Design, synthesis, in vitro evaluation and molecular docking study of N'-Arylidene imidazo [1,2-a] pyridine -2-carbohydrazide derivatives as novel Tyrosinase inhibitors

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Design, Synthesis, In Vitro Evaluation and Molecular Docking Study

of N’-Arylidene imidazo [1,2-a] pyridine -2-Carbohydrazide

Derivatives as Novel Tyrosinase Inhibitors

Tahereh Damghani , Saba Hadaegh , Mahsima khoshneviszadeh ,

Somayeh Pirhadi , Razieh Sabet , Mehdi Khoshneviszadeh ,

Najmeh Edraki

PII:

S0022-2860(20)31201-1

DOI:

Reference:

https://doi.org/10.1016/j.molstruc.2020.128876

MOLSTR 128876

To appear in:

Journal of Molecular Structure

Received date:

Revised date:

Accepted date:

20 April 2020

8 July 2020

9 July 2020

Please cite this article as: Tahereh Damghani , Saba Hadaegh , Mahsima khoshneviszadeh ,

Somayeh Pirhadi , Razieh Sabet , Mehdi Khoshneviszadeh , Najmeh Edraki , Design, Synthesis,

In Vitro Evaluation and Molecular Docking Study of N’-Arylidene imidazo [1,2-a] pyridine -2-

Carbohydrazide Derivatives as Novel Tyrosinase Inhibitors, Journal of Molecular Structure (2020),

doi: https://doi.org/10.1016/j.molstruc.2020.128876

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2020 Published by Elsevier B.V.

Highlights



A series of imidazo[1,2-a]pyridine 2-carbohudrazide derivatives bearing different

arylidene pendantrs were designed and synthesized (10 compounds).



Compo

unds bearing 3-nitro (6j) and 4-hydroxy (6g) moieties on the arylidene pendant exhibited

the best Tyrosinase inhibitory activity with IC50 values of 7.19 and 8.11μM, respectively.



Compo

unds 6j, 6h and 6g showed the potential of two critical pi-pi interactions with His263 and

Phe264 in the active site of Tyrosinase.

The results indicated that 6j and 6g could be introduced as potent Tyrosinase inhibitors.

1

Design, Synthesis, In Vitro Evaluation and Molecular Docking

Study of N'-Arylidene imidazo [1,2-a] pyridine -2-Carbohydrazide

Derivatives as Novel Tyrosinase Inhibitors

1

,2

1,2

1

Tahereh Damghani , Saba Hadaegh , Mahsima khoshneviszadeh , Somayeh Pirhadi , Razieh

2

1,2*

1*

Sabet , Mehdi Khoshneviszadeh , Najmeh Edraki .

1

. Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

. School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

2

*

Corresponding authors:

Najmeh Edraki, PhrmD, PhD

Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences, Shiraz, Iran

Tel: +98 713 230 3872; Fax: +98 713 230 2225

E-mail: edrakin@sums.ac.ir, najmeh_edraki@yahoo.com

Mehdi Khoshneviszadeh, PhrmD, PhD

Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences, Shiraz, Iran

School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

E-mail: m.khoshneviszadeh@gmail.com

2

Graphical abstract

O

K2CO3

N

Br

O

+

N

NH2

O

EtOH, Reflux

O

NH2NH2.H2O

Reflux, 3hrs

N

O

R

EtOH

Reflux, 3hrs

N

O

NH

N

X

+

NH

H

NH2

O

Synthesis route of Tyrosinase inhibitors

Molecular docking interaction inside

Tyrosinase active site

^N^O2

N

NH

N

O

Compound 6j with IC50= 7.19 μM

3

Abstract

A novel series of imidazo[1,2-a]pyridine 2-carbohudrazide derivatives bearing different

arylidene pendantrs were designed, synthesized and evaluated for their inhibitory activity against

mushroom Tyrosinase. It was found that compounds bearing 3-nitro (6j) and 4-hydroxy (6g)

moieties on the arylidene pendant exhibited the best Tyrosinase inhibitory activity with IC50

values of 7.19 and 8.11μM, respectively. These results were comparable to that of kojic acid as

the reference drug (IC50 = 9.64±0.5 μM). Additionally, molecular docking analysis was

performed to study the interactions and binding modes of compounds 6j, 6h and 6g which are

showing the potential of two critical pi-pi interactions with His263 and Phe264 in the active site

of Tyrosinase. The results indicated that 6j and 6g could be introduced as potent Tyrosinase

inhibitors that might serve as promising candidates in medicine, cosmetics or food industry.

Keywords: Tyrosinase inhibitor; imidazo [1,2-a]pyridine; docking; Kojic acid

4

1

. Introduction

Melanin, the key pigment primarily responsible for the hair, eyes and skin pigmentation of

human, is produced by melanocytes through melanogenesis. In regard to photoprotection,

melanogenesis and skin pigmentation are the most significant factors in damage response to

ultraviolet radiation from the sun and skin photocarcinogenesis.

