Synthesis and biological evaluation of N-naphthoyl-phenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors

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Kumar, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB00298F.

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Synthesis and Biological Evaluation of N-Naphthoyl-Phenylglyoxamide-Based

Small Molecular Antimicrobial Peptide Mimics as Novel Antimicrobial agents

and Biofilm Inhibitors

Shashidhar Nizalapur^a, Kitty K. K. Ho^a, Önder Kimyon^b, Eugene Yee^a,e, Thomas Berry^c, Mike

Manefield^b, Charles G. Cranfield^c, Mark Willcox^d, David StC Black^aand Naresh Kumar^a*

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

www.rsc.org/

Abstract

Antimicrobial peptides (AMPs) are a key component of the human immune system. Synthetic AMP mimics represent a

novel strategy to counteract the increasing incidence of antimicrobial resistance. Here, we describe the synthesis of novel

glyoxamide derivatives via ring-opening reactions of N-hexanoyl, N-benzoyl and N-naphthoylisatins with N,N-

dimethylethane-1,2-diamine and N,N-dimethylpropane-1,3-diamine. These were converted to both the hydrochloric acid

(HCl) or quaternary ammonium iodide (MeI) salts and their antibacterial activity against Staphylococcus aureus was

investigated by their zone-of-inhibition and minimum inhibitory concentration (MIC). The HCl salt 22b exhibited the lowest

MIC of 16 µg mL^-1, whereas the corresponding MeI salt 22c had a MIC of 39 µg mL^-1. We also investigated the in vitro

toxicity of active compounds against the MRC-5 normal human lung fibroblasts and their activity against established

biofilm in S. aureus.

*E‒mail: n.kumar@unsw.edu.au*Tel: +61 29385 4698; Fax:+61 29385 6141.

Introduction

antibiotics than planktonic bacteria,¹¹and are also protected

fromthe host immune system.¹⁴In this context, it is very important

to develop drugs that eradicate the biofilms through novel

therapeutic actions.

The evolution of bacterial resistance towards antibiotics is a rapidly

increasing problem.¹In the US alone, two million people are

stricken with serious bacterial infections and more than 23,000

people die due to drug-resistant bacteria on an annual basis.^{2, 3}This

resistance arises from the selective pressure applied by the

mechanisms of action employed by traditional antibiotics, such as

killing bacteria by the inhibition of cell wall synthesis, DNA and RNA

synthesis, protein synthesis and folate synthesis, or the

depolarization of membrane potential.^{4, 5}Furthermore, the misuse

and overuse of broad-spectrum antibiotics has accelerated the

development of antibiotic resistance.⁶

Antimicrobial peptides (AMPs) are an important constituent in

the immune defenses of most living organisms.¹⁵AMPs exhibit a

broad range of activities against Gram-positive and Gram-negative

bacteria, protozoa, yeast, fungi and viruses.¹⁶AMPs are generally

between 12 and 100 amino acid residues long and are amphipathic

in nature, i.e., they possess both hydrophobic and hydrophilic

18

regions.^17,

Unlike traditional antibiotics, most AMPs act by

Another problem with bacteria is the formation of bacterial

biofilms, which are a protective barrier formed by pathogenic

colonies, increasing their resistance to antibiotics and immune

defenses.⁷Bacteria cooperatively communicate with each other

through a quorum sensing (QS) mechanisms producing various

phenotypes including biofilms.^{8, 9}These are a major contributor to

the fouling of medical implants such as catheters, artificial hips and

contact lenses.^{10, 11}In addition, 60-80% of major chronic infections

disrupting the membranes, which leads to cell death. It is difficult

for bacteria to develop resistance against AMPs.¹⁹One remarkable

feature of AMPs is that they are selective to microbes and have

little or no effect on mammalian cells.¹⁹However, despite their

many advantages, AMPs have a few limitations, such as protease

liability, non-selective action on microbial strains and high

manufacturing costs.¹⁵

In order to overcome these limitations, research groups have

synthesized various types of antimicrobial peptidomimetics (AMP

mimetics), such as α-peptides,²⁰β-peptides,²¹peptoides,²²α-

AApeptides,²³and γ-AApeptides.²⁴In addition, several small

molecular antimicrobial peptidomimics (SMAMP mimics) have also

been investigated and showed significant antimicrobial activity

against a range of bacterial strains (Figure 1).^25-33Ghosh et al. have

synthesized small molecule AMPmimics using one, two and three-

aromatic rings combined with hydrophobic chains of variable length

13

are due to bacterial biofilms.^12,Notably, bacterial colonies that

have formed a biofilm are 10-1000 times more resistant to

^aSchool of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia.

^bSchool of Biotechnology and Biomolecular Sciences.

^cMolecular Biosciences Team, School of Life Sciences, University of Technology Sydney.

^dSchool of Optometry and Vision Science, UNSW Australia, Sydney, NSW 2052,

Australia.

^eChildren’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New

South Wales, Sydney, Australia

.

UNSW Australia, Sydney, NSW 2052, Australia.

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Figure 1: SMAMP mimics synthesized by various research groups.

as a hydrophobic core. Lysine was used as the cationic motif and Interestingly, the synthesis of N-naphthoylisatins and their

these novel molecules were tested against different glyoxamide derivatives have not yet been explored. For the first

bacterialstrains, with the most promising candidate compound 1 time, we report here the synthesis of N-naphthoylisatins using a

possessing excellent antibacterial activity with a minimal inhibitory simple and effective method. In addition, we also report the

concentration (MIC) of 2.2 µg mL^-1towards Staphylococcus synthesis of novel glyoxamides via the ring-opening reaction of N-

aureus.³³Tew designed the aryl-based compound 2, which was naphthoyl, N-benzoyl and N-hexanoylisatins with N,N-

found to mimic the mechanism of action of natural AMPs, showing dimethylethane-1,2-diamine and N,N-dimethylpropane-1,3-diamine

excellent activity against S. aureus and Escherichia coli with a MIC of to give novel SMAMP mimics. The N-substituents confer excellent

3.13 µg mL^-1against both bacterial strains.³¹Furthermore, the hydrophobicity, while hydrophilicity is introduced through the

group of DeGrado synthesized several SMAMP mimics, among formation of quaternary ammonium salts at the dimethylamine

which compound 3 exhibited excellent antimicrobial activity against functionalities, thus making the compounds amphipathic in nature

S. aureus and E. coli with a MIC of 6.25 µg mL^-1against both (Figure 2). The biological evaluation of these compounds was

strains.²⁵

performed against Gram-positive and Gram-negative bacterial

It is well known that N-acyl and N-benzoylisatins undergo ring- strains and normal mammalian cells. Finally, the effectiveness of

opening reactions with alcohols, amines and amino acids to yield these compounds to disrupt the biofilm formation in Gram-positive

the corresponding glyoxylic esters and amides. These reactions are bacteria was also investigated.

irreversible, clean and high yielding. Recently, our group has

reported glyoxamide derivatives of N-acyl and N-benzoylisatins that

Results and Discussion

Synthesis

were ring-opened with amines and aminoacids.^{34, 35, 36}Owing to the

resemblance of N-phenylglyoxylamides to peptide bonds, they are

Initially, Isatin 4 was reacted with NaH followed by hexanoyl

chloride in THF according to a known method,³⁷generating N-

ideal candidate for the developments of peptide mimics.

hexanoylisatin 5 in 65% yield (Scheme 1). Similarly, 4 was treated

with benzoyl chloride and pyridine,³⁸giving N-benzoylisatin 6 in

90% yield (Scheme 1).

For the synthesis of N-naphthoylisatins, methods similar to

those used to generate 5 and 6 were found to be ineffective (Table

1, entries 1 and 2). Optimization of the method using various base

and solvent combinations identified the combination of NaH and

DMF (Table 1, entry 5) as the most suitable system, giving N-

naphthoylisatin 11 in 90% yield (Scheme 2). This suggests that the

reaction of isatin with naphthoyl chloride requires a highly polar

solvent and strong base. Using these optimized conditions, a series

of 5-substituted naphthoylisatins (12-15) were synthesized in good

yields (65-92%). Recrystallization from MeOH was found to be the

best purification method, being more favourable than column

chromatography in terms of ease and time required.

Figure 2: General structure of glyoxamide-based SMAMP mimics.

2 | J. Name., 2012, 00, 1-3

The ring-opening reactions of N-substituted isatins (5-6) and

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compounds were then converted to the corresponding

quaternaryammonium salts by two methods. Firstly, treatment with

4M HCl in dioxane gave the hydrochloride (HCl) salts (16b-25b) in

70-98% yield (Scheme 3). Alternatively, treatment with

methyliodide in DCM/THF gave the quaternary ammonium iodide

(MeI) salts (16c-25c) in 54-95% yields (Scheme 3). The identity of

the synthesized compounds is detailedin Table 2.

In general, the N-naphthoylisatins 11-15 were synthesized in

good to excellent yields (65-90%). With the exception of compound

13, these compounds were isolated as pure compounds without

additional purification, by simply precipitating from the reaction

mixture after the addition of ice water. Similarly, the glyoxamide

derivatives (16a-25a) did not require any additional purification

before being carried to the next step. Purification of compounds

16b-25b and 16c-25c was achieved by the addition of diethyl ether

to remove baseline impurities or unreacted starting materials.

Proton NMR was found to be the best method to confirm the

formation of the ring-opened glyoxamide derivatives, which

showed the appearance of prominent signals corresponding to the

amide NH proton (δ 10-11) and glyoxylamide proton (δ 8-9). This

novel methodology opens a new avenue for the synthesis of N-

naphthoylisatins and their glyoxamide derivatives, allowing for the

expansion of structure-activity relationship (SAR) data concerning

isatin-based antimicrobial agents.

Scheme 1: Synthesis of N-hexanoyl and N-benzoyl isatins. Reaction

conditions: Compound 5: 4 (1 equiv), hexanoyl chloride (1.2 equiv),

THF, 0 °C to rt, 16h; Compound 6: 4 (1 equiv), benzoyl chloride (1.1

equiv), pyridine, 0 °C, 2h.

Antibacterial activity

The antimicrobial activity of the synthesized compounds (16a-25a;

16b-25b and 16c-25c) was evaluated by zone-of-inhibition and their

minimum inhibitory concentrations values (MICs, the concentration

of compounds required to completely inhibit the growth of

bacteria).

Scheme 2: Synthesis of N-naphthoylisatins. Reaction condition: 4, 7-

10(1.0 equiv), naphthoyl chloride (1.2 equiv), DMF, 0 °C, 2 h.

Table 1: Conditions for the reaction of naphthoyl chloride with

isatins.

Disk diffusion assay (Zone-of-inhibition)

Entry Reactant Conditions

Results^a

NR

The antibacterial activity of the synthesized compounds 16a-21a,

16b-21b and 16c-21c was first evaluated against Staphylococcus

aureus (Saur 38), Pseudomonas aeruginosa (PAO1) and Escherichia

coli (K-12) by the disk diffusion assay (Table 3).³⁹In this assay, the

bacterial cultures were incubated overnight in tryptone soya broth

medium at 37 °C and the resulting cultures were spread over

nutrient agar plates. The compounds to be tested were dissolved in

DMSO and dried on sterile 6 mm paper disks (0.1 mg per disk).

Zone-of inhibition was measured after incubation at 37 °C for 24 h.

12 (92%) A well-known antibiotic, Gentamicin (0.01 mg per disk), was used as

1

4

Pyridine, 0 °C, 5h.

2

3

4

NaH, THF, 0 °C, 16h.

NaH, THF, 0 °C to 60 °C, 16h.

NR

NaH, 1,4-dioxane, 0 °C to 100 °C, NR

16h.

