Synthesis, biological evaluation and docking studies of some novel isatin-3-hydrazonothiazolines

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Synthesis, biological evaluation and docking studies of some novel isatin-3-

hydrazonothiazolines

a

b

a

Maqbool Ahmad , Humayun Pervez *, Sumera Zaib , Muhammad Yaqub , Muhammad Moazzam

c

b

Naseer , Shafi Ullah Khan , Jamshed Iqbal *

a

Institute of Chemical Sciences, Organic Chemistry Division, Bahauddin Zakariya University,

Multan 60800, Pakistan

b

Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad

2

2060, Pakistan

c

Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

Eꢀmail addresses: pdhpervez@hotmail.com (H. Pervez); drjamshed@ciit.net.pk (J. Iqbal)

Abstract

New series of thirty nine 5ꢀtrifluoromethoxy/fluoro/chloroꢀisatin 3ꢀhydrazonothiazolines 5a-n, 6a-o

4

and 7a-j were synthesized by cyclization of the corresponding intermediate N ꢀarylꢀsubstituted

4

isatinꢀ3ꢀthiosemicarbazones 3 (prepared by condensation of appropriate isatin 1 with appropriate N ꢀ

arylꢀsubstituted 3ꢀthiosemicarbazides 2) with 4ꢀchlorophenacyl bromide 4 in absolute ethanol or

ethanolꢀbenzene mixture and screened for their cytotoxicity, phytotoxicity, antifungal and urease

inhibitory potential. All the synthesized compounds were found to be almost inactive in the brine

ꢀ4

shrimp (Artemia salina) bioassay, demonstrating IC50 values >1.62 x 10 − 2.17 x 10 M. In

phytotoxicity assay, out of thirty nine compounds tested, six i.e. 5i, 6h, 6i, 6k, 7c and 7h proved to

be active, showing weak or nonꢀsignificant (5ꢀ30%) activity at the highest tested concentration (500

µg/mL). Similarly, in antifungal assay, twenty six compounds i.e. 5a, 5b, 5d-f, 5hꢀj, 5m, 6a, 6b, 6d,

6

j, 6lꢀo, 7a, 7b and 7d-j were found to be active against one, two, or three selected fungal strains,

exhibiting weak or nonꢀsignificant inhibition (10ꢀ30%). Of these, 6d and 6o displayed relatively

better activity profile in terms of the number of organisms inhibited. On the other hand, in urease

inhibition bioassay, all the synthesized hydrazonothiazolines proved to be potent enzyme inhibitors,

demonstrating inhibitory activity with IC50 values ranging from 3.70 ± 0.62 to 849 ± 2.26 ꢁM.

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Compounds 5c, 5g-i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be

relatively very much potent, displaying outstanding enzymatic activity (IC50 = 3.70 ± 0.62 – 20.9 ±

0

.57 ꢁM) even better than the reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM) and may thus act

as valid leads for further studies. Molecular docking studies of the synthesized isatinꢀthiazolines 5a-

n, 6a-0 and 7a-j were also carried out to elucidate their relationship with the binding pockets of the

enzyme. This study offers the first example of exhibition of urease inhibitory potential by isatinꢀ

thiazolines and as such provides a solid basis for further research on these compounds to develop

more potent antiurease compounds of medicinal /agricultural interest.

Keywords: Cytotoxicity, Hydrazones, Hydrazonothiazolines, Isatin, Phytotoxicity, Thiazoline,

Urease inhibition, Urease inhibitors

Introduction

The literature survey indicates that isatin and its derivatives possess a wide spectrum of biological

1ꢀ20

properties.

Among isatin derivatives, isatinꢀthiosemicarbazones are reported to possess diverse

1

1,7

pharmacological activities, including antiulcer,

analgesic and antiꢀinflammatory,

1

,4,7,12,21,22

1,2,3,5,7,22

23ꢀ25

1,2,5,7,12,25

anticancer,

antimicrobial,

antituberculosis,

antiviral,

and enzymatic

1

,4,11,17

inhibition.

Stimulated by this and as a part of our research work aiming to the synthesis of

isatinꢀbased organic compounds of medicinal/agricultural interest, we recently reported the synthesis

4

of a number of 5ꢀ(un)ꢀN ꢀarylꢀsubstituted isainꢀ3ꢀthiosemicarbazones as antimicrobial, cytotoxic,

2

6ꢀ33

phytotoxic and more importantly antiurease compounds.

Investigation of the structureꢀactivity

relationship (SAR) studies in the synthesized compounds revealed that in certain cases, the type and

4

position of different substituents about the phenyl ring attached to N of the thiosemicarbazone

2

6,27,29

part

positionꢀ5 of the isatin moiety

different activities. Moreover, it has been reported by some other authors that certain 5ꢀbromoisatinꢀ

and/or the presence of an inductively electronꢀwithdrawing group (NO

2

, F

3

CO, F, or Cl) at

28,30ꢀ33

played a key role in the inducement and/ or increment of

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3

ꢀhydrazonothiazolines, prepared by cyclization of the respective thiosemicarbazones with phenacyl

bromides, exhibited high cytotoxicity at low concentrations when tested for antiviral activity against

MDCK cells. The presence of more than one lipophilic functions in these compounds was considered

34

responsible for such

a

display of activity.

Likewise, 5ꢀbromo isatinꢀderived 3ꢀ

hydrazonothiazolidinones, obtained by cyclization of the corresponding thiosemicarbazones with

21

ethyl 2ꢀbromopropionate, have been found to show favourable cytotoxicity. Also, certain 5ꢀ

fluoroisatinꢀderived 3ꢀhydrazonothiazolidinones, obtained by condensation of 5ꢀfluoroisatin with

35

different thiazolidinꢀ4ꢀones, have been claimed to possess antimicrobial activity. In view of these

points, it was envisioned that the simultaneous presence of different lipophilic groups at positionꢀ5 of

the isatin scaffold and at the N atom of thiazoline moiety in the isatinꢀthiazoline hybrids would result

in an increase in the selected activities. Thus, the present work to synthesize three new series of

4

thirty nine title hydrazonothiazolines (derived by cyclization of avariety of N ꢀarylꢀsubstituted 5ꢀ

trifluoromethoxy/fluoro/chloroꢀisatin 3ꢀthiosemicarbazones with 4ꢀchlorophenacyl bromide) and

screen them for their cytotoxicity, phytotoxicity, antifungal and specially urease inhibitory potential

was accomplished. It is pertinent to mention here that such a category of isatin derivatives has been

scarcely investigated before by other researchers for their antiurease potential. The work presented

herein illustrates the influences of the type of aryl groups (modified by placing one, two, or three

functionalities on the phenyl ring) attached to N atom of the thiazoline moiety as well as the presence

of inductively electronꢀattracting/lipophilic trifluoromethoxy, fluoro and chloro groups at positionꢀ5

of the isatin part on the cytotoxic, phytotoxic, antifungal and urease inhibitory properties of the

synthsized isatinꢀthiazoline hybrids. This study furnishes some motivating and attentionꢀgrabbing

results, which are reported herein.

