Design and synthesis of lactams derived from mucochloric and mucobromic acids as pseudomonas aeruginosa quorum sensing inhibitors

molecules

Article

Design and Synthesis of Lactams Derived from

Mucochloric and Mucobromic Acids as

Pseudomonas aeruginosa Quorum Sensing Inhibitors

Basmah Almohaywi ¹, Aditi Taunk , Daniel S. Wenholz , Shashidhar Nizalapur

Nripendra N. Biswas

1

1

3

1

, Kitty K. K. Ho , Scott A. Rice , George Iskander , David StC. Black ,

Renate Grifﬁth ⁴and Naresh Kumar ¹^,*

1

School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia; b.almohaywi@unsw.edu.au (B.A.);

a.taunk@unsw.edu.au (A.T.); d.wenholz@unsw.edu.au (D.S.W.); s.nizalapur@unsw.edu.au (S.N.);

nnathbiswas@gmail.com (N.N.B.); kitty.ho@unsw.edu.au (K.K.K.H.); g.iskander@unsw.edu.au (G.I.);

d.black@unsw.edu.au (D.S.B.)

School of Pharmacy, King Khalid University, Abha 62529, Saudi Arabia

The Singapore Centre of Environmental Life Sciences Engineering, Nanyang Technological University,

2

3

Singapore 639798, Singapore; RSCOTT@ntu.edu.sg

School of Medical Science, UNSW Australia, Sydney, NSW 2052, Australia; r.grifﬁth@unsw.edu.edu.au

4

*

Correspondence: n.kumar@unsw.edu.au; Tel.: +61-29385-4698; Fax: +61-29385-6141

Received: 18 April 2018; Accepted: 5 May 2018; Published: 7 May 2018

Abstract: Bacterial infections, particularly hospital-acquired infections caused by Pseudomonas

aeruginosa, have become a global threat with a high mortality rate. Gram-negative bacteria including

P. aeruginosa employ N-acyl homoserine lactones (AHLs) as chemical signals to regulate the expression

of pathogenic phenotypes through a mechanism called quorum sensing (QS). Recently, strategies

targeting bacterial behaviour or QS have received great attention due to their ability to disarm rather

than kill pathogenic bacteria, which lowers the evolutionary burden on bacteria and the risk of

resistance development. In the present study, we report the design and synthesis of N-alkyl- and

N-aryl 3,4 dichloro- and 3,4-dibromopyrrole-2-one derivatives through the reductive amination

of mucochloric and mucobromic acid with aliphatic and aromatic amines. The quorum sensing

inhibition (QSI) activity of the synthesized compounds was determined against a P. aeruginosa MH602

reporter strain. The phenolic compounds exhibited the best activity with 80% and 75% QSI at 250 µM

and were comparable in activity to the positive control compound Fu-30. Computational docking

studies performed using the LasR receptor protein of P. aeruginosa suggested the importance of

hydrogen bonding and hydrophobic interactions for QSI.

Keywords: quorum sensing; Pseudomonas aeruginosa; lactam; mucochloric acid; mucobromic acid

1

. Introduction

Exploring new directions to combat bacterial infections has become critically important with

the rising incidence of hospital-acquired bacterial infections and the global prevalence of bacterial

resistance. Traditional antibiotics are either bactericidal (kill bacteria) or bacteriostatic (inhibit the

growth of bacteria) [

1]. Therefore, the selective evolutionary pressures exerted by these antibiotics

on microorganisms have resulted in the rise and spread of antibiotic resistance [2]. Other factors that

have contributed to increased drug resistance include the expanded use of medical devices, treatments

for infections in immune-compromised patients and the overuse or mishandling of antibiotics either

intentionally or inadvertently [

2]. Therefore, novel therapeutic approaches to combat bacterial infection

and resistance are required [3].

Molecules 2018, 23, 1106; doi:10.3390/molecules23051106

www.mdpi.com/journal/molecules

Molecules 2018, 23, 1106

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Bacteria possess an adaptive intracellular mechanism that aids in their communications in

a cell density-dependent manner, allowing them to synchronize gene expression as a group

using a process termed quorum sensing (QS) [4]. Bacteria sense changes in their population

density via the production of diffusible small molecules known as autoinducers, such as

the N-acylated homoserine lactones (AHLs) N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)

1

in Vibrio ﬁscheri, and N-butyryl-L-homoserine lactone

N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL, also known as 3-oxo-C12-HSL) in P. aeruginosa

Figure 1) [5].

2 (C4-HSL, also known as BHL) and

3

(

Figure 1. Bacterial N-acylated homoserine lactones (AHLs) used for QS and synthetic QS inhibition.

P. aeruginosa is an opportunistic and ubiquitous human pathogen and is amongst the most

common causative agents of nosocomial and life-threatening infections [6]. In P. aeruginosa, QS is

coordinated via a triumvirate of LuxR homologues, namely the LasR, RhlR and QscR systems [

7].

These overlapping receptors have signiﬁcant roles in the regulation of gene expression and QS signals.

In P. aeruginosa, QS mediates and controls the gene expressions and phenotypes responsible for its

pathogenicity and resistance against the host immune system. It does so by utilizing autoinducers,

which trigger the production of virulence factors (e.g., elastase, protease, pyocyanin) and bioﬁlm

formation. These phenotypes, however, are not vital to the growth of this pathogen, and thus,

their inhibition does not have bacteriostatic or bactericidal effects. Hence, the interference with

and antagonism of QS comprise an attractive strategy to overcome and prevent virulence and

pathogenicity with minimal likelihood of resistance. Antagonists possessing the lactone head of natural

AHLs, but with non-native acyl chains, represent the most extensively-studied class of synthetic QS

modulators. For instance, synthetic analogues of the natural autoinducer OdDHL

as AHL-based LasR antagonists (Figure 1) [ ]. However, QS antagonists derived directly from AHLs

are sensitive to enzymatic and chemical hydrolysis of the lactone ring at physiological pH, giving

ring-opened products that lack QS activity [ ]. Hence, several research groups have investigated

replacement of the lactone group with saturated or unsaturated cyclic and heterocyclic structures [ 10].

Our research group has led the development of both ﬁmbrolide-based analogues [11] and

their lactam analogues [12 13]. The lactam ﬁmbrolide analogue exhibited good QS activity and

3 were developed

8

9

9,

,

6

was the most active lactam-based ﬁmbrolide derivative tested against AHL-mediated signaling in

Escherichia coli [13]. In line with our continuing efforts to develop new QS inhibitors, we explored

the potential use of mucochloric acid

7 and mucobromic acid 8 as precursors that could provide

access to functionalized lactams [14]. These compounds are inexpensive, commercially available,

highly functionalized and possess multiple sites for reactivity, particularly the two halogen atoms

situated across one double bond adjacent to a pseudo acid functionality. Mucohalic acids have been

used for the synthesis of furanones with antibacterial and antibioﬁlm activities [15

activity [17] and anti-inﬂammatory activity [18]. Mucohalic acids have also been used for the synthesis

of furanone-based natural products such as rubrolide [19 20], as well as their lactam analogues,

,16], anticancer

,

showing herbicidal [20] and antibioﬁlm activities [15]. They have also been used as precursors of the

antiseizure agent levetiracetam [21]. In this work, a library of 34 lactam compounds was prepared

using the reductive amination of mucochloric and mucobromic acids with selected aliphatic and

Molecules 2018, 23, 1106

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aromatic amines to furnish N-alkyl- and N-aryl 3,4 dichloro- and 3,4-dibromopyrrole-2-one derivatives.

The QS inhibitory activities of these lactam derivatives were determined.

2

. Results

.1. Chemical Synthesis

.1.1. Synthesis of N-alkyl and N-aryl Lactams

In order to generate a diverse array of lactam analogues, various amines were selected including

aliphatic, arylated and heterocyclic amines. Lactams

method using sodium triacetoxyborohydride as a reducing agent in acetic acid and mucohalic acids

mucochloric acid and mucobromic acid ) (Scheme 1) [14]. Most of the products were puriﬁed

easily by either trituration from methanol or ﬂash column chromatography (ethyl acetate/hexane)

if required). Compounds with a relatively acidic functional group, such as those derived from

carboxyaniline (15 16) or aminophenol (12 14), precipitated out from the reaction mixture as pure

9–31 were prepared following a literature

(

7

8

(

–

solids. Products were obtained in reasonable yields. The yields obtained are shown in Scheme 1 and

were dependent on both the identity of the starting mucohalic acid and the type of functional group

introduced. In general, reactions with mucochloric acid gave higher yields compared to those with

mucobromic acid. The proposed mechanism for this reaction [14] depends on nucleophilic attack of the

amine onto a protonated carbonyl group. The higher electrophilicity of chlorine compared to bromine

facilitates this. When aliphatic groups were introduced, butylamine produced a higher percentage

yield (79%) for 9 compared to hexylamine (39%) for 10. In the phenolic Compounds 12–14, the yield

was lower when the hydroxyl group was installed on the ortho position compared to the meta and para

analogues. The yields for these phenols follow the relative nucleophilicity of the nitrogen.

Scheme 1. Reductive amination of mucochloric acid

Compounds 9–31.

7 and mucobromic acid 8 to generate lactam

In the ortho derivative, the internal hydrogen bond resulted in the lower nucleophilicity of

2, which required longer reaction times. The introduction of para-carboxyphenyl (16) resulted

1

in a higher yield compared to the meta analogue 15, suggesting the impact of the position of the

electron-withdrawing group on the nucleophilicity of the aniline group. However, in the reaction of

Molecules 2018, 23, 1106

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mucobromic acid with carboxy substituted amines, the meta-carboxyphenyl (26) produced a relatively

higher yield than the para analogue (27) of 26% and 7%, respectively. Furthermore, the formation of

◦

the para analogue (27) required more forcing reaction conditions by heating at 30 C instead of room

temperature. Compounds 19 and 20, and their mucobromic analogue 29, containing aminophenyl

groups, were prepared from protected meta- and para-phenylenediamines via the Boc Compounds 17

8 and 28.

Products were fully characterized using H and ¹³C NMR spectroscopy and high-resolution

mass spectrometry. Compounds 31 could possibly undergo nucleophilic substitution at the vinylic

,

1

7

–

carbon with the loss of a chlorine or bromine. However, high-resolution mass spectrometry conﬁrmed

1

the correct mass to charge ratio of the anticipated products without the loss of halogens. In H

NMR (in DMSO-d ), the loss of the 5-CH and 5-hydroxyl groups, which resonate at 6.24 ppm and

6

8

.63 ppm, respectively, for mucochloric acid and 6.21 ppm and 8.52 ppm, respectively, for mucobromic

13

acid conﬁrmed the disappearance of the starting materials. Likewise, C NMR spectra showed

the disappearance of the C5 signal, which resonates at 96.76 ppm and 100.13 ppm in mucochloric

and mucobromic acids, respectively. The H NMR spectra of the products showed a distinctive

1

singlet peak for the ring C5 methylene (CH ) protons. For compounds derived from aliphatic amines

2

including the butyl

9, hexyl 10 and allyl 22 analogues, this peak appeared at 4.01–4.03 ppm. In the

tryptamine-derived Compound 30, the methylene peak appeared as a singlet at 4.30 ppm (in DMSO-d₆).

The methylene peak of the tyramine-derived Compound 31 (in CDCl ) resonated upﬁeld at 3.81 ppm,

3

presumably due to an electron-donating ability of these analogues. In contrast, compounds with

an aromatic substituent (e.g., 12

to the deshielding effect of the aromatic group. The hydroxyl proton of the phenolic Compounds

14 was observed as a singlet at 9.5–9.8 ppm. The correct numbers of carbons were observed for

Compounds

to 31 as assigned by ¹³C NMR. Differences between the N-alkyl and N-aryl lactams

31 were also observed in the distinctive carbon C5 peaks. The C5 of the alkyl compounds resonated

–20, 23–29) exhibited the C5 methylene protons at 4.6–4.8 ppm due

12–

9

–

at 52.9 ppm, except for the N-allyl, which resonated at 53.4 ppm due to the subtle deshielding effect

of the allylic double bond. Compounds with N-aryl substituents were also characterized by their C5

peak, which resonated downﬁeld at 53.4 ppm–55.4 ppm. Substituents on the N-phenyl had an effect

on the resonance of C5. For example, the C5 in Compound 23 (N-phenyl) resonated at 53.4 ppm,

while installing the hydroxyl group at the ortho position of the N-phenyl resulted in a chemical shift

of 55.4 ppm (Compound 12), compared to 54.0 ppm for Compound 13 (N-3-hydroxyphenyl) and

5

4.5 ppm for Compound 14 (N-4-hydroxy phenyl). Their mucobromic analogues 24 (N-phenyl) and 25

(3-hydroxyphneyl) had a chemical shift of 57.4 ppm (supplementary materials NMR Spectra).