5

6

7

8

9

4

NaH, DMF, 0 °C, 2h.

11 (90%)

7

a positive control. Compounds 21b and 21c showed significant

8

13 (65%)

zones-of-inhibition of 15 and 19 mm against S. aureus and good

9

14 (89%)

inhibition (≥7mm) against the Gram-negative bacteria. Whereas

10

15 (90%)

compounds 16-19(a-c) did not show any activity against the tested

strains.The compounds, as well as their salts, which showed a zone-

of-inhibition ≥15 mm were further examined by determining their

minimum inhibitory concentrations (MIC).

a: Isolated yields, NR: no reaction.

(11-15) were performed with either N,N-dimethylethane-1,2-

diamine or N,N-dimethylpropane-1,3-diamine in an acetonitrile/

dichloromethane solvent mixture to yield the glyoxamide

derivatives (16a-25a) in yields of 76-99% (Scheme 3). These

Minimal inhibitory concentration (MIC) determination assay

The antimicrobial activity of compounds (21a-c to 25a-c) were

examined by determination of their minimal inhibitory

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Scheme 3: General synthetic scheme for the synthesis of glyoxamide derivatives. Reaction conditions: (a) N-acylisatins (1.0 equiv), amine

(1.0 equiv), MeCN/DCM, rt, 10 min; (b) 4M HCl/ dioxane (5 equiv), DCM, 0 °C, 15 min; (c) CH₃I (2.0 equiv), DCM/THF, 0 °C, 2h.

Table 2: Glyoxamide derivatives and their yields.

Entry

R

R¹

n

Glyoxamides

(yield)^a

Hydrochloride salts (yield)^b

Quaternary ammonium

iodide salts (yield)^c

1

2

3

4

5

6

7

8

H

Br

Cl

F

C₅H₁₁

C₆H₅

1

2

1

2

1

2

16a (76%)

17a (82%)

18a (76%)

19a (85%)

20a (99%)

21a (99%)

22a (92%)

23a (90%)

24a (90%)

25a (90%)

16b (70%)

17b (90%)

18b (98%)

19b (95%)

20b (73%)

21b (83%)

22b (93%)

23b (90%)

24b (90%)

25b (92%)

16c (54%)

17c (85%)

18c (86%)

19c (71%)

20c (84%)

21c (67%)

22c (93%)

23c (94%)

24c (95%)

25c (90%)

C₆H₅

C₁₀H₇

9

10

CH₃

^{a, b and c}Isolated yields.

concentration (MIC) by following a previously published protocol Biofilm disruption assay

(Table 4).⁴⁰In this assay, S. aureus was incubated with compounds The biofilm disruption activity of the compounds (21-25a-c) was

at 37 °C for 24 h. The MIC was recorded by measuring the OD determined as their ability to disrupt the established biofilms of S.

valueat 600 nm using a Wallac Victor (Perkin-Elmer) microplate aureus. In this pre-established biofilm assay, the S. aureus cultures

reader. The MIC value was determined as the lowest concentration were grown in 96-well plate wells overnight prior to treatment with

of compound that inhibits the complete growth of the bacteria (i.e. the synthetic compounds. The biofilms adhered on polystyrene

OD value equal or less than the control having no bacteria). The substratum were quantified by crystal violet staining by following

results showed that the synthesized glyoxamide derivatives the previously published protocol.⁴¹The anti-biofilm activity results

possessed moderate to excellent MIC values. The most potent showed that compounds 22b and 22c exhibited the greatest biofilm

compound 22b (HCl salt) showed a MIC of 16 µg/mL and its disruption activity in S. aureus, with 50% and 46% dispersion at 250

quaternary iodide salt (22c) exhibited a MIC of 39 µg/mL against S. µM, respectively.

aureus.

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Table 3: Zone-of-inhibition diameter (in millimeter) against corresponding 2-carbon linked derivatives, displaying greater zones-

different types of bacteria

Compound

of-inhibition and broad-spectrum activity.

As the N-naphthoyl derivatives (21a-25a) exhibited a moderate

zone-of-inhibition, these compounds were further characterized by

determining their MIC. Since their activity was greatest against S.

aureus, this strain was selected for the MIC determination (Table 4).

We compared the MIC data of all our compounds with MSI-78

(also known as pexiganan),⁴³which is an antibiotic currently in

Phase-III clinical trials and has the amino acid sequence of Gly-Ile-

Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-

Leu-Lys-Lys-NH2. It was found that the neutral compounds (21a-

25a) all had MIC values greater than 350 µg/mL. Additionally, it was

interesting to observe that in the case of the substituted isatin

derivatives (22a-25a) the quaternary ammonium iodide salts 22c-

25c had MIC values 2-3 times higher than the corresponding

hydrochloride salts 22b-25b (39-210 µg/mL vs. 16-57 µg/mL,

respectively). However, in the case of the unsubstituted isatin

derivatives (21b-c), this trend was reversed with the hydrochloride

salt 21b having a MIC of 329 µg/mL, compared to 204 µg/mL for the

corresponding quaternary iodide salt 21c.

Zone-of-inhibition (mm)

S.aureus

12

P. aeruginosa

E. coli

20a

-

21a

14

13

15

7

-

20b

-

21b

11

-

9

-

17c

20c

11

19

23

-

21c

8

25

7

21

Gentamicin

Toxicity assay

To assess the specificity of the SMAMP mimics, the more potent

compounds (22b-25b and 22c-25c) were tested for their in vitro

toxicity against the MRC-5 normal human lung fibroblasts using the

An interesting structure-activity relationship was observed upon

modifying the phenyl ring substituent, which was consistent across

both the HCl and quaternary iodide salts. The replacement of the

proton (21b-c) with fluorine (24b-c), a common replacement in

Alamar blue (Resazurin) assay.⁴²

A dose-response curve was

generated for each compound (Figure 3) at concentrations ranging

from 16-400 µg/mL and the IC₅₀values determined (Table 4).

Table 4: Antibacterial activity (MIC) of compounds against S.aureus

and their toxicity (IC₅₀) in vitro toxicity against the MRC-5 normal

human lung fibroblasts

Activity discussion

It was found that derivatives bearing N-hexanoyl (16a, 17a) and N-

benzoyl (18a, 19a) chains showed no significant activity across the

panel of bacterial strains (data not shown). The only exception to

this was the N-hexanoyl ammonium iodide salt compound 17c,

which showedasmall 7 mm zone-of-inhibition against S. aureus, but

also no inhibition against P. aeruginosa (PAO1) and E.coli K-12.

Interestingly, all of the N-naphthoyl compounds (20a, 20b, 20c)

exhibited moderate to good zones-of-inhibition against S.

aureus.Compound 20a, which has a two-carbon linker and neutral

charge, showed a 12 mm of zone-of-inhibition against S. aureus, but

did not show activity against P. aeruginosa or E. coli. Compound

21a, which has a three-carbon linker and neutral charge, showed an

increased zone-of-inhibition (relative to 20a) of 14 mm against S.

aureus, however this compound was also inactive against P.

aeruginosa or E. coli. Compounds 20b (2 carbon linker), 21b (3

carbon linker) and 20c (2 carbon linker) with positive charge in the

cationic region showed 13, 15 and 11 mm zones-of-inhibition

against S. aureus, respectively, and 21b also showed 11 and 9 mm

zones-of-inhibition against P. aeruginosa and E. coli, respectively. In

addition, compound 21c (3 carbon linker with positive charge)

showed the highest zone-of-inhibition against S. aureus (19 mm)

and also showed 8 and 7 mm of zones-of-inhibition against P.

aeruginosa and E. coli, respectively.

Minimum inhibitory concentration

Toxicity

Compound

MIC (µg mL^-1)

IC₅₀(µg mL^-1)

21a

22a

23a

24a

25a

21b

22b

23b

24b

25b

21c

>350

329

16

ND

20

29

20

57

34

56

17

204

39

ND

>400

352

>400

ND

22c

23c

72

24c

105

210

8-16 ^a

25c

MSI-78⁴³

Based on these zone-of-inhibition results, compounds with an

N-naphthoyl group and a positive charge are necessary for the

antimicrobial activity. Furthermore, it was found that compounds

with 3-carbon linkers were generally more active than the

ND: Not determined

^aLiterature MIC value of MSI78 was taken from ref.43.

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corresponding quaternary iodide salt (22c) showed good activity,

with a MIC of 39 µg/mLagainst S. aureus and low mammalian cell

toxicity.

MRC-5

22b

23b

24b

25b

22c

23c

24c

25c

100

Biofilm disruption activity discussion

Having determined the antibacterial activity of glyoxamides 21-25a-

c

, the ability of these compounds to disrupt established S. aureus

50

biofilms was measured at 250µM (Figure 4). In general, the

compounds displayed low to moderate levels of inhibition, with the

bromo (22a-c) and fluoro (24a-c) substituted derivatives performing

the best. Despite having poor antibacterial activity, the free-base

0

10⁰

10¹

10²

10³

compounds

disruptionactivity. The greatest levels of disruption were observed

for compounds 21a 22a and 24a, which showed 41%, 35% and 37%

(

21a-25a

)

showed good levels of biofilm

Drug concentration [ µM]

Figure 3.In vitro anti-proliferative properties of compounds 22b-25b

and 22c-25c against the MRC-5 normal human lung fibroblast cells

after 72 h incubation, relative to a DMSO control. Points represent

the mean of at least three individual experiments ± Standard error

of the mean (SEM).

,

inhibition at 250 µM, respectively. The hydrochloride salts (21b-

25b) displayed 5-50% biofilm disruptionactivity against S. aureusat

250 µM. Compound 24b, with a fluoro-substituent, showed the

highest inhibition activity amongst all the compounds, with 50 %

dispersion at 250 µM against S. aureus. Finally, the glyoxamide

derivatives with quaternary iodide salts (21c-25c) exhibited low to

moderate biofilm disruption activity. Compounds 22c with bromo-

substituent, and 24c with fluoro-substituent exhibited 46% and 31%

at 250µM against S. aureus respectively. In order to confirm the

biofilm disruption activity, the fluorescence images of the untreated

and treated biofilms were obtained by epifluorescence microscopy.

medicinal chemistry⁴⁴, resulted in a marked (2-5 fold) improvement

in MIC values. Increasing the steric bulk of this substituent further

improved the MIC values, with the chloro derivatives (23b-c) being

more potent than the fluoro derivatives

(24b-c), or their

isosterically related methyl derivatives (25b-c). The most potent

compounds were those bearing a bromo substituent (22b-c), with

MIC values of 16.0 and 39 µg/mL, respectively. The activity of the

HCl salt 22b is also highly comparable to that of the control MSI-78

.

In general, the hydrochloride salts 22b-25b showed higher

levels of toxicity (IC₅₀: 17-34 µg/mL) compared to the corresponding

quaternary iodide salts 22c-25c (IC₅₀: 352 - >400 µg/mL; Table 4).

Although the hydrochloride salts have approximately 2 times more

potent anti-microbial activity than the quaternary ammonium

iodide salts, they are >11 times as toxic towards the normal cells.

Furthermore, the MIC value was typically higher than the IC₅₀value

of these hydrochloride salts, meaning that they are not useful as

anti-bacterials for human disease. However, the quaternary

ammonium iodide salt 22b had a moderate MIC of 39 µg/mL and

was not toxic to normal cells at concentrations of up to 400 µg/mL.

This suggests that the quaternary ammonium iodide salts are a

potentially useful scaffold for the treatment of bacterial infections

in humans.

Figure 4: Percentage of remaining biofilm of S.aureus after 24 h

treatment with the synthesized compounds at 250 µM. Pre-

established biofilm without any addition of compound was used as

control. Error bars represent the standard error of three

independent experiments (n=3).