Results and discussion

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The present study describes the synthesis and in vitro evaluation of cytotoxic, phytotoxic, antifungal

3

6,37

and urease inhibitory potential of thirty seven currently and two previously reported

isatinꢀ

hydrazonothiazolines 5a-n, 6a-d, 6f-h, 6j-o, 7a-j and 6e, 6i, respectively.

Chemistry of compounds 5a-n, 6a-o and 7a-j

The designed isatinꢀhydrazonothiazolines 5-7 were prepared in 73ꢀ91% yield from 5ꢀsubstituted

4

isatins 1 by first reacting them with equimolar quantities of appropriate N ꢀsubstituted 3ꢀ

thiosemicarbazides 2 in aqueous ethanol containing a catalytic amount of glacial acetic acid, and

then by treating the resultant intermediate thiosemicarbazones 3 with 4ꢀchlorophenacyl bromide 4 in

absolute ethanol or ethanolꢀbenzene mixture at reflux temperature (Scheme 1).

The structures of the synthesized isatinꢀ3-hydrazonothiazolines 5a-n, 6a-o and 7a-j were established

1

on the basis of elemental (CHN) and spectral (IR, HNMR, EIMS) analyses. Satisfactory elemental

analyses were obtained for all the compounds synthesized in this study. Also, the spectral data were

21,34,38

in ageement with the relevant literature.

The IR spectra of isatinꢀthiazolines 5a-n, 6a-o and 7a-j

ꢀ1

showed a single band resulting from NH stretching of indole in the 3261ꢀ3008 cm region. The

ꢀ1

lactam C=O and azomethine C=N were observed in the 1739ꢀ1693 and 1631ꢀ1558 cm regions,

2

1,34,38

1

respectively.

of thiosemicarbazone N ꢀH and N ꢀH,and displayed one or two separate singlets at δ 10.39ꢀ10.75

and δ 10.35ꢀ10.77, respectively, for indole NH. The thiazoline ring C ꢀH resonated either as a singlet

The HꢀNMR spectra of 5a-n, 6a-o and 7a-j did not show the down field signals

4

2

5

21,34,38

at δ 7.01ꢀ7.22 or two separate singlets at δ 6.90ꢀ7.12 or a multiplet at δ 6.95ꢀ7.13.

Furthermore,

1

the HꢀNMR spectra of compounds 6c, 6d, 6f, 6o, 7a, 7c, 7d and 7f-j exhibited duplicate signals,

confirming the presence of two isomers. It is suggested that in these compounds, the restricted

rotation about the azomethine (C=N) linkage and the partial double bond character of the lactam CꢀN

bond, induced by delocalization of the nitrogen lone pair of electrons onto the carbonyl oxygen, led

21,38

to the formation of E and Z isomers.

The EI mass spectra of 5a-n, 6a-o and 7a-j showed

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molecular ions of different intensity peculiar to the isatin and thiazoline moieties. The key

38

fragmentation pathways involved the NꢀN, NꢀC, and NꢀCO bonds fission. The proposed

fragmentation patterns of compounds 5c, 6g and 7f, representing each series of isatinꢀthiazoline

hybrids, are illustrated in Figures 1, 2 and 3, respectively. Xꢀray structures of two representative

examples 6e and 6i were determined in order to prove the allocated structures and to substantiate

conformations of the synthesized isatinꢀhydrazonothiazolines. The related crystallographic data and

3

6,37

refinement details of 6e and 6i have been reported somewhere else.

R₁

NH

S

R₁

O

NNH

C

R

NH

S

EtOHꢀHOAc

Reflux, 2h

O

H NHN C

O

2

N

H

1

2

3

Cl

^R1

Cl

N

S

N N

C

O

CH Br

2

R

4

O

N

EtOH (abs.)

Reflux, 12ꢀ15h

or

H

5-7

EtOHꢀBenzene

Reflux, 12ꢀ15h

5

a

b

c

d

e

f

R=OCF

3

; R

1

=2ꢀCH

=3ꢀCH

3

5n

6a

6b

6c

6d

6e

6f

R=OCF

3

; R

1

=2,4,6ꢀ(Cl)

3

6m

6n

60

7a

7b

7c

R=F ; R

1

=2,5ꢀ(Cl)

2

5

1

R=F ; R

1

=2ꢀCH

=3ꢀCH

=4ꢀCH

3

=2,6ꢀ(Cl)

2

=4ꢀ CH

3

=2,4,6ꢀ(Cl)

3

=2ꢀO CH

3

R=Cl ; R

1

=2ꢀCH

=3ꢀCH

=4ꢀCH

3

=3ꢀOCH

=4ꢀOCH

=2ꢀF

3

=2ꢀOCH

=3ꢀOCH

=4ꢀOCH

3

g

7d

=2ꢀOCH

3

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5

h

R=OCF

3

; R

1

=3ꢀF

6g

6h

6i

R=F ; R

1

=2ꢀF

7e

7f

7g

7h

7i

R=Cl ; R

1

=3ꢀOCH

=4ꢀOCH

=2ꢀF

3

i

=4ꢀF

=3ꢀF

3

j

=2,4ꢀ(Cl)

=2,5ꢀ(Cl)

=2,6ꢀ(Cl)

=3,4ꢀ(Cl)

2

=4ꢀF

k

l

6j

=2,4ꢀ(F)

2

=3ꢀF

6k

6l

=2,6ꢀ(F)

=4ꢀF

m

=2,4ꢀ(Cl)

2

7j

=2,4ꢀ(F)

2

Scheme 1. Synthesis of title hydrazonothiazolines 5a-n, 6a-o and 7a-j.

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CH₃

N

S

N

S

Cl

^F3^C^O

N N

O

N

ꢀ

CO

^F3^C^O

H

NH

m/z 500

m/z 528 (M⁺)

N

H

CH₃

N

F₃CO

N

S

Cl

O

N

H

m/z 229

m/z 299

ꢀ

^N2

F₃CO

CH₃

N

NH

H

m/z 201

N

S

Cl

H

CH₃

m/z 286

^F3^C^O

ꢀH

O

N

C S

N

m/z 149

H

m/z 215

CH₃

ꢀCO

F₃CO

NH

S

N

CH

Cl

m/z 118

m/z 187

ꢀ

HCN

Cl

ꢀHNC

m/z168

ꢀHC C

Cl

CH₃

^F3^C^O

HC C

m/z 136

m/z 160

m/z 111

m/z 91

Figure 1. The proposed fragmentation pattern of compound 5c.

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F

NH

N

F

O

F

N

H

m/z 136

H

m/z 163

N

Cl

N

S

m/z 303

F

N

Cl

N

N N

O

Cl

F

ꢀ

CO

N

S

H

S

NH

N

H

+

m/z 438

Cl

m/z 466

H

(

M )

ꢀ

N₂

F

N

S

F

Cl

H

O

m/z 290

N

H

ꢀH

CH

C

m/z 149

m/z 136

2

H

ꢀ

HC C

ꢀ

HNCO

F

ꢀ

CO

Cl

F

N

C S

m/z 108

m/z 153

m/z 111

F

NH

ꢀHCN

m/z 121

F

ꢀHNC

CH

N

m/z 95

ꢀ

F

m/z 122

F

S

Cl

m/z 76

m/z 168

m/z 94

Figure 2. The proposed fragmentation pattern of compound 6g.

