Design, synthesis and biological evaluation of 1,2,3-triazole based 2-aminobenzimidazoles as novel inhibitors of LasR dependent quorum sensing in: Pseudomonas aeruginosa

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Design, synthesis and biological evaluation of 1,2,3-

triazole based 2-aminobenzimidazoles as novel

inhibitors of LasR dependent quorum sensing in

Pseudomonas aeruginosa†

Cite this: RSC Adv., 2019, 9, 29273

a

b

Singireddi Srinivasarao, Adinarayana Nandikolla, Shashidhar Nizalapur, Tsz Tin Yu,

c

d

b

Sravani Pulya, Balaram Ghosh, Sankaranarayanan Murugesan, Naresh Kumar

and Kondapalli Venkata Gowri Chandra Sekhar

a

*

Bacteria regulate their phenotype, growth and population via a signalling pathway known as quorum sensing. In

this process, bacteria produce signalling molecules (autoinducers) to recognize their population density.

Inhibiting this quorum sensing signalling pathway is one of the potential methods to treat bacterial infection.

2-Aminobenimdazoles are reported to be the strongest inhibitors of quorum sensing against wild-type P.

aeruginosa. 1,2,3-Triazole based acyl homoserine lactones are found to be good inhibitors of the quorum

sensing LasR receptor. Hence, in our current study, forty 1,2,3-triazole based 2-aminobenzimdazoles were

synthesized and characterized using IR, NMR, MS and elemental analysis. A single crystal was developed for

N-(1H-benzo[d]imidazol-2-yl)-2-(4-nonyl-1H-1,2,3-triazol-1-yl)acetamide (6d). All ﬁnal compounds were

screened for in vitro quorum sensing inhibitory activity against Pseudomonas aeruginosa. The quorum

sensing inhibitory activity was determined in the LasR expressing P. aeruginosa MH602 reporter strain by

measuring green ﬂuorescent protein production. Among the title compounds, N-(1H-benzo[d]imidazol-2-yl)-

2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)acetamide (6i) exhibited good quorum sensing inhibitory activity

of 64.99% at 250 mM. N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-nitrophenyl)-1H-1,2,3-triazol-1-yl)acetamide (6p)

exhibited the most promising quorum sensing inhibitory activity with 68.23, 67.10 and 63.67% inhibition at

2

50, 125 and 62.5 mM, respectively. N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-

triazol-1-yl)acetamide (6o) and N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(4-(triﬂuoromethyl)phenyl)-

H-1,2,3-triazol-1-yl)acetamide (7l) also exhibited 64.25% and 65.80% quorum sensing inhibition at 250 mM.

1

Received 4th July 2019

Accepted 6th September 2019

Compound 6p, the most active quorum sensing inhibitor, also displayed low cytotoxicity at the tested

concentrations (25, 50 and 100 mM) against normal human embryonic kidney cell lines. Finally, a docking

study using Schrodinger Glide elucidated the possible putative binding mode of the signiﬁcantly active

compound 6p at the active site of the target LasR receptor (PDB ID: 2UV0).

DOI: 10.1039/c9ra05059k

rsc.li/rsc-advances

1

. Introduction

Many pathogenic bacteria use signaling strategies to multiply

their number in a process known as Quorum sensing (QS). In

this process, bacteria produce and release chemical signaling

molecules, known as auto-inducers (AIs). These signaling

a

Department of Chemistry, Birla Institute of Technology and Science, Pilani,

Hyderabad Campus, Jawahar Nagar, Kapra Mandal, Hyderabad

– 500078,

Telangana, India. E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs.bits@gmail.com; chemical molecules play a vital role in both bacterial virulence

Tel: +91 40 66303527

and symbiosis and have a noteworthy impact on human health,

b

1–3

School of Chemistry, UNSW Sydney, NSW 2052, Australia

cultivation and the environment.

At high bacterial cell

c

Department of Pharmacy, Birla Institute of Technology and Science, Pilani,

density, bacteria alter gene expression levels to coordinate

assorted behaviors, including biolm formation, virulence

factor production, bioluminescence, conjugation, sporulation,

Hyderabad Campus, Jawahar Nagar, Kapra Mandal, Hyderabad-500078,

Telangana, India

d

Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of

4

–7

etc. Gram-negative bacteria cause several nosocomial infec-

Technology and Science, Pilani, 333031, India

†

Electronic supplementary information (ESI) available: Experimental procedures tions including bloodstream infections, pneumonia, wound or

8,9

for intermediates (chemistry) and spectroscopic/analytical data. CCDC 1904042. surgical site infections. P. aeruginosa, an opportunistic path-

For ESI and crystallographic data in CIF or other electronic format see DOI:

ogen and second leading cause of ventilator associated

1

0.1039/c9ra05059k

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pneumonia is responsible for several serious infections in inhibition at 100 mM. In conclusion, this group noticed that

immune compromised patients, chronic lung infection (cystic compounds with methylene group between N-acyl group and

3

1



brosis) and for the most common fatal communicable 1,2,3-triazole nucleus is indispensable for QSI activity, while

10–13

disease.

As P. aeruginosa develops resistant to available compounds with absence of methylene group and N-acyl group

32,33

carbapenem and other antibiotics, it is listed as critical by the exhibited poor QSI activity (compounds

V

and VI).

14

World Health Organization. Bacterial resistance may cause Compounds VII and VIII are active QSIs with 50.7% and 49.7%

prolonged hospitalization, expensive medical costs and inhibition at 250 mM against P. aeruginosa MH602 respectively.

34

increase in mortality and hence, there is an urgent need to look In another recent work, two methylene linkers retained between

out for non-antibiotic solutions to manage invasive bacterial triazole nucleus and N-acyl-2-phenyl indole gave the most active

infections and QS could be a potential solution. QSIs are QSI with 60.8% inhibition at 250 mM against P. aeruginosa

24

attractive alternatives to replace the conventional antibiotics to MH602 (compound IX). Compound X, with one methylene

1

5,16

minimize the development of bacterial resistance.

So far, linker, is an another active QSI with 74.2% inhibition at 250

35

many QSIs have been reported but only very few entered clinical mM. Based on these results, we retained at least one methylene

trials. Many proteobacteria which includes P. aeruginosa use linker between 1,2,3-triazole and N-acyl cycloalkane and

N-acylated-L-homoserine lactones (AHLs/AIs) as QS signaling designed the compounds of the current scheme (Fig. 3). 1,2,3-

17

18

molecules. (Fig. 1).

Triazole based QSIs are summarized in Table 1.

P. aeruginosa uses LasI/LasR, RhlI/RhlR and PQS/MvfR QS

Enthused by the QSI active molecules of 2-amino-

systems to alter gene expression and is responsible for pro- nobenzimdazoles and 1,2,3-triazole analogues, we incorporated

longed hospitalization and high mortality in hospital these active pharmacophores into one framework, and synthe-

19–21

patients.

N-(3-Oxododecanoyl)-L-homoserine

lactone sized non-AHL based QSIs, as shown in Fig. 3. In this work, we

(

OdDHL) and N-butanoyl-L-homoserine lactone (BHL) are the synthesized various substituted 1,2,3-triazoles of N-(1H-benzo

22

AIs of P. aeruginosa (Fig. 2).

[d]imidazol-2-yl) and N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)

acetamide, propanamide and butamides and screened them

for their QSI activity against P. aeruginosa MH602 strain. Several

N-acyl analogues of cyclopentyl, cyclohexyl and amino pyridines

Design

Several AHL and non-AHL based QSIs against P. aeruginosa are

reported till date.

occurs at high cell-density and higher cell density is responsible

for QS.

active biolm inhibitors, 2-aminobenzimidazole (I) and 5,6-

dimethyl-2-aminobenzimidazole (II) and evaluated for QSI

activity against wild-type P. aeruginosa QS reporter strains.

3

4

also exhibited promising QSI activity against P. aeruginosa.

2

3,24

Biolm formation of pathogenic bacteria

Hence, as a part of this work, we also introduced triazole based

N-acyl cyclopentane, cyclohexane and pyridine as the tail groups

of aminobenzimdazole to gauge their importance in exhibiting

QS inhibition.

25–27

Based on this, Blackwell group identied two most

Among these, compound II possesses higher inhibition against 2. Results and discussion

Las and Rhl QS systems with IC50 values 37.5 mM and 12.5 mM

Chemistry

28,29

respectively.

Furthermore, the same group reported that

As shown in Scheme 1, rst compounds 2a and 2b were

prepared according to literature procedure from starting

materials 1a and 1b respectively. 2a and 2b upon treatment

with chloroacetyl chloride yielded N-acyl compounds 3a and 3b

respectively. To obtain fourth intermediate, compounds 5a and

compound II is a potential inhibitor of LasR activity with IC₅₀

2

.3 mM against P. aeruginosa PAO-JP2 and 1.4 mM against P.

3

6

29

aeruginosa PAO-JG21 (Fig. 3).

1,2,3-Triazoles are reported as QSIs, antitubercular agents,



uorinated hydrogels and their applications in radiochemistry

5b from second 3a and 3b, we initially performed several reac-

30–35

was also studied previously.

In 2013, Stacy et al. reported

tions by varying the reaction conditions, which includes the use

of diﬀerent solvents (acetonitrile, tetrahydrofuran, dime-

thylformamide etc.) and temperatures. All these conditions

required high temperatures and seal tube/microwave irradia-

tion with long time. Furthermore, KI was used as the catalyst

AHL based 1,2,3-triazoles as QSIs, in which, they retained the

lactone ring of AHL and its amide bond and modied the side

chain with various substituted 1,2,3-triazoles and reported

eﬀective QSIs against P. aeruginosa. Further, they have clearly

highlighted that inclusion of triazoles led to superior activity

(Finkelstein reaction) and the reaction was carried out in an

31

than their counter parts against LasR. Compounds III and IV

ꢀ

aprotic solvent in seal tube at 120 C. It took 72 h for the reac-

tion to proceed but only gave 35% yield. Since satisfactory yields

were not obtained even aer varying the conditions, we decided

to synthesize 5a and 5b in two steps. 3a and 3b were dissolved in

acetone and water to give a clear solution, followed by the

addition of 1.2 equivalent of KI and the reaction mixture was

are the most active QS inhibitors (Fig. 3), with 75% QS

ꢀ

heated up to 55 C for 12 h. On completion of reaction, as

indicated by TLC, 4a and 4b were isolated. Then pre-nal azides

5

a and 5b were prepared from 4a and 4b using NaN . Final

3

compounds 6a–q from 5a and 7a–n from 5b were obtained on

treatment with various acetylenes in 45–91% yield (Scheme 1).

Fig. 1 Autoinducers in proteobacteria.

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Fig. 2 Auto-inducers in P. aeruginosa.

As shown in Scheme 2, in order to get amides 9a–c, we per-

The purity of the synthesized compounds was assessed by

formed acid-amine coupling reaction using EDC$HCl and HOBt LC-MS and elemental analyses. Structures of the compounds

1

13

as coupling reagents. Compound 9a was prepared from 1H- were conrmed by spectral data. In H NMR and C NMR, the

benzo[d]imidazol-2-amine (2a) and pent-4-ynoic acid (8a). signals of the corresponding protons and carbon atoms were

Similarly, 2a and 2b on treatment with hex-5-ynoic acid (8b) veried based on their chemical shi, multiplicities and

yielded compounds 9b and 9c respectively. Finally, the 9a, 9b coupling constant values. The results of elemental analysis were

and 9c on treatment with various substituted azides using within ꢁ0.05 of the theoretical values.

1

copper sulphate pentahydrate as catalyst gave 10a–b, 11a–d and

H NMR spectra of compounds 6a–q and 7a–n from Scheme

1 showed singlet which integrates to two protons at 5–6 ppm

12a–c in 45–68% yield.

2

Most of the azides required for triazole preparation were due to the active methylene CH , whereas the methylene

procured commercially. However, azides A, B and C required for protons for compounds 10–12 from Scheme 2 were observed at

the preparation of the nal derivatives of Series-III were more up eld (at around 1.5 to 3.5 ppm) due to a larger sepa-

accomplished via Scheme 3.

ration of methylene protons from N-acyl group and triazole

nucleus. All nal compounds from Schemes 1 and 2 have

Fig. 3 Design strategy for the synthesis of titled quorum sensing inhibitors.

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Table 1 1,2,3-Triazole based QSIs against P. aeruginosa reported from literature

Entry

Compound number

Structures of QSIs

% Inhibition against P. aeruginosa

Reference no.

