Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents

European Journal of Medicinal Chemistry xxx (2017) 1e21

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Design and synthesis of short amphiphilic cationic peptidomimetics

based on biphenyl backbone as antibacterial agents

Rajesh Kuppusamy ^a, Muhammad Yasir ^b, Thomas Berry ^c, Charles G. Cranﬁeld ^c,

Shashidhar Nizalapur ^a, Eugene Yee ^a, Onder Kimyon ^d, Aditi Taunk ^a, Kitty K.K. Ho ^a,

,

*

Bruce Cornell ^e, Mike Maneﬁeld ^d, Mark Willcox ^b, David StC Black ^a, Naresh Kumar ^a

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

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

^cSchool of Life Sciences, University of Technology Sydney, PO Box 123, Ultimo 2007, Australia

^dSchool of Civil and Environmental Engineering, UNSW, Sydney, NSW 2052, Australia

^eSDx Tethered Membranes Pty Ltd, 30-32 Barcoo St, Roseville 2069, Australia

a r t i c l e i n f o

a b s t r a c t

Article history:

Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alter-

natives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many mi-

crobes. AMPs are part of the natural defenses of most living organisms and they also have a unique

mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics

were synthesized by incorporating the 3⁰-amino-[1,1⁰-biphenyl]-3-carboxylic acid backbone to mimic the

essential properties of natural AMPs. By altering hydrophobicity and charge, we identiﬁed the most

Received 29 August 2017

Received in revised form

5 October 2017

Accepted 24 October 2017

Available online xxx

potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC ¼ 15.6

mM)

Keywords:

and Gram-negative Escherichia coli (MIC ¼ 7.8

m

M) bacteria. Cytoplasmic permeability assay results

Antimicrobial peptide

Peptidomimetics

Membrane disruption

Antibioﬁlm activity

revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active

compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using

tethered bilayer lipid membranes (tBLMs) and their activity against established bioﬁlms of S. aureus and

E. coli.

1. Introduction

[3e5]. AMPs such as PMX30063 and LTX109 are currently in clinical

trials [6e8]. Although some promising AMPs are in the pipeline,

Antibiotic resistance in bacteria is a major concern facing global

public health. Multidrug resistant strains of Gram-positive bacteria

such as methicillin-resistant Staphylococcus aureus (MRSA),

methicillin-resistant Staphylococcus epidermidis (MRSE), and

vancomycin-resistant Enterococci faecalis (VRE), and Gram-negative

bacteria such as Escherichia coli, Klebsiella pneumoniae, and Pseu-

domonas aeruginosa, have emerged as major causes of hospital and

community-acquired infections [1]. Particularly, Gram-positive

bacteria such as MRSA account for a high percentage of hospital-

acquired infections [1,2]. Due to the increasing resistance of bac-

terial strains against conventional antibiotics, efforts have been

made to investigate naturally-occurring antimicrobial peptides

(AMPs) and their derivatives as alternative antimicrobial agents

there is still an urgent need for the development of new antibiotic

scaffolds.

AMPs are widespread in nature and serve as the ﬁrst-line of

defense against microbial attack in insects, plants, amphibians and

mammals [9e12]. Unlike conventional antibiotics, AMPs act via

non-receptor interactions, which make it difﬁcult for bacteria to

develop resistance to AMPs. Most cationic antimicrobial peptides

possess a rigid secondary structure and adopt an amphipathic

conformation such that their positively-charged face interacts

electrostatically with the negatively-charged membrane surface,

while their hydrophobic face inserts into the lipophilic interior of

the membrane [13,14]. The broad-spectrum antimicrobial peptide

pexiganan (MSI-78), which acts via disruption of bacterial cell

membranes, has reached phase III clinical trials [15]. Only a few

naturally-occurring AMPs have been used clinically, including

polymyxin B and colistin (polymyxin E), due to their in vivo toxicity,

* Corresponding author.

E-mail address: n.kumar@unsw.edu.au (N. Kumar).

https://doi.org/10.1016/j.ejmech.2017.10.066

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

2

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

susceptibility to proteolytic degradation, poor activity in the pres-

ence of salts and cytotoxicity to the host cells [16]. Produced

through solid-phase synthesis, AMPs also have high manufacturing

costs [17].

The drawbacks of conventional AMPs have stimulated the

development of peptidomimetics [18], which are synthetic non-

peptidic molecules designed to mimic the properties of peptides.

as arginine and lysine. This demonstrated the importance of short

non-natural amine and guanidine groups over cationic amino acids.

Arginine and 1-(2-aminoethyl)guanidine were chosen because the

guanidine group exhibits a stronger electrostatic interaction and

more extensive hydrogen bonding with the negatively-charged

phospholipids of the bacterial cell membrane [47].

The synthesized compounds were evaluated for biological ac-

tivity against Gram-positive and Gram-negative bacterial strains. In

addition, cytotoxicity was also investigated for selected potent

antimicrobial compounds. To study how the active compounds

interacted with lipid membranes, tethered bilayer lipid membranes

(tBLMs) in association with electrical impedance spectroscopy was

employed. In addition, the ability of the compounds to alter cyto-

plasmic permeability was assessed by using the membrane

potential-sensitive cyanine dye diSC3-5. Finally, the ability of the

compounds to inhibit the bioﬁlm formation of S. aureus and E. coli

was also evaluated.

The various kinds of peptidomimetics include

peptides [20], peptoids [21e23], -turn mimetics [24], cationic

^3R3-peptides [25] and lipopeptides [26]. In particular, a number of

a-peptides [19], b-

b

structurally simple, cationic peptidomimetics possessing natural or

unnatural amino acids and with amphipathic character have been

investigated as antibacterial agents [27,28]. Svendsen and co-

workers have synthesized a range of peptides of variable length

utilizing arginine and tryptophan amino acids, and showed good

minimal inhibitory concentration (MIC) of 2.5

Staphylococcus aureus and 5

g mL^ꢀ1against Escherichia coli [29].

An ultra-short pyrazole-based peptidomimetics showed a MIC of

g mL^ꢀ1against MRSA and was four times more potent than

m

g mL^ꢀ1against

m

4

m

2. Results and discussion

melittin [30]. Pyne and co-workers have developed C2-symmetric

binaphthyl-containing peptidomimetics that showed excellent

antimicrobial activity against both Gram-positive and Gram-

negative bacterial pathogens [31]. Haldar and co-workers

designed aryl-alkyl-lysine-based peptide mimics that mimicked

the membrane-active properties of natural AMPs [32]. Their group

also investigated cationic small molecules with spatial control of

hydrophobicity to minimize toxicity against human erythrocytes

while still maintaining antibacterial activity [33].

Our research group has recently synthesized short glyoxamide-

based peptidomimetics via the ring-opening reaction of N-naph-

thoylisatins with amines and amino acids [34,35]. As part of an

ongoing program to develop short antimicrobial peptidomimetics,

we were interested in utilizing the biphenyl backbone due to its

frequent presence in medicinal chemistry [36,37]. In an analysis of

scaffolds of pharmacologically active molecules, biphenyl was

found to be present in 2.1% of reference drug molecules [38,39].

Furthermore, the importance of the biphenyl unit is shown by their

presence in several natural products [38e40], such as the anti-

bacterial compounds MC21-A (1) and MC21-B (2) isolated from

marine bacterium Pseudoalteromonas phenolica [41,42]. The

biphenyl-containing antibacterial compounds biphenomycin A (3)

and B (4) were isolated from the cultured broth of Streptomyces

griseorubiginosus 43608 [43e45]. Antibiotics such as arylomycin A2

(5), arylomycin B2 (6) and vancomycin also contain biphenyl moi-

eties (Fig. 1).

2.1. Design and synthesis of biphenyl derived peptidomimetics

The synthesis of biphenyl based peptidomimetic compounds is

shown in Scheme 1. Suzuki-Miyaura cross-coupling of commer-

cially available methyl 3-bromobenzoate (7) and 3-

nitrophenylboronic acid (8) gave the 3,3’-substituted biphenyl 9,

and subsequent reduction of the nitro compound 9 using Pd/C

resulted in the formation of amine 10 in good yield. The synthesis of

amide 12 was achieved by coupling 10 with 11 using EDCI$HCl,

HOBt, and DIEA in DMF. Finally, methylester hydrolysis using

aqueous sodium hydroxide resulted in the formation of the desired

acid 13 which on coupling with different amines (18a-18e) gave the

respective amides 22a e 22e. Deprotection of Boc group using tri-

ﬂuoroacetic acid (TFA) yielded the series I compounds (23a-23e).

The acid 17 was synthesized by deprotecting the Boc group of 12

with TFA, followed by amide formation with 15 and subsequent

ester hydrolysis using aqueous sodium hydroxide (Scheme 1). On

coupling 17 with different amines (18a-18g), respective amides

24a-24g were yielded and the deprotection of 24a-24g gave the

series II compounds (25a-25g). The synthesis of amines 18b and

18d-18g were shown in Scheme 2. The guanylation reaction using

N,N⁰-Di-Boc-1H-pyrazole-1-carboxamidine provides the corre-

sponding amine 18b. The amino ester 18d was prepared from NH₂-

Arg(Mtr)-OH using thionyl chloride and methanol. The acids 21(X′),

21(X′), 20(Y′) were used to synthesize the amides 21a, 20b, 21b

using PyBOP/CH₂Cl₂coupling agent, and subsequent Fmoc cleavage

using piperidine/DMF to afford 18e-18g.

In this work, we designed a unique scaffold for developing short

antimicrobial peptidomimetics by utilizing

a

3,3⁰-substituted

biphenyl unit as the key hydrophobic backbone to mimic the

structural and biological properties of many AMPs. The segregation

of hydrophobic (R¹) and cationic (R²) groups via the biphenyl core

confers amphipathic character to the entire molecule (Fig. 2).

Importantly, the modular design of this scaffold allows for ready

optimization of the biphenyl-based peptidomimetics simply by

varying the nature of the hydrophobic (R¹) and cationic (R²) groups.

The biphenyl-based peptidomimetics synthesized in this work

were classiﬁed into two series based on the length of the hydro-

phobic group (R¹) attached to the molecule. Series I (23a-23e)

incorporated a single tryptophan (Trp) group at the 3-position of

biphenyl, whereas series II (25a-25g) incorporated a tryptophan-

phenylalanine (Trp-Phe) dipeptide at the same position. The se-

lection of Trp was based on its ability to interact with the interfacial

region of the bacterial membranes, thereby anchoring the biphenyl

derivatives to lipid bilayers [46]. The cationic groups (R²) were

incorporated at the 3⁰-position of biphenyl, and they included 1,2-

diaminoethane, 1-(2-aminoethyl)guanidine and amino acids such

The biphenyl derivative 29 attached with guanidine without any

hydrophobic group was synthesized as shown in Scheme 3.

The biphenyl ester 9 was hydrolyzed to acid 26 and coupled

with 18b to afford the amide 27, subsequent deprotection with TFA

gave 29. Finally, 35 without the biphenyl backbone but intact with

hydrophobic and cationic group was synthesized from L-Trypto-

phan 30. The acid 33 was obtained by the sequential formation of

dipeptide 32 from 30, followed by ester hydrolysis. The amide 34

was obtained on coupling 33 with 18b, and it was deprotected with

TFA to yield 35 (Scheme 4).

2.2. Antimicrobial activity study

The antimicrobial activities of the newly synthesized peptido-

mimetics are summarized in Table 1. The activities were assessed

against the Gram-positive bacterium Staphylococcus aureus [SA38],

and two Gram-negative bacteria, Pseudomonas aeruginosa [PA01],

and Escherichia coli [K12]”.

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

3

Fig. 1. Biphenyl-containing antibacterial compounds, with the biphenyl motif highlighted in blue. (For interpretation of the references to colour in this ﬁgure legend, the reader is

referred to the web version of this article.)

Fig. 2. General structure of biphenyl based peptidomimetics.

To understand the role of cationic group on antibacterial activ-

ity, series I compounds 23a-23e containing the biphenyl backbone

with tryptophan hydrophobic group and various cationic groups

were determined for their MICs. For S. aureus, compound 23b

hydrophobicity along with the cationic character. All the com-

pounds 25a-25g were tested against the S. aureus and as expected,

25a (MIC ¼ 62.5

m

M) and 25b (MIC ¼ 15.6

mM) containing an

additional hydrophobic phenylalanine group showed greater ac-

(MIC ¼ 62.5

which mimic the arginine amino acid found in natural AMPs dis-

M)

with a simple amine group. When the amine group was replaced

with a Lys-ester (compound 23c), a four-fold increase in antibac-

m

M) containing a simple guanidium cationic moiety

tivity compared to the less hydrophobic compounds 23a

(MIC ¼ 125

ingly, the increase in hydrophobicity of 23c (31.2

improve the activity of 25c (31.2 M). Whereas in compound 25d

M), the activity was increased by two-fold compared to 23d

M). This may be due to increase in the hydrophobic bulki-

mM) and 23b (MIC ¼ 62.5

m

M) respectively. Interest-

played higher activity compared to compound 23a (MIC ¼ 125

m

M) did not

m

(31.2

(62.5

m

terial activity was observed (MIC ¼ 31.2

m

M) even though the total

net charge and hydrophobicity of the compound was maintained.

This suggests the pendant ester group and the length of the cationic

group may play a role in antibacterial activity. However when an

Arg-ester is in place of the amine (compound 23d), only a two-fold

ness along with the extensive hydrogen bonding of guanidine

group. Compound 25e (MIC ¼ 31.2 M) retained the same activity

compared to 23e (MIC ¼ 31.2 M) though the hydrophobicity is

m

increased, which could be due to the net positive charge equalized

to the number of hydrophobic groups. To further investigate the

effect of increase in net positive charge, one or two of the amine

groups on compound 25e were replaced with guanidine to yield

compounds 25f and 25g respectively. Interestingly, compound 25f

increase in activity was observed (MIC ¼ 62.5

mM), and the activity

remained the same when compared with compound 23b with a

simple guanidium group. The similar antibacterial activity of 23b

and 23d revealed that the simple guanidine cationic group is

enough to mimic the arginine. The antibacterial activity of com-

(31.2

25e with guanidine group. On the other hand, compound 25g

(MIC ¼ 15.6 M) with two guanidine groups showed a two-fold

M). This may

mM) showed the same activity after replacing one amine of

pound 23e (MIC ¼ 31.2

mM) and 23c remained the same even

though the net cationic charge is increased. This could be due to the

imbalance of the peptide hydrophobicity and charge distribution.

Although, the series I compounds 23a-23e were found to be active

against S. aureus, these compounds did not have signiﬁcant activity

against the two Gram-negative bacteria, P. aeruginosa and E. coli.

Based on the results from series I, increase in the net cationic charge

did not show any profound antibacterial effect against Gram-

positive and Gram-negative bacterial strains.

m

increase in activity compared to 25e (MIC ¼ 31.2

m

be explained by the increase in hydrogen bonding with the nega-

tively charged phospholipids of bacterial cell membrane resulting

in higher antibacterial activity.

Compound 25a-25g were also tested against Gram-negative

bacteria E. coli. While compounds 25a, 25c and 25d did not show

notable antibacterial activity, compound 25b (MIC ¼ 31.2

mM)

The series II compounds 25a-25g were made based on hy-

pothesis that the increase in hydrophobicity will enhance the

antibacterial activity and to understand the effect of

showed a dramatically four-fold improvement against E. coli. The

antibacterial activity of cationic peptides on Gram-negative bacte-

ria is achieved by two steps to overcome the outer-membrane

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

4

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

Scheme 1. Synthesis of compounds 13 and 17. Reagents and conditions: a) 8 (1.1 equiv), Pd(PPh₃)₄(0.03 equiv), aq. 2M Na₂CO₃(3.0 equiv), 1,4-dioxane, reﬂux, overnight, 65%; b) 10%

Pd/C, H₂balloon, THF, rt, overnight, 90%; c) EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt, 8 h, 63%; d) aq. 1N NaOH(2.0 equiv), THF, MeOH, rt, overnight, 90% e) TFA,

CH₂Cl₂, RT, 4 h, 89%; f) EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt, 8 h, 54%; g) aq. 1N NaOH (2.0 equiv), THF, MeOH, rt, overnight, 89%; h) EDCI (1.2 equiv), HOBt (1.0

equiv), DIEA (2.5 equiv), DMF, rt, 8 h-overnight, 52e84%; i) TFA, CH₂Cl₂, RT, 4 h-overnight, 49e89%.

barrier. The high membrane bound concentration of the cationic

peptides facilitates the outer-membrane permeabilization, and in

the ﬁnal step the inner membrane damage causes the cell lysis [48].

In this step, the hydrophobic interactions become dominant. This

clearly suggests the importance of hydrophobicity of active com-

pounds for outer membrane permeabilization of Gram-negative

Finally, upon the removal of biphenyl backbone from the active

compound 23b, the antibacterial activity was completely lost

(compound 35; MIC >250

mM). This result conﬁrms that the

biphenyl backbone plays a major role in balancing the hydropho-

bicity and amphipathicity which are considered as the key pa-

rameters for the antibacterial activity against Gram-positive and

Gram-negative bacterial strains.

bacteria. Compounds 25f (MIC

(MIC ¼ 7.8 M) containing guanidine cationic groups displayed

superior activity against E. coli compared to 25c (MIC ¼ 125 M)

M) containing the amine cationic groups.

¼

15.6

mM) and 25g

m

Overall, the structure activity relationship (SAR) draws the

inference regarding the antibacterial activity of the biphenyl

backbone peptidomimetic derivatives against S. aureus and E. coli.