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OCH₃

Cl

NH

N

Cl

O

N

H

m/z 152

H

m/z 179

N

Cl

N

S

OCH₃

m/z 315

N

S

Cl

N

S

N N

O

Cl

ꢀ

CO

N

H

^O^C^H3

NH

_m_/_z₄₉₄₍_M+₎

m/z 466

N

H

ꢀN₂

H

OCH

3

N

S

ꢀH

Cl

H

O

m/z 302

N

S

ꢀ

N

C S

H

^O^C^H3

m/z 165

OCH₃

ꢀ

HC C

Cl

ꢀ

CO

CH

N

m/z 136

N

m/z 244

S

Cl

ꢀ HC C

NH

ꢀ

m/z 168

m/z 137

Cl

ꢀHNC

Cl

m/z 111

OCH₃

Cl

ꢀHCN

ꢀHCHO

HC

N

m/z 110

m/z 134

m/z 107

m/z 77

Figure 3. The proposed fragmentation pattern of compound 7f.

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Biology

Cytotoxicity (in vitro) of compounds 5a-n, 6a-o and 7a-j

The synthesized isatinꢀ3-hydrazonothiazolines 5a-n, 6a-o and 7a-j were screened for their cytotoxic

effects by a brine shrimp (Artemia salina) lethality bioassay using etoposide, a standard anticancer

drug as a reference point for comparison to the trial compounds. All the compounds of this series

ꢀ4

gave LD50 values >1.62 x 10 − 2.17 x 10 M in the present assay when tested at 100, 10 and 1

µg/mL concentrations and thus can be considered to be almost inactive.

Phytotoxicity (in vitro) of compounds 5a-n, 6a-o and 7a-j

The synthetic compounds 5a-n, 6a-o and 7a-j were further screened for their phytotoxicity potential

at 500, 50 and 5 µg/mL concentrations. Out of thirty nine compounds tested, only six i.e. 5i, 6h, 6i,

6

k, 7c and 7h appeared to be active, exhibiting weak or nonꢀsignificant (5ꢀ30%) plant growth

inhibition at the highest tested concentration (500 µg/mL) compared to the standard drug, paraquat,

which displayed 100% inhibition at 0.015 µg/mL concentration (Table 1).

Table 1. Growth inhibition of Lemna aequinocitalis by compound 5a-n, 6a-o and

,

7

a-j* ** at different concentrations

Cl

R₁

N

S

R

O

N

H

Compound

R

1

% Growth inhibition

5

00 µg/mL

05

50 µg/mL

00

5 µg/mL

00

5i

OCF

3

4ꢀF

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6

h

F

3ꢀF

05

15

18

30

00

6i

4ꢀF

k

F

2,6ꢀ(F)

2

7

c

Cl

4ꢀCH

3ꢀF

3

7

h

*The reference compound, paraquat, shows 100% growth inhibition at 0.015 µg/mL

Antifungal activity (in vitro) of compounds 5a-n, 6a-o and 7a-j

Antifungal activity of the synthesized isatinꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j was

determined against five fungal cultures i.e. Candida albicans, Aspergillus flavus, Microsporum

canis, Fusarium solani and Candida glabrata at 200 µg /mL in DMSO (Table 2). Of the thirty nine

compounds tested, twenty six i.e. 5a, 5b, 5d-f, 5hꢀj, 5m, 6a, 6b, 6d, 6j, 6lꢀo, 7a, 7b and 7d-j were

found to be active against one, two, or three selected pathogens, displaying varied but low or nonꢀ

significant inhibition (10ꢀ30%). Among these, 6d and 6o bearing fluoro group at positionꢀ5 of the

isatin scaffold and methoxy and chloro functions, respectively, at positionsꢀ2 ( ortho) and ꢀ2,4,6 (

ortho, para, ortho) of the phenyl ring attached to N atom of the thiazoline moiety exhibited relatively

better activity profile in terms of the number of organisms affected.

,

Table 2. Antifungal activity (in vitro) of compounds 5a-n, 6a-o and 7a-j* ** (% inhibition)

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Cl

R₁

N

S

R

O

N

H

Microbial species

C. albicans A. flavus M. canis F. solani C. glabrata

Compound

R

R₁

5

a

OCF

F

Cl

3

2ꢀCH

3ꢀCH

2ꢀOCH

3ꢀOCH

4ꢀOCH

3ꢀF

4ꢀF

2,4ꢀ(Cl)

3,4ꢀ(Cl)

3

00

A

00

20

00

20

00

10

00

20

00

B

20

00

20

00

25

00

20

10

30

10

30

20

00

20

25

20

25

00

C

00

25

10

00

10

00

30

25

10

25

00

20

00

25

00

20

D

00

E

5

b

d

3

5

e

f

5

h

5

i

j

2

5m

2

6

a

b

d

2ꢀCH

3ꢀCH

3

6

3

2ꢀOCH

2,4ꢀ(F)

2

2,4ꢀ(Cl)

2,5ꢀ(Cl)

2,6ꢀ(Cl)

3

6

j

l

2

6m

6

n

6

7

o

a

2,4,6ꢀ(Cl)

3

2ꢀCH

3ꢀCH

3

7

b

d

3

2ꢀOCH

3ꢀOCH

4ꢀOCH

2ꢀF

3ꢀF

4ꢀF

2,4ꢀ(F)

2

3

7

e

f

7

g

7

h

7

i

j

Standard

Drug**

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*

Concentration used 200 µg/mL; ** Standard drugs (MIC in µg/mL): A = Miconazole (110.8

µg/mL); B= Amphotericin B (20 µg/mL); C = Miconazole (98.4 µg/mL); D = Miconazole (73.25

µg/mL); E= Miconazole (110.8 µg/mL); 00: absence of measurable inhibitory action

Urease inhibition (in vitro) of compounds 5a-n, 6a-o and 7a-j

The synthesized 5ꢀtrifluoromethoxy/fluoro/chloroꢀisatin 3ꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j,

respectively, were tested for their antiurease activity against Jack bean urease. Thiourea served as

reference standard in this assay. All the compounds proved to be potent inhibitors of the enzyme,

exhibiting inhibitory activity with IC50 values of 3.70 ± 0.62 – 849 ± 2.26 ꢁM. Compounds 5c, 5g-i,

5

k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be relatively very much

potent, displaying excellent activity (IC50 values 3.70 ± 0.62 – 20.9 ± 0.57 ꢁM) even better than the

reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM) and may thus act as compelling leads for further

studies. The remaining compounds i.e. 5a, 5b, 5d-f, 5j, 5l, 5m, 6a, 6d-h, 6j, 6m, 7b-d and 7f-h

showed a varying degree of activity with IC50 values of 24.6 ± 2.14 – 849 ± 2.26 ꢁM (Table 3).

