Evaluation and Docking Study of Pyrazine Containing 1, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class of Potential Antimicrobial and Antitubercular

Published online: 2020-10-07

Original Article

Thieme

Evaluation and Docking Study of Pyrazine Containing

, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one:

1

A New Class of Potential Antimicrobial and Antitubercular

Authors

Rina Das , Dinesh Kumar Mehta

AbSꢀRAcꢀ

Aﬃliation

M.M. College of Pharmacy, Maharishi Markandeshwar

Deemed to be University), Mullana, Ambala, Haryana

Background Tuberculosis (TB) caused by Mycobacterium

tuberculosis is one of the main killers of people all over the

(

world. The major hurdles with existing therapy are the lengthy

regimen and appearance of multi drug resistant (MDR) and

extensively drug resistant (XDR) strains of M.tuberculosis.

Aims The present work was aimed to synthesize and deter-

mine antitubercular and antimicrobial potential of some novel

Key words

antibacterial drugs, antifungal drugs, chemistry, drug

research

received

accepted

29.07.2020

30.08.2020

3

2

-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-

-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(a-h) from

published online 07.10.2020

pyrazinoic acid as precursor, which is a well-established antitu-

bercular agent. Here we report the synthesis of a new class of

heterocyclic molecules in which pyrazine, 1, 3, 4-oxadiazole

and azetidinone moieties were present in one frame work.

Methods Pyrazinoic acid (1) was esteriﬁed ﬁrst (2) followed by

amination to produce hydrazide (3) which was reﬂuxed with POCl3

toobtain2-chloromethyl-5pyrazino-1, 3, 4-oxadiazole(4). Thiswas

then further reacted with 4-amino phenol to obtain 4-[5-pyrazi-

no-1, 3, 4-oxadiazol-2-yl-methoxy]-phenyl amine (5) which on

condensation with various aromatic aldehydes aﬀorded a series

Schiﬀ’s bases6(a-h). Dehydrativeannulationsof6(a-h)inthepres-

ence of chloroacetyl chloride and triethylamine yielded 3-chloro-

Bibliography

Drug Res 2021; 71: 26–35

DOI 10.1055/a-1252-2378

ISSN 2194-9379

©

Georg Thieme Verlag KG, Rüdigerstraße 14,

0469 Stuttgart, Germany

7

Correspondence

Dr. Rina Das

Associate Professor, M.M.College of Pharmacy, Maharishi

4

-aryl-1-[4-(5-pyrazin-2-yl-[1, 3, 4]oxadiazole-2-ylmethoxy)-

Markandeshwar (Deemed to be) University, Mullana,

phenyl]-azetidin-2-onederivatives7(a-h). Antibacterial, antifungal

andantitubercularpotentialofallthesynthesizedcompoundswere

assessed. Docking study was performed using the software VLife

EnginetoolsofVlifemds4.6ontheproteinlumazinesynthaseofM.

tuberculosis (PDB entry code 2C92).

Ambala, HR 133207

India

Tel.: 0805993017

rinammu@gmail.com

Results The present studies demonstrated that synthesized

oxadiazole derivatives have good antimicrobial activity against

the various microorganisms. Among the synthesized derivative,

Supporting Information for this article is available online at

https://doi.org/10.1055/a-1252-2378

7

ꢁ and 7g were found to be prominent compounds which have

potential antibacterial, antifungal and antitubercular activity

with MIC 3.12µg/ml and high dock score ranging from −59.0

(

to −54.0) against Mycobacterium tuberculosis.

Conclusions Derivatives 7ꢁ and 7g would be eﬀective lead

candidates for tuberculosis therapy.

Introduction

Microbial infections remain the chief cause of mortality worldwide.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of

the main killers of people all over the world. The major hurdles with

existing therapy are the lengthy regimen and appearance of multi

drug resistant (MDR) and extensively drug resistant (XDR) strains

of M.tuberculosis and this made the condition most frightening

[1, 2]. Therefore, there is an urgent demand for a new class of an-

2

6

timicrobial agent with a diﬀerent mode of action and it led medic-

inal chemists to explore a wide variety of chemical structures. In

pursuit of this goal, our research eﬀorts herein have been directed

towards the discovery of new chemical entities that are eﬀective

antimicrobial and antitubercular agents.

Nitrogen containing heterocyclic compounds are well studied for

their broad spectrum of activities [3–7]. Among them 1, 3, 4-oxadi-

azole and their derivatives constitute a crucial class of organic com-

pounds which have been reported to possess versatile activities. Ox-

adiazole is a cyclic compound containing one oxygen and two nitro-

gen atoms in a ﬁve-member ring [8]. Oxadiazoles have occupied a

unique place in the ﬁeld of medicinal chemistry due to its wide range

of activities [9]. From the literature survey, oxadiazole nucleus has

been found to possess anti-inﬂammatory [10–12], anticonvulsant

bercular drugs. Various pyrazine derivatives and pyrazinamide ana-

logs also exhibit high antibacterial activity, e. g. pyrazinoic acid es-

ters [37], pyrazine thiocarboxamide and N-hydroxymethyl pyrazine

thiocarboxamide [38], and ring substituted pyrazinylchalcones [39].

It is also reported 4-mono- and 4-disubstituted 1-pyrazinoyl thio-

semicarbazides exhibit high tuberculostatic activity [40,41].

After extensive literature search, it was observed that, till date

enough eﬀorts have not been made to combine these moieties as

a single molecular scaﬀold and to study its biological activity [42–

44]. This initiated us to synthesizing compounds containing the

oxadiazole, azetidinone and pyrazine ring systems in the same ma-

trix to serve as a new scaﬀold. In this paper, we have reported the

synthesis of a new class of heterocyclic molecules in which pyra-

zine, 1, 3, 4-oxadiazole and azetidinone moieties were present in

one frame work. Further this new class of heterocyclic molecules

was explored for their antitubercular, antibacterial and antifungal

potential.

[

13], antifungal and antibacterial [14,15], antiviral [16], antioxidant

17], analgesic [12], and antitubercular activity [18].

β-Lactams are the most successful antimicrobials [19–22] till

recent days, unless microorganisms producing β- lactamase. The

-azetidinone (β-lactam) ring system is the common structural fea-

2

Results and Discussion

Pyrazine-2-carboxylic acid methyl ester (2) was prepared in quantita-

tive yield according to a known method [45]. This compound on am-

ination with hydrazine hydrate aﬀorded pyrazine-2-carboxylic acid hy-

ture of a number of broad spectrum β-lactam antibiotics, includ-

ing penicillins, cephalosporins, carbapenems, nocardicins, mono-

bactams, clavulanic acid, sulbactams and tazobactams, which have

been widely used as chemotherapeutic agents to treat bacterial in-

fections and microbial diseases [23–30]. A large number of 3-chlo-

ro- 2-azetidinones possesses potent antimicrobial [31], antituber-

cular [32, 33], antioxidant [34], inhibitors of cholesterol absorption

drazide (3). Compound 3 reﬂuxed with POCl to obtain 2-chloro me-

3

thyl-5-pyrazyl-1, 3, 4-oxadiazole (4). This was then further reacted

with 4-amino phenol to obtain 2-[4aminophenyloxymethyl]-5-pyra-

zyl-1, 3, 4-oxadiazole (5).The condensation of compound (5) with var-

ious aromatic aldehydes yielded aryl methylene-[4-(5-pyrazin-

2-yl-[1,3,4] oxadiazole-2-ylmethoxy)-phenyl]-amine derivatives 6(a-

h). Compounds 6(a-h), on reaction with chloroacetyl chloride in the

presence of triethylamine underwent dehydrative annulation

[

35], vasopressin V1a antagonists [36] activity.