Firstly, the series II compounds with additional hydrophobic group

showed good to excellent antibacterial activity against S. aureus and

E. coli. Secondly, the amphipathic nature played a crucial role in

determining the antibacterial activity. The antibacterial activity of

compounds 25c-25f remain intact against S. aureus even after

increasing the net positive charge. Among all the synthesized

biphenyl derivatives, 25b and 25g displayed the most potent anti-

m

and 25e (MIC ¼ 31.2

m

These results evidently showed that the electrostatic attraction also

play a decisive role for activity against E. coli. Compound 29

(62.5 mM) with the biphenyl backbone showed a similar activity to

23b against S. aureus and E. coli even though the hydrophobic

tryptophan group was removed. This suggests the hydrophobicity

of the biphenyl backbone is maintaining the amphiphilic nature of

the compound. From the MIC results, it is evident that the increase

in hydrophobicity will enhance the activity of compounds against

Gram-negative bacteria.

bacterial activity against S. aureus (15.6

mM). Finally, 25g revealed

that an increase in cationic charge by utilizing guanidine groups

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

5

Scheme 2. Synthesis of 18b, 18d-18g. Reagents and conditions: a) N,N⁰-Di-Boc-1H-pyrazole-1-carboxamidine (0.1 equiv), THF, rt, 30 min, crude; b) SOCl₂(1.5 equiv), MeOH, rt,

overnight; c) PyBOP (1.1 equiv), HOBt (1.0 equiv), DIEA (3.0 equiv), CH₂Cl₂, rt, overnight; d) Piperidine (2.0 equiv), DMF, RT, overnight.

Scheme 3. Synthesis of compound 29. Reagents and conditions: a) aq. 1N NaOH (2.0 equiv), THF, MeOH, rt, overnight, 90%; b) EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv),

DMF, rt, overnight, 80%; c) TFA, CH₂Cl₂, rt, 4 h-overnight, 90%; d) N,N⁰-Di-Boc-1H-pyrazole-1-carboxamidine (1.0 equiv), Et₃N (2.0 equiv), THF, RT, 6 h, 85% e) 10% Pd/C, H₂balloon, rt,

5 h, 40%.

Scheme 4. Synthesis of compound 35. Reagents and conditions: a) SOCl₂(1.5 equiv), MeOH, RT, overnight, 90%; b) EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt,

overnight, 65%; c) 1N NaOH_(aq)(2.0 equiv), THF, MeOH, rt, overnight, 91%; d) EDCI (1.2 equiv), HOBt (1.0 equiv), DIEA (2.5 equiv), DMF, rt, overnight, 45%; e) TFA, CH₂Cl₂, RT,

overnight, 47%.

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

6

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

Table 1

Antibacterial activities of biphenyl peptidomimetic derivatives.

MIC (mM)

Gram þve

Gram ꢀve

S. aureus

P. aeruginosa/*E. coli

NT-Not tested

125

>250/>125

62.5

31.2

62.5

31.2

>250/>125

62.5

15.6

250/>125

250/31.2

31.2

250/125

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

7

Table 1 (continued )

MIC (mM)

Gram þve

Gram ꢀve

S. aureus

P. aeruginosa/*E. coli

NT-Not tested

31.2

250/125

250/31.2

31.2

NT/15.6

15.6

NT/7.8

62.5

>250/>125

NT/>125

>250

MSI-78 [15,52,53]

3.2e6.45 (NT)

*3.2e6.4

(NT)

* MIC for E. coli.

along with increase in hydrophobicity produced excellent activity

the most well-studied amphipathic antimicrobial peptides. Resis-

tant strains of bacteria did not generated against MSI-78 even after

repeated exposure to sub-inhibitory concentrations [15], and many

studies have shown that the antimicrobial action of the peptide

involve disruption of bacterial membranes [50,51]. It is hypothe-

sized that the amphiphilic biphenyl derivatives in this study act via

a similar mechanism of action and therefore MSI-78 was selected

for comparison.

against Gram-negative E. coli (7.6

mM) compared to the corre-

sponding amine compound.

The activity of the peptidomimetic compound 25g which con-

tains less number of amino-acids is comparable with the MIC of

MSI-78. MSI-78 (also known as pexiganan) is a chimera of magainin

and melittin peptides [49]. This peptide was chosen as a reference

compound as it is currently in Phase-III clinical trials and is one of

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

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2.3. Toxicity against human cells

2.5. Cytoplasmic membrane depolarization

To further evaluate the utility of these biphenyl derivatives as

antimicrobial agents, their speciﬁcity for bacterial cells over human

cells was determined. Therefore, the in vitro toxicity of the most

active compounds (23b, 23d, 25b-25g) was assessed against MRC-5

normal human lung ﬁbroblasts using the Alamar Blue (Resazurin)

assay [54]. A dose-response curve was generated for each com-

pound (shown in supplementary material Fig. S2) at concentrations

We evaluated the disruption effect of the biphenyl derivatives

on the bacterial cytoplasmic membrane using the membrane

potential-sensitive dye diSC3-5 (3,3⁰-dipropylthiadicarbocyanine

iodide). The distribution of diSC3-5 between the cell membrane

and periphery medium is dependent on the cytoplasmic membrane

potential gradient. The dye readily partitions into the bacterial cell

membrane and aggregates within the membrane, causing self-

quenching. If the antimicrobial compounds perturb the cell mem-

brane, it can lead to the loss of the membrane potential gradient,

causing the dye to be released into the medium. As a result, the

ﬂuorescence intensity of the dye increases. As shown in Fig. 5,

compounds 23b, 23d, 25b, 25e, 25f and 25g (added at 0.5 ꢁ , 1 ꢁ ,

2 ꢁ or 4 ꢁ MIC) induced disruption of the cytoplasmic membrane of

S. aureus in a time and concentration-dependent manner. Inter-

estingly, the most active compound 25g showed an increase in

ﬂuorescence intensity even at sub-MIC levels within 3 min. Simi-

larly, increases in ﬂuorescence intensity of 25e and 25f also

occurred at 1 ꢁ MIC within 3 min.

ranging from 1 to 350

(shown in supplementary material Fig. S3). All of the tested com-

pounds displayed very low toxicity (IC₅₀> 300 M) towards human

mM and their IC₅₀values were determined

m

cells. Although amphipathic antibacterial agents are often cyto-

toxic, our compounds did not show noticeable cytotoxicity up to

concentration of 350 mM.

2.4. Lipid bilayer membrane interactions

The compounds ability to interact with lipid bilayers was

assessed using tethered bilayer lipid membranes (tBLMs) in asso-

ciation with AC electrical impedance spectroscopy [55,56]. Com-

pounds which have shown membrane conduction at concentration

We also conducted the cytoplasmic permeability assay on

Gram-negative bacterium E. coli (K12) with the most active com-

pound 25g. 25g also perturbs the cell membrane led to similar

disruption of diSC3-5 ﬂuorescence intensity (Fig. 5G), indicating

that the cell membrane of both Gram-positive and Gram-negative

bacteria can be disrupted. Taken together, these observations

indicate that the biphenyl amphiphilic compounds can readily

permeabilize the bacterial membrane, thus resulting in bacterial

death.

of 10

mM are discussed. Membrane conduction responses of a

zwitterionic phospholipid membrane to compounds 25b-25e

increased with increasing concentration of compounds (Fig. 3A).

Signiﬁcant conduction changes are evident at concentrations equal

to or higher than 10 mM. Interestingly, these responses are more

muted in negatively-charged phospholipid membranes which

mimic the negatively charged bacterial cell membranes (Fig. 3B).

The changes in membrane conduction are modest when

compared to that produced by

a

-hemolysin or gramicidin-A [56,57]

2.6. Antibioﬁlm activity

which produce ion channels in the membranes. This would indicate

that it is unlikely that these compounds form membrane spanning

ion channel-like pores. Alternatively, the changes in membrane

conduction could be the result of the formation of toroidal pore-like

structures in the membrane. Either the compounds form a part of

the lipidic toroidal pore themselves, or they independently induce

phospholipidic toroidal pore formation by altering the critical

packing parameter of the membrane [58].

The membrane capacitance changes increased with compound

concentration in both zwitterionic (Fig. 3C) and negatively-charged

(Fig. 3D) tBLMs, with the exception of compound 25d which

exhibited a slight decrease in capacitance at high concentrations in

negatively-charged membranes. An increase in capacitance is

indicative of a thinning of the lipid membrane and/or an increase in

membrane dielectric caused by the presence of water molecules.

This indicates these compounds may follow mechanism of pore

formation like AMPs by membrane thinning effect [59].

The ability of the amphiphilic biphenyl derivatives to disrupt the

established S. aureus or E. coli bioﬁlms was measured (Fig. 6).

Compound 23b showed the highest level of disruption against

S. aureus and E. coli of 41% and 39% respectively. Meanwhile,

compound 25e displayed 30% inhibition against S. aureus bioﬁlms

but only 8% activity against bioﬁlms of E. coli. The established

bioﬁlms are harder to eradicate and have limited data available on

novel AMPs with anti-bioﬁlm properties [60]. Compound 23b

containing less hydrophobic and cationic groups moderately dis-

rupted both the S. aureus or E. coli bioﬁlms compared to active

antibacterial compounds. These results revealed that the increase

in cationic charge and hydrophobicity elevates the activity against

planktonic cells, but they are unable to disrupt the large aggregates

of bacteria surrounded by an extracellular matrix. Previous studies

have used a similar method for measuring the antibioﬁlm proper-

ties of cationic peptides, that is allowing the bioﬁlm to form in the

presence of the peptide [61]. Cirioni et al. found that at a concen-

tration of half the MIC, MSI-78 (pexiganan) was able to prevent

bioﬁlm formation by P. aeruginosa by 27.5% [61]. This is higher than

the activity of the compounds in the current study which showed

39% inhibition of bioﬁlm production by another Gram-negative

bacterium E. coli but at a concentration of 31 times the MIC. In an

alternative bioﬁlm study, where bioﬁlms were formed prior to the

addition of MSI-78 (pexiganan) was unable to minimize the

amount of bioﬁlm produced by with P. aeruginosa, E. coli or S. aureus

even at a concentration of 128 times its MIC. However, in a similar

preformed bioﬁlm assay, another naturally occurring peptide, LL-37

which is also a membrane disrupting peptide [62], reduced the

bioﬁlm mass by approximately 40% at 4 times its MIC [63]. Thus, the

current compounds show similar antibioﬁlm capabilities to cationic

antimicrobial peptides, but future studies should examine whether

the current compounds are able to prevent bioﬁlms formation.

Notably, the change in membrane conduction at 10

mM was

correlated with antimicrobial activity, with compounds having

lower MIC values (compounds 25c-25e) showing greater positive

changes in membrane conduction in zwitterionic lipids than

compounds with higher MIC values 25b (Fig. 4A). In negatively-

charged lipids, however, the results were mixed and there was no

clear relationship between MIC and conductance change (Fig. 4B).

This would suggest that the effectiveness of these compounds is not

so much related to their overall net cationic charge, rather their

ability to insert into the bilayer proper. To test the hypothesis that

the tryptophan moiety plays a signiﬁcant role in membrane

disruption, the change of conductance induced by compound 29,

which lacks any Trp residue, was tested. As expected, compound 29

exhibited no change in membrane conduction in either zwitter-

ionic or negatively-charged lipids, demonstrating the importance

of the Trp residue.

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

9

Fig. 3. Change in conduction in (A) zwitterionic and (B) negatively-charged tBLMs in response to increasing concentrations of compounds 25b-25e. Change in membrane

capacitance in (C) zwitterionic and (D) negatively-charged tBLMs in response to increasing concentrations of compounds 25b-25e.

Fig. 4. The change in tBLM conduction of the various antimicrobial compounds at 10 mM in (A) zwitterionic and (B) negatively-charged membranes.

3. Conclusion

activity against S. aureus (15.6 mM) and E. coli (7.8 mM) without

cytotoxicity against mammalian cells. Based on the results of

cytoplasmic permeability assay and tBLMs-AC impedance spec-

troscopy, we propose that the biphenyl peptidomimetics exhibit

bacterial cell membrane disruption mechanism similar to most

AMPs. The importance of tryptophan in mechanism of action was

also revealed by compound 29. In addition, 23b biphenyl attached

with hydrophobic Trp and cationic aminoethyl guanidine could

disrupt bioﬁlms of S. aureus and E. coli. Collectively, our results

In conclusion, we have developed novel peptidomimetics based

on the 3,3⁰-biphenyl structural scaffold. The systematic tuning of

hydrophobicity and cationic charge of the peptidomimetics resul-

ted in moderate to excellent antibacterial activities. Compound

25g, the amphipathic peptidomimetic compound in which the

hydrophobic Trp-Phe and cationic Arg-aminoethylguanindine

segregated by biphenyl backbone, showed excellent antibacterial

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

Fig. 5. (A-F) S. aureus cytoplasmic membrane disruption promoted by 23b, 23d, 25b, 25e, 25f, 25g. (G) E. coli cytoplasmic membrane disruption promoted by 25g. Error bars

represent the standard error of triplicates (n ¼ 3).

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

11

residue subjected to ﬂash chromatography (2e5% MeOH/CH₂Cl₂as

the eluent) to afford the desired compound.

4.2.2. Procedure 2: peptide formation method b

To a stirred solution of an acid (1 equiv), amine (1.0 equiv), HOBt

(1.0 equiv), PyBOP (1.1 equiv) in CH₂Cl₂(3e5 mL) DIEA (2.0 equiv)

was added dropwise. The reaction was stirred overnight before the

solvent was removed under reduced pressure and the resultant

residue subjected to ﬂash chromatography (2e5% MeOH/CH₂Cl₂as

the eluent) to afford the desired compound.

4.2.3. Procedure 3: N-fmoc deprotection

To a stirred solution of the Fmoc-protected peptide in DMF

(3.0e5.0 mL)was added piperidine (2.0 equiv). The resultant solu-

tion was then stirred at rt for overnight. The solvent was then

removed under reduced pressure and puriﬁed using ﬂash chro-

matography (5% of 100:10:1; CHCl₃:MeOH: aqueous NH₃and 95%

of CH₂Cl₂) as eluent.

Fig. 6. Percentage of remaining bioﬁlm of S. aureus and E. coli after 24 h treatment

with the synthesized compounds at 250 M. The control represents the pre-

established bioﬁlms without any compounds. Error bars represent the standard er-

ror of triplicates (n ¼ 3).

m

4.2.4. Procedure 4: N-boc and Pbf deprotection

To a stirred solution of the boc/Pbf protected peptide in CH₂Cl₂

(3.0 mL) was added TFA (3.0 mL). The reaction mixture was stirred

at room temperature overnight before the solvent was removed

under reduced pressure. After triturating with diethyl ether, the

residue was concentrated to dryness. For Pbf and deboc of guani-

dine the residue was dissolved in CH₂Cl₂and CH₃CN and precipi-

tated by addition of diethyl ether and ﬁltered and dried. Some of

the compounds were puriﬁed using reverse phase chromatography

with 40% CH₃CN/H₂O as eluent utilizing GRACE instrument. The

resultant compounds isolated as TFA salt.

suggest biphenyl is a versatile core about which hydrophobic and

cationic amino acids can be arranged in order to generate short

cationic amphipathic peptidomimetics that mimic natural AMPs.

4. Experimental section

4.1. General notes

All chemical reagents were purchased from commercial sources

(Combi-Blocks, Chem-Impex and Sigma Aldrich) and used without

further puriﬁcation. Solvents were commercial and used as ob-

tained. Reactions were performed using oven-dried glassware un-

der an atmosphere of nitrogen and in anhydrous conditions (as

required). Room temperature refers to the ambient temperature.

Yields refer to chromatographically and spectroscopically pure

compounds unless otherwise stated. Reactions were monitored by

thin layer chromatography (TLC) plates pre-coated with Merck

silica gel 60 F254. Visualization was accomplished with UV light, a

ninhydrin staining solution in n-butanol. Flash chromatography

and silica pipette plugs were performed under positive air pressure

4.3. Preparation of derivatives

13

4.3.1. ¹H NMR and C NMR spectra of Methyl 3⁰-nitro-[1,1⁰-

biphenyl]-3-carboxylate (9)

1-Bromo-3-nitrobenzene 7 (5.0 g, 24.7 mmol), (3-(Methox-

ycarbonyl)phenyl)boronic acid 8 (5.34 g, 29.7 mmol), tetrakis-

triphenylphosphinepalladium(0) (1.40 g, 1.23 mmol), sodium

carbonate (2 M) (37 mL, 74.1 mmol), and 1,4-dioxane (120 mL) were

combined and the reaction mixture was heated at reﬂux under a

nitrogen atmosphere for 16 h. The reaction mixture was allowed to

cool at room temperature and partitioned between ethyl acetate

and water. The organic phase was separated, dried over magnesium

sulfate, ﬁltered, and the ﬁltrate was concentrated to give a liquid.