The structureꢀactivity relationship (SAR) studies in the case of 5ꢀtrifluoromethoxyisatin derivatives

5

aꢀn revealed that compound 5k having chloro substituents at positionsꢀ2 and ꢀ5 of the phenyl ring

attached to N atom of the thiazoline moiety was the most potent urease inhibitor of the series,

demonstrating several fold more activity (IC50 = 3.70 ± 0.62 ꢁM) than the reference inhibitor,

thiourea (IC50 = 22.3 ± 1.12 ꢁM). Comparison of the urease inhibitory potential of compound 5k

with that of closely related dichloroꢀsubstituted compounds 5j, 5l and 5m indicated that chloro

substitution at positionsꢀ2,5 (ortho ,meta) of the phenyl ring was more favourable than at positionsꢀ

2

,4 (ortho, para), ꢀ2,6 (ortho, ortho) and ꢀ3,4 (meta, para), respectively. The dichloro–substituted

derivatives 5j, 5l and 5m displayed relatively much lower activity (IC50 = 44.5 ± 3.15, 129 ± 5.91

and 134 ± 2.21 ꢁM, respectively) in the present assay. This clearly showed that compound 5k

compared to 5j, 5l and 5m interfered with the enzyme in a different fashion. Interestingly, the only

trichloro–substituted compound tested in this assay i.e. 5n bearing the substituents at positionsꢀ2,4,6

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(

ortho, para, ortho) of the phenyl ring demonstrated much higher activity in contrast to the dichloroꢀ

substituted compounds 5j and 5l with the substituents at positionsꢀ2,4 (ortho, para) and ꢀ2,6 (ortho,

ortho) of the phenyl ring, respectively (IC50 value 14.1 ± 1.31 vs. 44.5 ± 3.15 and 129 ± 5.91 ꢁM).

Chlorine is both electronꢀdonating by mesomeric effect (+M) and electronꢀwithdrawing by inductive

effect (ꢀI). Much higher enzyme inhibitory activity presented by compound 5n in the present assay

clearly indicated that the ultimate or overall electronꢀwithdrawing effect of the three chloro functions

increased its inhibitory potential, though not exclusively. The next most potent urease inhibitor was

compound 5g having a fluoro group at positionꢀ2 of the phenyl ring, showing inhibitory activity with

IC value of 7.27 ± 0.33 ꢁM. This compound was found to be three fold more active than the

5

0

reference inhibitor, thiourea, but two fold less active than the most potent urease inhibitor 5k. The

other highly active monofluoroꢀsubstituted derivatives 5h and 5i, possessing the substituent at

positionsꢀ3 and ꢀ4 of the phenyl ring exhibited enzymatic inhibition with IC50 values of 12.0 ± 0.83

and 11.7 ± 0.07 ꢁM, respectively. This clearly indicated that compound 5g in comparison to 5h and

5

i meddled with the enzyme differently and more competently. The remaining relatively very much

potent urease inhibitor was compound 5c with a methyl substituent at positionꢀ4 of the phenyl ring

attached to N atom of the thiazoline moiety. This compound was found to be two fold more active

than the reference inhibitor (thiourea) but three times less active than the most potent urease inhibitor

of the series i.e. compound 5k (IC50 value 10.3 ± 0.51 vs. 22.3 ± 1.12 and 3.70 ± 0.62 ꢁM,

respectively). The other methylꢀsubstituted compounds 5a and 5b bearing the substituent at

positionsꢀ2 and ꢀ3 of the phenyl ring displayed relatively much lower inhibitory activity (IC values

50

3

36 ± 5.45 and 427 ± 2.89 ꢁM, respectively). These results showed that steric hindrance played a

significant role in reducing the inhibitory potential of the compounds.

Among 5ꢀfluoroisatin derivatives 6aꢀo, compounds 6i and 6l having fluoro and chloro substituents at

positionsꢀ4 (para) and ꢀ2,4 (ortho, para) of the phenyl ring attached to N atom of the thiazoline

moieties were the most potent urease inhibitors of the series, presenting excellent and almost the

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same inhibitory activity (IC50 values 8.20 ± 0.01 and 8.40 ± 0.23 ꢁM, respectively). Comparison of

the urease inhibitory potential of compound 6i with that of closely related fluoroꢀsubstituted

derivatives 6g and 6h showed that fluoro substitution at positionꢀ4 of the phenyl ring was more

favourable than at positionsꢀ2 and ꢀ3. The fluoroꢀsubstituted compounds 6g and 6h exhibited

relatively much lower activity (IC₅₀values 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM, respectively) in the

present assay. Also, comparison of the antiurease potential of compound 6l with that of 6m and 6n

indicated that chloro substitution at positionsꢀ2,4 (ortho, para) was more favourable than at ꢀ2,5

(ortho, meta) and ꢀ2,6 (ortho, ortho). The dichloroꢀsubstituted derivative 6n compared to 6l

displayed lower but still exciting activity (IC₅₀value 12.7 ± 0.62 vs. 8.40 ± 0.23 ꢁM), while 6m

demonstrated relatively much lower activity with IC₅₀value of 33.1 ± 2.88 ꢁM. Similarly, the

trichloroꢀsubstituted compound 6o having the chloro substituents at positionsꢀ2,4,6 (ortho, para,

ortho) of the phenyl ring exhibited lower but still stimulating inhibitory activity (IC50 = 14.3 ± 0.12

ꢁM) in comparison to the corresponding dichloroꢀsubstituted compounds 6l and 6n, showing enzyme

inhibition with IC₅₀values of 8.40 ± 0.23 and 12.7 ± 0.62 ꢁM, respectively. In case of difluoroꢀ

substituted compounds tested in this assay, compound 6k bearing the fluoro substituents at positionsꢀ

2

,6 (ortho, ortho) of the phenyl ring was found to show much higher inhibitory potential compared to

compound 6j having the substituents at positionsꢀ2,4 (ortho, para) (IC50 value 11.0 ± 0.86 vs. 66.6 ±

.40 ꢁM). This indicated that compound 6k in contrast to 6j intermingled with the enzyme in a

5

different manner, resulting into much marked enhancement in enzyme inhibitory potential. Amongst

methylꢀsubstituted compounds 6a-6c, compound 6c possessing the substituent at positionsꢀ4 of the

phenyl ring was found to be the most active one, displaying enzyme inhibition with IC50 value of

1

1.4 ± 1.51 ꢁM. The next most active derivative was 6b with the substituent at positionꢀ3 of the ring,

which showed inhibitory potential with IC50 value of 15.0 ± 1.92 ꢁM. The remainder derivative 6a

having the substituent at positionꢀ2 of the phenyl ring exhibited much lower inhibitory activity with

IC value of 275 ± 4.99 ꢁM. This indicated that in case of 6a, steric hindrance played a pivotal role

5

0

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in decreasing the inhibitory potential of the compound. The above results demonstrated that

compounds 6g and 6h in contrast to 6i, and 6m in comparison to 6l, 6n and 6o, interacted with the

enzyme differently, resulting into reduction in the inhibitory potential to a smaller or greater extent.

