Design, Synthesis, Antibacterial Evaluation and Molecular Docking Studies of Some Newer Baenzothiazole Containing Aryl and Alkaryl Hydrazides

Title: Design, synthesis, antibacterial evaluation and molecular docking

studies of some newer benzothiazole containing aryl and alkaryl

hydrazides

Authors: Rani gurram Swarupa and Afzal Azam Mohammed

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To be cited as: Chem. Biodiversity 10.1002/cbdv.202100117

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10.1002/cbdv.202100117

Chemistry & Biodiversity

Design, synthesis, antibacterial evaluation and molecular docking studies of some newer

benzothiazole containing aryl and alkaryl hydrazides

Swarupa Rani gurram¹, Mohammed Afzal Azam^1*

¹Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ooty- 643001, Nilgiris, Tamil Nadu (JSS

Academy of Higher Education and Research), India

Corresponding author: Mohammed Afzal Azam

E-mail: afzal9azam@hotmail.com;afzal@jssuni.edu.in;

Tel: +91-423-2443393; Fax: +91-4232442937

ORCID: https://orcid.org/0000-0001-9770-9371

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Abstract

The alarming rise of bacterial resistance is occurring worldwide and endangering the efficacy of antibiotics.

Therefore, development of new and efficient antibacterial agents remains paramount. In the present work we

designed and synthesized a series of N'-(1,3-benzothiazol-2-yl)-substituted aryl/aralkyl hydrazides C1-C27 and

evaluated them in vitro for their antibacterial activity. Among all tested compounds, C10, C15, and C24 showed

potent activity against Staphylococcus aureus ATCC 43300 (MRSA). Minimum bactericidal concentration studies

of synthesized compounds are performed against selected bacterial strains. Time kill kinetics showed that the

compounds C10 and C15 possess bactericidal activity against MRSA ATCC 43300, while compound C24 possess

bactericidal activity against S. aureus NCIM 5022. In the extra-precision docking compounds C1-C27 exhibited

interactions mainly with the N-terminal and central domains of S. aureus GyrB catalytic pocket. Binding free energy

(ΔG_bind) of compounds C1-C27/3U2K complexes were computed by MM-GBSA approach. Free energy components

indicated Coulomb energy term as favourable for binding, while van der Waals and electrostatic solvation energy

terms strongly disfavoured the binding. ADMET properties of synthesized compounds C1-C27 are also computed.

Key words Benzothiazole; arylhydrazides; Staphylococcus aureus, MIC; binding free energy

Introduction

The rapid rise of bacterial resistance to antibiotics is a worldwide problem and increases healthcare costs and

mortality.^[1]Specifically, infections caused by methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-

resistant Enterobacteriaceae (CRE), multi-drug-resistant Mycobacterium tuberculosis (MDR-TB), vancomycin-

resistant Enterococcus (VRE), and multidrug-resistance Streptococcus pneumonia are hard to treat.^[2]This has

necessitated an interest in the development of new and effective antibacterial agents to fight antibiotic resistant

bacterial strains.

Benzothiazoles (BT) are sulphur-containing heterocyclic constituted from a benzene ring fused to a thiazole

ring. Benzothiazole derivatives have been synthesized by different synthetic paths.^[3-5]Due to associated biological

and pharmacological activities, BTs played significant role in synthetic and medicinal chemistry. BT is present as

core nucleus in various therapeutic agents like ethoxazolamide, riluzole, viozan, zopolrestat, Tiaramide,

Halethazole, Frentizole, Thioflavin-T etc.^[6].Many patents have been published on benzothiazoles highlighting its

importance and few derivatives are in different phases of clinical trials.^[7-10]Compounds possessing 1,3-

benzothiazole nucleus^[11-13]gained attention due to their diverse biological activities like amyotrophic lateral

sclerosis^[11]

,

anti-inflammatory,^[12]antifungal,^[13]hypoglycemic,^[14]anthelmintics,^[15]anticancer,^[16]PPARα

antagonists^[17]and Jun N-terminal kinase inhibitors.^[18]

In addition, 2-hydrazinobenzothiazole linked to the arylidene moieties (1)^[19,20]exhibited promising

antibacterial activity. Functionalized 2-hydrazinobenzothizole (2)^[21]linked to the isatin and some carbohydrates is

also reported to possess antibacterial activity. In another investigation a series of 2-hydrazinobenzothiazoles have

been designed and synthesized using the molecular hybridization of 2-hydrazinyl benzothiazole and 4-

(aryloxy)benzaldehyde.^[22].Specifically, compound

3

exhibited promising activity against Mycobacterium

tuberculosis H37Rv (MIC, 1.5 μg/mL).

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On the same line, aryl/alkyl hydrazides are considered as an important scaffold in the drug discovery and

development processes because of their wide range pharmacological activities. In search of the potent antibacterial

agents synthesis and antimicrobial activity of 2-bromo-4-methoxy benzohydrazide linked to the substituted thiazole

moieties has been described by Raj et al.^[23]; in particular, compound 4 showed promising activity against

Pseudomonas aeruginosa with MIC of 6.25 μg/mL. In another study Peng et al.^[24]reported the antibacterial activity

of substituted benzohydrazide derivatives linked to the substituted benzylidene derivatives. Among all the tested

compounds, derivative 5 exhibited significant activity against Bacilus subtilis (MIC, 3.1 μg/mL). In another

research, Kumar et al.^[25]synthesized 3-ethoxy-4-hydroxybenzylidene hydrazides linked to the alkyl groups and

evaluated their antibacterial activity. Compound 6 showed higher antibacterial activity against Staphylococcus

aureus, Bacilus subtilis and Eschericia coli than that of ciprofloxacin. Further, 2-(2,3-dihydro-1-benzofuran-6-

yl)acetohydrazide linked to the benzylidene moiety in compound 7 showed significant activity against both Gram-

positive and Gram-negative bacteria.^[26]

Molecular hybridization is shown to be valuable medicinal chemistry strategy that aims to combine two

molecules in a new and single chemical entity. It is recognized as an effective approach to design ligands to

modulate the affinity against targets of interest.^[27,28]Based on these reported good performance of molecular

hybridization, combining benzothiazole ring with alkyl/aryl hydrazide moieties in a single molecular scaffold was

presumed to be a reasonable and promising approach. In view of this data, we aimed the development of new

antibacterial agents using molecular hybridization approach. In this context, various molecules were designed by

incorporating substituted alkyl/substituted aryl/aralkyl carbonyl parmacophore into the 2-hydrazinobenzothiazole to

combine the synergistic activity of both moieties. In the present study we carried out 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDCl) and N-hydroxybenzotriazole (HOBt) mediated synthesis

of novel N'-(1,3-benzothiazol-2-yl)-substituted aryl/aralkyl hydrazides C1-C27 and subsequent evaluated for their

activity against selected strain of bacteria.

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Figure 1. Designing of pharmacophoric groups into benzothiazole containing aryl and alkaryl hydrazides.

RESULTS AND DISCUSSION

Coupling of carboxylic acids with amines have been successfully carried out under the catalysis of EDCl/HOBt in

moderate to high yield.^[29-31]Amidation reaction was performed using EDCl/HOBt under ultrasound irradiation with

65-78% yield.^[32]Amides have also been synthesized in high yields (81-93%) with EDCl/HOBt using different

solvents like dichloromethane, acetone and tetrahydrofuran under reflux.^[33,34]Keeping in view of this versatile uses,

we carried out the coupling reaction of 2-hydrazino-1,3-benzothiazole with different aromatic carboxylic acids using

EDCl/HOBt in N,N-dimethyl formamide (DMF) at room temperature. We synthesized new N'-(1,3-benzothiazol-2-

yl)-substituted aryl/aralkylhydrazides C1-C27 as outlined in Scheme 1. 2-Hydrazino-1,3-benzothiazole (A) was

obtained by the reaction of 2-mercaptobenzothiazole with hydrazine hydrate (98% w/w) in water.^[35]Stirring of 2-

hydrazino-1,3-benzothiazole with appropriate substituted aromatic acids B1-B27 in the presence of HOBt and

EDCl^[36]in DMF afforded title compounds C1-C27 in 78.2-94.2% yield. Optimization of the reaction conditions for

the synthesis of N'-(1,3-benzothiazol-2-yl)-substituted benzohydrazides derivatives C1-C27 started by reacting 2-

hydrazino-1,3-benzothiazole (A) and 4-methyl benzoic acid as model substrates. Increase in the reaction time did

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not improve the reaction yield remarkably. To further improve the reaction yield other solvents including

chloroform and dichloromethane were also tested and but did not improve the yield.

