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

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

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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 Govindaiah^a*, Naresh Dumala^b, Irshad Mattan^a, Paramjit Grover^b, Jaya Prakash. M^a

^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 IC₅₀values 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 POCl₃were 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 POCl₃was 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% CO₂humidified 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,

5x10³exponentially 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 CO₂incubator 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). IC₅₀values 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 IC₅₀values 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.

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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

IC₅₀is 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

7

5

6

5

6

2

3

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

Inhibitor

inhibitor

Weak-inhibitor

23

inhibitor

6c

Non-inhibitor

12a

inhibitor

Weak-Inhibitor

12b

12c

18a

18b

18c

24a

24b

24c

Non-inhibitor

Non-Inhibitor

Non-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.

28

Article Doi

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

DOI: 10.1016/j.bioorg.2019.103143

Source and publish data:

Authors:

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

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

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