31

1

III

75% at 100 mM

2

3

IV

V

75% at 100 mM

31

32

33

Ranged from 0 to 49.9% at 1 mM concentration

Ranged from 1 to 22% at 100 mM

4

5

VI

n ¼ 1; VI

n ¼ 2; VII

50.7% at 250 mM

49.7% at 250 mM

34

VII & VIII

6

7

IX

60.8% at 250 mM

24

35

X

74.2% at 250 mM

Scheme 1 General synthetic route for compounds 6a–q and 7a–n. Reagents and conditions: (i) cyanogen bromide (1.1 eq.), methanol, reﬂux,

ꢀ

5

min; (ii) chloroacetyl chloride (1.0 eq.), pyridine (2.5 eq.), CH

2

Cl

2

, 0 C-rt, 12 h; (iii) potassium iodide (1.2 eq.), acetone, 55 C, 12 h; (iv) NaN

3

(1.5

ꢀ

eq.), DMF : water (8 : 2), 0 C; (v) substituted terminal alkynes (1.4 eq.), CuSO

4

$5H

2

O, (5 mol%), sodium ascorbate (10 mol%), DMF : water (8 : 2),

rt, 6–16 h.

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Scheme 2 General synthetic route for compounds 10a–b, 11a–d and 12a–c. Reagents and conditions: (vi) 2a or 2b (1.0 eq.), EDC$HCl (1.5 eq.)

HOBt, (1.5 eq.) diisopropyl ethylamine (2.5 eq.), DMF, rt, 16 h; (vii) 9a/9b/9c and substituted azides (1.5 eq.), CuSO

4 2

$5H O, (10 mol%), sodium

ascorbate (10 mol%), DMF : water (8 : 2), rt, 6–16 h.

triazole nucleus, benzimidazole N–H and amide N–H, in which signicant eﬀect on the QSI activities of the compound as

the proton of triazole nucleus was observed at around 8.2– compounds 6a–d possess similar percentage of QS inhibition

9.2 ppm as singlet, and the N–H protons of amide and imid- within diﬀerent tested concentrations. The eﬀect of attaching

azole were observed as broad singlets at 11–14 ppm. Further- a terminal heterocyclic group on the triazole partner of the

more, single crystal was developed for compound 6d.

molecule was then studied. While attaching a 2-methylthio-

benzimidazole onto the triazole ring (6e) gave a similar QSI

activity (41.31% inhibition at 250 mM) as compounds 6a–

d (42.76–50.50% inhibition at 250 mM), it was found that

attaching 5-chloro-pyridone on the triazole nucleus (6f)

enhanced the QSI activity of benzimidazole to 58.84% at 250

mM. The QSI activities of benzimidazoles having various

substituted aryl groups on triazole nucleus (6g–q) were also

investigated. Halo-substitution at the para-position of aryls on

the triazole nucleus (6h–j) showed promising QSI activity even

at low concentration (>45.00% inhibition at 62.5 mM) when

compared to its un-substituted parent molecule 6g, with only

Biological activity

Series of triazole based benzimidazole derivatives (6a–q, 7a–n,

10a–b, 11a–d and 12a–c) were analyzed for their QS-inhibition

activity at various concentrations (62.5, 125 & 250 mM) against

P. aeruginosa MH602 lasB reporter strain [PlasB: gfp(ASV)]

following the protocol developed by Hentzer et al. The

production of AHL signals by this reporter strain leads to an

increase in the production of unstable green uorescent protein

37

(GFP-ASV) as a function of an active QS system. Considering this

fact, synthesis of benzimidazole triazole analogues has been

prioritized to counteract the QS system. The results are

compiled in Table 2.

15.10% inhibition at 62.5 mM. Furthermore, among these halo-

aryl derivatives, chloro-derivative (6i) exhibited the highest QSI

activity at all concentrations. Alkyl groups substituted at the

para-position of aryl ring (6k–l) diminished the QSI activity of

the benzimidazoles. In fact, when a tert-butyl group was placed

on the aryl ring of the triazole nucleus, the corresponding

benzimidazole (6l) showed no QSI activity at lower concentra-

tions. To study the eﬀect of aryl rings carrying electron-releasing

groups on the QSI activity, benzimidazoles 6m and 6n bearing

Predictive structure–activity relationship study (SAR)

In the rst series (Series-I in Table 2), benzimidazoles 6a–q with

no substitution on the benzene ring were undertaken. The eﬀect

of alkyl chain length on the triazole partner of the molecule was



rst studied. It was found that changing the lipophilicity by

a

phenol and anisole group, respectively, were tested.

altering the length of the alkyl chain on triazole ring has no

Scheme 3 General synthetic route for intermediates A, B and C.

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Table 2 Percentage inhibition of GFP ﬂuorescence by the synthesized compounds against P. aeruginosa MH602

a

Concentration (mM)

Entry

R

1

X

n

250

125

62.5

Series-I

6

a

b

c

—

H

—

50.50 ꢁ 11.81

43.47 ꢁ 6.23

42.76 ꢁ 14.58

48.55 ꢁ 12.66

35.28 ꢁ 20.43

38.77 ꢁ 11.47

34.69 ꢁ 11.60

41.93 ꢁ 4.50

25.10 ꢁ 0.37

27.37 ꢁ 2.36

25.68 ꢁ 7.62

30.61 ꢁ 7.62

d

6

e

f

—

H

—

41.31 ꢁ 7.90

58.84 ꢁ 5.09

37.20 ꢁ 6.71

52.70 ꢁ 11.86

24.87 ꢁ 6.69

36.07 ꢁ 10.70

6

g

—

H

—

45.95 ꢁ 10.15

56.14 ꢁ 1.77

17.43 ꢁ 21.96

54.85 ꢁ 2.42

15.10 ꢁ 5.83

48.95 ꢁ 12.32

h

6

i

—

H

—

64.99 ꢁ 8.25

60.55 ꢁ 6.16

52.86 ꢁ 8.86

6

j

H

—

50.74 ꢁ 10.91

35.85 ꢁ 13.83

35.22 ꢁ 8.78

46.25 ꢁ 8.52

32.32 ꢁ 22.23

NA

45.68 ꢁ 5.44

36.82 ꢁ 6.63

NA

k

l

—

6

m

—

H

—

58.44 ꢁ 7.75

20.03 ꢁ 14.00

64.25 ꢁ 7.30

45.66 ꢁ 6.21

5.16 ꢁ 16.63

59.85 ꢁ 7.99

34.37 ꢁ 4.97

NA

n

o

57.55 ꢁ 7.73

6

p

q

—

H

—

68.23 ꢁ 3.87

39.37 ꢁ 5.14

67.10 ꢁ 5.45

36.04 ꢁ 6.13

63.67 ꢁ 7.51

28.21 ꢁ 10.23

Series-II

7

a

b

c

—

CH

3

—

31.76 ꢁ 10.99

26.59 ꢁ 20.80

NA

22.05 ꢁ 14.66

20.22 ꢁ 11.52

NA

19.85 ꢁ 3.80

16.92 ꢁ 5.74

NA

d

44.57 ꢁ 3.87

41.65 ꢁ 5.08

37.11 ꢁ 7.75

7

e

f

—

CH

3

—

16.00 ꢁ 26.10

36.80 ꢁ 7.90

NA

15.13 ꢁ 9.82

26.08 ꢁ 7.69

7

CH

29.03 ꢁ 13.57

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Table 2 (Contd.)

a

Concentration (mM)

Entry

R

1

X

n

250

125

62.5

7

g

—

CH

3

—

47.56 ꢁ 14.43

57.60 ꢁ 7.43

29.85 ꢁ 17.40

44.67 ꢁ 14.27

28.51 ꢁ 8.41

31.23 ꢁ 5.02

7

h

CH

7

i

j

—

CH

3

—

50.74 ꢁ 12.31

44.73 ꢁ 13.15

14.91 ꢁ 8.82

24.16 ꢁ 14.52

NA

7

CH

17.62 ꢁ 6.21

7

k

l

—

CH

3

—

40.82 ꢁ 23.41

65.80 ꢁ 1.91

53.85 ꢁ 9.78

24.30 ꢁ 23.26

56.44 ꢁ 5.85

31.46 ꢁ 16.18

20.38 ꢁ 2.59

36.13 ꢁ 7.67

21.02 ꢁ 10.29

m

7

n

—

CH

3

—

46.73 ꢁ 4.87

34.42 ꢁ 14.10

34.20 ꢁ 7.43

Series-III

10a

10b

11a

11b

—

H

1

2

47.40 ꢁ 2.69

28.46 ꢁ 17.54

51.23 ꢁ 7.10

12.87 ꢁ 20.18

42.35 ꢁ 8.46

15.64 ꢁ 7.17

45.93 ꢁ 8.18

NA

38.43 ꢁ 7.72

4.40 ꢁ 2.93

42.27 ꢁ 10.29

NA

11c

11d

12a

—

H

2

19.41 ꢁ 8.16

39.38 ꢁ 4.60

48.48 ꢁ 5.25

15.69 ꢁ 8.81

21.37 ꢁ 2.62

34.14 ꢁ 12.60

3.69 ꢁ 11.15

21.17 ꢁ 1.76

28.54 ꢁ 7.69

H

CH

3

1

2b

2c

—

CH

3

2

30.83 ꢁ 26.38

9.74 ꢁ 20.67

5.35 ꢁ 10.20

—

CH

3

2

48.26 ꢁ 13.42

86.86 ꢁ 1.18

7.07 ꢁ 11.80

83.02 ꢁ 3.15

NA

Furanone 30

—

77.33 ꢁ 2.00

a

Growth inhibition, ꢁstandard deviation of the mean from at least two independent experiments. In each independent experiment, compounds

were tested in triplicate. NA: no activity.

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Fig. 4 Cytotoxicity study of the most active QSIs in HEK-293 cells by MTT assay after 24 h compound treatment. BG45 was used as a positive

control. Data represents mean ꢁ SD (n ¼ 3).

Benzimidazoles 6m with a phenol group present on the triazole attempt, various aryl groups bearing diﬀerent substituents on tri-

nucleus possesses moderate QSI activity at low concentrations azole nucleus were investigated. The presence of an electron-

(34.37% at 62.5 mM), however, when the phenol group was donating methyl group at the 4-position of the aryl ring (7i) did

substituted by a diﬀerent electron-releasing anisole group (6n), not impact the inhibitory activity at higher doses (50.74% at 250

the QSI activity of the molecule was completely abolished. This mM) when compared to its un-substituted parent molecule 7g, in

suggested that QSI activity of a compound could not be corre- fact, it hampered the QSI inhibition at lower concentrations. The

lated by attaching an electron-releasing group at the para- presence of other electron-donating groups such as hydroxyl (7j)

position of the phenyl ring on the triazole nucleus. Following and alkoxy (7k) at the 4-position of the phenyl ring were also found

this nding, the eﬀect of substituting electron withdrawing to be eﬀective only at higher concentrations (>40% at 250 mM). In

groups on the aryl ring towards QS inhibition was studied. case of aryl rings possessing electron withdrawing groups, tri-

Benzimidazole compound 6o bearing a triuoromethyl group uoromethyl group at para-position enhanced the QSI activity of

on the aryl ring exhibited a signicantly higher QSI activity the dimethylbenzimidazole (65.80% at 250 mM), while other nitro

(57.55% inhibition at 62.5 mM) when compared to its parent substituted aryl partners were eﬀective only at higher concentra-

compound 6g (with only 15.10% inhibition at 62.5 mM) and the tions (7m & 7n). It can be concluded that aryl substituted triazole

QSI activity is further enhanced by substituting more electron- nucleus based dimethylbenzimidazole derivatives displayed better

withdrawing nitro group at para-position of the phenyl ring, QS inhibition activity when electron withdrawing groups are

with compound 6p showed 63.67% inhibition at 62.5 mM. present on the aryl ring.

However, when the nitro group was moved to the meta-position

6q), QSI activity was diminished (28.21% at 62.5 mM). From the aminobenzimidazole derivatives were designed and synthe-

Apart from the above moieties studied so far, few more 2-

(

QSI data of this series, it can be concluded that, QSI activity of sized by altering the length of carbon chain between the

the benzimidazole molecule is favored by the presence of aryl amido functional group and the triazole moiety as well as

ring with a para-substituted electron-withdrawing group on the placing diﬀerent functionalities on triazole nucleus (Series-

triazole nucleus.