Pyrazine derivatives are well-known and an important aromatic

heterocyclic compound having two-nitrogen-containing six-mem-

bered ring. They can carry substituents at one or more of the four

ring carbon atoms. Pyrazinamide is one of the most eﬀective antitu-

▶

Fig. 1 Synthesis of 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl [1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(a–h).

2

7

Original Article

Thieme

(

2

Staudinger’s ketene–imine reaction) to aﬀord 3-chloro-4-aryl-1-[4-

5-pyrazin-2-yl-[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-

-one derivatives 7(a-h).These reactions are summarized in ▶Fig. 1.

The antimicrobial activity of the synthesized derivatives was as-

sessed by agar cup plate method [46] and the average diameter of

zone of inhibition (mm) and MIC (μg/ml) [47] were determined in

comparison with standard drugs.

All the synthesized compounds were screened for their in vitro

antibacterial activity against two gram positive (S. aureus, B.subti-

lis) and two gram negative (P.aeruginosa, E. coli) bacteria taking

Amoxicillin as a standard drug and in vitro antifungal activity against

two fungus strains C.albicans and A. niger taking Miconazole as a

standard drug (▶ꢀaꢁle 4).

The results of zone of inhibition studies as shown in ▶ꢀaꢁle 4

showed that the compounds 7ꢁ, 7e and 7g have greater antibac-

terial potential than other derivatives against the gram positive (B.

subtilis and S.aureus) and gram negative (E.coli and P.aeruginosa)

bacterias. Similarly antifungal activity of 7ꢁ, 7e and 7g derivatives

displayed higher zone of inhibition as compared to other deriva-

tives against C.albicans and A.niger. The antimicrobial (antibacte-

rial and antifungal) potential of the synthesized compounds was

further conﬁrmed by determination of MIC of all the derivatives.

The MIC is deﬁned as the minimum concentration of compounds

required to completely inhibit the bacterial growth. It was clearly

observed from the results as shown in ▶ꢀaꢁle 4 that the deriva-

tives 7ꢁ, 7e and 7g showed greater antibacterial potential against

gram positive and gram negative bacterias as compared to other

derivatives.

The series synthesized was found to be more active against

S.aureus and E.coli compared to other bacterias used. In compari-

son to standard drug compound 7ꢁ and 7g exhibited two fold in-

crease in antibacterial activity against S.aureus and compound 7ꢁ

against E.coli. Compound 7g also exhibited two fold increase in ac-

tivity against P.aeruginosa compared to standard drug.

The oxadiazole derivatives are found to be more potent against

C.albicans compared to A. niger. The derivative 7g with electron do-

nating group in substitution and 7ꢁ and 7e with electron withdraw-

ing substitutions displayed potent activity against C.albicans.

In perticular the compounds 7g with MIC 12.5 μg/ml is endowed

with maximum potency against C. albicans, being two times more

active than the standard drug Miconazole.

The substituents of the compounds are given in ▶ꢀaꢁle 1. Yields of

the synthesized compounds were moderate to fair (54–70%). The

physical and molecular data are listed in ▶ꢀaꢁles 2 and ▶3. The puri-

ty of the compounds was monitored by TLC and the structure of the

compounds was deduced on the basis of their elemental analysis and

spectral data. Both analytical and spectral data of all the synthesized

derivatives were in full agreement with the proposed structures. The

structures assigned to 7(a-h) were supported by IR, NMR and MASS

spectra. The IR spectrum of titled compounds 7(a-h) revealed C=C

aromatic characteristic stretching absorption between 1492–

1

591cm⁻¹and aromatic C-H stretching absorption manifested be-

tween 2993–3065cm⁻¹. The aromatic C-H stretching for methyl moi-

ety manifested between 2807–2927cm⁻¹. The ring stretching mode

(C=O) of azetidinone for titled compounds 7(a-h), manifested fre-

quency between 1 730–1 789 cm⁻¹, that indicates formation of

β-lactam ring of azetidinone, while C-Cl bending for azetidinone is re-

vealed by characteristic bands between 2933–3065cm⁻¹. The oxa-

diazole ring manifested C-O-C stretching absorption between

1

053–1089cm⁻¹. The asymmetric bending absorptions of aromatic

NO function was manifested at 1531cm⁻¹and the symmetric bend-

2

ing absorption was manifested at 1394cm⁻¹for 7e and 7h. The aro-

matic C-O bending for ether moiety of 7g was manifested at

1

was manifested at 3208cm⁻¹. The C-Cl bending frequency for aro-

matic chloro function in 7aand 7ꢁmanifested at 642cm⁻¹. In H NMR

all the aromatic rings were observed as multiplets at δ scale between

162cm⁻¹and aromatic O-H stretching of hydroxyl function of 7d

1

6

.96–811ppm in the titled compounds 7(a-h) and three protons of

pyrazine moiety manifested as multiplets at δ scale between 8.22–

37ppm in the titled compounds 7(a-h). The one proton of CH-Cl of

8

azetidinone having one proton in its proximity, thus were observed as

doublet at δ scale between 5.75–5.77ppm. The two protons of a CH₂

function having no proton in its proximity thus, shows singlet peak

between 5.27–5.29 in the titled compounds 7(a-h), a singlet peak is

seen at 3.77ppm in 7gfor methoxy function and presence of one pro-

ton for aromatic C-OH manifested singlet at 5.15ppm in 7d. In the

MASS spectrum, the m/z 68 and m/z 79 due to formation of [C N O]

and [C H NOCl] depicted the presence of oxadiazole and azetidinone

ring in the titled compounds.

+

Compound 7ꢁ and 7g exhibited potential activity against

A. niger but not as potent as compared to the standard drug. Other

oxadiazole derivatives have shown fair to medium antibacterial and

antifungal activities. No inhibitory eﬀect was observed for DMSO.

2

+

3

2

▶

ꢀaꢁle 1 Various substitutions (R) used.

compound

R

compound

7e

R

7

a

7

ꢁ

ꢂ

7f

7g

7

d

7h

2

8

▶

ꢀaꢁle 2 Physical and molecular properties of synthesized aryl methylene-[4-(5-pyrazin-2-yl-[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-amine derivatives.

compound

Physiꢂal state

Mol. Formula

Mol. Weight

Perꢂentage

Yield (in %)

M.P (°c)

Soluꢁility

Rf value

6

a

ꢁ

ꢂ

Brownish crystals

C20N5O2ClH14

392

358

374

403

372

388

403

55

61

66

52

72

56

77

67

222 − 224

188 − 190

217 − 218

202 − 204

179 − 180

182 − 184

197 − 198

208 − 210

DMSO, MeOH, CHCl3

0.55

0.48

0.41

0.39

0.57

0.33

0.52

0.59

Pale Brownish crystals

Brownish crystals

C20N5O2H15

C20N5O3H15

C20N6O4H14

C21N5O2H17

C21N5O3H17

C20N6O4H14

d

e

f

Yellowish Brown crystals

Reddish Brown crystals

Dark Yellowish crystals

Pale Brownish crystals

Brown crystals

g

h

▶

ꢀaꢁle 3 Physical and molecular properties of synthesized 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl [1,3,4] oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one

derivatives.