R¹

HOBt 2.0 eq.

N R²

NH₂NH₂

NH₂

NH

O

N

S

N

S

N

S

EDCl 2.0 eq.

SH

NH

+

H₂O/reflux

R

OH

DMF/strring

B1 – B27

A

C1 – C27

R

R¹

R²

B1

C1

C2

C3

C4

C5

C6

C7

C8

C9

4-CH₃-C₆H₄-C(=O)

C₆H₅-C(=O)

H

4-CH₃-C₆H₄

C₆H₅

C₆H₅-(CH₂)₂

4-NO₂-C₆H₄

3-Cl-4-NO₂-C₆H₃

4-OH-C₆H₄

3,5-di-OH-C₆H₃

3,5-di-Cl-C₆H₃

4-pyridyl

B2

C₆H₅–C(=O)

B3

C₆H₅-(CH₂)₂-C(=O)

4-NO₂-C₆H₄-C(=O)

3-Cl-4-NO₂-C₆H₃-C(=O)

4-OH-C₆H₄-C(=O)

3,5-di-OH-C₆H₃-C(=O)

3,5-di-Cl-C₆H₃-C(=O)

pyridine-3-yl-C(=O)

H

B4

B5

B6

B7

B8

B9

B10

B11

B12

B13

B14

B15

B16

B17

B18

B19

B20

B21

B22

B23

B24

B25

B26

B27

C10 4-F-C₆H₄-C(=O)

C11 quinoline-2-yl-C(=O)

C12 2-I-C₆H₄-C-(=O)

4-F-C₆H₄

2-quinolinyl

2-I-C₆H₄

C13 3-CH₃O-2-NO₂-C₆H₃-C(=O)

C14 3-CH₃-2-NO₂-C₆H₃-C(=O)

C15 3,5-di-NO₂-C₆H₃-C(=O)

C16 C₅H₅-CH(OH)-CH₂-C(=O)

C17 2-F-C₆H₄-C(=O)

C18 3-Br-pyridine-4-yl-C(=O)

C19 2,4,6-tri-Cl-C₆H₂-C(=O)

C20 2,4-di-Cl-C₆H₃-C(=O)

C21 thiophene-2-yl-C(=O)

C22 thiophene-2-yl-C(=O)

C23 2-CH₃-C₆H₄-C(=O)

C24 4-C₄H₉O-C₆H₄-C(=O)

C25 4-CH₃O-C₆H₄-C(=O)

C26 C₆H₅-(CH₂)₂-C(=O)

C27 thiophene-2-yl-C(=O)

3-CH₃O-2-NO₂-C₆H₃

3-CH₃-2-NO₂-C₆H₃

3,5-di-NO₂-C₆H₃

C₆H₅-CH(OH)-CH₂

2-F-C₆H₄

2-Br-4-pyridyl

2,4,6-tri-Cl-C₆H₂

2,4-di-Cl-C₆H₃

2-thienyl

C₆H₅O-CH₂

2-CH₃-C₆H₄

4-C₄H₉O-C₆H₄

4-CH₃O-C₆H₄

C₆H₅-(CH₂)₂

2-thienyl

C₆H₅-(CH₂)₂-C(=O)

thiophene-2-yl-C(=O)

Scheme 1. The synthetic route of the target compounds C1-C27.

1

The structures of newly synthesized compounds C1-C27 is characterized by H NMR, ¹³C NMR and

HRMS spectral data (see Supplementary Figure S1-S27). In the Fourier-Transform Infrared (FT-IR) spectra of

compounds C1-C27 characteristics absorption bands of >C=O, >C=N and C-S-C were observed, respectively in the

range 1655 to 1678, 1611 to 1637 and 667 to 696 cm^-1. Characteristic NH and NHNH absorption bands were

1

observed in the range between 3253 to 3369 cm^-1. In the H NMR spectra of synthesized compounds absorption

bands due to NHNH/NH appeared in the range at δ 4.77-12.28 ppm. Moreover, in compound C24 NHNH signal has

not appeared may be due to the replacement of exchangeable protons with deuterium of residual DCl. The ¹H NMR

spectrum of representative compound C1 (Figure S1a) showed a sharp singlet at δ 4.99 ppm indicating the presence

of two -NHNH- protons. A singlet signal integrating for three protons is observed at δ 2.43 ppm and is assigned to -

CH₃group. Eight aromatic protons of this compound appeared in the region at δ 7.28 to 7.84 ppm. Further, in the ¹H

NMR spectra of compounds C1-C27 aromatic protons showed splitting patterns which agree with the substitution

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pattern of respective compounds. The chemical shift and coupling constant (J) values of aromatic protons were

assigned accordingly with their position. In compound C1, two protons present on position five and six of

benzothiazole ring appeared as double doublet at δ 7.84 ppm (J = 8.0, 2.3 Hz), while two protons present on position

four and seven of the same ring showed a doublet signal at δ 7.78 ppm (J = 8.1 Hz). A multiplet signal appeared at δ

7.49-7.32 ppm is assigned to two protons present at position two and six of the phenyl ring of 4-

methylphenylhydrazino moiety. Two protons present at position three and five of this phenyl ring appeared as

doublet signal at δ 7.28 ppm (J = 9.4 Hz). In the ¹³C NMR spectrum of compound C1 (Figure S1b) the carbonyl

carbon of hydrazide exhibited a singlet signal at δ 170.55 ppm, while the azomethine carbon of the benzothiazole

ring appeared as a singlet at δ 161.44 ppm. A singlet signal appeared at δ 29.38 ppm is ascribed to the -CH₃group.

Remaining ten SP²hybridized aromatic carbons appeared in their expected region between δ 7.28 to 7.84 ppm. Mass

spectrum of C1 (Figure S1c) showed [M + H]⁺peak at m/z 284.0845 (100%), which corresponds to its molecular

formula C₁₅H₁₃N₃OS.

Minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC)

MIC and MBC of synthesized compounds C1-C27 were determined by broth microdilution method (CLSI 2007)^[37]

and results are presented, respectively in Table 1 and Table 2. It can be seen from Table 1 that compounds C14

exhibited maximum activity against S. aureus NCIM 5021 with MIC of 20.75 μM, whereas compounds C4, C5,

C11, C14, C18-C20 showed moderate activity (MICs, 24.02 to 48.04 μM). High activity of C14 against this strain

may be attributed to the presence of bulky NO₂and CH₃groups, respectively on position two and three of the phenyl

ring attached to the hydrazide function. Against S. aureus NCIM 5022 compounds C4 and C24 showed high activity

with MICs of 24.81 and 20.75 μM, respectively. Whereas compounds C5, C8, C12-C15 and C18-C20 displayed

moderate activity (MICs, 39.21 to 46.12 μM) against this strain when compared to the standard drugs ciprofloxacin

(MIC, 6.33 μM) and gentamicin (MIC, 16.95 μM). Presence of bulky NO₂and n-butoxy groups on position four of

the phenyl rings, respectively in C4 and C24 may be attributed for their high activity. Five compounds i.e. C10,

C12, C15, C16 and C24 exhibited potent activity (MICs, 19.73 to 27.14 μM) against S. aureus ATCC 43300

(MRSA) in comparison to the standard drugs ciprofloxacin (MIC, 95.97 μM) and gentamycin (MIC, 41.24 μM).

Compounds C8, C11, C13, C14, C14 and C19 also displayed promising activity (MICs, 40.52 to 47.13 μM) against

this strain. It is evident that presence of electronegative atoms either on positions two or three of the phenyl ring

(C10, C12 and C24) or presence of electron withdrawing goups on position three and five of the phenyl ring (C15)

is optimum for activity against S. aureus ATCC 43300. In general presence of alkyl substituted phenyl ring (C1,

C23), unsubstitued phenyl ring (C2) or when phenyl ring is attached to the hydrazide moiety via alkyl groups (C3,

C26) markedly decreased activity against S. aureus ATCC 43300. Against Staphylococcus epidermidis NCIM 2493

compounds C13 and C21 possessing, respectively 3-methoxy-2-nitrophenyl and thiophene rings linked to the

hydrazide moiety showed maximum activity (MICs, 22.65 and 28.32 μM, respectively), while all other tested

compounds showed moderate to low activity. However, activity of compounds C13 and C21 is far low against this

strain in comparision to the tested standard drugs ciprofloxacin (MIC, 6.33 μM) and gentamycin (MIC, 17.16 μM).