The increased melanin synthesis and accumulation of these pigments occur in specific parts of

the skin disorders including hyperpigmentation, lentigines, neurodegeneration associated with

Parkinson’s disease and skin cancer risk[1-3]

Though melanogenesis (the synthesis of melanin) is a very complex process represented by

many enzymatic and chemical reactions, the enzymes such as Tyrosinase and other Tyrosinase-

related proteins (TYRP1 and TYRP2) have a key role in melanin synthesis [4].

Tyrosinase a multifunctional copper-containing metalloenzyme with dinuclear copper ions, is

responsible for the distinct reaction of melanin biosynthesis from tyrosine [5, 6]. Moreover

Tyrosinase is the main factor for undesired browning of fruits and vegetables which leads to

rapid degradation through the postharvest and handing process [7].

Considering the above mentioned points, controlling the activity of this enzyme by Tyrosinase

inhibitors is an important effort for the treatment of hyperpigmentation disorders and fruit

enzymatic browning. In addition, many Tyrosinase inhibitors are of great interest in the medical

and cosmetic products as well as in food and environmental industries [8-10]. However, due to

some limitations such as lack of safety, allergic reactions, low efficacy and poor bioavailability,

development of effective and safe Tyrosinase inhibitors is still a field of great interest [11, 12].

Many natural and synthetic products such as simple phenolic and hydrazine-containing

compounds can be classified as Tyrosinase inhibitors. for example vanillin [13] and its

derivatives [14], hydroquinone [15, 16] and compounds of this type [17, 18] such as resorcinol

(

or resorcin) [19], 4-n-butylresorcinol [20], tropolon and kojic acid have been reported as

possible phenolic inhibitors of the Tyrosinase in the scientific literature (Fig. 1).

Imidazo [1,2-a]pyridine have displayed a broad range of biological and pharmacological activities

including anti-inflammatory [21], anticancer [22], anti-ulcer [23, 24] and antifungal [25].

Additionally, a number of hydrazides, acylated derivatives of hydrazine have been reported to

5

possess a number pharmaceutically effects such as antitumoral, anticonvulsant, antioxidant, anti-

inflammatory and antimicrobial [26-29]. In this study, in order to the discovery of new

Tyrosinase inhibitors, we report the synthesis and biological evaluation of imidazo [1,2-

a]pyridine-2-carbohydrazide derivatives bearing various arylidene pendants. Additionally,

molecular docking analysis of the synthetic compounds was carried out to find a perception of

the ligand-receptor interactions of the compounds in the active site of Tyrosinase enzyme.

2

. Materials and methods

.1. Chemistry

2

All starting materials, reagents and solvents were purchased from the suppliers (Sigma-Aldrich,

Fluka and Merck) and were used without more purification. Reaction progress was monitored by

thin layer chromatography (TLC) on MERCK precoated silica gel 60-F254 (0.5 mm) aluminum

plates and visualized under UV light (254 nm).

The melting points of title compounds were determined with Thermo Scientific Electrothermal

digital apparatus (Thermo Fisher Scientific Inc.). The IR spectra obtained using Perkin-Elmer

1

13

Spectrum RXI FTIR spectrophotometer (KBr disks). H NMR (300 MHz) and C NMR (75 Hz)

spectra were measured using a Bruker 300 MHz NMR instrument by using DMSO-d6 as solvent

and TMS as an internal standard. The chemical shifts (흳) are expressed in parts per million

(

ppm). The MS spectra were recorded using Agilent 7000 triple quadrupole mass spectrometer at

an electron impact mode with an ionization voltage of 70 eV.

2

.2. Synthesis

.2.1. Procedure for the synthesis of ethyl imidazo [1,2-a]pyridine-2-carboxylate (3)

2

-Aminopyridine (5 mmol) was added to 10 ml of ethanol and the mixture was stirred on ice

bath. Then ethyl 3-bromo 2-oxopropanoat (7.5 mmol) was added and the mixture was refluxed

for about 24 h. After cooling, the solid was filtered and washed with cold diethyl ether and

recrystallized from ethanol.

°

−1

1

Pale yellow solid; Yield: 90%, mp: 135-142 C. IR (KBr, cm ): 3086, 2986, 1729. HNMR

300MHz, CD COCD ): δ (ppm) 8.41 (d, J = 6 Hz, 1H, Ar-H), 8.30 (s, 1H, Ar-H), 7.43 (d, J =

Hz, Ar-H), 7.20(t, J = 6 Hz, 1H, Ar-H), 6.84 (t, J = 6 Hz, 1H, Ar-H), 4.22 (q,

(

3

H

9

6

2

2 3 2 3

H,COOCH CH ), 1.23 (t, J = 9Hz, 3H, COOCH CH ) MS (EI) m/z (%): 190 (M+, 28), 145

(

34), 118 (100), 91 (19), 78 (40).

2

.2.2. Procedure for the synthesis of imidazo [1,2-a]pyridine-2-carbohydrazide (4)

A mixture of ethyl imidazo [1,2-a]pyridine-2-carboxylate (5 mmol) and hydrazine hydrate (4ml)

was refluxed for 3 h. After cooling, the mixture of reaction was washed with cold ethanol and

recrystallized from ethanol to give pure imidazo [1,2-a]pyridine-2-carbohydrazide.