compound

Mol. Formula

Mol. Weight

Physiꢂal state

Perꢂentage

Yield (in %)

M.P

Soluꢁility

Rf value

7

a

ꢁ

ꢂ

C22H15Cl2N5O3

468.29

433.85

449.85

478.84

479.96

463.87

494.89

Brownish crystals

Pale Brownish crystals

Brownish crystals

Light Brown crystals

Dark Brown crystals

Yellowish crystals

Pale Brownish crystals

Brown crystals

64

70

58

55

61

54

62

67

176 − 177

162 − 164

209 − 210

175 − 176

140 − 142

152 − 154

134 − 135

171 − 172

DMSO, MeOH, CHCl3

0.81

0.79

0.68

0.73

0.86

0.65

0.77

0.83

C22H16ClN5O3

C22H16ClN5O4

C22H15ClN6O5

C25H26ClN5O3

C23H18ClN5O4

C23H19ClN6O5

d

e

f

g

h

Protein Preparation

The encouraging results from the antibacterial and antifungal

studies impelled us to go for preliminary screening of synthesized

compounds against M. tuberculosis. Therefore, all the synthesized

compounds were screened for their in vitro antimycobacterial ac-

tivity against Mycobacterium tuberculosis H37Rv (MTCC200) by

Lowenstein-Jensen (L.J.) Agar (MIC) method [48] using isoniazid

and rifampicin as standard drugs. Results as shown in ▶ꢀaꢁle 5 de-

picted that out of eight screened compounds 7ꢁ and 7g having

para substituted aromatic ring displayed potential antitubercular

activity with MIC 3.12 µg/ml against the strain Mycobacterium tu-

berculosis H37Rv. Substitution at ortho position on the aromatic

ring in compound 7a have MIC value 6.25 µg/ml shows consider-

able antitubercular activity.

Compounds 7e, 7d, 7h showed good activity (MIC 25–50 mg/

ml) which is attributed to 3-nitro, 4-hydroxy and 2-nitro substitu-

ents whereas the MIC values of compounds 7ꢂ and 7f which having

phenyl alkyl substitution were found to be more than 100 µg/ml

and exhibited lesser antitubercular activity.

After conducting ample literature study lumazine synthase of M.

tuberculosis (PDB entry code 2C92), was selected as the biological

target for carrying out the docking study of our synthesized com-

pounds. The enzyme lumazine synthase is not present in the human

or animal host [50, 51]. Hence, they are promising candidates for

the inhibition of M. tuberculosis growth. Lumazine synthase inhibi-

tors can be considered as potential lead compounds for the design

of therapeutically useful antitubercular agents.

▪ The crystal structures of the target protein were obtained

from Protein Data Bank, downloaded from www.rcsb.org and

saved in standard 3D coordinate format. The protein Data

Bank (PDB) provides the 3-D structure of all the proteins by

NMR or by X-ray crystallography.

▪ The selected lumazine synthase was prepared using biopre-

dicta module of the VLife MDS 4.6.

▪ The enhancement of the crude PDB structure of receptor was

done by completing the incomplete residues. The water

molecules were removed and hydrogens were added in to the

protein structure to retain the geometry, the co-crystallized

ligand lying within the receptor was modiﬁed by assigning

missing bond order and hybridization states.

▪ The structure was then optimized by the optimization module

of the Vlife MDS 4.6 using Merck molecular force ﬁeld. The

optimized receptor was then saved as mol ﬁle and used for

docking study.

Although the synthesized derivatives speciﬁcally 7ꢁ, 7g and 7a

displayed potent antitubercular activity but they are still lesser ac-

tive as compared to standard drugs (isoniazid and rifampicin). The

results obtained reveals that the nature of substituents on the ar-

omatic ring have a great impact on the antitubercular activities of

the test compounds.

In silico Molecular Docking studies were performed with the syn-

thesized compounds using VLife MDS 4.6 [49].

▪

The assorted 3D view of the protein PDB 2C92 is shown in

Fig. 2

▶

2

9

Original Article

Thieme

▶

ꢀaꢁle 4 Antibacterial and Antifungal evaluation of synthesized 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1,3,4] oxadiazole-2-ylmethoxy)-phenyl]-azetidin-

2-one derivatives.

compound

Zone of inhiꢁition (mm) against miꢂroorganisms

G-ve ꢁaꢂteria

G+ve ꢁaꢂteria

S. aureus

Fungal speꢂies

A. niger

B. subtilis

23 ± 0.86

25 ± 0.66

19.3 ± 0.47

18.6 ± 0.26

24.3 ± 0.24

20 ± 0.86

26.3 ± 0.47

23 ± 0.06

NZ

E. coli

P.aeruginosa

21 ± 0.86

22.3 ± 0.24

16.6 ± 0.26

17 ± 0.63

22 ± 0.86

20 ± 0.63

24 ± 0.86

21.3 ± 0.47

NZ

C.albicans

21.3 ± 0.24

22 ± 0.66

19.3 ± 0.47

20.3 ± 0.24

22.3 ± 0.47

20.3 ± 0.47

23 ± 0.66

21 ± 0.86

NZ

7

a

ꢁ

ꢂ

25 ± 0.66

28.3 ± 0.47

20 ± 0.63

19.6 ± 0.26

26.3 ± 0.47

22 ± 0.86

27.3 ± 0.24

25 ± 0.66

NZ

25.3 ± 0.47

29 ± 0.63

20.3 ± 0.24

19.6 ± 0.26

26 ± 0.66

23.3 ± 0.24

28 ± 0.66

25 ± 0.66

NZ

22 ± 0.86

24 ± 0.66

21.3 ± 0.47

20 ± 0.63

23 ± 0.66

22.3 ± 0.47

25 ± 0.66

22 ± 0.86

NZ

d

e

f

g

h

Control

Amoxicillin

Miconazole

29.3 ± 0.47

ND

31.3 ± 0.24

ND

30 ± 0.86

ND

28.3 ± 0.24

ND

26.3 ± 0.47

28 ± 0.86

ND: Not Determined. NZ: No Zone of Inhibition.

▶

ꢀaꢁle 5 Antimicrobial activity of the synthesized 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl [1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives

*

(

MIC’s in μg/ml ).

compound

MIC (μg/ml)

baꢂterial strains

Fungal strains

C.albicans A.niger

M. tuberculosis

B.subtilis

100

50

S.aureus

E.ꢂoli

25

P.aeruginosa

7

a

ꢁ

ꢂ

100

50

200

400

200

100

200

−

50

6.25

3.12

> 100

50

6.25

100

12.5

100

12.5

25

200

100

200

50

200

100

200

50

100

50

100

50

d

e

f

25

50

> 100

3.12

50

g

h

12.5

50

25

100

−

100

−

50

Control

−

Amoxicillin

Miconazole

Rifampicin

Isoniazid

50

100

ND

12.5

ND

200

ND

25

ND

12.5

ND

0.25

0.20

ND

*

MIC- Minimum inhibitory concentration. ND: Not Determined.

Ligand Preparation [49]

▪

Energetically minimizing all the conformers up to the rms

gradient of 0.01 and then saved in separate folder.

Ligand preparation was done by

▪

Drawing the structures using ChemSketch 12 and Chem Draw

Ultra 7 in 2D and saved as MDL Molﬁle format.

Converting the ligands to 3D format using VLife Engine tools

of Vlifemds 4.6.