The results given in the Table revealed that compared to compounds 5a-n having trifluoromethoxy

substituent at positionꢀ5 of the isatin part, substitution of fluoro group at the same position in the case

of 6a-o caused either a decrement or an increment in the enzymatic activity in certain cases. For

example, compound 6c having a methyl substituent at positionꢀ4 of the phenyl ring attached to N

atom of the thiazoline moiety showed inhibition of the enzyme with IC50 value of 11.4 ± 1.51 ꢁM,

whereas the respective compound 5c bearing trifluoromethoxy function at positionꢀ5 of the isatin

moiety displayed more inhibitory activity (IC50 = 10.3 ± 0.51 ꢁM). Similarly, compounds 6f-h

possessing methoxy and fluoro substituents at positions ꢀ4 and ꢀ2, ꢀ3 of the phenyl ring, respectively,

were found to demonstrate reduced activity (IC50 values 849 ± 2.26, 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM)

in comparison to the corresponding 5ꢀtrifluoromethoxyisatin derivatives 5f-h, which exhibited

inhibitory activity with IC₅₀values of 102 ± 5.82, 7.27 ± 0.33 and 12.0 ± 0.83 ꢁM, respectively.

Also, compound 6m possessing chloro functions at positions ꢀ2 and ꢀ5 of the phenyl ring was found

to show decreased activity (IC50 = 33.1 ± 2.88 ꢁM) when compared with the respective compound

5

k having trifluoromethoxy substituent at positionꢀ5 of the isatin moiety, which displayed enzyme

inhibition with IC50 value 3.70 ± 0.62 ꢁM. Much marked decrement in enzyme inhibitory activity

was found to occur in case of compounds 6g, 6h and 6m, respectively, when compared with the

corresponding 5ꢀtrifuoromethoxyisatinꢀderived hydrazonothiazolines 5g, 5h and 5k (IC50 values 7.27

±

0.33 → 26.1 ± 1.11 ꢁM, 12.0 ± 0.83 → 32.6 ± 3.42 ꢁM and 3.70 ± 0.62 → 33.1 ± 2.88 ꢁM,

respectively). On the contrary, compounds 6a and 6b bearing methyl function at positions ꢀ2 and ꢀ3

of the phenyl ring, respectively, displayed increased inhibitory activity (IC50 values 275 ± 4.99 and

1

5.0 ± 1.92 ꢁM, respectively) in contrast to the respective compounds 5a and 5b, showing enzyme

inhibition with IC50 values of 336 ± 5.45 and 427 ± 2.89 ꢁM. Similarly, compounds 6d and 6e

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having methoxy substituent at positionsꢀ2 and ꢀ3 of the phenyl ring displayed increased enzyme

inhibition with IC50 values 40.1 ± 5.32 and 29.3 ± 1.17 ꢁM, respectively, when compared with the

corresponding compounds 5d and 5e bearing trifluoromethoxy group at positionꢀ5 of the isatin

scaffold, demonstrating inhibition of the enzyme with IC values of 42.9 ± 4.67 and 42.6 ± 1.40 ꢁM.

50

Furthermore, the monofluoroꢀsubstituted derivative 6i possessing the substituent at positionꢀ4 of the

phenyl ring showed enhanced enzyme inhibition with IC50 value of 8.20 ± 0.01 ꢁM in comparison to

the corresponding 5ꢀtrifluoromethoxyisatinꢀderived hydrazonothiazoline 5i, which displayed

inhibitory activity with IC50 value of 11.7 ± 0.07 ꢁM. Also, the dichloroꢀsubstituted compounds 6l

and 6n having the substituents at positionsꢀ2,4 (ortho, para) and ꢀ2,6 (ortho, ortho) exhibited

enhanced enzyme inhibition (IC values 8.40 ± 0.23 and 12.7 ± 0.62 ꢁM, respectively) in contrast to

50

the respective compounds 5j and 5l bearing trifluoromethoxy residue at positionꢀ5 of the isatin

moiety, demonstrating inhibitory activity with IC50 values of 44.5 ± 3.15 and 129 ± 5.91 ꢁM.

Relatively, marked increment in enzyme inhibitory activity (IC50 values 427 ± 2.89 → 15.0 ± 1.92

ꢁM, 44.5 ± 3.15 → 8.40 ± 0.23 ꢁM and 129 ± 5.91 → 12.7 ± 0.62 ꢁM, respectively) occurred in case

of compounds 6b, 6l and 6n. Among the remaining compounds of the series i.e. 6j and 6k,

compound 6j having fluoro substituents at positionsꢀ2 and ꢀ4 of the phenyl ring exhibited inhibitory

activity with IC50 value of 66.6 ± 5.40 ꢁM. In contrast, 6k possessing the same substituents at

positions ꢀ2 and ꢀ6 of the phenyl ring showed much enhanced activity (IC50 = 11.0 ± 0.86 ꢁM).

These results showed that the presence of fluoro group (exercising both –ve inductive and +ve

mesomeric effects) at positionꢀ5 of the isatin scaffold as well as the nature, number and position of

different functions present in the phenyl ring caused the molecules to meddle with the enzyme in a

different fashion.

In case of 5ꢀchloroisatinꢀderived hydrazonothiazolines 7a-j, 7a bearing methyl group at positionꢀ2 of

the phenyl ring attached to N atom of the thiazoline moiety was found to be the most potent

antiurease compound, showing enzymatic activity (IC50 = 9.20 ± 0.77 ꢁM) much higher than the

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reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM). Comparison of the antiurease activity of

compound 7a with that of closely related methylꢀsubstituted compounds 7b and 7c having the

substituent at positionsꢀ3 and ꢀ4 of the phenyl ring, respectively, revealed that methyl substitution at

positionꢀ2 of the phenyl ring was more favourable than at ꢀ3 and ꢀ4. Compound 7b was found to be >

two fold less active than 7a but slightly more active than 7c (IC value 24.6 ± 2.14 vs. 9.20 ± 0.77

50

and 25.3 ± 3.20 ꢁM, respectively). This showed that compounds 7b and 7c compared to 7a interfered

with the enzyme much less efficiently, resulting in a decrement in their inhibitory potential. The next

most potent antiurease compound was 7e possessing methoxy substituent at positionꢀ3 of the phenyl

ring. This compound exhibited slightly less inhibitory activity (IC =10.3 ± 0.38 ꢁM) than the most

50

potent inhibitor 7a (IC = 9.20 ± 0.77 ꢁM) but much more than the reference inhibitor, thiourea

5

0

(IC50 = 22.3 ± 1.12 ꢁM). The other potent methoxyꢀsubstituted compounds 7d and 7f having the

substituent at positionꢀ2 and ꢀ4 of the phenyl ring attached to N atom of the thiazoline moiety,

however, displayed relatively much lower activity with IC50 values of 38.6 ± 1.50 ꢁM and 81.3 ±