Overall, all the N-naphthoylglyoxamide derivatives

(21-25)

exhibited good to excellent antimicrobial activity against S. aureus.

The free-base compounds (21a-25a) showed moderate activity,

while converting these compounds into hydrochloride and

quaternary iodide salts significantly increased their antimicrobial

activity by 2-20 fold. Importantly, the HCl salts were 2-fold better

than the corresponding MeI salts and 20-fold better than the free-

base compounds. The 5-substituted compounds

(22-25) also

exhibited better antimicrobial activity than the non-substituted

compounds (21). Compound 22b with a bromo-substituent, N-

naphthoyl group, 3-carbon linker and hydrochloride salts exhibited

Figure 5: Images of established biofilms of S. aureus obtained by

fluorescence microscopy. Live (green), dead (red). Fluorescence

microscopy images of S. aureus showing (

a) untreated biofilms and

highest activity, with

a MIC of 16 µg/mL, whereas the

(

b

) pre-established biofilms treated with 250 µM of compound 24b.

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Overall, these novel synthesized glyoxamide derivatives were result of a thinning of the membrane, as is evident from the slight

able to disrupt the established biofilms. Compounds 24b and 22c increase in membrane capacitances. The presence of negatively

exhibited highest-level of disruption with 50% and 46% at 250 µM charged lipids did not noticeably enhance the actions of the cationic

against S. aureus respectively.

compounds so it is unlikely that any specificity towards bacterial

membranes is as a result of charge.

Membrane Conduction

To determine whether compounds 22b and 22c are lytic to cell

membranes, tethered bilayer lipid membranes (tBLMs), in

association with AC electrical impedance spectroscopy techniques,

Conclusion

This study identified an efficient method for the synthesis of N-

naphthoylisatins and their glyoxamide derivatives by subsequent

ring-opening reactions. We have synthesized 30 novel glyoxamide

based SMAMP mimics by the facile ring-opening of N-naphthoyl, N-

benzoyl and N-hexanoylisatins with N,N-dimethylethane-1,2-

diamine and N,N-dimethylpropane-1,3-diamine, followed by

formation of their hydrochloride and quaternary ammonium iodide

salts. Further, these compounds were evaluated for their

antimicrobial activity against Gram-positive and Gram-negative

bacteria by determining their zone-of-inhibition and MIC, and

toxicity to mammalian cells. The N-naphthoyl derivatives were

found to possess activity against both the Gram-negative and Gram-

positive bacterial strains, with substitution of the isatin ring

improving this activity. It was found that compound 22b

(hydrochloride salt) exhibited the highest antimicrobial activity with

an MIC of 16 µg/mL, whereas 22c (the corresponding quaternary

ammonium iodide salt) showed good activity with an MIC of 39

µg/mL. In addition, these compounds also showed biofilm

disruption activity in established biofilms against S.aureus; with

compound 22b giving 50% and 22c giving 46% inhibition at 250 µM.

We found that quaternary ammonium salts are nontoxic to

mammalian cells and selectively toxic towards bacterial cells. Thus,

this new class of glyoxamide based SMAMP mimics represent an

innovative avenue for the development of novel, cost-effective

antimicrobial agents.

46

were employed.^45,Sparsely tethered tBLMs were created using

47

the solvent-exchange technique described previously.^45,

An

example of the tBLM conduction response to varying

concentrations of 22b and 22c in zwitterionic membranes are

depicted in Figure 6 (A). Responses are compared to the LL-37, a

cathelicidin antimicrobial peptide produced in human neutrophils

that is known to permeabilise membranes.⁴⁸

Little response to 22b and 22c are visible until concentrations as

high as ~50 µM are reached, and even then the response is

negligible when compared to LL-37, and known pore-forming

peptides such as PGLa when used on an equivalent tBLM

architecture.⁴⁶There were also minimal changes in membrane

conduction in tBLMs composed of 30% POPG lipids (Figure 6 (B)).

These data suggest that the actions of these compounds against

bacteria are unlikely to be as a result of any lipid bilayer lytic

response. Because there is also a corresponding slight increase in

membrane capacitance as a result of adding high concentrations of

the compounds (Figures 6 (C&D)), this would indicate that there

has been a thinning of the tBLM and/or a high dielectric molecule

such as water has been introduced into the membrane.

Using tBLM technology, it was demonstrated that the two

compounds 22b and 22c with greatest antibacterial activity were

unlikely to exert their actions via the formation of lipid membrane

at higher concentrations, but this conduction is likely to be as a

Acknowledgements

We thank the NMR and BMSF facilities at UNSW Australia for

supporting the characterization of the synthesized compounds. This

work was supported by

a

Discovery Project from

AustralianResearch Council grant (DP 140102195). Shashidhar

Nizalapur is thankful to the University of New South Wales for a

Tuition Fee Scholarship (TFS) and to Naresh Kumar for a Living

Allowance Scholarship.

Experimental

Chemistry

All chemical reagents were purchased from commercial sources

(Alfa-Aesar and Sigma Aldrich) and used without further

purification. Solvents were commercial and used as obtained.

Reactions were performed using oven-dried glassware under an

atmosphere of nitrogen and in anhydrous conditions (as required).

Figure 6: Membrane conduction and capacitance measures as a Room temperature refers to the ambient temperature. Yields refer

result of increased concentrations of compounds 22b

,

22c and LL- to chromatographically and spectroscopically pure compounds

37 in tethered bilayer lipid membranes (tBLMs).

unless otherwise stated. Reactions were monitored by thin layer

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PAPER

OBC

chromatography (TLC) precoated with Merck silica gel 60 F₂₅₄

.

added a solution of isatin (1.0 mmol) in DMF (15 mL) under a

Visualization was performed by the quenching of short or long nitrogen atmosphere at 0 °C for 10 min. The reaction mixture was

wavelength UV fluorescence. Flash chromatography was carried out stirred at 0 °C for 15 min. Naphthoyl chloride (1.0 mmol) in DMF (5

using Grace Davison LC60A 6-35 µm silica gel. Infrared spectra were mL) was added dropwise to the purple mixture at 0 °C for 10 min.

recorded using a Cary 630 ATR spectrophotometer. High-resolution The reaction mixture was warmed to room temperature and stirred

mass spectrometry was performed by the Bioanalytical Mass for 1h. After completion, the reaction mixture was poured into ice-

Spectrometry facility, UNSW. Melting points were obtained using a cold water, and a yellowish compound precipitated out. This was

Mel-Temp melting point apparatus and are uncorrected. Proton filtered and dried in vacuo to give N-naphthoylisatinsin 65 – 90%

and Carbon NMR spectra were recorded in the solvents specified yields.

using a Bruker DPX 300 or a Bruker Avance 300 spectrometer as 1-(2-Naphthoyl)indoline-2,3-dione (11): The title compound 11 was

designated. Chemical shifts (δ) are quoted in parts per million prepared from isatin (5.0 g, 0.034 mmol) and naphthoyl chloride

(ppm), to the nearest 0.01ppm and internally referenced relative to (6.48 g, 0.034 mmol) according to the general procedure

A. The

the solvent nuclei. ¹H NMR spectral data are reported as follows: product 11 was obtained as a yellow solid (9.21 g, 90%); Mp 152

[chemical shift in ppm; multiplicity in br, broad; s, singlet; d, 162 °C;¹H NMR (CDCl₃, 300 MHz): δ 8.57 (bs, 1H), 8.07–8.01 (m, 3H),

doublet; t, triplet; q, quartet; quint, quintet; sext, sextet; sept, 8.00–7.90 (m, 2H), 7.88–7.75 (m, 2H), 7.75–7.60 (m, 2H), 7.46–7.34

septet; m, multiplet; or as a combination of these (e.g. dd, dtetc.)]; (m, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ 180.7, 168.4, 157.7, 148.5,

̶

coupling constant (J) in hertz, integration, proton count and 138.0, 135.4, 132.2, 131.7, 131.5, 129.6, 129.1, 128.2, 128.0, 127.4,

assignment.

125.9, 125.7, 124.9, 120.4, 116.5; IR (ATR): ν_max3275, 3055, 2928,

2341, 1736, 1693, 1528, 1597, 1459, 1331, 1284, 1163 cm^-1; HRMS

Synthetic procedure for 1-Hexanoylindoline-2,3-dione ³⁷(5):

To a suspension of sodium hydride (358 mg, 14.5 mmol) in THF (ESI): m/zcalcd for C₁₉H₁₁NNaO₃[M+Na]+ 324.0629, found

(5mL) was added a solution of isatin (2.0 g, 13.5 mmol) in THF (20 324.0631.

mL) under N₂atmosphere at 0 °C for 10 min and stirring was 1-(2-Naphthoyl)-5-bromoindoline-2,3-dione

continued for 30 min. Hexanoylchloride (2.1 g, 14.5 mmol) in THF compound 1 was prepared from isatin (2.0 g, 0.0088 mmol) and

(10 mL) was added dropwise to the purple mixture at 0 °C for 10 naphthoyl chloride (1.85 g, 0.0097 mmol) according to the general

min. The reaction mixture was warmed to room temperature and procedure . The product 12 was obtained as a yellow solid (3.40 g,

(12):

The

title

2

A

stirred for 4h. After completion, the reaction was quenched with 92%); Mp 134.6-140.8 °C;¹H NMR (CDCl₃, 300 MHz): δ 8.59 (bs, 1H),

ice-cold water and extracted with ethyl acetate. The organic layer 8.18–7.86 (m, 7H), 7.77–7.57 (m, 2H); ¹³C NMR (CDCl₃, 400 MHz): δ

was separated, dried over sodium sulfate, filtered and evaporated 179.4, 168.3, 157.1, 147.3, 139.8, 135.4, 132.2, 131.7, 131.3, 129.6,

under reduced pressure. The crude compound was purified by 129.2, 128.2, 128.1, 127.4, 127.1, 125.8, 122.3, 118.7, 117.8; 3302,

column chromatography by eluting in 15% ethyl acetate-hexane to 3068, 2318, 1747, 1683, 1594, 1506, 1455, 1386, 1310, 1189, 1156;

yield a brownish yellow solid (1.2g, 45%); Mp 78

(CDCl₃, 300 MHz): δ 8.50 8.41 (m, 1H), 7.84 7.70 (m, 2H), 7.39

7.32 (m, 1H), 1.86 1.71 (m, 2H), 1.50 1.32 (m, 4H), 1.86 1.71 C₁₉H₁₀BrNNaO₃[M+Na]+ 401.9730, found 401.9736.