−

1

White solid; Yield: 25%, mp: 192-198 ºC. IR (KBr, cm ): 3353, 3187, 3050, 1660. HNMR

300MHz, CDCl ): δ (ppm) 9.53 (s, 1H, CONH), 8.58 (d , J = 6 Hz, Ar-H), 8.36 (s, 1H, Ar-H),

.59 (d, J = 9 Hz, 1H, Ar-H), 7.33 (t, J = 9 Hz, 1H, Ar-H), 6.94 (t, J = 6 Hz, 1H, Ar-H), 4.48 (s,

H, CONHNH ) MS (EI) m/z (%): 176 (M+, 100), 145 (100), 117 (45), 97 (34), 78 (39) .

(

3

H

7

2

.2.3. Procedure for the synthesis of substituted derivative of imidazo [1,2-a]pyridine-2-

carboxylic acid benzylidene-hydrazide (6a-6j)

Different derivatives of (6a-6j) were prepared via the reaction of appropriate imidazo [1,2-

a]pyridine-2-carbohydrazide(4) (1.2 mmol) with benzaldehyde derivatives (5a-5j) (2 mmol) in

chloroform/ethanol (9:1). This reaction mixture was stirred under reflux for 24 h. After

completion of the reaction confirmed by TLC, the solid salts were separated by filtration and the

filtrate was washed with n-hexane and further purified with appropriate solvent.

2

.3.1 Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (2-methoxy-benzylidene)-hydrazide

(

6a)

−

1

White solid; Yield: 88%, mp: 110-114 ºC, IR (KBr, cm ): 3290, 3140, 3000, 2835, 2400, 1673,

1

8

381. HNMR (300MHz, DMSO-d6): δ

H

(ppm) 11.99 (s, 1H, CONH), 8.92 (s, 1H, Ar-H ),

.62(d, 1H, J=7 Hz, Ar-H), 8.53 (s, 1H, N=CH), 7.87 (d, 1H, J=7 Hz, Ar-H), 7.64 (d, 1H, J=9

Hz, Ar-H), 7.39 (m, 2H, Ar-H), 7.09 (d, 1H, J=7 Hz, Ar-H), 7.02 (t, 2H, J=7 Hz, Ar-H), 3.86 (s,

1

3

1

H, OCH3). CNMR (75MHZ):159.04, 158.29, 144.4, 144.08, 139.14, 131.87, 128.16, 127.01,

26.10, 123.19, 121.1, 177.78, 116.23, 113.79, 112.34, 56.22. MS (EI) m/z (%): 214 (M+, 9.9),

61 (68.82), 145 (66.6), 118 (100), 78 (42.21).

7

2

.3.2 Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (4-methoxy-benzylidene)-hydrazide

(

6b)

−

1

White solid; Yield: 83%, mp: 174-178 ºC, IR (KBr, cm ): 3405, 3141, 2999, 2838, 2400, 1655,

1

443. HNMR (300MHz, DMSO-d6): δ

H

(ppm) ) 11.83 (s, 1H, C=ONH), 8.63(d, 1H, J=6 Hz,

Ar-H), 8.51 (s, 2H, Ar-H and N=CH),7.81 (d,1H, J=9 Hz, Ar-H),7.64 (d, 2H, J=8 Hz, Ar-H),

1

3

7

.39 (t, 1H, J=8 Hz, Ar-H), 7.00-7.04 (m, 3H, Ar-H),3.80 (s, 3H, OCH

3

) . CNMR

(

75MHZ):161.23, 158.75, 148.37, 144.38, 139.19, 129.14, 128.18, 127.49, 127.0, 117.72,

1

16.20, 114.79, 113.83, 55.74. MS (EI) m/z (%): 294.1 (M+, 17.88), 161 (69.43), 145 (63.15),

18 (100), 78 (36.82).

2

.3.3. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (3,4,5-trimethoxy-benzylidene)-

hydrazide (6c)

−

1

White solid; Yield: 27%, mp: 221-225 ºC. IR (KBr, cm ): 3194, 3121, 2998, 2940, 2400, 1668,

1

360, 1380, 1433. HNMR (300MHz, DMSO-d6): δ

H

(ppm) 11.95 (s, 1H, C=ONH), 8.63 (d, 1H,

J=7 Hz, Ar-H), 8.54 (s, 1H, Ar-H),8.51 (s, 1H, N=CH), 7.64 (d, 1H, J=9 Hz, Ar-H), 7.39 (t, 1H,

1

3

J=7 Hz Ar-H), 6.99-7.05 (m, 3H, Ar-H), 3.85 (s, 6H, OCH3 3 ), 3.71(s, 3H, OCH

3

). CNMR

(

75MHZ): 158.96, 153.65, 148.34, 144.40, 139.61, 138.97, 130.50, 128.19, 127.15, 117.72,

1

16.35, 113.88, 104.66, 60.59, 56.45. MS (EI) m/z (%): 354.1 (M+, 21.5), 161 (53.75), 145

(

59.13), 118 (100), 78 (29.02).

