Design, synthesis and biological evaluation of biphenylglyoxamide-based small molecular antimicrobial peptide mimics as antibacterial agents

Article abstract of DOI:10.3390/ijms21186789

There has been an increasing interest in the development of antimicrobial peptides (AMPs) and their synthetic mimics as a novel class of antibiotics to overcome the rapid emergence of antibiotic resistance. Recently, phenylglyoxamide-based small molecular

Full text of DOI:10.3390/ijms21186789

Article
Design, Synthesis and Biological Evaluation
of Biphenylglyoxamide-Based Small Molecular
Antimicrobial Peptide Mimics
as Antibacterial Agents
Tsz Tin Yu 1, Rajesh Kuppusamy 1, Muhammad Yasir 2, Md. Musfizur Hassan 1,
Amani Alghalayini 3, Satyanarayana Gadde 1, Evelyne Deplazes 3, Charles Cranfield 3,
Mark D. P. Willcox 2, David StC Black 1,* and Naresh Kumar 1,*
1
School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia;
tsztin.yu@unsw.edu.au (T.T.Y.); r.kuppusamy@unsw.edu.au (R.K.); md.m.hassan@unsw.edu.au (M.M.H.);
s.gadde@unsw.edu.au (S.G.)
2
School of Optometry and Vision Science, University of New South Wales, Sydney, NSW 2052, Australia;
m.yasir@unsw.edu.au (M.Y.); m.willcox@unsw.edu.au (M.D.P.W.)
3
School of Life Sciences, University of Technology Sydney, PO Box 123, Ultimo 2007, Australia;
amani.alghalayini@uts.edu.au (A.A.); evelyne.deplazes@uts.edu.au (E.D.);
charles.cranfield@uts.edu.au (C.C.)
* Correspondence: d.black@unsw.edu.au (D.S.B.); n.kumar@unsw.edu.au (N.K.)
Received: 28 August 2020; Accepted: 14 September 2020; Published: 16 September 2020
Abstract: There has been an increasing interest in the development of antimicrobial peptides
(AMPs) and their synthetic mimics as a novel class of antibiotics to overcome the rapid emergence
of antibiotic resistance. Recently, phenylglyoxamide-based small molecular AMP mimics have been
identified as potential leads to treat bacterial infections. In this study, a new series of
biphenylglyoxamide-based small molecular AMP mimics were synthesised from the ring-opening
reaction of N-sulfonylisatin bearing a biphenyl backbone with a diamine, followed by the
conversion into tertiary ammonium chloride, quaternary ammonium iodide and guanidinium
hydrochloride salts. Structure–activity relationship studies of the analogues identified the
octanesulfonyl group as being essential for both Gram-positive and Gram-negative antibacterial
activity, while the biphenyl backbone was important for Gram-negative antibacterial activity. The
most potent analogue was identified to be chloro-substituted quaternary ammonium iodide salt 15c,
which possesses antibacterial activity against both Gram-positive (MIC against Staphylococcus aureus
= 8 μM) and Gram-negative bacteria (MIC against Escherichia coli = 16 μM, Pseudomonas aeruginosa =
63 μM) and disrupted 35% of pre-established S. aureus biofilms at 32 μM. Cytoplasmic membrane
permeability and tethered bilayer lipid membranes (tBLMs) studies suggested that 15c acts as a
bacterial membrane disruptor. In addition, in vitro toxicity studies showed that the potent
compounds are non-toxic against human cells at therapeutic dosages.
Keywords: 5-phenylisatin; antimicrobial peptide mimics; biphenylglyoxamide; quaternary
ammonium iodide; guanidinium hydrochloride; membrane disruption; antibiofilm
1. Introduction
Rapid emergence of antibiotic-resistant bacteria is a major global health concern. The Gram-
positive Staphylococcus aureus often has a high antibiotic-resistant rate, with approximately 65–85% of
nosocomial S. aureus infections associated with a beta-lactam-resistant strain [1,2]. When taking other
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bacterial pathogens into account, there are more than 2.8 million individuals seriously infected by
bacteria in the United States every year, and 700,000 individuals are killed by drug-resistant bacteria
around the world annually [3,4]. Since the late 1980s, there have been no new classes of antibiotics
entering the market, with new clinical drugs merely being the structural derivatives of existing
antibiotics with the same scaffolds [5–7]. These derivatives are prone to rapid resistance development
and so can only retard but not overcome the bacterial resistance dilemma. Hence, there is an urgent
need to develop novel classes of antibiotics to treat bacterial infection.
Another challenge encountered in battling bacterial infections is bacterial biofilms. The
resistance of bacteria in biofilms can be up to 1000 times higher than their planktonic from [8,9]. A
biofilm is a complex multicellular exopolysaccharide matrix that acts to protect the bacteria against
harmful conditions and also sequestrates a nutrient-rich area [8–10]. This exopolysaccharide matrix
also significantly reduces the penetrance of antibiotics to the bacteria embedded in the biofilm [11–
13]. Furthermore, the presence of different phenotypes of bacteria in a biofilm creates a heterogeneity
in the growth rate and metabolism of the bacteria [14]. As conventional antibiotics mainly target
metabolically active and growing cells, slow or non-growing bacteria that survive the antibiotic
treatment can then reproduce and pass their resistance genes to their offspring. Moreover, the
increased rate of horizontal gene transfer in biofilms compared to planktonic cells can accelerate the
speed of resistance spread [8,15]. Approximately 65–80% bacterial infections are associated with
biofilm formations, and one of the most common pathogens found in biofilms is S. aureus [14,16,17].
To combat biofilms, a few antibiofilm mechanisms have been developed in recent years such as to
disrupt or degrade the membrane potential of bacterial cells embedded in biofilms [18].
In recent years, there has been increasing interest in the development of antimicrobial peptides
(AMPs) as a new class of antibiotics. AMPs are naturally occurring peptides that serve as the first line
of the innate immune defence system in humans. They exhibit a broad spectrum of antimicrobial
properties against different microorganisms including bacteria, viruses and fungi [19–22]. The
mechanism of action of AMPs is mainly attributed to their facially amphiphilic structure. It is thought
that the cationic residues on one face of the AMP first bind electrostatically with the anionic bacterial
membrane surface. Then, the hydrophobic residues on the opposite face of the AMP aid in the
insertion of the entire AMP molecule by associating with the lipophilic interior of the bacterial cell
membrane. This disrupts the bacterial membrane, leading to the loss of membrane potential and
leakage of cellular contents, eventually killing the bacterial cell [19,23–25]. Unlike conventional
antibiotics, AMPs act via non-receptor interactions. Since a complete restructuring of the cell
membrane is required for the development of resistance, the chances of bacteria developing drug
resistance to AMPs is low [19,26–28]. While AMPs possess high potency against bacterial cells, their
deployment as drugs has been impeded by their poor bioavailability, low proteolytic stability, high
manufacturing cost and poor yield from multi-step syntheses [19,29,30].
The limitations of AMPs have stimulated the development of AMP mimics such as α-peptides
[31], β-peptides [32,33] and peptoids [34,35]. In addition, there have been reports of several small
molecular AMP mimics such as anthranilamides [36], cationic peptoids [35], cholic acid derivatives
[37] and phenyleneethynylenes [38]. Similar to natural AMPs, these AMP mimics possess an
amphiphilic structure with good spatial separation between the hydrophobic and cationic groups.
Among these AMP mimics, Lytixar (LTX-109) 1 and Brilacidin (PMX-30063) 2 (Figure 1) have
completed phase II human clinical trials, suggesting that AMP mimics could be potential therapeutic
agents for treating bacterial infections [39].

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Figure 1. Structures of Lytixar (LTX-109) 1 and Brilacidin (PMX-30063) 2.
Isatin (indoline-2,3-dione) is a natural product found in plants of the genus Isatis [40]. Its
derivatives have been reported to show a wide range of biological and pharmacological properties,
such as being antimicrobial, anti-inflammatory, anticancer, antiviral and acting as analgesics [41].
Interestingly, N-acyl, N-aryl and N-sulfonylisatins 3 (Figure 2) can act as electrophiles and be ring-
opened by amines and alcohols to afford the corresponding phenylglyoxamides and glyoxylic esters,
respectively [40,42]. Since ring-opened phenylglyoxamide derivatives possess an amide bond, they
are potential candidates for the development of AMP mimics. Our group has previously reported the
synthesis of phenylglyoxamides (e.g., 4–5) derived from N-acylisatins and N-sulfonylisatins [43–45].
Among
these
molecules,
N-sulfonylphenylglyoxamide
iodide
salt
4b
and
N-
naphthoylphenylglyoxamide guanidinium salt 5 had moderate to good minimum inhibitory
concentrations (MIC) of 63 and 12 μM respectively, against Gram-positive S. aureus [43,44]. However,
these compounds gave no antibacterial activity against Gram-negative bacteria. Hence, we were
interested to structurally modify these compounds as part of the optimisation process.
Figure 2. Structures of N-substituted isatin 3 and phenylglyoxamide derivatives 4–5.
Biphenyl is an important scaffold in drug development and is found in many drug molecules
and natural products [46,47]. Compounds containing the biphenyl moieties possess a wide variety of