(m, 2H), 0.99 (13):

0.91 (m, 3H); ¹³C NMR (CDCl₃, 400 MHz): δ 180.3, 1-(2-Naphthoyl)-5-chloroindoline-2,3-dione

̶

80 °C;¹H NMR IR (ATR): ν_max3056, 2920, 2837, 2645, 2566, 2342, 2111, 1922, 1745,

̶

1683, 1600, 1507, 1458, 1191; HRMS (ESI): m/zcalcd for

̶

The

title

173.1, 157.8, 148.9, 138.9, 126.0, 125.3, 119.2, 118.3, 38.2, 31.2, compound 13 was prepared from isatin (2.0 g, 0.0092 mmol) and

23.8, 22.4, 13.9;IR (ATR): ν_max3056, 2920, 2867, 2645, 2566, 2342, naphthoyl chloride (1.76 g, 0.0092 mmol) according to the general

2111, 1922, 1745, 1683, 1600, 1507, 1458, 1305, 1191; HRMS (ESI): procedure

A

. The product 13 was obtained by recrystallization from

149.6 °C;¹H

m/z calcd for C₁₄H₁₅NO₃Na [M+Na]+ 268.0940, found 268.0944. methanol to yield a yellow solid (2.0 g, 58%); Mp 147.3

̶

Synthetic procedure for 1-Benzoylindoline-2,3-dione³⁸(6): To a NMR (CDCl₃, 300 MHz): δ 8.57 (bs, 1H), 8.30–7.90 (m, 4H), 7.89–

solution of isatin (2.0 g, 13.5 mmol) in pyridine (15 mL) at 0 °C was 7.39 (m, 5H); ¹³C NMR (CDCl₃, 400 MHz): δ 179.4, 168.3, 157.1,

added benzoyl chloride (1.91 g, 13.5 mmol). The reaction mixture 147.3, 139.8, 135.4, 132.2, 131.7, 131.3, 129.6, 129.2, 128.2, 128.1,

was stirred at the same temperature for 2h. The reaction mixture 127.4, 127.1, 125.8, 122.3, 118.7, 117.8; IR (ATR): ν_max3302, 2954,

was poured into ice water, and a yellow colored compound 2298, 2099, 1921, 1735, 1649, 1578, 1520, 1457, 1166, 1398, 1333,

precipitated out. This was filtered and dried under reduced 1166, 1005; HRMS (ESI): m/z calcd for C₁₉H₁₀ClNNaO₃[M+Na]+

pressure to yield the product (3.2 g, 94%); Mp 145 ̶

150 °C;¹H NMR 358.0240, found 358.0241.

(CDCl₃, 300 MHz): δ 7.99–7.86 (m, 3H), 7.85–7.75 (m, 2H), 7.70– 1-(2-Naphthoyl)-5-fluoroindoline-2,3-dione

(14):

The

title

7.63 (m, 1H), 7.58–7.47 (m, 2H), 7.43–7.34 (m, 1H); ¹³C NMR (CDCl₃, compound 14 was prepared from isatin (2.0 g, 0.012 mmol) and

400 MHz): δ 191.2, 165.9, 165.4, 146.7, 140.3, 135.8, 134.3, 132.7, naphthoyl chloride (2.5 g, 0.013 mmol) according to the general

129.3, 127.8, 124.5, 122.6, 122.4; IR (ATR): ν_max3255, 2851, 2799, procedure

2650, 2507, 1731, 1637, 1573, 1528, 1447, 1300, 1258, 1190 cm^-1; 90%); Mp 171.9

HRMS (ESI): m/z calcd for C₁₅H₁₀NO₃[M+H]+ 252.0658, found 8.23–7.84 (m, 5H), 7.79–7.53 (m, 4H); ¹³C NMR (CDCl₃, 400 MHz): δ

A

. The product 14 was obtained as a yellow solid (3.5 g,

̶

175.0 °C;¹H NMR (CDCl₃, 300 MHz): δ 8.57 (bs, 1H),

252.0655.

183.6, 166.2, 162.1, 160.5, 157.3, 135.0, 133.8, 132.5, 131.0, 130.0,

General procedure A for synthesis of

N

-naphthoylisatins 11-15: To 129.9, 129.5, 128.7, 128.6, 128.2, 127.5, 126.6, 126.5, 124.4, 121.0,

a suspension of sodium hydride (1.1 mmol) in DMF (5mL) was 120.7, 116.8, 116.4; IR (ATR): ν_max3299, 3064, 2951, 2296, 2111,

8 | J. Name., 2012, 00, 1-3

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1741, 1683, 1622, 1538, 1482, 1319, 1153, 1089 cm^-1; HRMS (ESI): prepared from compound

View Article Online

DOI: 10.1039/C6OB00298F

ARTICLE

6

(240 mg, 1.0 mmol) and N¹,N¹-

m/z calcd for C₁₉H₁₀FNNaO₃[M+Na]+ 342.0532, found 342.0537.

dimethylethane-1,2-diamine (80 mg, 1.0 mmol) according to the

. Colorless liquid (240 mg, 76%); ¹H NMR

1-(2-Naphthoyl)-5-methylindoline-2,3-dione (15): The title general procedure

B

compound 15 was prepared from isatin (1.0 g, 0.006 mmol) and (CDCl₃, 300 MHz): δ 11.5 (bs, 1H), 8.76–8.59 (m, 1H), 8.01–7.94 (m,

naphthoyl chloride (1.30 g, 0.0068 mmol) according to the general 1H), 7.89–7.82 (m, 1H), 7.78–7.60 (m, 1H 7.68–7.56 (m, 3H), 7.36–

procedure

A

. The product 15 was obtained as a yellow solid (1.75 g, 7.28 (m, 1H), 3.26 (q, J = 6.71 Hz, 2H), 2.29 (t, J = 6.63 Hz, 4H), 2.13

229.4 °C;¹H NMR (CDCl₃, 300 MHz): δ 8.55 (bs, 1H), (s, 6H); ¹³C NMR (CDCl₃, 300 MHz): δ 193.7, 165.7, 164.6, 140.2,

89%); Mp 215.2

̶

8.04 (d, J = 7.73 Hz, 3H), 7.90 (t, J = 8.4 Hz, 2H), 7.76–7.58 (m, 4H), 135.4, 134.4, 133.1, 132.7, 129.3, 127.7, 124.1, 123.0, 121.9, 58.0,

2.40 (s, 3H); ¹³C NMR (CDCl₃, 400 MHz): δ 180.8, 168.3, 157.8, 45.4, 37.0; IR (ATR): ν_max3309, 3058, 2944, 2805, 2763, 2342, 2114,

146.5, 138.5, 135.4, 135.3, 132.2, 131.6, 131.5, 129.6, 129.0, 128.2, 1657, 1526, 1476, 1288, 1255, 1042 cm^-1;HRMS (ESI): m/z calcd for

128.0, 127.4, 125.9, 124.9, 120.4, 116.5, 20.7; IR (ATR): ν_max3129, C₁₉H₃₀N₃O₃[M+H]+ 340.1655, found340.1661.

3047, 2287, 2109, 1735, 1687, 1645, 1615, 1482, 1430, 1317, 1277,

1145 cm^-1; HRMS (ESI): m/z calcd for C₂₀H₁₃NNaO₃[M+Na]+ oxoacetyl)phenyl)benzamide(19a): The title compound 19a was

338.0785, found 338.0788. prepared from compound

(400 mg, 1.60 mmol) and N¹,N¹-

General procedure B for the synthesis of compounds 16a to 25a: dimethylpropane-1,3-diamine (163 mg, 1.60 mmol) according to the

To a solution of acyl isatins (5, 6 and 11-15) 1.0 mmol in acetonitrile general procedure

. Colorless liquid (480 mg, 85%); ¹H NMR

N-(2-(2-((3-(Dimethylamino)propyl)amino)-2-

6

B

or dichloromethane was added N¹,N¹-dimethylethane-1,2-diamine (CDCl₃, 300 MHz): δ 12.0 (bs, 1H), 8.95 (dd, J = 0.91, 8.45 Hz, 1H),

or N¹,N¹-dimethylpropane-1,3-diamine (1.0 mmol) at 0 °C. The 8.50 (bs, 1H), 8.41 (dd, 1.60, 8.09 Hz, 1H), 8.10–8.03 (m, 2H), 7.74–

reaction mixture was warmed to room temperature and stirred for 7.65 (m, 1H), 7.63–7.50 (m, 3H), 7.24–7.16 (m, 1H), 3.56 (q, J = 5.89

1h. After completion of the reaction, water was added and the Hz, 2H), 2.51 (t, J = 6.15 Hz, 2H), 2.30 (s, 6H), 1.81 (m, 2H); ¹³C NMR

mixture was extracted with dichloromethane. The organic layer was (CDCl₃, 300 MHz): δ 193.1, 165.9, 163.3, 142.5, 136.6, 134.6, 132.1,

separated and dried over sodium sulfate, filtered and concentrated 128.8, 127.4, 122.6, 120.8, 119.0, 58.5, 45.2, 39.7, 39.6, 25.4; IR

under reduced pressure to yield the products in 76-99% yields.

-(2-(2-((2-(Dimethylamino)ethyl)amino)-2-

(ATR): ν_max3252, 3057, 2942, 2341, 2113, 1640, 1578, 1574, 1444,

1300, 1211, 1161, 1027 cm^-1; HRMS (ESI): m/z calcd for C₂0H₂₄N₃O₃

N

oxoacetyl)phenyl)hexanamide (16a): The title compound 16a was [M+H]+ 354.1812, found 354.1813.

prepared from compound -(2-(2-((2-(Dimethylamino)ethyl)amino)-2-oxoacetyl)phenyl)-2-

(205 mg, 0.863 mmol) and N¹,N¹-

dimethylethane-1,2-diamine (73 mg, 1.2 mmol) according to the naphthamide(20a): The title compound 20a was prepared from

general procedure . Colorless liquid (211 mg, 76%); Mp 78

80 compound 11 (250 mg, 0.83 mmol) and N¹,N¹-dimethylpropane-1,3-

°C;¹H NMR (CDCl₃, 300 MHz): δ 11.0 (bs, 1H), 8.75 (d, J = 8.54 Hz, diamine (73 mg, 0.83 mmol) according to the general procedure

1H), 8.36 (t, J = 1.36, 8.10 Hz, 1H), 7.69–7.59 (m, 1H), 7.18–7.09 (m, Brown solid (320 mg, 99 %); Mp 119.7

129.7 °C;¹H NMR (CDCl₃, 300

5

N

B

̶

B

.

̶

1H), 3.50 (q, J = 3.51 Hz, 2H), 2.54 (t, J = 5.98 Hz, 2H), 2.45 (t, J = MHz): δ 11.6 (bs, 1H), 8.69 (t, J = 5.72 Hz, 1H), 8.62–8.58 (m, 1H),

7.41 Hz, 2H), 2.29 (s, 6H), 1.87–1.69 (m, 2H), 1.48–1.28 (m, 4H), 8.35–8.26 (m, 1H), 8.18–8.09 (m, 2H), 8.08–8.00 (m, 2H), 7.89–7.83

0.93 (t, J = 6.91 Hz, 3H); ¹³C NMR (CDCl₃, 300 MHz): δ 192.3, 172.5, (dd, J = 1.54, 7.91 Hz m, 1H), 7.79–7.72 (m, 1H), 7.72–7.62 (m, 2H),

163.2, 142.4, 136.5, 134.4, 122.3, 120.6, 118.4, 57.3, 45.1, 38.6, 7.40–7.29 (m, 1H), 3.24 (q, J = 6.62 Hz, 2H), 2.28–2.20 (m, 2H), 2.07

36.8, 31.3, 25.1, 22.3, 13.9; IR (ATR): ν_max3251, 3119, 3188, 3067, (s, 6H); ¹³C NMR (CDCl₃, 300 MHz): δ 193.5, 165.8, 164.5, 140.0,

2934, 2854, 2757, 2341, 2111, 1654, 1605, 1526, 1478, 1253, 1214, 135.3, 135.0, 132.9, 132.6, 131.7, 129.5, 129.0, 128.7, 128.6, 128.2,

1041 cm^-1; HRMS (ESI): m/z calcd for C₁₈H₂₈N₃O₃[M+H]+ 334.2121, 127.6, 124.3, 124.1, 123.6, 122.2, 67.4, 58.0, 45.4; IR (ATR): ν_max

found 334.2125.