The crude product was puriﬁed by ﬂash chromatography over silica

with a hexanes:ethyl acetate gradient (100:0 to 90:10) to give

(4.12 g, 65%) the title compound 9 as a yellow solid; m.p.:

using Silica Gel 60 of 230e400 mesh (40e63

mm) and also using

Grace Davison LC60A 6- m for reverse phase chromatography.

m

Infrared spectra were recorded using a Cary 630 ATR spectropho-

tometer. Melting points were obtained using an OptiMelt melting

point apparatus and are uncorrected. High-resolution mass spec-

trometry was performed by the Bioanalytical Mass Spectrometry

facility, UNSW. Proton and Carbon NMR spectra were recorded in

the solvents speciﬁed using a Bruker DPX 300 or a Bruker Avance

96.5e96.6 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d

8.45 (t, J ¼ 4.00 Hz,

1H), 8.27e8.24 (m, 2H), 8.19 (dd, J ¼ 4.00, 8.00 Hz, 1H), 8.08e8.01

400 or 600 MHz spectrometer as designated. Chemical shifts (d) are

(m, 2H), 7.79 (t, J ¼ 8.00 Hz, 1H), 7.68 (t, J ¼ 8.00 Hz, 1H), 3.90 (s,

3H),; ¹³C NMR (100 MHz, DMSO-d₆):

d 166.4, 148.9, 141.0, 138.8,

quoted in parts per million (ppm), to the nearest 0.01 ppm and

internally referenced relative to the solvent nuclei. ¹HNMR spectral

data are reported as follows [chemical shift in ppm; multiplicity in

br, broad; s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet;

sext, sextet; sept, septet; m, multiplet; or as a combination of these

(e.g. dd, dt etc.)]; coupling constant (J) in hertz, integration, proton

count and assignment.

133.9, 132.3, 131.1, 131.0, 130.2, 129.6, 127.9, 123.1, 121.8, 52.8; IR

(ATR): max 3312, 2953, 2342, 1722, 1520, 1429, 1348, 1301, 1236,

n

1191, 1104, 963, 883, 839, 801, 690; HRMS (ESI): m/z calcd for

C

₁₄H₁₁NO₄Na [M þ Na]^þ: 280.0586; found: 280.0579.

13

4.3.2. ¹H NMR and C NMR spectra of Methyl 3⁰-amino-[1,1⁰-

biphenyl]-3-carboxylate (10)

4.2. General methods

To a stirred solution of 3⁰-nitro-[1,1⁰-biphenyl]-3-carboxylic acid

methyl ester 9 (4.0 g, 15.5 mmol) in anhydrous THF (100 mL) under

nitrogen atmosphere 10% palladium on activated charcoal (1.0 g)

was added. The reaction was evacuated and placed under a

hydrogen atmosphere and stirred overnight. The reaction mixture

was ﬁltered through Celite, and the solvent was removed under

reduced pressure to yield gray oil. The residue was

4.2.1. Procedure 1: peptide formation method a

To a stirred solution of an acid (1 equiv), amine (1.0 equiv), HOBt

(1.0 equiv), DIEA (2.5 equiv) in DMF (3e5 mL) EDCI (1.2 equiv) was

added portion-wise. The reaction was stirred overnight before the

solvent was removed under reduced pressure and the resultant

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

chromatographed on silica, eluting with 3:1 hexane/EtOAc. Con-

centration of the appropriate fractions provided the product as an

off-white solid (3.17 g, 90% yield); m.p.: 84.9e85.1 ^ꢂC; ¹H NMR

1525, 1433, 1310, 1253, 1110, 974, 877, 791, 691; HRMS (ESI): m/z

calcd for C₂₅H₂₃N₃O₃[M þ H]^þ: 413.1739; found 414.1810.

(400 MHz, CDCl₃):

d

8.28 (t, J ¼ 4.00 Hz, 1H), 8.03 (dt, J ¼ 2.00,

4.3.6. ¹H NMR and ¹³C NMR spectra of Methyl 3'-((S)-2-((S)-2-

((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-3-(1H-indol-

3-yl)propanamido)-[1,1⁰-biphenyl]-3-carboxylate (16)

5.00 Hz, 1H), 7.79e7.76 (m, 1H), 7.51 (t, J ¼ 12.00 Hz, 1H), 7.27 (q,

J ¼ 4.00 Hz, 1H), 7.06e7.03 (m, 1H), 6.97 (t, J ¼ 4.00 Hz, 1H),

6.75e6.73 (m, 1H), 3.96 (s, 5H),; ¹³C NMR (100 MHz, CDCl₃):

The title compound was prepared via protocol 1 using 14 and 15

(1.0 g, 3.76 mmol) to afford the coupled product 16 as an off-white

d

167.1146.6,141.6, 141.3, 131.5, 130.6,129.9,128.7, 128.3, 128.2, 117.7,

114.6, 113.9, 52.2; HRMS (ESI): m/z calcd for C₁₄H₁₃NO₂Na [M þ

solid; m.p.: 116.3e117.2 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 10.85

Na]^þ250.0844; found: 250.0838.

(s, 1H), 10.20 (s, 1H), 8.16e8.14 (m, 2H), 7.98e7.95 (m, 1H),

7.90e7.88 (m, 2H), 7.66e7.62 (m, 3H), 7.44e7.38 (m, 2H), 7.32 (d,

J ¼ 8.00 Hz, 1H), 7.21e7.11 (m, 6H), 7.07e7.03 (m, 1H), 6.95 (dd,

J ¼ 8.00, 14.00 Hz, 2H), 4.75 (q, J ¼ 8.00 Hz, 1H), 4.22e4.16 (m, 1H),

3.89 (s, 3H), 3.24 (dd, J ¼ 8.00, 16.00 Hz, 1H), 3.12 (dd, J ¼ 8.00,

14.00 Hz, 1H), 2.93 (dd, J ¼ 4.00, 12.00 Hz, 1H), 2.75e2.69 (m, 1H),

4.3.3. ¹H NMR and ¹³C NMR spectra of Methyl (S)-3'-(2-((tert-

butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanamido)-[1,1⁰-

biphenyl]-3-carboxylate (12)

The title compound was prepared via protocol 1, using 10 (1.0 g,

4.4 mmol) and (tert-butoxycarbonyl)-

L

-tryptophan (1.33 g,

1.28 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆):

d 171.9, 170.9, 166.6,

4.4 mmol) to afford the coupled product 12 as an off-white solid

155.7, 140.9, 139.9, 138.4, 136.5, 131.8, 130.8, 130.1, 130.0, 129.7,

128.7, 128.4, 127.8, 127.4, 126.6, 124.1, 122.3, 121.4, 119.4, 119.0, 118.7,

(1.42 g, 63%); m.p.: 107.3e108.0 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d

10.82 (s, 1H), 10.17 (s, 1H), 8.17 (s, 1H), 7.98e7.91 (m, 3H),

118.1, 111.7, 110.0, 78.7, 56.4, 54.7, 52.8, 37.9, 28.6, 28.4; IR (ATR).n

7.69e7.62 (m, 3H), 7.45e7.38 (m, 2H), 7.32 (d, J ¼ 8.00 Hz, 1H), 7.19

(s, 1H), 7.05 (t, J ¼ 8.00 Hz, 1H), 6.99e6.97 (m, 2H), 4.41e4.39 (m,

1H), 3.90 (s, 3H), 3.18e2.99 (m, 2H), 1.34 (s, 9H),; ¹³C NMR

max 3293, 3055, 2320, 2096, 1649, 1492, 1433, 1310, 1250, 1259,

1110, 1012, 882; HRMS (ESI): m/z calcd for C₃₉H₄₀N₄O₆[M þ H]^þ:

683.2846; found: 683.2841.

(100 MHz, DMSO- d₆): d 171.8,166.5,155.8,140.9,140.2,139.9,136.5,

131.9, 130.8, 130.8, 128.7, 127.7, 127.4, 124.2, 122.1, 121.3, 119.4, 118.6,

118.1, 111.8, 118.1, 111.8, 110.4, 78.6, 56.4, 52.8, 28.6, 28.3; IR

4.3.7. ¹H NMR and ¹³C NMR spectra of 3'-((S)-2-((S)-2-((tert-

butoxycarbonyl)amino)-3-phenylpropanamido)-3-(1H-indol-3-yl)

propanamido)-[1,1⁰-biphenyl]-3-carboxylic acid (17)

(ATR).nmax 3304, 2330, 2099, 1665, 1605, 1528, 1491, 1423, 1303,

1250, 1160, 1111, 1082, 1010, 856, 733, 690; HRMS (ESI): m/z calcd

To a solution of 16 (1.2 g, 2.33 mmol) in THF (10.0 mL) and MeOH

(10.0 mL), was added a 1N NaOH_(aq)(4.66 mL, 4.66 mmol) and

stirred at room temperature for 16 h. Ethyl acetate was added and

the layers were separated. The aqueous layer was then acidiﬁed

with 1N HCl and then extracted with CH₂Cl₂(2 ꢁ 100 mL) and then

the solvent was removed under reduced pressure to yield 17 (1.05 g,

90%) as an off-white solid; m.p.: 172.6e173.6 ^ꢂC; ¹H NMR (400 MHz,

for C₃₀H₃₁N₃O₅Na [M þ Na]^þ536.2161; found: 536.2153.

4.3.4. ¹H NMR and ¹³C NMR spectra of (S)-3'-(2-((tert-

butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanamido)-[1,1⁰-

biphenyl]-3-carboxylic acid (13)

To a solution of 12 (1.2 g, 1.81 mmol) in THF (10.0 mL) and MeOH

(10.0 mL), was added a 1N NaOH_(aq)(3.63 mL, 3.63 mmol) and

stirred at room temperature for 16 h. Ethyl acetate was added and

the layers were separated. The aqueous layer was then acidiﬁed

with 1N HCl and then extracted with CH₂Cl₂(2 ꢁ 100 mL) and then

the solvent was removed under reduced pressure to yield 13

(1.00 g, 85%) as an off-whit solid; m.p.: 140e140.3 ^ꢂC; ¹H NMR

DMSO-d₆):

d 13.08 (br s, 1H), 10.84 (s, 1H), 10.20 (s, 1H), 8.16e8.14

(m, 2H), 7.96e7.85 (m, 3H), 7.69e7.58 (m, 3H), 7.44e7.38 (m, 2H),

7.32 (d, J ¼ 4.00 Hz, 1H), 7.21e7.10 (m, 6H), 7.08e7.03 (m, 1H), 6.96

(dd, J ¼ 8.00, 12.00 Hz, 2H), 4.75 (q, J ¼ 8.00 Hz, 1H), 4.21e4.16 (m,

1H), 3.26e3.21 (m, 1H), 3.14e3.10 (m, 1H), 2.95e2.90 (m, 1H),

2.75e2.69 (m, 1H), 1.28 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆):

(400 MHz, DMSO-d₆):

d

13.10 (s, 1H), 10.82 (s, 1H), 10.17 (s, 1H), 8.18

d 171.9, 170.8, 167.7, 155.7, 140.7, 140.1, 140.0, 138.4, 136.5, 132.0,

(t, J ¼ 4.00 Hz, 1H), 7.97e7.95 (m, 2H), 7.90e7.87 (m, 1H), 7.69e7.66

(m, 2H), 7.61 (t, J ¼ 8.00 Hz, 1H), 7.45e7.38 (m, 2H), 7.32 (d,

J ¼ 8.00 Hz, 1H), 7.19 (s, 1H), 7.06 (t, J ¼ 4.00 Hz, 1H), 6.98 (t,

J ¼ 8.00 Hz, 2H), 4.41 (q, J ¼ 8.00 Hz, 1H), 3.19e3.14 (m, 1H),

3.05e3.00 (m, 1H), 1.34 (s, 9H); ¹³C NMR (100 MHz, DMSO- d₆):

131.4, 130.0, 138.4, 136.5, 132.0, 131.3, 130.0, 129.8, 129.6, 128.8,

128.4, 127.8, 127.6, 126.6, 126.6, 124.1, 122.2, 121.4, 119.3, 119.0, 118.7,

118.1, 111.7, 110.0, 78.7, 56.4, 54.7, 37.9, 28.6, 28.4; IR (ATR).nmax

2048,1653,1492, 1434,1391, 1228,1158, 1058, 849; HRMS (ESI): m/z

calcd for C₃₈H₃₈N₄O₆Na [M þ Na]^þ: 669.2689; found: 669.2689.

d

171.7, 167.6, 155.8, 140.7, 140.2, 140.1, 136.5, 132.0, 129.9, 128.8,

127.8, 127.6, 124.3, 122.1, 121.4, 119.3, 119.1, 118.7, 118.1, 111.8, 110.4,

79.6, 78.6, 56.3, 28.6, 28.3; IR (ATR). max 3278, 3051, 2946, 2342,

4.3.8. ¹H NMR and ¹³C NMR spectra of 1-(2-aminoethyl)2,3-

bis(tert-butoxycarbonyl)guanidine (18b)

n

2117, 1917, 1712, 1661, 1589, 1524, 1432, 1310, 1252, 1109, 973, 876,

790, 690; HRMS (ESI): m/z calcd for C₂₉H₂₉N₃O₅Na [M þ Na]^þ

522.2005; found: 522.1996.

The title compound 18b was prepared by adding the solution of

N,N⁰-bis-(Boc)-1H-Pyrazole-1-carboxamidine (2 g, 6.45 mmol) in

THF (50 mL) dropwise to ethylene diamine (4 mL, 59.6 mmol) in

THF (100 mL). After 30 min of mixing at room temperature solvent

was evaporated then toluene (100 mL) was added and evaporated

in order to remove remaining traces of the ethylene diamine.The

crude reaction mixture taken for next step due to the instability of

4.3.5. ¹H NMR and ¹³C NMR spectra of Methyl (S)-3'-(2-amino-3-

(1H-indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carboxylate (14)

The title compound 14 was prepared via protocol 4 using 12

(1.4 g, 2.72 mmol) to yield an off-white solid (1.00 g, 89%); m.p.:

the compound.¹H NMR (400 MHz, CDCl₃):

s, 1H), 3.47 (q, J ¼ 4.00 Hz, 2H), 2.87 (t, J ¼ 4.00 Hz, 2H), 1.49 (s, 9H),

1.48 (s, 9H); ¹³C NMR (100 MHz, CDCl₃):

163.6, 156.5, 153.2, 83.1,

79.3, 43.4, 41.0, 28.3, 28.1; IR (ATR). max 3385, 3068, 2972, 2887,

d 11.50 (br s, 1H), 8.63 (br

86.4e86.7 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 11.06 (s, 1H), 10.73

(s, 1H), 8.33 (br s, 2H), 8.16e8.15 (m, 1H), 8.00e7.97 (m, 1H),

7.92e7.89 (m, 1H), 7.86e7.85 (m, 1H), 7.68e7.60 (m, 3H), 7.48e7.46

(m, 2H), 7.36 (d, J ¼ 8.00 Hz, 1H), 7.26 (d, J ¼ 4.00 Hz, 1H), 7.09e7.05

(m, 1H), 6.98e6.94 (m, 1H), 4.21 (br s, 1H), 3.89 (s, 3H), 3.40e3.25

d

n

2807, 1613, 1519, 1463, 1347, 1311, 1249, 1090, 1161, 884, 799.

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆):

d

167.9, 166.6, 140.7, 140.1,

4.3.9. ¹H NMR and ¹³C NMR spectra of Methyl N^w-((4-methoxy-

139.2, 136.7, 131.9, 130.9, 130.3, 130.1, 128.8, 127.5, 127.4, 125.4,

123.0, 121.7, 119.7, 119.0, 118.9, 118.4, 111.9, 107.1, 54.1, 52.8, 27.8; IR

2,3,6-trimethylphenyl)sulfonyl)-L-argininate (18d)

The title compound 18d was prepared via protocol 3 using 20a

(ATR):

n

max 3282, 3054, 2947, 2341, 2106, 1911, 1713, 1663, 1589,

(0.9 g, 1.44 mmol) to yield the desired product as an off-white solid

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

13

(450 mg, 77%); m.p.: 136.3e137.3 ^ꢂC; ¹H NMR (400 MHz, CDCl₃):

6.50 (s, 1H), 6.46 (br s, 1H), 6.38 (br s, 2H), 3.80 (s, 3H), 3.67 (s, 3H),

3.47e3.45 (m, 1H), 3.15 (br s, 2H), 2.65 (s, 3H), 2.97 (s, 3H), 2.10 (s,

3H), 1.74e1.55 (m, 4H); ¹³C NMR (100 MHz, CDCl₃):

175.7, 158.4,

156.4, 138.4, 136.4, 133.5, 124.8, 111.7, 55.5, 53.7, 52.2, 40.7, 31.2,

25.5, 24.1, 18.3, 11.9; IR (ATR). max 3428, 3329, 2934, 1733, 1541,

94.0e94.4 ^ꢂC; ¹H NMR (400 MHz, CDCl₃):

d

7.76e7.35 (m, 7H),

d

7.30e7.28 (m, 2H), 6.50 (s, 1H), 6.20 (br s, 2H), 5.74e5.72 (br d,

J ¼ 7.2 Hz), 4.38e4.29 (m, 2H), 4.19e4.14 (m, 2H), 3.79 (s, 3H), 3.71

(s, 3H), 3.22e3.21 (m, 2H), 2.69 (s, 3H), 2.62 (s, 3H), 2.11 (s, 3H),

d

1.70e1.57 (m, 4H); ¹³C NMR (100 MHz, CDCl₃):

d 172.7, 158.5, 156.3,

n

143.7, 143.6, 141.2, 138.5, 136.5, 133.5, 127.8, 127.1, 125.1, 124.8, 120.0,

111.7, 67.13, 55.4, 52.6, 47.1, 40.7, 30.1, 25.2, 24.1, 18.3, 17.9, 11.9; IR

1459, 1398, 1246, 1303, 1098, 1016, 911, 802; HRMS (ESI): m/z calcd

for C₁₇H₂₈N₄O₅S [M þ H]^þ: 401.1780; found: 401.1851.