Against Mycobacterium sp NCIM 2984 only one compound i.e. C24 displayed promising activity (MIC, 21.81 μM),

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while all other tested compounds were observed to less active against this strain. However, activity of C24 against

this strain was comparatively less compared to the standard drugs ciprofloxacin (MIC, 3.62 μM) and gentamycin

(MIC, 16.95 μM). When tested against K. pneumoniae NCIM 2706 two compounds i.e. C18 and C19 possessing,

respectively 3-bromopyridine and 2,4,6-trichlorobenzene rings linked to the hydrazide moiety displayed promising

activity (MICs, 20.24 and 21.19 μM). However, activity of these two compounds against this strain was observed to

be significantly less compared to the standard drugs ciprofloxacin (MIC, 6.63 μM) and gentamycin (MIC, 2.30 μM).

When tested against E. coli NCIM 2065 only three compounds C11, C18 and C20 showed promising activity with

MICs of 24.65, 20.61 and 22.17 μM, respectively. But activity of these three compounds is low compared to the

tested standard drugs ciprofloxacin (MIC, 6.94 μM) and gentamycin (MIC, 2.09 μM). It is evident that the presence

of electron rich heterocyclic ring linked to the hydrazide moiety is optimum for activity against Gram-negative K.

pneumoniae NCIM 2706 and E. coli NCIM 2065. Against another Gram-negative bacteria P. aeruginosa NCIM

2036 tested compounds exhibited moderate to less activity (MICs, 38.20 to 220.14 μM) compared to the standard

drugs. It is evident that most of the compounds studied here have shown broad spectrum antibacterial

activity which is agreement with earlier study.^[38,39]

Table 1. Minimum inhibitory concentration in μM of the synthesized compounds C1-C27

Comp.

No

Minimum inhibitory concentration (µM)*

^dS.e ^eM. t ^fK. p

112.36±0.89 112.36±0.77 220.84±0.68 220.14±0.47

41.23±0.98 84.08±0.49 167.36±0.89 83.81±0.86

210.84±0.56 104.91±0.49 210.84±0.84 105.92±0.74 210.16±0.47 107.27±0.76 104.24±0.89

^aS.a

^bS. a

^cMRSA

^gP. a

^hE. c

112.72±0.74 55.12±0.68

168.43±0.81 83.54±0.69

220.84±0.39 112.36±0.68

166.02±0.68 83.54±0.98

C1.

C2.

>420

C3.

48.04±0.25

45.01±0.89

24.81±0.76

43.00±0.69

50.58±0.71

91.18±0.49

101.17±0.75

45.11±0.82

199.79±0.49 48.04±0.49

90.89±0.64 45.30±0.69

48.04±0.68

44.15±0.23

98.62±0.68

89.17±0.49

C4.

C5.

218.00±0.76 109.70±0.72 219.75±1.06 217.29±1.19

207.42±0.49 105.53±0.21 207.42±0.89 104.545±0.91

109.35±0.41 219.75±0.23 111.80±0.74 111.80±0.86

C6.

104.87±0.76 105.86±0.93 103. ±0.63

208.41±0.68

91.65±0.59

115.79±0.39

53.94±0.57

24.65±0.89

39.72±0.76

90.89±0.79

45.98±0.78

86.54±1.19

C7.

92.84±0.09

56.97±0.87

52.55±0.76

49.00±0.79

78.43±0.49

92.34±0.67

24.02±0.49

87.38±0.87

46.12±0.76

56.60±0.59

54.29±0.89

49.31±0.76

39.21±0.96

43.85±0.85

44.37±0.47

43.69±0.97

44.64± 0.09 45.82±0.89

93.43±0.45

28.85±0.43

27.49±0.69

47.13±0.80

39.21±0.47

90.89±0.49

46.29±0.85

44.25±0.68

48.18±0.39

53.94±0.09

44.38±0.61

40.52±0.49

45.82±0.95

91.65±0.89

57.71±0.80

45.82±0.60

58.45±0.69

C8.

56.97±0.98

27.14±1.34

47.13±0.88

19.74±0.69

43.56±0.77

48.420.44

115.42±0.80

54.29 ±0.67

47.13±0.59

80.460.68

C9.

108.94±0.85 53.25±0.86

C10.

C11.

C12.

C13.

C14.

C15.

C16.

C17.

C18.

C19.

C20.

C21.

C22.

C23.

98.94±0.69

40.23±0.89

43.85±0.49

47.51±0.09

86.54±0.39

99.87±0.89

53.25±0.79

20.24±0.04

21.19±0.74

93.13±0.43

49.31±0.49

38.20±0.88

45.88±0.47

97.12±0.89

43.96±0.47

22.65±0.99

47.51±0.76

41.74±0.58

101.15±1.21

109.98±0.89

90.20±0.86

85.06±0.75

92.25±0.47

21.70±0.09

25.52±0.88

101.79±0.79 98.92±0.85

101.47±0.34 198.8±0.79

53.25±0.49

44.38±0.85

42.66±0.49

108.59±0.28 53.94±0.71

53.25±0.09

90.20±0.64

41.59±0.90

44.64±0.74

57.74±0.48

218.57±0.89

20.61±0.79

85.06±0.44

22.17±0.88

55.56±0.70

43.24±0.85

40.25±0.74

90.20±0.57

40.52±0.49

92.25±1.14

46.12 ±0.74 44.64±0.94

57.38±1.21

114.04±0.86 114.04±0.74 28.32±0.56

115.49±0.64 56.29±0.89

210.12±0.32 104.44±1.32 208.78±0.71 209.78±0.93

111.52±0.49 110.81±0.92 221.98±1.12 112.22±0.81

53.11±0.89

56.11±0.49

104.55±0.89 207.44±0.73 105.22±0.54

55.76±1.31

110.11±0.49 220.92±0.95

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167.34±0.85 20.75±0.74

106.23±0.59 53.11±0.12

73.56±0.49 37.01±0.92

163.17±0.85 82.49±1.14

20.75±0.89

84.86±0.74

21.81±0.82

51.44±0.52

84.86±0.74

42.30±0.85

41.23±0.75

C24.

C25.

C26.

106.56±0.74 207.78±0.68

144.57±0.85 36.55±0.88

106.56±0.42 106.56±0.09 208.45±0.89

145.03±0.69 73.56±0.88

72.86±0.90

144.80±0.62

160.84±0.76

6.94±0.52

81.19±9.37

95.97±0.78

161.36±0.94

6.33±0.52

162.65±1.02 161.36±0.49 82.49±0.62

C27.

Cipro

floxacin

Genta

micin

6.18±0.82

6.33±0.52

3.62±0.52

6.63±0.76

2.30±0.88

1.62±0.94

4.60±0.83

17.16±0.99

16.95±0.92

41.240.78

17.16±0.98

16.95±0.62

2.09±0.52

*Values are mean ± SEM (n = 3).

Ciprofloxacin and Gentamicin used as positive control

^[a]S. a: Staphylococcus aureus NCIM 5021; ^[b]S. a: Staphylococcus aureus NCIM 5022;^[c]S. a: Methicillin resistant

Staphylococcus aureus ATCC 43300 (MRSA); ^[d]S. e: Staphylococcus epidermidis NCIM 2493; ^[e]M.t. a :Mycobacterium sp

NCIM 2984; ^[f]K. p: Klebsiella pneumoniae NCIM 2706; ^[g]P. a: Pseudomonas aeruginosa NCIM 2036;^[h]E. c: Escherichia

coli NCIM 2065.

MIC: Minimum inhibitory concentration.

MBC of tested compounds is reported in Table 2. It is evident that all ten tested compounds (C4, C5, C9-C11, C14,

C17, C18, C21 and C26) are bactericidal (MBC/MIC ≥4) against S. aureus NCIM 5021. Moreover, all twelve

compounds tested against S. aureus NCIM 5022 showed bactericidal activity (MBC/MIC ≥4). On the other hand,

eight tested compounds (C4, C10, C12, C14, C15, C17, C19 and C24) displayed bactericidal activity (MBC/MIC

≥4) against S. aureus ATCC 43300 (MRSA), while compounds C9 exhibited bacteriostatic activity (MBC/MIC, 8)

against this strain. All six compounds (C5, C12, C13, C17, C18 and C21) tested against S. epidermidis NCIM 2493

were observed to be bactericidal (MBC/MIC ≥4). The results of MBC screening of eight tested compounds (C9-

C11, C14, C18, C19, C23 and C24) against Mycobacterium sp NCIM 2984 indicated the bactericidal activity

(MBC/MIC ≥4). Except compound C5 (MBC/MIC, 8), all other eleven tested compounds C1, C2, C4, C12-15 and

C18-C21 showed bactericidal activity against K. pneumoniae NCIM 2706. Against P. aeruginosa NCIM 2036 two

tested compounds C5 and C21 showed bactericidal activity, while six compounds C8, C10, C12, C17, C19 and

C24 exhibited bacteriostatic activity (MBC/MIC, 8). When tested against E. coli NCIM 2931, all six tested

compounds C10-C12, C14, C20 and C24 displayed bactericidal activity (MBC/MIC ≥4).