-(2-(2-((3-(Dimethylamino)propyl)amino)-2-

3275, 3051, 2941, 2886, 2782, 2302, 2104, 1639, 1585, 1533, 1447,

1375, 1206, 1038 cm^-1; HRMS (ESI): m/z calcd for C₂₃H₂₄N₃O₃

N

oxoacetyl)phenyl)hexanamide(17a): The title compound 17a was [M+H]+ 390.1811, found 390.1812.

prepared from compound -(2-(2-((3-(Dimethylamino)propyl)amino)-2-oxoacetyl)phenyl)-2-

(0.20 g, 0.836 mmol) and N¹,N¹-

dimethylpropane-1,3-diamine (85 mg, 0.836 mmol) according to the naphthamide (21a): The title compound 21a was prepared from

general procedure

. Colorless liquid (278 mg, 82%); ¹H NMR compound 11 (250 mg, 0.83 mmol) and N¹,N¹-dimethylpropane-1,3-

(CDCl₃, 300 MHz): δ 11.0 (bs, 1H), 8.75 (dd, J = 8.54, 0.94 Hz, 1H), diamine (84 mg, 0.83 mmol) according to the general procedure

8.48 (bs, 1H), 8.31 (dd, J = 8.07, 1.57 Hz, 1H), 7.65–7.56 (m, 1H), Brown solid (330 mg, 99%); Mp 106.1

110.2 °C;¹H NMR (CDCl₃, 300

5

N

B

.

̶

7.17–7.09 (m, 1H), 3.53 (q, J = 5.73 Hz, 2H), 2.46 (t, J = 5.53 Hz, 4H), MHz): δ 11.5 (bs, 1H), 8.82 (t, J = 5.57 Hz, 1H), 8.63–8.57 (m, 1H),

2.26 (s, 6H), 1.84–1.70 (m, 4H), 1.47–1.31 (m, 4H), 0.93 (t, J = 7.04 8.31–8.21 (m, 1H), 8.16–8.09 (m, 2H), 8.08–7.99 (m, 2H), 7.83–7.61

Hz, 3H); ¹³C NMR (CDCl₃, 300 MHz): δ 192.8, 172.5, 163.2, 142.2, (m, 4H), 7.39–7.31 (m, 1H), 3.17 (q, J = 6.72 Hz, 2H), 2.18–2.11 (m,

136.3, 134.3, 122.3, 120.6, 118.6, 58.7, 45.3, 39.7, 38.6, 31.3, 25.5, 2H), 1.60–1.44 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 191.7, 166.01,

25.1, 22.3, 13.9; IR (ATR): ν_max3281, 3063, 2937, 2868, 2808, 2753, 164.0, 139.0, 134.9, 134.7, 132.5, 132.2, 131.8, 129.4, 128.9, 128.7,

1665, 1602, 1522, 1460, 1622, 1213, 1041 cm^-1; HRMS (ESI): m/z 128.6, 128.2, 127.6, 125.0, 124.4, 124.2, 122.6, 52.6, 36.3, 22.5,

calcd for C₁₉H₃₀N₃O₃[M+H]+ 348.2281, found 348.2282.

-(2-(2-((2-(Dimethylamino)ethyl)amino)-2-

15.6; IR (ATR): ν_max3294, 3061, 2939, 2757, 2320, 2107, 1610, 1536,

1449, 1302, 1201 cm^-1; HRMS (ESI): m/z calcd for C₂₄H₂₅N₃NaO₃

N

oxoacetyl)phenyl)benzamide(18a): The title compound 18a was [M+Na]+ 426.1784, found 426.1788.

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-(4-Bromo-2-(2-((3-(dimethylamino)propyl)amino)-2-

OBC

N

1600, 1533, 1457, 1405, 1302, 1236, 1173 cm^-1IR; HRMS (ESI):

oxoacetyl)phenyl)-2-naphthamide (22a): The title compound 22a m/zcalcd for C₂₅H₂₈N₃O₃[M+H]+ 418.2124, found 418.2125.

was prepared from compound 12 (500 mg, 1.31 mmol) and N¹,N¹- General procedure C for the synthesis of compounds 16b to 25b:

dimethylpropane-1,3-diamine (134 mg, 1.2 mmol) according to the To a solution of 16a-25a (1 mmol) in dichloromethane was added

general procedure B. Grey solid (580 mg, 91.5%); Mp 146.4 ̶ 150.2 HCl in dioxane (4M solution) (5.0 equiv) at 0 °C. The reaction

°C; ¹H NMR (CDCl₃, 300 MHz): δ 11.2 (bs, 1H), 8.81 (t, J = 5.88 Hz, mixture was warmed to room temperature and stirred for 1h. After

1H), 8.58 (s, 1H), 8.13–7.97 (m, 4H), 7.96–7.80 (m, 3H), 7.71–7.60 completion of the reaction, excess solvent was removed under

(m, 2H), 3.12 (q, J = 6.75, 2H), 2.12 (t, J = 7.18 Hz, 2H), 2.00 (s, 6H), reduced pressure, treated with diethylether, and dried to yield the

1.56–1.37 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 189.8, 166.0, 162.9, product (90-95%).

137.8, 136.5, 135.0, 133.6, 132.5, 131.3, 129.5, 128.8, 128.7, 128.6, 2-(2-(2-Hexanamidophenyl)-2-oxoacetamido)-N,N-dimethylethan-

128.3, 128.2, 127.5, 125.2, 124.3, 116.2, 57.0, 40.8, 37.6, 26.8; IR 1-aminium chloride(16b): The title compound 16b was prepared

(ATR): ν_max3304, 3060, 2988, 2760, 2342, 2106, 1656, 1510, 1477, from compound 16a (65 mg, 0.19 mmol) and HCl in dioxane (4M

1385, 1235, 1198, 1155 cm^-1; HRMS (ESI): m/z calcd for solution) according to the general procedure

C₂₄H₂₅BrN₃O₃[M+H]+ 482.1073, found 482.1074. 53%); Mp 51.5

55.9 °C; ¹H NMR (CDCl₃, 300 MHz): δ 10.5 (bs, 1H),

-(4-Chloro-2-(2-((3-(dimethylamino)propyl)amino)-2- 10.3 (bs, 1H), 8.99 (t, J = 5.35 Hz, 1H), 7.80–7.70 (m, 1H), 7.69–7.54

B. White solid (50 mg,

̶

N

oxoacetyl)phenyl)-2-naphthamide(23a): The title compound 23a (m, 2H), 7.22 (t, J = 7.70 Hz, 1H), 3.57 (q, J = 5.96 Hz, 2H), 3.29–3.15

was prepared from compound 13 (500 mg, 1.35 mmol) and N¹,N¹- (m, 2H), 2.80 (s, 6H), 2.31 (t, J = 2.31 Hz, 2H), 1.65–1.45 (m, 2H),

dimethylpropane-1,3-diamine (138 mg, 1.35 mmol) according to the 1.42–1.18 (m, 4H), 0.88 (t, J = 6.63 Hz, 3H); ¹³C NMR (CDCl₃, 300

general procedure B. Grey solid (531 mg, 90 %); Mp 164.2 ̶ 173.1 °C; MHz): δ 189.7, 171.9, 163.3, 138.0, 133.8, 131.6, 125.1, 123.6,

¹H NMR (CDCl₃, 300 MHz): δ 11.2 (bs, 1H), 8.81 (t, J = 5.67 Hz, 1H), 121.9, 55.5, 42.7, 36.7, 34.5, 31.2, 24.9, 22.3, 14.3; IR (ATR): ν_max

8.58 (s, 1H), 8.13–7.92 (m, 5H), 7.81–7.71 (m, 2H), 7.71–7.61 (m, 3396, 3276, 3055, 2923, 2855, 2694, 2098, 1668, 1603, 1531, 1482,

2H), 3.11 (q, J = 6.74, 2H), 2.16–2.04 (m, 2H), 1.98 (s, 6H), 1.53–1.38 1365, 1312, 1266, 1211, 1160; HRMS (ESI): m/z calcd for C₁₈H₂₈N₃O₃

(m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 189.8, 166.0, 162.9, 137.3, [M+H]+ 334.2121, found 334.2125.

135.0, 133.6, 132.5, 131.3, 130.7, 129.4, 128.8, 128.7, 128.6, 128.4, 3-(2-(2-Hexanamidophenyl)-2-oxoacetamido)-N,N

-

128.2, 128.1, 127.5, 125.0, 124.3, 57.0, 45.4, 37.7, 26.9; IR (ATR): dimethylpropan-1-aminium chloride (17b): The title compound

ν_max3309, 3062, 2940, 2758, 2321, 2111, 1655, 1516, 1478, 1386, 17b was prepared from compound 17a (130 mg, 0.37 mmol) and

1293, 1235, 1063 cm^-1; HRMS (ESI): m/z calcd for C₂₄H₂₅ClN₃O₃HCl in dioxane (4M solution) according to the general procedure

[M+Na]+ 460.1394, found 460.1398.

Grey sticky solid (90 mg, 90%); ¹H NMR (CDCl₃, 300 MHz): δ 10.5 (s,

-(2-(2-((3-(Dimethylamino)propyl)amino)-2-oxoacetyl)-4- 1H), 10.1 (bs, 1H), 8.87 (t, J = 5.72 Hz, 1H), 7.85–7.77 (m, 1H), 7.68–

B.

N

fluorophenyl)-2-naphthamide(24a): The title compound 24a was 7.55 (m, 2H), 7.28–7.18 (m, 1H), 3.27 (q, J = 6.94 Hz, 2H), 3.12–3.00

prepared from compound 14 (500 mg, 1.56 mmol) and N¹,N¹- (m, 2H), 2.74 (s, 6H), 2.31 (t, J = 7.30 Hz, 2H), 2.00–1.80 (m, 2H),

dimethylpropane-1,3-diamine (160 mg, 1.56 mmol) according to the 1.66–1.48 (m, 2H), 1.40–1.20 (m, 4H), 0.88 (t, J = 6.85 Hz, 3H); ¹³C

general procedure B. Yellow solid (594 mg, 90 %); Mp 148.7 ̶ 161.7 NMR (CDCl₃, 300 MHz): δ 191.1, 172.0, 163.8, 138.5, 134.2, 131.8,

°C; ¹H NMR (CDCl₃, 300 MHz): δ 11.1 (bs, 1H), 8.77 (t, J = 5.72 Hz, 124.5, 123.7, 121.7, 54.7, 42.4, 36.9, 36.3, 31.2, 24.9, 24.2, 22.3,

1H), 8.58 (s, 1H), 8.11–7.97 (m, 4H), 7.93–7.85 (m, 1H), 7.71–7.50 14.3; IR (ATR): 3476, 3375, 3276, 2954, 2857, 2678, 2095, 1650,

(m, 4H), 3.00 (q, J = 6.53, 2H), 2.12–2.02 (m, 2H), 1.97 (s, 6H), 1.51– 1620, 1524, 1479, 1370, 1299, 1247, 1213, 1162; HRMS (ESI): m/z

1.34 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 189.7, 166.0, 162.9, calcd for C₁₉H₃₀N₃O₃[M+H]+ 348.2281, found 348.2282.

134.9, 134.7, 132.5, 131.4, 129.4, 128.9, 128.7, 128.6, 128.1, 127.5, 2-(2-(2-Benzamidophenyl)-2-oxoacetamido)-N,N-dimethylethan-1-

125.8, 124.4, 120.9, 120.6, 117.5, 117.2, 57.0, 45.3, 37.7, 26.8; IR aminium chloride (18b): The title compound 18b was prepared

(ATR): ν_max3313, 3055, 2942, 2761, 2321, 2111, 1656, 1621, 1526, from compound 18a (100 mg, 0.38 mmol) and HCl in dioxane (4M

1506, 1528, 1490, 1231, 1169 cm^-1; HRMS (ESI): m/z calcd for solution) according to the general procedure

B

. Yellow solid (140

1

C₂₄H₂₄FN₃NaO₃[M+Na]+ 444.1689, found 444.1694.