III). All these moieties were explored for their QSI at reported

In the second part of the series (Series-II), benzimidazoles 7a–n concentrations. In the rst part of this series, the chain

bearing di-methyl substitution on the benzene ring was considered length between the 2-amido benzimidazole and the triazole

as basic moiety and the eﬀect of substitution on the triazole nucleus was xed at two and two analogues 10a and 10b were

nucleus towards the QSI activity was investigated. When alkyl synthesized by retaining diﬀerent substitutions on the tri-

groups of diﬀerent lengths were placed on the triazole nucleus, di- azole nucleus. Among these, N-cyclohexyl acetamido substi-

methylbenzimidazole (compound 7d) with the longest carbon tution at the triazole (10a) showed good QSI activity

chain exhibited moderate activity (44.57% at 250 mM). The QSI regardless of the concentration (38–47% at 62.5–250 mM). In

activity of the compounds reduced while decreasing the alkyl chain the later series, the length of the carbon chain between the 2-

length. When introducing heterocyclic groups such as 2- amido benzimidazole and the triazole nucleus was increased

methylthio-benzimidazole (7e) and 5-chloro-pyridone (7f) onto the to three. Four benzimidazoles (11a–d) with the three carbon

triazole nucleus, the analogues only showed mild inhibitory chain length were synthesized and tested for their QSI

activity even at the highest tested concentration. In another activity. It was found that triazole carrying N-cyclopentyl

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Table 3 In silico predicted physico-chemical and pharmacokinetic parameters of the titled compounds

a

b

c

d

e

f

g

h

i

Entry

Mol. wt

SASA

HBD

HBA

log Po/w

PCaco

log BB

log S

Rot. bonds

6

7

1

a

b

c

d

e

f

g

h

i

j

k

284.32

567.436

636.971

711.635

743.435

584.577

530.836

564.382

567.032

586.704

589.559

594.968

636.388

570.734

629.418

598.845

610.671

630.124

631.212

671.361

753.598

826.137

774.439

546.498

593.336

623.302

620.804

591.857

617.631

700.508

639.666

559.1

0

1

0

1

0

1

0

1

0

2

1

2

3.5

5.0

6.5

3.5

4.25

3.5

4.5

3.5

5.0

6.5

3.5

4.25

3.5

4.5

8.0

9.0

8.0

9.0

6.0

8.0

9.0

3.038

3.92

343.769

429.085

498.442

305.705

605.209

267.446

357.559

353.871

351.095

354.108

375.045

358.01

ꢂ1.286

ꢂ1.412

ꢂ1.569

ꢂ1.907

ꢂ0.839

ꢂ0.934

ꢂ1.068

ꢂ0.952

ꢂ0.933

ꢂ0.911

ꢂ1.088

ꢂ1.138

ꢂ1.634

ꢂ1.094

ꢂ0.799

ꢂ2.146

ꢂ2.088

ꢂ1.228

ꢂ1.414

ꢂ1.573

ꢂ1.803

ꢂ1.994

ꢂ0.82

ꢂ4.161

ꢂ5.153

ꢂ6.242

ꢂ6.519

ꢂ4.174

ꢂ2.154

ꢂ4.428

ꢂ4.683

ꢂ5.163

ꢂ5.242

ꢂ5.008

ꢂ5.63

6

8

10

12

7

6

4

5

4

5

6

8

10

12

7

6

4

5

4

5

7

8

312.374

340.427

368.481

404.448

383.796

318.337

336.328

352.782

397.233

332.364

374.444

334.337

348.363

386.336

363.335

312.374

340.427

368.481

396.534

432.502

411.85

346.391

425.287

360.418

362.39

376.417

414.389

391.388

395.463

390.404

395.463

409.49

4.822

5.268

3.838

2.171

3.461

3.647

3.94

4.025

3.794

4.559

2.794

3.834

4.344

2.762

3.014

3.818

4.387

5.352

6.173

4.777

2.611

3.9

4.458

4.176

3.165

4.029

5.396

3.217

2.89

2.322

1.653

2.482

2.778

2.694

3.206

3.099

3.317

3.272

l

m

n

o

p

q

a

b

c

d

e

f

g

h

i

j

106.664

519.109

358.282

42.587

ꢂ4.336

ꢂ5.105

ꢂ5.59

ꢂ4.619

ꢂ4.988

ꢂ5.369

ꢂ5.715

ꢂ7.037

ꢂ8.086

ꢂ7.549

ꢂ2.398

ꢂ4.977

ꢂ5.879

ꢂ5.498

ꢂ4.71

61.546

475.547

413.083

506.822

496.042

146.42

358.622

361.81

ꢂ1.048

ꢂ0.935

ꢂ1.108

ꢂ1.59

341.091

339.567

104.014

420.983

514.904

43.007

93.403

85.992

229.916

90.546

87.077

104.37

49.823

110.476

87.762

107.993

k

l

ꢂ1.026

ꢂ0.833

ꢂ2.11

ꢂ4.882

ꢂ7.521

ꢂ5.169

ꢂ3.642

ꢂ4.778

ꢂ2.688

ꢂ4.838

ꢂ5.447

ꢂ5.789

ꢂ6.191

ꢂ6.492

ꢂ6.169

ꢂ6.752

m

n

0a

0b

1a

1b

1c

1d

2a

2b

2c

ꢂ1.454

ꢂ2.006

ꢂ1.278

ꢂ2.062

ꢂ2.169

ꢂ2.408

ꢂ2.531

ꢂ2.224

ꢂ2.177

ꢂ2.472

752.156

576.151

765.567

800.267

778.651

720.921

841.741

842.109

835.341

8

6

8

404.43

391.388

423.517

437.544

432.484

a

b

c

d

Mol. wt – molecular weight (130–725). SASA – solvent accessible surface area (300–1000). HBD – no. of hydrogen bond donors (0–6). HBA – no.

e

f

ꢂ1

of hydrogen bond acceptors (2 to 20). log Po/w – predicted octanol/water partition coeﬃcient (ꢂ2.0–6.5). Caco – 2 cell permeability in nm s (<25

g

h

ꢂ3

poor, >500 great). log BB – predicted brain/blood partition coeﬃcient (ꢂ3.0–1.2). log S – aqueous solubility of a compound (ꢂ6.5–0.5 mol dm ).

i

Rot – no. of rotatable bonds (0 to 15).

acetamido group (11a) was identied to be the most potent observed for compound 12b with a N-cyclohexyl acetamido

compound in this series and its potency was retained even at group placed at the triazole nucleus. On the other hand,

lower concentration (42.27% at 62.5 mM). On the other end, compound 12a exhibited moderate inhibition activity at 250

compound 11d with 4-nitrophenyl group on triazole nucleus mM. While its inhibition activity reduces with the decrease in

was active only at high doses (39.38% at 250 mM). The concentration, the magnitude of the reduction was smaller

remaining analogues 11b–c were less potent even at highest than that of compounds 12b–c. It can be concluded that N-(2-

tested concentration. Finally, by maintaining three carbon pyridyl), N-cyclopentyl and N-cyclohexyl acetamido

chain length, various dimethylbenzimidazoles were also substituted triazoles exhibited moderate QSI activity at high

investigated. Dimethylbenzimidazoles possessing N-(2- concentration within this series.

pyridyl)-acetamido group on triazole nucleus (12c) showed

moderate inhibition activity only at high concentration study possess moderate to high inhibition activity at high

48.26% at 250 mM) and its inhibition activity was diminished concentration and compounds 6i, 6o, 6p and 7l exhibited signi-

when its concentration was halved. The same trend was cant QSI activity at all the reported concentrations and were

Overall, it can be concluded that, most of the scaﬀolds in this

(

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Fig. 5 Superimposed view of best scoring native pose of co-crystallized (grey) ligand with its X-ray pose (green) inside the active site of 2UV0.

identied to be the most active compounds in this study. Most of Cytotoxicity studies

the compounds reported in the current work exhibited comparable

activity to the previously reported triazoles as shown in Table 2.

The most active compound from this study, 6p is better than the

previously reported compounds (V–IX of Table 1).

On the basis of QS inhibitory activity, the signicantly active

compounds (6a, 6f, 6h, 6i, 6m, 6o, 6p, 7h, 7i, 7l, 7m and 10a)

were evaluated for their cytotoxicity against Human

Embryonic Kidney cell lines (HEK-293) at 25, 50 and 100 mM

Fig. 6 Docked pose view of co-crystallized ligand with interactions and distances (color interpretation: white-hydrogen bond, blue pi–pi

stacking) inside the active site of 2UV0.

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Fig. 7 Docked pose view of compound 6p inside the 2UV0, showing interactions and distances (color interpretation: white-hydrogen bond,

blue pi–pi stacking).

concentrations using BG45 as a positive control. Most of the In silico prediction of drug likeness properties

compounds have low cytotoxicity at all tested concentra-

tions using MTT assay (Fig. 4).

Physicochemical parameters of the designed compounds were

in silico predicted using Qik-prop module of Schr ¨o dinger.

The in silico predicted values of physico-chemical parameters

3

8

39,40

Fig. 8 Docked pose view of compound 11b inside the 2UV0, showing interactions and distances (color interpretation: white-hydrogen bond,

blue pi–pi stacking).

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Table 4 Crystal data and structure reﬁnement for 6d

Identication code

Empirical formula

Formula weight

Temperature/K

Crystal system

Space group

6d (exp_399-AB-4R)

C H N O

20 28 6

368.48

293(2)

Monoclinic

P2 /c

1

˚

a/A

18.8646(7)

5.4019(2)

20.0357(6)

90

90.614(3)

90

2041.61(12)

4

1.199

˚

b/A

˚

c/A

ꢀ

a/

b/

ꢀ

g/

3

˚

Volume/A

Z

3

rcalcg/cm

ꢂ1

m/mm

0.619

F(000)

Crystal size/mm

Radiation

792.0

3

0.5 ꢃ 0.02 ꢃ 0.01

CuKa (l ¼ 1.54184)

8.828 to 159.794

ꢀ

2Q range for data collection/

Index ranges

Reections collected

ꢂ23 # h # 23, ꢂ6 # k # 3, ꢂ23 # l # 25

10 329

Independent reections

Data/restraints/parameters

Goodness-of-t on F

4308 [Rint ¼ 0.0482, Rsigma ¼ 0.0635]

4308/0/245

1.063

2

Final R indexes [I $ 2s (I)]

Final R indexes [all data]

Largest diﬀ. peak/hole/e A

R

1

¼ 0.0671, wR

2

¼ 0.1867

¼ 0.2036

1

¼ 0.0834, wR

ꢂ3

˚

0.42/ꢂ0.27

for the titled compounds (6a–q, 7a–n, 10a–b, 11a–d, 12a–c) were

The predicted values of these parameters revealed that all the

shown in Table 3. Furthermore, ranges of these parameters parameters like mol. wt., hydrogen bond donors (HBD),

followed by 95% of market approved drugs are given as foot- hydrogen bond acceptors (HBA), solvent accessible surface area

notes of Table 3 (ref. 41 and 42) for comparison.

(SASA), partition co-eﬃcient (log P), predicted apparent Caco-2

Fig. 9 ORTEP crystal structure diagram of compound.

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cell permeability (PCaco), predicted brain/blood partition Single crystal X-ray crystallographic structure of compound 6d

coeﬃcient (log BB), aqueous solubility (log S) and number of

The suitable crystals of the compound 6d for X-ray crystallo-

rotatable bonds (Rot) were found within acceptable range and

graphic study were grown from the mixture of methanol and

followed Lipinski rule of ve. So overall, based upon the pre-

dichloromethane (1 : 1) solution. The single crystal X-ray

diﬀraction measurement of the molecule (C20H N O) was on

28 6

Rigaku XtaLAB P200 diﬀract meter using graphite mono-

dicted values of these physico-chemical parameters, all the

titled compounds possessed the drug-likeness behavior.

˚

chromated Cu-Ka radiation (l ¼ 1.54184 A) on 0.5 mm ꢃ

0

.02 mm ꢃ 0.01 mm colorless crystal. Data were collected and

Docking studies

processed using CrysAlisPro (Rigaku Oxford Diﬀraction). The

data were collected at a temperature of 293 K to a maximum 2q

We performed molecular docking to investigate the binding

mode of most active QSI 6p with LasR receptor by the X-ray

structure (PDB ID: 2UV0). For the docking study, we selected

co-crystal structure as the source of the target taken is from the

organism P. aeruginosa LasR and its resolution is better

compared to other available PDB structures. Moreover, all the

titled compounds were screened against the strain of P. aeru-

ginosa and hence we took the same species for docking to get

better correlation.

ꢀ

value of 159.794 . Of the 10 329 reections were collected,

where 4308 were unique (Rint ¼ 0.0482) and equivalent reec-

tions were merged. The crystal was kept at 293(2) K during data

collection. Using Olex2, the structure was solved with the ShelX

structure solution program using Direct Methods and rened

with the ShelXL renement package using least squares mini-

mization. The basic crystallographic data are shown in Table 4.

The molecular structure of the compound with methanol and

dichloromethane solvents of crystallization is given as an

ORTEP diagram in Fig. 9. The chain of nine carbons with C12 is

directly bonded to C11 with more rigidity. As we move from C12

to C20, since the molecule is continuously undergoing vibration

the terminal C20 with more exibility experiences higher degree

of vibrations as compare to C12. Therefore, the thermal

parameters increase as we go from C12 to C20 and it is highest

for C20. Crystallographic data for the compound 6d have been

deposited to the Cambridge Crystallographic Data Center and

corresponding deposition number is CCDC 1904042.

Docking studies of the signicantly active compound (6p)

and least active compound (11b) was performed using Glide 5.9

(ref. 43) running on maestro version 9.4, to investigate their

putative binding mode with the target protein. Enzyme used for

the docking study was LasR receptor protein (PDB: 2UV0) of P.