7

.99 ꢁM, respectively. This clearly demonstrated that compound 7e intermingled with the enzyme

differently and much more competently. The remaining relatively much more potent urease inhibitor

was compound 7i bearing a fluoro substituent at positionꢀ4 of the phenyl ring. This compound was

found to be slightly less active than the most potent urease inhibitor of the series i.e. compound 7a

but two fold more active than the reference inhibitor, thiourea (IC50 value 11.3 ± 0.32 vs. 9.20 ± 0.77

and 22.3 ± 1.12 ꢁM, respectively). The other fluoroꢀsubstituted compound 7g possessing the

substituent at positionꢀ2 of the phenyl ring

Table 3. Inhibition of Jack been urease by compounds 5a-n, 6a-o and 7a-j

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Cl

R₁

N

S

R

O

N

H

Compound

R

1

IC50 ± SEM

µM)

Compound

R

1

IC50 ± SEM

(µM)

(

5

a

OCF

F

3

2ꢀCH

3ꢀCH

4ꢀCH

3

336 ± 5.45

427 ± 2.89

10.3 ± 0.51

42.9 ± 4.67

42.6 ± 1.40

102 ± 5.82

7.27 ± 0.33

12.0 ± 0.83

11.7 ± 0.07

44.5 ± 3.15

3.70 ± 0.62

129 ± 5.91

134 ± 2.21

14.1 ± 1.31

275 ± 4.99

15.0 ± 1.92

11.4 ± 1.51

40.1 ± 5.32

29.3 ± 1.17

849 ± 2.26

6g

F

2ꢀF

3ꢀF

26.1 ± 1.11

32.6 ± 3.42

8.20 ± 0.01

5

b

3

6h

5

c

6i

F

4ꢀF

5

d

2ꢀOCH

3ꢀOCH

4ꢀOCH

2ꢀF

3

6j

F

2,4ꢀ(F)

2,6ꢀ(F)

2

66.6 ± 5.40

11.0 ± 0.86

8.40 ± 0.23

33.1 ± 2.88

12.7 ± 0.62

14.3 ± 0.12

9.20 ± 0.77

24.6 ± 2.14

25.3 ± 3.20

38.6 ± 1.50

10.3 ± 0.38

81.3 ± 7.99

25.6 ± 2.91

32.0 ± 0.69

11.3 ± 0.32

20.9 ± 0.57

22.3 ± 1.12

5

e

6k

F

5

f

6l

F

2,4ꢀ(Cl)

2,5ꢀ(Cl)

2,6ꢀ(Cl)

2

5

g

6m

F

5

h

3ꢀF

6n

F

5

i

4ꢀF

6o

F

2,4,6ꢀ(Cl)

3

5

j

2,4ꢀ(Cl)

2,5ꢀ(Cl)

2,6ꢀ(Cl)

3,4ꢀ(Cl)

2

7a

Cl

2ꢀCH

3ꢀCH

4ꢀCH

3

5

k

7b

5

l

7c

5

m

7d

2ꢀOCH

3ꢀOCH

4ꢀOCH

2ꢀF

3

5

n

2,4,6ꢀ(Cl)

3

7e

6

a

2ꢀCH

3ꢀCH

4ꢀCH

3

7f

6

b

F

7g

6

c

F

7h

3ꢀF

6

d

F

2ꢀOCH

3ꢀOCH

4ꢀOCH

3

7i

7j

4ꢀF

6

e

F

2,4ꢀ(F)

2

6

f

F

Thiourea*

*

Reference inhibitor of urease enzyme

showed inhibitory activity with IC value of 25.6 ± 2.91 ꢁM. On the contrary, compound 7h with

50

the fluoro substituent at positionꢀ3 of the phenyl ring displayed reduced enzymatic activity with IC50

value of 32.0 ± 0.69 ꢁM. Finally, the only difluoroꢀsubstituted compound tested in the present assay

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i.e. 7j having fluoro substituents at positionsꢀ2,4 (ortho, para) of the phenyl ring, though found to be

markedly less active than the monofluoroꢀsubstituted compound 7i (20.9 ± 0.57 vs. 11.3 ± 0.32 ꢁM),

but was still more active than the monofluoroꢀsubstituted compound 7g (IC50 = 25.6 ± 2.91 ꢁM) as

well as the reference inhibitor, thiourea (IC = 22.3 ± 1.12 ꢁM). Fluorine is both strongly electronꢀ

5

0

attracting by inductive effect (ꢀI) and electronꢀdonating by mesomeric effect (+M). Noticeably higher

inhibitory activity shown by compounds 7g, 7i and 7j compared to 7h in the present assay is

attributed, though not exclusively, to the overall or ultimate electronꢀdonating influences of the

fluoro functions present at positionꢀ2 (ortho) and ꢀ4 (para) of the phenyl ring attached to N atom of

the thiazoline moiety.

The results given in the Table further revealed that compared with the 5ꢀfluoroisatinꢀderived

thiazolines 6a-o, thiazolines of this series exhibited either increased or decreased enzyme inhibition

in certain cases. For example, compound 7a possessing a methyl group at positionꢀ2 of the phenyl

ring attached to N atom of the thiazoline moiety showed inhibitory activity with IC50 value 9.20 ±

0

.77 ꢁM, whereas the respective compound 6a displayed enzymatic inhibition with IC value of 275

50

±

4.99 ꢁM. Similarly, compounds 7d-f bearing methoxy functions at positionꢀ2, ꢀ3 and ꢀ4 of the

phenyl ring, respectively, showed enhanced activity (IC50 values 38.6 ± 1.50, 10.3 ± 0.38 and 81.3 ±

.99 ꢁM) compared to the corresponding compounds 6d-f, demonstrating enzyme inhibition with

7

IC50 values of 40.1 ± 5.32, 29.3 ± 1.17 and 849 ± 2.26 ꢁM, respectively. Also, the monofluoroꢀ

substituted compounds 7g and 7h having the substituent at positionꢀ2 and ꢀ3 of the phenyl ring,

respectively, displayed enhanced activity (IC50 values 25.6 ± 2.91 and 32.0 ± 0.69 ꢁM) in

comparison to the respective compounds 6g and 6h, exhibiting enzyme inhibitory activity with IC50

values of 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM, respectively. Furthermore, the difluoroꢀsubstituted

compound 7j with the substituents at positionsꢀ2 and ꢀ4 of the phenyl ring showed increased activity

(

IC = 20.9 ± 0.57 ꢁM) in contrast to the corresponding compound 6j, displaying inhibition of the

50

enzyme with IC50 value of 66.6 ± 5.40 ꢁM. Marked increment was found to occur in case of

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compounds 7a, 7e, 7f and 7j. To the contrary, compounds 7b and 7c possessing the methyl

substituent at positionꢀ3 and ꢀ4 of the phenyl ring, respectively, exhibited decreased enzyme

inhibitory activity (IC50 values 24.6 ± 2.14 and 25.3 ± 3.20 ꢁM) compared to the corresponding

compounds 6b and 6c, showing activity with IC values of 15.0 ± 1.92 and 11.4 ± 1.51 ꢁM. Also,

50

compound 7i bearing the fluoro function at positionꢀ4 of the phenyl ring showed reduced inhibitory

activity (IC50 value 11.3 ± 0.32 ꢁM) in contrast to the respective compound 6i, exhibiting enzyme

inhibition with IC50 value of 8.20 ± 0.01 ꢁM. Relatively, pronounced reduction in the enzymatic

activity was observed in the case of 7c.