(ATR).nmax 2946, 1717, 1542, 1616, 1445, 1244, 1169, 1102; HRMS

(ESI): m/z calcd for C₃₂H₃₈N₄O₇SNa [M þ Na]^þ: 645.2359; found:

4.3.10. ¹H NMR and ¹³C NMR spectra of tert-butyl (S)-(5-amino-6-

((2-((tert-butoxycarbonyl)amino)ethyl)amino)-6-oxohexyl)

carbamate (18e)

The title compound was prepared via protocol 3, using 20a

(630 mg, 1.03 mmol) to yield the desired product 18e as an off-

white solid (0.32 g, 80%); m.p.: 206.2e207.5 ^ꢂC; ¹H NMR

645.2356.

4.3.14. ¹H NMR and ¹³C NMR spectra of (9H-ﬂuoren-9-yl)methyl

(S)-(1-((2-(2,3-diboc guanidino)ethyl)amino)-5-(3-((4-methoxy-

2,3,6-trimethylphenyl)sulfonyl)guanidino)-1-oxopentan-2-yl)

carbamate (20b)

(600 MHz, CDCl₃):

(m, 6H), 1.84e1.76 (m, 1H), 1.50e1.46 (m, 2H), 1.42 (s, 18H),

1.37e1.36 (m, 3H); ¹³C NMR (100 MHz, CDCl₃):

175.6, 156.5, 156.1,

79.4,79.1, 55.0, 40.6, 40.1, 39.7, 34.4, 29.8, 28.4, 28.4, 22.8; IR

d

7.63 (s, 1H), 5.09 (s, 1H), 4.67 (s, 1H), 3.34e3.08

The title compound 20b was prepared from compound 18b

(0.5 g, 1.64 mmol) and N_a-Fmoc-N_u-MTR-

1.64 mmol) according to the protocol 2. Off-white solid (0.73 g,

50%); m.p.: 148.0e148.6 ^ꢂC; ¹H NMR (400 MHz, CDCl₃):

11.40 (br s,

L

-arginine (1.0 g,

d

(ATR).

n

max 3313, 2930, 1683, 1518, 1452, 1246, 1162, 994, 859, 779;

1H), 8.61 (br s, 1H), 7.98 (br s, 1H), 7.73 (d, J ¼ 8.00 Hz, 2H), 7.57 (d,

J ¼ 8.00 Hz, 2H), 7.36 (t, J ¼ 8.00 Hz, 2H), 7.30e7.24 (m, 2H), 6.50 (br

s, 1H), 6.23 (br s, 2H), 5.99 (d, J ¼ 8.00 Hz, 1H), 4.34e4.22 (m, 2H),

4.18e4.14 (m, 2H), 3.79 (s, 3H), 3.54e3.15 (m, 7H), 2.67 (s, 3H), 2.60

(s, 3H), 2.10 (s, 3H), 1.84e1.57 (m, 4H), 1.45 (s, 18H); ¹³C NMR

HRMS (ESI): m/z calcd for C₁₈H₃₆N₄O₅[M þ H]^þ: 389.2686; found:

389.2759.

4.3.11. ¹H NMR and ¹³C NMR spectra of tert-butyl (S)-(5-amino-6-

((2-(2,3-dibocguanidino)ethyl)amino)-6-oxohexyl)carbamate (18f)

The title compound was prepared via protocol 3, using 20b

(850 mg,1.07 mmol) to yield the desired product 18f as an off-white

solid (0.45 g, 79%); m.p.: 184.4e185.6 ^ꢂC; ¹H NMR (400 MHz,

(100 MHz, CDCl₃):

d 163.3, 162.8, 159.7, 158.5, 157.2, 156.4, 156.3,

153.0,143.8,143.8,141.3,138.6,136.6,133.4,127.7,127.1,125.2,124.8,

120.0, 83.6, 80.4, 80.1, 67.1, 55.4, 47.1, 42.1, 40.1, 28.2, 28.2, 28.0, 25.2,

24.1, 18.4, 12.0; IR (ATR).nmax 3324, 2976, 2064, 1717, 1615, 1543,

CDCl₃):

d

11.43 (s, 1H), 8.56 (t, J ¼ 4.00 Hz, 1H), 7.91 (t, J ¼ 8.00 Hz,

1446, 1304, 1248, 1117, 840; HRMS (ESI): m/z calcd for

C

1H), 4.59 (br s, 1H), 3.59e3.55 (m, 2H), 3.45e3.41 (m, 2H),

3.33e3.30 (m, 1H), 3.13e3.08 (m, 2H), 1.82e1.77 (m, 1H), 1.52e1.51

(m, 1H), 1.49 (s, 18H), 1.49e1.45 (m, 4H), 1.43 (s, 9H), 1.39e1.37 (m,

₄₄H6₀N₈O₁₀S [M þ H]^þ: 893.4153; found: 893.4224.

4.3.15. ¹H NMR and ¹³C NMR spectra of (9H-ﬂuoren-9-yl)methyl

tert-butyl (6-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-6-

oxohexane-1,5-diyl)(S)-dicarbamate (21a)

2H); ¹³C NMR (100 MHz, CDCl₃):

d

175.6, 163.2, 157.1, 156.0, 153.1,

83.4, 79.5, 55.4, 40.2, 40.1, 40.0, 34.8, 30.0, 34.8, 30.0, 28.4, 28.3,

28.1, 23.0; IR (ATR). max 3326, 2931, 1714, 1612, 1521, 1409, 1362,

n

The title compound 21a was prepared from compound 18a

1324, 1246, 1128, 1047, 854; HRMS (ESI): m/z calcd for C₂₄H₄₆N₆O₇

(0.34 g, 2.13 mmol) and N_a-Fmoc-N_ε-Boc-

according to the protocol 2. Off-white solid (0.65 g, 50%); m.p.:

173.7e175.2 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

7.90e7.87 (m, 3H),

L

-lysine (1.0 g, 2.13 mmol)

[M þ H]^þ: 531.3428; found: 531.3500.

d

4.3.12. ¹H NMR and ¹³C NMR spectra of (S)-2-amino-N-(2-(2,3-

dibocguanidino)ethyl)-5-(3-((4-methoxy-2,3,6-trimethylphenyl)

sulfonyl)guanidino)pentanamide (18g)

The title compound was prepared via protocol 3, using 21b

(730 mg, 0.82 mmol) to yield the desired product 18g as an off-

white solid (0.48 g, 87%); m.p.: 102.2e103.6 ^ꢂC; ¹H NMR

7.72 (t, J ¼ 6.00 Hz, 2H), 7.42e7.38 (m, 3H), 7.32 (t, J ¼ 6.00 Hz, 2H),

6.73 (d, J ¼ 6.00 Hz, 2H), 4.28e4.27 (m, 1H), 4.24e4.20 (m, 2H),

3.88e3.87 (m, 1H), 3.12e3.00 (m, 2H), 2.99e2.96 (m, 2H),

2.90e2.86 (m, 2H), 1.74e1.46 (m, 3H), 1.36 (s, 18H), 1.28e1.19 (m,

3H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.5, 156.4, 156.1, 156.0,

144.4, 144.2, 141.2, 128.1, 127.5, 125.7, 120.6, 79.6, 79.4, 79.2, 79.2,

78.2, 77.8, 66.1, 55.2, 47.1, 46.3, 46.3, 32.1, 29.7, 28.7, 28.6, 26.4, 26.3,

(400 MHz, CDCl₃):

d 11.39 (br s, 1H), 8.56 (br s, 1H), 8.04 (t,

J ¼ 4.00 Hz, 1H), 6.51 (s, 1H), 6.26 (br s, 3H), 3.81 (s, 3H), 3.54e3.39

(m, 5H), 3.24e3.20 (m, 2H), 2.67 (s, 3H), 2.60 (s, 3H), 2.11 (s, 3H),

1.76e1.61 (m, 4H), 1.46 (s, 9H), 1.45 (s, 9H); ¹³C NMR (100 MHz,

23.3; IR (ATR).nmax 3356, 2943, 2265, 2106, 1526, 1447, 1389, 1526,

1240, 1167, 1025, 836, 735; HRMS (ESI): m/z calcd for C₃₃H₄₆N₄O₇Na

[M þ Na]^þ: 633.3264; found: 633.3261.

CDCl₃):

124.7, 111.7, 83.6, 79.8, 55.4, 54.3, 40.1, 31.7, 28.3, 28.0, 25.2, 24.1,

18.3, 11.9; IR (ATR). max 3326, 1719, 1611, 1543, 1439, 1409, 1326,

d 175.1, 163.0, 158.4, 157.1, 156.4, 153.0, 138.5, 136.6, 133.6,

4.3.16. ¹H NMR and ¹³C NMR spectra of (9H-ﬂuoren-9-yl)methyl

tert-butyl (6-((2-(2,3-dibocguanidino)ethyl)amino)-6-oxohexane-

1,5-diyl)(S)-dicarbamate (21b)

n

1248, 1115, 1046, 839, 803; HRMS (ESI): m/z calcd for C₂₉H₅₀N₈O₈S

[M þ H]^þ: 671.3540; found: 671.3540.

The title compound 21b was prepared from compound 18b

(0.643 g, 2.13 mmol) and N_a-Fmoc-N_ε-Boc-

2.13 mmol) according to the protocol 2. Off-white solid (0.89 g,

55%); m.p.: 102.2e103.6 ^ꢂC; ¹H NMR (400 MHz, CDCl₃):

11.43 (s,

L

-lysine (1.0 g,

4.3.13. ¹H NMR and ¹³C NMR spectra of Methyl N²-(((9H-ﬂuoren-9-

yl)methoxy)carbonyl)-N^w-((4-methoxy-2,3,6-trimethylphenyl)

d

sulfonyl)-

The title compound 20′ was prepared by adding thionyl chloride

(0.23 mL, 3.28 mmol) dropwise to N_a-Fmoc-N_u-MTR- -arginine

L

-argininate (20⁰)

1H), 8.65 (br s, 1H), 8.07 (br s, 1H), 7.75 (d, J ¼ 8.00 Hz, 2H), 7.59 (d,

J ¼ 8.00 Hz, 2H), 7.39 (t, J ¼ 8.00 Hz, 2H), 7.33e7.26 (m, 2H), 5.65 (d,

J ¼ 4.00 Hz, 1H), 4.59 (br s, 1H), 4.38e4.36 (m, 1H), 4.22e4.17 (m,

1H), 3.57e3.53 (m, 2H), 3.45e3.42 (m, 2H), 3.10e3.08 (m, 2H),

1.87e1.80 (m, 1H), 1.70e1.66 (m, 1H), 1.50 (s, 9H), 1.48 (s, 9H), 1.42

L

(1.0 g, 1.64 mmol) in methanol (20.0 mL) at 0 ^ꢂC and then stirred at

room temperature for 16 h. The solvents were removed and

reduced pressure and the residue was diluted with ethylacetate

(100.0 mL) and washed with saturated NaHCO₃(40 mL), saturated

brine (40 mL), then dried (Na₂SO₄) and concentrated under

reduced pressure to afford a white solid (0.95 g, 93%); m.p.:

(s, 9H), 1.48e1.32 (m, 6H); ¹³C NMR (100 MHz, CDCl₃):

d 171.6,

162.8, 157.6, 156.0, 155.9, 153.0, 143.9, 141.3, 127.7, 127.0, 125.2,

120.0, 83.7, 80.0, 79.1, 66.9, 54.8, 47.2, 41.5, 40.3, 32.9, 29.8, 28.4,

28.3, 28.0, 22.3; IR (ATR).nmax 3316, 2973, 1713, 1612, 1516, 1448,

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

14

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

1410, 1325, 1244, 1129, 1046, 857; HRMS (ESI): m/z calcd for

C

4.3.20. ¹H NMR and ¹³C NMR spectra of Methyl N²-(3'-((S)-2-((tert-

butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanamido)-[1,1⁰-

biphenyl]-3-carbonyl)-N^w-((4-methoxy-2,3,6-trimethylphenyl)

₃₉H₅₆N₆O₉Na [M þ Na]^þ: 775.4006; found: 775.4006.

sulfonyl)- -argininate (22d)

L

4.3.17. ¹H NMR and ¹³C NMR spectra of tert-butyl (S)-(1-((3'-((2-

((tert-butoxycarbonyl)amino)ethyl)carbamoyl)-[1,1⁰-biphenyl]-3-

yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (22a)

The title compound was prepared from compound 13 (100 mg,

0.20 mmol) and 18a (32 mg, 0.20 mmol) following the general

protocol 1 to afford 22a as an off-white solid (83 mg, 65%); m.p.:

The title compound was prepared from compound 13 (100 mg,

0.20 mmol) and 18d (80 mg, 0.2 mmol) following the general

protocol 1 to afford 22d as an off-white solid (104 mg, 59%); m.p.:

172.2e173.1 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 10.82 (s, 1H), 10.17

(s, 1H), 8.88 (d, J ¼ 12.00 Hz, 1H), 8.13 (br s, 1H), 7.92e7.88 (m, 2H),

7.79 (d, J ¼ 12.00 Hz,1H), 7.68e7.66 (m, 2H), 7.60 (t, J ¼ 6.00 Hz,1H),

7.47e7.41 (m, 2H), 7.33 (d, J ¼ 12.00 Hz, 1H), 7.19 (br s, 1H),

7.08e7.04 (m,1H), 7.00e6.96 (m, 2H), 6.74e6.66 (m, 2H), 6.48e6.26

(m, 2H), 4.48e4.40 (m, 2H), 3.77 (s, 3H), 3.65 (s, 3H), 3.15e2.99 (m,

4H), 2.59 (s, 3H), 2.51 (s, 3H), 2.02 (s, 3H), 1.82e1.74 (m, 2H),

1.52e1.49 (m, 2H), 1.34 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆):

130.5e131.2 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 10.82 (br s, 1H),

10.20 (br s, 1H), 8.60 (t, J ¼ 4.00 Hz, 1H), 8.10 (s, 1H), 7.90 (br s, 1H),

7.85 (d, J ¼ 4.00 Hz,1H), 7.75 (d, J ¼ 8.00 Hz, 1H), 7.67 (d, J ¼ 8.00 Hz,

2H), 7.57 (t, J ¼ 8.00 Hz, 1H), 7.46e7.40 (m, 2H), 7.33 (d, J ¼ 4.00 Hz,

1H), 7.20 (s, 1H), 7.08e6.92 (m, 4H), 4.44e4.40 (m, 1H), 3.18e2.99

(m, 6H), 1.37 (s, 18H); ¹³C NMR (100 MHz, DMSO-d₆):

d

166.7, 156.2,

d 173.0, 171.8, 166.9, 157.9, 156.6, 155.7, 140.7, 140.5, 140.1, 138.1,

140.6, 140.6, 136.5, 135.7, 129.8, 129.4, 127.8, 126.9, 125.9, 124.3,

122.3, 121.3, 119.2, 119.1, 118.7, 118.3, 111.8, 110.4, 79.6, 78.6, 78.2,

136.5, 134.8, 130.1, 129.8, 129.5, 127.7, 127.1, 126.1, 124.3, 122.3, 121.3,

119.2, 119.1, 118.6, 118.3, 112.2, 111.8, 110.4, 79.6, 78.6, 56.3, 55.9,

28.7, 28.6, 28.3; IR (ATR).

n

max 3298, 2974, 1669, 1492, 1453, 1247,

53.0, 52.3, 28.6, 28.4, 24.1, 18.5, 12.2; IR (ATR).nmax 3305, 2932,

1158, 1010, 853; HRMS (ESI): m/z calcd for C₃₆H₄₃N₅O₆Na [M þ

1669, 1541, 1456, 1364, 1303, 1227, 1159, 1114, 1013; HRMS (ESI): m/z

Na]^þ: 664.3111; found: 664.3102.

calcd for C₄₆H₅₅N₇O₉S [M þ H]^þ: 882.3782; found: 882.3854.