2

.1.2. Synthesis of Amide Analogues

Additional amide groups were introduced into the lactam compounds to elaborate the

structure-activity relationship (SAR) surrounding these compounds. We envisaged that the amide

functionality might offer additional capacity to form hydrogen bonds with the biological target, the LasR

receptor protein, and thus enhance quorum sensing inhibition (QSI). Therefore, the C-linked amides 32

–38

and the N-linked amides 39 42 were targeted (Scheme 2). Mucochloric acid derivatives 16 and 20 were

–

selected for the synthesis of the amides due to their high yields and ease of synthesis. To generate the

C-linked amides, various amide coupling reagents such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDC)/hydroxy benzotriazole (HOBT), N,N-diisopropyl ethylamine (DIPEA) and hexafluorophosphate

benzotriazole tetramethyl uronium (HBTU) were used to couple the 4-carboxylphenyl mucochloric acid

derivative 16 with various aliphatic amines; however, low yields of the products were obtained. Therefore,

the acid 16 was instead converted to the corresponding acid chloride using SOCl and then reacted with

2

the corresponding amines in the presence of triethylamine to give the target Compounds 32

–38 in variable

yields (Scheme 2). Difficulties in the purification of the products were the main cause for the lower yields.

Molecules 2018, 23, 1106

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Scheme 2. Reductive amination of mucochloric acid

7

and mucobromic acid

8 to generate lactam

Compounds

TEA, rt, 24 h.

9–31. Reagent and conditions: I, SOCl ; ii, R-NH , TEA, rt, THF, 24 h; iii, R-COCl, THF,

2

1

In the H NMR, the C5 methylene group was observed as a singlet at 4.88–4.91 ppm, and there

were no signiﬁcant changes in this peak for the C-linked amides 32

precursor acid (Compound 16). The NH peak of the amides was observed as a singlet at 8.41 and

.43 ppm for Compounds 32 and 33 containing an aliphatic chain, respectively. The amide peak of

–38 when compared with the

8

Compound 34 was observed, but overlapped with the aromatic protons at 7.21–7.46 ppm, and the NH

peak of Compound 35 was observed at 10.94 ppm, presumably due to the electron-withdrawing effect

of the indole moiety. The amide peak of Compounds 36 and 37 appeared as a broad signal at 8.87 and

7

.91 ppm under the aromatic protons. For Compound 38, the NH peak was observed upﬁeld as a singlet

at 5.3 ppm. Attempts to prepare amides from the acid chloride derivative of 16 with 2-aminothiophene,

-aminobenzothizole, 2-aminobenzimidazole, 2-aminobenzoxazole and meta-aminopyridine were

2

unsuccessful, giving either unreacted starting materials or intractable mixtures of products. It is

possible a stronger base is needed to overcome the reduced nucleophilicity of the heterocyclic amines.

To generate N-linked amide analogues 39–42, intermediate 20 (Scheme 2) was acylated with the

appropriate acyl chloride derivatives resulting in moderate yields. The acid chloride compounds were

either commercially available or made by treating the corresponding acid with thionyl chloride.

The completion of the reaction was conﬁrmed with the disappearance of amine peak at 5.13 ppm

and the appearance of amide peaks at 9.92, 7.18, 7.24 and 10.33 ppm for Compounds 39–42 with butyl,

hexyl, octyl and 4-bromobenzyl groups, respectively.

Molecules 2018, 23, 1106

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2

.2. Biological Activity of Lactam Compounds against P. aeruginosa

2

.2.1. QSI of Alkyl and Aryl Lactams

The synthesized lactams were evaluated for their QSI activity following an established literature

protocol [22]. In this assay, the P. aeruginosa MH602 (PAMH602) strain that expresses the luxR gene

and the luxI gene and a promoter fused to the green ﬂuorescent protein (GFP) reporter gene were

used. Hence, the level of GFP ﬂuorescence is a measure of AHL-mediated QS in this P. aeruginosa

strain. The PAMH602 culture was incubated with varying concentrations (62.5

µ

M, 125

µM and 250

◦

µM) of the synthesized compounds at 37 C for 15 h, and the ﬂuorescence of GFP at

λ

= 535 nm was

determined. The optical density (OD) of the cultures at 600 nm was also measured to assess the effect

of the compounds on bacterial growth. In these experiments, the halogenated furanone ( ) (Fu-30) and

5

another known QS antagonist (TP-5) were used as positive controls. The effects of the compounds on

GFP ﬂuorescence and OD are presented in Table 1.

Table 1. Percentage inhibition of GFP ﬂuorescence and the reduction of OD by the synthesized lactams

against P. aeruginosa MH602.

QSI Activity against P. aeruginosa MH602

Concentration (µM)

Compound

9

250

125

62.5

b

a

71.4 ± 1.0

68.0 ± 0.5

62.1 ± 3.4

83.0 ± 2.8

78.3 ± 2.3

58.0 ± 3.3

35.7 ±1.8

19.8 ± 3.3

80.4 ± 1.1

58.82 ± 5.8

17.23 ± 3.0

76.4 ± 1.2

81.7 ± 0.1

80.7 ± 3.0

55.8 ± 1.2

28.17 ± 3.1

13.33 ± 3.0

42.55 ± 3.3

74.0 ± 1.7

43.6 ± 6.5

52.5 ± 6.2

32.3 ± 4.6

54.2 ± 5.9

53.5 ± 5.6

33.5 ± 0.5

37.1 ± 2.1

30.0 ± 3.9

41.4 ± 2.8

36.0 ± 3.0

53.6 ± 3.1

34.5 ± 2.0

34.4 ± 5.2

84.8 ± 2.9

51.0 ± 1.8

50.6 ± 0.5

39.4 ± 2.5

82.0 ± 2.6

74.6 ± 0.8

47.8 ± 1.3

25.92 ± 2.5

13.25 ± 3.9

79.5 ± 0.6

36.97 ± 3.2

13.5 ± 3.9

60.5 ± 0.5

78.7 ± 0.5

77.5 ± 4.1

54.2 ± 2.8

16.25 ± 1.9

47.8 ± 0.8

44.3 ± 7.0

38.2 ± 5.0

69.4 ± 0.3

65.7 ± 0.3

35.2 ± 5.7

15.35 ± 0.8

6.85 ± 1.8

78.1 ± 1.6

22.82 ± 3.2

9.27 ± 2.3

48.2 ± 2.0

76.5 ± 0.4

69.7 ± 0.9

37.1 ± 1.6

9.23 ± 1.1

NA

10

11

12

13

14

15

16

19

20

21

22

23

24

25

26

27

29

30

31

32

33

34

35

36

37

38

39

40

41

42

a

b

a

6.2 ± 3.5

26.15 ± 3.1

43.1 ± 1.1

40.2 ± 6.2

41.7 ± 4.1

a

13.55 ± 2.6

32.1 ± 3.5

33.5 ± 2.8

NA

a

c

a

3.5 ± 5.8

30.9 ± 6.0

28.7 ± 3.2

20.8 ± 2.1

29.1 ± 2.7

21.5 ± 1.6

32.2 ± 3.7

31.7 ± 2.0

32.7 ± 4.0

30.2 ± 4.4

30.4 ± 3.2

82.5 ± 2.5

NA

a

c

a

25.3 ± 1.0

14.4 ± 4.7

NA

a

4.0 ± 1.88

NA

16.7 ± 3.9

a

4.6 ± 0.7

16.9 ± 5.5

12.2 ± 2.9

2.3 ± 0.3

74.4 ± 3.6

a

d

a

TP-5

Fu-30 (5)

Bacterial growth inhibition

d

c

a

15%; ^bgrowth inhibition between 15 and 30%; growth inhibition greater than 30%;

c

≤

d

positive control;

±

the standard deviation of the mean for three independent experiments. In each independent

experiment, compounds were tested in triplicate. NA = no activity and no growth inhibition (supplementary

materials Table S1).

Molecules 2018, 23, 1106

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The QS inhibition assay indicated that the synthesized lactams displayed promising QSI activity.

Compounds 12 13 19 22 24 and 30 exhibited the highest QSI of 71.4–83.0% at 250 M. The most

active compound, the ortho-hydroxyphenyl mucochloric analogue 12, reduced GFP ﬂuorescence by 83%

2.8) at 250 M and was comparable in potency to the positive control and better in activity than

the triphenyl antagonist (TP-5). The QSI activity of 12 was well maintained at lower concentrations,

with 82.0% ( 2.6) and 69.4% ( 0.3) inhibitions at 125 M and 62.5 M, respectively. There was a slight

reduction in activity although not signiﬁcant (78 2.3% at 250 M, p > 0.05) as the phenol group moved

to the meta position as in derivative 13. The para-phenolic lactam 14 was the least active (58.0 3.3%

at 250 M, p < 0.001) when compared to its ortho (12) and meta (13) counterparts, suggesting the

9,

–

,

–

µ

(

±

µ

5

±

µ

±

µ

±

µ

importance of the position of the phenolic hydroxyl group on activity. Interestingly, no signiﬁcant

difference (p > 0.05) in activity was observed when comparing Compound 12 with other potent N-aryl

analogues including Compound 19 (80.4

N-3-aminophenyl and N-phenyl groups, respectively, which both exhibited high inhibition at 250

±

1.1%) and Compound 23 (81.7

±

0.1%) containing the

M.

µ

Furthermore, the activity of Compounds 19 and 23 was also retained at lower concentrations, giving

a high percentage of inhibition of 78.1% (±1.6) and 76.5% (±0.4) at 62.5 µM, respectively.

Compound 20 containing a 4-aminophenyl group was less active compared to the meta-amino

phenyl analogue 19. These observations from both the N-phenolic and the N-aminophenyl compounds

indicate the importance of the position of the electron donating groups installed on the N-phenyl ring

of the lactam, with both para-substituted compounds showing reduced activity. Activity was also

retained with the mucobromic analogue 24 (80.7

±

3.0% at 250 µM), suggesting that both of the dichloro-

and dibromo-pyrrolone were acceptable for activity for this type of scaffold. The N-carboxyphenyl

lactams including Compounds 15–16 and Compound 21 (N-butanoic acid group), both derived from

mucochloric acid, were less active compared to other N-aryl lactams. The position of the carboxyl

group did not improve the activity, and the same was observed with their mucobromic analogues 26

and 27, which were even less active.

The N-alkyl compounds were relatively less active compared to the N-aryl compounds.

Compounds

and 76.4 1.2% at 250

adding two carbon atoms to the butyl chain of Compound

a reduction in activity (68.0 0.5% at 250 M). Compound 30, containing the N-2-(1H-indol-3-yl)ethyl

substituent had QSI activity (74 1.7% at 250 M), but the activity dropped at lower concentrations

9

and 22 containing N-butyl and N-allyl groups possessed QSI activity (71.4

M, respectively), but their activity was poor at lower concentrations. Moreover,

to give the N-hexyl lactam 10 showed

±

1.0%

±

µ

9

±

µ

±

µ

(32.1 ± 3.5% at 62.5 µM).