44

aeruginosa, retrieved from RCSB, Protein Data Bank. Protein

preparation wizard of Schr ¨o dinger suite was used for prepara-

tion of target protein.

In order to predict the nature of binding mode of signicant

and least active compounds (6p and 11b respectively) with the

target protein, docking studies were carried out. The value of

RMSD obtained between X-ray pose and re-docked pose (Fig. 5)

for co-crystallized ligand in target protein was found to be 1.55 3. Conclusion

A^˚ , suggesting that docking protocol could be relied on for the

In conclusion, series of novel 1,2,3-triazole derivatives of 2-

docking studies. The best docked pose (docking score ꢂ8.62)

view of the co-crystallized ligand inside the active site of the

target protein (PDB ID: 2UV0) is shown in Fig. 6. The gure

revealed that, the co-crystallized ligand exhibited four hydrogen

aminobenzimidazole and 2-amino-5,6-dimethylbenzimidazole

were designed and synthesized. All synthesized compounds

1

13

were characterised using H NMR, C NMR, mass spectrometry

and elemental analyses. The QSI activity of all 40 nal

compounds was evaluated against P. aeruginosa MH602. Among

all derivatives, three compounds, 6i, 6o, 6p and 7l exhibited

promising QSI activity of 64.99, 64.25, 68.23 and 65.80% against

P. aeruginosa MH602 at 250 mM, respectively. These three

compounds also exhibited signicant activity at low concen-

trations (125 and 62.5 mM). Like, previously reported triazole

based AHL analogues (III and IV), 6p exhibited greatest QSI

activity at low concentrations with 63.67% inhibition at 62.5

mM. These compounds were not found to be toxic when treated

in normal cell line (HEK cell line) at 25, 50 and 100 mM. Hence,

these compounds may become good starting point for further

renement in order to emerge as potent molecules with

maximum therapeutic eﬀect.

˚

bond interactions with TYR 56 (2.29 A), TRP 60 (2.12 A), ASP 73

˚

(

1.94 A) and SER 129 (2.22 A) amino acid residues of the protein

and two pi–pi stacking interactions (3.39 and 3.36A^˚ )

respectively.

The analysis of best docked pose (Fig. 7) of compound 6p

(

docking score ꢂ8.40) revealed that, the compound showed

˚

hydrogen bond interaction with TYR 56 (2.08 A) and ASP 73

˚

(

2.90 A) as that of co-crystallized ligand and also formed

˚

hydrogen bond interactions with ARG 61 (2.25 A), TYR-93 (3.76

˚

A), LEU-125 (3.25 A) and TRP-88 (2.80 A) residues of the target.

The compound also exhibited three pi–pi stacking interactions

˚

with TYR-47 (2.25 A), TRP-88 (2.80 A) and PHE-101 (3.22 A)

residues of the target protein and hence might stabilize its

binding aﬃnity with the target receptor, consequently may be

responsible for its signicant in vitro activity. The best docked

pose (Fig. 8) of compound 11b (docking score ꢂ9.06) displayed

4. Experimental section

˚

weak hydrogen bond interactions between TYR-64 (2.96 A), TYR-

Materials and methods

˚

9

3 (1.90 A) and ASP 73 (3.37 A) residues of target protein. The

compound also exhibited pi–pi stacking interactions with Trp- All chemical reagents and solvents are purchased from Aldrich,

˚

8

8 (3.16 A) residues. This weak binding aﬃnity of compound Alfa Aesar, Finar. The solvents and reagents were of LR grade.

11b may be responsible for its lower activity in the in vitro assay. All the solvents were dried and distilled before use. Thin-layer

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chromatography (TLC) was carried out on aluminium- by TLC, the reaction was diluted with ethyl acetate and washed

supported silica gel plates (Merck 60 F254) with visualization with water. Organic layer was dried over anhydrous sodium

of components by UV light (254 nm). Column chromatography sulphate, concentrated under reduced pressure and the crude

1

was carried out on silica gel (Merck 100–200 mesh). H NMR residue was puried by column chromatography using 60–90%

1

3

and C NMR spectra were recorded at 400 MHz and 101 MHz ethyl acetate in hexane as eluent to give 0.42 g of N-(1H-benzo[d]

respectively using a Bruker AV 400 spectrometer (Bruker CO., imidazol-2-yl)-3-(1-(2-(cyclohexylamino)-2-oxoethyl)-1H-1,2,3-

Switzerland) in CDCl

3 6

and DMSO-d solution with tetrame- triazol-4-yl)propanamide (10a; yield 46%). Similar procedure

thylsilane as the internal standard and chemical shi values (d) was followed in the synthesis of 10b, 11a–d and 12a–c from 9a,

1

3

were given in ppm. H NMR spectra were recorded in CDCl or 9b and 9c respectively.

DMSO-d . The following abbreviations are used to designate

6

multiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multi-

plet, br ¼ broad. Melting points were determined on an electro

thermal melting point apparatus (Stuart-SMP30) in open

capillary tubes and are uncorrected. Elemental analyses were 1-yl)acetamide (6a). Pale green; (88%); m.p. 238–240 C; IR

performed by Elementar Analysensysteme GmbH vario MICRO

Analytical data for the nal compounds

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-propyl-1H-1,2,3-triazol-

ꢀ

ꢂ1

1

n

(KBr, cm ) 3392, 3124, 2959, 1637, 1598; H NMR (400

max

cube CHN Analyzer. Mass spectra (ESI-MS) were recorded on MHz, DMSO) d 12.05 (s, 2H), 7.81 (s, 1H), 7.33 (dd, J ¼ 5.9,

Schimadzu MS/ESI mass spectrometer.

3.2 Hz, 2H), 6.98 (dd, J ¼ 5.9, 3.2 Hz, 2H), 5.27 (s, 2H), 2.54 (t,

13

2

H), 1.54 (sextet, 2H), 0.84 (t, 3H). C NMR (101 MHz, DMSO)

d 168.17 147.83, 147.04, 135.44, 124.02, 121.95, 114.13, 52.77,

7.47, 22.77, 14.0. ESI-MS: (m/z) calcd for C14 O: 284.32,

O: (%) C, 59.14;

H, 5.67; N, 29.56; O, 5.63 found: C, 59.18; H, 5.63; N, 29.60; O,

General synthetic procedure for compounds 6a–q and 7a–n

2

16 6

H N

2

A solution of azide (5a) (0.5 g, 2.3 mmol) in DMF : H O (8 : 2)

was reacted with various substituted acetylenes (0.24 g, 3.5

mmol) in the presence of sodium ascorbate (0.023 g,

+

16 6

found 283.00 (M–H) ; anal. calcd for C14H N

5

.59.

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-pentyl-1H-1,2,3-triazol-

5

mol%), CuSO $5H O (0.006 g, 1 mol%). The reaction

4 2

mixture was stirred at rt for 6–24 h. The reaction mixture was

monitored by TLC. Upon completion of the reaction, as

indicated by TLC, the reaction was diluted with ethyl acetate

and washed with water. Organic layer was dried over anhy-

drous sodium sulphate, concentrated under reduced pres-

sure and the crude residue was puried by column

chromatography using 60–90% ethyl acetate in hexane as

eluent to give 0.57 g of the title compound N-(1H-benzo[d]

imidazol-2-yl)-2-(4-propyl-1H-1,2,3-triazol-1-yl)acetamide

ꢀ

1

-yl)acetamide (6b). Oﬀ white; (76%); m.p. 224–226 C; IR

ꢂ1

1

n

max (KBr, cm ) 3386, 3141, 2957, 1639, 1592, 1403; H NMR

(

400 MHz, DMSO) d 12.13 (s, 2H), 7.89 (s, 1H), 7.43 (dd, J ¼

5

2

.9, 3.2 Hz, 2H), 7.12 (dd, J ¼ 5.9, 3.2 Hz, 2H), 5.36 (s, 2H),

.64 (t, 2H), 1.72–1.52 (m, 2H), 1.41–1.25 (m, 4H), 0.88 (t, 3H).

C NMR (101 MHz, DMSO) d 168.15, 147.81 (s), 147.19,

35.31, 123.89, 121.96, 114.12, 52.77, 31.22, 29.20, 25.39,

1

3

1

2

2.33, 14.36. ESI-MS: (m/z) calcd for C16

H

20

N

6

O: 312.37,

O: (%) C, 61.52;

H, 6.45; N, 26.90; O, 5.12 found: C, 61.54; H, 6.43; N, 26.92; O,

+

20 6

found 311.00 (M–H) ; anal. calcd for C16H N

(

6a) (yield: 88%). Similar procedure was followed to get the

compounds 6b–q and 7a–n from 5a and 5b respectively.

5

.10.

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-heptyl-1H-1,2,3-triazol-

General synthetic procedure for 9a–c

ꢀ

1

-yl)acetamide (6c). White; (63%); m.p. 218–220 C; IR nmax

To a solution of compound (2a) (0.5 g, 3.776 mmol) in DMF (8.0

ꢂ1

1

(

KBr, cm ) 3373, 3144, 2948, 1632, 1594, 1401; H NMR (400

ꢀ

mL) at 0 C was added EDC–HCl (1.08 g, 5.64 mmol), diiso-

MHz, DMSO) d 12.11 (s, 2H), 7.84 (s, 1H), 7.41 (dd, J ¼ 5.9,

propyl ethylamine (1.33 mL, 9.4 mmol), and HOBt (0.76 g, 5.664

mmol). The reaction mixture was stirred at rt for 16 h. Upon

completion of the reaction, as indicated by TLC, DCM was

added to the reaction mixture and washed with 10% bicar-

bonate and brine solution. The organic layer was dried over

3

2

.2 Hz, 2H), 7.032 (dd, J ¼ 5.9, 3.2 Hz, 2H), 5.35 (s, 2H), 2.53 (t,

13

H), 1.61–1.49 (m, 4H), 1.39–1.22 (m, 6H), 0.87 (t, 3H).

C

NMR (101 MHz, DMSO) d 168.03, 147.69, 147.24, 135.08,

1

2

3

23.45, 121.74, 114.11, 52.18, 31.34, 29.20, 28.12, 25.39,

4.46, 22.33, 14.36. ESI-MS: (m/z) calcd for C18 O:

O: (%)

24 6

H N

anhydrous Na

2

SO

4

and concentrated in vacuo. The crude

+

24 6

40.42, found 339.00 (M–H) ; anal. calcd for C18H N

product was puried by column chromatography using 50–70%

ethyl acetate in hexane as eluent to yield 0.66 g of N-(1H-benzo

C, 63.51; H, 7.11; N, 24.69; O, 4.70 found: C, 63.54; H, 7.17; N,

2

4.66; O, 4.64.

[

d]imidazol-2-yl)pent-4-ynamide (9a). Similar procedure was

followed for preparation of 9a–c from 2a and 2b. Yield: 9a: 82%;

b: 78% and 9c: 80% (pale brown solids).

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-nonyl-1H-1,2,3-triazol-

ꢀ

1

(

-yl)acetamide (6d). White; (55%); m.p. 215–217 C; IR nmax

9

ꢂ1

1

KBr, cm ) 3339, 3142, 2962, 1634, 1591, 1402; H NMR (400

MHz, DMSO) d 12.13 (s, 2H), 7.89 (s, 1H), 7.45–7.40 (dd, J ¼

General synthetic procedure for 10a–c, 11a–d and 12a–c

A solution of alkyne (9a) (0.5 g, 2.3 mmol) in DMF : H

5

.9, 3.2 Hz, 2H), 7.12 (dd, J ¼ 5.9, 3.2 Hz, 2H), 5.35 (s, 2H),

13

2

O (8 : 2) 2.66 (t, 2H), 1.59 (m, 2H), 1.36–1.23 (m, 12H), 0.86 (t, 3H).

C

was reacted with 2-azido-N-cyclohexylacetamide (B) (0.24 g, 2.34 NMR (101 MHz, DMSO) d 168.01, 147.62, 147.13, 134.19,

mmol) in the presence of sodium ascorbate (0.023 g, 5 mol%), 122.46, 121.21, 113.08, 52.47, 31.34, 29.81, 29.74, 29.11,

CuSO

4 2

$5H O (0.006 g, 1 mol%). The reaction mixture was stir- 28.12, 25.39, 24.46, 22.33, 14.36. ESI-MS: (m/z) calcd for

+

red at rt for 12 h. Upon completion of the reaction, as indicated

C

20

H

28

N

6

O: 368.48, found 367.00 (M–H) ; anal. calcd for

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C

20

H

28

N

6

O: (%) C, 65.19; H, 7.66; N, 22.81; O, 4.34 found: C,

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-bromophenyl)-1H-

1,2,3-triazol-1-yl)acetamide (6j). Brown; (82%); m.p. 292–

6

5.20; H, 7.69; N, 22.80; O, 4.31.