2

.3.4. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (3-hydroxy-4-methoxy-

benzylidene)-hydrazide (6d)

−

1

White solid; Yield: 36%, mp: 260-264 ºC IR (KBr, cm ): 3583, 3307, 3019, 2964, 2400, 1711.

1

HNMR (300MHz, DMSO-d6): δ

H

(ppm) 11.74(s, 1H, C=ONH), 9.32 (s, 1H, OH), 8.62 (d, 1H,

J=7 Hz, Ar-H), 8.51 (s,1H, N=CH), 8.45(s, 1H, Ar-H), 7.62 (d, 1H, J=9 Hz, Ar-H), 7.38 (t, 1H,

13

J=7Hz, Ar-H), 7.26(S, 1H, Ar-H), 6.88-7.04 (m, 3H, Ar-H), 3.81 (s, 3H, OCH

3

). CNMR

(

75MHZ):158.77, 150.17, 148.57, 147.33, 144.39, 139.18, 128.16, 127.83, 127.05, 120.64,

1

17.71, 116.14, 113.79, 112.83, 112.35, 56.03. MS (EI) m/z (%): 310 (M+, 31.57), 161 (63.12),

45 (86.26), 118 (100), 78 (38.92).

8

2

.3.5. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (3-ethoxy-4-hydroxy-benzylidene)-

hydrazide (6e)

−

1

White solid; Yield: 43%, mp: 139-143 ºC, IR (KBr, cm ): 3168, 2964, 2853, 2964, 2400, 1655.

1

H

HNMR (300MHz, DMSO-d6): δ (ppm) 11.76 (s, 1H, C=ONH), 9.52(s, 1H, Ar-H), 8.62 (d,

1

H, J=6 Hz, Ar-H), 8.53 (s, 1H, N=CH), 8.46 (s, 1H, Ar-H), 7.64 (d, 1H, J=9 Hz, Ar-H), 7.39 (t,

H, J=8 Hz, Ar-H), 7.29(s,1H, Ar-H ), 6.84-7.04 (m, 2H, Ar-H), 6.5 (d, 1H, J=7 Hz, Ar-H), 4.06

1

3

(

2 3

t, 2H, J=6 Hz, OCH ), 1.34 (broad, 3H, CH ). CNMR (75MHZ): 158.70, 149.61, 149.04,

1

6

47.64, 144.32, 138.97, 128.19, 127.21, 126.32, 122.55, 117.62, 116.14, 115.96, 113.87, 110.72,

4.32, 15.19. MS (EI) m/z (%): 324 (M+, 19), 161 (50), 145 (70), 118 (100), 78 (35).

2

.3.6. Synthesis of Acetic acid 4-[(imidazo [1,2-a]pyridine-2-carbonyl)-hydrazonomethyl]-2-

methoxy-phenyl ester (6f)

−

1

White solid; Yield: 40%, mp: 208-212 ºC. IR (KBr, cm ): 3451, 3228, 3146, 3076, 2400, 1759.

1

H

HNMR (300MHz, DMSO-d6): δ (ppm) 11.98 (s, 1H, C=ONH), 8.59-8.55 (m, 3H, Ar-H and

N=CH ), 7.64 (d, 1H, J=9 Hz, Ar-H), 7.45 (s, 1H, Ar-H), 7.39 (t, 1H, J=7 Hz, Ar-H), 7.26 (d, 1H,

J=7 Hz, Ar-H), 7.18 (d, 1H, J=7 Hz, Ar-H ), 7.03 (t, 1H, J=7Hz, Ar-H), 3.85 (s, 3H, OCH ), 2.28

O). CNMR (75MHZ): 168.91, 159.07, 151.68, 147.81, 144.42, 141.21, 138.90,

33.87, 128.19, 127.17, 123.76, 120.98, 117.74, 116.39, 113.09, 110.18, 56.31, 20.87. MS (EI)

m/z (%): 352 (M+, 8.8), 161 (86.65), 145 (71.11), 118 (100), 78 (38.88).

3

13

(

s, 3H, O=CH

3

1

2

.3.7. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (4-hydroxy-benzylidene)-

hydrazide (6g)

−

1

Wihte solid; Yield: 63%, mp: 236-240 ºC. IR (KBr, cm ): 3243, 3242.7, 2400, 1627

1

H

HNMR (300MHz, DMSO-d6): δ (ppm) 11.72 (s, 1H, C=ONH), 9.98 (s, 1H, OH), 8.62(d, 1H,

J=6Hz, Ar-H), 8.48-8.51(m, 2H, Ar-H and N=CH), 7.63(d, 1H, J=9 Hz, Ar-H), 7.54 (d, 2H, J=9

Hz, Ar-H), 7.39 (t, 1H, J=7 Hz, Ar-H), 7.02 (t, 1H, J=7 Hz, Ar-H ), 6.84 (d, 2H, J=7 Hz, , Ar-H).