Time-kill assay

As expected from the MIC values, tested compounds C10 and C15 displayed significant bactericidal activity

towards S. aureus ATCC 43300 (MRSA) in comparison to the untreated bacteria and bacteria treated with standard

drug ciprofloxacin. The time kill kinetic profiles of C10 and C15 (Figure 2a and 2b) displayed bactericidal activity

with a ≥3log10 reduction in viable cell count at tested concentrations of 13.75 and 27.14 μM in case of C10 and

10.85 and 21.70 μM in case of C15 after 24 h exposure. The killing rate of C24 (Figure 2c) towards S. aureus

NCIM 5022 was similar to that of C10 and C15 observed against S. aureus ATCC 43300. This compound showed

bactericidal activity at concentrations of 10.37 μM and 20.75 μM after 24 h exposure. However, all three tested

compounds exhibited lower bactericidal activity compared to the standard drug ciprofloxacin tested at 47.98 μM and

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Chemistry & Biodiversity

95.97 μM concentrations against S. aureus ATCC 43300 and also against S. aureus NCIM 5022 at 3.16 μM and 6.33

μM concentrations. It is important to note that time kill kinetics of compounds C10, C15 and C24 are consistent

with their respective MIC values.

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Table 2. Minimum bactericidal concentrations (MBC) and MBC/MIC ratio of some selected compounds.

Comp.

C1

MBC^$*&

MBC/MIC

MBC

MBC/MIC --

MBC --

MBC/MIC --

MBC

MBC/MIC

MBC

^[a]S.a

^[b]S. a

^[c]MRSA

^[d]S.e

^[e]M. t

^[f]K. p

^[g]P. a

^[h]E. c

--

111.1±0.87

2

--

220.5±0.85

2

334.7±0.99

4

198.8±0.93

4

357.8±0.91

8

--

C2

100.2±0.47 100.2±0.88

2

90.3±0.47

2

198.8±0.97

4

--

C4

4

90.3±0.59

2

93.2±0.94

2

89.4±0.73

2

--

179.9±0.80

4

367.1±1.04

8

C5

MBC/MIC

MBC

MBC/MIC --

--

C8

MBC

MBC/MIC

MBC

115.6±1.02 116.3±0.92

461.8±0.97

8

108.8±0.94

--

115.9±0.91 --

--

107.5±0.91 --

--

C9

2

4

107.6±0.80 --

433.5±0.68

107.4±0.87

C10

C11

C12

C13

C14

C15

C17

C18

C19

C20

C21

C23

MBC/MIC

MBC

MBC/MIC

MBC

MBC/MIC --

MBC --

MBC/MIC --

MBC

MBC/MIC

MBC

MBC/MIC --

MBC

MBC/MIC

MBC

MBC/MIC

MBC

MBC/MIC --

MBC --

MBC/MIC --

MBC

MBC/MIC

MBC

2

--

4

--

4

--

8

--

2

98.4±0.44

2

--

95.9±0.82

2

77.9±0.81

2

--

96.7±0.67

2

--

89.4±1.21

2

--

91.3±0.79

2

98.1±0.99

2

--

94.1±0.49

2

--

99.9±0.57

4

78.2±0.49

2

--

93.4±0.47

2

--

78.4±0.79

4

--

97.1±0.81

2

88.3±0.91

4

108.5±0.88

2

--

84.6±0.69

2

--

80.9±0.90

2

92.9±1.04

4

--

79.9±0.91

2

268.0±0.93

4

188.4±0.80

4

347.6±0.89

4

313.6±0.85

8

--

434.7±0.76

8

357.7±0.94

4

335.1±0.97

8

--

94.0±0.94

4

--

109.7±0.7

2

90.4±0.94

2

215.2±0.79 --

--

177.4±0.89 89.2±0.90

--

2

89.8±0.86

4

85.2±0.74

4

368.9±0.94

4

227.2±0.88

4

2

--

83.9±0.90

2

--

94.6±0.79

4

--

112.5±0.58 --

2

--

115.3±0.75 --

4

--

224.7±0.78

4

--

221.9±0.79 --

--

MBC/MIC --

4

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Chemistry & Biodiversity

MBC

--

82.3±0.77

4

106.5±0.99

2

81.6±0.94

4

--

84.6±0.78

4

--

331.9±0.82

8

--

82.1±0.67

2

--

C24

C25

C26

MBC/MIC --

MBC --

MBC/MIC --

MBC

144.0±0.98 --

--

MBC/MIC

2

--

Ciprofloxacin MBC

MBC/MIC

2.1±0.45

1

1.9±0.78

<1

2.1±0.89

<1

2.0±0.68

1

2.2±0.83

<1

1.9±0.88

<1

2.0±0.96

<1

2.0±0.86

<1

^$minimum bactericidal concentration (μM).

*Values are mean ± SEM (n = 3); --: not determined.

Ciprofloxacin used as positive control

^[a]S. a: Staphylococcus aureus NCIM 5021; ^[b]S. a: Staphylococcus aureus NCIM 5022;^[c]S. a: Methicillin resistant Staphylococcus aureus ATCC

43300 (MRSA); ^[d]S. e: Staphylococcus epidermidis NCIM 2493; ^[e]M.t. a :Mycobacterium sp NCIM 2984; ^[f]K. p: Klebsiella pneumoniae NCIM 2706;

^[g]P. a: Pseudomonas aeruginosa NCIM 2036;^[h]E. c: Escherichia coli NCIM 2065.

MIC: Minimum inhibitory concentration (μM).

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Figure 2. Time kill curves of C10 and C15 against S. aureus ATCC 43300 and C24 against S. aureus NCIM 5022.

Computational study

The ATP dependent bacterial DNA GryB, a type II topoisomerase is actively involved in introducing

negative supercoils into DNA.^[40]It is vital for the biosynthesis of peptidoglyan, an essential component of bacterial

cells. This enzyme is ubiquitous in bacteria but has not been found in humans, making it an attractive target for the

investigation of new antibacterial agents.^[41]Coumarins like novobiocin and naturally occurring cyclothialidines

binds to GyrB and competitively inhibit the binding of ATP.^[42,43]Based on these observations and activity of

synthesized compounds against S. aureus we investigated the binding modes of these compounds in the catalytic

pocket of S. aureus GyrB by extra-precision docking.

Docking studies revealed that compounds C1-27 occupied the same site of catalytic pocket of 3U2K as

co-crystal structure and exhibited hydrogen bonding, π-π stacking, π-cation and salt bridge interactions with the key

binging residues of catalytic pocket (Table 3, Figure 2a-c also Supplementary Figure S28 and Table S1). The high

active compounds C10 (G_score-5.72 kcal/mol), C15 (G_score-5.82 kcal/mol) and C24 (G_score-5.89 kcal/mol) well

occupied the catalytic pocket and exhibited interactions mainly with key binding residues of the N-terminal and

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central domains (Figure 2a-c). Precisely, carbonyl oxygen of the hydrazide function in compound C10 accepted two

hydrogen bonds one each from Gly85 and Thr173. One of nitrogen atom of NH-NH fragment established hydrogen

bonding interaction with charged residue Asp81. This compound is further stabilized in the catalytic pocket by π-

cation interaction of thiazole part of the benzothiazole ring with protonated NH₂of Arg84. In case of compound C15

carbonyl oxygen of the hydrazide function exhibited similar hydrogen bonding interaction with Gly85 and Thr173

as observed in compound C10. Nitrogen atom of one the nitro group established salt bridge interaction with charged

residue Glu58, while oxygen atom another nitro group exhibited coordination bond with Mg234. Like C10, this

compound is also stabilized in the catalytic pocket by π-cation interaction of phenyl ring of 3,5-dinitropheyl moiety

with Glu84. Like C10 and C15, compound C15 displayed similar hydrogen bonding interactions with Gly85 and

Thr173 and π-cation interaction with Arg84. Additionally, in this compound oxygen atom of butoxy group present

on the phenyl ring displayed hydrogen-bonding interaction with Asn54. In general other compounds also displayed

hydrogen bonding interactions with Asp81, Gly85 and Thr173; π-cation interactions with Gly 85 and Arg84; salt

bridge interactions with Gly85. It is evident from above results that apart from hydrogen bonding, π-cation and salt

bridge interactions also play crucial role for the stabilization of compounds C1-C27 within the catalytic pocket.