-(2-(2-((3-(Dimethylamino)propyl)amino)-2-oxoacetyl)-4-

mg, 99%); Mp 106.1

̶

110.2 °C H NMR (CDCl₃, 300 MHz): δ 11.2 (s,

N

1H), 10.4 (bs, 1H), 9.10 (t, J = 5.79 Hz, 1H), 8.18–8.05 (m, 1H), 8.02–

methylphenyl)-2-naphthamide(25a): The title compound 25a was 7.92 (m, 2H), 7.90–7.81 (m, 1H), 7.77–7.54 (m, 4H), 7.31 (t, J = 7.44

prepared from compound 15 (400 mg, 1.26 mmol) and N¹,N¹- Hz, 1H), 362–3.48 (m, 2H), 3.23–3.08 (m, 2H), 2.75 (s, 6H); ¹³C NMR

dimethylpropane-1,3-diamine (129 mg, 1.26 mmol) according to the (CDCl₃, 300 MHz): δ 191.0, 165.8, 163.7, 139.1, 134.7, 134.4, 132.7,

general procedure B. Grey solid (480 mg, 90%); Mp 147.1 ̶ 151.7 °C; 132.5, 129.2, 127.9, 124.6, 124.2, 122.5, 55.4, 42.7, 34.5; IR (ATR):

¹H NMR (CDCl₃, 300 MHz): δ 11.4 (bs, 1H), 8.79 (t, J = 5.74 Hz, 1H), ν_max3236, 2942, 2658, 2463, 2342, 2110, 1667, 1602, 1441, 1293,

8.60–8.56 (m, 1H), 8.17–8.07 (m, 3H), 8.06–7.98 (m, 2H), 7.71–7.61 1250, 1205, 1159, 1076 cm^-1; HRMS (ESI): m/z calcd for C₁₉H₂₂N₃O₃

(m, 2H), 3.17 (q, J = 6.74 Hz, 2H), 2.37 (s, 3H), 2.18–2.09 (m, 2H), [M+H]+ 340.1654, found 340.1656.

1.97 (s, 6H), 1.59–1.43 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 193.3, 3-(2-(2-Benzamidophenyl)-2-oxoacetamido)-N,N,N

-

165.7, 164.4, 137.4, 135.7, 134.9, 133.6, 132.6, 132.5, 131.8, 129.5, trimethylpropan-1-aminium chloride (19b): The title compound

128.9, 128.6, 128.5, 128.1, 127.5, 124.1, 122.5, 57.0, 45.5, 37.4, 19b was prepared from compound 19a (250 mg, 0.70 mmol) and

27.0, 20.7; IR (ATR): ν_max3280, 3057, 2938, 2756, 2320, 2111, 1640, HCl in dioxane (4M solution) according to the general procedure B.

10 | J. Name., 2012, 00, 1-3

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ARTICLE

yellow solid (250 mg, 95%); Mp 199.3

̶

200.7 °C; ¹H NMR (CDCl₃, 300 HCl in dioxane (4M solution) according to the general procedure

191.2 °C; ¹H NMR (CDCl₃, 300

B.

MHz): δ 11.3 (s, 1H), 10.1 (bs, 1H), 8.97 (t, J = 5. 92 Hz, 1H), 8.21– Grey solid (97 mg, 90%); Mp 180.6

̶

8.11 (m, 1H), 8.01–7.92 (m, 2H), 7.83–7.76 (m, 1H), 7.76–7.54 (m, MHz): δ 11.2 (s, 1H), 9.95 (bs, 1H), 8.93 (t, J = 6.18 Hz, 1H), 8.65–

4H), 7.37–7.28 (m, 1H), 3.23 (q, J = 6.50 Hz, 2H), 3.08–2.95 (m, 2H), 8.53 (m, 1H), 8.17–7.95 (m, 4H), 7.92–7.82 (m, 1H), 7.81–7.59 (m,

2.73–2.65 (s, 6H), 1.94–1.76 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 4H), 3.19 (q, J = 6.56 Hz, 2H), 3.05–2.92 (m, 2H), 2.70–2.59 (m, 6H),

192.3, 165.9, 164.2, 139.3, 135.0, 134.3, 132.7, 132.5, 129.3, 127.8, 1.86–1.70 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 188.5, 166.0, 162.6,

124.3, 124.1, 122.3, 54.8, 42.5, 36.3, 24.2; IR (ATR): ν_max3273, 3079, 136.7, 134.9, 133.2, 132.5, 131.4, 130.4, 129.5, 128.8, 128.7, 128.6,

2955, 2590, 2473, 2110, 1641, 1587, 1537, 1449, 1296, 1210 cm^-1; 128.3, 128.2, 127.5, 125.1, 124.6, 54.6, 42.3, 36.4, 24.0; IR (ATR):

HRMS (ESI): m/z calcd for C₂₀H₂₄N₃O₃[M+H]+ 354.1813, found 3210, 3036, 2946, 2632, 2483, 2342, 2110, 1919, 1638, 1601, 1504,

354.1812.

1395, 1288, 1193, 1110; HRMS (ESI): m/z calcd for C₂₄H₂₅ClN₃O₃

2-(2-(2-(2-Naphthamido)phenyl)-2-oxoacetamido)-N,N

-

[M+H]+ 438.1574, found 438.1579.

dimethylethan-1-aminium chloride (20b): The title compound 20b 3-(2-(2-(2-Naphthamido)-5-fluorophenyl)-2-oxoacetamido)-N,N

was prepared from compound 20a (130 mg, 0.33 mmol) and HCl in dimethylpropan-1-aminium chloride (24b): The title compound

dioxane (4M solution) according to the general procedure . Yellow 4b was prepared from compound 24a (100 mg, 0.23 mmol) and

solid (104 mg, 73%); Mp 153.4

158.2 °C; ¹H NMR (CDCl₃, 300 MHz): HCl in dioxane (4M solution) according to the general procedure

δ 11.4 (s, 1H), 10.5 (bs, 1H), 9.15 (t, J = 5. 80 Hz, 1H), 8.69–8.62 (m, Grey solid (98 mg, 90%); Mp 193.1

200.2 °C; ¹H NMR (CDCl₃, 300

-

B

2

̶

B.

̶

1H), 8.23–8.01 (m, 5H), 7.97–7.86 (m, 1H), 7.83–7.64 (m, 3H), 7.37 MHz): δ 11.1 (s, 1H), 10.3 (bs, 1H), 9.01–8.83 (m, 1H), 8.67–8.51 (m,

(t, J = 7.70 Hz, 1H), 3.58 (q, J = 6.02 Hz, 2H), 3.27–2.10 (m, 2H), 1H), 8.18–7.93 (m, 4H), 7.92–7.77 (m, 1H), 7.73–7.49 (m, 4H), 3.26–

2.88–2.69 (s, 6H); ¹³C NMR (CDCl₃, 300 MHz): δ 191.3, 166.3, 164.1, 3.08 (m, 2H), 3.04–2.84 (m, 2H), 2.60 (s, 6H), 1.86–1.70 (m, 2H); ¹³C

139.3, 135.3, 134.9, 133.0, 132.8, 132.1, 129.9, 129.3, 129.1, 128.6, NMR (CDCl₃, 300 MHz): δ 188.6, 166.0, 162.7, 156.8, 134.9, 134.1,

128.0, 125.4, 124.7, 123.1, 55.8, 43.1, 34.9; IR (ATR): ν_max3404, 132.5, 131.5, 129.4, 129.1, 128.8, 128.7, 128.6, 128.2, 127.5, 125.8,

3292, 3031, 2957, 2578, 2452, 2112, 1931, 1661, 1624, 1577, 1523, 125.7, 124.4, 120.5, 117.1, 54.6, 42.3, 36.4, 24.0; IR (ATR): ν_max

1445, 1331, 1296, 1212, 1002 cm^-1; HRMS (ESI): m/z calcd for 3311, 2954, 2588, 2468, 2111, 1541, 1473, 1414, 1215, 1169, 1333

C₂₃H₂₄N₃O₃[M+H]+ 390.1814, found 390.1812.

cm^-1; HRMS (ESI): m/z calcd for C₂₄H₂₅FN₃O₃[M+H]+ 422.1873,

3-(2-(2-(2-Naphthamido)phenyl)-2-oxoacetamido)-N,N

-

found 422.1874.

dimethylpropan-1-aminium chloride (21b): The title compound 3-(2-(2-(2-Naphthamido)-5-methylphenyl)-2-oxoacetamido)-N,N

21b was prepared from compound 21a (133 mg, 0.33 mmol) and dimethylpropan-1-aminium chloride (25b): The title compound

HCl in dioxane (4M solution) according to the general procedure 25b was prepared from compound 25a (0.10 g, 0.23 mmol) and HCl

Orange solid (120 mg, 83%); Mp 192.0 . Grey

205.2 °C; ¹H NMR (CDCl₃, in dioxane (4M solution) according to the general procedure

300 MHz): δ 11.4 (bs, 1H), 8.95 (t, J = 5. 81 Hz, 1H), 8.59 (s, 1H), solid (100 mg, 92 %); Mp 223.3

233.1 °C; ¹H NMR (CDCl₃, 300

-

B.

̶

B

̶

8.23–7.94 (m, 5H), 7.88–7.58 (m, 4H), 7.44–7.26 (m, 1H), 3.33–3.15 MHz): δ 11.3 (s, 1H), 10.1 (bs, 1H), 8.94 (t, J = 5.98 Hz, 1H), 8.62–

(m, 4H), 2.98 (s, 9H), 1.97–1.78 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): 8.56 (m, 1H), 8.15–8.08 (m, 2H), 8.07–7.98 (m, 3H), 7.72–7.62 (m,

δ 191.7, 166.0, 164.0, 139.0, 134.9, 134.7, 132.5, 132.3, 131.8, 2H), 7.60–7.51 (m, 2H), 3.21 (q, J = 6.63 Hz, 2H), 3.06–2.92 (m, 2H),

129.4, 128.9, 128.7, 128.6, 128.2, 128.6, 124.9, 124.4, 124.2, 122.6, 2.65 (s, 6H), 2.38 (s, 3H), 1.89–1.73 (m, 2H); ¹³C NMR (CDCl₃, 300

96.7, 52.6, 36.3, 22.9; IR (ATR): 3394, 3277, 3030, 2941, 2341, 2106, MHz): δ 192.1, 165.8, 164.2, 136.8, 135.3, 134.9, 133.7, 132.5,

1920, 1734, 1638, 1586, 1539, 1448, 1307, 1201, 1160, 1091; HRMS 132.5, 131.1, 131.8, 129.5, 128.9, 128.6, 128.5, 128.2, 127.5, 124.9,

(ESI): m/z calcd for C₂₄H₂₆N₃O₃[M+H]+ 404.1968, found 404.1969.

3-(2-(2-(2-Naphthamido)-5-bromophenyl)-2-oxoacetamido)-N,N

124.2, 122.7, 54.6, 42.4, 36.3, 24.2, 20.7; IR (ATR): ν_max3275, 3071,

-

2954, 2586, 2464, 2107, 1645, 1601, 1534, 1474, 1305, 1176, 1061

dimethylpropan-1-aminium chloride (22b): The title compound cm^-1; HRMS (ESI): m/z calcd for C₂₅H₂₈N₃O₃[M+H]+ 418.212, found

22b was prepared from compound 22a (100 mg, 0.20 mmol) and 418.2125.

HCl in dioxane (4M solution) according to the general procedure

B.