4.3.18. ¹H NMR and ¹³C NMR spectra of tert-butyl (S)-(1-((3'-((2-

(2,3-dibocguanidino)ethyl)carbamoyl)-[1,1⁰-biphenyl]-3-yl)amino)-

3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (22b)

4.3.21. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-5-(3'-((S)-2-

((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanamido)-

[1,1⁰-biphenyl]-3-carboxamido)-6-((2-((tert-butoxycarbonyl)

amino)ethyl)amino)-6-oxohexyl)carbamate (22e)

The title compound was prepared from compound 13 (100 mg,

0.20 mmol) and 18b (61 mg, 0.2 mmol) following the general

protocol 1 to afford 22b as an off-white solid (94 mg, 60%); m.p.:

The title compound was prepared from compound 13 (100 mg,

0.20 mmol) and 18e (77 mg, 0.20 mmol) following the general

protocol 1 to afford 22e as an off-white solid (139 mg, 80%); m.p.:

127.2e127.6 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 11.49 (br s, 1H),

10.81 (br s, 1H), 10.15 (br s, 1H), 8.67 (t, J ¼ 6.00 Hz, 1H), 8.47 (t,

J ¼ 6.00 Hz, 1H), 8.08 (br s, 1H), 7.91 (s, 1H), 7.82 (d, J ¼ 12.00 Hz,

1H), 7.75 (d, J ¼ 12.00 Hz,1H), 7.67e7.66 (m, 2H), 7.56 (t, J ¼ 6.00 Hz,

1H), 7.32 (d, J ¼ 6.00 Hz, 1H), 7.19 (br s, 1H), 7.05 (t, J ¼ 6.00 Hz, 1H),

6.99e6.95 (m, 2H), 4.40 (q, J ¼ 6.00 Hz,1H), 3.52 (q, J ¼ 6.00 Hz, 2H),

3.46 (q, J ¼ 6.00 Hz, 2H), 3.17e3.13 (m, 1H), 3.04e3.00 (m, 1H), 1.43

(s, 9H), 1.37 (s, 9H), 1.33 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆):

131.5e131.7 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 10.82 (s, 1H), 10.16

(s, 1H), 8.55 (d, J ¼ 6.00 Hz, 1H), 8.16 (s, 1H), 8.01 (t, J ¼ 6.00 Hz, 1H),

7.92e7.91 (m, 2H), 7.77e7.75 (m, 1H), 7.68e7.67 (m, 1H), 7.57 (t,

J ¼ 12.00 Hz,1H), 7.45e7.43 (m, 2H), 7.32 (d, J ¼ 6.00 Hz,1H), 7.20 (s,

1H), 7.06 (t, J ¼ 6.00 Hz, 1H), 6.99e6.96 (m, 2H), 6.77e6.74 (m, 2H),

4.43e4.37 (m, 2H), 3.18e2.86 (m, 8H), 1.77e1.68 (m, 2H), 1.37e1.36

(m, 2H), 1.36 (s, 9H), 1.34 (s, 18H), 1.24e1.15 (m, 2H); ¹³C NMR

d

171.1, 166.9, 163.7, 163.5, 156.2, 155.7, 152.3, 140.6, 140.5, 140.1,

136.5, 135.6, 129.8, 129.7, 129.4, 129.4, 127.7, 126.7, 125.9, 124.2,

122.2, 121.3, 119.2, 119.0, 118.6, 118.2, 111.7, 110.4, 83.3, 78.6, 78.5,

67.4, 56.3, 40.5, 39.2, 31.7, 28.7, 28.6, 28.4, 28.2, 28; IR (ATR).nmax

(150 MHz, DMSO-d₆): d 172.4, 171.8, 166.7, 156.1, 156.0, 155.8, 140.6,

140.6, 136.5135.3, 129.8, 129.3, 127.7, 127.2, 126.3, 124.3, 121.3, 119.2,

119.1, 118.7, 118.3, 111.7, 110.4, 78.6, 78.1, 77.7, 56.3, 54.1, 40.5, 39.3,

2977, 1612, 1535, 1364, 1326, 1229, 1133, 875; HRMS (ESI): m/z calcd

31.8, 31.4, 29.7, 23.7, 22.5; IR (ATR).nmax 2972, 2930, 1683, 1521,

for C₄₂H₅₃N₇O₈[M þ H]^þ: 784.3956; found: 784.4031.

1455, 1246, 1163, 1010, 861; HRMS (ESI): m/z calcd for

C

₄₇H₆₃N₇O₉Na [M þ Na]^þ: 892.4585; found: 892.4575.

4.3.19. ¹H NMR and ¹³C NMR spectra of methyl N⁶-(tert-

butoxycarbonyl)-N²-(3'-((S)-2-((tert-butoxycarbonyl)amino)-3-

(1H-indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carbonyl)-

L

-

4.3.22. ¹H NMR and ¹³C NMR spectra of (S)-3'-(2-amino-3-(1H-

indol-3-yl)propanamido)-N-(2-aminoethyl)-[1,1⁰-biphenyl]-3-

carboxamide (23a)

The title compound was prepared from compound 22a (75 mg,

0.116 mmol) following the general protocol 4 to afford 23a as a

white solid (45 mg, 89%); m.p.: 164.5e164.7 ^ꢂC; ¹H NMR (600 MHz,

lysinate (22c)

The title compound was prepared from compound 13 (100 mg,

0.20 mmol) and 18c (52 mg, 0.2 mmol) following the general

protocol 1 to afford 22c as an off-white solid (109 mg, 74%); m.p.:

235.5e236.1 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 10.82 (s, 1H), 10.17

(s, 1H), 8.84 (d, J ¼ 8.00 Hz, 1H), 8.14 (s, 1H), 7.91e7.88 (m, 2H), 7.79

(d, J ¼ 8.00 Hz, 1H), 7.68 (t, J ¼ 4.00 Hz, 1H), 7.59 (t, J ¼ 8.00 Hz, 1H),

7.47e7.41 (m, 2H), 7.32 (d, J ¼ 8.00 Hz, 1H), 7.20 (s, 1H), 7.06 (t,

J ¼ 8.00 Hz, 1H), 7.00e6.97 (m, 2H), 6.78 (t, J ¼ 8.00 Hz, 1H),

4.47e4.38 (m, 2H), 3.66 (s, 3H), 3.17e2.89 (m, 4H), 1.81 (t,

J ¼ 8.00 Hz, 1H), 1.41e1.37 (m, 2H), 1.34 (s, 18H), 1.24e1.18 (m, 2H);

DMSO-d₆):

d

11.06 (d, J ¼ 6.00 Hz, 1H), 10.70 (s, 1H), 8.78 (t,

J ¼ 6.00 Hz, 1H), 8.30 (br s, 3H), 8.13 (t, J ¼ 6.00 Hz, 1H), 7.90e7.87

(m, 5H), 7.79e7.77 (m, 1H), 7.68 (d, J ¼ 12.00 Hz, 1H), 7.62e7.60 (m,

2H), 7.50e7.49 (m, 2H), 7.37 (d, J ¼ 12.00 Hz, 1H), 7.26 (d,

J ¼ 6.00 Hz, 1H), 7.10e7.07 (m, 1H), 6.99e6.97 (m, 1H), 4.21e4.20

(m, 1H), 3.55 (q, J ¼ 6.00 Hz, 2H), 3.39e3.35 (m, 2H), 3.28e3.25 (m,

2H), 3.03 (q, J ¼ 6.00 Hz, 2H); ¹³C NMR (150 MHz, DMSO-d₆):

¹³C NMR (100 MHz, DMSO-d₆):

d173.2, 166.9, 156.0, 155.7, 140.7,

140.5, 140.1, 136.5,134.8,130.0,129.8,129.4,127.7, 127.1, 126.1, 124.2,

122.3, 121.3, 119.0, 118.6, 118.2, 111.7, 110.4, 79.6, 78.5, 77.8, 53.2,

d

167.9, 167.2, 140.6, 140.4, 139.1, 136.7, 135.3, 130.1, 129.9, 129.6,

127.5, 127.1, 126.1, 125.4, 123.1, 121.7, 119.6, 119.0, 118.9, 118.5, 111.9,

107.1, 54.1, 40.5, 39.1, 37.6, 27.8; IR (ATR). max 3041, 2890, 1774,

52.3, 30.7, 29.5, 28.7, 28.6, 28.2, 23.5; IR (ATR).

nmax 2929, 2312,

n

1992, 1671, 1521, 1433, 1363, 1228, 1160, 1010, 860; HRMS (ESI): m/z

1664, 1525, 1429, 1318, 1179, 835, 795, 744; HRMS (ESI): m/z calcd

calcd for C₄₁H₅₁N₅O₈Na [M þ Na]^þ: 764.3635; found: 764.3630.

for C₂₆H₂₇N₅O₂[M þ H]^þ: 442.2165; found: 442.2234.

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

15

4.3.23. ¹H NMR and ¹³C NMR spectra of (S)-3'-(2-amino-3-(1H-

indol-3-yl)propanamido)-N-(2-guanidinoethyl)-[1,1⁰-biphenyl]-3-

carboxamide (23b)

The title compound was prepared from compound 22b (90 mg,

0.122 mmol) following the general protocol 4 to afford 23b as a

white solid (29 mg, 50%); m.p.: 236.7e238.0 ^ꢂC; ¹H NMR (600 MHz,

7.60e7.58 (m, 2H), 7.51e7.47 (m, 2H), 7.36 (d, J ¼ 8.00 Hz, 1H), 7.25

(d, J ¼ 4.00 Hz, 1H), 7.08 (t, J ¼ 4.00 Hz, 1H), 6.97 (t, J ¼ 4.00 Hz, 1H),

4.44e4.41 (m,1H), 4.21e4.20 (m,1H), 3.37e3.24 (m, 4H), 2.88e2.77

(m, 4H), 1.86e1.70 (m, 2H), 1.60e1.52 (m, 2H), 1.44e1.31 (m, 2H);

d 172.8, 167.9, 166.8, 158.9, 158.7,

158.5, 140.7, 140.3, 139.1, 136.7, 135.2, 130.0, 129.4, 127.5, 127.3,

126.3, 125.4, 123.2, 121.7, 119.6, 118.9, 118.6, 116.7, 111.9, 107.1, 54.1,

¹³C NMR (150 MHz, DMSO-d₆):

DMSO-d₆):

d

11.05 (br s, 1H), 10.69 (s, 1H), 8.77 (t, J ¼ 6.00 Hz, 1H),

8.28 (br s, 3H), 8.12 (t, J ¼ 6.00 Hz, 1H), 7.89e7.87 (m, 2H), 7.78e7.77

(m, 1H), 7.70e7.67 (m, 2H), 7.62e7.59 (m, 2H), 7.49e7.48 (m, 3H),

7.38e7.36 (m, 2H), 7.26 (d, J ¼ 6.00 Hz, 2H), 7.10e7.07 (m, 1H),

6.99e6.97 (m, 1H), 4.20 (t, J ¼ 6.00 Hz, 1H), 3.45 (q, J ¼ 6.00 Hz, 2H),

3.39e3.36 (m, 1H), 3.28e3.24 (m, 1H); ¹³C NMR (150 MHz, DMSO-

53.8, 40.5, 39.1, 38.9, 36.9, 31.2, 27.8, 27.2, 23.2; IR (ATR).nmax 3257,

3052, 2930, 2099, 1664, 1524, 1431, 1317, 1170, 797, 744, 720; HRMS

(ESI): m/z calcd for C₃₂H₃₉N₇O₃[M þ H]^þ: 570.3114; found:

570.2820.

d₆):

127.5, 126.9, 126.0, 125.4, 123.1, 121.7, 119.6, 119.0, 118.9, 118.5, 112.0,

107.1,54.1, 40.8, 39.1, 27.8; IR (ATR). max 3178, 3080, 2340, 2110,

d

167.9, 167.1, 157.5, 140.6, 140.4, 139.1, 135.3, 130.1, 129.8, 129.6,

4.3.27. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-1-

((3'-((2-((tert-butoxycarbonyl)amino)ethyl)carbamoyl)-[1,1⁰-

biphenyl]-3-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)

amino)-1-oxo-3-phenylpropan-2-yl)carbamate (24a)

n

1657, 1542, 1430, 1316, 1200, 1148, 835, 797, 743; HRMS (ESI): m/z

calcd for C₂₇H₂₉N₇O₂[M þ H]^þ: 484.2383; found: 484.2455.

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18a (25 mg, 0.15 mmol) following the general

protocol 1 to afford 24a as an off-white solid (94 mg, 80%); m.p.:

4.3.24. ¹H NMR and ¹³C NMR spectra of Methyl (3'-((S)-2-amino-3-

(1H-indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carbonyl)-

L

-

126.7e128.0 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 10.82 (s, 1H), 10.15

lysinate (23c)

(s, 1H), 8.57e8.56 (m, 1H), 8.43 (d, J ¼ 8.00 Hz, 1H), 8.10 (s, 1H),

7.81e7.67 (m, 7H), 7.52 (t, J ¼ 8.00 Hz, 1H), 7.30 (d, J ¼ 8.00 Hz, 1H),

7.17e6.91 (m, 9H), 6.76 (d, J ¼ 8.00 Hz, 1H), 4.75 (q, J ¼ 8.00 Hz, 1H),

4.26e4.21 (m, 1H), 3.29e3.03 (m, 6H), 2.73e2.72 (m, 1H),

2.60e2.54 (m, 1H), 1.36 (s, 9H), 1.29 (s, 9H); ¹³C NMR (100 MHz,

The title compound was prepared from compound 22c (50 mg,

0.067 mmol) following the general protocol 4 to afford 23c as a

gummy solid (36 mg, 70%); ¹H NMR (600 MHz, DMSO-d₆):

d 11.00

(s, 1H), 10.70 (s, 1H), 8.89 (d, J ¼ 6.00 Hz, 1H), 8.29 (br s, 3H),

8.14e8.13 (m, 1H), 7.93e7.91 (m, 1H), 7.87e7.86 (m, 1H), 7.80e7.75

(m, 4H), 7.68 (d, J ¼ 12.00 Hz, 1H), 7.62e7.60 (m, 2H), 7.50e7.48 (m,

2H), 7.37e7.36 (m, 1H), 7.26 (d, J ¼ 6.00 Hz, 1H), 7.10e7.07 (m, 1H),

6.99e6.97 (m, 1H), 4.50e4.46 (m, 1H), 4.20 (br s, 1H), 3.67 (s, 3H),

3.35e3.25 (m, 2H), 2.80e2.79 (m, 2H), 1.86e1.39 (m, 6H); ¹³C NMR

(100 MHz, DMSO-d₆): d 173.1, 167.9, 166.9, 158.6, 158.4, 140.6, 140.4,

139.1, 136.7, 134.8, 130.2, 130.1, 129.6, 127.5, 127.2, 126.2, 125.4,

123.2, 121.6, 119.6, 118.95, 118.9, 118.6, 111.9, 107.1, 54.1, 53.0, 52.4,

DMSO-d₆):

d 171.9, 166.7, 160.0, 156.2, 155.7, 155.1, 140.0, 139.8,

138.9, 138.3, 137.1, 136.5, 135.6, 134.9, 129.7, 129.3, 129.3, 128.3,

127.7, 127.5, 126.5, 125.3, 124.2, 121.4, 120.2, 119.0, 118.7, 111.7, 110.1,

78.5, 78.1, 56.0, 54.5, 37.8, 28.7, 28.5, 28.2; IR (ATR).nmax 3299,

2976, 1663, 1518, 1455, 1246, 1160, 1118, 1021, 806; HRMS (ESI): m/z

calcd for C₄₅H₅₂N₆O₇Na [M þ Na]^þ: 811.3795; found: 811.3789.

4.3.28. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-1-

((3'-((2-(2,3-dibocguanidino)ethyl)carbamoyl)-[1,1⁰-biphenyl]-3-yl)

amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-1-oxo-3-

phenylpropan-2-yl)carbamate (24b)

40.5, 40.4, 39.0, 30.4, 27.8, 27.0, 23.1; IR (ATR).nmax 3251, 3044,

2932, 2098, 1666, 1524, 1432, 1338, 1178, 1118, 835, 796, 743, 719;

HRMS (ESI): m/z calcd for C₃₁H₃₅N₅O₄[M þ H]^þ: 542.2689; found:

542.2157.

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18b (46 mg, 0.15 mmol) following the general

protocol 1 to afford 24b as an off-white solid (95 mg, 68%); m.p.:

4.3.25. ¹H NMR and ¹³C NMR spectra of Methyl (3'-((S)-2-amino-3-

(1H-indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carbonyl)-

L

-

206.3e206.5 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 11.50 (s, 1H),

argininate (23d)

10.80 (s, 1H), 10.20 (s, 1H), 8.41e8.45 (m, 1H), 8.14 (br s,1H), 8.07 (br

s, 1H), 7.88 (s, 1H), 7.82 (d, J ¼ 4.00 Hz, 1H), 7.73 (d, J ¼ 4.00 Hz, 1H),

7.62 (t, J ¼ 8.00 Hz, 2H), 7.55 (t, J ¼ 4.00 Hz, 1H), 7.42e7.40 (m, 2H),

7.32e7.31 (m, 1H), 7.20e7.13 (m, 7H), 7.05 (t, J ¼ 4.00 Hz, 1H),

6.97e6.92 (m, 2H), 4.75e4.74 (m, 1H), 4.18e4.17 (m, 1H), 3.50 (t,

J ¼ 4.00 Hz, 2H), 3.45 (q, J ¼ 4.00 Hz, 2H), 3.25e3.21 (m, 1H),

3.13e3.09 (m, 1H), 2.93e2.90 (m, 1H), 2.73e2.69 (m, 1H), 1.42 (s,

The title compound was prepared from compound 22d (90 mg,

0.10 mmol) following the general protocol 4 to afford 23d as a

gummy solid (50 mg, 54%).¹H NMR (600 MHz, DMSO-d₆):

d 11.04 (s,

1H), 10.66 (s, 1H), 8.93 (d, J ¼ 6.00 Hz, 1H), 8.27 (br s, 3H), 8.13 (br s,

1H), 7.92 (d, J ¼ 6.00 Hz, 2H), 7.79 (d, J ¼ 12.00 Hz, 1H), 7.73e7.45

(m, 6H), 7.38e7.25 (m, 2H), 7.10e6.97 (m, 3H), 4.52e4.49 (m, 1H),

4.19e4.18 (m, 1H), 3.68 (s, 3H), 3.35e3.12 (m, 4H), 1.92e1.54 (m,

9H), 1.37 (s, 9H), 1.28 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆):

d 171.9,

4H); ¹³C NMR (150 MHz, DMSO-d₆):

d

172.9, 167.9, 166.9, 162.2,

170.8, 166.9, 163.6, 156.2, 155.7, 152.3, 140.6, 140.5, 139.9, 138.4,

136.5, 135.7, 129.8, 129.6, 129.4, 128.4, 127.8, 126.7, 126.6, 125.9,

124.1, 122.4, 121.4, 119.2, 118.9, 118.7, 118.3, 111.7, 110.0, 83.3, 78.6,

158.7,158.5,157.2,140.6,140.4,139.1,136.7,134.8,130.2,130.1,129.6,

127.5, 127.2, 126.2, 125.4, 123.2, 121.7, 119.6, 118.9, 118.9, 118.8, 118.5,

112.0, 107.1, 54.1, 52.8, 52.5, 40.7, 40.5, 28.1, 27.8, 25.8; IR (ATR).

n

56.4, 54.6, 40.5, 39.2, 37.9, 31.7, 28.7, 28.6, 28.0; IR (ATR).nmax 2974,

max, 3192, 3065, 2118, 1660, 1539, 1434, 1315, 1227, 1159, 836, 797,

744; HRMS (ESI): m/z calcd for C₃₁H₃₅N₇O₄[M þ H]^þ: 570.3114;

found: 570.2820.