2

.2.2. QSI of C-Linked and N-Linked Amide Analogues

In general, amides 32 42 displayed only low to moderate activity, as their QSI activity reduced

by 30–54% at 250 M, with low to no activity at 62.5 M. The C-linked amides 32 38 were generally

more active than the N-linked amides 39 42. This indicates the possible importance of the position

or the orientation of the amide group. The C-linked amides had higher activity, which ranges from

–

µ

–

3

0–53.5% inhibition at 250

µ

M when compared to their parent acid lactam 16 (QSI = 19.8%

6.2) and 54.2% ( 5.9), respectively.

5.8 at

M) showed reduced QSI activity of these amides, which ranges from 53.6–34.5%. The octyl group

produced inhibition of 53.6% (±3.1), which was relatively higher than the butyl and hexyl groups.

±

3.3 at

250 M). Compounds 32 and 34 had a moderate QSI of 52.5% (

µ

±

A comparison of the N-linked amides to their parent 4-aminophenyl lactam 20 (QSI = 58.8%

50

±

2

µ

2

.2.3. Evaluation of Growth Inhibition

To ensure that the decrease in GFP ﬂuorescence was related to QS inhibition and not a result of

toxicity or a decline in the population of the bacteria, the OD of the cultures were also measured and

the degree of growth inhibition noted along with the QS inhibition data in Table 1 (full results are

presented in the Supplementary Information). Overall, our synthesized library of lactams displayed

low growth inhibition against P. aeruginosa. At 62.5 µM, all synthesized compounds had little to no

Molecules 2018, 23, 1106

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effect on the growth of bacteria. Although the synthesized compounds have minimal effect on bacterial

growth, it should be noted that there may be other factors that can affect the QS activity [23]. The best

compounds in this study and TP-5 reduced bacterial growth by less than 30%, whereas the positive

control compound Fu-30 (5) inhibited P. aeruginosa growth by 84% at 250 µM. Therefore, this study

showed that the tested compounds inhibit QS with minimal effect on the growth of P. aeruginosa.

2

.2.4. Pyocyanin Inhibition

The virulence factor pyocyanin is produced when cell density is high in response to the AHL

molecule interacting with LasR. Since LasR is one of the determinant factors for the production of

pyocyanin in P. aeruginosa [24], the ability of our compounds to inhibit pyocyanin production was

investigated. Wild-type P. aeruginosa (PAO1) were grown in the presence of Compounds 12

, 13, 19,

2

3

and 24, and the amount of pyocyanin in the culture supernatants was quantiﬁed based on its

absorbance at 695 nm following a reported protocol [25]. The ability of the compounds to reduce

pyocyanin levels was determined with respect to the levels of pyocyanin in the DMSO-treated positive

control (Figure 2). Growth inhibition at OD₆₀₀was monitored, and the compounds showed moderate

reduction in bacterial growth (22–30%).

Figure 2. Pyocyanin inhibition in P. aeruginosa PAO1 by the synthesized compounds. Compounds were

added to the bacteria at 250 µM or 31.25 µM and were incubated for 17 h with shaking. Pyocyanin

production was measured at 695 nm in cell-free culture ﬂuids. Error bars represent the standard error

of two independent experiments.

The compounds were effective in inhibiting pyocyanin in the range of 90–94% at 250

µM.

The compounds also showed potent activity at a lower concentration of 32 µM (80–89% inhibition).

Our QS-based inhibitors showed comparable pyocyanin inhibition activity to those reported by

others [24]. The outcome of this assay correlates well with the potent QS activity of these compounds.

2

.3. Docking Studies

Lactams 42 were docked into the binding site of an X-ray crystal structure of the LasR receptor

9–

protein (supplementary materials Table S2) in complex with OdDHL (PDB Code 2UV0) using the

Genetic Optimisation for Ligand Docking (GOLD) algorithm through the Accelrys Discovery Studio

software package. Before docking, the compounds were minimized using a CHARMM force ﬁeld.

The crystal structure consisted of four subunits (two sets of dimers); however, compounds were

only docked into the binding site of subunit E following the results of previous control dockings.

Molecules 2018, 23, 1106

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The co-crystallised OdDHL ligand (

3) was docked back into the protein giving an acceptable root mean

square of deviation (RMSD) of 0.94 Å (heavy atoms) with the X-ray crystal structure.

The ligand-LasR interactions of Compounds 42 were analysed based on the highest scoring

docked pose of the largest cluster. The predicted binding poses of Compounds 10 11 13 15 22 26, and

42 were observed to be very similar to OdDHL, with the lactam carbonyl group of most compounds

forming a hydrogen bond with Trp60 from the same position as the lactone ring of the natural ligand

Figure 3A,B). Interestingly, the remaining compounds were predicted to bind in a different pose, with

the molecule ﬂipped (Figure 3C). This alternate pose still allowed the carbonyl group of the lactam

ring to form a hydrogen bond with Trp60, as was predicted for Compounds 12 14 and 25; however,

9–

,

31–

(

9

,

it also allowed the lactam CO to form a hydrogen bond with Arg61, which was observed for the

remaining compounds.

Figure 3. Predicted interactions for ligands 13 and 23 in the binding pocket of LasR compared to

N-(3-oxododecanoyl)-L-homoserine lactone

interactions and orientation. Hydrogen bonds are in pink. OdDHL is in yellow. (

shows a ﬂipped orientation relative to the lactone ring of OdDHL. ( ) describe the interactions of

and 23 with LasR. Hydrogen bonds are green. Halogen interactions are blue. Hydrophobic and

electrostatic interactions are red.

3

(OdDHL) (

3). (

A

) Compound 13 shows OdDHL-like

C) Compound 23

B,D

1

3

It was further observed that most of the compounds with high QSI were predicted to bind in

a pose different from OdDHL. One explanation for this may be similar to that proposed by Bottomley

et al., in that this pose severely limits the interaction with the hydrophobic pocket of the receptor

and thus inhibits the formation of a stable protein conformation [26]. Previous studies by Gerdt

et al. and Bottomley et al. have identiﬁed that hydrogen bonds to residues Ser129 and Thr115 are

important for agonistic activity of ligands [26

,27]. Notably, none of the QS inhibitors in this study were

observed to form these hydrogen bonds, which is consistent with the antagonistic activity seen for

Molecules 2018, 23, 1106

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these compounds. The predicted binding poses for Compounds 12 and 14 both shared the lactam ring

in the same position, resulting in the main difference between the two being the interactions of the

hydroxyl group of each compound. In Compound 12, the ortho hydroxyl group was predicted to form

a hydrogen bond to Tyr56, whilst in the less active Compound 14, the para hydroxyl group formed

a hydrogen bond with Tyr93. Hydrogen bonding to Tyr56 has been identiﬁed to be a key interaction

in determining the agonistic or antagonistic behaviour of a compound, which may explain the much

greater QSI activity observed for 12 [27].

The potent QS inhibitors 19, 23 and 24 form the same hydrogen bond with Arg61 and have

a ﬂipped orientation relative to the lactone head, but they form different interactions between the

phenyl and the LasR binding pocket. The 4-aminophenyl group in Compound 19 forms an electrostatic

π

-anion with Asp73. This suggested that NH is not required for activity since Compounds 23

2

and 24 lack the amino group and yet have potent QS inhibition. Although there are no signiﬁcant

differences in QS activity between 23 and 24, the phenyl groups differ in the type of interactions they

produce. Compound 23 makes hydrophobic interactions as shown in Figure 3C,D, while Compound

24 only forms an electrostatic π-anion interaction with Asp73. For the majority of docked compounds,

the halogen at the 3-position of the lactam ring was not predicted to make any interactions with the

LasR pocket except halogen interaction with Leu110 for Compound 13 and the least active compounds

including 15, 33, 35–38 and 40–41. In contrast, the halogen at the 4-position was predicted to form

hydrophobic interactions with the pocket for every docked compound including the most active ones.

To further investigate the importance of these observations, a new series of compounds similar to those

detailed in this study should be synthesized that lack a halogen at the 3-position of the lactam ring

and have various hydrophobic alkyl or aromatic groups at the 4-position. Detailed docking results are

provided in the Supplementary Information.

3

. Conclusions

A small library of 34 lactam compounds was synthesized and evaluated for QS inhibition against

P. aeruginosa. Compounds 42 were prepared in moderate to high yields via the reductive amination

9–

of mucochloric and mucobromic acid with a wide range of amines, including aliphatic, aromatic and

heteroaromatic amines. In biological testing, several compounds possessed promising activities, with

1

2

,

13

controls TP-5 and Fu-30 (

to Fu-30. Amides were also introduced to Compounds 16 and 20 to give the C-linked amides 32

and the N-linked amides 39 42, respectively. Generally, the amides 32 42 were less active compared

to lactams 31, although the C-linked amides 32 38 were more potent than the N-linked amides

42. Several compounds showed high efﬁcacy in pyocyanin inhibition, and the results are consistent

,

23 and 24 being the most active and showing comparable or even superior activity to the positive

). The tested compounds showed low bacterial growth inhibition in contrast

38

5

–

9–

–

39–

with their potent QS activity. Docking of the synthesized compounds to the LasR receptor protein

predicted favourable intermolecular interactions similar to OdDHL, including a hydrogen bond with

the conserved polar Arg61 and Trp60 residues. The most active compounds were docked in a different

orientation compared to OdDHL and did not form hydrogen bonds implicated in the stabilization

of LasR by agonists. Overall, the results obtained from this study suggest that lactams derived from

mucochloric and mucobromic acid could serve as new lead compounds for the development of potent

QSI compounds that are unlikely to exert selective pressure on bacteria.

4

. Experimental

4

.1. Chemistry

Commercially-available reagents were purchased from Aldrich, Acros Organics, Alfa Aesar.

The synthetic procedures have been reported for all compounds as general methods and appropriate

references have been given for known compounds. Melting points were measured using a Mel-Temp

melting point apparatus and were used uncorrected. High-resolution [+ESI] mass spectra were

Molecules 2018, 23, 1106

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recorded by the Bioanalytical Mass Spectrometry Facility, UNSW, on an Orbitrap LTQ XL ion trap

1

13

mass spectrometer using a nanospray (nano-electrospray) ionization source. H and C NMR spectra

were determined in the designated solvent on a Bruker DPX 300 spectrometer or a Bruker Avance

4

00 spectrometer unless otherwise stated. Chemical shifts (

δ

) are quoted in parts per million (ppm),

1

to the nearest 0.01 ppm and internally referenced relative to the solvent nuclei. H NMR spectral

data are reported with their chemical shift in parts per million (ppm). The multiplicity in H NMR is

1

abbreviated as follows: brs, broad; s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; sext, sextet;

m, multiplet; or as a combination (e.g., dd, dt, etc.). The coupling constant (J) in hertz, integration and

proton count were determined.

4

.2. General Procedure

Procedure A: Reductive amination

Mucohalic acid (1 eq) was added to a solution of 5:3 v/v dichloromethane/glacial acetic acid.

Then, an amine (1 eq) was added, and the mixture was stirred for 10 min. To that mixture, sodium

triacetoxyborohydride (3 eq) solution in 5:3 v/v dichloromethane and glacial acetic acid was added.

The mixture was left to stir at room temperature for 24 h unless otherwise stated.

Procedure B: Amide coupling 1

◦

Acid 16 (1 eq) was treated with thionyl chloride (3 mL) and reﬂuxed for 3 h at 70 C. The mixture

was then cooled and left to stir overnight at room temperature. Thionyl chloride was removed under

high vacuum, and the resultant acid chloride was used without further puriﬁcation. The acid chloride

was dissolved in 10 mL of dry tetrahydrofuran, and triethylamine (1 eq) and amine (1 eq) were then

added. The reaction mixture was left to stir at room temperature for 24 h.

Procedure C: Amide coupling 2

The amine was dissolved in 10 mL of dry tetrahydrofuran, and then, triethylamine (1 eq) and

acid chloride (1.0 eq) were added. The reaction mixture was left to stir at room temperature for 24 h.