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biological properties, including being antibacterial and antifungal [48]. However, the low solubility
of biphenyl compounds in both water and common organic solvents is a major drawback which
impedes their synthesis and development for pharmaceutical applications. To address this issue,
Ol’khovik et al. incorporated the quaternary ammonium group in the development of antimicrobial
biphenyl molecules [49].
Recently, our group has demonstrated the importance of the biphenyl moiety in the
development of AMP mimics [50]. As the synthesis of biphenylglyoxamide derivatives has not been
explored, we report for the first time the synthesis of novel biphenylglyoxamide-based antimicrobial
peptide mimics from 5-phenylisatins. The antibacterial and antibiofilm activities of these compounds
were evaluated against S. aureus, Pseudomonas aeruginosa and Escherichia coli. The mechanism of action
and in vitro cytotoxicity of these compounds were also explored.
2. Results and Discussion
2.1. Design and Synthesis of Biphenyl Glyoxamide-Based Antimicrobial Peptide Mimics
In this work, a phenyl ring was installed at the 5-position of the phenylglyoxamide scaffold,
giving the biphenylglyoxamide scaffold. The terminal phenyl ring of this scaffold was modified with
electron-withdrawing halogen substituent (F and Cl) or a bulky naphthalenyl substituent to
investigate their effect on antibacterial activity. Four series of compounds with different cationic or
hydrophilic groups, namely glyoxamide derivatives (Series I), tertiary ammonium hydrochloride
salts (Series II), quaternary ammonium iodide salts (Series III) and guanidinium hydrochloride salts
(Series IV), were synthesised to compare the effect of cationic functionality on the biological activity
of the AMP mimics. Moreover, the N-octanesulfonyl group was appended to these AMP mimics as
the hydrophobic group, and this hydrophobic group was modified to the N-butanesulfonyl group or
N-naphthoyl group to study their effect on antibacterial activity.
These biphenylglyoxamide-based antimicrobial peptide mimics were synthesised according to
the pathways described in Schemes 1–4.
The biphenyl scaffold was incorporated into the molecules by the Suzuki-Miyaura cross-
coupling reaction. This was achieved by reacting 5-bromisatin 6 with different commercially
available arylboronic acids to afford 5-arylisatins 7 in good yields (57–86%) (Scheme 1).
Scheme 1. Synthesis of 5-arylisatins 7a–7d.
A hydrophobic group was introduced into the scaffold by reacting 5-arylisatins 7a–7d with an
alkylsulfonyl chloride and triethylamine as base, furnishing N-alkylsulfonyl compounds 8a–8d and
9a in good yields (52–72%) (Scheme 2). Subsequent nucleophilic ring-opening reaction of N-
alkylsulfonyl compounds 8a–8d and 9a with 3-dimethylaminopropylamine 10 afforded the
corresponding glyoxamides 11a–11d and 12a as series I compounds in excellent yields (94–99%). The
cationic group was installed by treating glyoxamides 11a–11d and 12a with 4 M HCl in dioxane to
give tertiary ammonium chloride salts 13a–13d and 14a as series II compounds in 92–99% yields or

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with methyl iodide in tetrahydrofuran (THF) to give quaternary ammonium iodide salts 15a–15d and
16a as series III compounds in 90–95% yields. The analogous reactions with 5-butylisatin 7e as the
starting material generated the corresponding tertiary ammonium chloride salt 13e and quaternary
ammonium iodide salt 15e in 99% and 85% yields, respectively.
Scheme 2. General synthetic scheme for the synthesis of series I glyoxamide derivatives 11–12, series
II tertiary ammonium chloride salts 13–14 and series III quaternary ammonium iodide salts 15–16.
Alternatively, N-octanesulfonyl compounds 8a–8d were also ring-opened with N-Boc-1,3-
propanediamine 17 to give Boc-protected glyoxamides 18a–18d in excellent yields (95–97%) (Scheme
3). Boc-protected glyoxamides 18a–18d were treated with 4 M HCl in dioxane to yield
aminoglyoxamides 19a–19d in good yields (64–90%). The subsequent guanylation reaction using
N,N’-di-Boc-1H-pyrazole-1-carboxamide and triethylamine afforded the corresponding Boc-
protected guanidine glyoxamides 21a–21d in moderate to good yields (31–77%). Finally, treating the
Boc-protected guanidine glyoxamides 21a–21d with trifluoroacetic acid in dichloromethane (DCM)
followed by 4 M HCl in dioxane provided the guanidinium hydrochloride salts 22a–22d as series IV
compounds in 50–77% yields.

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Scheme 3. General synthetic scheme for the synthesis of series IV guanidinium hydrochloride salts
22.
Biphenyl derivatives 25–27 bearing a naphthoyl hydrophobic group in place of the alkylsulfonyl
group were also synthesised. Naphthoylation was achieved by treating 5-phenylisatin 7a with
sodium hydride and 2-naphthoyl chloride 23 to give the 5-phenyl-N-naphthoylisatin 24 in 35% yield
(Scheme 4). The nucleophilic ring-opening reaction of 5-phenyl-N-naphthoylisatin 24 with 3-
dimethylaminopropylamine 10 gave amine 25, which was followed by salt conversion into the
corresponding tertiary ammonium chloride salt 26 and quaternary ammonium iodide salt 27 in 84%
and 79% yields, respectively.

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O
O
H2N
N
O
Cl
O
NaH,
N
23
10
DCM, r.t., 6 h
O
O
DMF, r.t., 3 h
N
H
7a
24
O
H
N
NH
Cl
4 M HCl in dioxane
DCM, r.t., 20 min
O
H
O
N
N
NH
O
O
NH
O
26
25
O
H
N
N
CH3I
THF, r.t., 24 h
O
I
NH
O
27
Scheme 4. Synthesis of N-naphthoylglyoxamide derivative 25 and its corresponding tertiary
ammonium chloride salt 26 and quaternary ammonium iodide salt 27.
2.2. Structure–Activity Relationship Study
The antibacterial activities of the synthesised antimicrobial peptide mimics were evaluated by
determining their minimum inhibitory concentration (MIC) against the Gram-positive S. aureus
(SA38). Moreover, quaternary ammonium iodide salts 15a–15e and guanidinium hydrochloride salts
22a–22d were also tested against Gram-negative P. aeruginosa (PA01) and E. coli (K12) (Table 1).
Generally, the tested compounds showed lower antibacterial activity against Gram-negative strains
compared to Gram-positive S. aureus.
Table 1. Antibacterial activity (minimum inhibitory concentration, MIC) of biphenylglyoxamide
derivatives against different strains of bacteria.
MIC (μM)
Compound
S. aureus
24
P. aeruginosa
E. coli
ND
ND
ND
ND
ND
ND
ND
ND
ND
11a
11b
11c
11d
11e
12a
25
ND
ND
ND
ND
ND
ND
ND
ND
ND
16
16
Glyoxamide derivatives
(Series I)
16
63
> 250
> 250
16
Tertiary ammonium chloride salts
(Series II)
13a
13b
16

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13c
13d
13e
14a
26
16
8
ND
ND
ND
ND
ND
125
63
ND
ND
ND
ND
ND
32
32
> 250
> 250
16
16
8
15a
15b
15c
15d
15e
16a
27
32
Quaternary ammonium iodide
63
16
salts
8
250
> 250
ND
ND
63
63
(Series III)
16
> 250
32
8
> 250
ND
ND
63
22a
22b
22c
22d
Guanidinium hydrochloride salts
(Series IV)
8
63
16
8
250
> 250
63
8
> 250
pexiganan
(MSI-78) a
3.2–6.5
3.2–6.5
3.2–6.5
ND = Not determined; a previously reported values. [51].
In the structure–activity relationship (SAR) analysis, the biphenyl system was beneficial for the
antibacterial activity of the analogues. Against Gram-positive S. aureus, the previously synthesised
unsubstituted parent and 5-bromosubstituted quaternary ammonium iodide salt 4 had an MIC value
of 250 and 63 μM respectively [43]. When a phenyl ring was substituted at the 5-position to give the
corresponding biphenyl analogue 15a, the MIC value decreased to 16 μM, indicating that this
analogue was nearly sixteen and four times as potent as the unsubstituted and 5-bromosubstituted
compound, respectively. Interestingly, having an n-butyl group at the 5-position of the phenyl ring,
as in analogue 15e, also gave strong activity against S. aureus (MIC = 16 μM). Moreover, the biphenyl
analogue 15a showed MIC values of 125 and 32 μM against the Gram-negative P. aeruginosa and E.
coli respectively, while the unsubstituted parent compound 4a, 5-bromosubstituted 4b and 5-
butylsubstituted 15e analogues showed no antibacterial activity against these strains even at the
highest concentration tested (250 μM). This suggested that the biphenyl moiety is essential for the
antibacterial activity against Gram-negative bacteria, as the antibacterial ability was lost once the
phenyl ring was removed.
After the biphenyl moiety was identified to be essential for antibacterial activity, modifications
were made to the terminal phenyl ring to investigate the effect of incorporating an electron-
withdrawing halogen atom at the para-position of the terminal phenyl ring as well as replacing the
terminal phenyl ring with a bulky naphthalene ring. Neither modification had a significant influence
on the antibacterial activity of the analogues against Gram-positive S. aureus, as all cationic analogues
(13a–13d, 15a–15d, 22a–22d) showed MIC values of 8 or 16 μM. Against the Gram-negative P.
aeruginosa and E. coli, the introduction of an electron-withdrawing halogen atom had no significant
influence or only slightly increased the antibacterial activity of the analogues. However, when the
terminal phenyl ring was replaced by a bulky naphthalene ring, the antibacterial activity of the
analogue against Gram-negative bacteria was significantly reduced (Table 1). Specifically, the activity
of the naphthalenyl-substituted quaternary ammonium iodide salt 15d was halved compared to 15a,
while the activity of the corresponding guanidinium hydrochloride salt 22d was completely lost. This
suggested that the steric hinderance arising from the bulky naphthalene group may reduce the
activity of the analogues against Gram-negative bacteria.
The effect of modifying the terminal group of the glyoxamide chain was also studied. In general,
Gram-positive antibacterial activity was weakest for the non-charged glyoxamide compounds 11a–
11e. Among the cationic compounds, the guanidinium hydrochloride salts 22a–22d were slightly
more potent against Gram-positive bacteria compared to the corresponding tertiary ammonium