Table 3. Extra-precision docking result and contribution of binding free energy (MM-GBSA) (kcal/mol) between

synthesized compounds C1-C27 in the catalytic pocket of S. aureus GyrB enzyme (pdb.3U2K).

[a]

Comp.

g

score

^[b]ΔG_Bind^[c]ΔG_Coul^[d]ΔG_Cov^[e]ΔG_HB^[f]ΔG_Lipo

^[g]ΔG_Solv^[h]ΔG_vdW

^[i]ΔG_packing

-5.75

-4.35

-5.09

-5.91

-5.10

-4.86

-5.87

-6.09

-4.73

-5.72

-5.15

-5.45

-5.72

-5.29

-5.82

-5.66

-4.78

-5.06

-5.40

-4.85

-4.26

-6.30

-5.12

-5.89

-4.44

-5.72

-4.72

-57.26

-46.51

-67.30

-50.37

-54.02

-36.81

-41.28

-57.03

-48.23

-71.99

-54.56

-55.74

-62.14

-42.85

-67.87

-59.42

-36.12

-48.63

-54.43

-48.71

-51.18

-59.19

-44.19

-71.28

-54.51

-45.91

-42.3

-34.01

-25.59

-33.88

-41.61

-17.75

-63.55

-72.8

-33.45

-32.06

-29.43

-17.96

-22.92

-31.03

-25.91

-45.31

-36.8

-1.72

-12.45

-2.22

0.13

-8.64

-3.14

-7.45

-11.16

-7.71

-2.80

-5.16

-5.22

0.75

-1.75

1.76

-11.93

-14.33

-13.7

-11.96

-10.48

-5.46

-4.10

-12.85

-9.91

33.86

41.25

19.82

47.61

28.71

77.49

84.86

34.51

38.84

33.34

26.7

24.11

24.98

44.89

43.8

26.56

47.4

23.05

31.82

39.66

26.88

29.52

21.54

21.07

27.13

43.50

26.88

33.86

41.25

19.82

47.61

28.71

77.49

84.86

34.51

38.84

33.34

26.70

24.11

24.98

44.89

43.80

26.56

47.40

23.05

31.82

39.66

26.88

29.52

21.54

21.07

27.13

43.50

26.88

-2.43

-3.29

-1.84

-4.34

-4.58

-3.38

-2.96

-3.50

-5.35

-4.02

-6.41

-3.52

-2.89

-4.71

-2.10

-1.32

-5.68

-4.90

-3.70

-4.04

-4.70

-4.48

-3.26

-6.56

-4.88

-1.83

-5.18

C1

C2

C3

C4

C5

C6

C7

C8

-0.73

-2.55

-1.49

-0.56

-2.01

-0.84

-1.09

-1.27

-0.27

-1.30

0.04

-0.12

-2.16

1.55

-0.91

-0.02

0.48

-2.05

-1.8

-2.33

1.88

-2.54

-1.16

2.64

C9

-11.98

-7.56

C10

C11

C12

C13

C14

C15

C16

C17

C18

C19

C20

C21

C22

C23

C24

C25

C26

C27

-11.61

-13.59

-12.34

-13.73

-12.72

-9.00

-12.03

-17.23

-12.99

-10.37

-10.97

-9.77

-4.08

0.42

-4.85

-5.06

-5.54

-0.81

-1.33

-3.17

-4.5

-13.77

6.91

-5.09

-1.53

-0.70

-37.62

-7.26

-44.64

-30.07

-22.42

-27.1

-3.55

-22.64

-20.85

-29.51

-11.43

-19.21

-12.36

-17.38

-15.55

1.29

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^[a]glide score; ^[b]free energy of binding; ^[c]Coulomb energy; ^[d]covalent energy (internal energy); ^[e]hydrogen bonding

energy; ^[f]hydrophobic energy (non-polar contribution estimated by solvent accessible surface area); ^[g]electrostatic

solvation energy; ^[h]van der Waals energy; ^[i]packing energy.

Binding free energy (ΔG_bind) of compounds C1-27/3U2K ranges between -36.12 to -71.99 kcal/mol

showing high binding affinity for most compounds with S. aureus GyrB (Table 3). Among all compounds C10, C15

and C24 exhibited high negative value of ΔG_bind(-71.99, -67.87 and -71.28 kcal/mol, respectively) which is

observed to be in correlation with MIC values against S. aureus ATCC 43300. It is evident from the computed

energy components that Coulomb (ΔG_Coul) and hydrophobic (ΔG_Lipo) energy terms are favourable for the binding of

ligands to S. aureus GyrB, whereas electrostatic solvation (ΔG_Solv, 19.82 to 84.86 kcal/mol) and van der Waals

(ΔG_vdW, 19.82 to 77.49 kcal/mol) energy terms strongly disfavours the binding. Except for compounds C5, C11,

C18, C23-C25 and C27 (ΔG_Coul, -3.55 to -22.64 kcal/mol), Coulomb energy (ΔG_Coul, -3.55 to -17.96 kcal/mol) term

is observed to be strongly favourable for the binding of all other compounds. The high negative value in these

compounds indicated it as the driving force for binding. In most of the compounds covalent energy (-0.70 to -13.77

kcal/mol) term is observed to either weakly unfavourable or moderately unfavourable for the binding.

b

a

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Chemistry & Biodiversity

c

Figure 3 Plot represent interaction of compounds (a) C10 (b) C15 and (c) C24 with the catalytic pocket residues of

S. aureus GyrB (pdb.3U2K).

Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties prediction

ADMET properties were computed to assess the pharmacokinetic and safety profiles of synthesized compounds C1-

C27. It is evident from supplementary Table S2 that none of the synthesized molecules exhibited blockage of human

ether-a-go-go (HERG) K⁺channels and obeyed Lipinski’s rule of five (0 to 1) indicating drug-like property of these

molecules. These compounds are observed to be non toxic as predicted by the AMES test. Predicted Caco-2 cell

permeability (a model for the gut-blood barrier) for C1-C27 is within the range 0.22 to 1.43 cm/sec showing the

non-active transport of these compounds. Total polar surface area (TPSA) of compounds ranges between 82.2 to

173.9 Å²and are well within the recommended range (7 to 200 Å²). The predicted central nervous system (CNS)

permeability (-1.34 to -2.07) is well within the recommended range of -2 to +2. The human intestinal absorption

ranges between 88.54 to 97.88% indicating the favourable kinetic profile of these compounds. All compounds are

observed to be inhibitors of some of the Cytochrome P450s. In addition, all synthesized compounds exhibit zero

Pains alert (PA), indicating absence of PAN assay interference structure.

Conclusions

In the present work we synthesized new benzothiazole containing aryl and alkaryl hydrazides C1-C27. All

compounds were characterized by spectral data and screened for their antibacterial activity. Compounds C10, C15

and C24 showed high activity against S. aureus ATCC 43300 (MRSA) and also exhibited bactericidal activity

against this strain in MBC determination. In time kill kinetics C10 and C15 displayed bactericidal activity towards

S. aureus ATCC 43300 (MRSA), respectively at concentrations of 13.75 and 10.85 μM after 24 h exposure.

Compound C24 exhibited bactericidal activity towards S. aureus NCIM 5022 at concentrations of 10.37 μM after 24

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h exposure. Among all tested compounds, C20 and C24 showed maximum activity, respectively against

Mycobacterium sp NCIM 2984 (MICs, 21.81 μM) and E. coli NCIM 2065 (MIC, 22.17 μM). On the other hand,

tested compounds were observed to less active against P. aeruginosa NCIM 2036. In extra-precision docking, C1-

C27 showed interactions mainly with the N-terminal and central domains of the catalytic pocket. Components of

binding free energy calculation revealed Coulomb energy term strongly favourable for binding of most of the

compounds with S. aureus GyrB. In ADMET prediction all compounds obeyed Lipinski’s rule of five. Results show

that compound C24 exhibited promising activity against S. aureus NCIM 5022 (MIC, 20.75 μM), S. aureus ATCC

43300 (MIC, 20.75 μM) and Mycobacterium sp NCIM 2984 (MIC, 21.81 μM). This compound also exhibited

bactericidal activity against S. aureus NCIM 5022 (MBC/MIC, 4). In addition, the binding free energy of compound

C24 as computed by the MM-GBSA approach is -71.28 kcal/mol, indicating high binding affinity of this compound

towards S. aureus GyrB protein. Further, we can see from Figure 3c that n-butyl fragment of n-butoxy group is

located near to the hydrophobic pocket. This n-butyl fragment may further be exchanged with other hydrophobic

groups to increase the hydrophobic interactions and binding affinity towards S. aureus GyrB. Hence chemical

structure of his compound may further be optimized to increase the antibacterial activity of this compound against S.

aureus and also against Mycobacterium sp.