(1) 1-butyl-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (9)

Cl

N

O

The title compound was prepared according to Procedure A from mucochloric acid (400 mg,

.3 mmol), butyl amine (170 mg, 270 mL, 2.3 mmol) and sodium triacetoxyborohydride (1.46 g,

.9 mmol). The reaction mixture was left to react for 24 h. The crude mixture was extracted with

2

6

dichloromethane (30 mL) and washed with water (15 mL) followed by brine (15 mL), dried over

sodium sulphate, and the solvent was evaporated. A residue of oil was obtained, which was puriﬁed

by ﬂash column chromatography using a gradient eluent of 25–50% ethyl acetate in hexane. A yellow

1

oil was obtained (390 mg; 79%). H NMR (CDCl , 400MHz):

δ

0.95 (t, J = 7.3, 14.6 Hz, 3H, CH₃),

3.50 (t, J = 7.4, 15.0,

4.04 (s, 2H, CH₂). ¹³C NMR (CDCl , 101): 13.7 (CH ), 19.9 (CH ), 30.4 (CH ), 42.8 (CH ),

3

δ

2

5

1.36 (sext, J = 7.2, 15.0 Hz, 2H, CH ), 1.58 (quint, J = 7.2, 15.0 Hz, 2H, CH ), δ

2 2

H, CH₂),

δ

3

2

3.4 (C5-CH ), 125.9 (C), 139.0 (C), 164.2 (C); IR (ATR): υmax 2957.6, 2870.7, 1700.9, 1397.7, 1341.6,

269.6, 1188.5, 1130.0, 843.9; UV (MeOH) λmax 230.0 nm (

C H Cl N O Na 230.0110 [M + Na] , found 230.0108.

2

7488 cm ¹

−

M

−1

)HRMS (ESI) m/z calcd. for

1

ε

+

8

11

2

1

(2) 3,4-dichloro-1-hexyl-1,5-dihydro-2H-pyrrol-2-one (10)

Molecules 2018, 23, 1106

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Cl

N

O

The title compound was prepared according to Procedure A from mucochloric acid (400 mg,

.3 mmol), hexyl amine (300 mg, 270 mL, 2.3 mmol) and sodium triacetoxyborohydride (1.46 g,

.9 mmol). The reaction mixture was left to react for 24 h. The crude mixture was extracted with

2

6

dichloromethane (30 mL) and washed with water (15 mL) followed by brine (15 mL), dried over

sodium sulphate, and the solvent was evaporated and a residue oil obtained, which was puriﬁed by

ﬂash column chromatography using a gradient eluent of 25–50% ethyl acetate in hexane. A yellow oil

1

was obtained (220 mg; 39%); H NMR (CDCl , 400 MHz):

6

δ

0.90 (t, J = 6.9, 13.5 Hz, 3H, CH ),

δ

1.24 (m,

4.04 (s, 2H, C5-CH₂). ¹³C NMR (CDCl₃,

01): 14.0 (CH ), 22.5 (CH ), 26.3 (CH ), 28.3 (CH ), 31.4 (CH ), 43.1 (CH ), 53.4 (C5-CH ), 125.7 (C),

3

H, CH₂), δ 1.60 (m, 2H, CH₂), δ 3.49 (t, J = 7.4, 14.6 Hz, CH₂), δ

1

3

2

39.2 (C), 164.2 (C=O); IR (ATR): υmax 2927.2, 2857.2, 1702.3, 1438.2, 1397.4 1367.5, 1131.0, 1171.0, 953.7;

4248 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Cl N O 236.0603

UV (MeOH) λmax 235.0 nm (

ε

10

16

2

1

+

[M + H] , found 236.0601.

(3) 1-benzyl-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (11)

The title compound was made according to Procedure A following the reported method [14]. m.p.

◦

02.1 C; H NMR (CDCl , 400 MHz): δ 3.92 (s, 2H, CH ), 4.67 (s, 2H, CH ), 7.26–7.39 (m, 5H, ArH);

3 2 2

1

3

C NMR (CDCl , 101):

δ 47.0 (CH ), 52.8 (CH ), 125.5 (C), 128.2 (ArCH), 129.0 (ArCH), 135.8, 139.9

2 2

3

(C), 164.28 (C=O); IR (ATR): υmax 2917.9, 2850.9, 1693.6, 1437.5, 1266.9, 966.1, 846.2, 739.1.

(4) 3,4-dichloro-1-(2-hydroxyphenyl)-1,5-dihydro-2H-pyrrol-2-one (12)

Cl

N

O

OH

The title compound was prepared according to Procedure A from mucochloric acid (600 mg, 3.55

mmol), ortho-aminophenol (388 mg, 3.55 mmol) and sodium triacetoxyborohydride (2.26 g, 10.65 mmol).

The reaction mixture was left to react for 48 h. The precipitated solid was ﬁltered by vacuum ﬁltration,

◦

1

and a white solid was obtained (280 mg; 32%). m.p. 171.6 C; H NMR (DMSO-d , 300 MHz):

6

13

δ

4.61 (s, 2H, CH₂),

δ 6.85–6.97 (m, 2H, ArH), δ 7.17–7.28 (m, 2H, ArH), 9.86 (s, OH); C NMR

(

DMSO, 75.5 MHz): 55.4 (CH ), 117.2 (ArCH), 119.6 (ArCH), 124.3 (C), 124.4 (ArCH), 129.3 (ArCH),

2

Molecules 2018, 23, 1106

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1

29.6 (ArCH), 142.0 (ArC), 153.5 (C), 163.1 (C=O); IR (ATR): υmax 3105.4, 1666.0, 1591.4, 1509.6, 1445.4,

2928 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for

1

275.3, 1198.2, 930.5, 839.1; UV (MeOH): λmax 280.0 nm (

ε

+

C H Cl N O Na 265.9746 [M + Na] , found 265.9744.

10

7

2

1

2

1

(5) 3,4-dichloro-1-(3-hydroxyphenyl)-1,5-dihydro-2H-pyrrol-2-one (13)

Cl

N

O

OH

The title compound was prepared according to Procedure A following the reported method [14

]

from mucochloric acid (600 mg, 3.55 mmol), meta-aminophenol (388 mg, 3.55 mmol) and sodium

triacetoxyborohydride (2.26 g, 10.65 mmol). The reaction mixture was left to react for 24 h. As the

reaction progresses, a yellow precipitated solid was evident. The solid was ﬁltered by vacuum ﬁltration

◦

1

and a yellow solid was obtained (578 mg; 66%); m.p. 163.6 C; H NMR (DMSO-d , 400 MHz): δ 4.80

6

(

s, 2H, CH₂),

δ 6.59 (d, J = 8.0 Hz, 1H, ArH), 7.04 (d, J = 9.3 Hz, 1H, ArH), 7.18 (t, J = 8.0 Hz, 1H,

13

ArH), 7.28 (s, 1H, ArH), 9.71 (brs, OH); C NMR (DMSO, 100 MHz): 54.0 (CH ), 106.6 (ArCH), 109.8

2

(

ArCH), 112.3 (ArCH), 124.3 (C), 130.2 (ArCH), 139.8 (C), 141.9 (C), 158.4 (C), 162.4 (C=O); UV (MeOH):

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 265.9746 [M + Na] ,

10 7 2 1 2 1

+

λmax 280.0 nm (

ε

3854 cm

M

found 265.9745.

(6) 3,4-dichloro-1-(4-hydroxyphenyl)-1,5-dihydro-2H-pyrrol-2-one (14)

Cl

N

O

OH

The title compound was prepared according to Procedure A from mucochloric acid (600 mg,

.55 mmol), 4-aminophenol (388 mg, 3.55 mmol) and sodium triacetoxyborohydride (2.26 g,

3

10.65 mmol). The reaction mixture was left to react for 24 h. As the reaction progressed, a yellow

precipitated solid was evident. The solid was ﬁltered by vacuum ﬁltration, and a yellow solid

◦

1

was obtained (500 mg; 58%); m.p. 126.0 C; H NMR (DMSO-d , 400 MHz):

δ 4.77 (s, 2H, CH ),

2

6

13

δ

6.81 (d, J = 8.9 Hz, 2H, ArH),

δ 7.44 (d, J = 8.9 Hz, 2H, ArH), 9.8 (brs, 1H, OH); C NMR (DMSO,

1

00 MHz): 54.5 (CH ), 115.8 (ArCH), 122.2 (ArCH), 124.3 (C), 130.3 (ArC), 141.2 (ArC), 155.4 (C), 162.1

2

(

C=O), IR (ATR): υmax 3277.8, 1682.9, 1630.2, 1510.6, 1270.8, 1219.3, 1046.9, 934.0, 830.7; UV (MeOH):

1090.2 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 265.9746 [M + Na] ,

10 7 2 1 2 1

+

λmax 280.0 nm (

ε

found 265.9745.

(7) N-(3-carboxyphenyl)-3,4-dibromo-1,5-dihydro-2H-pyrrol-2-one (DHP phenyl acid-2) (15)

Molecules 2018, 23, 1106

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The title compound was synthesized according to Procedure A from mucochloric acid (1 g,

.91 mmol), 3-aminobenzoic acid (0.81 g, 5.91 mmol) and sodium triacetoxyborohydride (3.76 g,

5

17.75 mmol) in 5:3 v/v dichloromethane/glacial acetic acid (12 mL). The reaction mixture was left to

stir at room temperature for 18 h, during which time, a yellow precipitate was evident. The mixture

was ﬁltered under vacuum, and the ﬁltered solid was puriﬁed by ﬂash chromatography. The solid was

then recrystallized in methanol after chromatography to yield the pure title product as a white solid

◦

1

(

1

0.54g; 34%); m.p. 214–216 C; H NMR (300 MHz, DMSO-d )

δ

4.93 (s, 2H, CH₂),

7.89–7.93 (m, 1H, ArH), 8.34 (t, J = 1.8 Hz, 1H,

13.13 (brs, 1H, COOH); ¹³C NMR (75.5 MHz, DMSO-d₆)

54.0 (CH ), 120.0 (ArCH), 123.4

ArCN), 125.8 (ArC), 129.9 (ArCH), 132.1 (ArCH), 138.9 (ArCH), 142.4 (2

δ 7.55 (t, J = 7.98 Hz,

6

H, ArH),

δ

7.74 (tt, J = 7.98 and 1.48 Hz, 1H, ArH),

δ

ArH),

(

8

δ

2

×

CCl), 162.7 (C=O), 167.3

C=O); IR (ATR): υmax 2969, 2824, 2654, 2539, 1699, 1586, 1491, 1433, 1382, 1313, 1272, 1158, 1051, 938,

99, 818, 757, 739, 677 cm ¹; UV (THF): λmax 236 nm (

−

ε

20671 cm M ), 288 (32,346); HRMS (ESI)

−1 −1

+

m/z calcd. for C H Cl NO Na 293.9695 [M + Na] , found 293.9698.

11

7

2

3

(8) N-(4-carboxyphenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (16)

Cl

N

O

COOH

The title compound was synthesized according to Procedure A, by ﬁrst dissolving mucochloric

acid (1 g, 5.91 mmol) in 5:3 v/v dichloromethane/glacial acetic acid (12 mL). To this mixture, a solution

of p-aminobenzoic acid (0.81 g, 5.91 mmol) in dichloromethane (8 mL) was added followed by sodium

triacetoxyborohydride (3.76 g, 17.75 mmol). The reaction mixture was stirred at room temperature for

1

8 h, during which time a yellow precipitate was evident. The mixture was ﬁltered under vacuum and

◦

1

washed with dichloromethane and distilled water to yield a yellow solid (0.6 g; 37%). m.p. 229 C; H

NMR (300 MHz, DMSO-d ) 4.91 (s, 2H, CH₂), 7.84–7.88 (m, 2H, ArH), 7.96–8.01 (m, 2H, ArH),

12.8 (brs, 1H, COOH); ¹³C NMR (75.5 MHz, DMSO-d₆)

53.9 (CH ), 118.3 (2 ArCH), 124.2 (ArCN),

26.7 (ArC), 130.9 (2 ArCH), 142.4 (CCl), 142.8 (CCl), 162.8 (C=O), 167.1 (C=O); IR (ATR): υmax 2939,

δ

6

δ

×

2

1

2

×

814, 2659, 2537, 1679, 1604, 1516, 1425, 1375, 1279, 1042, 934, 860, 769, 741, 718 cm ¹; UV (THF): λmax

−

1

−1

), 289 (2736); HRMS (ESI) m/z calcd. for C H Cl NO Na 293.9695 [M + Na] ,

11 7 2 3

+

36 nm (

ε

5338 cm

M

found 293.9696.