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chloride salts 13a–13d and quaternary ammonium iodide salts 15a–15d. In this study, only
quaternary ammonium iodide salts 15a–15e and guanidinium hydrochloride salts 22a–22d were
tested against Gram-negative P. aeruginosa and E. coli owing to their lower cytotoxicity, while the
corresponding glyoxamide derivatives 11a–11e and tertiary ammonium chloride salts 13a–13e were
not tested against these Gram-negative strains due to their cytotoxicity against mammalian cells (see
below). Against Gram-negative strains, the quaternary ammonium iodide salts 15a–15e displayed
slightly higher activities compared to their corresponding guanidinium hydrochloride salts 22a–22d
in most cases.
As the N-naphthoyl-phenylglyoxamide derivative 5 was previously reported to possess
moderate to high antibacterial activity [45], the effect of replacing the octanesulfonyl group by a
naphthoyl group was investigated. Upon replacing the octanesulfonyl group by a naphthoyl group,
the antibacterial activity of the ammonium chloride salt 26 was lost, while the corresponding
quaternary ammonium iodide salt 27 showed a two-fold decrease in activity (MIC = 32 μM against
S. aureus; Table 1) compared to the corresponding octanesulfonyl compound 15a. The tertiary
ammonium chloride salt 14a and quaternary ammonium iodide salt 16a bearing a butanesulfonyl
group were also synthesised in order to investigate the effect of alkyl chain length on antibacterial
activity. Upon shortening the octanesulfonyl group to butylsulfonyl (12a, 14a, 16a), the antibacterial
activity was completely lost (MIC > 250 μM against S. aureus; Table 1). These results show that the
octanesulfonyl group was the preferred hydrophobic group for high antibacterial activity.
Overall, the SAR analysis for these biphenylglyoxamide-based AMP mimics has demonstrated
the importance of the octanesulfonyl group for high antibacterial activity against Gram-positive S.
aureus (Figure 3). These biphenylglyoxamide-based AMP mimics showed excellent antibacterial
activity against S. aureus regardless of the substitution or bulkiness of the terminal aryl ring. Against
Gram-negative bacteria, the biphenyl scaffold is also essential for antibacterial activities against P.
aeruginosa and E. coli. Out of the four series of compounds, the guanidinium hydrochloride series
(series IV) showed the highest antibacterial activity against Gram-positive bacteria, while the
quaternary ammonium iodide series (series III) showed the highest antibacterial activity against
Gram-negative bacteria.
Figure 3. Summary of structure–activity relationships (SARs) for antibacterial activity.
2.3. Antibiofilm Activity
The ability of the more potent biphenyl-based antimicrobial peptide mimics (15c–15d, 22a–22d)
to disrupt established S. aureus biofilms was investigated at 1×, 2× and 4× MIC of the mimics using a
crystal violet staining assay (Figure 4). The naphthalene-bearing guanidinium hydrochloride salt 22d
showed the highest level of biofilm disruption among the tested compounds at 4× MIC (32 μM),
disrupting 50% of preformed S. aureus biofilms, whereas the fluoro-substituted guanidinium
hydrochloride salt 22b and the chloro-substituted quaternary ammonium iodide salt 15c disrupted

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39% and 35% respectively, of preformed S. aureus biofilms at 4× MIC. The biofilm disruption ability
of these three compounds (15c, 22b, 22d) are comparable to that of LL-37, a natural antimicrobial
peptide that is being tested in phase II clinical trials that can disrupt mature S. aureus biofilms by
approximately 40% at 4× its MIC (32 μM) [52].
Figure 4. Percentage of remaining S. aureus biofilms after 24 h treatment with compounds 22d (top),
22b (middle) or 15c (bottom) at 1×, 2× and 4× of their MIC. Error bars represent the standard error of
triplicates (n = 3).

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The remaining three tested antimicrobial peptide mimics (15d, 22a, 22c) failed to disrupt any
performed S. aureus biofilms at any tested concentrations (Supplementary Figure S1), suggesting that
they are active against planktonic S. aureus cells but are unable to disrupt bacteria that are densely
packed in matrices of extracellular polymeric communities.
The chloro-substituted quaternary ammonium iodide salt 15c and the fluoro-substituted
guanidinium hydrochloride salt 22b, which had high antibacterial activity against E. coli, were also
tested for their ability to disrupt preformed E. coli biofilm at 4× MIC (64 μM). However, these
compounds only managed to disrupt an insignificant amount of the preformed E. coli biofilm
(Supplementary Figure S2). This difference may be due to the different extracellular polymeric
substances used by these bacteria to form biofilms. The biofilms of S. aureus are composed of β-1,6-
N-acetyl-D-glucosamine polymer and extracellular DNA, whereas the biofilms of E. coli are
composed of β-1,6-N-acetyl-D-glucosamine polymer, colanic acid and cellulose [53–57].
Overall, the guanidinium hydrochloride salts 22b, 22d and the quaternary ammonium iodide
salt 15c were the most active antimicrobial peptide mimics against S. aureus biofilms.
2.4. Cytoplasmic Membrane Depolarisation
The mechanisms of action of the most active compounds were further explored using a
membrane dye release assay. It was hypothesised that the mechanism of action of cationic AMP
mimics arises from the electrostatic interactions between the cationic head group of the compounds
and the negatively charged bacterial cell membrane. In order to verify this hypothesis, 3,3′-
dipropylthiadicarbocyanine iodide (diSC3-5), a membrane potential sensitive dye, was employed to
monitor bacterial cytoplasmic membrane integrity in the presence of the compounds. This dye
readily partitions to and aggregates in the bacterial cell membrane, causing self-quenching of
fluorescence when the bacterial cell membrane is intact. However, if the test compound disrupts or
induces pore formation in the bacterial cell membrane, the membrane potential gradient would be
lost and an increase in fluorescence intensity would be observed due to the release of the dye from
the bacterial cell membrane.
As shown in Figure 5, compounds 15c, 22b and 22d induced disruption of the cytoplasmic
membrane of S. aureus, as indicated by the increase of dye fluorescence in a time- and concentration-
dependent manner. Out of these three compounds, the quaternary ammonium iodide salt 15c was
the most effective bacterial cytoplasmic membrane disruptor, as evidenced by the largest increase in
fluorescence intensity at 1× and 2× MIC within 5 min. The increase in fluorescence intensity for the
other two guanidinium hydrochloride salts 22b and 22d was modest when compared to that of the
quaternary ammonium iodide salt 15c, suggesting they are less effective in disrupting bacterial
cytoplasmic membrane.

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Figure 5. Cytoplasmic membrane disruption of S. aureus promoted by compounds 15c, 22b and 22d
at 1× and 2× of their MIC. 10% DMSO was used as positive control.
In addition to the cytoplasmic membrane dye release assay, the time-kill kinetic assay was
utilised to further investigate the mechanism responsible for the bactericidal effect of the AMP
mimics (Figure 6). The time-kill kinetic assay measures bacterial cell viability after the treatment of
bacteria with a compound, and hence can indicate the bactericidal activity of a compound over time.
The bacterial cell viability of all test compounds, 15c, 22b, 22d, against S. aureus was observed to be
time- and concentration-dependent in this assay, which resembled the results observed in the
cytoplasmic membrane dye release assay. In this assay, quaternary ammonium iodide salt 15c
showed the highest reduction (2-log and 1-log reductions, respectively) in bacterial numbers at 2×
and 1× MIC, while the other two guanidinium hydrochloride salts, 22b and 22d, showed smaller
reductions (less than 1-log reductions) of bacterial numbers. The trend in the activity of these
compounds are consistent with what was observed in the cytoplasmic membrane dye release assay.
Figure 6. Cell viability count of S. aureus in the presence of compound 15c, 22b and 22d at 1× and 2×
their MIC.

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Overall, these results suggested that the AMP mimics could exert their antibacterial action via
permeating bacterial membranes, with the quaternary ammonium iodide salt 15c being the most
active AMP mimic. However, there might be other mechanisms of action as well, such as effect on
intracellular components, and these will be explored in future studies [58].
2.5. Lipid Bilayer Membrane Conduction
Tethered bilayer lipid membranes (tBLM), in conjunction with AC electrical impedance
spectroscopy, were used to assess the ability of selected potent compounds: 15c–15d, 22a, and 22c–
22d, to interact with cell membranes. [59,60] In zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphorylcholine (POPC) tBLMs, quaternary ammonium iodide salts 15c and 15d produced a 2.5×
shift in membrane conductance at concentrations as low as 100 nM (Figure 7A). In contrast, these
changes are notably absent for 15c and 15d in membranes that contain 30% negatively charged
palmitoyl-oleoyl-phosphatidyglycerol (POPG) lipids, compared to other tested compounds (Figure
7B). Responses to low concentrations of the compounds (<1 μM) in zwitterionic membranes are
thought to be due to the compounds producing changes in lipid packing as they insert. Such
alterations in the area per lipid, according to the critical packing parameter model of antimicrobial-
membrane interactions [61], are associated with changes in the diameter of intrinsic pores in the lipid
bilayer.
At higher concentrations of the compounds, the changes of membrane conduction were more
pronounced, especially for compounds 15c, 15d and 22a in zwitterionic membrane (Figure 7C).
Interestingly, these responses were lower in negatively charged membranes (Figure 7D). The
significant increases in membrane conduction suggested that these compounds possess lytic or
surfactant-like properties on membranes, in which these compounds sequester lipids from the lipid
membrane bilayer via micellisation and eventually disintegrate the membrane [61].
These findings are supported by the large increase of membrane capacitance in both zwitterionic
(Figure 7E) and negatively charged tBLMs (Figure 7F) upon treatment of the compounds 15c and 15d,
suggesting that these membranes have been disintegrated due to the saturating concentrations of
these particular compounds. Increases in membrane capacitance indicate a thinning of the lipid
membrane and/or the incorporation of a high dielectric, such as water, into the membrane. These
results suggest a diminishing of the membrane due to the removal of lipids from the lipid membrane
bilayer brought by the surfactant-like effect of compounds.