ꢀ

ꢂ1

1

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-nonyl-1H-1,2,3-triazol-

294 C; IR n

max

(KBr, cm ) 3354, 3112, 1629, 1594, 1403; H

ꢀ

1

-yl)acetamide (6e). White; (92%); m.p. 201–203 C; IR n

NMR (400 MHz, DMSO) d 12.20 (s, 2H), 8.38 (s, 1H), 7.85 (d, J

max

ꢂ1

1

(

KBr, cm ) 3391, 3136, 1641, 1583, 1399; H NMR (400 MHz, ¼ 8.5 Hz, 2H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.42 (dd, J ¼ 6.7,

13

DMSO) d 12.62 (s, 1H), 12.14 (s, 2H), 8.13 (s, 1H), 7.52 (m, 1H), 3.3 Hz, 2H), 7.18–7.08 (m, 2H), 5.46 (s, 2H). C NMR (101

7

4

1

4

.41 (dd, J ¼ 5.9, 3.1 Hz, 3H), 7.24–6.99 (m, 4H), 5.38 (s, 2H), MHz, DMSO) d 168.63, 148.38, 145.63, 134.63, 132.40,

13

.68 (s, 2H). C NMR (101 MHz, DMSO) d 168.27, 150.05, 131.85, 130.52, 127.58, 123.86, 122.13, 121.28, 113.90, 53.41.

47.99, 144.11, 143.56, 125.67, 122.03, 121.65, 117.94, 114.01, ESI-MS: (m/z) calcd for C₁₇H₁₃BrN O: 397.23, found 395.00

6

+

10.87, 53.04, 28.28. ESI-MS: (m/z) calcd for C H N OS: (M–2) ; anal. calcd for C₁₇H₁₃BrN O: (%) C, 51.40; H, 3.30;

1

9

16

8

6

+

04.45, found 403.00 (M–H) ; anal. calcd for C H N OS: (%) Br, 20.12; N, 21.16; O, 4.03 found: C, 51.43; H, 3.32; Br,

1

9

16

8

C, 56.42; H, 3.99; N, 27.71; O, 3.96; S, 7.93 found: C, 56.38; H, 20.10; N, 21.15; O, 4.04.

4

.12; N, 27.69; O, 3.97; S, 7.97.

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-((5-chloro-2-oxopyridin-1(2H)-

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-

ꢀ

yl)acetamide (6k). Oﬀ white; (70%); m.p. 281–283 C; IR n

max

ꢂ1

1

yl)methyl)-1H-1,2,3-triazol-1-yl)acetamide (6f). White; (86%); m.p. (KBr, cm ) 3376, 3132, 1645, 1597, 1405; H NMR (400 MHz,

ꢀ

ꢂ1

1

2

76–278 C; IR nmax (KBr, cm ) 3395, 3135, 1648, 1583, 1398; H DMSO) d 12.19 (s, 2H), 8.56 (s, 1H), 7.77 (d, J ¼ 8.0 Hz, 2H), 7.42

NMR (400 MHz, DMSO) d 12.15 (s, 2H), 8.14 (s, 1H), 8.10 (dd, J ¼ (dd, J ¼ 6.6, 3.3 Hz, 2H), 7.28 (d, J ¼ 8.0 Hz, 2H), 7.17–7.09 (m,

1

3

7

.5, 3.0 Hz, 1H), 7.50 (dt, J ¼ 8.9, 4.4 Hz, 1H), 7.45–7.35 (m, 2H), 2H), 5.44 (s, 2H), 2.34 (s, 3H). C NMR (101 MHz, DMSO)

7.19–7.06 (m, 2H), 6.46 (t, J ¼ 8.9 Hz, 1H), 5.40 (s, 2H), 5.16 (s, 2H). d 168.51, 148.23, 146.73, 137.60, 129.73, 129.50, 128.50, 125.54,

13

C NMR (101 MHz, DMSO) d 168.21, 160.26, 147.99, 142.43, 141.19, 123.06, 122.08, 113.95, 53.26, 21.30. ESI-MS: (m/z) calcd for

+

137.22, 134.97, 126.12, 122.02, 121.37, 114.02, 110.86, 53.02, 44.02. C H N O: 332.36, found 331.00 (M–H) ; anal. calcd for

1

8

16 6

+

ESI-MS: (m/z) calcd for C17

7 2

18 16 6

H14ClN O : 383.79, found 382.00 (M–H) ; C H N O: (%) C, 65.05; H, 4.85; N, 25.29; O, 4.81 found: C,

anal. calcd for C17

5.55; O, 8.34 found: C, 53.25; H, 3.65; Cl, 9.28; N, 25.53; O, 8.30.

H14ClN O : (%) C, 53.20; H, 3.68; Cl, 9.24; N, 65.11; H, 4.82; N, 25.30; O, 4.83.

7 2

2

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-(tert-butyl)phenyl)-1H-

1,2,3-triazol-1-yl)acetamide (6l). Oﬀ white; (67%); m.p. 265–

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-phenyl-1H-1,2,3-triazol-1-

ꢀ

ꢂ1

1

yl)acetamide (6g). Brown; (45%); m.p. 272–274 C; IR n_m_a_x267 C; IR n

(KBr, cm ) 3362, 3123, 1642, 1599, 1404; H

max

ꢂ1

1

(

KBr, cm ) 3385, 3101, 1646, 1595, 1401; H NMR (400 MHz, NMR (400 MHz, DMSO) d 12.07 (s, 2H), 8.51 (s, 1H), 7.76 (d, J ¼

DMSO) d 12.15 (s, 2H), 8.62 (m, 2H), 8.13 (s, 1H), 7.79–7.40 (m, 8.0 Hz, 2H), 7.41 (dd, J ¼ 6.6, 3.3 Hz, 2H), 7.18 (d, J ¼ 8.0 Hz,

1

3

13

4

H), 7.31–6.83 (m, 3H), 5.46 (s, 2H). C NMR (101 MHz, DMSO) 2H), 7.15–7.05 (m, 2H), 5.42 (s, 2H), 1.33 (s, 9H). C NMR (101

d 168.51, 147.23, 142.71, 141.38, 136.48, 135.82, 125.21, 124.98, MHz, DMSO) d 168.43, 148.24, 146.71, 137.41, 129.01, 129.67,

1

C

22.02, 121.37, 114.02, 54.01. ESI-MS: (m/z) calcd for 128.61, 125.55, 123.09, 121.22, 114.05, 53.22, 35.79, 32.41. ESI-

+

17

H

14

N

6

O: 318.33, found 317.00 (M–H) ; anal. calcd for MS: (m/z) calcd for C H N O: 374.44, found 373.00 (M–H) ;

21 22 6

17

14

6

O: (%) C, 64.14; H, 4.43; N, 26.40; O, 5.03 found: C, anal. calcd for C H N O: (%) C, 67.36; H, 5.92; N, 22.44; O,

2

1

22 6

64.19; H, 4.42; N, 26.37; O, 5.01.

4.27 found: C, 67.39; H, 5.90; N, 22.41; O, 4.29.

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-uorophenyl)-1H-1,2,3-

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-hydroxyphenyl)-1H-

ꢀ

triazol-1-yl)acetamide (6h). White; (54%); m.p. 283–285 C; IR 1,2,3-triazol-1-yl)acetamide (6m). Brown gummy solid;

ꢂ1

1

ꢂ1

1

n

max (KBr, cm ) 3381, 3135, 1632, 1589, 1406. H NMR (400 (46%); IR n_m_a_x(KBr, cm ) 3365, 3142, 1634, 1596, 1403; H

MHz, DMSO) d 12.09 (s, 2H), 8.36 (s, 1H), 7.87 (d, J ¼ 8.5 Hz, 2H), NMR (400 MHz, DMSO) d 12.13 (s, 2H), 8.38 (s, 1H), 7.72 (d, J

7

3

1

.64 (m, 2H), 7.43 (dd, J ¼ 6.7, 3.3 Hz, 2H), 7.12 (dd, J ¼ 6.7, ¼ 8.5 Hz, 2H), 7.41 (dd, J ¼ 6.7, 3.3 Hz, 2H), 7.16–7.11 (m, 2H),

13

.3 Hz, 2H), 5.45 (s, 2H). C NMR (101 MHz, DMSO) d 168.63, 6.91 (d, J ¼ 8.5 Hz, 2H), 6.7 (bs, 1H), 5.57 (s, 2H). C NMR

57.42, 148.37, 145.71, 134.89, 131.87, 130.16, 128.56, 124.14, (101 MHz, DMSO) d 168.43, 156.75, 148.23, 146.61, 135.42,

22.10, 121.26, 113.91, 54.45. ESI-MS: (m/z) calcd for 131.09, 130.81, 126.44, 123.88, 122.12, 121.23, 113.49, 54.98.

+

C H FN O: 336.32, found 335.00 (M–1) ; anal. calcd for ESI-MS: (m/z) calcd for C H N O : 334.33, found 333.00 (M–

C H FN O: (%) C, 60.71; H, 3.90; F, 5.65; N, 24.99; O, 4.76 2) ; anal. calcd for C H N O : (%) C, 61.07; H, 4.22; N,

1

7

13

6

17 14

6

2

+

1

7

13

6

17 14

6

2

found: C, 60.74; H, 3.92; F, 5.66; N, 24.96; O, 4.73.

25.14; O, 9.57 found: C, 61.10; H, 4.21; N, 25.15; O, 9.54.

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-chlorophenyl)-1H-1,2,3-

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-methoxyphenyl)-1H-

ꢀ

triazol-1-yl)acetamide (6i). White; (52%); m.p. 269–271 C; IR 1,2,3-triazol-1-yl)acetamide (6n). Pale yellow; (48%); m.p.

ꢂ1

1

ꢀ

ꢂ1

n

max (KBr, cm ) 3348, 3137, 1644, 1597, 1403; H NMR (400 242–243 C; IR n

(KBr, cm ) 3343, 3107, 1646, 1598, 1405;

max

1

MHz, DMSO) d 12.08 (s, 2H), 8.37 (s, 1H), 7.72 (d, J ¼ 8.5 Hz, 2H),

H NMR (400 MHz, DMSO) d 12.16 (s, 2H), 8.36 (s, 1H), 7.75

7

(

.54 (d, J ¼ 8.5 Hz, 2H), 7.41 (dd, J ¼ 6.7, 3.3 Hz, 2H), 7.16–7.11 (d, J ¼ 8.4 Hz, 2H), 7.62 (d, J ¼ 8.4 Hz, 2H), 7.40 (dd, J ¼ 6.7,

13

m, 2H), 5.47 (s, 2H). C NMR (101 MHz, DMSO) d 168.65, 3.3 Hz, 2H), 7.16–7.07 (m, 2H), 5.46 (s, 2H), 3.78 (s, 3H).

C

1

C

48.24, 146.52, 135.51, 132.45, 131.24, 130.86, 127.42, 123.87, NMR (101 MHz, DMSO) d 168.73, 159.41, 147.63, 135.13,

22.26, 121.27, 113.83, 53.41. ESI-MS: (m/z) calcd for 132.42, 131.87, 130.47, 127.64, 123.26, 122.14, 121.38, 113.91,

+

17

H

13ClN

6

O: 397.23, found 395.00 (M–2) ; anal. calcd for 53.42, 54.47. ESI-MS: (m/z) calcd for C H N O : 348.36,

1

8

16

6

2

+

17

6

O: (%) C, 51.40; H, 3.30; Br, 20.12; N, 21.16; O, 4.03 found 347.00 (M–2) ; anal. calcd for C H N O : (%) C,

1

8

16

6

2

found: C, 51.43; H, 3.32; Br, 20.10; N, 21.15; O, 4.04.

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ꢂ1

1

6

2

2.06; H, 4.63; N, 24.12; O, 9.19 found: C, 62.11; H, 4.61; N,

4.11; O, 9.17.

n

max (KBr, cm ) 3369, 3124, 1691, 1594, 1403; H NMR (400 MHz,

DMSO) d 12.12 (s, 2H), 7.87 (s, 1H), 7.18 (s, 2H), 5.29 (s, 2H), 2.36 (t,

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-(triuoromethyl)phenyl)- 2H), 2.26 (s, 2H), 1.62–1.50 (m, 6H), 1.38–1.23 (m, 8H), 0.84 (t, 3H).

1

3

1

2

H-1,2,3-triazol-1-yl)acetamide (6o). White; (72%); m.p. 286–

C NMR (101 MHz, DMSO) d 168.90, 147.96, 147.11, 132.91,

ꢀ

ꢂ1

1

88 C; IR nmax (KBr, cm ) 3383, 3107, 1654, 1599, 1404; H NMR 130.22, 123.81, 114.15, 53.16, 31.24, 29.13, 28.95, 25.41, 22.23,

(

400 MHz, DMSO) d 12.06 (s, 2H), 8.66 (s, 1H), 7.96 (d, J ¼ 8.1 Hz, 20.29, 19.18, 14.36. ESI-MS: (m/z) calcd for C20

H

28

N

6

O: 368.48,

+

2

5

1

3

H), 7.69 (d, J ¼ 8.3 Hz, 2H), 7.31–7.24 (m, 2H), 7.02–6.95 (m, 2H), found 367.00 (M–H) ; anal. calcd for C20

H N O: (%) C, 65.19; H,

28 6

1

3

.33 (s, 2H). C NMR (101 MHz, DMSO) d 168.69, 148.44, 145.30, 7.66; N, 22.81; O, 4.34 found: C, 65.23; H, 7.64; N, 22.77; O, 4.36.