(9) N-(3-tert-butylphenylcarbamate)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (17)

Molecules 2018, 23, 1106

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The title compound was prepared from mucochloric acid (1.0 g, 5.91 mmol),

N-Boc-m-phenylenediamine (1.2 g, 5.91 mmol) and sodium triacetoxyborohydride (3.7 g, 17.75 mmol)

in 5:3 v/v dichloromethane/glacial acetic acid (12 mL). The mixture was stirred at room temperature

for 3.5 h. The reaction mixture was washed with water and brine and then extracted into ethyl

acetate. The organic layer was dried over sodium sulphate and evaporated in vacuo. The solid was

recrystallized in DCM and hexane to yield the title compound as a yellow solid (1.36 g, 67.17%). m.p.

◦

1

31–132 C; H NMR (300 MHz, DMSO-d )

δ

1

1.48 (s, 9H, 3

×

CH₃),

δ

4.80 (s, 2H, CH₂),

28.6 (3

, 129.6 (ArCH), 139.0 (ArCH), 140.7 (ArC), 153.2

C=O), 162.4 (C=O); IR (ATR): υmax 3305, 2977, 1695, 1604, 1498, 1421, 1388, 1285, 1227, 1151, 945, 876,

δ

7.29 (m, 3H,

6

3

ArH), 7.97 (s, 1H, ArH),

CH₂), δ 79.6.0 (C-O), 113.2 (ArC), 115.1 (ArC), 124.2 ©

δ

9.45 (s, 1H, NH); C NMR (100 MHz, DMSO-d )

δ

×

CH₃), 54.1

δ

6

(

6

−

83, cm ; HRMS (ESI) m/z calcd. for C H Cl N O Na 365.0430 [M + Na] , found 381.0428.

15 16 2 2 3

1

+

0

10) N-(4 -tert-butylphenylcarbamate)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (18)

(

The title compound was prepared according to Procedure A from mucochloric acid (0.9 g,

5

.36 mmol), N-Boc-p-phenylenediamine (1.1 g, 5.36 mmol) and sodium triacetoxyborohydride (3.38 g,

1

5.98 mmol) in 5:3 v/v dichloromethane/glacial acetic acid (12 mL). The mixture was stirred at room

temperature for 3 h. The reaction mixture was washed with water and brine and then extracted into

ethyl acetate. The organic layer was dried over sodium sulphate and evaporated in vacuo to yield the

◦

1

title compound as a dark red solid (1.16 g, 64%). m.p. 130–131 C; H NMR (400 MHz, DMSO-d )

δ

6

1

.48 (s, 9H, 3

C NMR (100 MHz, DMSO-d )

×

CH₃),

δ

4.8 (s, 2H, CH₂),

28.5 (3

ArCH), 136.9 (CCl), 141.5 (CCl), 153.2 (C=O), 162.2 (C=O); IR (ATR):

δ

7.46 (dd, J = 41.9 and 9 Hz, 4H, ArH),

δ

9.38 (s, 1H, NH);

13

δ

×

CH₃), 54.1 (CH₂), 82.0 (C-O), 118.9 (2

δ

×

ArCH), 120.3

6

(ArC), 124.2 (ArC), 133.1 (2

×

−

1

υmax 3345, 2923, 2321, 1698, 1589, 1519, 1385, 1312, 1228, 1151, 1025, 932, 831, 758 cm ; UV (ACN):

λmax 245 nm (

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 365.0430 [M + Na] ,

15 16 2 2 3

+

ε

13,990 cm

M

found 381.0426.

(11) N-(3-aminophenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (19)

Molecules 2018, 23, 1106

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The title compound was prepared according to Procedure A using Compound 17. The Boc group

was cleaved by treating 17 (1.36 g) with triﬂuoroacetic acid (6 mL) at room temperature for 1 h followed

by evaporation of TFA under high vacuum. The residue was neutralized and washed with saturated

solution of sodium bicarbonate, and the solid obtained was ﬁltered under vacuum to yield a red solid

◦

0.75 g, 78%). m.p. 109 C; H NMR (300 MHz, DMSO-d ) δ 4.75 (s, 2H, CH ), 5.21 (brs, 2H, NH ),

6 2 2

1

(

δ

6.43 (dd, J = 6.42, 7.7 Hz, 1H, ArH), 6.78 (dd, J = 6.4, 7.7 Hz, 1H, ArH), 7.04–7.12 (m, 2H, ArH);

13

C NMR (75.5 MHz, DMSO-d₆)

δ 54.0 (CH₂), δ 105.4 (ArCH), 107.3 (ArCH), 111.4 (ArCH), 124.3 (C),

1

29.8 (ArCH), 139.4 (ArC), 141.6 (ArC), 149.0 (C), 162.2 (C=O); IR (ATR): υmax 3340, 1687, 1466, 1389,

1

+

268, 949, 758, HRMS (ESI) m/z calcd. for C H Cl N O 243.0086 [M + 1] , found 243.0084.

10

8

2

(12) N-(4-aminophenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (20)

The title compound was prepared according to Procedure A from the Boc group, which was

cleaved by treating intermediate 18 (0.53 g) with triﬂuoroacetic acid (5 mL) at room temperature for 1

h followed by evaporation of TFA under high vacuum. The residue was neutralized and washed with

saturated solution of sodium bicarbonate, and the solid obtained was ﬁltered under vacuum to yield

◦

1

a red solid (0.25 g, 64%). m.p. 106 C; H NMR (300 MHz, DMSO-d )

δ

4.7 (s, 2H, CH₂),

δ

5.13 (brs,

54.6

6

1

H, NH₂),

δ

6.55–6.6 (m, 2H, ArH),

δ

7.24–7.29 (m, 2H, ArH); ¹³C NMR (75.5 MHz, DMSO-d₆)

δ

(

CH ), δ 114.2 (2 × ArCH), 121.5 (ArC), 122.3 (2 × ArCH), 127.5 (ArC), 140.6 (CCl), 146.9 (CCl), 163.0

2

−

1

C=O); IR (ATR): υmax 3297, 3205, 2921, 1697, 1636, 1515, 1400, 1389, 1300, 1175, 1043, 928, 812 cm

;

−

1

−1

UV (ACN): λmax 247 nm (

2

ε

10,817 cm

M ), 307 (5783); HRMS (ESI) m/z calcd. for C H Cl N ONa

10

8

2

+

64.9906 [M + Na] , found 264.9909.

(13) 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (21)

The title compound was synthesized according to Procedure A, by ﬁrst dissolving mucochloric

acid (1.0 g, 5.9 mmol) in 5:3 v/v dichloromethane/glacial acetic acid (12 mL). To this mixture, a solution

of aminobutanoic acid (0.61 g, 5.9 mmol) in dichloromethane (8 mL) was added followed by sodium

◦

1

triacetoxyborohydride (3.76 g, 17.75 mmol) to yield a white solid (0.41 g, 30%). m.p. 106 C; H

NMR (300 MHz, DMSO-d ) 1.91 (m, CH₂), 2.42 (m, CH₂), 3.56 (t, J = 7.0 Hz), 4.07 (s, C5-CH₂);

C NMR (75.5 MHz, DMSO-d₆) 23.42 (CH ), 30.82 (CH ), 42.40 (CH ), 53.56 (C5-CH ), 125.7 (C),

δ

6

13

δ

2

−

1

39.7 (C), 164.7 (C=O), 176.9 (C=O); IR (ATR): υmax 3936, 2641, 1735, 1339, 1289, 1195, 859, 745 cm

;

+

HRMS (ESI) m/z calcd. for C H Cl N O Na 259.9852 [M + Na] , found 259.9850.

8

9

2

1

3

1

(14) 1-allyl-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (22)

Molecules 2018, 23, 1106

17 of 29

Cl

N

O

The title compound was prepared according to Procedure A from mucochloric acid (600 mg,

.55 mmol), allylamine (202.7 mg, 3.55 mmol) and sodium triacetoxyborohydride (2.26 g, 10.65 mmol).

3

The reaction mixture was left to react for 3 days. The crude mixture was extracted with dichloromethane

30 mL) and washed with water (15 mL) followed by brine (15 mL), dried over sodium sulphate,

(

and the solvent was evaporated and a residue oil obtained, which was puriﬁed by ﬂash column

chromatography using a gradient eluent of 5% to 25% ethyl acetate and hexane. A yellow oil was

1

obtained (244 mg, 36%); H NMR (CDCl , 300 MHz): 4.01 (s, 2H, C5-CH ), 4.11 (d, J = 6.1 Hz, 2H,

3

2

N-CH ), 5.20–5.80 (m, 2H, HC=CH₂), 5.74–5.80 (m, 1H,

H

C=CH₂); ¹³C NMR (CDCl , 75.5MHz):

2

3

4

5.5 (CH ), 52.9 (C5-CH ), 119.0 (HC=CH ), 125.6 (CCl), 132.0 (HC=CH ), 139.8 (C) 164.0 (C); IR (ATR):

2 2 2 2

υmax 3290.1, 1685.5, 1621.0, 1402.1, 1270.5, 1157.0, 1100.6, 929.8, 841.8; UV (MeOH): λmax 280.0 nm

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 213.9797 [M + Na] , found 213.9794.

7 7 2 1 1 1

+

(ε

4928 cm

M

(15) 3,4-dichloro-1-phenyl-1,5-dihydro-2H-pyrrol-2-one (23)

Cl

N

O

The title compound was prepared according to Procedure A from mucochloric acid (1g,

.92 mmol), aniline (551.2 mg, 5.92 mmol) and sodium triacetoxyborohydride (3.76 g, 17.76 mmol).

5

The reaction mixture was left to react for 48 h. The reaction mixture was extracted with dichloromethane

30 mL) and washed with water (15 mL) followed by brine (15 mL), dried over sodium sulphate, and

(

the solvent was evaporated. The obtained semi solid was triturated with methanol, and the precipitated

solid was collected by vacuum ﬁltration and dried over silica in the desiccator. A white small needle

◦

1

solid was obtained (680 mg, 99%). m.p. 201.2 C; H NMR (DMSO-d , 300 MHz):

7

δ 4.86 (s, 2H, CH ),

2

6

13

.20 (t, J = 9.0 Hz, 1H, ArH), 7.43 (t, J = 9.00 Hz, 2H, ArH), 7.73 (d, J = 9.0 Hz, 2H, ArH); C NMR

(

DMSO, 75.5MHz): 54.0 (CH ), 119.5 (ArCH), 124.3 (C), 125.1 (ArC)), 129.6 (ArCH), 139.0 (C), 142.0 (C),

2

1

7

2

62.5 (C=O); IR (ATR): υmax 3060.4, 2918.3, 1688.4, 1628.9, 1500.0, 1437.1, 1298.7, 1153.3, 1049.2, 927.4,

−

1

−1

M ); HRMS (ESI) m/z calcd. for C H Cl N O Na

55.6; UV (MeOH): λmax 280.0 nm (

ε

3854.4 cm

10

7

2

1

+

49.9797 [M + Na] , found 249.9794.

(16) 3,4-dibromo-1-phenyl-1,5-dihydro-2H-pyrrol-2-one (24)

Br

N

O

The title compound was prepared according to Procedure A from mucobromic acid (1.20 g,

.65 mmol), aniline (433.3 mg, 4.65 mmol) and sodium triacetoxyborohydride (3.60 g, 13.95 mmol).