1

3

CNMR (75MHZ): 159.80, 158.70, 148.80, 144.33, 138.99, 129.32, 128.19, 127.20, 125.91,

17.62, 116.28, 113.86. MS (EI) m/z (%): 280 (M+, 14.7), 161 (73.5), 145 (71.56), 118 (100), 78

1

(

53.9).

9

2

.3.8. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (2,4-dihydroxy-benzylidene)-

hydrazide (6h)

−

1

White solid; Yield: 21%, mp: 300-305 ºC, IR (KBr, cm ): 3583, 3307, 3019, 2964, 2400, 1711.

1

HNMR (300MHz, DMSO-d6): δ

H

(ppm) 12.15 (s, 1H, C=ONH), 11.67 (s, 1H, OH), 10.02 (s,

1

H, OH), 8.54-8.64 (m, 3H, Ar-H and N=CH), 7.65 (d, 1H, J=8 Hz, Ar-H), 7.40 (t, 1H, J=7 Hz,

1

3

Ar-H), 7.22 (d, 1H, J=7 Hz, Ar-H), 7.03 (s, 1H, Ar-H), 6.35(m, 2H, OH). CNMR (75MHZ):

1

61.10, 160.06, 158.54, 150.30, 144.42, 138.49, 132.13, 128.24, 127.30, 117.63, 116.31, 113.93,

11.01, 108.13, 103.17. MS (EI) m/z (%): 296 (M+, 14), 145(99), 118 (80), 78 (79).

2

.3.9. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (3-hydroxy-benzylidene)-

hydrazide (6i)

−

1

White solid; Yield: 48%, mp: 143-145 ºC. IR (KBr, cm ): 3544, 3148, 3019, 2400, 1644.

1

H

HNMR (300MHz, DMSO-d6): δ (ppm) 11.90 (s, 1H, C=ONH), 9.67 (s, 1H, OH), 8.62 (d, 1H,

J=6, Ar-H), 8.52-8.54 (m, 2H, Ar-H and N=CH), 7.63 (d, 1H, J=9 Hz, Ar-H), 7.38(t, 1H, J=7 Hz,

Ar-H), 7.25 (t, 1H, J=7 Hz, Ar-H), 7.19 (broad, 1H, Ar-H), 7.08 (d, 1H, J=6 Hz, Ar-H), 7.02 (t,

1

3

1

H, J=6 Hz, Ar-H), 6.84 (d, 1H, J=7Hz, Ar-H). CNMR (75MHZ): 158.98, 158.11, 148.59,

44.39, 138.89, 136.22, 130.34, 128.19, 127.19, 119.21, 117.82, 117.69, 116.31, 113.88, 113.14.

MS (EI) m/z (%): 280 (M+, 9.25), 161 (84.05), 145 (97.12), 118 (100), 78 (41.62).

2

.3.10. Synthesis of Imidazo [1,2-a]pyridine-2-carboxylic acid (3-nitro-benzylidene)-hydrazide

(

6j)

−

1

Light yellow solid; Yield: 44%, mp: 258-262 ºC. IR (KBr, cm ): 3446, 3154, 3029, 2400, 2400,

1

8

1

7

1

9

658. HNMR (300MHz, DMSO-d6): δ

H

(ppm) 12.20 (s, 1H, C=ONH), 8.70 (s, 1H, Ar-H),

.57-8.62-(m, 2H, Ar-H and N=CH), 8.48 (s, 1H, Ar-H), 8.23 (d, 1H, J=7 Hz, Ar-H), 8.10 (d,

H, J=6 Hz, Ar-H), 7.73 (t, 1H, J=6 Hz, Ar-H), 7.65 (d, 1H, J=8 Hz, Ar-H ), 7.38 (s, 1H, Ar-H),

13

.02 (s, 1H, Ar-H). CNMR (75MHZ): 159.30, 148.63, 146.01, 144.45, 138.65, 136.77, 133.70,

30.91, 128.19, 127.21, 124.59, 121.25, 117.75, 116.59, 113.92. MS (EI) m/z (%): 309 (M+,

.7), 161 (70.59), 145 (80.43), 118 (100), 78 (49.95).

2

.3. Tyrosinase assay

10

Mushroom Tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was tested according to a previous

study with slight modifications using LDOPA as substrate [30]. Enzyme activity was checked by

detecting dopachrome formation at 475 nm. The stock solutions of all test samples and kojic acid

were prepared by dissolving in DMSO at 40mM and then diluted with phosphate buffer

-

1

(

pH=6.8) to the required concentrations. Initially, 10 μL of Tyrosinase (0.5 mg.ml ) was mixed

with 160 μL of 50 mM phosphate buffer (pH = 6.8) and then 10 μL of the test sample was added

in 96-well microplates. After the mixture was pre-incubated at 28 ºC for 20 min, 20 μL of L-

DOPA solution (0.5 mM) was added to the phosphate buffer. DMSO in the absence of the test

compounds was applied as the control, and kojic acid was used as a positive control. Each

concentration was analyzed in three separate replicates. The inhibitory activity of the test

compounds was expressed as the concentration that inhibited 50% of the enzyme activity (IC50).