35.23, 134.59, 128.62, 128.31, 126.44, 124.78, 123.38, 122.15,

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-nonyl-1H-

13.87, 53.50. ESI-MS: (m/z) calcd for C H F N O: 386.33, found 1,2,3-triazol-1-yl)acetamide (7d). Oﬀ white; (55%); m.p. 194–

1

8

13 3 6

+

ꢀ

ꢂ1

1

85.00 (M–H) ; anal. calcd for C H F N O: (%) C, 55.96; H, 3.39; 196 C; IR n (KBr, cm ) 3358, 3112, 1684, 1598, 1406; H

max

1

8

13

3

6

F, 14.75; N, 21.75; O, 4.14 found: C, 60.01; H, 3.40; F, 14.72; N, NMR (400 MHz, DMSO) d 12.03 (s, 2H), 7.87 (s, 1H), 7.18 (s,

1.71; O, 4.15. 2H), 5.29 (s, 2H), 2.63 (t, 2H), 2.25 (s, 6H), 1.60 (m, 2H), 1.31–

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(4-nitrophenyl)-1H-1,2,3- 1.28 (m, 4H), 1.27–1.23 (s, 8H), 0.85 (t, 3H). C NMR (101

triazol-1-yl)acetamide (6p). Pale yellow; (68%); m.p. 283– MHz, DMSO) d 168.72, 148.47, 147.50, 133.55, 130.26, 123.66,

2

1

3

ꢀ

ꢂ1

1

2

85 C; IR nmax (KBr, cm ) 3358, 3104, 1638, 1595, 1403; H 114.06, 53.13, 31.77, 29.54, 29.44, 29.29, 29.19, 29.05, 25.45,

O:

.9 Hz, 2H), 8.23 (d, J ¼ 8.8 Hz, 2H), 7.48 (dd, J ¼ 5.9, 3.2 Hz, 396.53, found 395.00 (M–H) ; anal. calcd for C H N O: (%)

NMR (400 MHz, DMSO) d 12.28 (s, 2H), 8.96 (s, 1H), 8.40 (d, J ¼ 22.57, 20.30, 14.42. ESI-MS: (m/z) calcd for C22

32 6

H N

+

8

2

32

6

1

3

H), 7.25–7.12 (m, 2H), 5.56 (s, 2H). C NMR (101 MHz, C, 66.64; H, 8.13; N, 21.19; O, 4.03 found: C, 66.66; H, 8.11; N,

DMSO) d 168.79, 148.54, 147.07, 147.78, 137.66, 126.37, 21.17; O, 4.05.

25.63, 124.93, 124.36, 122.19, 113.81, 53.62. ESI-MS: (m/z) 2-(4-(((1H-Benzo[d]imidazol-2-yl)thio)methyl)-1H-1,2,3-tri-

1

+

calcd for C17

for C17

C, C, 56.24; H, 3.59; N, 26.96; O, 13.22.

H

13

N

7

O

3

: 363.33, found 362.00 (M–H) ; anal. calcd azol-1-yl)-N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)

ꢀ

H

13

N

7

O

3

: (%) C, 56.20; H, 3.61; N, 26.99; O, 13.21 found: acetamide (7e). Oﬀ white; (84%); m.p. 194–196 C; IR nmax

ꢂ1

1

(KBr, cm ) 3371, 3132, 1691, 1594, 1405; H NMR (400 MHz,

N-(1H-Benzo[d]imidazol-2-yl)-2-(4-(3-nitrophenyl)-1H-1,2,3- DMSO) d 12.62 (s, 2H), 11.19 (s, 1H), 8.16 (s, 1H), 7.60 (m, 2H),

ꢀ

triazol-1-yl)acetamide (6q). Yellow; (64%); m.p. 240–242 C; IR 7.43 (dd, J ¼ 5.9, 3.1 Hz, 2H), 7.19 (s, 2H), 5.37 (s, 2H), 4.43 (s,

ꢂ1

1

13

n

(KBr, cm ) 3357, 3120, 1642, 1596, 1403; H NMR (400 2H), 2.31 (s, 6H). C NMR (101 MHz, DMSO) d 168.26, 150.24,

max

MHz, DMSO) 12.26 (s, 2H), 8.91 (s, 1H), 8.32 (m, 2H), 8.21 (d, J 148.01, 144.08, 143.53, 125.67, 122.04, 121.68, 117.91, 114.00,

¼

6.8 Hz, 1H), 7.79 (t, J ¼ 7.8 Hz, 1H), 7.42 (dd, J ¼ 5.5, 3.1 Hz, 110.67, 53.06, 28.48, 20.18. ESI-MS: (m/z) calcd for

1

3

+

2

H), 7.13 (dd, J ¼ 5.4, 3.2 Hz, 2H), 5.49 (s, 2H). C NMR (101

C

21

H

20

N

8

OS: 432.50, found 431.00 (M–H) ; anal. calcd for

MHz, DMSO) d 168.25, 148.44, 148.18, 147.13, 137.52, 1345.41,

21

20

8

OS: (%) C, 58.32; H, 4.66; N, 25.91; O, 3.70; S, 7.41

1

33.46, 126.77, 125.47, 124.82, 124.53, 122.67, 113.22, 53.41. found: C, 58.35; H, 4.68; N, 25.89; O, 3.69; S, 7.40.

ESI-MS: (m/z) calcd for C17 : 363.33, found 362.00 (M– 2-(4-((5-Chloro-2-oxopyridin-1(2H)-yl)methyl)-1H-1,2,3-tri-

H) ; anal. calcd for C H N O : (%) C, 56.20; H, 3.61; N, 26.99; azol-1-yl)-N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)

13 7 3

H N O

+

1

7

13 7 3

ꢀ

O, 13.21 found: C, 56.22; H, 3.57; N, 26.97; O, 13.21.

acetamide (7f). White; (83%); m.p. 281–283 C; IR n_m_a_x

ꢂ1

1

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-propyl-1H-1,2,3- (KBr, cm ) 3369, 3131, 1694, 1593, 1409; H NMR (400 MHz,

ꢀ

triazol-1-yl)acetamide (7a). White; (73%); m.p. 241–243 C; IR nmax DMSO) d 12.01 (s, 2H), 8.11 (m, 2H), 7.51 (dd, J ¼ 9.7, 2.9 Hz,

ꢂ1

1

(

KBr, cm ) 3367, 3134, 1692, 1598, 1401; H NMR (400 MHz, 1H), 7.17 (s, 2H), 6.47 (d, J ¼ 9.7 Hz, 1H), 5.33 (s, 2H), 5.17 (s,

13

DMSO) d 11.99 (s, 2H), 7.88 (s, 1H), 7.18 (s, 2H), 5.30 (s, 2H), 2.62 (t, 2H), 2.26 (s, 6H). C NMR (101 MHz, DMSO) d 160.16, 142.36,

13

2

H), 2.26 (s, 6H), 1.69–1.56 (m, 2H), 0.93 (t, 3H). C NMR (101 141.51, 141.51, 137.22, 136.84, 126.05, 121.37, 121.23, 113.99,

MHz, DMSO) d 168.95, 148.07, 146.92, 132.79, 130.27, 123.90, 110.86, 53.41, 44.00, 20.29. ESI-MS: (m/z) calcd for

+

1

14.12, 53.13, 27.51, 22.79, 20.30, 14.05. ESI-MS: (m/z) calcd for C H ClN O : 411.84, found 410.00 (M–H) ; anal. calcd for

19 18 7 2

+

C H N O: 312.37, found 300.00 (M–H) ; anal. calcd for C H ClN O : (%) C, 55.41; H, 4.41; Cl, 8.61; N, 23.81; O, 7.77

16

20

6

19 18

7

2

C H N O: (%) C, 61.52; H, 6.45; N, 26.90; O, 5.12 found: C, 61.56; found: C, 55.44; H, 4.43; Cl, 8.60; N, 23.80; O, 7.73.

16 20 6

H, 6.43; N, 26.86; O, 5.14.

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-phenyl-1H-

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-pentyl-1H-1,2,3- 1,2,3-triazol-1-yl)acetamide (7g). Dark brown gummy solid;

ꢀ

ꢂ1

1

triazol-1-yl)acetamide (7b). Oﬀ white; (66%); m.p. 200–202 C; IR (47%); IR nmax (KBr, cm ) 3358, 3137, 1671, 1584, 1404; H

n

ꢂ1

1

max (KBr, cm ) 3374, 3132, 1682, 1584, 1407; H NMR (400 MHz, NMR (400 MHz, DMSO) d 12.03 (s, 2H), 8.37 (s, 1H), 7.73 (d, J ¼

DMSO) d 12.09 (s, 2H), 7.87 (s, 1H), 7.18 (s, 2H), 5.29 (s, 2H), 2.64 (t, 7.8 Hz, 2H), 7.41–7.32 (m, 3H), 7.17 (s, 2H), 5.34 (s, 2H), 2.23 (s,

13

2

H), 2.26 (s, 6H), 1.64–1.56 (m, 2H), 1.32 (m, 4H), 0.87 (t, 3H).

C

6H). C NMR (101 MHz, DMSO) d 168.43, 147.18, 146.45,

NMR (101 MHz, DMSO) d 168.92, 147.98, 147.16, 132.91, 130.26, 136.89, 129.84, 1298.40, 127.51, 125.59, 122.34, 113.91, 53.25,

1

23.85, 114.12, 53.14, 31.23, 29.19, 25.40, 22.33, 20.29, 14.35. ESI- 21.30. ESI-MS: (m/z) calcd for C H N O: 346.39, found 345.00

19 18 6

+

MS: (m/z) calcd for C18

H N

24 6

O: 340.42, found 339.00 (M–H) ; (M–H) ; anal. calcd for C19

O: (%) C, 63.51; H, 7.11; N, 24.69; O, 4.70 24.26; O, 4.62 found: C, 65.90; H, 5.28; N, 24.24; O, 4.58.

found: C, 63.54; H, 7.13; N, 24.67; O, 4.67. 2-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)-N-(5,6-dimethyl-

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-heptyl-1H-1,2,3- 1H-benzo[d]imidazol-2-yl)acetamide (7h). White; (76%); m.p.

18 6

H N O: (%) C, 65.88; H, 5.24; N,

24 6

anal. calcd for C18H N

ꢀ

ꢂ1

1

triazol-1-yl)acetamide (7c). Grey colour; (57%); m.p. 209–211 C; IR 273–275 C; IR nmax (KBr, cm ) 3349, 3137, 1691, 1588, 1407; H

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NMR (400 MHz, DMSO) d 12.04 (s, 2H), 8.55 (s, 1H), 7.41 (dd, J ¼ 8.89 (s, 1H), 8.31 (d, J ¼ 7.9 Hz, 2H), 8.16 (d, J ¼ 7.9 Hz, 2H),

1

3

6

(

.7, 3.3 Hz, 2H), 7.12–7.09 (m, 2H), 7.18 (s, 2H), 5.37 (s, 2H), 2.26 7.18 (s, 2H), 5.42 (s, 2H), 2.26 (s, 6H). C NMR (101 MHz,

1

3

s, 6H). C NMR (101 MHz, DMSO) d 168.51, 148.17, 146.25, DMSO) d 169.68, 145.28, 135.45, 131.07, 128.67, 128.38,

1

5

37.11, 129.69, 129.41, 128.22, 125.69, 123.63, 122.34, 114.12, 127.02, 126.01, 124.75, 123.43, 113.76, 53.92, 20.29. ESI-MS:

+

3.24, 21.29. ESI-MS: (m/z) calcd for C19

H

17BrN

17BrN O: (%) C, 53.66; anal. calcd for C19

H, 4.03; Br, 18.79; N, 19.76; O, 3.76, found: C, 53.71; H, 4.00; Br, O, 12.26 found: C, 58.33; H, 4.42; N, 25.01; O, 12.24.

6

O: 425.28, (m/z) calcd for C19

H

17

N

7

O

3

: 391.38, found 390.00 (M–H) ;

+

found 424.00 (M–H) ; anal. calcd for C19

H

6

H

17

N

7

O

3

: (%) C, 58.31; H, 4.38; N, 25.05;

1

8.77; N, 19.78; O, 3.74.