4

Molecules 2018, 23, 1106

18 of 29

The reaction mixture was left to stir for 48 h. The precipitated solid was ﬁltered by vacuum ﬁltration,

◦

1

and a white solid was obtained (440 mg; 30%); m.p. 160.7 C; H NMR (DMSO-d , 300 MHz):

δ

4.88

6

(

s, 2H, CH ), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.43 (t, J = 7.5 Hz, 2H, ArH), 7.72 (d, J = 8.7 Hz, 1H, ArH);

2

1

3

C NMR (DMSO, 75.5 MHz): 57.3 (CH ), 119.4 (ArCH), 119.8 (ArC), 125.0 (C), 129.5 (ArCH), 136.4 (C),

2

1

38.8 (C), 163.3 (C); IR (ATR): υmax 3059.5, 1692.5, 1589.5, 1421.5, 1374.4, 1145.1, 1035.1, 885.4, 753.7; UV

7829 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Br N O Na 337.8787

(MeOH): λmax 280.0 nm (

ε

10

7

2

1

+

[M + Na] , found 337.8789.

(17) 3,4-dibromo-1-(3-hydroxyphenyl)-1,5-dihydro-2H-pyrrol-2-one (25)

Br

N

O

OH

The title compound was prepared according to Procedure A from mucobromic acid (1.20 g,

4.65 mmol), meta-aminophenol (507.8 mg, 4.65 mmol) and sodium triacetoxyborohydride (3.60 g,

1

3.95 mmol). The reaction mixture was left to stir for 48 h. The precipitated solid was ﬁltered by

◦

1

vacuum ﬁltration, and a light brown solid was obtained (780 mg, 50%); m.p. 164.0 C; H NMR

DMSO-d , 300 MHz): 4.80 (s, 2H, CH ), 6.57 (m, 2H, ArH), 7.05–7.27 (m, 2H, ArH), 9.59 (s, 1H,

57.4 (CH ), 106.6 (ArCH), 109.8 (ArCH), 112.2 (ArCH), 119.8 (C),

(

δ

6

2

OH); ¹³C NMR (DMSO, 75.5 MHz):

δ

2

1

30.2 (ArCH), 136.4 (C), 139.9 (C), 158.3 (C), 163.2 (C=O); IR (ATR): υmax 3257.5, 1664.0, 1597.9, 1456.1,

−

1

−1

M ); HRMS (ESI)

392.4, 1255.0, 1213.9, 1039.0, 929.8, 867.1; UV (MeOH): λmax 290.0 nm (

ε

5893 cm

+

m/z calcd. for C H Br N O Na 353.8736 [M + Na] , found 353.8733.

10

7

2

1

2

1

(18) N-(3-carboxyphenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (26)

The title compound was prepared from mucobromic acid (2.0 g, 7.75 mmol), meta-aminobenzoic

acid (1.06 g, 7.75 mmol) and sodium triacetoxyborohydride (4.93 g, 23.26 mmol). The reaction mixture

was left to stir for 24 h. The precipitated solid was ﬁltered by vacuum ﬁltration to afford the product

◦

1

as pale yellow solid (0.36 g, 26%). m.p. 186 C; H NMR (300 MHz, DMSO-d )

δ

4.93 (s, 2H, CH₂),

7.89–7.93 (m, 1H, ArH),

13.09 (brs, 1H, COOH); ¹³C NMR (75.5 MHz, DMSO-d₆)

57.3(CH₂),

19.9 (ArCH), 123.3 (ArCN), 125.7 (ArC), 129.8 (ArCH), 136.9 (ArCH), 139.0 (ArCH), 146.0 (2 CBr),

63.5 (C=O), 167.4 (C=O); IR (ATR): υmax 2821, 2551, 2321, 1698, 1584, 1490, 1425, 1380, 1312, 1289, 1227,

6

δ

7.55 (t, J = 8.0 Hz, 1H, ArH),

8.33 (t, J = 1.8 Hz, 1H, ArH),

δ 7.73 (tt, J = 8.0 and 1.47 Hz, 1H, ArH), δ

δ

1

×

−

1

−1 −1

151, 1032, 939, 901, 840, 757, 673 cm ; UV (THF): λmax 239 nm (

ESI) m/z calcd. for C H Br NO Na 381.8685 [M + Na] , found 381.8685.

ε

15,683cm M ), 291 (6639); HRMS

+

(

11

7

2

3

(19) N-(4-carboxyphenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (27)

Molecules 2018, 23, 1106

19 of 29

The title compound was synthesized from mucochloric acid (3 g, 11.63 mmol), p-aminobenzoic

acid (1.59 g, 11.63 mmol) and sodium triacetoxyborohydride (7.39 g, 34.9 mmol) in 5:3 v/v

◦

dichloromethane/glacial acetic acid (30 mL). The reaction mixture was stirred and heated at 30 C

for 3 h during which time a precipitate was evident. The mixture was ﬁltered under vacuum, and the

solid was recrystallized in 1:9 acetone/methanol to get the desired product as a white solid (0.296 g,

◦

1

7

%). m.p. 221 C; H NMR (300 MHz, DMSO-d )

δ

4.9 (s, 2H, CH₂),

12.8 (brs, 1H, COOH); ¹³C NMR (75.5 MHz, DMSO-d₆)

57.2 (CH ), 118.2 (2

ArCN), 126.6 (ArC), 130.9 (2 ArCH), 137.4 (CBr), 142.5 (CBr), 163.7 (C=O), 167.1 (C=O); IR (ATR):

υmax2811, 2659, 2535, 2112, 1679, 1601, 1516, 1423, 1371, 1275, 1188, 1145, 1017, 929, 889, 757, 704 cm

δ

7.83 (dd, J = 37.8 and 8.7 Hz, 4H,

6

ArH),

(

δ

×

ArCH), 120.0

2

×

−

1

;

−

1

−1

UV (THF): λmax 245 nm (

81.8685 [M + Na] , found 381.8684.

ε

14,965cm

M ), 288 (15,575); HRMS (ESI) m/z calcd. for C H Br NO Na

11

7

2

3

+

3

0

20) N-(4 -tert-butylphenylcarbamate)-3,4-dibromo-1,5-dihydro-2H-pyrrol-2-one (28)

(

The title compound was synthesized by reacting mucobromic acid (1 g, 3.87 mmol),

N-Boc-p-phenylenediamine (0.8 g, 3.87 mmol) and sodium triacetoxyborohydride (2.46 g, 11.63 mmol)

in 5:3 v/v dichloromethane/glacial acetic acid (10 mL). The reaction mixture was left to stir at room

temperature for 18 h. The mixture was washed with water and brine and then extracted into ethyl

acetate. The organic layer was dried over sodium sulphate and chromatographed on silica gel to yield

1

the desired product as a yellow solid (0.6 g, 36%). H NMR (300 MHz, CDCl )

δ

1.54 (s, 9H, 3

×

CH₃),

7.54–7.59 (m, 2H, ArH). ¹C NMR

ArCH), 119.9 (ArC), 121.5 (ArC),

ArCH), 133.3 (CBr), 135.5 (CBr), 152.6 (C=O), 163.1 (C=O); IR (ATR): υmax 3348, 3099, 2973,

3

δ

4.50 (s, 2H, CH₂),

δ

6.51 (s, 1H, NH),

δ 7.39–7.42 (m, 2H, ArH), δ

28.3 (3 CH ), 57.2 (CH ), 80.8 (C-O), 119.2 (2

(

1

75.5 MHz, CDCl₃)

δ

×

3

2

32.9 (2

×

−

1

768, 1688, 1605, 1518, 1430, 1364, 1283, 1232, 1146, 1020, 846, 739, 680 cm ; HRMS (ESI) m/z calcd.

for C H Br N O Na 452.9420 [M + Na] , found 452.9422.

+

15

16

2

3

(21) N-(4-aminophenyl)-3,4-dibromo-1,5-dihydro-2H-pyrrol-2-one (29)

Molecules 2018, 23, 1106

20 of 29

The title compound was synthesized by following the same method used to synthesize Compound

◦

1

9

to afford a yellow solid (0.13 g, 28%). m.p. 163 C; H NMR (300 MHz, DMSO-d )

δ

4.70 (s, 2H,

6.55–6.6 (m, 2H, ArH), 7.25–7.28 (m, 2H, ArH); ¹³C NMR (75.5 MHz,

57.0 (CH ), 115.4 (2 ArCH), 121.6 (2 ArCH), 127.0 (ArC), 129.4 (ArC), 132.4 (CBr),

44.2 (CBr), 164.2 (C=O); IR (ATR): υmax 3305, 3208, 2920, 2287, 1693, 1611, 1512, 1442, 1383, 1280, 1150,

6

CH₂),

DMSO-d₆)

1

δ 5.16 (brs, 1H, NH₂), δ

δ

×

2

−

1

−1 −1

023, 900, 830 cm ; UV (ACN): λmax 243 (

ε

8973 cm M ), 306 (3430); HRMS (ESI) m/z calcd. for

+

C H Br N O 330.9076 [M + H] , found 330.9075.

1

0

9

2

(22) 3,4-dichloro-1-(4-hydroxyphenethyl)-1,5-dihydro-2H-pyrrol-2-one (30)

Cl

N

O

OH

The title compound was prepared according to Procedure A from mucochloric acid (700 mg,

.14 mmol) and tyramine (568.4 mg, 4.14 mmol) and was left to react for 3 days. The crude mixture was

poured into icy water, and the solid was collected and washed twice with ether. A light green solid

4

◦

1

was obtained (280 mg; 25%). m.p. 157.8 C; H NMR (CDCl , 300 MHz):

δ

2.72 (t, J = 7.2 Hz, 2H, CH₂),

4.2 (s, 2H, C5-CH ), 6.67 (d, J = 8.4 Hz, 2H, ArH), 7.00 (d, J = 8.4 Hz,

H, ArH), 9.22 (s, 1H, OH). ¹³C NMR (DMSO, 75.5): 33.3 (CH ), 44.6 (CH ), 53.7 (C5-CH ), 115.7

3

δ

3.55 (t, J = 7.2 Hz, 2H, CH₂),

δ

2

(

1

3

ArCH), 124.1

©

, 129.0 (C), 130.0 (ArCH), 140.9 (C), 156.3 (C), 163.4 (C=O); IR (ATR): υmax 3237.6,

19075.9 cm ¹

−

M

−1

),

670.5, 1513.3, 1451.15, 1279.3, 1219.1, 1169.4, 827.6; UV (MeOH): λmax 277.0 nm (

20 (2585.1); HRMS (ESI) m/z calcd. for C H Cl N O Na 294.0059 [M + Na] , found 294.0060.

ε

+

12

11

2

1

2

1

(23) 1-(2-(1H-indol-3-yl)ethyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-one (31)

The title compound was prepared according to Procedure A from mucochloric acid (200 mg,

.18 mmol) and tryptamine (189.7 mg, 1.18 mmol) and was left to react for 24 h. The crude mixture was

1

extracted with dichloromethane (30 mL) and washed with water (15 mL) followed by brine (15 mL),

dried over sodium sulphate, and the solvent was evaporated. The oil residue was then triturated

with methanol, and the precipitated solid was ﬁltered to yield a green solid (145 mg; 42%). m.p.

Molecules 2018, 23, 1106

21 of 29

◦

1

59.3 C; H NMR (DMSO, 400 MHz,):

ArH), 7.16 (t, J = 7.7, 14.8 Hz, 1H, ArH),

H),

3.6 (CH ), 54.0 (C5-CH ), 111.3

δ

2.96 (t, J = 7.3 Hz, CH ), 3.67 (t, J = 7.3 > N-CH ), 7.07 (s, 1H,

2

δ

7.22 (t, J = 7.0, 14.8 Hz, 1H, ArH),

δ

7.40 (d, J = 8.1 Hz,

1

4

(

1

δ

7.61 (d, J = 8.1 Hz, 1H, ArH), 10.84 (1H, NH). ¹³C NMR (DMSO, 100 MHz):

δ

24.2 (CH₂),

, 111.9 (ArCH), 118.6 (ArCH), 118.8 (ArCH), 121.5 (ArCH), 123.3

ArCH), 124.2 (C), 127.5 (ArC), 136.7 (ArC), 140.9 (C), 163.5 (C=O); IR (ATR): υmax 3277.5, 3058.0, 2918.1,

772.8, 1678.8, 1452.1, 1364.8, 1298.4, 1099.1, 1036.7, 974.5, 852.9, 738.8; UV (MeOH): λmax 275.0 nm

©

2

−

1

−1

(ε

3463.2 cm

M + Na] , found 317.02188.