Then energetically minimizing the 3D structures up to the rms

gradient of 0.01 using Merck Molecular Force Field (MMFF) by

using Vlifemds 4.6 software.

▪ Molecular alignment: Using the template-based technique

the molecules of the dataset were aligned, for alignment of

the molecules the most active molecule was used as a

template. The alignment of all the molecules on the template

shown in ▶Fig. 3.

Docking Studies [49]

▪

Then generating the conformers of all the synthesized ligands

by Monte Carlo method. For doing this, all rotatable bonds of

the ligands were selected and number of seeds used for

searching the conformational space was set as 5.

Docking simulations were performed using Biopredicta tool in the

grip docking mode.

▪ The active site selection was done by choosing the cavity

having maximum hydrophobic surface area.

3

0

▪

The docking simulation was done using GRIP batch docking. In

this, all generated conformers of one ligand were put as one

batch in GRIP docking wizard. Likewise, the batches for all

other ligands were put.

▪

All the conformers were virtually docked at the deﬁned cavity

of the receptor.

The parameters considered for docking study were – rotation

angle: 30 ° (the ligand gets rotated for diﬀerent poses by

rotation angle), number of placements: 30 (by placements,

the method will check all the 30 possible placements into the

active site pocket and will result out few best placements out

of 30), scoring function: dock score, exhaustive method.

For each ligand, all the conformers with their best placements

and their dock score will be saved in output folder. The

method also highlights the best placement of best conformer

of one particular ligand which is having best (minimum) dock

score.

▪

▶Fig. 2 Structure of PDB (2C9B) with cavity assorted view.

Diﬀerent types of interactions were studied between docked

3

D ligands and 3D macromolecule target as shown in ▶Fig. 4.

Here we have listed only best ligands and their dock score in ▶ꢀaꢁle 6.

Theligandformingmoststabledrug-receptorcomplexistheonewhich

is having minimum dock score. After docking simulation, the best

docked conformer of each ligand and receptor were merged and were

checked for various interactions of ligand with receptor like hydrogen

bonding, hydrophobic bonding and van der waal’s interaction. Com-

pounds were found to exhibit hydrogen bonding, hydrophobic bond-

ing and van der Waal’s interaction with the receptor. ▶Fig. 4 shows the

docking position and interactions of the active compounds in the re-

ceptorcavity. Allthecompoundswerefoundtoexhibitlargenumberof

van der Waal’s bonding with wide range of residues. Some of the com-

monresiduesinvolvedinthistypeofinteractionareASP74A, ARG154A,

SER109A, ARG157A, ASN72A and THR110A. However ARG154A and

ARG157A were found to be the common residues involved in docking

of all the ligands.

▶

Fig. 3 Alignment of all the molecules.

From the dock score, compounds 7ꢁ, 7a and 7g were found to

have highest negative dock score ranging from − 59.0 to − 54.0

(

▶ꢀaꢁle 6). It means that these formed most stable drug-receptor

complex amongst other compounds.

also required to set the Max Distance Allowed between two fea-

tures, the value is set to 10. Pharmacophore feature and Distance

between features are shown in ▶Fig. 5 and ▶Fig. 6. ▶Fig 5 shows

pharmacophore for synthesized molecules indicating the pharma-

cophoric features like hydrogen bond donor (green) and hydropho-

bic (orange), Hydrogen bond acceptor (blue), Aliphatic (buﬀ) which

are required for activity. The larger tessellated spheres are indica-

tive of the common pharmacophores identiﬁed in the molecules;

the smaller solid features are of the individual molecules.

Literature survey indicates that electron-withdrawing groups or

electron releasing groups increases lipophilicity of the test com-

pounds, which in turn enhances permeability across the mycobac-

terial cell membrane. The presence of electronegative atom such

as chloro group in 7ꢁ has shown better activity than 7ꢂ and 7fwhich

have alkyl substitutions on the phenyl ring. This clearly indicates

the importance of electronegative atom at para position of aro-

matic ring for better activity. Also 7g bearing a methoxy group at

para position have shown good antitubercular activity. The antitu-

Pharmacophore Modeling [49]

Pharmacophore modeling was carried out in the molsign module

of Vlife MDS 4.6 software. A series of derivatives was ﬁrst aligned

on the active molecule. Alignment of small organic molecules is

one of the important tasks in drug design and bioactivity predic-

tion. A pharmacophore model is a set of three dimensional features

that are necessary for bioactive ligands. Six diﬀerent pharmacoph-

ore types are currently supported include H bond acceptor (HBA)

and donor (HBD), hydrophobic feature, aromatic ring and positive

and negative ionizable centers (Ku). Three ﬁelds are required to set

the parameters for pharmacophore identiﬁcation. In the ﬁrst ﬁeld

Primary Pharmacophore Feature Count, value 3 indicates the min-

imum number of pharmacophore features generated for an align-

ment. The next is a number for Tolerance which indicates the ﬂex-

ibility in percentage allowed while comparing two feature combi-

nations across two molecules. Here the value entered is 10 Å. It is

3

1

Original Article

Thieme

Compo Ligandꢀshowingꢀinteractionꢀwithꢀlumazineꢀsynthase 2DꢀinteractionꢀofꢀLigandꢀwithꢀwithꢀlumazineꢀ

undꢀ

of M.ꢀ

synthaseꢀofꢀM.ꢀtuberculosisꢀ(PDBꢀ2C92)

tuberculosisꢀ(PDBꢀ2C92)

a

b

e

g

▶

Fig. 4 Binding modes (docking view) and 2D interaction of 7a,7ꢁ,7e and 7g with lumazine synthase of M. tuberculosis (PDB 2C92), where dark

blue and light blue lines represent various hydrogen bonding and hydrophobic interactions with amino acid residues.

bercular activities of the synthesized derivatives were found in the

following order: para chloro or para methoxy substitution on aro-

matic ring > ortho chloro substitution on aromatic ring > 3-nitro

substitution on aromatic ring > para hydroxy or ortho nitro substi-

tution on aromatic ring>compounds with alkyl substitution or un-

substituted aromatic ring. These results make the synthesized

novel oxadiazole-substituted azitidinone derivatives interesting

lead molecule for more synthetic and biological evaluation. It can

be concluded that this class of compounds certainly hold great

promise towards pursuit to discover novel class of antimicrobial

and antimycobacterial agents. The compounds 7ꢁ and 7g with po-

tential activity against the selected microorganisms may be regard-

ed as precursor compounds for searching for new derivatives show-

ing potent antimicrobial and antimycobacterial activity.

3

2

▶

ꢀaꢁle 6 Ligand-receptor interaction of target compounds.

compound

Doꢂk sꢂore

Distance (in ˚A)

Amino aꢂid

ASP74A

binding group

7

a

− 62.923

4.876

-C–Cl of azatidinone ring

-C–N of azatidinone ring

-C–Cl of azatidinone ring

-C–N of azatidinone ring

-C–Cl of azatidinone ring

-C–N of azatidinone ring

-C- of –CH2O-

4

.9181

.3814

.7966

ASP74A

ARG154A

ARG157A

ASN72A

7

ꢁ

− 69.449

4.1296

3

4

3

4

2

1

2

.8212

.564

.645

.213

.207

.598

.299

.051

ASN72A

ARG71A

THR110A

ASP74A

-C- of –CH2O-

ARG157A

ARG154A

ARG19A

SER109A

ARG157A

ARG71A

ARG154A

THR110A

ARG157A

–N– of Oxadiazole ring

–N– of Pyrazine

7

e

− 56.415

− 64.705

1.906

O of C = O of azatidinone ring

–N– of Pyrazine

2

4

.277

.097

.402

.762

-N- of -NO2 group

-C- of –CH2O-

g

3.99

–C– of OCH3

4

2

.99

-C- of –CH2O-

.511

.54

–N– of Oxadiazole ring

–N– of Pyrazine

.449

▶

Fig. 5 Pharmacophore for synthesized molecules indicating the pharmacophoric features.