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(p-tolyl)-

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(3-nitro-

phenyl)-1H-1,2,3-triazol-1-yl)acetamide (7n). Yellow; (67%);

ꢀ

ꢂ1

1

2

1

7

5

H-1,2,3-triazol-1-yl)acetamide (7i). Pale yellow; (55%); m.p. m.p. 277–279 C; IR n

(KBr, cm ) 3328, 3137, 1649, 1598,

max

ꢀ

ꢂ1

1

58–260 C; IR n_m_a_x(KBr, cm ) 3386, 3127, 1639, 1592, 1389; H NMR (400 MHz, DMSO) d 12.08 (s, 2H), 8.89 (s, 1H),

1

404; H NMR (400 MHz, DMSO) d 12.06 (s, 2H), 8.54 (s, 1H), 8.73–8.63 (m, 1H), 8.33 (d, J ¼ 7.9 Hz, 1H), 8.21 (d, J ¼ 7.8,

.76 (d, J ¼ 7.8 Hz, 2H), 7.27 (d, J ¼ 7.8 Hz, 2H), 7.18 (s, 2H), 1H), 7.78 (t, J ¼ 8.0 Hz, 1H), 7.18 (s, 2H), 5.41 (s, 2H), 2.26 (s,

1

3

13

.37 (s, 2H), 2.34 (s, 3H), 2.26 (s, 6H). C NMR (101 MHz, 6H). C NMR (101 MHz, DMSO) d 169.67, 145.21, 135.34,

DMSO) d 168.53, 148.17, 146.45, 137.89, 129.86, 129.40, 134.81, 131.18, 130.72, 128.69, 128.38, 127.13, 126.11,

28.52, 125.59, 123.67, 122.33, 113.94, 53.25, 21.30, 20.24. 124.74, 123.23, 113.77, 53.82, 20.27. ESI-MS: (m/z) calcd for

1

+

ESI-MS: (m/z) calcd for C20

H

20

N

6

O: 360.41, found 359.00 (M–

19 17 7 3

C H N O : 391.38, found 390.00 (M–H) ; anal. calcd for

+

H) ; anal. calcd for C₂₀H₂₀N O: (%) C, 66.65; H, 5.59; N, C₁₉H₁₇N O : (%) C, 58.31; H, 4.38; N, 25.05; O, 12.26 found:

6

7

3

2

3.32; O, 4.44 found: C, 66.68; H, 5.56; N, 23.34; O, 4.42.

C, 58.32; H, 4.43; N, 25.02; O, 12.23.

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(4-hydrox-

N-(1H-Benzo[d]imidazol-2-yl)-3-(1-(2-(cyclohexylamino)-2-

yphenyl)-1H-1,2,3-triazol-1-yl)acetamide (7j). Brown; (65%); oxoethyl)-1H-1,2,3-triazol-4-yl)propanamide (10a). White;

ꢀ

ꢂ1

ꢀ

ꢂ1

m.p. 326–328 C; IR nmax (KBr, cm ) 3378, 3136, 1684, 1594, (46%); m.p. 328–330 C; IR nmax (KBr, cm ) 3384, 3137, 1648,

1

7

6

409; H NMR (400 MHz, DMSO) d 11.85 (s, 2H), 8.49 (s, 1H), 1579, 1408; H NMR (400 MHz, DMSO) 12.04 (s, 1H), 11.54 (s,

.83 (d, J ¼ 8.1 Hz, 2H), 7.66 (d, J ¼ 8.2 Hz, 2H), 7.19 (s, 2H), 1H), 8.32 (d, 1H), 7.81 (s, 1H), 7.48 (dd, J ¼ 5.9, 3.2 Hz, 2H),

1

3

.83 (bs, 1H) 5.22 (s, 2H), 2.23 (s, 6H). C NMR (101 MHz, 7.05 (dd, J ¼ 5.9, 3.2 Hz, 2H), 4.98 (s, 2H), 3.84 (p, 1H), 2.76 (t,

1

3

DMSO) d 168.41, 148.34, 136.21, 131.69, 128.53, 128.24, 2H) 2.61 (t, 2H) 2.14–1.18 (m, 10H). C NMR (101 MHz,

26.19, 126.37, 123.82, 122.39, 113.77, 53.58, 20.35. ESI-MS: DMSO) d 172.75, 165.22, 146.48, 124.96, 124.49, 121.61,

1

(

+

m/z) calcd for C H N O : 362.39, found 361.00 (M–H) ; 121.67, 121.44, 51.96, 50.83, 32.64, 31.11, 25.06, 24.38, 23.86.

1

9

18

6

2

anal. calcd for C19

H

18

N

6

O

2

: (%) C, 62.97; H, 5.01; N, 23.19; O, ESI-MS: (m/z) calcd for C20

H

25

N

7

O

2

: 395.46, found 394.00 (M–

+

8

.83 found: C, 62.99; H, 5.04; N, 23.16; O, 8.81.

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(4-methox-

yphenyl)-1H-1,2,3-triazol-1-yl)acetamide (7k). Pale yellow;

H) ; anal. calcd for C20

H

25

N

7

O

2

: (%) C, 60.74; H, 6.37; N,

24.79; O, 8.09 found: C, 60.78; H, 6.39; N, 24.77; O, 8.05.

N-(1H-Benzo[d]imidazol-2-yl)-3-(1-(2-oxo-2-(pyridin-2-yla-

ꢀ

ꢂ1

(

58%); m.p. 283–285 C; IR nmax (KBr, cm ) 3338, 3127, 1672, mino)ethyl)-1H-1,2,3-triazol-4-yl)propanamide (10b). White;

598, 1402; H NMR (400 MHz, DMSO) d 12.03 (s, 2H), 8.49 (s, (63%); m.p. 286–288 C; IR n

1

ꢀ

ꢂ1

1

2

(KBr, cm ) 3378, 3131, 1643,

max

1

H), 7.96 (d, J ¼ 8.1 Hz, 2H), 7.81 (d, J ¼ 8.2 Hz, 2H), 7.00 (s, 1569, 1405; H NMR (400 MHz, DMSO) d 12.14 (s, 1H), 11.30

1

3

H), 5.36 (s, 2H), 3.78 (s, 3H) 2.23 (s, 6H). C NMR (101 MHz, (s, 1H), 10.96 (s, 1H), 8.36 (s, 1H), 7.95–7.79 (m, 3H), 7.68–

DMSO) d 168.33, 148.42, 136.93, 131.17, 128.73, 126.81, 7.57 (m, 1H), 7.53–7.11 (m, 4H), 5.38 (s, 2H), 3.04–2.60 (m,

1

3

1

26.73, 123.64, 121.82, 113.37, 53.71, 51.47, 20.32. ESI-MS: (m/ 4H). C NMR (101 MHz, DMSO) d 171.92, 165.73, 151.89,

+

z) calcd for C20 : 376.41, found 375.00 (M–H) ; anal. 148.61, 145.99, 145.84, 139.15, 138.87, 124.38, 124.30, 121.69,

H

20

N

6

O

2

calcd for C20H N O : (%) C, 63.82; H, 5.36; N, 22.33; O, 8.50 120.34, 113.96, 52.55, 31.13, 21.07. ESI-MS: (m/z) calcd for

20 6 2

+

found: C, 63.87; H, 5.34; N, 22.32; O, 8.49.

C

19

H

18

N

8

O

2

: 390.40, found 389.00 (M–H) ; anal. calcd for

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(4-(tri-

C H N O : (%) C, 58.45; H, 4.65; N, 28.70; O, 8.20 found: C,

1

9

18

8

2



uoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)acetamide (7l). 58.49; H, 4.62; N, 28.66; O, 8.23.

ꢀ

ꢂ1

Pale yellow; (66%); m.p. 273–275 C; IR n

(KBr, cm

)

N-(1H-Benzo[d]imidazol-2-yl)-4-(1-(2-(cyclopentylamino)-

max

1

3

385, 3142, 1694, 1527, 1402; H NMR (400 MHz, DMSO) 2-oxoethyl)-1H-1,2,3-triazol-4-yl)butanamide (11a). White;

ꢀ

ꢂ1

d 11.93 (s, 2H), 8.64 (s, 1H), 7.96 (d, J ¼ 8.1 Hz, 2H), 7.68 (d, (62%); m.p. 261–263 C; IR nmax (KBr, cm ) 3372, 3131, 1660,

1

3

1

J ¼ 8.2 Hz, 2H), 7.03 (s, 2H), 5.22 (s, 2H), 2.10 (s, 6H).

C

1577, 1420; H NMR (400 MHz, DMSO) 12.07 (s, 1H), 11.50 (s,

NMR (101 MHz, DMSO) d 169.69, 145.24, 135.29, 130.54, 1H), 8.30 (d, 1H), 7.84 (s, 1H), 7.47 (dd, J ¼ 5.9, 3.2 Hz, 2H),

1

5

28.56, 128.24, 126.45, 126.09, 124.74, 123.39, 113.78, 7.07 (dd, J ¼ 5.9, 3.2 Hz, 2H), 4.99 (s, 2H), 4.00 (p, 1H), 2.81 (t,

13

3.91, 20.28. ESI-MS: (m/z) calcd for C₂₀H₁₇F N O: 314.38, 2H) 2.79 (m, 1H), 2.08–1.29 (m, 11H). C NMR (101 MHz,

3

6

+

found 313.00 (M–H) ; anal. calcd for C₂₀H₁₇F N O: (%) C, DMSO) 172.70, 165.23, 146.47, 124.16, 124.00, 121.61, 121.52,

3

6

5

7.97; H, 4.14; F, 13.75; N, 20.28; O, 3.86 ﬀound: C, 57.99; 121.39, 51.98, 51.02, 32.69, 31.14, 25.06, 24.91, 23.85. ESI-MS:

+

H, 4.18; F, 13.74; N, 20.26; O, 3.83.

(m/z) calcd for C20

anal. calcd for C20

H

25

N

7

O

2

: 396.46, found 395.00 (M–H) ;

N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-2-(4-(4-nitro-

H

25

N

7

O

2

: (%) C, 60.74; H, 6.37; N, 24.79; O,

phenyl)-1H-1,2,3-triazol-1-yl)acetamide (7m). Pale yellow; 8.09 found: C, 60.79; H, 6.34; N, 24.78; O, 8.08.

ꢀ

ꢂ1

(

1

71%); m.p. 288–289 C; IR nmax (KBr, cm ) 3346, 3148,

N-(1H-Benzo[d]imidazol-2-yl)-4-(1-(2-(cyclohexylamino)-2-

1

686, 1585, 1406; H NMR (400 MHz, DMSO) d 12.09 (s, 2H), oxoethyl)-1H-1,2,3-triazol-4-yl)butanamide (11b). White;

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ꢀ

ꢂ1

+

(

58%); m.p. 199–201 C; IR nmax (KBr, cm ) 3383, 3133, 1637,

C

23

H

31

N

7

O

2

: 437.54, found 436.00 (M–H) ; anal. calcd for

1

7

2

566, 1402; H NMR (400 MHz, DMSO) 12.09 (s, 1H), 11.57 (s, C H N O : (%) C, 63.14; H, 7.14; N, 22.41; O, 7.31 found: C,

2

3

31

7 2

H), 8.31 (d, 1H), 7.86 (s, 1H), 7.51 (dd, J ¼ 5.9, 3.2 Hz, 2H), 63.17; H, 7.11; N, 22.43; O, 7.29.

.14 (dd, J ¼ 5.9, 3.2 Hz, 2H), 4.97 (s, 2H), 3.89 (p, 1H), 2.69 (t, N-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-4-(1-(2-oxo-2-

H) 2.62 (t, 2H), 2.14–1.18 (m, 12H). C NMR (101 MHz, (pyridin-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)butanamide

1

3

ꢀ

ꢂ1

DMSO) d 172.71, 165.82, 146.73, 124.83, 124.47, 121.61, (12c). White; (45%); m.p. 273–275 C; IR nmax (KBr, cm

)

1

2

3

6

7

21.66, 121.49, 51.95, 50.82, 32.66, 31.17, 27.53, 25.26, 24.40, 3314, 3127, 1639, 1571, 1407; H NMR (400 MHz, DMSO)

3.83. ESI-MS: (m/z) calcd for C21 : 409.48, found d 12.45 (s, 1H), 11.54 (s, 1H), 10.96 (s, 1H), 8.44 (s, 1H), 7.47

: (%) C, 61.60; H, (s, 2H), 5.18 (s, 2H), 2.27 (t, 2H), 2.59 (s, 6H), 2.48 (t, 2H), 1.78

H

27

N

7

O

2

+

94.00 (M–H) ; anal. calcd for C21

.65; N, 23.94; O, 7.81 found: C, 61.64; H, 6.61; N, 23.92; O, (p, 2H). C NMR (101 MHz, DMSO) d 174.84, 165.71, 150.91,

.83. 148.66, 146.19, 133.43, 124.46, 123.27, 121.45, 121.43, 120.37,

N-(1H-Benzo[d]imidazol-2-yl)-4-(1-(2-oxo-2-(pyridin-2-yla- 116.14, 113.88, 52.54, 33.54, 31.54, 24.97. ESI-MS: (m/z) calcd

H

27

N

7

O

2

1

3

+

mino)ethyl)-1H-1,2,3-triazol-4-yl)butanamide (11c). White; for C22

68%); m.p. 258–259 C; IR nmax (KBr, cm ) 3394, 3125,

652, 1563, 1404; H NMR (400 MHz, DMSO) d 12.24 (s, 1H), 61.14; H, 5.57; N, 25.87; O, 7.38.