General procedure C for the synthesis of compounds 16c to 25c:

Yellow solid (100 mg, 93%); Mp 159.9

̶

MHz): δ 11.2 (s, 1H), 10.0 (bs, 1H), 8.94 (t, J = 5. 93 Hz, 1H), 8.68– CH₃I (2.5 equiv) at 0 °C. The reaction mixture was warmed to room

8.52 (m, 1H), 8.18–7.95 (m, 5H), 7.94–7.76 (m, 3H), 7.74–7.60 (m, temperature and stirred for another 2h. After completion of

2H), 3.26–3.11 (q, J = 6.55 Hz, 2H), 3.05–2.91 (m, 2H), 2.70–2.58 (s, reaction, excess solvent was removed under reduced pressure.The

6H), 1.92–1.68 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 188.5, 166.0, residue was treated with diethylether, and dried to yield the

162.6, 137.1, 136.1, 134.9, 133.3, 132.5, 131.4, 129.5, 128.9, 128.8, product (90-95%).

128.7, 128.2, 127.6, 125.3, 124.4, 116.2, 54.6, 42.3, 36.4, 24.1; IR 2-(2-(2-Hexanamidophenyl)-2-oxoacetamido)-N,N,N

-

(ATR): ν_max3211, 3034, 2950, 2586, 2484, 2109, 1669, 1638, 1572, trimethylethan-1-aminium iodide (16c): The title compound 16c

1503, 1389, 1289, 1192, 1114 cm^-1; HRMS (ESI): m/z calcd for was prepared from compound 16a (65 mg, 0.18 mmol) and CH₃I (30

C₂₄H₂₅BrN₃O₃[M+H]+ 482.1072, found 482.1074.

µL, 046 mmol ) according to the general procedure

C. Grey solid (50

3-(2-(2-(2-Naphthamido)-5-chlorophenyl)-2-oxoacetamido)-N,N

-

mg, 54 %); Mp 103.7

̶

110.2 °C; ¹H NMR (CDCl₃, 300 MHz): δ 10.4

dimethylpropan-1-aminium chloride (23b): The title compound (bs, 1H), 9.02 (t, J = 5.64 Hz, 1H), 7.72–7.55 (m, 3H), 7.29–7.18 (m,

23b was prepared from compound 23a (100 mg, 0.22 mmol) and 1H), 7.22 (t, J = 7.70 Hz, 1H), 3.64 (q, J = 6.30 Hz, 2H), 3.49 (t, J =

J. Name., 2013, 00, 1-3 | 11

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PAPER

OBC

6.19 Hz, 2H), 3.14 (s, 9H), 2.30 (t, J = 7.29 Hz, 2H), 1.67–1.47 (m, (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 190.7, 166.0, 163.8, 138.6,

2H), 1.38–1.20 (m, 4H), 0.88 (t, , J = 6.74 Hz, 3H); ¹³C NMR (CDCl₃, 134.9, 134.5, 132.5, 131.9, 131.6, 129.5, 128.9, 128.7, 128.2, 127.6,

300 MHz): δ 189.6, 172.0, 163.5, 137.8, 133.9, 131.3, 125.1, 123.8, 125.2, 124.4, 124.2, 122.7, 63.7, 53.0, 33.8; IR (ATR): ν_max3388,

121.8, 63.8, 53.1, 36.7, 33.8, 31.1, 24.9, 22.3, 14.3; IR (ATR): ν_max3336, 3258, 3006, 2737, 2321, 2104, 1653, 1577, 1526, 1446, 1198,

3445, 3252, 3052, 2922, 2855, 2340, 2109, 1664, 1602, 1531, 1479, 1061 cm^-1; HRMS (ESI): m/z calcd for C₂₄H₂₆N₃O₃[M]+ 404.1965,

1309, 1214 cm^-1; HRMS (ESI): m/z calcd for C₁₉H₃₀N₃O₃[M]+ found 404.1969.

348.2280, found 348.2282.

3-(2-(2-(2-Naphthamido)phenyl)-2-oxoacetamido)-N,N,N

-

3-(2-(2-Hexanamidophenyl)-2-oxoacetamido)-N,N,N

-

trimethylpropan-1-aminium iodide (21c): The title compound 21c

trimethylpropan-1-aminium iodide (17c): The title compound 17c was prepared from compound 21a (133 mg, 0.33 mmol) and CH₃I

was prepared from compound 17a (130 mg, 0.37 mmol) and CH₃I (51 µL, 0.82 mmol) according to the general procedure . Grey solid

182.2 °C; H NMR (CDCl₃, 300 MHz): δ

72.2 °C; H NMR (CDCl₃, 300 MHz): δ 10.5 11.4 (s, 1H), 8.95 (t, J = 5. 98 Hz, 1H), 8.63–8.55 (m, 1H), 8.21–7.96

C

1

(57 µL, 0.92 mmol) according to the general procedure C. Grey solid (120 mg, 67 %); Mp 179.1 ̶

1

(154 mg, 85%); Mp 64.7

̶

(s, 1H), 8.88 (t, J = 5.83 Hz, 1H), 7.79–7.71 (m, 1H), 7.67–7.56 (m, (m, 5H), 7.87–7.62 (m, 4H), 7.41–7.29 (m, 1H), 3.33–3.16 (m, 4H),

2H), 7.31–7.17 (m, 1H), 3.27 (q, J = 6.46 Hz, 2H), 3.14–3.08 (m, 2H), 2.98 (s, 9H), 2.68–2.62 (s, 6H), 1.95–1.78 (m, 2H); ¹³C NMR (CDCl₃,

3.06 (s, 9H), 2.30 (t, J = 7.45 Hz, 2H), 2.03–1.49 (m, 2H), 1.68–1.48 300 MHz): δ 193.2, 165.8, 164.3, 139.8, 135.1, 135.0, 132.7, 132.6,

(m, 2H), 1.40–1.20 (m, 4H),0.88 (t, J = 6.81 Hz, 3H); ¹³C NMR (CDCl₃, 131.7, 129.5, 128.9, 128.7, 128.6, 128.2, 127.6, 124.5, 124.3, 124.1,

300 MHz): δ 190.7, 172.0, 163.6, 138.2, 134.1, 131.7, 124.8, 123.7, 124.0, 122.3, 67.4, 57.0, 37.4, 25.5; IR (ATR): ν_max3331, 3031, 2946,

121.8, 63.8, 52.8, 36.9, 36.2, 31.2, 25.0, 23.0, 22.3, 14.3; IR (ATR): 2603, 2477, 2112, 1679, 1629, 1605, 1524, 1447, 1198, 1299, 1161,

ν_max3462, 3241, 3068, 1660, 1528, 1451, 1373, 1283, 1091 cm^-1; 1091 cm^-1; HRMS (ESI): m/z calcd for C₂₅H₂₈N₃O₃[M]+ 418.2125,

HRMS (ESI): m/z calcd for C₂₀H₃₂N₃O₃[M]+ 362.2435, found found 418.2121.

362.2438.

3-(2-(2-(2-naphthamido)-5-bromophenyl)-2-oxoacetamido)-N,N,N

-

3-(2-(2-Benzamidophenyl)-2-oxoacetamido)-N,N,N

-

trimethylpropan-1-aminium iodide (22c): The title compound 22c

trimethylpropan-1-aminium iodide (18c): The title compound 18c was prepared from compound 22a (100 mg, 0.207 mmol) and CH₃I

was prepared from compound 18a (110 mg, 0.31 mmol) and CH₃I (33 µL, 0.51 mmol) according to the general procedure . Grey solid

(112 µL, 0.79 mmol) according to the general procedure . Grey (120 mg, 93%); Mp 202.4

210.2 °C ¹H NMR (CDCl₃, 300 MHz): δ 11.1

solid (133 mg, 86%); Mp 156.7

162.5 °C ¹H NMR (CDCl₃, 300 MHz): (s, 1H), 8.93 (t, J = 6. 03 Hz, 1H), 8.63–8.52 (m, 1H), 8.16–7.94 (m,

C

̶

δ 11.2 (s, 1H), 9.10 (t, J = 5.68 Hz, 1H), 8.10–8.00 (m, 1H), 7.99–7.90 4H), 7.93–7.84 (m, 2H), 7.83–7.74 (m, 1H), 7.73–7.62 (m, 2H), 3.30–

(m, 2H), 7.88–7.46 (m, 6H), 7.33 (t, J = 7.58 Hz, 1H), 367–3.54 (m, 3.12 (m, 4H), 2.94 (s, 9H), 1.92–1.73 (m, 2H); ¹³C NMR (CDCl₃, 300

2H), 3.43 (t, J = 6.26 Hz, 1H), 3.09 (s, 9H); ¹³C NMR (CDCl₃, 300 MHz): MHz): δ 188.3, 166.1, 162.5, 136.9, 136.1, 135.0, 133.3, 132.5,

δ 190.8, 165.9, 163.8, 138.8, 134.7, 134.3, 132.7, 132.1, 129.3, 131.5, 129.4, 129.0, 128.8, 128.7, 128.2, 127.6, 125.4, 124.3, 116.3,

127.8, 124.8, 124.3, 122.5, 63.7, 53.1, 33.7; IR (ATR): ν_max3254, 63.7, 52.6, 36.4, 22.9; IR (ATR): ν_max3311, 2945, 2760, 2321, 2111,

3069, 2341, 2111, 1640, 1541, 1449, 1301, 1211, 1079 cm^-1; HRMS 1648, 1569, 1505, 1395, 1293, 1195, 1092; HRMS (ESI): m/z calcd

(ESI): m/z calcd for C₂₀H₂₆N₃O₃[M]+ 354.1812, found 354.1808.

3-(2-(2-Benzamidophenyl)-2-oxoacetamido)-N,N,N

for C₂₅H₂₇BrN₃O₃[M]+ 496.1231, found 496.1230.

-

3-(2-(2-(2-Naphthamido)-5-chlorophenyl)-2-oxoacetamido)-N,N,N-

trimethylpropan-1-aminium iodide (19c): The title compound 19c trimethylpropan-1-aminium iodide iodide

was prepared from compound 19a (240 mg, 0.68 mmol) and CH₃I compound 23c was prepared from compound 23a (100 mg, 0.21

(105 µL, 3.4 mmol) according to the general procedure . Grey solid mmol) and CH₃I (34 µL, 0.52 mmol) according to the general

100.2 °C H NMR (CDCl₃, 300 MHz): δ 11.3 procedure . Grey solid (124 mg, 94%); Mp 227.7

236.2 °C; ¹H NMR

(23c): The title

C

1

(240 mg, 71%); Mp 96.9

̶

B

̶

(s, 1H), 9.09–8.76 (m, 1H), 8.19–8.05 (m, 1H), 8.02–7.88 (m, 2H), (CDCl₃, 300 MHz): δ 11.1 (s, 1H), 8.93 (t, J = 5.80 Hz, 1H), 8.57 (bs,

7.85–7.77 (m, 1H), 7.76–7.51 (m, 4H), 7.33 (t, J = 7.40 Hz, 1H), 3.36– 1H), 8.17–7.93 (m, 4H), 7.88–7.60 (m, 5H), 3.29–3.11 (m, 4H), 2.94

3.16 (m, 4H), 3.02 (s, 9H), 2.03–1.77 (m, 2H); ¹³C NMR (CDCl₃, 300 (s, 9H), 1.94–1.70 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 188.3,

MHz): δ 192.0, 166.0, 164.1, 139.2, 134.9, 134.4, 132.8, 132.4, 166.1, 162.5, 136.9, 136.1, 135.0, 133.3, 132.5, 131.5, 129.4, 129.0,

129.3, 127.8, 124.4, 122.4, 63.7, 52.7, 36.2, 22.9; IR (ATR): ν_max128.8, 128.7, 128.2, 127.6, 125.4, 124.3, 116.3, 63.7, 52.6, 36.4,

3580, 3261, 3036, 2956, 2321, 2072, 1640, 1611, 1302, 1211, 1094 22.9; IR (ATR): ν_max3310, 2946, 2343, 2104, 1648, 1572, 1518, 1398,

cm^-1; HRMS (ESI): m/z calcd for C₂₁H₂₆N₃O₃[M]+ 368.1969, found 1294, 1196 cm^-1; HRMS (ESI): m/z calcd for C₂₅H₂₇ClN₃O₃[M]+

368.1967.