The above results showed that the simultaneous presence of varied inductively electronꢀwithdrawing

groups at positionꢀ5 of the isatin scaffold and the variously substituted aryl functions at N atom of

the thiazoline moiety caused the isatinꢀthiazoline hybrids to meddle with the enzyme differently and

sometimes relatively much more competently.

Molecular docking studies of compounds 5a-n, 6a-o and 7a-j

Molecular docking studies of isatinꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j were performed using

LeadIT to investigate the binding modes of inhibitors as well as interactions in the active site. The

structures were docked to the crystallographic structure of Jack bean urease PDB ID: 3LA4 having

biꢀnickel center in its active site. After reproducing the coꢀcrystallized reference ligand into the

active site of receptor, all the derivatives were docked inside the active site. Most of the compounds

were found interacting deep inside with the active site residues and showed coordinated interactions

with the nickel ions in the active site of the receptor. The active site of the receptor contains the

amino acids residues His409, Ala436, Arg439, Ala440, His492, Asp494, His519, His593, His594,

Arg609, Ala636 and Met637.

Overall, the structures of the compounds are too large to be accommodated in the active pocket of

the enzyme; therefore, some part lies within the bottom, while the rest interacts with the mid gorge

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area. The putative binding modes of the most active compounds among the three series i.e. 5a-n, 6a-

o and 7a-j are given below:

Binding mode analysis of compounds 5a-n, 6a-o and 7a-j

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Among 5ꢀtrifluoromethoxyisatinꢀhydrazonothiazolines 5a-n, 5k was found to be the most potent

inhibitor of Jack bean urease. It was directed deep towards the nickel ion present inside the active

site of the receptor, as shown in Figure 4. Its 5ꢀtrifluoromethoxyisatin part was located towards

bottom of the binding pocket, whereas the 4ꢀchlorophenyl and 2,5ꢀdichlorophenyl substituents

attached to C and N atoms of the thiazoline moiety, respectively, were directed more towards

entrance of the active site. In all the compounds studied, the 5ꢀtrifluoromethoxy isatin scaffold did

not form direct bond with the Ni center. The 2,5ꢀdichlorophenyl substituent attached to C atom of the

thiazoline moiety showed πꢀπ interactions with the imidazole ring of His593. Additionally, nonꢀ

covalent molecular interactions were found between 5ꢀtrifluoromethoxy isatin moiety and Arg609 in

the active site, while the hydrogen bonding was noticed between imidazole ring of His492 and the

carbonylꢀO of the 5ꢀtrifluoromethoxy isatin part of compound 5k. Figure 4 reports a prioritized

binding pose of the potent inhibitor 5k, and it is selected after Hyde assessment and visualization.

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Figure 4. The putative binding mode of compound 5k bound to the active site of Jack bean urease.

Carbon atoms of the compound are golden coloured and that of protein are blue. Dark green spheres

are two nickel ions.

Binding mode analysis of compounds 6a-o

As evident from the in vitro results, amongst 5ꢀfluoroisatinꢀhydrazonothiazolines 6a-o, 6i was the

most active inhibitor of Jack bean urease. The docking studies revealed that it adopts best orientation

inside the active pocket by forming hydrogen bonding with the amino acid residues of His492,

His593 and His594. The 5ꢀfluoroisatin part of the compound was directed into the pocket towards

the nickel ions. Furthermore, the 4ꢀchlorophenyl substituent attached to C and 4ꢀfluorophenyl one to

N atoms of the thiazoline moiety was aligned towards His594 and Asp494, respectively.

Additionally, πꢀcharge interactions were found with amino acid Asp494, which stabilized the pose.

The thiazoline part of the compound showed πꢀπ interactions with the imidazole ring of His594. The

putative binding pose of compound 6i was selected after Hyde assessment and visualization, and is

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depicted in Figure 5.

Figure 5. The putative binding mode of compound 6i bound to the active site of Jack bean urease.

Carbon atoms of the compound are magenta coloured and that of protein are blue. Dark green

spheres are two nickel ions.

Binding mode analysis of compounds 7a-j

Docking studies supported the experimental results that potent inhibitors inhibit the catalytic site of

the enzyme. The docking studies revealed the stable hydrogen bonding interactions between the

oxygen and hydrogen atoms of the inhibitors and the amino acid residues Arg439, His593 and

Arg609. Figure 6 reports the prioritized binding mode of the most potent inhibitor 7a; the pose was

selected after Hyde assessment and visualization of 30 lowest energy docked conformations. The 5ꢀ

chloroisatin part of the molecule was oriented towards amino acids His409, Ala436, Arg439, Ala440

and His593 where πꢀπ interactions were observed with His593 in the active pocket; the 4ꢀ

chlorophenyl and 2ꢀmethylphenyl substituents attached to C and N atoms of the thiazoline moiety,

respectively, were inclined towards Asp494.

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Figure 6. The putative binding mode of compound 7a bound to the active site of Jack bean urease.

Carbon atoms of the compound are light green coloured and that of protein are blue. Dark green

spheres are two nickel ions.

(5k)

(6i)

(7a)

Figure 7. 2D Interaction diagrams of the selected docked conformations for most potent inhibitors.

Hydrogen bond interactions are indicated with dotted lines and hydrophobic interactions are shown

with green lines. Metal interactions were observed by Nickel ions.

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Conclusions

Conclusively, three new series of thirty nine isatinꢀ3ꢀhydrazonothiazolines have been synthesized in

this study and evaluated for their in vitro cytotoxic, phytotoxic, antifungal and urease inhibitory

effects. All the compounds proved to be almost inactive in the brine shrimp (Artemia salina) lethality

bioassay. However, in phytotoxicity bioassay, six compounds were found to be active, exhibiting

weak or nonꢀsignificant (5ꢀ30%) activity at the highest tested concentration (500µg/mL). Similarly,

in antifungal assay, out of thirty nine compounds tested, twenty six proved to be active, showing

weak or nonꢀsignificant activity (10ꢀ30%) against one, two, or three fungal species. On the other

hand, the urease inhibition testing results have demonstrated that all the thirty nine compounds tested

appeared as potent urease inhibitors; seventeen of these i.e. 5c, 5g-i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o,

7

a, 7e, 7i and 7j proved to be relatively highly potent ones and may act as convincing leads for

further studies. These compounds, being non toxic, could be potent candidates for orally effective

therapeutic agents used for the treatment of certain clinical conditions induced by microbial ureases.