Conclusion

The present research work reports the successful synthesis of various

-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1,3,4] oxadiazole-2-ylmethoxy)-

3

phenyl]-azetidin-2-one derivatives 7(a-h) and assessment of their anti-

microbial and anti-tuberculosis activity. On the basis of results of bio-

logical studies, better antimicrobial activity was observed with deriv-

atives having electron withdrawing or electron releasing groups on

the aromatic ring. It was found that the para position of such substit-

uents is responsible for better activity. Also, some of the derivatives

were found to be potential antimycobacterial agent. The docking score

▶

Fig. 6 Distance between pharmacophoric features.

3

Original Article

Thieme

[

12] Narayana B, Vijayaraj KK, Ashlatha BV et al. Synthesis of some new

of the synthesized compounds correlated with the antitubercular ac-

tivity study. Another important feature of the study was pharmaco-

phore modeling which provided diﬀerent features of the compounds

helpful in binding. So, compound 7ꢁ and 7g due to their better activ-

ity against M.tuberculosis H37Rv strain are the best choice for the prep-

aration of new derivatives in order to further improve antimicrobial

and antitubercular activity in future.

2

-(6-methoxy-2-naphthyl)- 5-aryl-1,3,4-oxadiazoles as possible

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Weinheim) 2005; 338: 373–378

(

[

13] Zarghi A, Tabatabai SA, Faizi M et al. Synthesis and anticonvulsant

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Acknowledgement

15] Gaonkar SL, Rai KM, Prabhuswamy B. Synthesis and antimicrobial

studies of a new series of 2-[4-[2-(5-ethylpyridin-2-yl) ethoxy]

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The authors wish to thank M.M. College of Pharmacy, Maharishi

Markandeshwar (Deemed to be University), Mullana, India for all

necessary facilities and sincerely thank SAIF/CIL, Punjab University,

Chandigarh, India for providing spectroscopic data and Microcare

laboratory, Surat, India for antitubercular study data. We are also

highly thankful to VLife Sciences, Pune, India for providing the trial

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8

41–847

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16] Tan TM, Chen Y, Kong KH et al. Synthesis and the biological evaluation

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3

5

Original Article

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Supporting Information

Evaluation and Docking Study of Pyrazine Containing 1, 3,

4

-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class

of Potential Antimicrobial and Antitubercular

Authors

Rina Das, Dinesh Kumar Mehta

Affiliation

M.M. College of Pharmacy, Maharishi Markandeshwar (Deemed to be University),

Mullana, Ambala, Haryana

Correspondence

Dr. Rina Das Associate Professor,

M.M.College of Pharmacy,

Maharishi Markandeshwar (Deemed to be) University, Mullana,

Ambala, Haryana 133207

India

Tel.: 0805993017

rinammu@gmail.com

1

Original Article

Thieme

Material and Methods

Experimental

Reagents: Starting materials, reagents and solvents were purchased from Sigma-Aldrich,

USA and Merck, Germany and were used further without any purification.

Equipments: The progress of reaction was confirmed by using thin layer chromatography

(

TLC) method on silica gel plates (Merck, Germany) of 3x15 cm coated with silica gel G.

The spots on TLC plates were visualized by using UV lamp (254nm) and R values were

f

calculated. Melting points were determined on digital melting point apparatus (Flora;

Perfit, India) and were uncorrected. Elemental analysis (C, H, N) was conducted using

Carlo Erba analyzer model 1108. The IR spectra (in NaCl prism) were recorded on

1

Schimadzu FT-IR spectrophotometer using Nujol method. The H NMR spectra were

recorded (in DMSO) on a BRUKER AVANCE-400 MHz spectrometer using TMS as an

internal standard, values of chemical shift were expressed in ppm. The spin

multiplicities are indicated by the symbols, s (singlet), d (doublet), t (triplet), m

(

multiplet) and br (broad). Mass spectra (ESI, 70ev) were recorded on Water, Q-TOF

Micromass (LC-MS).

Methodology

The scheme for the synthesis of 5-pyrazyl-2-substitutedsulfanyl-1, 3, 4-oxadiazole

derivatives from pyrazinoic acid is shown in Figure 1 and corresponding R for 5a–h is

given in Table 1

2

Synthesis and spectral data of compounds 2-7

Pyrazine-2-carboxylic acid methyl ester (2): Pyrazine-2-carboxylic acid methyl ester was

synthesized from pyrazinoic acid following the procedure as reported earlier with slight

42

modifications. Pyrazinoic acid (0.1mol), 60ml methanol and 1.4ml conc. H SO were

2

4

refluxed for 7hrs. The progress of reaction was monitored via TLC till single spot. The

mixture was then cooled and poured in crushed ice, was made alkaline, which was

followed by extraction with ether. Ether layers were evaporated and kept aside to obtain

shiny pale brownish crystals of compound 2 in good yield. Recrystallisation was done

using methanol.

2

-pyrazylhydrazide (3): Mixture of ester 2 and hydrazine hydrate in 1:1 portion and 30

42

ml of ethanol were taken in a round bottom flask and refluxed for 4-6 hrs. The

progress of reaction was monitored via TLC till single spot. Excess of ethanol was

removed by distillation. Product 3 separates out upon cooling in good yield which was

filtered and was recrystallised using methanol.

2

-chloromethyl-5pyrazino-1, 3, 4-oxadiazole (4): The compound 4 was synthesized by

refluxing a mixture of compound 3 (0.001mole), POCl (15ml) and chloroacetic acid

3

(

0.002mole) on oil bath at 110°C for 7hrs. Progress of the reaction was checked by TLC

using toluene: ethyl acetate: methanol (70:20:10) as mobile phase. The excess of POCl₃

was distilled off and cooled residue was poured into ice cold water. The content was

neutralized with ammonia to obtain crude product 4 which was filtered, dried and

recrystallized by using ethanol.

4

-[5-pyrazino-1,3,4-oxadiazol-2-yl-methoxy]-phenyl amine (5): Solution of compound

4

(0.01mole) in 15ml methanol part wise was added in 15ml methanolic solution of

4-amino phenol (0.012mole) and KOH (0.15mole). After addition, reaction mixture was

3

Original Article

Thieme

refluxed on water bath for 12hrs. Progress of the reaction was checked by TLC using

toluene: ethyl acetate (75:25) as mobile phase. Then the reaction mixture was cooled

and poured on crushed ice with stirring. The product compound 5 was filtered, washed

well with cold water, then dried and recrystallized with ethanol.