The percent inhibition ratio was calculated according to the following equation:

Inhibition (%) = 100 - (Abscontrol - Abscompound) / Abscontrol

2

.4. Molecular docking study

All compounds were sketched using MarvinSketch 18.20.0 [31] and energy of compounds was

optimized using steepest descent algorithm by Open Babel 2.4.0 software [32]. GOLD 2018

docking program [33] was applied to dock all compounds using PDB code of 2Y9X. This

structure is a Tyrosinase from Agaricus bisporus having tropolone as the native ligand in the

2

+

binding site. Two Cu metal ions exist in the binding site of Tyrosinase and were included for

docking calculations. Protein structure was prepared using Discovery studio client [34] and all

hydrogens were added. Binding site of the enzyme for docking was defined automatically by

setting 8 Å around the coordinates of the native ligand tropolone. All available scoring functions

in GOLD, namely CHEMPLP, ChemScore, ASP, and GoldScore were tried to re-dock tropolone

inside the 2Y9X and then RMSD measure was calculated to validate and find the most

appropriate scoring function for docking analyses.

3

. Results and discussion

.1. Design strategy

11

We designed the target compounds bearing imidazo [1,2-a]pyridine-carbohydrazid scaffold

based on the structures of some potent Tyrosinase inhibitors reported in the literature [35, 36].

Some benzimidazole derivatives such as 2-(3-Methylbenzyl)-4(7)-phenyl-1H benzo[d]imidazole

(

I) illustrated Tyrosinase inhibitory activity with IC50 value of 37.86 μM [37]. The benzothiazole

analogs like compound (II) have been reported as potential Tyrosinase inhibitors which

demonstrated higher inhibition than kojic acid at 50 μM [38]. Additionally, the hydrazine-

containing compounds such as compound (III) have been reported as effective Tyrosinase

inhibitors (IC50=1.93 μM) which exhibited better Tyrosinase inhibitor activity than kojic acid

(

Fig. 2) [39]. Based on these findings, herein we designed a series of imidazo [1,2-a]pyridine-2-

carbohydrazide derivatives containing various arylidene pendants as tyroinase inhibitors via

hybridization of imidazo pyridine core with hydrazine moiety as copper chelator. Furthermore,

various phenolic moieties and its derivatives were introduced as a substituted benzylidene

pendant to meet the essential requirements of Tyrosinase inhibitors and constructing structure–

activity relationships (SARs).

3

.2. Synthesis

The synthetic route to target compounds 6a–6j is represented in Scheme 1. Reaction of 2-

aminopyridine (1) and ethyl bromopyruvate (2) in refluxing ethanol resulted in ethyl imidazo

[

1,2-a]pyridine-2-carboxylate (3). In the next step, reaction of compound (3) with hydrazine

hydrate under reflux condition afforded imidazo [1,2-a]pyridine-2-carbohydrazide (4). Finally

different substituted benzaldehyde derivatives (5a-j) were reacted with 2 in chloroform/ethanol

(

9:1). The resulting mixture was stirred under reflux and completion of the reaction was

monitored by TLC to give compounds 6a–6j.

3

.3. Tyrosinase inhibitory activity

The inhibitory effect of all the synthesized derivatives were conducted on mushroom Tyrosinase

enzyme by evaluation of their potential to inhibit enzymatic oxidation of L-Dopa. Kojic acid was

used as the reference inhibitor according to the literature protocol [30, 40]. The chemical

structure of the synthesized compounds and results are presented in Table 1.

12

The most potent compounds 6j and 6g exhibited considerable inhibitory potential with IC50

values of 7.19±2.56 μM and 8.11±1.25 μM, respectively against mushroom Tyrosinase

compared to kojic acid with IC50 value of 9.64±0.50 μM. Compounds 6c, 6h, 6b, 6f and 6e

showed moderate to weak inhibitory activities (IC50s = 11.1-81.9 μM) while the rest of

compounds (6a, 6d and 6i) were almost inactive.

Considering the obtained results from the SAR, there is a strong relation between the nature and

position of the phenyl ring substitutions and Tyrosinase inhibition that can be shown as follows:

It seems that the presence of an electron donating group such as hydroxy and methoxy at the para

position of the phenyl ring enhanced the inhibitory activity (compounds 6g and 6b). In addition,

introduction of some groups such as nitro into the meta position of the phenyl moiety increased

the anti-Tyrosinase potential of synthesized compound (compound 6j). In contrast, compound 6i

bearing hydroxyl group on meta position of the phenyl ring was completely inactive.

Furthermore, it was found that compound 6a bearing 2-methoxy group on the phenyl ring was

completely inactive while compound 6h bearing 2,4 di hydroxyl residues exhibited the moderate

Tyrosinase inhibitory activities. The comparison of substituents on different positions of phenyl

ring indicated that compounds 6f, 6e and 6d bearing different substitutes such as 3-methoxy-4-

acetoxy, 3-ethoxy-4-hydroxy and 3-hydroxyl-4methoxy showed weak to inactive inhibitory

activities; however, increasing the number of electrondonating group as in compound 6c bearing

3

,4,5 tri methoxy improved the activity. Consequently, the anti-Tyrosinase activity of 6j and 6g

are higher than kojic acid and we hope that the imidazopyridine derivative would overcome

some limitations of kojic acid such as formulation stability and skin penetration difficulties [41].

