 4 , 7-dihydroxycoumarin derivatives as anticancer agents,
Bioorganic & Medicinal Chemistry Letters. 29 (2019) 1819–1824.
[27] M.B. Hansen, S.E. Nielsen, K. Berg, Re-examination and further development of a
precise and rapid dye method for measuring cell growth/cell kill, Journal of
Immunological Methods. 119 (1989) 203–210.
[28] B. Munguía, R. Teixeira, V. Veroli, E. Melian, J. Salda, M. Minteguiaga, M. Se,
Experimental Parasitology Puri fi cation of native M . vogae and H . contortus tubulin
by TOG af fi nity chromatography, 182 (2017).
[29] S. Rampogu, M. Son, C. Park, H.H. Kim, J.K. Suh, K.W. Lee, Sulfonanilide
derivatives in identifying novel aromatase inhibitors by applying docking, virtual
screening, and MD simulations studies, BioMed Research International. 2017 (2017).
[30] E.A. Wydysh, A. Vadlamudi, S.M. Medghalchi, C.A. Townsend, Design, synthesis,
and biological evaluation of conformationally constrained glycerol 3-phosphate
acyltransferase inhibitors, Bioorganic and Medicinal Chemistry. 18 (2010) 6470–6479.
[31] Z. Yu, S. Wu, W. Zhao, L. Ding, Y. Fan, D. Shiuan, J. Liu, F. Chen, Anti-Alzheimers
activity and molecular mechanism of albumin-derived peptides against AChE and
BChE, Food & Function. (2018).
[32] I.M. Ayoub, M. Korinek, T.-L. Hwang, B.-H. Chen, F.-R. Chang, M. El-Shazly,
A.N.B. Singab, Probing the Antiallergic and Anti-inflammatory Activity of
Biflavonoids and Dihydroflavonols from Dietes bicolor, Journal of Natural Products.
81 (2018) 243–253.
[33] B.P. Repsold, S.F. Malan, J. Joubert, D.W. Oliver, Multi-targeted directed ligands for
Alzheimer’s disease: design of novel lead coumarin conjugates, SAR and QSAR in
Environmental Research. 29 (2018) 231–255.
[34] Y.H. Chu, Y. Li, Y.T. Wang, B. Li, Y.H. Zhang, Investigation of interaction modes
17

involved in alkaline phosphatase and organophosphorus pesticides via molecular
simulations, Food Chemistry. 254 (2018) 80–86.
[35] Osiris Property Explorer. Available at: http://www.organic-chemistry.org/prog/peo/.
[36] Pre ADMET server available at: https://preadmet.bmdrc.kr/adme.
[37] P. Di, L. Guan, L. Sun, W. Li, Y. Tang, G. Liu, H. Yang, Y. Cai, ADMET-score – a
comprehensive scoring function for evaluation of chemical drug-likeness,
MedChemComm. 10 (2018) 148–157.
18

Scheme-I
6a; R=CH3, Ar= Benzoyl
6b; R=CH3, Ar= p-Tosyl
6c; R=CH3, Ar=4-pyridinoyl
12a; R=OH, Ar= Benzoyl
12b; R=OH Ar= p-Tosyl
12c; R=OH, Ar= 4-Pyridinoyl
18a; R=H, Ar= Benzoyl
18b; R=H Ar= p-Tosyl
18c; R=H, Ar= 4-Pyridinoyl
19

Scheme-II:
24a; Ar= Benzoyl
24b; Ar= p-Tosyl
24c; Ar= 4-Pyridinoyl
20

Table-1: In vitro cytotoxicity of target compounds against A549, HeLa, SKNSH, MCF-7 and
NRK 49F cells,
S. No.
Sample
IC50 (in µMol)
A549
HeLa
SKNSH
MCF7
NRK 49F
1
2
3
4
5
6
7
8
9
6a
33.94 ± 0.34
21.47 ± 0.18
40.72 ± 0.27
8.83 ± 0. 82
16.55 ± 0.86
19.35 ± 0.12
7.78 ± 0.14
12.47 ± 0.32
15.08± 0.42
25.49 ± 0.46
29.35 ± 0.18
29.97 ± 0.42
60.45 ± 0.84
38.17 ± 0.45
6.07 ± 0.12
14.17 ± 0.31
13.98 ± 0.18
12.36 ± 0.38
15.28 ± 0.12
13.02 ± 0.64
39.13 ± 0.34
28.43 ± 0.24
42.96 ± 0.12
22.08 ± 0.61
8.24 ± 0.03
17.79 ± 0.16
9.08 ± 0.92
13.59 ± 0.23
21.58 ± 0.16
18.45 ± 0.85
26.98 ± 0.18
31.83 ± 0.62
28.49 ±0.09
45.44 ± 1.18
6b
29.06 ±0.34
31.12 ± 0.46
7.79 ± 0.41
12.31 ± 1.12
14.81 ± 1.04
11.93 ± 0.98
12.12 ± 0.34
16.26 ± 0.16
28.73 ± 1.23
32.66 ±0.86
40.86 ± 1.15
46.11 ± 2.36
89.53 ± 1. 82
133.75 ± 1.16
44.27 ± 0.48
186.56 ± 1.68
47.26 ± 0.16
52.85 ± 0.24
43.05 ± 0.36
47.47 ± 1.21
6c
12a
12b
12c
18a
18b
18c
24a
24b
10
11
12
35.022 ±
0.09
28.41 ± 0.16
24c
21.76 ± 0.28
6. 22 ± 0.03
31.77 ± 0.54
6.42 ± 0.56
35.59 ± 1.68
9.86 ± 0.12
45.79 ± 0.46
14.22 ± 0.18
13 Dox
7.01 ± 0.015
IC50 is the 50% inhibitory concentration of the samples, and the results were represented as
average values ± standard deviation
A-549, human lung adenocarcinoma cells: HeLa, human cervical cancer cells: SK-N-SH,
human neuroblastoma cells: MCF7, mammary gland adenocarcinoma cells and NRK-49F,
normal rat kidney cells.
Dox = Doxorubicin used as positive control (Standard)
Table-2: Tubulin polymerisation inhibition assay IC50 (µM)
Sample
12a
IC50 (µM)
14.25 ± 1.25
21