1641, 1539, 1364, 1326, 1250, 1133, 1049, 876; HRMS (ESI): m/z calcd

for C₅₁H₆₂N₈O₉Na [M þ Na]^þ: 953.4537; found: 953.4533.

4.3.29. ¹H NMR and ¹³C NMR spectra of Methyl N⁶-(tert-

butoxycarbonyl)-N²-(3'-((S)-2-((S)-2-((tert-butoxycarbonyl)

amino)-3-phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-

4.3.26. ¹H NMR and ¹³C NMR spectra of N-((S)-6-amino-1-((2-

aminoethyl)amino)-1-oxohexan-2-yl)-3'-((S)-2-amino-3-(1H-

indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carboxamide (23e)

The title compound was prepared from compound 22c (100 mg,

0.114 mmol) following the general protocol 4 to afford 23e as a

white solid (45 mg, 70%); m.p.: 237.6e238.0 ^ꢂC; ¹H NMR (600 MHz,

[1,1⁰-biphenyl]-3-carbonyl)-

-lysinate (24c)

L

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18c (39 mg, 0.15 mmol) following the general

protocol 1 to afford 24c as an off-white solid (106 mg, 80%); m.p.:

DMSO-d₆):

d

11.05 (d, J ¼ 4.00 Hz, 1H), 10.71 (s, 1H), 8.68 (d,

132.5e133.9 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d 10.85 (s,1H),10.20

J ¼ 4.00 Hz, 1H), 8.29e8.23 (m, 4H), 8.16 (s, 1H), 7.94 (d, J ¼ 4.00 Hz,

(s, 1H), 8.83 (d, J ¼ 8.00 Hz, 1H), 8.16e8.12 (m, 2H), 7.90e7.88 (m,

1H), 7.85 (br s, 4H), 7.77e7.75 (m, 4H), 7.67 (d, J ¼ 8.00 Hz, 1H),

2H), 7.76 (d, J ¼ 8.00 Hz, 1H), 7.65e7.56 (m, 3H), 7.46e7.43 (m, 2H),

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

16

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

7.32 (d, J ¼ 8.00 Hz, 1H), 7.21e7.14 (m, 6H), 7.06 (t, J ¼ 4.00 Hz, 1H),

6.99e6.93 (m, 2H), 6.78 (t, J ¼ 4.00 Hz, 1H), 4.76 (q, J ¼ 8.00 Hz, 1H),

4.44 (q, J ¼ 8.00 Hz, 1H), 4.22e4.16 (m, 1H), 3.66 (s, 3H), 3.27e3.21

(m, 1H), 3.15e3.09 (m, 1H), 2.95e2.94 (m, 3H), 2.75e2.69 (m, 1H),

1.83e1.80 (m, 2H), 1.40e1.37 (m, 2H), 1.34 (s, 9H), 1.29 (s, 9H),

4.3.32. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-1-

((3'-(((S)-6-amino-1-((2-(2,3-dibocguanidino)ethyl)amino)-1-

oxohexan-2-yl)carbamoyl)-[1,1⁰-biphenyl]-3-yl)amino)-3-(1H-

indol-3-yl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)

carbamate (24f)

1.18e1.14 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆):

d

173.2, 171.9,

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18f (80 mg, 0.15 mmol) following the general

protocol 1 to afford 24f as an off-white solid (128 mg, 74%); m.p.:

170.8, 166.9, 156.0, 155.7, 140.6, 140.5, 139.8, 138.4, 136.5, 134.8,

130.0, 129.8, 129.6, 129.5, 128.4, 127.8, 127.2, 126.6, 126.1, 124.1,

122.4, 121.4, 119.2, 118.9, 118.7, 118.3, 118.7, 118.3, 111.7, 110.0, 78.7,

192.5e194.0 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 11.50 (s, 1H), 10.80

77.7, 56.4, 54.6, 53.2, 37.9, 31.4, 30.7, 29.6, 28.7, 28.6; IR (ATR).

n

max

(s, 1H), 10.19 (s, 1H), 8.52 (d, J ¼ 12.00 Hz, 1H), 8.35 (t, J ¼ 6.00 Hz,

1H), 8.13 (br s, 3H), 7.90e7.74 (m, 2H), 7.73 (d, J ¼ 12.00 Hz, 1H),

7.64e7.62 (m, 2H), 7.55 (t, J ¼ 12.00 Hz, 1H), 7.44e7.41 (m, 2H), 7.31

(d, J ¼ 12.00 Hz, 1H), 7.20e7.12 (m, 6H), 7.07e7.03 (m, 1H),

6.98e6.92 (m, 2H), 6.73 (t, J ¼ 6.00 Hz, 1H), 4.75 (q, J ¼ 6.00 Hz, 1H),

4.40 (q, J ¼ 12.00 Hz, 1H), 4.20e4.15 (m, 1H), 3.34e3.36 (m, 2H),

3.30e3.10 (m, 4H), 2.94e2.86 (m, 3H), 2.74e2.71 (m, 1H), 1.75e1.68

(m, 2H), 1.43 (s, 9H), 1.38 (s, 9H), 1.37e1.34 (m, 2H), 1.28 (s, 9H), 1.32

3305, 3047, 2925, 1652, 1509, 1453, 1363, 1245, 1161, 1012, 855;

HRMS (ESI): m/z calcd for C₅₀H₆₀N₆O₉Na[M þ Na]^þ: 911.4319;

found: 911.4316.

4.3.30. ¹H NMR and ¹³C NMR spectra of Methyl N²-(3'-((S)-2-((S)-

2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-3-(1H-

indol-3-yl)propanamido)-[1,1⁰-biphenyl]-3-carbonyl)-N^w-((4-

(s, 9H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.6, 172.0, 170.8, 166.5,

163.5, 156.1, 155.7, 152.3, 151.8, 140.6, 140.5, 139.8, 136.5, 135.3,

129.8, 129.6, 129.3, 128.4, 127.8, 127.2, 126.6, 126.2, 124.1, 121.3,

119.3, 118.9, 118.7118.4, 111.7, 110.0, 83.3, 79.6, 78.6, 77.7, 56.4, 54.6,

methoxy-2,3,6-trimethylphenyl)sulfonyl)- -argininate (24d)

L

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18d (60 mg, 0.15 mmol) following the general

protocol 1 to afford 24d as an off-white solid (115 mg, 75%); m.p.:

54.1, 38.3, 31.9, 29.7, 28.7, 28.5, 28.4, 28.0, 23.6; IR (ATR).nmax 3284,

3066, 2973,1635,1540,1363,1322,1247,1131,1022, 856, 741; HRMS

(ESI): m/z calcd for C₆₂H₈₂N₁₀O₁₂Na[M þ Na]^þ: 1181.6011; found:

1181.6010.

179.5e179.7 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d 10.85 (s, 1H),10.20

(s, 1H), 8.87 (d, J ¼ 4.00 Hz, 1H), 8.15e8.12 (m, 2H), 7.89e7.88 (m,

2H), 7.77 (d, J ¼ 4.00 Hz, 1H), 7.64e7.57 (m, 3H), 7.45e7.41 (m, 2H),

7.33 (d, J ¼ 4.00 Hz, 1H), 7.21e7.13 (m, 6H), 7.07e7.04 (m, 1H),

6.98e6.93 (m, 2H), 6.66 (br s, 2H), 4.75 (q, J ¼ 4.00 Hz, 1H),

4.46e4.42 (m, 1H), 4.21e4.17 (m, 1H), 3.77 (s, 3H), 3.65 (s, 3H),

3.25e3.22 (m,1H), 3.14e3.06 (m, 3H), 2.94e2.91 (m,1H), 2.74e2.70

(m, 1H), 2.59 (s, 3H), 2.02 (s, 3H), 1.84e1.59 (m, 2H), 1.52e1.47 (m,

4.3.33. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-1-

((3'-(((S)-1-((2-(2,3-dibocguanidino)ethyl)amino)-5-(3-((4-

methoxy-2,3,6-trimethylphenyl)sulfonyl)guanidino)-1-oxopentan-

2-yl)carbamoyl)-[1,1⁰-biphenyl]-3-yl)amino)-3-(1H-indol-3-yl)-1-

oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate

(24g)

The title compound was prepared from compound 17 (50 mg,

0.075 mmol) and 18g (50 mg, 0.15 mmol) following the general

protocol 1 to afford 24g as a gummy solid (50 mg, 49%).¹H NMR

2H), 1.29 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.9, 171.9,

166.9, 157.9, 155.7, 140.7, 140.5, 139.8, 138.4, 138.1, 136.5, 136.0,

134.8, 130.1, 129.8, 129.6, 129.5, 128.4, 127.8, 127.2, 126.6, 126.1,

124.1, 122.5, 121.4, 119.3, 118.9, 118.7, 118.3, 112.1, 111.7, 110.0, 79.6,

78.7, 56.4, 55.9, 52.9, 52.4, 40.5, 37.9, 28.6, 28.4, 24.1, 18.5, 12.1; IR

(600 MHz, DMSO-d₆): d 11.40 (s, 1H), 10.84 (s, 1H), 10.19 (s, 1H), 8.56

(ATR).nmax 2931, 1649, 1541, 1454, 1363, 1304, 1227, 1158, 1114,

(d, J ¼ 12.00 Hz, 1H), 8.33 (t, J ¼ 6.00 Hz, 1H), 8.18e8.14 (m, 3H),

7.91e7.89 (m, 2H), 7.75e7.89 (m, 2H), 7.74 (d, J ¼ 12.00 Hz, 1H),

7.69e7.62 (m, 2H), 7.56 (t, J ¼ 12.00 Hz, 1H), 7.43e7.40 (m, 2H), 7.32

(d, J ¼ 12.00 Hz,1H), 7.21e7.09 (m, 6H), 7.06 (t, J ¼ 6.00 Hz,1H), 6.95

(dd, J ¼ 12.00, 24.00 Hz, 2H), 6.78e6.66 (m, 2H), 6.56e6.36 (m, 2H),

4.75 (q, J ¼ 6.00 Hz, 1H), 4.43 (q, J ¼ 12.00 Hz, 1H), 4.21e4.15 (m,

1H), 3.75 (s, 3H), 3.27e3.05 (m, 8H), 2.94e2.90 (m, 1H), 2.74e2.68

(m, 1H), 2.57 (s, 3H), 2.49 (s, 3H), 2.00 (s, 3H), 1.80e1.53 (m, 4H),

1.41 (s, 9H), 1.38 (s, 9H), 1.28 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆):

1013; HRMS (ESI): m/z calcd for

1029.4466; found: 1029.4540.

C₅₅H₆₄N₈O₁₀

S

[M

þ

H]^þ:

4.3.31. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-3-

(1H-indol-3-yl)-1-oxo-1-((3'-(((S)-2,2,18,18-tetramethyl-4,9,16-

trioxo-3,17-dioxa-5,8,15-triazanonadecan-10-yl)carbamoyl)-[1,1⁰-

biphenyl]-3-yl)amino)propan-2-yl)amino)-1-oxo-3-phenylpropan-

2-yl)carbamate (24e)

d

172.4, 170.8, 166.6, 163.5, 157.9, 156.6, 156.0, 152.3, 140.5, 140.53,

The title compound was prepared from compound 17 (100 mg,

0.15 mmol) and 18e (58 mg, 0.15 mmol) following the general

protocol 1 to afford 24e as an off-white solid (106 mg, 70%); m.p.:

139.8, 138.4, 138.1, 136.5, 136.0, 135.1, 129.8, 129.6, 129.3, 128.4,

127.7, 126.6, 126.2, 124.1, 121.4, 118.9, 112.1, 111.7, 110.0, 83.3, 79.6,

78.7, 56.4, 55.9, 54.6, 53.6, 40.5, 38.3, 29.5, 28.5, 28.4, 28.0, 24.0,

131.9e132.4 ^ꢂC; ¹H NMR (600 MHz, DMSO-d₆):

d

10.84 (s, 1H), 10.19

18.4, 12; IR (ATR).nmax 3310, 2305, 2095, 1868, 1609, 1543, 1363,

(s, 1H), 8.53 (d, J ¼ 6.00 Hz, 1H), 8.13 (br s, 2H), 8.00 (t, J ¼ 6.00 Hz,

1H), 7.91e7.88 (m, 2H), 7.73 (d, J ¼ 12.00 Hz, 1H), 7.63e7.62 (m, 2H),

7.56 (t, J ¼ 6.00 Hz, 1H), 7.44e7.42 (m, 2H), 7.31 (d, J ¼ 12.00 Hz, 1H),

7.21e7.13 (m, 6H), 7.06e7.03 (m, 1H), 6.97e6.92 (m, 2H), 6.76e6.73

(m, 2H), 4.75 (q, J ¼ 6.00 Hz, 1H), 4.39e4.37 (m, 1H), 4.20e4.16 (m,

1H), 3.25e3.05 (m, 4H), 3.00e2.86 (m, 5H), 2.73e2.69 (m, 1H),

1.75e1.67 (m, 2H),1.34 (s, 9H),1.33 (s, 9H),1.28 (s, 9H),1.16e1.13 (m,

1304, 1246,1112, 740, 683; HRMS (ESI): m/z calcd for C₆₇H₈₆N₁₂O₁₃

S

[M þ H]^þ: 1299.6158; found: 1299.6229.

4.3.34. ¹H NMR and ¹³C NMR spectra of 3'-((S)-2-((S)-2-amino-3-

phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-N-(2-

aminoethyl)-[1,1⁰-biphenyl]-3-carboxamide (25a)

The title compound was prepared from compound 24a (80 mg,

0.101 mmol) following the general protocol 4 to afford 25a as a

white solid (38 mg, 65%); m.p.: 206.9e207.5 ^ꢂC; ¹H NMR (400 MHz,

2H), 0.96e0.94 (m, 2H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.4,

171.9, 170.8, 166.7, 156.1, 156.0, 155.7, 140.6, 140.5, 139.8, 138.4,

136.5, 135.3, 129.8, 129.6, 129.3, 128.4, 127.8, 127.2, 126.6, 126.2,

124.1, 122.5, 121.4, 118.9, 118.3, 111.7, 110.0, 78.7, 78.1, 77.7, 56.4, 54.6,

54.0, 56.4, 54.6, 54.0, 40.5, 39.3, 37.9, 31.8, 29.7, 28.7, 28.6, 28.5,

DMSO-d₆):

d

10.86 (d, J ¼ 4.00 Hz, 1H), 10.40 (s, 1H), 8.94 (t,

J ¼ 4.00 Hz, 1H), 8.60 (d, J ¼ 8.00 Hz, 1H), 8.27 (br s, 3H), 8.16 (br s,

1H), 7.84e7.83 (m, 2H), 7.76e7.67 (m, 5H), 7.55 (t, J ¼ 16.00 Hz, 1H),

7.32 (d, J ¼ 8.00 Hz, 1H), 7.20e7.05 (m, 6H), 7.02e6.98 (m, 3H),

4.77e4.76 (m, 2H), 3.70 (q, J ¼ 4.00 Hz, 1H), 3.54 (q, J ¼ 8.00 Hz, 1H),

3.20e3.17 (m,1H), 3.07e2.99 (m, 3H), 2.89e2.83 (m, 1H), 2.63e2.57

23.6; IR (ATR).nmax 3292, 2973, 1650, 1510, 1453, 1247, 1163, 1045,

856, 741; HRMS (ESI): m/z calcd for C₅₆H₇₂N₈O₁₀Na[M þ Na]^þ:

1039.5269; found: 1039.5261.

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

17

(m, 1H); ¹³C NMR (100 MHz, DMSO-d₆):

d

172.3, 170.9, 167.2, 164.7,

717.3435; found: 717.3501.

140.1,139.1,137.6,136.6,135.2,134.8,129.8,129.5,129.4,128.5,127.7,

127.5, 126.8, 125.5, 124.4, 121.4, 120.3, 119.1, 118.7, 111.8, 109.9, 55.3,

54.6, 39.1, 37.9, 28.6; IR (ATR).nmax 2919, 2848, 2250, 1654, 1537,

4.3.38. ¹H NMR and ¹³C NMR spectra of N-((S)-6-amino-1-((2-

aminoethyl)amino)-1-oxohexan-2-yl)-3'-((S)-2-((S)-2-amino-3-

phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-[1,1⁰-

biphenyl]-3-carboxamide (25e)

1397, 1299, 1173, 1067, 743; HRMS (ESI): m/z calcd for C₃₅H₃₆N₆O₃

[M þ H]^þ: 589.2849; found: 589.2911.