452.1732, found 452.1735.

2-(2-(2-(2-Naphthamido)phenyl)-2-oxoacetamido)-N,N,N

-

3-(2-(2-(2-Naphthamido)-5-fluorophenyl)-2-oxoacetamido)-N,N,N-

trimethylethan-1-aminium iodide (20c): The title compound 20c trimethylpropan-1-aminium iodide (24c): The title compound 24c

was prepared from compound 20a (130 mg, 0.33 mmol) and CH₃I was prepared from compound 24a (100 mg, 0.23 mmol) and CH₃I

(51 µL, 0.83 mmol) according to the general procedure

C

. Yellow (37 µL, 0.59 mmol) according to the general procedure

C. Grey solid

solid (150 mg, 84%); Mp 158.7

̶

162.2 °C; ¹H NMR (CDCl₃, 300 MHz): (125 mg, 94 %); Mp 79.4

̶

83.2 °C; ¹H NMR (CDCl₃, 300 MHz): δ 11.0

δ 11.3 (s, 1H), 9.11 (t, J = 5.71 Hz, 1H), 8.61–8.55 (m, 1H), 8.16–8.09 (s, 1H), 8.91 (t, J = 5.74 Hz, 1H), 8.60–8.53 (m, 1H), 8.16–7.94 (m,

(m, 2H), 8.08–7.96 (m, 3H), 7.84–7.61 (m, 4H), 7.40–7.30 (m, 1H), 4H), 7.85–7.75 (m, 1H), 7.73–7.62 (m, 2H), 7.62–7.51 (m, 2H), 3.29–

3.68–3.53 (m, 2H), 3.42 (t, J = 6.13 Hz, 2H), 3.07 (s, 9H), 2.54–2.47 3.11 (m, 4H), 2.93 (s, 9H), 1.90–1.71 (m, 2H); ¹³C NMR (CDCl₃, 300

12 | J. Name., 2012, 00, 1-3

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MHz): δ 188.5, 166.6, 162.6, 156.9, 134.9, 134.0, 132.4, 131.5, than 0.07). Each experiment was performed in triplicate and was

129.4, 129.3, 128.7, 128.6, 128.2, 128.2, 127.6, 125.8, 125.7, 120.6, repeated in three independent experiments.

120.3, 117.4, 117.1, 63.7, 52.6, 36.3, 22.8; IR (ATR): ν_max3404, 3276,

3014, 2320, 2098, 1922, 1649, 1521, 1477, 1408, 1288, 1214, 1164; Toxicity assay

HRMS (ESI): m/z calcd for C₂₅H₂₇FN₃O₃[M]+ 436.2033, found The normal human lung fibroblasts MRC-5 were cultured in minimal

436.2031.

essential medium (MEM, Invitrogen) supplemented with 10% foetal

calf serum (FCS), 1% L-glutamine-penicillin-streptomycin 2%

(25c): The title sodium bicarbonate, 1% non-essential amino acids (NEAA) and 1%

3-(2-(2-(2-

N

aphthamido)-5-methylphenyl)-2-oxoacetamido)-

,

N,N,N-trimethylpropan-1-aminium iodide

compound 25c was prepared from compound 25a (100 mg, 0.23 sodium pyruvate. The cell line was maintained at 37 °C in 5% CO₂as

mmol) and CH₃I (37 µL, 0.59 mmol) according to the general an adherent monolayer and was passaged upon reaching

procedure B. Grey solid (100 mg, 92%); Mp 212.5 ̶

221.4 °C; ¹H NMR confluence by standard cell culture techniques. MRC-5 cells were

(CDCl₃, 300 MHz): δ 11.3 (s, 1H), 8.92 (t, J = 6.03 Hz, 1H), 8.60–8.56 seeded at 2 x 10⁴cells per well in 96-well plates to ensure full

(m, 1H), 8.14–8.07 (m, 2H), 8.07–7.93 (m, 3H), 7.27–7.62 (m, 2H), confluence (quiescence). Cells were treated 24 h after seeding with

7.61–7.51 (m, 2H), 3.31–3.13 (m, 4H), 2.96 (s, 9H), 2.38 (s, 3H), 0.1 to 1000 μM of compounds. After 72 h drug incubation, the

1.93–1.76 (m, 2H); ¹³C NMR (CDCl₃, 300 MHz): δ 191.7, 165.9, 164.0, treated media was replaced with fresh media containing 10%

136.9, 135.2, 134.9, 133.7, 132.5, 132.0, 131.8, 129.4, 128.8, 128.6, Alamar blue and the cells were incubated for another 6 h. The

128.5, 128.2, 127.6, 125.3, 124.2, 122.9, 63.7, 52.6, 36.3, 22.9, 22.0; metabolic activity was detected by spectrophotometric analysis by

IR (ATR): ν_max3263, 3069, 2944, 2878, 2320, 2104, 1731, 1642, assessing the absorbance of Alamar blue as previously described by

1596, 1532, 1418, 1305, 1177 cm^-1IR; HRMS (ESI): m/z calcd for Pasquieret. al.⁴⁹Cell proliferation was determined and expressed

C₂₆H₃₀N₃O₃[M]+ 432.2280, found 432.2282

.

as a percentage of untreated control cells. The determination of IC₅₀

values was performed using GraphPad Prism 6 (San Diego, CA, USA).

Disk diffusion assay

A disk diffusion method was used to evaluate the antibacterial Biofilm disruption assay

activity of the synthesized compounds against S. aureus (strain 38), S. aureus was grown in YPD broth (0.5% yeast extract, 1.0% peptone

P. aeruginosa (PAO1) and E. coli (K-12). A single colony of each test and 0.5% dextrose) overnight at 37 °C with shaking at 150 r.p.m.

strain was grown overnight in tryptone soya broth (TSB; Oxoid, UK) Cultures were diluted (1:20) in YPD medium and 200 µl aliquots

at 37 °C. The resulting bacterial culture (200 µL) was spread onto were dispensed to flat bottom 96-well plate wells (Sarstedt

nutrient agar plates. The compounds to be tested were dissolved in Australia). For pre-established biofilm assay, cultures were grown in

DMSO and dried on sterile 6 mm diameter paper disks (0.1 mg 96-well plate wells overnight prior to supplementation with

compound per disc). Disks with absorbed compound were then synthetic compounds. Control cultures were supplemented with an

placed onto the nutrient agar plates. After incubation at 37 °C for equal amount of DMSO. Plates were sealed with self-adhesive

24 h, the diameter of the zone of inhibition was measured. microplate sealers (TopSeal-A, PerkinElmer) to allow air diffusion

Gentamicin (0.01 mg per disk) was used as positive control. The and to prevent condensation. Cultures were incubated overnight at

assay was performed in duplicate and repeated twice.

37 °C with shaking at 150 r.p.m. Biofilms adhered on polystyrene

substratum were quantified by crystal violet staining as described

previously.⁴¹All cultures were prepared in triplicates. Error bars

Minimal inhibitory concentration (MIC) determination

The minimal inhibitory concentration (MIC) of the active represent the standard error of three independent experiments (n =

compounds was determined by following the previously published 3).

protocol.⁴⁰The compounds to be tested were dissolved and diluted

in autoclaved Millipore water. A single colony of S. aureus was Epifluorescence Microscopy Experiment

cultured overnight in TSB at 37 °C. The resulting bacteria were S. aureus biofilms were grown by adding 1.5 mL of adjusted

collected by centrifugation and re-suspended in the same volume of bacterial culture (OD₆₀₀~ 0.600) in fresh YPD medium into 35 × 10

TSB twice. Optical density (OD) of the resulting culture was adjusted mm polystyrene petri dishes and incubated over 24 h at 37 °C in a

to 0.1 at 600 nm (10⁸CFU/mL) in TSB, and the adjusted culture was shaking incubator at 120 r.p.m. After overnight incubation, cultures

further diluted to 10⁵CFU/mL in TSB. Then, 150 µL of the bacterial were supplemented with varying concentrations of synthetic

solution was added to wells of a 96-well plate containing 50 µL of compounds dissolved in DMSO and incubated another 24 hours.

serially diluted compound. Two controls were also prepared: one Control cultures were supplemented with an equal amount of

containing 150 µL of TSB media and 50 µL steriled water, and the DMSO. After the incubation, the biofilm growth medium was

other containing 150 µL of bacterial solution and 50 µL steriled discarded and the biofilms were gently rinsed 3 times with PBS (pH:

water. The plates were then incubated at 37 °C for 24 h, and the 7.0) in order to remove any planktonic bacterial cells. The remaining

MIC was recorded by measuring the OD value at 600 nm using a substratum adhered biofilms were then stained with Live/dead

Wallac Victor (Perkin-Elmer) microplate reader. The MIC value was stain (Invitrogen, Oregon, USA) for 20 mins in the dark and then

determined as the lowest concentration of compound that subjected to epifluorescence microscopy. Stained biofilms were

inhibited the complete growth of the bacteria (i.e. OD₆₀₀value less

J. Name., 2013, 00, 1-3 | 13

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OBC

visualized with an Olympus BX51 fixed stage fluorescence 10.

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To determine if the compounds are lytic to cell membranes,

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electrical impedance spectroscopy techniques were employed.^{45, 46}

13.

14.

15.

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benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl tethers

benzyl-disulfide-tetra-ethyleneglycol-OH spacers in the ratio of 1:10

were coated onto a gold patterned polycarbonate slide (SDx

Tethered Membranes Pty Ltd, Australia). Using

cartridge chamber, a 3 mM solution of a standard mobile lipid

phase [70% zwitterionic C20 Diphytanyl-Glycero-

a specialised

17.

18.

19.

20.

R. E. W. Hancock, Lancet Infect Dis, 2001, 1, 156-164.

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12630-12631.

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ether] was added to the tethering chemistries. All lipids were

dissolved in 100% ethanol. Alternatively, to create negatively

charged membranes like those present in bacterial species, the

same mobile lipid phase were instead supplemented with 30%

21.

palmitoyl-oleoyl-phosphatidylglycerol (POPG) (Avanti Lipids, USA). 22.

Lipids were left for 2 minutes to associate with the tethering

chemistries before being washed with 3 x 200 mL phosphate

N. P. Chongsiriwatana, J. A. Patch, A. M. Czyzewski, M. T.

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buffered saline (PBS). AC impedance spectrometry was then

employed to verify the presence of the tethered lipid bilayers and

to report on changes in membrane conduction as a result of adding

24.

the compounds.

AC impedance spectroscopy measures were performed using a

TethaPod™ operated with TethaQuick™ software (SDx Tethered

Membranes Pty Ltd, Australia). This employed a 60 mV peak-to-

25.

D. Liu, S. Choi, B. Chen, R. J. Doerksen, D. J. Clements, J. D.

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peak AC excitation at 0.1, 0.2, 0.5, 1, 2, 5, 40, 100, 200, 500, and

1000 Hz. The data were fitted to a Constant Phase Element in series

with a Resistor/Capacitor network using a proprietary adaptation of

26.

a Lev Mar fitting routine as described previously.

S. Choi, A. Isaacs, D. Clements, D. Liu, H. Kim, R. W. Scott,

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Synthesis and biological evaluation of N-naphthoyl-phenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors

DOI: 10.1039/c6ob00298f

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