Arylmethylene-[4-(5-pyrazin-2-yl-[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-amine

derivatives 6(a-h): The compound 6 was synthesized by adding solution of compound 5

(

0.1mole), methanol, substituted aldehyde(0.1mole) and few drops of glacial acetic

acid followed by reflux on water bath for 4hrs. Progress of the reaction was checked by

TLC. The reaction mixture was cooled, poured on ice, recrystallized with methanol to

obtain compound 6(a-h).

a]2-[4-[2-chlorobenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6a):

-

1

Obtained in 55% as Brownish crystals; mp (°C): 222-224; IR (Nujol, cm ): 3062 (Ar C-H

str), 2900 (C-H str), 1462,1541(Ar C=C str), 1259 (C-O bend),1059 (C-O-C bend in

oxadiazole),1672 (C=N), 642(C-Cl mono substituted in benzene), 1574 (CH=N str,

1

azomet); H NMR (DMSO, 400MHz, ppm): 8.24-8.39 (m,3H,CH of pyrazine), 5.27(s, 2H,

CH ), 7.01- 7.12 (m,4H, Ar), 7.34-7.71(m,4H, Ar-Cl), 8.54(s1H,N=CH, azomet).

2

b]2-[4-[4-chlorobenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6b):

-1

Obtained in 61% as Pale Brownish crystals; mp (°C): 188-190; IR (Nujol, cm ): 3026 (Ar

C-H str ), 2928 (C-H str), 1490,1518(Ar C=C str), 1265(C-O bend),1055 (C-O-C bend. in

oxadiazole),1677 (C=N), 715(C-Cl monosubstituted in benzene), 1571(CH=N str,

1

azomet); H NMR (DMSO, 400MHz, ppm): 8.24-8.38 (m,3H,CH of pyrazine ), 5.27(s, 2H,

CH ), 7.01 -7.12 (m,4H, Ar), 7.34-7.71 (m, 4H, Ar-Cl) , 8.54(s1H,N=CH,azomet).

2

c]2-[4-[benzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6c): Obtained in

-1

6

6% as Brownish crystals; mp (°C): 217-218; IR (Nujol, cm ): 3018 (Ar C-H str ), 2920

(

1

C-H str), 1492, 1565(Ar C=C str ), 1235 (C-O bend.),1084 (C-O-C bend. in oxadiazole),

640 (C=N), 1574 (CH=N str, azomet); H NMR (DMSO, 400MHz, ppm): 8.20-8.35

1

(

m,3H,CH of pyrazine ), 5.27(s, 2H, CH ), ), 7.01-7.78 (m,9H, ArH), 7.34,

2

8.52(s1H,N=CH, azomet).

d]2-[4-[4-hydroxybenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6d):

-

1

Obtained in 52% as Yellowish Brown crystal; mp (°C): 202-204; IR (Nujol, cm ): 3030

Ar C-H str ), 2906 (C-H str), 1541, 1579(Ar C=C str ), 1263 (C-O bend.), 1070 (C-O-C

(

1

bend. in oxadiazole),1635 (C=N), 3205 (Ar-OH str), 1569(CH=N str, azomet); H NMR

DMSO, 400MHz, ppm): 8.21-8.36 (m,3H,CH of pyrazine ), 5.27(s, 2H, CH ), ),

(

2

7.01-7.12 (m,4H, ArH), 7.34-7.69 (m, 4H, Ar-OH), 8.52 (s1H,N=CH,azomet), 5.12

4

(

s,1H,C-OH).

e]2-[4-[3-nitrobenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6e):

-1

Obtained in 72% as Reddish Brown crystals; mp (°C): 179-180; IR (Nujol, cm ): 3021 (Ar

C-H str ), 2910 (C-H str),1525(Ar C=C str ), 1202 (C-O bend.),1073 (C-O-C str in

oxadiazole),1672 (C=N), 1530 (asymAr N-O str.), 1289 (sym Ar N-O bend..), 1571

1

(

CH=N str, azomet); H NMR (DMSO, 400MHz, ppm): 8.23-8.39 (m,3H,CH of pyrazine ),

5

.27 (s, 2H, CH ), 7.01 -7.12 (m,4H, ArH), 7.34-7.88, (m, 4H, Ar-NO ), 8.52

2

(

s1H,N=CH,azomet).

f]2-[4-[4-methylbenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6f):

-1

Obtained in 56% as Dark Yellowish crystals; mp (°C): 182-184; IR (Nujol, cm ): 3069 (Ar

C-H str ), 2928 (C-H str), 1360(C-H bend.) 1501, 1581(Ar C=C str ), 1263(C-O), 1055

1

(

C-O-C str in oxadiazole), 1672 (C=N), 1573 (CH=N str, azomet); H NMR (DMSO,

4

00MHz, ppm): 8.24-8.39 (m,3H,CH of pyrazine ), 5.26(s, 2H, CH ), 7.01 -7.12 (m,4H,

2

Ar), 7.19-7.85 (m, 4H, Ar- CH ), 8.55 (s,1H, N= CH azomet),2.38 (s,3H, CH ).

3

g]2-[4-[4-methoxybenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6g):

-1

Obtained in 77% as Pale Brownish crystals; mp (°C): 197-198; IR (Nujol, cm ): 3190 (Ar

C-H str ), 2922 (C-H str.), 1455, 1490 (Ar C=C str ), 1221(C-O bend.), 1063 (C-O-C str

1

in oxadiazole), 1640 (C=N), 1155(C-O bend. in ether),1571(CH=N str, azomet); H NMR

(

DMSO, 400MHz, ppm): 8.21-8.36 (m,3H,CH of pyrazine ), 5.27 (s, 2H, CH ), ), 7.01-

2

7

.12 (m,4H, ArH), 6.90-7.86 (m, 4H, Ar- OCH ), 8.52 (s1H,N=CH,azomet.),3.77 (s,3H,

3

CH₃).

h]2-[4-[2-nitrobenzylidene]aminophenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole(6h):

-1

Obtained in 67% Brown crystals; mp (°C): 208-210; IR (Nujol, cm ): 3010 (Ar C-H str ),

904 (C-H str), 1527(Ar C=C str ), 1070 (C-O-C bend. in oxadiazole),1652 (C=N), 1510

2

1

(

asym Ar N-O str.), 1369 (sym Ar N-O bend.), 1574(CH=N str, azomet); H NMR (DMSO,

00MHz, ppm): 8.21-8.35 (m,3H,CH of pyrazine ), 5.27(s, 2H, CH ), ), 7.01 & 7.12

4

2

(

m,4H, ArH), 7.34-8.52 (m, 4H, Ar-NO ), 8.52(s1H,N=CH, azomet).

2

3

-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-

one derivatives 7(a-h): Triethylamine (0.005 mol, 0.795 ml) was added to a solution of

Schiff’s base 6(a-h) (0.01 mol) in acetone followed by drop wise addition of a solution

of chloroacetyl chloride (0.01 mol, 1.13 ml) with stirring. The mixture was refluxed upto

6

hrs and the reaction progress was monitored via TLC till single spot. The contents

were then poured onto the crushed ice. The solid material was filtered, washed with

petroleum ether and recrystallized by ethanol to obtain compounds 7(a-h).

a]2-[4-[4-[2-chlorophenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4

oxadiazole (7a): Obtained in 64% as Brownish crystals; mp (°C): 176-177; IR (Nujol,

5

Original Article

Thieme

-1

cm ): 3026 (Ar C-H str ), 2928 (C-H str), 1517(Ar C=C str), 1275(C-O),1055 (C-O-C str

in oxadiazole), 1677 (C=N), 642(C-Cl mono substituted in benzene), 1751(C=O str in

1

azetidinone), 734 (C-Cl); H NMR (DMSO, 400MHz, ppm): 8.23-8.36 (m,3H,CH of

pyrazine ), 5.27 (s, 2H, CH ), 7.05-7.14 (m,4H, phenyl), 7.19-7.42 (m,4H,CH in phenyl),

2

+

5

.14 (s,1H,CH-Ar), 5.75-5.76 (d,1H, CH-Cl of azetidinone). Ms (m/z): 468(M ), 470

(

M+2) other fragments peak are 320, 290, 214, 196, 179, 163, 147, 103, 79, 69. Anal.