Experimental

Material and Methods

All the glass wares were oven dried prior to use. The purification and drying of solvents were carried wherever

required. All reactions were examined by thin layer chromatography (TLC) using pre-coated aluminum back thin

layer chromatography (TLC) Silica Gel F254 plates (Merck, Ltd., Germany). All the chemicals and solvents were

purchased from Sigma Aldrich, Merck, Finar, Alfa aesar and LOBA Chemie. The melting point of the synthesized

compounds was determined using Veego VMP-1 melting point apparatus expressed in °C and are uncorrected.

Fourier transform infrared (FT-IR) spectroscopy was performed either by Shimadzu or Perkin Elmer-Spectrum Two

1

spectrophotometers. H and ¹³C-NMR spectra were recorded either in deuterated chloroform (CDCl₃) or dimethyl

sulfoxide ((D₆)DMSO) at 300, 400 or 500 and 101 or 125 MHz, respectively using Bruker AV-III 400 spectrometer

(Germany). Chemical shifts were recorded in ppm using the solvent as internal standard. High resolution mass

spectra (HRMS) were recorded using Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometer (USA)

with positive electrospray ionization (ESI) mode at 70 ev.

2-Hydrazino-1,3-benzothiazole (A) was synthesized from 2-mercaptobenzothizole (A) following the procedure

described in the literature (m.p. 192 ⁰C, lit. 189-193 ⁰C).^[35]

General procedure for the synthesis of N'-(1,3-benzothiazol-2-yl)-substituted aryl/aralkyl hydrazides (C1-

C27)

2-Hydrazino-1,3-benzothiazole (A) (0.242 g, 0.0014 mol) and hydroxybenzotriazole (HOBt) (0.450 g, 0.0029 mol)

was successively added to the corresponding substituted benzoic acid (0.2 g, 0.0014 mol) in N,N-

dimethylformamide (DMF) (15 mL). The mixture was cooled to 0 °C in an ice bath with stirring and then 1-ethyl-3-

(3-dimethylaminopropyl)carbodiimde hydrochloride (EDCl) (0.563 gm, 0.00294 mol) was added. The reaction

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mixture was then slowly allowed to reach the room temperature over 1 h and then stirring was further continued at

this temperature till completion of reaction. Progress of reaction was monitored with TLC using n-

hexane:ethylacetate (1:4) as eluent. The reaction was quenched by saturated NaHCO₃solution and then extracted

with EtOAc (20 mL x 3). The organic layer was dried with anhydrous Na₂SO₄, filtered and concentrated under

reduced pressure. The residue thus obtained was purified by column chromatography on silica gel Merck (100-200

mesh) in glass columns (2 or 3 cm diameter) using 25-30 grams of silica gel per one gram of the residue. The elution

of column was started with n-hexane and then eluent polarity was gradually increased with ethyl acetate Compounds

C_1-27thus obtained in yield of 78-95%.

Synthetic route for compounds C1-C27 is presented in Scheme 1. Characterization data of compounds C1-C27 is

given in supplementary.

Antibacterial activity assay

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The newly synthesized compounds C1-C27 were screened against seven bacterial strains, Staphyloccocus aureus

NCIM 5021, Staphyloccocus aureus NCIM 5022, methicillin resistant Staphyloccocus aureus ATCC 43300 (MRSA

strain), Staphylococcus epidermidis NCIM 2493, Mycobacterium tuberculosis NCIM 2984, Klebsiella pneumoniae

NCIM 2706, Pseudomonas aeruginosa NCIM 2036 and Escherichia coli NCIM 2065 following the guidelines of

Clinical Laboratories Standard Institute (CLSI 2007)^[37]. The tests were performed in Mueller Hinton medium (Hi-

media) by broth microdilution method, in 96-well microtiter plates. Compounds C1-C27 was dissolved in sterile

dimethyl sulfoxide (DMSO) to screen their antibacterial activity. Ciprofloxacin and gentamicin were dissolved in

sterile DMSO and used as positive control, while sterile DMSO served as a negative control. The final volume for

MIC protocols was 100 μL, whereas DMSO concentration in assays well was less than 1%. Synthesized compounds

were tested in the concentration range of 19.74 to 221.98 μM. The bacterial suspensions at 10⁵Colony Forming

Unit/mL (CFU/mL) concentrations were inoculated to the corresponding wells. Following inoculation 96-well

microtitre plates was incubation at 37 °C for 24 h. Plates were then agitated and read for the absorbance at a

wavelength of 600 nm. All tests were performed in triplicate and the results were taken as a mean. After MIC

determination, 50 μL aliquot from each well was sub-cultured on Mueller-Hinton agar plates and were further

incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration of the tested compound responsible

for 99.9% reduction in bacterial viable count. All experiments were repeated in triplicate for each strain. The ratio

MBC/MIC ≤2 indicates bactericidal activity, while MBC/MIC ratio ≥4 shows bacteriostatic activity of test

compound. MIC and MBC values (μM) of the tested compounds are presented in Table 1 and Table 2, respectively.

Time-kill assay

Compounds C10, C15 and C24 exhibited promising MIC values against S. aureus ATCC 43300. Compound C24

also exhibited high activity against S. aureus NCIM 5022. These three compounds are selected for further

antibacterial evaluation by the broth macro-dilution method following the guidelines of Clinical Laboratories

Standard Institute (CLSI 2007).^[37]Inoculum suspension of S. aureus NCIM 5022 S. aureus ATCC 43300 and was

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serially diluted to achieve a concentration of 10⁵CFU/mL and treated with respective test compounds (C10, C15

and C24) respectively at 13.57, 10.85 and 10.37 μM, and also at 27.14, 21.70 and 20.75 μM concentrations.

Ciprofloxacin was used as positive control against S. aureus ATCC 43300 at 47.98 μM and 95.97 μM

concentrations and against S. aureus NCIM 5022 at 3.16 μM and 6.33 μM concentrations. The inoculum cultures

were incubated at 37 °C and 50 μL from the corresponding cultures were collected at timed intervals (0, 2, 4, 6, 8,

12, and 24 h) and sub-cultured on nutrient agar medium. After incubation at 37 °C for 24 h CFUs were determined.

Data were analyzed by plotting the log₁₀CFU per millilitre versus time (h). A reduction of ≥3 log₁₀cfu/mL

compared to the initial inoculums is defined as the bactericidal activity, whereas <3 log₁₀cfu/mL decrease compared

to the initial inoculum corresponds to bacteriostatic activity.

Computational Studies

Molecular docking and binding free energy calculation

It was of particular interest to investigate the mechanism by which title compounds exerted their antibacterial

activities. Grid-based extra-precision glide docking was performed for compounds C1-C12 in the catalytic pocket of

S. aureus GyrB. For this purpose 3D-crystal structure of S. aureus GyrB 24-kDa N-terminal domain (pdb.3U2K,

resolution 1.64 Å)^[44]was retrieved from the protein data bank and prepared by the Protein Preparation Wizard^[45]

using prime^[46]and OPLS3e force field.^[47]Around centre of the co-crystal ligand a 10 Å glide grid was generated.

Synthesized compounds C1-C27 structures were sketched with the builder panel of Maestro version 12.0 and

optimized with LigPrep module using OPLS3e force field. Low energy conformations of all compounds were

docked into the prepared catalytic pocket of 3U2K in extra-precision mode using Glide^[48]without applying any

constraint. Further, binding free energy (ΔG_bind) of docked complexes was computed by the MM-GBSA approach

using VSGB 2.0 energy model^[49]and OPLS3e force field.

Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties prediction

ADMET properties of compounds play vital roles in design and development of drug. ADMET properties of the

synthesized compounds were computed by SwissADME and (http://www.swissadme.ch/) and pkCSM

(http://biosig.unimelb.edu.au/pkcsm/prediction) online tools. ADMET properties, principle descriptors of

compounds C1-C27 including hERG inhibition potential, AMES toxicity, Lipinski number of violations and

PAINS number of alerts were taken into consideration (Supplementary Table S2) to assess the acceptability for

rational drug design.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

One of the authors would like to thank All India Council for Technical Education (AICTE) Quality Improvement

Programme for providing fellowship.