The title compound was prepared from compound 24e (90 mg,

0.088 mmol) following the general protocol 4 to afford 25e as a

4.3.35. ¹H NMR and ¹³C NMR spectra of 3'-((S)-2-((S)-2-amino-3-

phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-N-(2-

guanidinoethyl)-[1,1⁰-biphenyl]-3-carboxamide (25b)

The title compound was prepared from compound 24b (80 mg,

0.08 mmol) following the general protocol 4 to afford 25b as a

white gummy solid (27 mg, 49%).¹H NMR (600 MHz, DMSO-d₆):

gummy solid (45 mg, 49%).¹H NMR (600 MHz, DMSO-d₆):

d 10.91 (s,

1H), 10.36 (s, 1H), 8.97 (d, J ¼ 6.00 Hz, 1H), 8.68 (d, J ¼ 6.00 Hz, 1H),

8.24 (t, J ¼ 6.00 Hz,1H), 8.16e7.67 (m, 12H), 7.68 (d, J ¼ 6.00 Hz, 1H),

7.62e7.58 (m, 2H), 7.46e7.45 (m, 2H), 7.33 (d, J ¼ 12.00 Hz, 1H),

7.26e7.15 (m, 6H), 7.06 (t, J ¼ 6.00 Hz, 1H), 6.98 (t, J ¼ 6.00 Hz, 1H),

4.81 (q, J ¼ 6.00 Hz, 1H), 4.44e4.41 (m, 1H), 4.09 (t, J ¼ 6.00 Hz, 1H),

3.31e3.30 (m, 2H), 3.27 (dd, J ¼ 6.00, 12.00 Hz, 1H), 3.15e3.09 (m,

2H), 2.97 (dd, J ¼ 12.00, 15.00 Hz, 1H), 2.87 (t, J ¼ 6.00 Hz, 2H), 2.77

(br s, 2H), 1.84e1.81 (m, 1H), 1.76e1.72 (m, 1H), 1.59e1.52 (m, 2H),

d

10.89 (br s, 1H), 10.35 (br s, 1H), 8.94 (d, J ¼ 4.00 Hz, 1H), 8.75 (t,

J ¼ 4.00 Hz,1H), 8.11e7.93 (m, 4H), 7.87e7.85 (m, 1H), 7.77e7.75 (m,

1H), 7.68 (d, J ¼ 8.00 Hz, 1H), 7.63e7.58 (m, 4H), 7.46e7.42 (m, 4H),

7.26e7.15 (m, 6H), 7.08e6.97 (m, 4H), 4.82 (q, J ¼ 4.00 Hz, 1H), 4.07

(t, J ¼ 4.00 Hz, 1H), 3.43 (q, J ¼ 4.00 Hz, 2H), 3.28e3.24 (m, 1H),

3.14e3.08 (m, 2H), 2.97e2.93 (m, 1H); ¹³C NMR (150 MHz, DMSO-

1.42e1.32 (m, 2H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.8, 170.3,

168.5, 166.9, 158.5, 158.3, 140.5, 139.8, 136.6, 135.1, 135.12, 130.0,

129.9, 129.4, 128.8, 127.7, 127.5, 127.2, 126.2, 124.2, 122.6, 121.5,

119.3, 118.9, 118.7, 118.3, 118.8, 109.7, 55.03, 53.8, 53.7, 40.5, 39.5,

d₆): d 169.8,166.6,158.1,157.9,156.9,140.2,140.0,139.3,136.0,134.8,

129.5, 129.4129.41, 129.1, 128.4, 127.2, 127.0, 126.3, 125.5, 123.7,

122.1, 121.0, 118.9, 118.4, 118.3, 117.8, 111.4, 109.2, 87.8, 54.5, 53.3,

39.2, 39.9, 37.4, 36.9, 31.3, 28.4, 27.1, 23.2; IR (ATR).nmax 3274,

40.3, 38.6, 37.1, 28.0; IR (ATR).

n

max 3276, 3189, 3064, 1651, 1538,

3056, 2926, 2119, 1661, 1526, 1431, 1200, 1121, 836, 797, 743, 721;

HRMS (ESI): m/z calcd for C₄₁H₄₈N₈O₄[M þ H]^þ: 717.3435; found:

717.3501.

1433, 1318, 1201, 1150, 836, 798, 742 HRMS (ESI): m/z calcd for

C

₃₆H₃₈N₈O₃[M þ H]^þ: 631.3067; found: 631.3141.

4.3.36. ¹H NMR and ¹³C NMR spectra of Methyl (3'-((S)-2-((S)-2-

amino-3-phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-

4.3.39. ¹H NMR and ¹³C NMR spectra of N-((S)-6-amino-1-((2-

guanidinoethyl)amino)-1-oxohexan-2-yl)-3'-((S)-2-((S)-2-amino-

3-phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-[1,1⁰-

biphenyl]-3-carboxamide (25f)

[1,1⁰-biphenyl]-3-carbonyl)-

-lysinate (25c)

L

The title compound was prepared from compound 24c (90 mg,

0.112 mmol) following the general protocol 4 to afford 25c as a

white gummy solid (46 mg, 60%).¹H NMR (600 MHz, DMSO-d₆):

The title compound was prepared from compound 24f (100 mg,

0.086 mmol) following the general protocol 4 to afford 25f as a

d

10.89 (br s, 1H), 10.35 (s, 1H), 8.94 (d, J ¼ 4.00 Hz, 1H), 8.87 (d,

gummy solid (41 mg, 40%).¹H NMR (600 MHz, DMSO-d₆):

d 10.90 (d,

J ¼ 8.00 Hz, 1H), 8.13e7.90 (m, 5H), 7.79e7.68 (m, 5H), 7.63e7.59

(m, 2H), 7.46e7.44 (m, 2H), 7.33 (d, J ¼ 4.00 Hz, 1H), 7.26e7.15 (m,

6H), 7.07e7.05 (m, 1H), 6.99e6.97 (m, 1H), 4.81 (q, J ¼ 4.00 Hz, 1H),

4.49e4.45 (m, 1H), 4.07 (t, J ¼ 4.00 Hz, 1H), 3.66 (s, 3H), 3.28e3.24

(m, 1H), 3.14e3.09 (m, 2H), 2.99e2.94 (m, 1H), 2.79e2.77 (m, 2H),

1.85e1.80 (m, 2H), 1.60e1.53 (m, 2H), 1.47e1.38 (m, 2H); ¹³C NMR

J ¼ 6.00 Hz, 1H), 10.35 (s, 1H), 8.93 (br s, 1H), 8.65 (d, J ¼ 6.00 Hz,

1H), 8.20e8.13 (m, 4H), 7.96e7.91 (m, 2H), 7.84e7.53 (m, 7H),

7.51e7.43 (m, 2H), 7.37e7.10 (m, 8H), 7.10e7.04 (m, 1H), 7.00e6.97

(m, 1H), 6.54 (br s, 1H), 4.82 (q, J ¼ 6.00 Hz, 1H), 4.47e4.44 (m, 1H),

4.06 (br s, 1H), 3.46e3.39 (m, 2H), 3.27e2.77 (m, 8H), 1.91e1.24 (m,

6H); ¹³C NMR (150 MHz, DMSO-d₆):

d 172.9, 170.3, 166.7, 158.5,

(150 MHz, DMSO-d₆):

d

173.1, 170.3, 168.6, 167.0, 158.5, 158.3, 140.6,

158.3, 157.4, 140.5, 139.7, 136.5, 135.1, 130.0, 129.9, 129.4, 128.9,

127.7, 127.5, 126.3, 124.2, 123.4, 122.63, 122.6, 121.5, 119.4, 118.9,

118.7, 118.4, 118.3, 116.7, 111.8, 109.7, 54.9, 54.0, 53.7, 40.8, 40.5, 39.2,

140.5, 139.9, 136.6, 135.2, 134.8, 130.0, 130.0, 129.5, 128.9, 127.7,

127.5, 127.1, 126.2, 124.2, 122.6, 121.5, 119.4, 118.9, 118.8, 118.3, 111.8,

109.7, 55.0, 53.7, 52.9, 52.4, 39.1, 37.6, 30.4, 28.5, 27.0, 23.1; IR

38.4, 31.4, 28.5, 27.2, 23.2; IR (ATR).nmax 3265, 3065, 1655, 1537,

(ATR).

n

max 3276, 3195, 3081, 2109, 1540,1433, 1318,1198, 798, 743;

1428, 1170, 1128, 1001,787; HRMS (ESI): m/z calcd for C₄₂H₅₀N₁₀O₄

HRMS (ESI): m/z calcd for C₄₀H₄₄N₆O₅[M þ H]^þ: 689.3373; found:

[M þ H]^þ: 759.4017; found: 759.4084.

689.3448.

4.3.40. ¹H NMR and ¹³C NMR spectra of 3'-((S)-2-((S)-2-amino-3-

phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-N-((S)-5-

guanidino-1-((2-guanidinoethyl)amino)-1-oxopentan-2-yl)-[1,1⁰-

biphenyl]-3-carboxamide (25g)

4.3.37. ¹H NMR and ¹³C NMR spectra of Methyl (3'-((S)-2-((S)-2-

amino-3-phenylpropanamido)-3-(1H-indol-3-yl)propanamido)-

[1,1⁰-biphenyl]-3-carbonyl)-

-argininate (25d)

L

The title compound was prepared from compound 24d (90 mg,

0.087 mmol) following the general protocol 4 to afford 25d as a

brown gummy solid (25 mg, 40%).¹H NMR (600 MHz, DMSO-d₆):

The title compound was prepared from compound 24g (48 mg,

0.036 mmol) following the general protocol 4 to afford 25g as a

gummy solid (20 mg, 40%).¹H NMR (600 MHz, DMSO-d₆):

d 10.84 (s,

d

10.89 (s, 1H), 10.35 (s, 1H), 8.94 (dd, J ¼ 4.00, 12.00 Hz, 2H),

1H),10.27 (s,1H), 8.84 (s,1H), 8.62 (d, J ¼ 6.00 Hz,1H), 8.16e8.11 (m,

2H), 7.94 (d, J ¼ 6.00 Hz, 1H), 7.76 (d, J ¼ 12.00 Hz, 1H), 7.67 (d,

J ¼ 12.00 Hz, 1H), 7.62e7.56 (m, 3H), 7.46 (d, J ¼ 6.00 Hz, 2H), 7.34

(d, J ¼ 12.00 Hz, 1H), 7.28e7.14 (m, 8H), 7.08e7.06 (m, 2H),

7.01e6.97 (m, 1H), 4.82 (q, J ¼ 6.00 Hz, 1H), 4.51e4.46 (m, 1H), 4.07

(br s, 1H), 3.25e3.19 (m, 6H), 3.15e3.12 (m, 3H), 2.96e2.94 (m, 1H),

1.89e1.84 (m, 1H), 1.76e1.72 (m, 1H), 1.60e1.50 (m, 2H); ¹³C NMR

8.13e8.12 (m, 4H), 7.93e7.90 (m, 2H), 7.79e7.78 (m, 1H), 7.69 (d,

J ¼ 8.00 Hz,1H), 7.64e7.60 (m, 3H), 7.48e7.33 (m, 4H), 7.27e7.16 (m,

7H), 7.08e7.06 (m, 1H), 7.00e6.98 (m, 1H), 4.82 (q, J ¼ ꢀ4.00 Hz,

1H), 4.52e4.49 (m, 1H), 4.10 (br s, 1H), 3.67 (s, 3H), 3.29e3.25 (m,

1H), 3.15e3.09 (m, 4H), 2.99e2.95 (m, 1H), 1.91e1.54 (m, 4H); ¹³C

NMR (150 MHz, DMSO-d₆): d 172.9, 170.3, 168.4, 166.9, 158.2, 157.2,

140.6, 140.4, 139.8, 136.6, 135.1, 134.8, 130.1, 130.0, 129.9, 129.5,

128.9,129.6,128.9,127.7, 126.1, 124.2, 122.6, 121.5, 119.4,118.9, 118.9,

118.3, 111.8, 109.7, 55.0, 53.7, 52.8, 52.4, 40.7, 40.5, 37.5, 28.5, 28.1,

(150 MHz, DMSO-d₆): d 172.6, 170.3, 166.8, 158.6, 158.4, 158.2, 157.5,

157.2, 140.6, 140.5, 139.8, 136.6, 135.1, 130.0, 130.0, 129.8, 129.4,

128.8, 128.8, 127.7, 127.4, 127.2, 126.2, 124.1, 122.6, 121.4, 119.4, 118.9,

118.7, 118.4, 116.8, 111.8, 109.8, 55.0, 53.5, 40.98, 40.9, 40.6, 38.4,

25.8; IR (ATR).nmax 3272, 3191, 3063, 2103, 1529, 1434, 1340, 1029,

835, 798, 744, 720; HRMS (ESI): m/z calcd for C₄₀H₄₄N₈O₅[M þ H]^þ:

29.1, 28.4, 25.8; IR (ATR).nmax 3279, 3186, 3086, 1646, 1529, 1431,

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

18

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

1175, 1125, 798, 743; HRMS (ESI): m/z calcd for C₄₂H₅₀N₁₂O₄[M þ

1351, 1290, 1222, 1104, 1012, 945, 890, 805, 737; HRMS (ESI): m/z

calcd for C₁₂H₁₄N₂O₂[M þ H]^þ: 219.1055; found: 219.1125.

H]^þ: 787.4078; found: 787.4152.

13

4.3.41. ¹H NMR and C NMR spectra of 3⁰-nitro-[1,1⁰-biphenyl]-3-

4.3.45. ¹H NMR and ¹³C NMR spectra of Methyl

L-tryptophanate

carboxylic acid (26)

(31)

To the solution of 9 (1.2 g, 3.88 mmol) in THF (10.0 mL) and

MeOH (10.0 mL), was added a 1N NaOH_(aq)(7.77 mL, 7.77 mmol)

and stirred at room temperature for 16 h. Ethyl acetate was added

and the layers were separated. The aqueous layer was then acidiﬁed

with 1N HCl and then extracted with CH₂Cl₂(2 ꢁ 100 mL) and then

the solvent was removed under reduced pressure to yield 26

(0.84 g, 90%) as a pale-yellow solid; m.p.: 233.0e233.6 ^ꢂC; ¹H NMR

The title compound 31 was prepared by adding thionyl tem-

perature for 16 h. Ethyl acetate was added and the layers were

separated. The aqueous layer was then acidiﬁed with 1N HCl and

then extracted with CH₂Cl₂(2 ꢁ 100 mL) and then the solvent was

removed under reduced pressure to yield 26 (0.84 g, 90%) as a pale-

yellow solid; m.p.: 209.4e210.6 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d

13.19 (br s,1H), 8.44 (s, 1H), 8.25 (br s, 2H), 8.18 (d, J ¼ 7.60 Hz,1H),

(400 MHz, DMSO-d₆):

8.18 (d, J ¼ 7.60 Hz, 1H), 8.02 (t, J ¼ 8.00 Hz, 1H), 7.78 (t, J ¼ 8.00 Hz,

1H); ¹³C NMR (100 MHz, DMSO-d₆):

167.5, 148.9, 141.3, 138.6,

133.9, 132.2, 131.9, 131.1, 130.1, 129.8, 128.1, 123.1, 121.8; IR (ATR).

d

13.19 (br s, 1H), 8.44 (s, 1H), 8.25 (br s, 2H),

8.02 (t, J ¼ 8.00 Hz, 1H), 7.78 (t, J ¼ 8.00 Hz, 1H); ¹³C NMR (100 MHz,

DMSO-d₆): d 167.5, 148.9,141.3,138.6,133.9,132.2,131.9,131.1,130.1,

d

129.8, 128.1, 123.1, 121.8; IR (ATR).nmax 2965, 2840, 2541, 2099,

n

1681, 1590, 1514, 1421, 1189, 1095, 928, 853; HRMS (ESI): m/z calcd

max 2965, 2840, 2541, 2099, 1681, 1590, 1514, 1421, 1189, 1095, 928,

853; HRMS (ESI): m/z calcd for C₁₃H₉NO₄Na [M þ Na]^þ: 266.0429;

found: 266.0424.

for C₁₃H₉NO₄Na [M þ Na]^þ: 266.0429; found: 266.0424.