M ), 220.0 nm (15204.8); HRMS (ESI) m/z calcd. for C H Cl N O Na 317.02189

14

12

2

1

+

[

(24) N-butyl-4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide (32)

The title compound was prepared from acid 19 (200 mg, 0.74 mmol) and butylamine (53.8 mg,

0.74 mmol) according to the general Procedure B. The solvent of the reaction mixture was evaporated

then triturated with methanol, and the precipitated solid was ﬁltered. A beige solid was obtained

◦

40 mg; 17%). m.p. 168.5 C; H NMR (DMSO-d , 600 MHz): δ 0.99 (t, J = 7.4 Hz, 3H, CH ), 1.23–1.34 (m,

6 3

1

(

4

2

5

H, 2

×

CH ), 1.50 (m, 2H, CH ), 4.89 (s, 2H, C5-CH ), 7.80 (d, J = 8.9 Hz, 2H, ArH), 7.89 (d, J = 8.9 Hz,

2 2 2

13

H, ArH), 8.41 (s, 1H, NH). C NMR (DMSO-d , 150.9 MHz):

3.92 (C5-CH ), 118.26 (ArCH), 124.2 (C), 129.60 (ArCH), 130.8 (C), 141.0 (C), 142.5 (C), 162.7 (C=O),

δ

14.11 (CH ), 20.11 (CH ), 31.71 (CH ),

3 2 2

6

2

1

65.7 (C=O); IR (ATR): υmax 3314.7, 3078.0, 2955.3, 1696.7, 1606.9, 1508.1, 1381.5, 1152.3, 835.7, 763.2;

−

1

−1

M ); HRMS (ESI) m/z calcd. for C H Cl N O Na

UV (MeOH): λmax 300.0 nm (

3

ε

10,897.6 cm

15

16

2

1

+

49.0481 [M + Na] , found 349.0482.

(25) 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-hexylbenzamide (33)

Molecules 2018, 23, 1106

22 of 29

The title compound was prepared from acid 19 (200 mg, 0.74 mmol mmol) and hexylamine

(74.4 mg, 0.74 mmol) according to the general Procedure B. The solvent of the reaction mixture was

evaporated then triturated with methanol, and the precipitated solid was ﬁltered. A white solid

◦

1

was obtained (75 mg; 29%); m.p. 159.5 C; H NMR (DMSO-d , 400 MHz):

δ

0.88 (t, J = 6.9 Hz,

6

3

H, CH ), 1.18–1.21 (m, 6H, 3

×

CH ), 1.28–1.53 (m, 2H, CH ), 3.06–3.09 (m, 2H, CH ), 4.90 (s, 2H,

3

2

13

CH ), 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.90 (d, J = 8.4 Hz, 2H, ArH), 8.43 (s, NH). C NMR (DMSO-d₆,

2

1

(

00 MHz):

ArCH), 124.2 (C), 128.6 (ArCH), 130.8 (C), 140.9 (C), 142.4 (C), 162.7 (C=O, 165.7 (C=O). IR (ATR):

υmax 3331.8, 2929.2, 1691.9, 1630.4, 1503.6, 1377.3, 1270.6, 1151.8, 927.3, 846.7; UV (MeOH): λmax

δ 22.5 (CH ), 26.7 (CH ), 29.6 (CH ), 31.5 (CH ), 39.7 (CH ), 45.9 (CH ), 54.0 (C5-CH ), 120.0

3 2 2 2 2 2 2

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 377.0794 [M + H] ,

17 20 2 2 2 1

+

2

80.0 nm (

ε

6522.5 cm

M

found 377.0792.

(26) N-benzyl-4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide (34)

The title compound was prepared from 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic

acid (19) (200 mg, 0.74 mmol mmol) and benzylamine (117.8 mg, 1.1 mmol) according to the general

Procedure B. The precipitated solid was collected by ﬁltration and washed with 1 mL of water, followed

◦

by hexane, then triturated from methanol. A white solid was obtained (230 mg; 76%). m.p. 175.3 C;

1

H NMR (DMSO-d , 400 MHz):

NH), 7.80 (d, J = 8.8 Hz, 2H, ArH), 7.96 (d, J = 8.8, 2H, ArH). C NMR (DMSO-d , 400 MHz):

δ 3.96 (s, 2 H, CH ), 4.90 (s, 2H, C5-CH ), 7.21–7.46 (m, 6H, ArH and

2 2

6

13

δ

54.0

6

(

C5-CH ), 67.5 (CH ), 118.2 (ArCH), 124.2 (C), 127.7 (ArC), 128.3 (ArC), 128.4 (ArCH), 128.7 (ArCH),

2

1

28.9 (ArCH), 130.8 (C), 142.4 (C), 162.8 (C=O), 167.8 (C=O). IR (ATR): υmax 2824.5, 2641.0, 1699.5, 1604,

3272.6 cm ¹

−

M

−1

); HRMS (ESI) m/z

510.0, 1366.2, 1305.9, 1152.1, 782.0; UV (MeOH): λmax 285.0 nm (

ε

+

calcd. for C H Cl N O 361.0505 [M + H] , found 361.0501.

18

15

2

(27) Synthesis of N-(2-(1H-indol-3-yl)ethyl)-4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)

benzamide (35)

Molecules 2018, 23, 1106

23 of 29

The title compound was prepared from 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic

acid (19) (300 mg, 1.10 mmol) and tryptamine (176.7 mg, 1.1 mmol) according to the general Procedure

B. The precipitated solid was collected by ﬁltration and washed with (1 mL) water, followed by

hexane, then the solid was triturated from methanol to yield a pale pink solid (400 mg, 88%); m.p.

◦

1

49.8 C; H NMR (DMSO-d , 400 Hz):

δ

2.52 (m, 2H, CH ), 3.32 (m, 2H, CH ), 4.88 (S, 2H, C5-CH ),

6

2

6

.99 (t, J = 7.0 Hz, 1H, ArH),

H, ArH),

0.94 (s, CONH). ¹³C NMR (DMSO-d , 100 MHz):

δ

7.08 (t, J = 7.0 Hz, 1H, ArH)),

δ

7.22 (s, 1H, NH),

δ

7.36 (d, J = 8.0 Hz,

1

δ

7.55 (d, J = 8.0 Hz, 1H, ArH), 7.75 (d, J = 8.8 Hz, 2H, ArH), 7.94 (d, J = 8.8 Hz, 2H, ArH),

23.4 (CH ), 46.1 (CH ), 53.8 (CH ), 83.01 (ArC),

δ

6

2

09.82 (ArCH), 112.04 (ArCH), 118.53 (ArCH), 119.03 (ArCH), 120.15 (ArCH), 121.7 (ArCH), 123.8

30.6 (ArCH), 136.7 (ArCH), 141.2 (ArC), 142.37 (ArC), 142.85 (ArC), 153.0 (C), 166.35 (C=O), 167.4

©,

(

2

C=O); IR (ATR): υmax 3223.4, 2902.4, 1700.1, 1603.1, 1370.0, 1248, 1038.6, 843.4, 781.0, UV (MeOH): λmax

3802.5 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 436.0590 [M + Na] ,

21 17 2 3 2 1

+

80.0 nm (

ε

found 436.0583.

(28) 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-morpholinoethyl)benzamide (36)

The title compound was prepared from 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic

acid (19) (200 mg, 0.74 mmol mmol) and 2-morpholinoethan-1-amine (95.7 mg, 0.74 mmol) according to

Procedure B. After the completion of the reaction, the crude mixture was dissolved in ethanol, and few

drops of diethyl ether were added dropwise. Then, the black solid impurity was ﬁltered and discarded.

The yellow ﬁltrate was evaporated to dryness then recrystallized from ethanol, and a yellow small

◦

1

needle crystal was obtained (7 mg; 3%). m.p. 129.1 C; H NMR (DMSO-d , 600 MHz):

δ

3.05–3.13

6

(

m, 4H, 2 X CH ), 3.55 (d, J = 12 Hz, 2H, CH ), 3.68 (d, J = 6.0 Hz, 2H, CH ), 3.79 (t, J = 12.0 Hz, 2H,

2

CH ), 3.98 (d, J = 12.0, 2H, CH ), 4.91 (s, 2H, C5-CH ), 7.84 (d, J = 8.6, 2H, ArH), 7.98 (d, J = 8.6 Hz, 2H,

2

13

ArH), 8.87 (s, NH). C NMR (DMSO-d , 100 MHz):

δ 34.2 (CH ), 46.0 (CH ), 51.7 (CH ), 54.0 (CH ),

2 2 2 2

6

3.6 (CH ), 118.3 (ArCH), 124.2

2

(

ATR): υmax 3307.4, 2923.8, 1699.1, 1603.7, 1509.0, 1370.2, 1110.5, 782.0; UV (MeOH): λmax 285.0 nm

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O 384.0876 [M + H] , found 384.0879.

17 20 2 3 3

+

ε 6378.6 cm

M

(29) 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-morpholinopropyl)benzamide (37)

Molecules 2018, 23, 1106

24 of 29

The title compound was prepared from 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic

acid (19) (300 mg, 1.1 mmol mmol) and 3-morpholinopropan-1-amine (160 mg, 1.1 mmol) according to

the general Procedure B. The precipitated solid was ﬁltered, washed with ether and hexane to yield

◦

1

a light beige solid (180 mg; 50%). m.p. 129.1 C; H NMR (DMSO-d , 400 MHz):

δ 1.70–1.74 (m, 2H,

6

CH ), 2.34–2.41 (m, 10 H, 5

×

CH ), 2.53 (t, J = 5.4, 10.0, 2H, CH ), 4.88 (s, 2H, C5-CH ), 7.72 (d, J = 8.8,

2

13

2H, ArH), 7.91 (d, J = 8.8, 2H ArH and NH), C NMR (DMSO-d , 100 MHz):

δ

23.91 (CH ), 38.8

6

2

(

CH ), 53.3 (CH ), 55.97 (CH ), 66.34 (CH ), 117.9 (ArCH), 124.8

2

(

1

C), 141.44 (C), 163.18 (C=O), 173.09 (C=O). IR (ATR): υmax 3372.1, 3078.2, 2934.0, 1695.2, 1531.4, 1370.7,

2695.1 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for

110.8, 1042.2, 778.6; UV (MeOH): λmax 285.0 nm (

ε

+

C H Cl N O 398.1033 [M + H] , found 398.1030.

18

22

2

3

(

30) 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-(piperidin-1-yl)propyl)benzamide (38)

The title compound was prepared from 4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic

acid (19) (200 mg, 0.74 mmol mmol) and 3-(piperidin-1-yl)propan-1-amine (104.6 mg, 0.74 mmol)

according to the general Procedure B. The solid product was collected by ﬁltration, washed with

◦

1

water and hexane to yield a beige solid (64 mg; 27%). m.p. 165.5 C; H NMR DMSO-d , 600 MHz):

6

δ

1.24–1.38 (m, 8H, 4

×

CH ), 1.46–1.48 (m, 4H, 2

×

CH ), 1.98–2.00 (m, 2H, CH ), 2.01–2.3 (m, 2H,

2 2

2

CH ), 4.90 (s, 2H, C5-CH ), 5.32 (s, NH), 7.79 (d, J = 8.2 Hz, 2H, ArH), 7.95 (d, J = 8.2 Hz, 2H, ArH).

2

13

C NMR (DMSO-d , 100 MHz):

δ 22.3 (CH ), 25.5 (CH ), 26.4 (CH ), 29.1 (CH ), 31.3 (CH ), 39.7

2 2 2 2 2

6

(

2 2

Molecules 2018, 23, 1106

25 of 29

1

67.8 (C=O). IR (ATR): υmax 3355.8, 2936.3, 16887.5, 1540.6, 1375.5, 1042.7, 850.0, 775.0; UV (MeOH):

7022.6 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Cl N O 396.1240 [M + H] ,

19 24 2 3 2

+

λmax 285.0 nm (

found 396.1234.