Calcd. for C H Cl N O C, 56.43; H, 3.23; N, 14.96 Found: C,56.47; H, 3.24; N, 14.99.

22

15

2

5

3

b]2-[4-[4-[4-chlorophenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazo

-1

le (7b): Obtained in 70% as Pale Brownish crystals; mp (°C): 162-164; IR (Nujol, cm ):

015 (Ar C-H str ), 2900 (C- H str), 1492,1573 (Ar C=C str ), 1235 (C-O),1084 (C-O-C

3

str in oxadiazole), 1645 (C=N), 710 (C-Cl monosubstituted in benzene), 1738 (C=O str

1

in azetidinone), 1107.19, 782 (C-Cl); H NMR (DMSO, 400MHz, ppm): 8.23-8.37

(

m,3H,CH of pyrazine ), 5.29 (s, 2H, CH ), 6.98 -7.14 (m,4H, phenyl), 7.16-7.39

2

m,4H,CH in phenyl), 5.18 (s,1H,CH-Ar), 5.76-7.77 (d,1H, CH-Cl of azetidinone). Ms

+

m/z): 468(M ), 470(M+2) other fragments peak are 320, 290, 214, 196, 179, 163, 147,

1

03, 79, 69. Anal. Calcd. for C H Cl N O C, 56.43; H, 3.23; N,14.96 Found: C,56.47;

22 15 2 5 3

H, 3.27; N, 15.00.

c] 2-[4-[4-phenyl-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole (7c):

-

1

Obtained in 58% as Brownish crystals; mp (°C): 209-210; IR (Nujol, cm ): 3050 (Ar C-H

str ), 2926 (C- H str), 1511, 1571(Ar C=C str ), 1273(C-O), 1086 (C-O-C str in

1

oxadiazole), 1637 (C=N), 1739 (C=O str in azetidinone), 678 (C-Cl); H NMR (DMSO,

4

00MHz, ppm): 8.23-8.37 (m,3H,CH of pyrazine), 5.29 (s, 2H, CH ), 6.99 -7.14 (m,4H,

2

phenyl), 7.16-7.37 (m,5H,CH in phenyl), 5.19 (s,1H,CH-Ar), 5.75-5.76 (d,1H, CH-Cl of

+

azetidinone). Ms (m/z): 433(M ), 435 (M+2) other fragments peak are 253, 180, 177,

1

61, 152,147, 103, 79, 68. Anal. Calcd. for C H ClN O C, 60.91; H, 3.72; N,16.14

22 16 5 3

Found: C,60.89; H, 3.75;N,16.18.

d]2-[4-[4-[4-hydroxyphenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadia

-1

zole (7d): Obtained in 55% as Light Brown crystals; mp (°C): 175-176; IR (Nujol, cm ):

026 (Ar C-H str ), 2919 (C- H str), 1498 (Ar C=C str ), 1192(C-O), 1093 (C-O-C str in

3

oxadiazole), 1670 (C=N), 1756 (C=O str in azetidinone), 732(C-Cl), 3208 (Ar-OH str). ;

1

H NMR (DMSO, 400MHz, ppm): 8.23-8.36 (m,3H,CH of pyrazine ), 5.29(s, 2H, CH₂),

6

.89-7.15 (m,4H, phenyl), 7.17-7.38 (m,4H,CH in phenyl), 5.18 (s,1H,CH-Ar), 5.75-5.76

+

(

d,1H, CH-Cl of azetidinone), 5.15 (s,1H,aromatic C-OH). Ms (m/z): 449(M ), 451 (M+2)

other fragments peak are 302, 272, 253,196, 168, 177, 161, 147, 103, 79, 68. Anal.

Calcd. for C H ClN O C, 58.74; H, 3.59; N, 15.57 Found: C,58.77; H, 3.63; N,15.55.

22

16

5

4

e]2-[4-[4-[3-nitrophenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole

-

1

(

7e): Obtained in 61% as Dark Brown crystals; mp (°C): 140-142; IR (Nujol, cm ): 3065

Ar C- H str ), 2922 (C-Haliph), 1531, 1591(Ar C=C str ), 1273 (C-O), 1053 (C-O-C str

in oxadiazole), 1754 (C=O str in azetidinone), 710 (C-Cl), 1531 (asymAr N-Ostr.), 1394

1

(

5

(

sym Ar N-Ostr.) ; H NMR (DMSO, 400MHz, ppm): 8.23-8.36 (m,3H,CH of pyrazine ),

.29 (s, 2H, CH ), 6.88 -7.14 (m,4H, phenyl), 7.57-8.11 (m,4H,CH in phenyl), 5.18

2

+

s,1H,CH-Ar), 5.76-7.77 (d,1H, CH-Cl of azetidinone). Ms (m/z): 478 (M ), 480 (M+2)

other fragments peak are 331, 301, 253, 225, 177, 161, 147, 122, 103, 79, 68. Anal.

Calcd. for C H ClN O C, 55.18; H, 3.16; N, 17.55 Found: C, 55.22; H, 3.19; N, 17.59.

22

15

6

5

6

f]2-[4-[4-[4-methylphenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxa

-1

diazole (7f): Obtained in 54% Yellowish crystals; mp (°C): 152-154; IR (Nujol, cm ):

2993 (Ar C-H str), 2807 (C- H str), 1367(C-Hstr.) 1455, 1498 (Ar C=C str ), 1201(C-O),

1

089 (C-O-C str in oxadiazole), 1646 (C=N), 1737 (C=O str in azetidinone), 720

1

(

C-Cl).; H NMR (DMSO, 400MHz, ppm): 8.22-8.37 (m,3H,CH of pyrazine) , 5.29(s, 2H,

CH ), 6.96- 7.18 (m,4H, phenyl), 7.19-7.21 (m,4H, CH in phenyl), 5.16 (s,1H,CH-Ar)

2

+

5

.76-7.77 (d,1H, CH-Cl of azetidinone), 2.39 (s,3H, CH ). Ms (m/z): 447(M ), 449(M+2)

3

other fragments peak are 300, 270, 253, 194, 177, 166, 161, 147, 103, 91, 79, 68. Anal.

Calcd. for C H ClN O C, 62.56; H, 5.46; N, 14.59 Found: C, 62.60; H, 5.49; N, 14.63.

25

26

5

3

g]2-[4-[4-[4-methoxyphenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadia

-1

zole (7g): Obtained in 62% as Pale Brownish crystals; mp (°C): 134-135; IR (Nujol, cm ):

010 (Ar C-H str ), 2944 (C- H str), 1494, 1527 (Ar C=C str ), 1081 (C-O-C str in

3

oxadiazole), 1651 (C=N), 1789(C=O str in azetidinone), 724 (C-Cl), 1162 (C-O str. in

1

ether); H NMR (DMSO, 400MHz, ppm): 8.23-8.36 (m,3H,CH of pyrazine ), 5.29 (s, 2H,

CH ), 6.99 -7.14 (m,4H, phenyl), 7.16-7.36 (m,4H,CH in phenyl), 5.20 (s,1H,CH-Ar),

2

+

5

.74-5.75 (d,1H, CH-Cl of azetidinone), 3.77 (s,3H, CH ). Ms (m/z): 463(M ), 465(M+2)

3

other fragments peak are 320, 290, 253, 214, 196, 177, 161, 147, 103, 79, 68. Anal.