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Figure 7. (A) Changes in membrane conductance caused by the addition of the compounds at
concentrations less than 1 μM in (A) zwitterionic POPC tBLMs, and (B) tBLMs containing 30%
negatively charged POPG lipids. Large increases in membrane conduction due to select compounds
are also visible at higher concentrations (1–100 μM) in (C) zwitterionic POPC tBLMs, and (D) tBLMs
containing 30% negatively charged POPG lipids. Large increases in membrane capacitance in (E)
zwitterionic tBLMs, and (F) negatively charged tBLMs, suggests that high concentrations of
compounds lead to massive disruption of the lipid bilayers. Error bars represent the standard errors
from n = 3 replicates for each compound, with the exception of 15c, which is n = 2.
2.6. Cytotoxicity Activity
The cytotoxicity of biphenyl-based AMP mimics was determined in order to evaluate their
utility as antimicrobial agents. The in vitro toxicity of selected compounds (11a, 11d, 13a, 13c–13e,
15a–15e, 22a–22d) was assessed against MRC-5 normal human fibroblasts using the MTT assay. A
dose-response curve for each test compound was generated and their IC50 values were determined.
The IC50 values obtained were then used to calculate the therapeutic indices (IC50 value divided by

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MIC value) against S. aureus, P. aeruginosa and E. coli for each the compound (Table 2). A higher
therapeutic index corresponds to a more selective antimicrobial agent.
Table 2. IC50 value of compounds against MRC-5 normal human lung fibroblasts and their therapeutic
indices with respect to different strains of bacteria.
Therapeutic index
Compound IC50 (μM)
S. aureus
1.18
0.89
1.56
1.59
2.46
0.74
3.17
11.9
12.0
14.0
6.31
9.44
9.50
11.9
6.19
P. aeruginosa
N/A
N/A
N/A
N/A
N/A
N/A
0.41
E. coli
N/A
N/A
N/A
N/A
N/A
N/A
1.59
5.94
5.99
1.79
N/A
1.21
4.75
1.52
N/A
Glyoxamide derivatives
11a
11d
13a
13c
13d
13e
15a
15b
15c
15d
15e
22a
22b
22c
22d
28.2
14.2
25.0
25.5
19.7
23.6
50.7
190
Tertiary ammonium
chloride salts
3.04
Quaternary ammonium
iodide salts
95.8
112
1.53
0.45
101
N/A
1.21
75.5
76.0
95.0
49.5
Guanidinium
1.21
hydrochloride salts
0.38
N/A
N/A = Not applicable.
The glyoxamide compounds 11a and 11d were found to possess high toxicity (IC50 < 30 μM)
towards human cells, resulting in therapeutic indices of below 1.20 against S. aureus. This could be
due to the lack of cationic charge and the higher hydrophobicity of the compounds allowing them to
bind to the zwitterionic human cell membranes more easily [62]. The conversion of the glyoxamide
compounds 11a and 11d into their corresponding tertiary ammonium chloride salts 13a and 13d gave
no improvement in the toxicity of the compounds as they possess similar toxicity (IC50 < 30 μM)
towards human cells. However, owing to their lower MIC values, their therapeutic indices slightly
increased to 1.56–2.46 against S. aureus compared to the corresponding glyoxamide compounds. In
contrast, quaternary ammonium iodide salts 15a–15d and guanidinium hydrochloride salts 22a–22d
showed lower toxicity (IC50 > 50 μM), with therapeutic indices of 3.17–13.98 against S. aureus. In
particular, compounds 15b, 15c, 15d, 22a, 22b and 22c with therapeutic indices above 9 against S.
aureus are promising antimicrobial agents as they are likely to be non-toxic to human cells at the
therapeutic dosages required to inhibit bacterial growth.
Owing to the lower potency of the quaternary ammonium iodide salts 15a–15d and guanidinium
hydrochloride salts 22a–22d against Gram-negative bacteria, their therapeutic indices against P.
aeruginosa and E. coli were significantly lower compared to that of S. aureus. Among these compounds,
the fluoro- and chloro-substituted quaternary ammonium iodide salts 15b–15c possessed the highest
therapeutic indices of around 6 against E. coli. Meanwhile, the fluoro-substituted quaternary
ammonium iodide 15b showed a therapeutic index of 3.04 against P. aeruginosa. Notably, compound
15b was also the least toxic (IC50 = 190 μM) against human cells among the compounds tested.
3. Materials and Methods
3.1. Synthesis of Analogues
3.1.1. General Information
All commercially available reagents were purchased from standard suppliers (Sigma Aldrich, St
Louis, MO, USA and Alfa-Aesar, Ward Hill, MA, USA) and used without further purification. All

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reactions were performed under anhydrous condition with anhydrous solvent unless otherwise
specified, and anhydrous solvents were obtained using the PureSolv MD Solvent Purification System.
Reactions were monitored by thin-layer chromatography precoated with Merck silica gel 60 F254 and
visualisation was performed by using short or long wavelength of ultraviolet light. Flash
chromatography was carried out using Grace Davisil LC60A silica.
Melting points were measured using an OptiMelt melting point apparatus and are uncorrected.
1H and 13C NMR spectra were obtained in the specified solvents on a Bruker Avance III HD 400
(Bruker, Sydney, NSW, Australia) or Bruker Avance III 600 Cryo spectrometer (Bruker, Sydney,
NSW, Australia). Chemical shift (δ) are in parts per million (ppm) internally referenced to the solvent
nuclei. Multiplicities are assigned as singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q),
multiplet (m) or a combination of these (e.g., dd, dt, td), and coupling constants (J) are reported in
Hertz (Hz). Infrared (IR) spectra were recorded using a Cary 630 FTIR spectrometer or NicoletTM iSTM
10 FTIR spectrometer (Thermo Nicolet, Waltham, MA, USA) fitted with a diamond attenuated total
reflectance (ATR) sample interface. Low-resolution mass spectrometry was performed using a
Thermo Fisher LCQ Mass Spectrometer (Thermo Scientific, Waltham, MA, USA), while high-
resolution mass spectrometry (HRMS) was performed using a Thermo LTQ Orbitrap XL instrument
(Thermo Scientific, Waltham, MA, USA).
3.1.2. Synthetic Procedures and Experimental Characterisation Data
General Synthetic Procedure A for 5-arylisatins
To a solution mixture of 5-bromoisatin (1.0 equivalent) and the appropriate boronic acid (1.1
equivalents) in degassed (for 30 min) 1:1 toluene/ethanol solution (40 mL), 2 M potassium carbonate
solution (2.0 equivalents; degassed for 30 min prior to addition) was added. The dark brown solution
was degassed for 30 min. Pd(PPh3)4 (0.01 equivalents) was then added to the solution mixture and
the reaction was heated at 90 °C under nitrogen atmosphere for 24 h. The brownish-black solution
was concentrated in vacuo. Water was then added to the reaction mixture and the resulting solution
was then acidified to pH 1 with HCl (2 M). The reddish-orange organic layer was extracted thrice
with dichloromethane (3 × 30 mL), washed with brine, dried over sodium sulphate and concentrated
in vacuo to give the crude product as a red solid. The crude product was purified by flash column
chromatography on silica to afford the product.
5-Phenylindoline-2,3-dione (7a)
The titled compound was synthesised from 5-bromoisatin (2.02 g, 8.92 mmol), phenylboronic
acid (1.20 g, 9.83 mmol), potassium carbonate (2.50 g, 18.06 mmol) and Pd(PPh3)4 (116 mg, 0.100 mmol)
following general synthetic procedure A. The product was obtained as a red solid (1.72 g, 86%); mp
250.3–250.4 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.29 (bs, 1H, NH), 7.91 (dd, J = 8.2, 2.0 Hz, 1H, ArH),
7.76 (d, J = 1.9 Hz, 1H, ArH), 7.67–7.63 (m, 2H, ArH), 7.48–7.42 (m. 2H, ArH), 7.39–7.33 (m, 1H, ArH),
7.01 (d, J = 8.2 Hz, 1H, ArH); 13C NMR (100 MHz, DMSO-d6): δ 184.4 (CO), 159.6 (CO), 150.0 (ArC),
138.7 (ArC), 136.5 (ArCH), 134.9 (ArC), 129.0 (ArCH), 127.5 (ArCH), 126.2 (ArCH), 122.5 (ArCH),
118.4 (ArC), 112.7 (ArCH); IR (ATR): νmax 3273, 1759, 1729, 1617, 1508, 1473, 1432, 1308, 1254, 1203,
1183, 1099, 1022, 966, 910, 846, 770, 752, 730, 697, 654, 576, 517, 456, 424 cm-1; MS (+ ESI): m/z 246.08,
[M + Na]+.
5-(4-Fluorophenyl)indoline-2,3-dione (7b)
The titled compound was synthesised from 5-bromoisatin (1.06 g, 4.70 mmol), 4-
fluorophenylboronic acid (0.76 g, 5.40 mmol), potassium carbonate (1.31 g, 9.45 mmol) and Pd(PPh3)4
(55 mg, 0.048 mmol) following general synthetic procedure A. The product was obtained as a red
solid (0.68 g, 60%); mp 238.9–239.3 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.13 (bs, 1H, NH), 7.87 (dd,
J = 8.2, 2.0 Hz, 1H, ArH), 7.74 (d, J = 1.8 Hz, 1H, ArH), 7.72–7.65 (m, 2H, ArH), 7.31–7.23 (m, 2H, ArH),
6.99 (d, J = 8.2 Hz, ArH); 13C NMR (100 MHz, DMSO-d6): δ 184.3 (CO), 161.8 (ArC), 159.5 (CO), 149.9
(ArC), 136.4 (ArCH), 135.2 (ArC), 133.9 (ArC), 128.3 (ArCH), 122.5 (ArCH), 118.4 (ArC), 115.8 (ArCH),