3

.4. Molecular docking analysis

Molecular docking analysis was carried out to investigate interactions and binding poses of all

synthesized compounds inside the binding site of Tyrosinase enzyme. RMSD value of redocking

process of tropolone inside the active site of Tyrosinase with all available scoring functions in

the GOLD was calculated in such a way that for ChemScore was 2.00 Å and for other scoring

function algorithms was higher than 2.00 Å. Therefore, score values and binding modes of

Tyrosinase inhibitors with the enzyme with PDB code 2Y9X was investigated by ChemScore

fitness function. ChemScore fitness value of docking of all compounds along with their

important interactions with amino acid residues inside the Tyrosinase active site were shown in

13

Table 1. ChemScore fitness values ranged from 34.73 in compound 6j to 20.37 in compound 6c.

D interaction patterns of two active compounds 6j and 6g with IC50 values 7.19 and 8.11 µM

3

respectively, and two inactive compounds 6a and 6i with IC50 values more than 100 µM, were

depicted in Fig. 3.

Fig. 3a shows the docking interactions of compound 6j. Docking score of this compound is 34.73

which is the highest score value among all compounds. Backbone structure of the compound

made two pi-pi interactions via its aromatic rings: one pi-pi t-shaped interaction from imidazo

[

1,2‐a]pyridine ring to Phe264 and one pi-pi stacked interaction from nitrobenzene group to

His263. These two pi-pi interactions with these residues were among important ones. Other

interaction types comprised pi-sigma and pi-alkyl interactions with Val283 and Ala286

respectively and pi-sigma interaction from imidazo [1,2‐a]pyridine ring toVal283.

Docking interaction analysis of compound 6g, Fig. 3b, shows the same pattern of pi-pi

interactions with Phe264 and His263 from imidazo [1,2‐a]pyridine ring and phenyl ring,

respectively. Replacement of 3-nitro with 4-hydroxy group changed the orientation of compound

6

g in such a way that one hydrogen bond was made between carbonyl group and residue Val283.

This additional hydrogen bond interaction did not cause any significant difference in activity

against Tyrosinase in comparison to compound 6j. Chemscore fitness value of this compound is

a high value of 29.29. Compound 6g made other interaction types such as a pi-cation from

imidazo [1,2‐a]pyridine ring to residue Arg268, a pi-alkyl interaction and a pi-sigma interaction

between phenol and Ala286, and Val283 respectively that all further strengthened its binding

towards Tyrosinase enzyme.

Another moderate to active compound is 6h with IC50 value of 12.6 µM having ChemScore

fitness value of 31.53. This compound made two important pi-pi interactions of the backbone

with Phe264 and His263. In comparison to compound 6g bearing 4-hydroxy moiety, compound

6

h has an additional hydroxyl in position 2 that donated a hydrogen bond to Asn260. This

2

+

hydroxyl also made a metal-acceptor interaction with one Cu . Another hydrogen bond

interaction was formed between carbonyl group and Val283. A pi-alkyl interaction between

Val283 and phenyl ring was also made, see Fig 3c.

In inactive compound 6i, 3-hydroxy replaced 4-hydroxy in compound 6g. This substitution got

the backbone away from Phe264 in such a way that it was not able to make this critical pi-pi

14

interaction and just made pi-pi stacked interaction with His263. ChemScore fitness value of 6i is

0.17 which might be resulted from other not that much important hydrophobic interactions it

3

made like pi-alkyl with Ala286 and pi-sigma with Val248 inside the active site of Tyrosinase,

see Fig. 3d.

In overall, inactive compounds, 6a, 6d and 6i, lacked one or two critical pi-pi interactions with

His263 and Phe264 that resulted from a different orientation of these molecules in the enzyme

active site. Presence of an electrondonating group such as hydroxyl in the para position of the

+

2

phenyl ring makes compound able to form a metal-acceptor interaction with Cu ion and also

changes the orientation of the molecule to be able to form a hydrogen bond from carbonyl group

to residue Val283.

4

. Conclusions

In conclusion, we developed a series of imidazo [1,2-a]pyridine-2-carbohydrazide derivatives

bearing various arylidene pendants as novel Tyrosinase inhibitors. Based on the obtained data,

compounds 6j and 6g containing 3-nitro and 4-hydroxyl moieties were the most potent

compounds against Tyrosinase with respective IC50 values of 7.19 μM and 8.11 μM. The results

indicated that the inhibitory potential was dependent on the substituted moiety on the phenyl

ring. Therefore, derivatives bearing an electrondonating such as hydroxyl and electron-

withdrawing group such as nitro at the para and meta position of the phenyl ring exhibited

considerable inhibitory activity, respectively. Molecular docking analysis results demonstrated

that potent compounds 6g, 6h and 6j had high docking score values and made two critical pi-pi

interactions with Phe264 and His263. These results indicate that the compounds can serve as

structural outlines and promising leads in order to design and develop novel effective Tyrosinase

inhibitors.