Calcd. for C H ClN O C, 59.55; H, 3.91;N, 15.10 Found: C, 59.59; H, 3.90;N, 15.13.

23

18

5

4

h]2-[4-[4-[2-nitrophenyl]-3-chloroazetidinonyl]phenyloxymethyl]-5-pyrazyl-1,3,4-oxadiazole

-

1

(

7h): Obtained in 67% as Brown crystals; mp (°C): 171-172; IR (Nujol, cm ): 3065 (Ar

C-H str ), 2922 (C- H str), 1531, 1581(Ar C=C str ), 1273(C-O),1053 (C-O-C str in

oxadiazole), 1655 (C=N), 1757 (C=O str in azetidinone), 701(C-Cl), 1531(asym Ar

1

N-Ostr.), 1384(sym Ar N-Ostr.).; H NMR (DMSO, 400MHz, ppm): 8.23-8.37 (m ,3H,CH

of pyrazine ), 5.27(s, 2H, CH ), 6.91-7.18 (m, 4H, phenyl), 7.51-8.21 (m,4H,CH in

2

+

phenyl), 5.18 (s,1H,CH-Ar), 5.76-7.77 (d,1H, CH-Cl of azetidinone). Ms (m/z): 478(M ),

4

6

1

80(M+2) other fragments peak are 316, 286, 253, 210, 182, 177, 161, 147, 103, 79,

8. Anal. Calcd. for C H ClN O C, 55.82; H, 3.87; N, 16.98 Found: C, 55.86; H, 3.91; N,

23

19

6

5

7.02.

Antimicrobial Evaluation

In vitro anti-bacterial activity

Test strain

The various test microorganisms Bacillus subtilis (MTCC121), Staphylococcus aureus

MTCC737), Escherichia coli (MTCC1687) and Pseudomonas aeruginosa (MTCC1688) were

(

obtained from the Department of Microbiology, MM Institute of Medical Sciences and

Research, MMU, Ambala, India, and used to evaluate the antibacterial potential of the

synthesized compounds. Cultures of the test bacteria were grown on Nutrient agar

7

Original Article

Thieme

(

NA) slant and incubated at 37°C for 1-3 days, depending upon the growth period of

bacteria.

Evaluation technique

43,44

The antibacterial evaluation was carried out by using agar cup-plate method.

Sample solution used was prepared by dissolving each test compound (50 mg) in DMSO

50 ml, 1000 μg /ml). For all compounds the sample size was fixed at 0.1 ml. Cups of 6

(

mm diameter were scooped out using a sterilized cork borer in agar medium contained

in a petri dish which was previously inoculated with the microorganisms. The test

compound solution (0.1 ml) was added in the cups and petri dishes were incubated at

37°C for 48 hours. Ampicillin was used as reference drugs and dimethylformamide as a

negative control. Zone of inhibition produced by each compound was measured in mm

and the averages based on triplicate measurements were recorded.

In vitro anti-fungal activity

Test strain

The clinical isolates of Aspergillus niger (MTCC281) and Candida albicans (MTCC183) were

obtained from the Department of Microbiology, MM Institute of Medical Sciences and

Research, MMU, Ambala, India. The isolate was subcultured on Sabouraud Dextrose

Agar (Hi-Media) at 37°C for 48-72 hours.

Evaluation technique

All the synthesized compounds were assessed for their in vitro anti-fungal activity

against pathogenic fungi using cup plate method with Sabouraud’s dextrose agar

8

43, 44

media.

Different concentrations of compounds in DMSO were added into each

labeled well (DMSO, solvent as a control) and incubated at 37°C for 72 hours.

Experiment was carried in triplicate. The zone of inhibition was calculated by measuring

the diameter of the zone using Miconazole as standard drug.

Minimum Inhibitory Concentration (MIC)

Minimum Inhibitory Concentration (MIC) was calculated using a method as reported

43, 44

earlier

Nutrient agar was prepared first, sterilized, and then cooled to 45°C with

gentle shaking. It was inoculated with microorganism culture before pouring into the

sterilized petri dishes. The culture media were allowed to set and thereafter the cups

were made by punching into the agar surface with sterile cork borer and scooping out

the punched part of the agar. Each test compounds (0.1ml) was added into the cups.

Two fold diluted solutions of the synthesized compounds (6.5, 12.5, 25, 50, 100, 200,

4

00, 800, and 1600 µg/ml) and reference drugs were used. The drug solutions were

allowed to diffuse into the medium for some time. The plates were incubated at

0-35°C for 24-48 hours. MIC values were determined at the end of the incubation

3

period.

Anti-tubercular activity:

Drug susceptibility of MIC of the synthesized compounds against M. tuberculosis H RV

37

45

(

MTCC200) were performed by Lowenstein-Jensen (L.J) Agar (MIC) method, where

primary 1000, 500 and 250 mg/ml and secondary 200, 100, 62.5, 50, 25, 12.5, 6.25

and 3.25 mg/ml dilutions of each test compound were added to liquid L. J. Medium and

then media were sterilized by inspissations method. A culture of M. tuberculosis H37Rv

9

Original Article

Thieme

growing on L. J. medium was harvested in 0.85% saline in bijou bottles. First stock

solution of 2000 mg/ml concentration of all test compounds were prepared in

DMSO.These tubes were then incubated at 37°C for 24 h followed by streaking of M.

tuberculosis H37Rv (5×104 bacilli per tube). Incubation of these tubes was then done at

37°C. After 12 days, 22 days and finally 28 days of incubation growth of bacilli was seen.

Tubes having the compounds were compared with control tubes where medium alone

was incubated with M.tuberculosis H RV (MTCC200) bacillus. The concentration at

37

which no development of colonies occurred or <26 colonies was taken as MIC

concentration of test compound. Also the strain M.tuberculosis H RV (MTCC200) was

37

tested with drug isoniazide and rifampicin as standard drugs.

List of Abbreviations

Aliph-

Anal.-

Bend-

Aliphatic

Analysis

Bending

Calcd- Calculated

CDCl Deuterated chloroform

DMSO- Dimethyl sulfoxide

3

-

FT-IR-

Fourier transform infra red

INH-Isoniazide

L.J.- Lowenstein-Jensen

LC-MS- Liquid chromatography–mass spectrometry

m/z-Mass / charge

MDR-

MeOH- Methanol

MHz- Megahertz

Multi drug resistant

MIC-Minimum inhibitory concentration

MP- Melting point

Ms- Mass spectrum

NA- Nutrient agar

NMR-

PAS-p-aminosalicylic acid

ppm- Parts per million

Nuclear magnetic resonance

PZA-Pyrazinamide

Str- Stretching

TB- Tuberculosis

TLC- Thin layer chromatography

TMS-

UV- Ultra violet

XDR- Extensively drug resistant

Tetramethylsilane

1

0

Article Doi

Evaluation and Docking Study of Pyrazine Containing 1, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class of Potential Antimicrobial and Antitubercular

DOI: 10.1055/a-1252-2378

Source and publish data:

Authors:

Article abstract of DOI:10.1055/a-1252-2378

Full text of DOI:10.1055/a-1252-2378

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