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112.7 (ArCH); IR (ATR): νmax 3324, 3273, 1765, 1739, 1621, 1601, 1589, 1505, 1475, 1454, 1366, 1349, 1307,
1275, 1225, 1192, 1159, 1127, 1098, 1013, 967, 935, 914, 891, 843, 821, 752, 703, 652, 590, 566, 514, 497,
460, 448 cm-1; MS (+ ESI): m/z 264.00, [M + Na]+.
5-(4-Chlorophenyl)indoline-2,3-dione (7c)
The titled compound was synthesised from 5-bromoisatin (1.10 g, 4.87 mmol), 4-
chlorophenylboronic acid (0.84 g, 5.40 mmol), potassium carbonate (1.37 g, 9.91 mmol) and Pd(PPh3)4
(78 mg, 0.068 mmol) following general synthetic procedure A. The product was obtained as a red
solid (0.72 g, 57%); mp 244.2–244.3 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.15 (bs, 1H, NH), 7.90 (dd,
J = 8.2, 2.0 Hz, 1H, ArH), 7.78 (d, J = 1.9 Hz, 1H, ArH), 7.71–7.67 (m, 2H, ArH), 7.52–7.47 (m, 2H, ArH),
7.00 (d, J = 8.2 Hz, 1H, ArH); 13C NMR (100 MHz, DMSO-d6): δ 184.3 (CO), 159.5 (CO), 150.2 (ArC),
137.6 (ArC), 136.4 (ArCH), 133.5 (ArC), 132.3 (ArC), 128.9 (ArCH), 128.0 (ArCH), 122.5 (ArCH), 118.5
(ArC), 112.7 (ArCH); IR (ATR): νmax 3327, 3279, 1763, 1742, 1620, 1506, 1474, 1452, 1367, 1349, 1308,
1272, 1258, 1220, 1194, 1160, 1122, 1090, 1012, 966, 915, 842, 821, 811, 753, 742, 703, 655, 584, 567, 541,
513, 497, 460, 448 cm−1; MS (+ESI): m/z 280.08, [M + Na]+.
5-(Naphthalen-2-yl)indoline-2,3-dione (7d)
The titled compound was synthesised from 5-bromoisatin (1.05 g, 4.66 mmol), 4-
naphthyllboronic acid (0.94 g, 5.19 mmol), potassium carbonate (1.32 g, 9.51 mmol) and Pd(PPh3)4 (58
mg, 0.050 mmol) following general synthetic procedure A. The product was obtained as a red solid
(0.58 g, 46%); mp 293.9–294.0 °C; 1H NMR (600 MHz, DMSO-d6): δ 11.17 (bs, 1H, NH), 8.24 (d, J = 1.7
Hz, 1H, ArH), 8.07 (dd, J = 8.2, 2.1 Hz, 1H, ArH), 8.01–7.97 (m, 2H, ArH), 7.95–7.92 (m, 2H, ArH), 7.84
(dd, J = 8.6, 1.9 Hz, 1H, ArH), 7.56-7.50 (m, 2H, ArH), 7.05 (dd, J = 8.2, 0.5 Hz, 1H, ArH); 13C NMR (150
MHz, DMSO-d6): δ 184.4 (CO), 159.6 (CO), 150.0 (ArC), 136.7 (ArCH), 136.0 (ArC), 134.7 (ArC), 133.3
(ArC), 132.2 (ArC), 128.6 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 126.5 (ArCH), 126.2 (ArCH), 124.7
(ArCH), 124.6 (ArCH), 122.7 (ArCH), 118.6 (ArC), 112.8 (ArCH); IR (ATR): νmax 3252, 1753, 1732, 1616,
1490, 1459, 1429, 1389, 1344, 1305, 1270, 1250, 1195, 1154, 1119, 980, 905, 891, 863, 846, 808, 752, 698,
636, 622, 598, 577, 560, 525, 496, 474, 460, 443 cm-1; HRMS (+ ESI): Found m/z 296.0682 [M + Na]+,
C18H11NO2Na required 296.0682.
General Synthetic Procedure B for N-sulfonylisatins
To a solution of 5-substituted isatin (1.0 equivalent) in dichloromethane (20 mL), triethylamine
(1.1 equivalents) was added at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at
0 °C for 20 min. 1-Octanesulfonyl chloride or 1-butanesulfonyl chloride (1.0 equivalent) was then
added slowly dropwise to the reaction mixture at 0 °C with stirring. The reaction mixture was then
stirred at room temperature for 3 h. After completion of the reaction, the resulting mixture was
concentrated in vacuo and washed with methanol to afford the product.
1-(Octylsulfonyl)-5-phenylindoline-2,3-dione (8a)
The titled compound was synthesised from 5-phenylindoline-2,3-dione 7a (1.70 g, 7.62 mmol),
triethylamine (1.20 mL, 8.61 mmol) and 1-octanesulfonyl chloride (1.50 mL, 7.67 mmol) following
general synthetic procedure B. The product was obtained as a yellow solid (1.58 g, 52%); mp 131.9-
132.2 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.08 (dd, J = 8.5, 2.1 Hz, 1H, ArH), 7.98 (d, J = 2.0 Hz, 1H,
ArH), 7.80 (d, J = 8.5 Hz, 1H, ArH), 7.75-7.71 (m, 2H, ArH), 7.50 (t, J = 7.8 Hz, 2H, ArH), 7.41 (t, J = 7.4
Hz, 1H, ArH), 3.67–3.59 (m, 2H, CH2), 1.86–1.76 (m, 2H, CH2), 1.43–1.15 (m, 10H, CH2), 0.83 (t, J = 7.0
Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 178.7 (CO), 156.6 (CO), 146.1 (ArC), 138.0 (ArC), 137.0
(ArC), 135.9 (ArCH), 129.1 (ArCH), 128.1 (ArCH), 126.5 (ArCH), 122.4 (ArCH), 120.1 (ArC), 114.6
(ArCH), 53.7 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.2 (CH2), 22.0 (CH2), 13.9 (CH3);
IR (ATR): νmax 3326, 3274, 2915, 2049, 1765, 1737, 1615, 1505, 1472, 1454, 1373, 1349, 1308, 1259, 1222,
1191, 1175, 1160, 1120, 1096, 1012, 967, 945, 913, 850, 821, 762, 754, 703, 651, 609, 592, 566, 532, 513, 497,
461, 448 cm-1; HRMS (+ ESI): Found m/z 422.1398 [M + Na]+, C22H25NO4SNa required 422.1397.

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5-(4-Fluorophenyl)-1-(octylsulfonyl)indoline-2,3-dione (8b)
The titled compound was synthesised from 5-(4-fluorophenyl)indoline-2,3-dione 7b (0.54 g, 2.23
mmol), triethylamine (0.35 mL, 2.51 mmol) and 1-octanesulfonyl chloride (0.44 mL, 2.25 mmol)
following general synthetic procedure B. The product was obtained as a yellow solid (0.65 g, 70%);
mp 142.3–142.7 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.06 (dd, J = 8.6, 2.2 Hz, 1H, ArH), 7.98 (d, J = 2.0,
1H, ArH), 7.82–7.74 (m, 3H, ArH), 7.36–7.28 (m, 2H, ArH), 3.67–3.59 (m, 2H, CH2), 1.86–1.75 (m, 2H,
CH2), 1.43–1.15 (m, 10H, CH2), 0.83 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 178.7
(CO), 162.2 (ArC), 156.6 (CO), 146.1 (ArC), 136.0 (ArC), 135.8 (ArCH), 134.5 (ArC), 128.7 (ArCH), 122.5
(ArCH), 120.1 (ArC), 115.9 (ArCH), 114.6 (ArCH), 53.7 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3
(CH2), 22.2 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3675, 2970, 2900, 1766, 1739, 1615, 1573, 1519,
1475, 1406, 1393, 1373, 1308, 1292, 1232, 1175, 1139, 1066, 1057, 1027, 944, 891, 856, 831, 784, 763, 726,
714, 701, 619, 605, 590, 567, 532, 497, 484, 466, 447 cm-1; HRMS (+ ESI): Found m/z 440.1303 [M + Na]+,
C22H24FNO4SNa required 440.1302.
5-(4-Chlorophenyl)-1-(octylsulfonyl)indoline-2,3-dione (8c)
The titled compound was synthesised from 5-(4-chlorophenyl)indoline-2,3-dione 7c (0.36 g, 1.41
mmol), triethylamine (0.22 mL, 1.58 mmol) and 1-octanesulfonyl chloride (0.28 mL, 1.43 mmol)
following general synthetic procedure B. The product was obtained as a yellow solid (0.32 g, 52%);
mp 171.7–172.1 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.08 (dd, J = 8.6, 2.2 Hz, 1H, ArH), 8.01 (d, J = 2.0
Hz, 1H, ArH), 7.82–7.75 (m, 3H, ArH), 7.54 (d, J = 8.6 Hz, 2H, ArH), 3.67–3.59 (m, 2H, CH2), 1.86–1.75
(m, 2H, CH2), 1.43–1.15 (m, 10H, CH2), 0.83 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ
178.6 (CO), 156.6 (CO), 146.3 (ArC), 136.8 (ArC), 135.8 (ArCH), 135.6 (ArC), 132.9 (ArC), 129.0 (ArCH),
128.4 (ArCH), 122.5 (ArCH), 120.2 (ArC), 114.6 (ArCH), 53.7 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2),
27.3 (CH2), 22.2 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3675, 2970, 2900, 1772, 1746, 1614, 1587,
1568, 1468, 1405, 1394, 1369, 1306, 1287, 1241, 1183, 1168, 1139, 1076, 1066, 1057, 1011, 945, 892, 851,
818, 804, 765, 727, 707, 688, 601, 533, 509, 463, 454, 437 cm-1; HRMS (+ ESI): Found m/z 456.1007 [M +
Na]+, C22H24ClNO4SNa required 456.1007.
5-(Naphthalen-2-yl)-1-(octylsulfonyl)indoline-2,3-dione (8d)
The titled compound was synthesised from 5-(naphthalen-2-yl)indoline-2,3-dione 7d (0.42 g,
1.54 mmol), triethylamine (0.24 mL, 1.72 mmol) and 1-octanesulfonyl chloride (0.30 mL, 1.54 mmol)
following general synthetic procedure B. The product was obtained as a yellow solid (0.50 g, 73%);
1
mp 125.0-125.4 °C; H NMR (400 MHz, DMSO-d6): δ 8.33 (s, 1H, ArH), 8.23 (dd, J = 8.6, 2.1 Hz, 1H,
ArH), 8.16 (d, J = 2.0 Hz, 1H, ArH), 8.05–8.00 (m, 2H, ArH), 7.98–7.94 (m, 1H, ArH), 7.93–7.88 (m, 1H,
ArH), 7.85 (d, J = 8.6 Hz, 1H, ArH), 7.59–7.52 (m, 2H, ArH), 3.69–3.61 (m, 2H, CH2), 1.88-1.77 (m, 2H,
CH2), 1.44–1.15 (m, 10H, CH2), 0.83 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 178.7
(CO), 156.7 (CO), 146.2 (ArC), 136.8 (ArC), 136.0 (ArCH), 135.2 (ArC), 133.3 (ArC), 132.4 (ArC), 128.7
(ArCH), 128.3 (ArCH), 127.5 (ArCH), 126.6 (ArCH), 126.5 (ArCH), 125.3 (ArCH), 124.6 (ArCH), 122.7
(ArCH), 120.2 (ArC), 114.7 (ArCH), 53.7 (CH2), 31.1 (CH2), 28.4 (CH2), 28.4 (CH2), 27.3 (CH2), 22.2
(CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 2923, 2852, 1766, 1737, 1615, 1578, 1488, 1460, 1424, 1374,
1306, 1293, 1270, 1259, 1235, 1198, 1173, 1136, 1110, 1094, 1038, 1017, 998, 953, 937, 922, 893, 872, 852,
824, 783, 763, 751, 744, 721, 700, 659, 632, 613, 589, 560, 532, 497, 474, 446 cm-1; HRMS (+ ESI): Found
m/z 472.1554 [M + Na]+, C26H27NO4SNa required 472.1553.
5-Butyl-1-(octylsulfonyl)indoline-2,3-dione (8e)
The titled compound was synthesised from 5-butylindoline-2,3-dione 7e (0.40 g, 1.95 mmol),
triethylamine (0.30 mL, 2.15 mmol) and 1-octanesulfonyl chloride (0.39 mL, 1.99 mmol) following
general synthetic procedure B. The product was obtained as a yellow solid (0.41 g, 55%); mp 113.2–
113.6 °C; 1H NMR (400 MHz, DMSO-d6): δ 7.64–7.53 (m, 3H, ArH), 3.62–3.56 (m, 2H, CH2), 2.62 (t, J =
7.7 Hz, 2H, CH2), 1.82–1.72 (m, 2H, CH2), 1.59–1.49 (m, 2H, CH2), 1.41–1.16 (m, 12H, CH2), 0.89 (t, J =
7.5 Hz, 3H, CH3), 0.84 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 178.9 (CO), 156.7 (CO),