1.54 (s, 1H), 10.97 (s, 1H), 8.37 (s, 1H), 7.97 (m, 2H), 7.74

H

N

24

N

8

O

2

: 432.48, found 431.00 (M–H) ; anal. calcd for

: (%) C, 61.10; H, 5.59; N, 25.91; O, 7.40 found: C,

ꢀ

ꢂ1

(

1

C

22

H

24

8

O

2

1

(

2

dd, J ¼ 5.8, 3.5 Hz, 3H), 7.15 (m, 1H), 7.08 (m, 2H), 5.37 (s,

1

3

H), 2.73 (t, 2H), 2.59 (t, 2H), 1.93 (p, 2H). C NMR (101

Quorum sensing inhibition assay

MHz, DMSO) d 176.80, 165.75, 151.90, 148.61, 146.79,

Antibacterial activities of the synthesized compounds were

evaluated by using P. aeruginosa MH602 lasB reporter strain

1

C

33.93, 124.47, 124.29, 121.65, 121.45, 120.37, 116.09,

13.98, 52.51, 33.55, 31.27, 24.93. ESI-MS: (m/z) calcd for

(

PlasB: gfp(ASV)) following the protocol developed by Hentzer

+

20

H

20

N

8

O

2

: 304.43, found 403.00 (M–H) ; anal. calcd for

37

et al. The production of AHL signals by this reporter strain

leads to an increase in product of unstable green uorescent

protein (GFP-ASV) as a function of an active QS system.

: (%) C, 59.40; H, 4.98; N, 27.71; O, 7.91 found: C,

5

9.43; H, 4.96; N, 27.68; O, 7.93.

N-(1H-Benzo[d]imidazol-2-yl)-4-(1-(4-nitrophenyl)-1H-1,2,3-

AHL dependent GFP expressing P. aeruginosa MH602

PlasB-gfp(ASV) harbouring a chromosomal fusion of the lasB

promoter expressing Gfp(ASV) in response to 3-oxo-

dodecanoyl HSL strains were cultured overnight in LB10.

Strains were diluted (1 : 100) in ABT medium supplemented

with 0.25% tryptone and 0.13% yeast extract and 200 mL of

aliquots were dispensed to at bottom 96-well plate wells

ꢀ

triazol-4-yl)butanamide (11d). Yellow; (59%); m.p. 263–265 C;

ꢂ1

1

IR n_m_a_x(KBr, cm ) 3396, 3143, 1654, 1566, 1405; H NMR (400

MHz, DMSO) d 12.08 (s, 1H), 11.61 (s, 1H), 8.93 (s, 1H), 8.51 (d,

J ¼ 8.9 Hz, 2H), 8.32 (d, J ¼ 8.8 Hz, 2H), 7.43 (dd, J ¼ 5.9, 3.2 Hz,

2

H), 7.25–7.12 (m, 2H), 2.83 (t, 2H), 2.61 (t, 2H), 1.98 (p, 2H).

C NMR (101 MHz, DMSO) d 168.72, 148.57, 147.37, 147.71,

37.26, 126.57, 125.63, 124.98, 124.31, 122.14, 113.88, 33.69,

1.73, 24.28. ESI-MS: (m/z) calcd for C19H N O : 391.38,

17 7 3

found 390.00 (M–H) ; anal. calcd for C H N O : (%) C, 58.31;

1

3

1

3

(

Sarstedt Australia). Cultures were supplemented with

varying concentrations of synthetic compounds dissolved in

DMSO. Control cultures were supplemented with equal

amount of DMSO (1%). Plates were sealed with self-adhesive

microplate sealers (TopSeal-A, PerkinElmer) to allow air

diﬀusion and to prevent condensation. Cultures were incu-

bated in a plate reader (EnSight Multimode Plate Reader,

+

1

9

17 7 3

H, 4.38; N, 25.05; O, 12.26 found: C, 58.35; H, 4.41; N, 25.02; O,

2.22.

-(1-(2-(Cyclopentylamino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)-

N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)butanamide (12a).

White; (47%); m.p. 252–254 C; IR nmax (KBr, cm ) 3377, 3148,

1

4

ꢀ

ꢂ1

ꢀ

PerkinElmer) at 37 C with shaking briey prior to each

1

3

647, 1582, 1404; H NMR (400 MHz, DMSO) d 12.08 (s, 1H),

1.57 (s, 1H), 10.91 (s, 1H), 8.34 (s, 1H), 7.44 (s, 2H), 4.94 (s, 2H),

.61 (m, 1H), 2.65 (t, 2H) 2.4–1.86 (m, 12H), 1.67–1.33 (m, 6H).

C NMR (101 MHz, DMSO) d 172.74, 165.37, 146.38, 124.91,

24.80, 121.49, 121.24, 121.45, 51.96, 51.02, 35.64, 34.45, 32.57,

reading and uorescence (excitation, 485 nm; emission, 535

nm) and OD600 of cultures were measured every 30 min over

ꢂ1

2

0 h. Ampicillin (100 mg mL ) and gentamicin (20 mg mL

)

1

3

were supplemented to P. aeruginosa MH602 cultures.

1

3

2.29, 25.05, 24.83. ESI-MS: (m/z) calcd for C22

found 422.00 (M–H) ; anal. calcd for C22H N O

29 7 2

H

29

N

7

O

2

: 423.51,

: (%) C, 62.39;

H, 6.90; N, 23.15; O, 7.56 found: C, 62.37; H, 6.88; N, 23.17; O,

.58.

-(1-(2-(Cyclohexylamino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)- was determined using MTT assay. 45 7.5 ꢃ 10 cells were seeded

+

Cytotoxicity assay

7

MTT assay. Cytotoxicity of the novel promising compounds

3

4

N-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)butanamide (12b). in 96 well plates and incubated overnight. Cells were treated

ꢀ

ꢂ1

White; (46%); m.p. 282–285 C; IR n

(KBr, cm ) 3393, 3127, with synthesized compounds at three concentrations (50 mM, 25

max

1

2

8

1

3

682, 1582, 1408; H NMR (400 MHz, DMSO) d 12.05 (s, 1H), mM and 10 mM) in duplicates and incubated for 24 h. 50 mL of

ꢂ1

1.53 (s, 1H), 10.78 (s, 1H), 8.32 (s, 1H), 7.82 (s, 2H), 4.98 (s, 5 mg mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

H), 3.54 (m, 1H), 2.64 (t, 2H) 2.4–1.5 (m, 12H), 1.31–1.12 (m, bromide (MTT; Himedia Laboratories Pvt. Ltd., Mumbai, India)

1

3

H). C NMR (101 MHz, DMSO) d 172.69, 165.37, 146.42, was added and incubated for 4 h. Formazan crystals were dis-

24.11, 124.08, 121.19, 121.22, 121.49, 51.98, 51.02, 35.37, solved using DMSO and absorbance was measured using

4.27, 32.69, 32.14, 25.06, 24.92, 23.86. ESI-MS: (m/z) calcd for Spectramax M4 (Molecular Devices, USA).

29290 | RSC Adv., 2019, 9, 29273–29292

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Paper

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Cell culture and assay procedure

4 M. E. Mattmann and H. E. Blackwell, J. Org. Chem., 2010, 75,

6

737–6746.

All the compounds tested for their cytotoxicity against Human

Embryonic Kidney (HEK) cell lines (procured from National

Centre for Cell Science, Pune, India) were cultured in DMEM

5

6

7

A. Eberhard, A. L. Burlingame, C. Eberhard, G. L. Kenyon,

K. H. Nealson and N. J. Oppenheimer, Biochemistry, 1981,

20, 2444–2449.

(high glucose media: AL007S, Dulbecco's modied eagle

D. A. Higgins, M. E. Pomianek, C. M. Kraml, R. K. Taylor,

M. F. Semmelhack and B. L. Bassler, Nature, 2007, 45, 883–

medium) with 10% fetal bovine serum (FBS) and 1% antibiotic

ꢀ

(

Pen strep: A001) incubated at 37 C and 5% CO

2

atmosphere.

886.

All reagents were purchased from Himedia Laboratories Pvt.

Ltd., Mumbai, India. Cells were seeded in a 96-well plate

M. M. Marketon, M. R. Gronquist, A. Eberhard and

J. E. Gonz ´a lez, J. Bacteriol., 2002, 184, 5686–5695.

8 https://www.cdc.gov/hai/organisms/gram-negative-

bacteria.html.

H. A. Khan, F. K. Baig and R. Mehboob, Asian Pac. J. Trop.

Med., 2017, 7, 478–482.

(

Eppendorf 0030730119) with 100 mL of cell suspension con-

4

taining 10 cells per well and incubated overnight. The DMSO

solutions of the synthesized compounds and BG45 were

prepared and they were further diluted to their respective

concentrations of 25 mM, 50 mM, and 100 mM with the DMEM

complete media. The cells were then treated with 100 mL of the

compound solutions made in media along with a blank control

containing DMSO in medium and BG45 as positive control and

were incubated for 24 h. The culture medium was aspirated and

9

1

0 N. A. Bhawsar and M. Singh, Int. J. Adv. Res., 2014, 6, 778–

83.

7

1

1 R. Manfredi, A. Nanetti, M. Ferri and F. Chiodo, Eur. J. Clin.

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12 A. R. Hauser and J. Rello, Severe Infections Caused by

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ꢂ1

subsequently, 50 mL of 5 mg mL concentrated solution of

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) in phenol red free DMEM was prepared and added

in each well and further incubated for 3 h for the formation of

formazan crystals. Later, 100 mL DMSO was added to the culture

aer aspirating media in the wells to dissolve the formazan

crystals and the absorbance was measured using multi-well

plate reader Spectramax (Molecular Devices, USA) at two

diﬀerent wavelengths of 570 nm and 650 nm. The % cell

viability was calculated as a fraction of absorbance obtained

from the treated cells from the absorbance of untreated control

1

3 C. Van Delden and B. H. Iglewski, Emerging Infect. Dis., 1998,

, 551–560.

4

4 http://www.who.int/medicines/publications/WHO-PPL-

Short_Summary_25FebET_NM_WHO.pdf.

5 http://www.who.int/news-room/fact-sheets/detail/antibiotic-

resistance.

6 L. Fulghesu, C. Giallorenzo and D. Savoia, J. Chemother.,

2

007, 19, 388–391.

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016, 8, 1–15.

8 N. Ni, M. Li, J. Wang and B. Wa, Med. Res. Rev., 2009, 29, 65–

24.

37

cells.

2

1

Conﬂicts of interest

9 C. Y. Chang, T. Krishnan, H. Wang, Y. Chen, W. F. Yin,

Y. M. Chong, L. Y. Tan, T. M. Chong and K. G. Chan, Sci.

Rep., 2014, 4, 1–8.

There are no conicts of interest to declare.

2

0 Y. Peng, J. Bi, J. Shi, Y. Li, X. Ye, X. Chen and Z. Yao, Am. J.

Infect. Control, 2014, 42, 1308–1311.

1 L. C. Antunes, R. B. Ferreira, M. M. Buckner and B. B. Finlay,

Microbiology, 2010, 156, 2271–2282.

2 P. Williams and M. C ´a mara, Curr. Opin. Microbiol., 2009, 12,

Acknowledgements

KVGCS and SM thank DBT, New Delhi [BT/IN/Spain/39/

SMl2017-18] for providing nancial support. The nancial

assistance provided by DIST FIST grant (SR/FST/CSI-240/2012),

New Delhi is gratefully acknowledged. SS thanks CSIR for

providing SRF fellowship. Central analytical lab facilities of

BITS Pilani Hyderabad campus are gratefully acknowledged. We

are also thankful to UNSW School of Biotechnology and

Biomolecular Sciences and UNSW School of Medical Sciences

for their support.

182–191.

2

3 https://www.springer.com/in/book/9788132219811.

4 S. Srinivasarao, S. Nizalapur, T. T. Yu, D. S. Wenholz,

P. Trivedi, B. Ghosh, K. Rangan, N. Kumar and

K. V. G. Chandra Sekhar, ChemistrySelect, 2018, 3, 9170–

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006, 13, 2163–2177.

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6 H. O. Sintim, J. A. Smith, J. Wang, S. Nakayama and L. Yan,

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Article Doi

Design, synthesis and biological evaluation of 1,2,3-triazole based 2-aminobenzimidazoles as novel inhibitors of LasR dependent quorum sensing in: Pseudomonas aeruginosa

DOI: 10.1039/c9ra05059k

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Article abstract of DOI:10.1039/c9ra05059k

Full text of DOI:10.1039/c9ra05059k

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