Also, by demonstrating weak or nonꢀsignificant phytotoxicity at the highes tested concentration, they

invite much attention to their utility as potent inhibitors of soil ureases, as they could be mixed with

fertilizers in small quantities to enhance the overall effectiveness of nitrogen utilization. Generally,

urease inhibitory potential of the sunthesized isatinꢀthiazolines was found to be dependent upon

electronic effects of the substituents at positionꢀ5 of the isatin scaffold and on the phenyl ring

attached to N atom of the thiazoline moiety. In order to understand the binding mechanism,

molecular docking studies were performed for compounds 5a-n, 6a-o and 7a-j, which elucidated

their relationship with the binding pockets of urease. Based on the data presented in Table 3 and in

terms of further development and SAR studies, simultaneous substitution of diverse inductively

electronꢀwithdrawing groups at positionꢀ5 of the isatin scaffold and the aryl substituents (modified

by placing one or more lipophilic functions about the phenyl ring) at N as well as Cꢀ4 atoms of the

thiazoline moiety in the hybrid molecules certainly warrants investigations.

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Experimental

General procedure

Chemical materials and solvents were obtained from MerckꢀSchuchatdt, Fluka and SigmaꢀAldrich.

Melting points were determined on a FisherꢀJohns melting point apparatus and are not corrected.

Elemental analyses were performed by using a Leco CHNSꢀ9320 (USA) elemental analyzer. The

data obtained from all the synthesized compounds were found to be satisfactory. FTꢀIR spectra

(

KBr) were run on a Shimadzu 8400 or Thermo Scientific Nicolet 6700 FTIR spectrophotometer

1

using ATR facility. HꢀNMR spectra were recorded in C

2

D

6

SO on Bruker (RhenistettenꢀForchheim,

Germany) AM 300, Bruker Avance AV300 and Bruker Spectrospin 300 spectrometers, operating at

00 MHz and using Si (CH as an internal standard. Mass spectra were obtained on MATꢀ312 and

3

3 4

)

JEOL JMSꢀ600H mass spectrometers.

Synthesis

General procedure for the synthesis of isatin-3-thiosemicarbazones 3

4

An appropriate 5ꢀsubstututed isatin 1 (0.01mol), N ꢀsubstituted thiosemicarbazide 2 (0.01mol) and

50% aqueous ethanol (40 mL) containing a catalytic amount of acetic acid were mixed and heated

under reflux for 2h. The solid obtained in each case during heating was filtered and washed

thoroughly with hot 50% aqueous ethanol to give the desired thiosemicarbazones 3, which were used

30,32,33,39,40

as such in the next step without further purification.

General procedure for the synthesis of isatin-3-hydrazonothiazolines 5-7

An appropriate isatinꢀ3ꢀthiosemicarbazone 3 (0.005 mol) was mixed with 4ꢀchlorophenacyl bromide

4

(0.005 mol) in absolute ethanol (25 mL) or in a mixture of ethanolꢀbenzene and heated under reflux

for 12ꢀ15 h.The refluxate was concentrated on a rotary evaporator and the precipitare thus formed in

Page 29 of 51

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DOI: 10.1039/C6RA10043K

each case was filtered. Thorough washing with warm diethyl ether or nꢀhexane followed by

crystallization from ethanolꢀwater furnished the target compounds 5-7 in pure form.

The different compounds are characterized as under:

(3

Z)-5-(Trifluoromethoxy)-1

H

-indole-2,3-dione-3-{[(2

Z)-4-(4-chlorophenyl)-3-(2-

methylphenyl)-1,3-thiazol-2(3

H

)-ylidene]hydrazone} 5a

ꢀ1

Yield 82% as orange brown crystals; m.p. 248ꢀ250 °C; IR (KBr, cm ): 3122 (NH stretching), 1717

1

(

C=O), 1618 (C=N); HꢀNMR (DMSOꢀd

6

, δ, ppm): 2.08 (s, 3H, CH

ꢀH, C ꢀH), 7.17 (s, 1H, thiazoline =CH), 7.27 (d, J = 9.1

ꢀH) 7.29 (dd, J = 9.1 Hz, 1H, indole C ꢀH), 7.31ꢀ7.38 (m, 4H, Nꢀphenyl C

ꢀH, indole C ꢀH), 7.42 (d, J = 7.4 Hz, 1H, Nꢀphenyl C ꢀH), 10.66 (s, 1H, indole

NH); EIMS (70eV): m/z (%): 530 (M +2, 41), 529 (M +1, 31), 528 (M , 100), 499 (5), 301 (7), 300

19), 299 (39), 298 (40), 297 (77), 288 (8), 287 (12), 286 (48), 285 (23), 284 (81), 240 (14), 228 (7),

87 (6), 174 (6), 170 (5), 168 (12), 162 (19), 139 (5), 134 (6), 133 (7), 91 (14), 77 (5); Anal calcd. for

S (528): C 56.76, H 3.03, N 10.06; found: C 56.77, H 2.98, N 10.11.

3

), 6.84 (d, J = 9.1 Hz, 1H,

indole C

Hz, 2H, phenyl C

H, phenyl C ꢀH, C

7

ꢀH), 7.13ꢀ7.16 (m, 2H, Nꢀphenyl C

4

5

2

ꢀH, C

6

ꢀ

3

5

4

3

+

(

1

25 3 4 2

C H16ClF N O

(3

Z)-5-(Trifluoromethoxy)-1

H

-indole-2,3-dione-3-{[(2

Z)-4-(4-chlorophenyl)-3-(3-

methylphenyl)-1,3-thiazol-2(3

H

)-ylidene]hydrazone} 5b

ꢀ1

Yield 88% as orange yellow fluffy crystals; m.p. 268ꢀ270 °C; IR (KBr, cm ): 3111 (NH stretching),

1

709 (C=O), 1622 (C=N); HꢀNMR (DMSOꢀd

6

3

, δ, ppm): 2.28 (s, 3H, CH ), 6.83 (d, J = 8.2 Hz, 1H,

indole C

ꢀH, C

7

ꢀH), 7.05 (s, 1H, Nꢀphenyl C

2

ꢀH), 7.09 (s, 1H, thiazoline =CH), 7.13ꢀ7.37 (m, 9H, Nꢀphenyl

C

4

5

ꢀH, C

6

ꢀH, phenyl C

2

ꢀH, C

3

ꢀH, C

5

ꢀH, C

6

ꢀH, indole C

4

6

ꢀH, C ꢀH), 10.59 (s, 1H, indole NH);

+

EIMS (70eV): m/z (%): 530 (M +2, 47), 529 (M +1, 34), 528 (M , 100), 501 (6), 500 (16), 500 (7),

3

00 (6), 299 (11), 298 (6), 288 (10), 287 (7), 286 (32), 285 (6), 284 (30), 228 (6), 187 (4), 170 (5),

68 (12), 149 (13), 133 (4), 91 (9), 69 (9); Anal calcd. for C25 S (528): C 56.76 H 3.03

1

H

3 4 2

16ClF N O

N 10.06; found: C 56.70, H 3.00, N 10.00.

(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-

Article Doi

Synthesis, biological evaluation and docking studies of some novel isatin-3-hydrazonothiazolines

DOI: 10.1039/c6ra10043k

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Article abstract of DOI:10.1039/c6ra10043k

Full text of DOI:10.1039/c6ra10043k

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