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145.1 (ArC), 139.5 (ArC), 137.8 (ArCH), 124.4 (ArCH), 119.4 (ArC), 114.0 (ArCH), 53.6 (CH2), 33.7
(CH2), 32.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.2 (CH2), 22.0 (CH2), 21.6 (CH2), 13.9
(CH3), 13.7 (CH3); IR (ATR): νmax 3854, 3675, 2968, 2911, 2362, 1762, 1737, 1615, 1584, 1483, 1467, 1406,
1393, 1372, 1293, 1250, 1241, 1223, 1177, 1152, 1134, 1116, 1077, 1066, 1057, 1027, 954, 868, 851, 782, 724,
706, 653, 605, 565, 532, 484, 462, 446 cm-1; HRMS (+ ESI): Found m/z 402.1711 [M + Na]+, C20H29NO4SNa
required 402.1710.
1-(Butylsulfonyl)-5-phenylindoline-2,3-dione (9a)
The titled compound was synthesised from 5-phenylindoline-2,3-dione 7a (0.45 g, 1.92 mmol),
triethylamine (0.30 mL, 2.15 mmol) and 1-butanesulfonyl chloride (0.25 mL, 1.93 mmol) following
1
general synthetic procedure B. The product was obtained as a yellow sticky solid (0.23 g, 34%); H
NMR (400 MHz, DMSO-d6): δ 8.08 (dd, J = 8.6, 2.2 Hz, 1H, ArH), 7.98 (d, J = 2.1 Hz, 1H, ArH), 7.80 (d,
J = 8.6 Hz, 1H, ArH), 7.75–7.71 (m, 2H, ArH), 7.50 (t, J = 7.8 Hz, 2H, ArH), 7.41 (t, J = 7.3 Hz, 1H, ArH),
3.67–3.60 (m, 2H, CH2), 1.85–1.76 (m, 2H, CH2), 1.47–1.36 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6): δ 178.7 (CO), 156.6 (CO), 146.1 (ArC), 138.0 (ArC), 137.0 (ArC), 135.9
(ArCH), 129.1 (ArCH), 128.0 (ArCH), 126.5 (ArCH), 122.4 (ArCH), 120.1 (ArC), 114.6 (ArCH), 53.5
(CH2), 24.1 (CH2), 20.7 (CH2), 13.3 (CH3); IR (ATR): νmax 3196, 2961, 2873, 1777, 1736, 1648, 1615, 1588,
1508, 1485, 1472, 1455, 1399, 1368, 1339, 1310, 1293, 1270, 1241, 1183, 1171, 1139, 1117, 1040, 1000, 982,
949, 917, 842, 758, 734, 694, 674, 651, 620, 602, 579, 556, 532, 516, 464, 426 cm-1; HRMS (+ ESI): Found
m/z 366.0771 [M + Na]+, C18H17NO4SNa required 366.0770.
General Synthetic Procedure C for Glyoxamide Derivatives
To
a solution of N-sulfonylisatin (1.0 equivalent) in dichloromethane (5 mL), 3-
dimethylaminopropylamine (1.0 equivalent) was added at 0 °C. The reaction mixture was stirred at
room temperature for 6 h. After completion of the reaction, water was added to the reaction mixture
and the product was extracted into dichloromethane (3 × 30 mL), washed with brine, dried over
anhydrous sodium sulphate and concentrated in vacuo to afford the product.
N-(3-(Dimethylamino)propyl)-2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide (11a)
The titled compound was synthesised from 1-(octylsulfonyl)-5-phenylindoline-2,3-dione 8a
(0.11 g, 0.28 mmol) and 3-dimethylaminopropylamine (35 μL, 0.28 mmol) following general synthetic
procedure C. The product was obtained as a yellow oil (0.13 g, 96%); 1H NMR (400 MHz, CDCl3): δ
8.78 (bs, 1H, NH), 8.72 (d, J = 1.9 Hz, 1H, ArH), 7.87–7.80 (m, 2H, ArH), 7.60–7.55 (m, 2H, ArH), 7.47–
7.41 (m, 2H, ArH), 7.39–7.34 (m, 1H, ArH), 3.53 (t, J = 6.0 Hz, 2H, CH2), 3.21-3.15 (m, 2H, CH2), 2.55 (t,
J = 6.2 Hz, 2H, CH2), 2.33 (s, 6H, CH3), 1.86–1.76 (m, 4H, CH2), 1.43–1.15 (m, 10H, CH2), 0.85 (t, J = 6.6
Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 192.0 (CO), 162.8 (CO), 140.9 (ArC), 139.1 (ArC), 135.8
(ArC), 134.9 (ArCH), 133.6 (ArCH), 129.1 (ArCH), 127.9 (ArCH), 127.0 (ArCH), 120.0 (ArC), 118.6
(ArCH), 58.6 (CH2), 52.7 (CH2), 45.2 (CH3), 39.8 (CH2), 31.8 (CH2), 29.1 (CH2), 29.0 (CH2), 28.2 (CH2),
25.3 (CH2), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3327, 3274, 2924, 2854, 1765, 1738, 1616,
1508, 1476, 1395, 1367, 1308, 1261, 1225, 1195, 1143, 1098, 1013, 967, 935, 821, 760, 698, 655, 585, 566,
514, 497, 460, 448 cm-1; HRMS (+ ESI): Found m/z 502.2734 [M + H]+, C27H40N3O4S required 502.2734.
N-(3-(Dimethylamino)propyl)-2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-
oxoacetamide (11b)
The titled compound was synthesised from 5-(4-fluorophenyl)-1-(octylsulfonyl)indoline-2,3-
dione 8b (0.13 g, 0.32 mmol) and 3-dimethylaminopropylamine (40 μL, 0.32 mmol) following general
1
synthetic procedure C. The product was obtained as a yellow oil (0.16 g, 99%); H NMR (400 MHz,
CDCl3): δ 8.86 (bs, 1H, NH), 8.71 (d, J = 2.2 Hz, 1H, ArH), 7.84 (d, J = 8.7 Hz, 1H, ArH), 7.77 (dd, J =
8.7, 2.2 Hz, 1H, ArH), 7.56–7.50 (m, 2H, ArH), 7.16–7.09 (m, 2H, ArH), 3.53 (t, J = 6.0 Hz, 2H, CH2),
3.20–3.14 (m, 2H, CH2), 2.52 (t, J = 6.1 Hz, 2H, CH2), 2.30 (s, 6H, CH3), 1.86–1.74 (m, 4H, CH2), 1.43–
1.16 (m, 10H, CH2), 0.85 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 191.8 (CO), 162.8 (ArC),