18a
16.96 ± 0.92
7.35 ± 1.05
Colchicine
Table-3: Molecular Properties (Lipinski Rule). (Results were predicted by using the Osiris
property explorer software available at http://www.organic-chemistry.org/prog/peo/..)
Sample
MW
Log P
HBA
HBD
6a
6b
336.34
386.42
337.33
338.31
388.39
339.30
332.31
372.40
323.30
336.34
386.42
337.33
3.35
2.77
2.35
2.66
2.08
1.66
3.01
2.43
2.01
3.127
2.54
2.12
5
6
6
6
7
7
5
6
6
5
6
6
2
2
2
3
3
3
2
2
2
2
2
2
6c
12a
12b
12c
18a
18b
18c
24a
24b
24c
MW=Molecular weight, LogP= Partition coefficient, TPSA= Topological polar surface area,
HBA= Hydrogen bond acceptors, HBD= Hydrogen bond donors.
Table-4: Absorption and distribution properties of the synthesized compounds (Results
were predicted by using the server https://preadmet.bmdrc.kr/adme.)
Absorption
Human
intestinal
Distribution
Plasma
Protien
CaCo-2
cell
MDCK cell Skin
permeabilit permeability
Blood-Brain
Barrier
sample
absorptio permeabilit
y
(logkp,cm/hour) Binding
penetration
22

n
(HIA y (nm/sec)
(nm/sec)
(%)
(c.brain/c.blood)
%)
6a
93.89
96.78
93.65
89.47
90.10
85.99
93.77
94.30
93.22
93.89
94.40
93.66
16.06
12.48
12.23
15.59
0.38
9.27
-3.27
-1.45
-3.90
-4.19
-3.02
-4.67
-3.40
-2.90
-4.01
-3.20
-2.67
-3.83
81.79
88.30
68.03
76.57
75.75
59.99
78.31
76.85
62.42
84.50
89.50
74.71
0.23
0.098
0.04
0.11
0.05
0.02
0.18
0.44
0.048
0.125
0.04
0.018
6b
0.05
6c
37.78
35.70
0.63
12a
12b
12c
18a
18b
18c
24a
24b
24c
11.25
14.93
0.52
23.47
34.44
2.24
10.21
18.97
0.64
19.67
3.08
0.081
23.23
16.86
Table-5: Metabolism of all synthesized compounds via hepatic microsomal isoforms (Results
were predicted by using the server https://preadmet.bmdrc.kr/adme.)
Sample
CYP_2C9
Inhibition
Inhibitor
CYP_2C9 CYP_2D6
inhibition inhibition
CYP_2D6
substrate
CYP_3A4
inhibition
CYP_3A4
substrate
Pgp_
inhibition
Inhibitor
inhibitor
6a
6b
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Inhibitor
inhibitor
Weak-inhibitor
23

inhibitor
inhibitor
inhibitor
inhibitor
6c
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
12a
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
Weak-Inhibitor
12b
12c
18a
18b
18c
24a
24b
24c
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-Inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Weak-Inhibitor
Weak-Inhibitor
Weak-Inhibitor
24

Figure 1: Plausible binding mode of the compound 12a with tubulin protein A) stick model
interactions with tubulin protein B) 2D representation interactions C) 3D view of proposed
binding mode.
25

Figure 2: Plausible binding mode of the compound 18a with tubulin protein A) stick model
interactions with tubulin protein B) 2D representation interactions C) 3D view of proposed
binding mode.
26

Graphical abstract
27

Highlights
 Syntheiszed different sets of coumarin based hydrazone derivatives.
 All the compounds were characterised by FT-IR, NMR and ESI-MS spectroscopic
methods.
 Evaluated for antiproliferative activity against different cancer cell lines (A549,
HeLa, SKNSH, and MCF7).
 Tubulin polymerization inhibition assay was performed by sedimentation method.
The results were compared with standard drug colchicine.
 The tubulin polymerization inhibition assay results were validated with molecular
docking simulation study with the tubulin protein retrieved from protein data bank
(PDB ID: 4YJ3).
 All the synthesized compounds molecular properties and pharmacokinetic properties
were predicted by using some online servers and software’s.
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