Declaration of competing interest

The authors report no conﬂict of interest.

15

Credit Author Statement

Najmeh Edraki : principle investigator, supervision of the team in all research steps including

data gathering, data analysis, and manuscript preparation/revision

Mehdi Khoshneviszadeh: Supervision of the design, synthetic steps and biological evaluation,

manuscript preparation.

:

Tahereh Damghani contribution in the data gathering, design and synthesis of compounds, data

analysis, preparation of results, writing and revising of this manuscript.

Saba Hadaegh : contribution in the synthesis of compounds.

Mahsima khoshneviszadeh: Contribution in the biological and enzymatic assay..

Somayeh Pirhadi : Supervision and performance of computational docking and data analysis of

this study.

Razieh Sabet : Supervision of synthetic parts.

Acknowledgment

The authors wish to thank the support of the Vice-Chancellor for Research, Shiraz University of

Medical Sciences (Grant Number: 14006). This work was the PharmD thesis of Saba Hadaegh.

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1

20

Table 1

Chemical structure, Tyrosinase inhibitory effects and molecular docking studies of synthesized

compounds 6a-6j.

ChemScore

Binding interactions with

Tyrosinase active site

Mw

fitness

value

Yields*

compounds

R

IC50(μm )±SE**

6

a

2- methoxy

4- methoxy

294

22.62

23.93

Hydrogen bonds: Val283

88%

83%

> 100

b

Hydrogen bonds: Val283

Pi-pi interactions: His263,

14.3±3.11

6

c

3,4,5-trimethoxy

354

20.37

Hydrogen bonds: Arg268

Pi-pi interactions: Phe264

27%

11.10±1.55

6

d

3-hydroxy-4-

methoxy

310

324

ꢀ52

28ꢁ

28.47

27.29

28.49

29.29

Hydrogen bonds: Asn260, Val283

36%

43%

40%

63%

> 100

6

e

3-ethoxy-4-

hydroxy

Pi-pi interactions: His263,

Metal acceptor interaction: 1 Cu

Metal acceptor interaction: 2 Cu

81.9±9.75

41.70±2.40

8.11±1.25

+

2

+

6

f

3-methoxy-4-

acetoxy

6

g

4-hydroxy

Hydrogen bonds: Ala283

Pi-pi interactions: His263, Phe264

6

h

2,4-dihydroxy

29ꢂ

31.53

Hydrogen bonds: Asn260, Val283

Pi-pi interactions: His263, Phe264

Metal acceptor interaction: 1 Cu

Pi-pi interactions: His263

21%

12.60±3.67

6

i

3-hydroxy

28ꢁ

309

-

30.17

34.73

48%

44%

> 100

6

j

3-nitro

-

Pi-pi interactions: His263, Phe264

7.19±2.56

9.64±0.50

Kojic Acid***

*

Reported yields are based on the yield of final step of synthesis.

* Values represent means ± SE of 3 independent experiments.*** Kojic Acid was used as a positive control.

21

Highlights



A series of imidazo[1,2-a]pyridine 2-carbohudrazide derivatives bearing different

arylidene pendantrs were designed and synthesized (10 compounds).



Compo

unds bearing 3-nitro (6j) and 4-hydroxy (6g) moieties on the arylidene pendant exhibited

the best Tyrosinase inhibitory activity with IC50 values of 7.19 and 8.11μM, respectively.



Compo

unds 6j, 6h and 6g showed the potential of two critical pi-pi interactions with His263 and

Phe264 in the active site of Tyrosinase.

The results indicated that 6j and 6g could be introduced as potent Tyrosinase inhibitors.

22

Design, Synthesis, In Vitro Evaluation and Molecular Docking

Study of N'-Arylidene imidazo [1,2-a] pyridine -2-Carbohydrazide

Derivatives as Novel Tyrosinase Inhibitors

1

,2

1,2

1

Tahereh Damghani , Saba Hadaegh , Mahsima khoshneviszadeh , Somayeh Pirhadi , Razieh

2

1,2*

1*

Sabet , Mehdi Khoshneviszadeh , Najmeh Edraki .

1

. Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

. School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

2

*

Corresponding authors:

Najmeh Edraki, PhrmD, PhD

Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences, Shiraz, Iran

Tel: +98 713 230 3872; Fax: +98 713 230 2225

E-mail: edrakin@sums.ac.ir, najmeh_edraki@yahoo.com

Mehdi Khoshneviszadeh, PhrmD, PhD

Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences, Shiraz, Iran

School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

E-mail: m.khoshneviszadeh@gmail.com

23

Graphical abstract

O

K2CO3

N

Br

O

+

N

NH2

O

EtOH, Reflux

O

NH2NH2.H2O

Reflux, 3hrs

N

O

R

EtOH

Reflux, 3hrs

N

O

NH

N

X

+

NH

H

NH2

O

Synthesis route of Tyrosinase inhibitors

Molecular docking interaction inside

Tyrosinase active site

^N^O2

N

NH

N

O

Compound 6j with IC50= 7.19 μM

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