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162.6 (CO), 140.9 (ArC), 135.3 (ArC), 134.8 (ArC), 134.7 (ArCH), 133.5 (ArCH), 128.6 (ArCH), 119.6
(ArC), 118.6 (ArCH), 116.1 (ArCH), 58.8 (CH2), 52.7 (CH2), 45.3 (CH3), 40.0 (CH2), 31.8 (CH2), 29.1
(CH2), 29.0 (CH2), 28.2 (CH2), 25.3 (CH2), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3675, 2970,
2923, 1659, 1635, 1571, 1491, 1393, 1338, 1261, 1222, 1200, 1139, 1066, 1057, 921, 821, 770, 724, 677, 595,
563, 520, 488, 419 cm-1; HRMS (+ ESI): Found m/z 520.2641 [M + H]+, C27H39FN3O4S required 520.2640.
2-(4’-Chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-(dimethylamino)propyl)-2-
oxoacetamide (11c)
The titled compound was synthesised from 5-(4-chlorophenyl)-1-(octylsulfonyl)indoline-2,3-
dione 8c (0.10 g, 0.24 mmol) and 3-dimethylaminopropylamine (30 μL, 0.24 mmol) following general
1
synthetic procedure C. The product was obtained as a yellow oil (0.12 g, 94%); H NMR (400 MHz,
CDCl3): δ 8.88 (bs, 1H, NH), 8.75 (d, J = 2.2 Hz, 1H, ArH), 7.85 (d, J = 8.8 Hz, 1H, ArH), 7.78 (dd, J =
8.8, 2.3 Hz, 1H, ArH), 7.54–7.47 (m, 2H, ArH), 7.43-7.38 (m, 2H, ArH), 3.52 (t, J = 6.0 Hz, 2H, CH2),
3.21–3.14 (m, 2H, CH2), 2.53–2.48 (m, 2H, CH2), 2.29 (s, 6H, CH3), 1.86–1.74 (m, 4H, CH2), 1.42–1.16
(m, 10H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 191.7 (CO), 162.5 (CO),
141.2 (ArC), 137.6 (ArC), 134.6 (ArCH), 134.4 (ArC), 134.0 (ArC), 133.5 (ArCH), 129.3 (ArCH), 128.2
(ArCH), 119.6 (ArC), 118.6 (ArCH), 58.9 (CH2), 52.8 (CH2), 45.4 (CH3), 40.1 (CH2), 31.8 (CH2), 29.1
(CH2), 29.0 (CH2), 28.2 (CH2), 25.3 (CH2), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3675, 2969,
2922, 1773, 1746, 1636, 1615, 1507, 1482, 1467, 1394, 1338, 1260, 1198, 1140, 1076, 1066, 1012, 919, 852,
817, 773, 697, 600, 564, 539, 509, 492, 436, 426 cm-1; HRMS (+ ESI): Found m/z 536.2344 [M + H]+,
C27H39ClN3O4S required 536.2344.
N-(3-(Dimethylamino)propyl)-2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamide
(11d)
The titled compound was synthesised from 5-(naphthalen-2-yl)-1-(octylsulfonyl)indoline-2,3-
dione 8d (0.15 g, 0.33 mmol) and 3-dimethylaminopropylamine (42 μL, 0.33 mmol) following general
1
synthetic procedure C. The product was obtained as a yellow oil (0.18 g, 96%); H NMR (400 MHz,
CDCl3): δ 8.87 (d, J = 2.1 Hz, 1H, ArH), 8.85 (bs, 1H, NH), 8.02 (s, 1H, ArH), 7.98–7.84 (m, 5H, ArH),
7.71 (dd, J = 8.5, 1.8 Hz, 1H, ArH), 7.55–7.47 (m, 2H, ArH), 3.55 (t, J = 5.9 Hz, 2H, CH2), 3.23–3.16 (m,
2H, CH2), 2.52 (t, J = 6.2 Hz, 2H, CH2), 2.30 (s, 6H, CH3), 1.87–1.75 (m, 4H, CH2), 1.43–1.17 (m, 10H,
CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 191.9 (CO), 162.7 (CO), 141.0 (ArC),
136.4 (ArC), 135.7 (ArC), 135.2 (ArCH), 133.9 (ArCH), 133.7 (ArC), 132.9 (ArC), 128.9 (ArCH), 128.4
(ArCH), 127.8 (ArCH), 126.7 (ArCH), 126.4 (ArCH), 125.7 (ArCH), 125.1 (ArCH), 119.7 (ArC), 118.7
(ArCH), 58.9 (CH2), 52.7 (CH2), 45.4 (CH3), 40.1 (CH2), 31.8 (CH2), 29.1 (CH2), 29.0 (CH2), 28.2 (CH2),
25.3 (CH2), 23.6 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3675, 2924, 2855, 1767, 1739, 1641, 1616,
1577, 1492, 1462, 1394, 1333, 1262, 1234, 1201, 1142, 1098, 918, 892, 815, 747, 720, 670, 593, 561, 516, 475
cm-1; HRMS (+ ESI): Found m/z 552.2895 [M + H]+, C31H42N3O4S required 552.2891.
2-(5-Butyl-2-(octylsulfonamido)phenyl)-N-(3-(dimethylamino)propyl)-2-oxoacetamide (11e)
The titled compound was synthesised from 5-butyl-1-(octylsulfonyl)indoline-2,3-dione 8e (0.15
g, 0.40 mmol) and 3-dimethylaminopropylamine (50 μL, 0.40 mmol) following general synthetic
1
procedure C. The product was obtained as a yellow oil (0.18 g, 95%); H NMR (400 MHz, CDCl3): δ
8.65 (bs, 1H, NH), 8.21 (d, J = 1.8 Hz, 1H, ArH), 7.67 (d, J = 8.5 Hz, 1H, ArH), 7.41 (dd, J = 8.6, 2.0 Hz,
1H, ArH), 3.56–3.48 (m, 2H, CH2), 3.14-3.08 (m, 2H, CH2), 2.63–2.52 (m, 4H, CH2), 2.34 (s, 6H, CH3),
1.86–1.71 (m, 4H, CH2), 1.63-1.52 (m, 2H, CH2), 1.41–1.17 (m, 12H, CH2), 0.92 (t, J = 7.3 Hz, 3H, CH3),
0.85 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 192.1 (CO), 163.0 (CO), 139.5 (ArC), 137.6
(ArC), 136.7 (ArCH), 134.6 (ArCH), 119.6 (ArC), 118.5 (ArCH), 58.6 (CH2), 52.4 (CH2), 45.2 (CH3), 39.6
(CH2), 34.9 (CH2), 33.6 (CH2), 31.8 (CH2), 29.1 (CH2), 29.0 (CH2), 28.2 (CH2), 25.3 (CH2), 23.5 (CH2), 22.7
(CH2), 22.4 (CH2), 14.2 (CH3), 14.0 (CH3); IR (ATR): νmax 3674, 2968, 2922, 1609, 1497, 1458, 1405, 1393,
1331, 1256, 1141, 1066, 1057, 934, 834, 781, 732, 668, 562, 520 cm-1; HRMS (+ ESI): Found m/z 482.3047
[M + H]+, C25H44N3O4S required 482.3047.

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2-(4-(Butylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-(dimethylamino)propyl)-2-oxoacetamide (12a)
The titled compound was synthesised from 1-(butylsulfonyl)-5-phenylindoline-2,3-dione 9a
(0.15 g, 0.44 mmol) and 3-dimethylaminopropylamine (55 μL, 0.44 mmol) following general synthetic
1
procedure C. The product was obtained as a yellow oil (0.18 g, 93%); H NMR (400 MHz, CDCl3): δ
8.81 (bs, 1H, NH), 8.73 (d, J = 2.0 Hz, 1H, ArH), 7.87–7.80 (m, 2H, ArH), 7.60–7.55 (m, 2H, ArH), 7.44
(t, J = 8.0 Hz, 2H, ArH), 7.39–7.33 (m, 1H, ArH), 3.53 (t, J = 6.2 Hz, 2H, CH2), 3.22–3.15 (m, 2H, CH2),
2.51 (t, J = 6.2 Hz, 2H, CH2), 2.29 (s, 6H, CH3), 1.84–1.74 (m, 4H, CH2), 1.47–1.36 (m, 2H, CH2), 0.90 (t,
J = 7.3 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 192.0 (CO), 162.7 (CO), 140.9 (ArC), 139.1 (ArC),
135.8 (ArC), 134.9 (ArCH), 133.6 (ArCH), 129.1 (ArCH), 127.9 (ArCH), 127.0 (ArCH), 119.6 (ArC),
118.6 (ArCH), 58.8 (CH2), 52.4 (CH2), 45.3 (CH3), 40.0 (CH2), 25.5 (CH2), 25.3 (CH2), 21.5 (CH2), 13.6
(CH3); IR (ATR): νmax 2960, 2871, 2363, 2345, 2183, 2160, 2049, 1978, 1870, 1773, 1734, 1710, 1701, 1685,
1670, 1663, 1654, 1647, 1636, 1617, 1578, 1570, 1560, 1541, 1534, 1522, 1508, 1482, 1466, 1459, 1449, 1395,
1330, 1259, 1194, 1142, 1096, 1025, 921, 794, 761, 733, 697, 681, 669, 616, 583, 555, 535, 477, 428 cm-1;
HRMS (+ ESI): Found m/z 466.2109 [M + H]+, C23H32N3O4S required 466.2108.
General Synthetic Procedure D for Tertiary Ammonium Chloride Salts
To a solution of glyoxamide derivative (1.0 equivalent) in diethyl ether (5 mL), 4 M HCl/dioxane
(5.0 equivalents) was added. The reaction mixture was stirred at room temperature for 20 min. After
completion of reaction, the reaction mixture was concentrated in vacuo, washed thrice with diethyl
ether and freeze-dried to afford the product.
N,N-Dimethyl-3-(2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)propan-1-aminium
chloride (13a)
The titled compound was synthesised from N-(3-(dimethylamino)propyl)-2-(4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide 11a (33 mg, 0.066 mmol) and 4 M
HCl/dioxane (0.10 mL, 0.40 mmol) following general synthetic procedure D. The product was
obtained as a yellow sticky solid (33 mg, 93%); 1H NMR (600 MHz, DMSO-d6): δ 10.25 (bs, 2H, NH),
9.03 (t, J = 5.8 Hz, 1H, NH), 8.01–7.98 (m, 2H, ArH), 7.67-7.60 (m, 3H, ArH), 7.52–7.48 (m, 2H, ArH),
7.40 (t, J = 7.4 Hz, 1H, ArH), 3.32 (t, J = 6.5 Hz, 2H, CH2), 3.24-3.20 (m, 2H, CH2), 3.12–3.07 (m, 2H,
CH2), 2.73 (s, 6H, CH3), 1.97–1.90 (m, 2H, CH2), 1.70–1.64 (m, 2H, CH2), 1.37–1.16 (m, 10H, CH2), 0.82
(t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.1 (CO), 163.8 (CO), 138.3 (ArC), 137.9
(ArC), 135.9 (ArC), 132.9 (ArCH), 130.3 (ArCH), 129.2 (ArCH), 127.9 (ArCH), 126.5 (ArCH), 125.8
(ArC), 122.3 (ArCH), 54.4 (CH2), 51.3 (CH2), 42.0 (CH3), 36.0 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2),
27.3 (CH2), 23.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3382, 2924, 2854, 2703, 1647,
1581, 1508, 1483, 1395, 1331, 1267, 1197, 1144, 1075, 974, 919, 842, 761, 697, 681, 616, 586, 509 cm-1;
HRMS (+ ESI): Found m/z 502.2731 [M + H]+, C27H40N3O4S required 502.2734.
3-(2-(4’-Fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N-dimethylpropan-1-
aminium chloride (13b)
The titled compound was synthesised from N-(3-(dimethylamino)propyl)-2-(4’-fluoro-4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide 11b (30 mg, 0.058 mmol) and 4 M
HCl/dioxane (0.10 mL, 0.40 mmol) following general synthetic procedure D. The product was
obtained as a yellow sticky solid (32 mg, 99%); 1H NMR (600 MHz, DMSO-d6): δ 10.43 (bs, 1H, NH),
10.18 (bs, 1H, NH), 9.02 (t, J = 6.0 Hz, NH), 8.00–7.95 (m, 2H, ArH), 7.73–7.68 (m, 2H, ArH), 7.60 (d, J
= 9.0 Hz, 1H, ArH), 7.36–7.31 (m, 2H, ArH), 3.33-3.29 (m, 2H, CH2), 3.21 (t, J = 7.9 Hz, 2H, CH2), 3.12–
3.07 (m, 2H, CH2), 2.73 (s, 6H, CH3), 1.98–1.90 (m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.37–1.15 (m, 10H,
CH2), 0.82 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.0 (CO), 163.7 (CO), 162.1
(ArC), 137.8 (ArC), 135.0 (ArC), 134.8 (ArC), 132.8 (ArCH), 130.1 (ArCH), 128.6 (ArCH), 126.1 (ArC),
122.5 (ArCH), 116.0 (ArCH), 54.4 (CH2), 51.3 (CH2), 42.0 (CH3), 42.0 (CH3), 36.0 (CH2), 31.1 (CH2), 28.4
(CH2), 28.3 (CH2), 27.3 (CH2), 23.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3358, 2925,