ε

(31) N-(4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)butyramide (39)

The title compound was prepared from 1-(4-aminophenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-

one (23) (200 mg, 0.82 mmol) and butyryl chloride (87.7 mg, 0.82 mmol) according to the general

Procedure C. After completion of the reaction, the crude mixture was evaporated to complete dryness,

followed by trituration from methanol, then washed with 1 mL of ether twice; the solid was collected

◦

by ﬁltration and dried in high vac. An off-white solid was obtained (89 mg, 31%). m.p. 195.5 C;

1

H NMR (DMSO-d , 400 MHz):

δ 0.91 (t, J = 7.3 Hz, 3H, CH ), 1.58–1.64 (m, 2H, CH ), 2.26 (t, J = 7.3 Hz,

3 2

6

13

2

H), 4.82 (s, 2H, C5-CH ), 7.62 (brs, 4 H, ArH), 9.92 (s, NH). C NMR (DMSO-d , 100 MHz):

δ

14.1

, 134.0 (C), 136.6 (C), 141.6 (C), 162.2

C), 171.5 (C=O); IR (ATR): υmax 3343.2, 2961.6, 1677.3, 1522.0, 1408.4, 1283.0, 820.6; UV (MeOH): λmax

2

6

(

2 2 2

(

2

−

1

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 335.0325 [M + Na] ,

14 24 2 2 2 1

+

90.0 nm (

ε

8377.5 cm

M

found 335.0323.

(32) N-(4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)hexanamide (40)

The title compound was prepared from 1-(4-aminophenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-

one (23) (200 mg, 0.82 mmol) and hexanoyl chloride (110.7 mg, 0.82 mmol) according to Procedure C.

After completion of the reaction, the crude mixture was evaporated to complete dryness, followed

by trituration from methanol, then the solid was washed with 1 mL of ether twice. The solid was

collected by ﬁltration and dried in high vac. An off-white solid was obtained (164 mg; 52%). m.p.

◦

25.7 C; H NMR (DMSO-d , 300 MHz): δ 0.93 (t, J = 7.0 Hz, 3H, CH ), 1.38 (d, J = 3.6 Hz, 4H,

6 3

1

2

7

3

×

CH ), 1.73–1.79 (m, 2H, CH ), 2.38 (t, J = 7.47 Hz, 2H, CH ), 4.49 (s, 2H, C5-CH ), 7.18 (s, NH),

2 2 2 2

13

.59 (brs, 4H, ArH). C NMR (CDCl , 75.5 MHz): 14.3 (CH ), 22.4 (CH ), 25.2 (CH ), 31.4 (CH ),

3

2

6.8 (CH ), 54.1 (CH ), 119.9 (ArCH), 120.1 (ArCH), 124.3 (C), 133.8 (C), 136.7 (C), 141.6 (C), 162.2

2

(C), 171.7 (C); IR (ATR): υmax 3358.6, 2949.4, 1677.3, 1515.9, 1413.1, 1515.9, 1284.3, 1042.8, 853.0; UV

Molecules 2018, 23, 1106

26 of 29

8394.3 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for C H Cl N O Na 363.0638

(

[

MeOH): λmax 290.0 nm (

M + Na] , found 363.0636.

ε

16

18

2

1

+

(33) N-(4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)octanamide (41)

The title compound was prepared from 1-(4-aminophenyl)-3,4-dichloro-1,5-dihydro-2H-pyrrol-2-

one (23) (200 mg, 0.82 mmol) and octanoyl chloride (133.8 mg, 0.82 mmol) according to Procedure C.

After completion of the reaction, the crude mixture was evaporated to complete dryness, followed

by trituration from methanol and then washed with 1 mL of ether twice. The solid was collected by

◦

1

ﬁltration and dried in high vac. A white solid was obtained (140 mg; 44%). m.p. 194.1 C; H NMR

(

CDCl , 300 MHz): 0.91 (t, J = 6.8 Hz, 3H, CH ), 1.31–1.40 (m, 4H, 2

×

CH ), 1.58 (brs, 4H, 2

×

CH₂),

3

2

1

.75 (m, 2 H, CH ), 2.38 (t, J = 7.7 Hz, 2H, CH ), 4.50 (s, 2H, C5-CH ), 7.24 (s, NH), 7.58 (brs, 4H, ArH).

2

1

3

C NMR (CDCl , 75.5 MHz): 14.4 (CH ), 22.5 (CH ), 25.6 (CH ), 28.9 (CH ), 29.1 (CH ), 31.6 (CH ),

3

2

3

6.8 (CH ), 54.1 (CH ), 119.9 (ArCH), 120.1 (ArCH), 124.3 (C), 133.8 (C), 136.7 (C), 141.6 (C), 162.2 (C),

2 2

1

2

71.7 (C=O); IR (ATR): υmax 3338.9, 2915.7, 1678.1, 1521.3, 1285.3, 1156.1, 931.4, 822.9; UV (MeOH): λmax

5982.4 cm ¹M ); HRMS (ESI) m/z calcd. for C H Cl N O Na 391.0951 [M + Na] ,

− −1

90.0 nm (ε

18 22 2 2 2 1

+

found 391.0952.

(

34) 2-(4-bromophenyl)-N-(4-(3,4-dichloro-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide (42

)

The title compound was prepared according to method C from amine 1-(4-aminophenyl)-3,

4

-dichloro-1,5-dihydro-2H-pyrrol-2-one (23) (200 mg, 0.82 mmol) and 2-(4-bromophenyl)acetic acid

(

110.7 mg, 0.82 mmol). The latter was ﬁrst converted to the corresponding acid chloride prior to amide

coupling by treatment with thionyl chloride (2 h) on reﬂux, followed by complete dryness in high vac.

After completion of the reaction, the crude mixture was evaporated to complete dryness, followed by

trituration from methanol, and the obtained solid was then washed twice with 1 mL of ether. The solid

was collected by ﬁltration and dried in high vac. A white solid (100 mg; 32%) was obtained; m.p. 159.2

◦

1

C; H NMR (CDCl , 300 MHz): 3.64 (s, 2H, CH ), 4.82 (s, 2H, C5-CH ), 7.30 (d, J = 8.2 Hz, 2H, ArH),

3

2

Molecules 2018, 23, 1106

27 of 29

7

.52 (d, J = 8.2 Hz, 2H, ArH), 7.63 (brs, 4H, ArH), 10.33 (s, NH). ¹³C NMR (CDCl , 75.5 MHz): 45.95

3

(

CH ), 54.09 (CH ), 120.06 (ArCH), 120.21 (ArCH), 124.3 (C), 131.6 (ArCH), 131.9 (ArCH), 134.1 (C),

2 2

1

35.85 (C), 136.40 (C), 141.7 (C), 162.3 (C), 169.0 (C); IR (ATR): υmax 3279.8, 2978.7, 1690.5, 1516.5, 1379.5,

3375.7 cm ¹

−

M

−1

); HRMS (ESI) m/z calcd. for

153.7, 927.8, 824.9; UV (MeOH): λmax 285.0 nm (

ε

+

C H Br Cl N O Na 460.9430 [M + Na] , found 460.9431.

18

13

1

2

1

4

.3. Biology

4

.3.1. Quorum Sensing Inhibition Assay for PAMH602

The P. aeruginosa MH602 PlasB::gfp (ASV) reporter strain was used. An overnight culture was

prepared in Luria-Bertani (LB10) media supplemented with gentamycin (40 M). This bacterial culture

solution was diluted (1 in 100) with LB10 supplemented with gentamycin (15 M). Stock solutions of

the synthesized compounds were prepared at 20 mM in DMSO. Compounds were pipetted into each

well with ﬁnal concentrations of 250 M, 125 M and 62.5 M (in triplicate) with a ﬁnal volume of

00 L with the prepared bacterial culture. The negative control was prepared containing 200 L of the

and TP-5 were

µ

2

µ

bacterial culture without the tested compounds. The halogenated furanone (Fu-30)

5

◦

used as positive controls. The plates were incubated at 37 C for 15 h. The plates were measured for

GFP expression (ﬂuorescence: excitation 485 nm, emission 535 nm) using a microplate reader (Wallac

Victor, Perkin-Elmer), and the cell growth was also assessed by recording the OD at 600 nm.

4

.3.2. Pyocyanin Assay

An overnight culture of P. aeruginosa PAO1 was diluted 1 in 100 with LB10 medium. To a 5-mL

test tube were added the tested compounds (compounds prepared from a DMSO stock of 20 mM),

and 2.5 mL of the prepared culture was added, giving ﬁnal concentrations of 250 M and 32 M.

µ

An equivalent amount of DMSO was prepared for the positive control containing the bacterial culture

without the tested compounds. The same medium without bacterial culture in an equivalent amount

◦

of DMSO was used as the background reading. The cultures were grown with shaking at 37 C for 17 h.

The ﬁnal cell density was measured by reading the absorbance at 600 nm (OD₆₀₀), and the solutions

were then centrifuged for 5000 rpm. The clear supernatant was then transferred by pipetting into

a plastic 96-well plate, and the absorbance was measured at 695 nm.

4

.4. Docking

The crystal structure of LasR complexed with OdDHL (PDB: 2UV0) was used. The protein

was prepared before docking by removing the other subunits, and only subunit E was used for

docking based on a previous control docking. The binding site and cavity were prepared as follows.

Water molecules and ligands including the LasR bound ligand in the binding pocket were removed,

and the crystal structure was protonated. The binding pocket was chosen from the ‘receptor cavities’

tool, and this deﬁned the binding site sphere in the proposed pocket. Alternate conformers were

identiﬁed (Ser20 and Ser131), but were outside the binding pocket sphere. The geometry of the

protonated OdDHL ligand was optimized with the CHARMM force ﬁeld [28], and it was docked

back to the prepared protein using the Genetic Optimization for Ligand Docking (GOLD) algorithm,

Versions 5.2.1, 5.2.2, and 5.4.0 (Cambridge Crystallographic Data Centre, U.K.) [29]. The number of

the docking runs was set to 100, while the ‘detect cavity’ and ‘early determination’ were set to ‘false’,

but all other parameters were set to their defaults. The GoldScore was speciﬁed as the ﬁtness score

function. After the docking run, ligand poses were analysed based on their clusters using the RMSD

of heavy atoms and within each cluster; poses were ranked in order of decreasing GoldScore value.

The largest clusters at RMSD of around 2 Å were usually considered. The best pose of the largest

cluster for OdDHL when docked back to the prepared protein was selected. The ligand from the

crystal structure was superimposed with the selected pose, and an acceptable RMSD was obtained of

0

.94 Å (heavy atoms).

Molecules 2018, 23, 1106

28 of 29

The tested compounds were sketched, protonated and their energy minimized as above with the

CHARMm force ﬁeld [28]. Docking was then performed and analysed as described above. Receptor

ligand interactions of selected poses were analysed using the ‘view interactions’ tool. Different types of

interactions including hydrogen bonds, hydrophobic and π-interactions were examined and compared

to the crystal structure. Any other attractive forces, repulsive or unfavourable interactions were noted.

Supplementary Materials: The growth inhibition, full docking and spectral data are provided in

the Supplementary.

Author Contributions: N.K., R.G., D.S.B., G.I. and K.K.K.H. planned and designed the experiments. B.A., S.N.

and N.N.B. provided the data from biology screening. B.A. and D.S.W. performed the docking study and

analysed and interpreted the data. B.A. and A.T., performed the chemistry experiments and analysed and

interpreted the data. B.A. wrote the paper. S.A.R. provided the bacterial strains and assistance with biology. N.K.

supported ﬁnancially.

Acknowledgments: We thank the NMR and BMSF facility, UNSW. The authors would like to acknowledge the

Saudi Ministry of Education and King Khalid University for an Endowment Fund and ﬁnancial support given to

B. Almohaywi.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of Compounds 12, 13, 19, 23 and 24 are available from the authors.

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Design and synthesis of lactams derived from mucochloric and mucobromic acids as pseudomonas aeruginosa quorum sensing inhibitors

DOI: 10.3390/molecules23051106

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Full text of DOI:10.3390/molecules23051106

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