Authors Contribution

18

10.1002/cbdv.202100117

Chemistry & Biodiversity

Swarupa Rani gurram was the author, who synthesized the literature and drafted the paper. Dr. Md. Afzal Azam

provided conceptual inputs and revised the manuscript. All authors read and approved the final paper.

References

[1] C. L. Ventola, The antibiotic resistance crisis Part 1: Causes and Threats. Pharm. Ther. 2015, 40, 277-283.

[2] Friedman ND, Temkin E, Carmeli Y. The negative impact of antibiotic resistance. Clin. Microbiol. Infect.

2016, 22, 416-422 https://doi.org/10.1016/j.cmi.2015.12.002

[3] S. S. Panda, M.A. Ibrahim, A. A. Oliferenko, A.M. Asiri, A. R. Katritzky, Catalyst-free facile synthesis of 2-

substituted benzothiazoles, Green Chem. 2013, 15, 2709-2712. https://doi.org/10.1039/C3GC41255E

[4]. P. C. Sharma, A. Sinhmar, A. Sharma, H. Rajak, D. P. Pathak, Medicinal significance of benzothiazole

scaffold: an insight view, J. Enz. Inhib. Med. Chem. 2013, 28, 240-266. https://doi.org/10.3109/147563

66.2012.720572

[5]. Y. Riadi, R. Mamouni, R. Azzalou, M. E. Haddadc, S. R. G. Guillaumetd, S. Lazara, An efficient and reusable

heterogeneous catalyst animal bone meal for facile synthesis of benzimidazoles, benzoxazoles, and benzothiazoles,

Tetrahedron. Lett. 2011, 52, 3492-3495. https://doi.org/10.1016/j.tetlet.2011.04.121

[6]. K. Bahrami, M. M. Khodaei, F. Naali, Mild and highly efficient method for the synthesis of 2-

arylbenzimidazoles and 2-arylbenzothiazoles, J. Org. Chem. 2008, 73, 6835-6837. https://doi.org/10.1021/jo80

10232

[7]. P. Raje, W. E. Klunk, C. A. Mathis, Jr., Y. Wang, Benzothiazole derivative compounds, compositions and

uses. US Patent 8691185 B2, 2014, Application No. US 13/548014, July 12, 2012. https://patents.google.com/

patent/US8236282B2

[8]. R. Dahl, A. Y. Lee, C. Kho, R. J. Hajjar, Benzothiazole or benzoxazole compounds as sumo activators.

WO2014036242 A3, July 7, 2015, Publication No. PCT/US2013/057264, Aug’ 29, 2013.

[9]. W. E. Klunk, C. A. Mathis, J. Y. Wang, Benzothiazole derivative compounds, compositions and uses, US

Patent 20130004422 A1. April 8, 2013, Application No. US13/548,014, July 12, 2012.

[10]. J. C. Sutton, M. Wiesmann, W. Wang, M. Lindvall, J. Lan, et al., 6-O-substituted benzoxazole and

benzothiazole compounds and methods of inhibiting CSF-1R signalling, US Patent 7553854 B2, June 30, 2009.

Application No.: US11/737069, April 18, 2007.

[11]. M. Bryson, B. Fulton, P. Benfield, Riluzole a review of its pharmacodynamic and pharmacokinetic properties

and therapeutic potential in amyotrophic lateral sclerosis. Drugs 1996, 52, 549-563.

https://doi.org/10.2165/00003495-199652040-00010

[12]. S. Shafi, M. M. Alam, N. Mulakayala, C. Mulakayala, G. Vanaja, A. M. Kalle, R. Pallu, M. S. Alam,

Synthesis of novel 2-mercaptobenzothiazole and 1,2,3-triazole based bis-heterocycles: Their anti-inflammatory

and anti-nociceptive activities. Eur. J. Med. Chem. 2012, 49, 324-333. https://doi.org/10.1016/j.ejmech.2012

.01.032

19

10.1002/cbdv.202100117

Chemistry & Biodiversity

[13]. Y. Liu, Y. Wang, G. Dong, Y. Zhang, S. Wu, et al., Novel benzothiazole derivatives with a broad antifungal

spectrum: design, synthesis and structure–activity relationships. Med. Chem. Commun. 2013, 4, 1551-1561.

https://doi.org/10.1039/C3MD00215B

[14]. M. E. Mats, B. G. Shani, L. Pasternak, et al., Synthesis and mechanism of hypoglycemic activity of

benzothiazole derivatives. J. Med. Chem. 2013, 56, 5335–5350. https://doi.org/10.1021/jm4001488

[15]. M. I. Hussain, V. Kumar, Synthesis and studies of (2-enzothiazolyl/benzimidazolyl/ benzoxazolylthio)-acetic

acid[(3-pyridyl)[substitutedphenyl)azo]methylenehydrazides as possible anthelmintics. Indian J. Chem. 1993, 32B,

905-907. https://doi.org/10.1016/j.ejmech.2012.03.033

[16]. L. W. Yurttas, K. B. Cavusoglu, A. Sever, G. A. Ciftci, A preliminary investigation of anticanceractivity of

novel benzothiazole derivatives against A549 lung carcinoma cell line, Turk J. Biochem. 2017, 42, 535-544.

https://doi.org/10.1515/tjb-2017-0015

[17]. A. Ammazzalorso, G. A. Antonella, A. D’Angelo, B. De Filippis, M. Fantacuzzi, et al., Benzothiazole-based

N-(phenylsulfonyl)amides as a novel family of PPARα antagonists, Bioorg. Med. Chem. Lett. 2011, 21, 4869-

4872. https://doi.org/10.1016/j.bmcl.2011.06.028

[18]. S. K. De, L. H. Chen, J. L. Stebbins, T. Machleidt, M. Riel-Mehan, et al., Discovery of 2-(5-nitrothiazol-2-

ylthio) benzo[d]thiazoles as novel c-Jun N-terminal kinase inhibitors. Bioorg. Med. Chem. 2009, 17, 2712–2717.

https://doi.org/ 10.1016/j.bmc.2009.02.046

[19]. V. Asati, N. K. Sahu, A. Rathore, S. Sahu, D.V. Kohli, Synthesis, characterization and antimicrobial

evaluation of some 1,3-benzothiazole-2-yl-hydrazone derivatives, Arabian J. Chem. 2015, 8, 495-499.

https://doi.org /10.1016/j.arabjc.2011.01.036

[20]. M. T. Gabr, N. S. El-Gohary, E. R. El-Bendary, M. M. El-Kerdawy, N. Ni, M. I. Shaaban, Synthesis,

antimicrobial, antiquorum-sensing and cytotoxic activities of new series of benzothiazole derivatives, Chinese

Chem. Lett. 2015, 26, 1522-1528. https://doi.org/10.1016/j.cclet.2015.09.004

[21]. K. O. Badahdah, H. M. Abdel Hamid, S. A. Noureddin, Functionalized 2-hydrazinobenzothiazole with isatin

and some carbohydrates under conventional and ultrasound methods and their biological activities, J. Het. Chem.

2015, 52, 67-74. https://doi.org/10.1002/jhet.1986

[22]. V. N. Telvekar, V. K. Bairwa, K. Satardekar, A. Bellubi, “Novel 2-(2-(4-aryloxybenzylidene)

hydrazinyl)benzothiazole derivatives as anti-tubercular agents,” Bioorg. Med. Chem. Lett. 2012, 22, 649-652.

https://doi.org/10.1016/j.bmcl.2011.10.064

[23]. T. Karabasanagouda, A. V. Adhikari, N. S. Shetty, Synthesis and antimicrobial activities of some novel

1,3,4-oxadiazoles carrying alkylthio and alkylsulphonyl phenoxy moieties, Phosphorus, Sulfur Silicon Rel. Elem.

2007, 182, 2925-2941. https://doi.org/10.1080/10426500701542981

[24]. H. A. Abuelizz, R. A. El-Dib, M. Marzouk, R. Al-Salahi, In vitro evaluation of new 2-phenoxy-

benzo[g][1,2,4]triazolo[1,5-a]quinazoline derivatives as antimicrobial agents, Microb. Pathogen. 2018, 117, 60-

67. https://doi.org/10.1016/j.micpath.2018.02.018

20

10.1002/cbdv.202100117

Chemistry & Biodiversity

[25]. U. A. More, S. D. Joshi, T. M. Aminabhavi, V. H. Kulkarni, A. M. Badiger, C. Lherbet, Discovery of target

based novel pyrrolyl phenoxy derivatives as antimycobacterial agents: An in silico approach, Eur. J. Med. Chem.