4.3.46. ¹H NMR and ¹³C NMR spectra of Methyl (tert-

butoxycarbonyl)-

The title compound was prepared via protocol 1, using N-(tert-

Butoxycarbonyl)- -phenylalanine (1.21 g, 4.6 mmol) and 31 (1.00 g,

L-phenylalanyl-L-tryptophanate (32)

4.3.42. ¹H NMR and ¹³C NMR spectra of N-(2-(2,3-dibocguanidino)

ethyl)-3⁰-nitro-[1,1⁰-biphenyl]-3-carboxamide (27)

L

The title compound 27 was prepared from compound 18b

(0.621 g, 2.00 mmol) and 33 (0.5 g, 2.00 mmol) according to the

protocol 2. Off-white solid (0.47 g, 45%); m.p.: 117.6e118.9 ^ꢂC; ¹H

4.6 mmol) to afford the coupled product 32 as an off-white solid

(1.40 g, 65%); m.p.: 135.3e135.7 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

d

10.89 (s, 1H), 8.29 (d, J ¼ 8.00 Hz,1H), 7.49 (d, J ¼ 8.00 Hz, 1H), 7.34

NMR (400 MHz, CDCl₃):

d

11.51 (br s, 1H), 8.83e8.82 (m, 1H),

(d, J ¼ 8.00 Hz, 1H), 7.23e7.18 (m, 6H), 7.07 (t, J ¼ 8.00 Hz, 1H), 6.99

(t, J ¼ 8.00 Hz, 1H), 6.87 (d, J ¼ 12.00 Hz, 1H), 4.55 (q, J ¼ 8.00 Hz,

1H), 4.23e4.18 (m, 1H), 3.55 (s, 3H), 3.20e3.07 (m, 2H), 2.95e2.90

(m, 1H), 2.72e2.66 (m, 1H), 1.28 (s, 9H); ¹³C NMR (100 MHz, DMSO-

8.26e8.16 (m, 3H), 8.00e7.97 (m, 1H), 7.84e7.82 (m, 1H), 7.66e7.63

(m, 1H), 7.56 (t, J ¼ 8.00 Hz, 1H), 3.80e3.77 (m, 2H), 3.71e3.67 (m,

2H), 1.51 (s, 9H), 1.49 (s, 9H); ¹³C NMR (100 MHz, CDCl₃):

d 167.1,

162.7, 157.9, 153.0, 148.8, 142.0, 139.1, 135.4, 133.2, 129.9, 129.8,

129.1, 126.9, 126.3, 122.4, 122.0, 83.8, 79.9, 79.7, 42.5, 39.7, 28.3,

d₆):

124.2, 121.4, 118.9, 118.4, 111.8, 109.6, 78.5, 56.0, 53.5, 52.2, 37.8,

28.5, 28.2, 27.5; IR (ATR). max 3295, 3001, 2952, 1694, 1648, 1525,

d 172.6, 172.2, 155.6, 138.5, 136.5, 129.6, 128.4, 127.5, 126.6,

28.2, 28.0; IR (ATR).

n

max 3196, 2985, 2930, 1784, 1719, 1532, 1473,

n

1268, 1139, 869; HRMS (ESI): m/z calcd for C₄₁H₅₁N₅O₈[M þ Na]^þ:

1433, 1288, 1164, 1112, 922, 846; HRMS (ESI): m/z calcd for

550.2278; found: 550.2273.

C

₂₆H₃₁N₃O₅[M þ H]^þ: 465.23; found: 337.14340.

13

4.3.43. ¹H NMR and C NMR spectra of 3⁰-amino-N-(2-

4.3.47. ¹H NMR and ¹³C NMR spectra of (tert-butoxycarbonyl)-

phenylalanyl- -tryptophan (33)

L-

guanidinoethyl)-[1,1⁰-biphenyl]-3-carboxamide (28)

L

The title compound 28 was prepared from 27 (0.25 g,

0.47 mmol) according to the protocol 4. Pale brown solid (139 mg,

90% yield); m.p.: 183.8e184.5 ^ꢂC; ¹H NMR (400 MHz, DMSO-d₆):

To a solution of 31 (1.4 g, 3.00 mmol) in THF (10.0 mL) and MeOH

(10.0 mL), was added a 1N NaOH_(aq)(6.0 mL, 6.00 mmol) and stirred

at room temperature for 16 h. Ethyl acetate was added and the

layers were separated. The aqueous layer was then acidiﬁed with

1N HCl and then extracted with CH₂Cl₂(2 ꢁ 100 mL) and then the

solvent was removed under reduced pressure to yield 33 (1.23 g,

91%) as an off-white solid; m.p.: 133.2e134.0 ^ꢂC; ¹H NMR

d

8.93 (t, J ¼ 5.20 Hz, 1H), 8.54 (t, J ¼ 2.00 Hz, 1H), 8.28e8.25 (m,

3H), 7.98 (t, J ¼ 7.60 Hz, 2H), 7.83e7.75 (m, 2H), 7.65 (t, J ¼ 8.00 Hz,

1H), 7.26 (br s, 3H), 3.48e3.43 (m, 2H), 3.39e3.35 (m, 2H); ¹³C NMR

(100 MHz, DMSO-d₆):

131.1,130.3,129.8,128.0,126.1,123.0,121.7, 40.7, 39.1; IR (ATR).

d

166.8, 157.5, 148.9, 141.5, 138.3, 135.5, 133.9,

max

n

(400 MHz, DMSO-d₆):

d

10.89 (s, 1H), 8.08 (d, J ¼ 8.00 Hz, 1H), 7.54

3280, 3155, 2341, 2112, 1638, 1522, 1457, 1344, 1299, 1192, 1114, 996,

864, 798, 675, 730; HRMS (ESI): m/z calcd for C₁₆H₁₇N₅O₃[M þ H]^þ:

327.1331; found: 328.1401.

(d, J ¼ 8.00 Hz, 1H), 7.33 (d, J ¼ 8.00 Hz, 1H), 7.24e7.17 (m, 6H),

7.08e7.04 (m, 1H), 7.00e6.96 (m, 1H), 6.87 (d, J ¼ ꢀ8.00 Hz, 1H),

4.51 (q, J ¼ 8.00 Hz, 1H), 4.21e4.15 (m, 1H), 3.25e3.17 (m, 1H),

3.11e3.06 (m, 1H), 2.96e2.92 (m, 1H), 2.71e2.65 (m, 1H), 1.28 (s,

13

4.3.44. ¹H NMR and C NMR spectra of 3⁰-amino-N-(2-

9H); ¹³C NMR (100 MHz, DMSO-d₆):

d

173.6, 172.1, 155.6, 138.5,

136.5, 129.6, 128.4, 127.7, 126.5, 124.1, 121.3, 118.8, 118.6, 111.8, 110.3,

110.0, 78.5, 56.1, 53.3, 37.9, 31.4, 28.5, 27.5, 22.5, 14.4; IR (ATR). max

guanidinoethyl)-[1,1⁰-biphenyl]-3-carboxamide (29)

To a stirred solution of 28 (0.1 g, 0.305 mmol) in anhydrous-

n

chloride (0.79 mL, 9.80 mmol) dropwise to

L

-Tryptophan (1.0 g,

3310, 3046, 2926, 1650, 1509, 1436, 1364, 1247, 1158, 1011, 848;

HRMS (ESI): m/z calcd for C₂₅H₂₉N₃O₅Na[M þ H]^þ: 474.2005;

found: 474.1998.

4.90 mmol) in methanol (20.0 mL) at 0 ^ꢂC and then stirred at room

temperature for 16 h. The solvents were removed and reduced

pressure and the residue was diluted with ethylacetate (100.0 mL)

and washed with saturated NaHCO₃(40 mL), saturated brine

(40 mL), then dried (Na₂SO₄) and concentrated under reduced

pressure to afford a white solid (1.00 g, 94%); m.p.: 89.4e89.9 ^ꢂC; ¹H

4.3.48. ¹H NMR and ¹³C NMR spectra of tert-butyl ((S)-1-(((S)-1-

((2-(2,3-dibocguanidino)ethyl)amino)-3-(1H-indol-3-yl)-1-

oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate

(34)

The title compound 34 was prepared from compound 18b

(0.333 g, 1.1 mmol) and 33 (0.5 g, 1.1 mmol) according to the pro-

tocol 2. Off-white solid (0.36 g, 45%); m.p.: 180.6e181.2 ^ꢂC; ¹H NMR

NMR (400 MHz, DMSO-d₆):

d

10.87 (s, 1H), 7.48 (d, J ¼ 8.00 Hz, 1H),

7.33 (d, J ¼ 8.00 Hz, 1H), 7.12 (br s, 1H), 7.05 (t, J ¼ 8.00 Hz, 1H), 6.97

(t, J ¼ 8.00 Hz, 1H), 3.63 (t, J ¼ 8.00 Hz, 1H), 3.55 (s, 1H), 3.05e2.91

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆):

d

176.1, 136.5, 127.8, 124.1,

max

3356, 3292, 3096, 3051, 2916, 2871, 2338, 2095, 1727, 1567, 1448,

121.3, 118.8, 118.7, 111.8, 110.3, 55.6, 51.8, 40.6, 31.1; IR (ATR).

n

(400 MHz, DMSO-d₆):

8.37 (t, J ¼ 4.00 Hz, 1H), 8.13 (t, J ¼ 4.00 Hz, 1H), 7.91 (d, J ¼ 8.00 Hz,

d

11.47 (br s, 1H), 10.80 (d, J ¼ 4.00 Hz, 1H),

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R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

19

1H), 7.57 (d, J ¼ 8.00 Hz, 1H), 7.30 (d, J ¼ 8.00 Hz, 1H), 7.20e7.06 (m,

6H), 7.07e7.02 (m, 1H), 6.98e6.94 (m, 1H), 6.88 (d, J ¼ 8.00 Hz, 1H),

4.48 (q, J ¼ 4.00 Hz, 1H), 4.15e4.09 (m, 1H), 3.27e2.95 (m, 6H),

2.90e2.85 (m, 1H), 2.69e2.63 (m, 1H), 1.44 (s, 9H), 1.38 (s, 9H), 1.28

d 172.0, 171.6, 163.5, 156.0,

155.6, 152.3, 138.5, 136.5, 129.6, 128.4, 127.8, 126.6, 124.0, 121.3,

118.9, 118.6, 111.7, 110.3,83.3, 78.7, 78.6, 56.4, 53.8, 38.5, 37.9, 37.8,

2% sodium bicarbonate, 1% non-essential amino acids (NEAA) and

1% sodium pyruvate. The cell line was maintained at 37 ^ꢂC in 5% CO₂

as an adherent monolayer and was passaged upon reaching

conﬂuence by standard cell culture techniques. MRC-5 cells were

seeded at 2 ꢁ 10⁴cells per well in 96-well plates to ensure full

conﬂuence (quiescence). Cells were treated 24 h after seeding with

(s, 9H); ¹³C NMR (100 MHz, DMSO-d₆):

0.1e1000 mM of compounds. After 72 h drug incubation, the treated

28.6, 28.5, 28.4, 28.1; IR (ATR).

n

max 2974, 1637, 1523, 1363, 1248,

media was replaced with fresh media containing 10% Alamar blue

and the cells were incubated for another 6 h. The metabolic activity

was detected by spectrophotometric analysis by assessing the

absorbance of Alamar blue as previously described by Pasquier et al.

[65] Cell proliferation was determined and expressed as a per-

centage of untreated control cells. The determination of IC₅₀values

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

experiment was performed in triplicate and was repeated in three

independent experiments.

1132, 1021; HRMS (ESI): m/z calcd for C₃₈H₅₃N₇O₈[M þ H]^þ:

736.3956; found: 736.4023.

4.3.49. ¹H NMR and ¹³C NMR spectra of (S)-2-amino-N-((S)-1-((2-

guanidinoethyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-3-

phenylpropanamide (35 TFA salt)

The title compound was prepared via protocol 4, using 34

(0.150 g, 0.203 mmol) to yield the desired product 35 as a brown

gummy solid (40 mg, 47%). ¹H NMR (400 MHz, DMSO-d₆):

d 10.87

(br s,1H), 8.81 (d, J ¼ 8.00 Hz,1H), 8.30 (t, J ¼ 4.00 Hz,1H), 8.06 (br s,

3H), 7.64e7.62 (m, 2H), 7.34e7.24 (m, 9H), 7.16 (d, J ¼ 4.00 Hz, 1H),

7.08e7.04 (m, 1H), 7.00e6.96 (m, 1H), 4.58e4.52 (m, 1H), 4.03 (br s,

4.6. Tethered bilayer lipid membranes

The ability of the biphenyl derivatives to interact with lipid bi-

layers was tested using tethered bilayer lipid membranes (tBLMs)

in association with AC electrical impedance spectroscopy tech-

niques [55,56]. Lipid bilayers were anchored to a gold substrate

which were created using the solvent exchange technique

described previously [56,57]. In short, tethered benzyl-disulﬁde

1H), 3.26e2.88 (m, 8H); ¹³C NMR (100 MHz, DMSO-d₆):

d

172.0,

168.3, 157.5, 136.6, 135.2, 130.0, 129.0, 127.64, 127.6, 124.3, 121.4,

118.9, 118.7, 111.8, 110.0, 54.2, 53.6, 38.5, 37.5, 28.6; IR (ATR). max

n

3060, 2919, 1670, 1542, 1421, 1199, 1128, 1002, 825; HRMS (ESI): m/z

calcd for C₂₃H2₉N₇O₂[M þ H]^þ: 436.2383; found: 436.2451.

(tetra-ethyleneglycol)

n

¼

2C20-phytanyl tethers: benzyl-

4.4. Antibacterial activity

disulﬁde-tetra-ethyleneglycol-OH spacers were pre-prepared in

the ratio of 1:10 and were coated onto a gold patterned poly-

carbonate slide containing a large gold return electrode (SDx

Tethered Membranes Pty Ltd, Australia). Attached to this slide was a

specialised cartridge chamber which allows for easy addition of

reagent between tethering and reference electrodes. A 3 mM so-

lution of a standard mobile lipid phase [70% zwitterionic C20

The antimicrobial activity of compounds was evaluated through

the broth micro dilution assay using the procedure described by

CLSI [64]. Brieﬂy, Staphylococcus aureus [SA38] were grown to mid-

log phase in Muller-Hinton broth (MHB) with shaking at 120 rpm

and incubated at 37 ^ꢂC for 18e24 h. Following incubation, bacteria

were washed three times using PBS pH 7.4 with centrifugation at

3500 g for 10 min after each wash. After washing, bacteria were

diluted with fresh MHB. The turbidity of the bacterial suspensions

were adjusted so that OD_600nmwas 0.1, which gave 1 ꢁ 10⁸cfu/mL,

and then further diluted to achieve 1e2 ꢁ 10⁵cfu/ml as a ﬁnal

bacterial concentration. Each compound was added at concentra-

Diphytanyl-Glycero-Phosphatidylcholine

lipid:

30%

C20

Diphytanyl-diglyceride-OH ether] was added to the tethering

chemistries. All lipids were dissolved in 100% ethanol. Lipids were

left for 2 min to associate with the tethering chemistries before

being washed with 3 ꢁ 200 mL phosphate buffered saline (PBS).

The membranes formed from these lipids are designated as the

“zwitterionic membranes”. Alternatively, negatively charged

membranes, that resemble those present in many bacterial species,

were produced using the same mobile lipid phase which was

tions ranging from 250 to 3.9

mM through serial two-fold dilution.

Wells in microtitre plates were loaded with 100

ml of inoculum

containing 1e2 ꢁ 10⁵cfu/mL bacteria. Wells containing only bac-

teria and without any compound were used as a negative controls

(i.e. no inhibition of growth). Wells without bacteria but containing

compound acted as another control. The microtitre plate was

wrapped with parafﬁn to prevent evaporation and incubated with

shaking at 120 rpm and 37 ^ꢂC for 18e24 h. After incubation,

spectrophotometric reading of the wells was taken at 600 nm. The

minimal inhibitory concentration (MIC) was deﬁned as the lowest

concentration of the biphenyl compounds that causes 100% inhi-

bition of microbial growth. The same procedure was followed for

the two Gram-negative bacteria, Pseudomonas aeruginosa [PA01],

and Escherichia coli [K12]. Each experiment was performed in

triplicate and was repeated in three independent experiments. The

MICs of the compounds were compared to published MIC of MSI-78

[15,52,53]. The MIC of MSI-78 [52,53], was determined according to

the CLSI (Clinical and Laboratory Standards Institute) guidelines

and the same experimental conditions were used to determine the

MIC for compounds in this study.

instead

supplemented

with

30%

palmitoyl-oleoyl-

phosphatidylglycerol (POPG) (Avanti Lipids, USA). The presence of

a lipid bilayer was veriﬁed using AC electrical impedance spec-

trometry. These measures were done using a TethaPod™ operated

with TethaQuick™ software (SDx Tethered Membranes Pty Ltd,

Australia). The signal employed was 50 mV peak-to-peak AC exci-

tation at 0.1e2000 Hz with four steps per decade. Impedance data

were ﬁtted to a constant phase element (CPE) in series with a

resistor/capacitor. The CPE represents the capacitance of the gold

electrode interface, whist the resistor and capacitor represent the

resistance and capacitance of the tethered membrane respectively.

Fitting was done using a proprietary adaptation of a Lev Mar ﬁtting

routine as described previously [56]. All tethered membrane assays

were performed in triplicate. Individual results shown are of typical

responses to the antimicrobial compounds.

4.7. Cytoplasmic membrane permeability assay

4.5. Toxicity assay

The method was adopted from Wu et al. [66] with slight

modiﬁcation. Bacterial cytoplasmic membrane permeability was

determined using membrane potential sensitive dye diSC3-5 (3,3⁰-

dipropylthiadicarbocyanine iodide) which penetrates inside bac-

terial cells depending on the membrane potential gradient of the

Normal human lung ﬁbroblasts MRC-5 were cultured in mini-

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

foetal calf serum (FCS), 1% L-glutamineepenicillinestreptomycin,

Please cite this article in press as: R. Kuppusamy, et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl

backbone as antibacterial agents, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.066

20

R. Kuppusamy et al. / European Journal of Medicinal Chemistry xxx (2017) 1e21

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were washed with 5 mM HEPES containing 20 mM glucose pH (7.2)

and resuspended in the same buffer to an OD₆₀₀0.05e0.06 which

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mM to the bacterial

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Acknowledgments

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 Australian

Research Council grant (DP140102195 and DP160101664). Rajesh

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

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

Allowance Scholarship. We declare that Bruce Cornell is a share-

holder of SDx Tethered Membranes Pty Ltd.

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Appendix A. Supplementary data

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