2015, 94, 317-339. https://doi.org/10.1016/j.ejmech.2015.03.013

[26]. S. Gaonkar, K. L. Rai, B. Prabhuswamy, 'Synthesis of novel 3-[5-ethyl-2-(2-phenoxy-ethyl)-pyridin]-5-

substituted isoxazoline libraries via 1,3-dipolar cycloaddition and evaluation of antimicrobial activities', Med.

Chem. Res. 2007, 15, 407-417. https://doi.org/10.1007/s00044-006-0015-z

[27]. G. F. Makhaeva, N. V. Kovaleva, E.V. Rudakova, N. P. Boltneva, S. V. Lushchekina, et al., New

multifunctional agents based on conjugates of 4-amino-2,3-polymethylenequinoline and butylated hydroxytoluene

for alzheimer's disease treatment, Molecules 2020, 25, 5891. https://doi.org/10.3390/molecules25245891

[28]. V. S. Gontijo, F. P. D. Viegas, C. J. C. Ortiz, M. de Freitas Silva, C. M. Damasio et al., Molecular

hybridization as a tool in the design of multi-target directed drug candidates for neurodegenerative diseases. Curr.

Neuropharmacol. 2020, 18, 348-407. https://doi.org/10.2174/1385272823666191021124443

[29]. A. R. Das, A. Medda, R. Singha, A. Samanta, N. Guchhait, Synthesis of novel 3-substituted coumarin

carboxamides with biological interest and their spectral studies, J. Indian Chem. Soc. 2008, 85, 1124-1129.

https://doi.org/ 10.1002/chin.200925125

[30]. Y. Han, L. M. Xu, C. Y. Nie, S. Jiang, J. Sun, C. G. Yan, Synthesis of diamido-bridged bis-pillar[5]arenes

and tris-pillar[5]arenes for construction of unique [1]rotaxanes and bis-[1]rotaxanes, Beilstein J. Org. Chem. 2018,

14, 1660-1667. https://doi.org/10.3762/bjoc.14.142.

[31]. L. L. Zhao, Y. Han, C. G. Yan, Construction of [1]rotaxanes with pillar[5]arene as the wheel and terpyridine

as the stopper, Chinese Chem. Lett. 2020, 31, 81-83. https://doi.org/10.1016/j.cclet.2019.04.024

[32]. R. S. Joshi, P. G. Mandhane, P. V. Badadhe, H. Charansingh, C. H. Gill, Development of practical

methodologies for the synthesis of novel3(4-oxo-4H-chromen-3-yl)acrylic acid hydrazides. Ultrason. Sonochem.

2011, 18, 735-738 https://doi.org/10.1016/j.ultsonch.2010.11.001.

[33]. D. Cai, Z. H. Zhang, Y. Meng, K. L. Zhu, L. Y. Chen, C. X. Yu, C. W. Yu, Z. Y. Fu, D. S. Yang, Y. X.

Gong, Efficient synthesis of piperazinyl amides of 18β-glycyrrhetinic acid. Beilstein J. Org. Chem. 2020, 16, 798-

808. https://doi.org/10.1016/10.3762/bjoc.16.73

[34]. N. Sanz Del Olmo, C. E. Pena González, J. D. Rojas, R. Gomez, P. Ortega, A. Escarpa, F. J. de la Mata,

Antioxidant and antibacterial properties of carbosilane dendrimers functionalized with polyphenolic moieties.

Pharmaceutics. 2020, 12, 698. https://doi.org/10.3390/pharmaceutics12080698

[35]. A. F. Halasa, Hydrazinium salts of mercaptobenzothazole, US Patent 3,860,602A, Jan’ 14, 1975, Application

No. 159464, July 2, 1971.

[36]. F. Larsen, T. Ulven, Efficient synthesis of 4,7-diamino substituted 1,10-phenanthroline-2,9-dicarboxamides,

Org. Lett. 2011, 13, 3546-3548. https://doi.org/10.1021/ol201321z

[37]. Clinical and Laboratory Standard Institute (CLSI) Methods for dilution antibacterial susceptibility test for

bacteria that grow aerobically, 7th ed. Approved Standard (MA7-A7), Clinical and Laboratory Standard Institute:

Wayne, 2007, 27, pp. 13.

21

10.1002/cbdv.202100117

Chemistry & Biodiversity

[38]. M. Saini, P. Kumar, M. Kumar^,K. Ramasamy, V. Mani,^,R. K. Mishra, A. Bakar, A. Majeed, B. Narasimhan,

Synthesis, in vitro antimicrobial, anticancer evaluation and QSAR studies of N′-(substituted)-4-(butan-2-

lideneamino) benzohydrazides. Arabian J. Chem. 2014, 7, 448-460 https://doi.org/10.1016/j.arabjc.2013.05.010

[39]. S. Saidugari;L. Rao, K. Vidya, B. Ram, B. Balram, Synthesis, characterization and antibacterial activity of

(E)-4-((3-methyl-4-(methylsulfonyl)pyridin-2-yl)methoxy)-N'-(substitutedbenzylidene)benzohydrazide

derivatives. Indian J. Chem. 2017, 56B:177-182.

[40]. J. J. Champoux, DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001, 70,

369-413 10.1146/annurev.biochem.70.1.369 https://doi.org/10.1007/s00253-011-3557-z

[41]. F. Collin, S. Karkare, A. Maxwell, Exploiting bacterial DNA gyrase as a drug target: current state and

perspectives. Appl. Microbiol. Biotechnol. 2011, 92, 479–497 https://doi.org/10.1007/s00253-011-3557-z

[42]. E. J. Gilbert, A. Maxwell, The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin

drugs. Mol. Microbiol. 1994, 12, 365–373. https://doi.org/10.1111/j.1365-2958.1994.tb01026.x

[43]. R. J. Lewis, O. M. Singh, C. V. Smith, T. Skarzynski, A. Maxwell, A. J. Wonacott, D. B. Wigley, The nature

of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography. EMBO

J. 1996, 15, 1412-1420

[44]. E. Eakin, O. Green, N. Hales, G. K. Walkup, S. Bist et al., Pyrrolamide DNA gyrase inhibitors: fragment-

based nuclear magnetic resonance screening to identify antibacterial agents. Antimicrob. Agents Chemother. 2012,

56, 1240-1246. https://doi.org/10.1128/AAC.05485-11

[45]. G. M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju, W. Sherman, Protein and ligand preparation:

parameters, protocols, and influence on virtual screening enrichments, J. Comput. Aid. Mol. Des. 2013, 27, 221-

234. https://doi.org/10.1007/s10822-013-9644-8

[46]. M. P. Jacobson, D. L. Pincus, C. S. Rapp, T. J. Day, B. Honig, D. E. Shaw, R. A. Friesner, A hierarchical

approach to all-atom protein loop prediction, Proteins 2004, 55, 351-367. https://doi.org/10.1002/prot.10613

[47]. K. Roos, C. Wu, W. Damm, M. Reboul, J. M. Stevenson et al., OPLS3e: extending force field coverage for

drug-like small molecules, J. Chem. Theo. Comput. 2019, 15, 1863-1874. https://doi.org/10.1021/acs.jctc.8b01026

[48]. R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren et al., Glide: a new approach for rapid, accurate

docking and scoring. 1. Method and assessment of docking accuracy, J. Med. Chem. 2004, 47, 1739-1749.

https://doi.org/10.1021/jm0306430

[49]. J. Li, R. Abel, K. Zhu, Y. Cao, S. Zhao, R. A. Friesner, The VSGB 2.0 Model: a next generation energy

model

for

high resolution protein

structure

modeling,

Proteins

2011, 79, 2794-2812.

https://doi.org/10.1002/prot.23106

22

10.1002/cbdv.202100117

Chemistry & Biodiversity

Graphical Abstract

N'-(1,3-benzothiazol-2-yl)-substituted benzohydrazides were synthesized by direct coupling of 2-hydrazino-1,3-

benzothiazole with different aromatic carboxylic acids under catalysis of HOBt/EDCl in DMF.

23

Article Doi

Design, Synthesis, Antibacterial Evaluation and Molecular Docking Studies of Some Newer Baenzothiazole Containing Aryl and Alkaryl Hydrazides

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