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2855, 2690, 2360, 1647, 1603, 1515, 1488, 1400, 1332, 1222, 1196, 1143, 1099, 1012, 975, 919, 828, 725, 670,
597, 559, 520, 418 cm−1; HRMS (+ESI): Found m/z 520.2641 [M + H]+, C27H39FN3O4S required 520.2640.
3-(2-(4’-Chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N-dimethylpropan-1-
aminium chloride (13c)
The titled compound was synthesised from 2-(4’-chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-
yl)-N-(3-(dimethylamino)propyl)-2-oxoacetamide 11c (30 mg, 0.056 mmol) and 4 M HCl/dioxane
(0.10 mL, 0.40 mmol) following general synthetic procedure D. The product was obtained as a yellow
1
sticky solid (29 mg, 92%); H NMR (600 MHz, DMSO-d6): δ 10.37 (bs, 1H, NH), 10.25 (bs, 1H, NH),
9.02 (t, J = 6.0 Hz, 1H, NH), 8.02–7.98 (m, 2H, ArH), 7.71–7.68 (m, 2H, ArH), 7.63–7.60 (m, 1H, ArH),
7.57–7.54 (m, 2H, ArH), 3.34–3.30 (m, 2H, CH2), 3.21 (t, J = 7.8 Hz, 2H, CH2), 3.12–3.07 (m, 2H, CH2),
2.73 (s, 6H, CH3), 1.98-1.90 (m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.36–1.15 (m, 10H, CH2), 0.82 (t, J =
7.2 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 191.9 (CO), 163.6 (CO), 138.1 (ArC), 137.1 (ArC),
134.6 (ArC), 132.8 (ArC), 132.7 (ArCH), 130.2 (ArCH), 129.2 (ArCH), 128.3 (ArCH), 126.1 (ArC), 122.5
(ArCH), 54.4 (CH2), 51.4 (CH2), 42.1 (CH3), 36.0 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2),
23.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3372, 2924, 2854, 2704, 2359, 1643, 1578,
1507, 1481, 1400, 1333, 1273, 1196, 1143, 1092, 1012, 974, 918, 818, 758, 697, 668, 593, 511, 488 cm-1;
HRMS (+ ESI): Found m/z 536.2346 [M + H]+, C27H39ClN3O4S required 536.2344.
N,N-Dimethyl-3-(2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamido)propan-1-
aminium chloride (13d)
The titled compound was synthesised from N-(3-(dimethylamino)propyl)-2-(5-(naphthalen-2-
yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamide 11d (32 mg, 0.058 mmol) and 4 M HCl/dioxane (0.10
mL, 0.40 mmol) following general synthetic procedure D. The product was obtained as a yellow
sticky solid (33 mg, 96%); 1H NMR (600 MHz, DMSO-d6): δ 10.26 (bs, 2H, NH), 9.03 (t, J = 5.9 Hz, 1H,
NH), 8.23 (s, 1H, ArH), 8.16–8.13 (m, 2H, ArH), 8.06–8.01 (m, 2H, ArH), 7.96 (d, J = 7.6 Hz, 1H, ArH),
7.83 (dd, J = 8.5, 1.4 Hz, 1H, ArH), 7.65 (d, J = 9.1 Hz, 1H, ArH), 7.60–7.52 (m, 2H, ArH), 3.35–3.29 (m,
2H, CH2), 3.22 (t, J = 7.7 Hz, 2H, CH2), 3.14–3.09 (m, 2H, CH2), 2.74 (s, 6H, CH3), 1.99-1.92 (m, 2H, CH2),
1.72-1.64 (m, 2H, CH2), 1.39–1.14 (m, 10H, CH2), 0.82 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 192.0 (CO), 163.7 (CO), 137.8 (ArC), 135.9 (ArC), 135.6 (ArC), 133.2 (ArC), 133.0 (ArCH),
132.4 (ArC), 130.3 (ArCH), 128.7 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 126.6 (ArCH), 126.6 (ArC),
126.4 (ArCH), 125.2 (ArCH), 124.6 (ArCH), 122.8 (ArCH), 54.5 (CH2), 51.3 (CH2), 42.0 (CH3), 36.0
(CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 23.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR
(ATR): νmax 3675, 2924, 2854, 2698, 1641, 1494, 1466, 1397, 1331, 1269, 1236, 1201, 1143, 1086, 919, 892,
861, 815, 747, 720, 670, 557, 516, 476 cm−1; HRMS (+ ESI): Found m/z 552.2893 [M + H]+, C31H42N3O4S
required 552.2891.
3-(2-(5-Butyl-2-(octylsulfonamido)phenyl)-2-oxoacetamido)-N,N-dimethylpropan-1-aminium
chloride (13e)
The titled compound was synthesised from 2-(5-butyl-2-(octylsulfonamido)phenyl)-N-(3-
(dimethylamino)propyl)-2-oxoacetamide 11e (33 mg, 0.069 mmol) and 4 M HCl/dioxane (0.10 mL,
0.40 mmol) following general synthetic procedure D. The product was obtained as a yellow sticky
solid (35 mg, 99%); 1H NMR (600 MHz, DMSO-d6): δ 10.29 (bs, 1H, NH), 10.04 (bs, 1H, NH), 8.92 (t, J
= 6.0 Hz, 1H, NH), 7.54–7.50 (m, 2H, ArH), 7.41 (dd, J = 7.7, 0.9 Hz, 1H, ArH), 3.32–3.27 (m, 2H, CH2),
3.15–3.06 (m, 4H, CH2), 2.74 (s, 6H, CH3), 2.61 (t, J = 7.8 Hz, 2H, CH2), 1.96–1.89 (m, 2H, CH2), 1.66–
1.50 (m, 4H, CH2), 1.34–1.15 (m, 12H, CH2), 0.89 (t, J = 7.3 Hz, 3H, CH3), 0.84 (t, J = 7.3 Hz, 3H, CH3);
13C NMR (150 MHz, DMSO-d6): δ 192.6 (CO), 164.2 (CO), 138.6 (ArC), 136.4 (ArC), 134.8 (ArCH), 131.7
(ArCH), 125.6 (ArCH), 122.0 (ArCH), 54.5 (CH2), 51.0 (CH2), 42.1 (CH3), 35.9 (CH2), 33.8 (CH2), 32.9
(CH2), 31.1 (CH2), 28.3 (CH2), 28.3 (CH2), 27.3 (CH2), 23.9 (CH2), 22.9 (CH2), 22.0 (CH2), 32.7 (CH2), 13.9
(CH3), 13.7 (CH3); IR (ATR): νmax 3379, 2953, 2922, 2855, 2674, 2360, 1670, 1578, 1524, 1496, 1465, 1443,

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1400, 1334, 1253, 1179, 1153, 1086, 978, 918, 906, 874, 836, 799, 759, 677, 604, 570, 544, 516, 475, 432 cm−1;
HRMS (+ ESI): Found m/z 482.3046 [M + H]+, C25H44N3O4S required 482.3047.
3-(2-(4-(Butylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N-dimethylpropan-1-aminium
chloride (14a)
The titled compound was synthesised from 2-(4-(butylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-
(dimethylamino)propyl)-2-oxoacetamide 12a (35 mg, 0.079 mmol) and 4 M HCl/dioxane (0.10 mL,
0.40 mmol) following general synthetic procedure D. The product was obtained as a yellow sticky
solid (34 mg, 91%); 1H NMR (600 MHz, DMSO-d6): δ 10.30 (bs, 1H, NH), 10.18 (bs, 1h, NH), 9.03 (t, J
= 6.0 Hz, 1H, NH), 8.01–7.98 (m, 2H, ArH), 7.67–7.64 (m, 2H, ArH), 7.63–7.60 (m, 1H, ArH), 7.52–7.48
(m, 2H, ArH), 7.42–7.38 (m, 1H, ArH), 3.34–3.30 (m, 2H, CH2), 3.24–3.20 (m, 2H, CH2), 3.13–3.07 (m,
2H, CH2), 2.74 (s, 6H, CH3), 1.97–1.91 (m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.41–1.33 (m, 2H, CH2),
0.85 (t, J = 7.5 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.1 (CO), 163.8 (CO), 138.3 (ArC),
137.8 (ArC), 136.0 (ArC), 132.9 (ArCH), 130.2 (ArCH), 129.2 (ArCH), 127.9 (ArCH), 126.5 (ArCH),
125.9 (ArC), 122.4 (ArCH), 54.5 (CH2), 51.1 (CH2), 42.1 (CH3), 36.0 (CH2), 24.9 (CH2), 23.9 (CH2), 20.7
(CH2), 13.4 (CH3); IR (ATR): νmax 3363, 2960, 2872, 2690, 2361, 1643, 1581, 1508, 1483, 1394, 1330, 1266,
1239, 1196, 1144, 1076, 974, 921, 843, 800, 761, 698, 681, 616, 585, 539 cm−1; HRMS (+ ESI): Found m/z
446.2109 [M + H] +, C23H32N3O4S required 446.2108.
General Synthetic Procedure E for Quaternary Ammonium Iodide Salts
To a solution of glyoxamide derivative (1.0 equivalent) in THF (5 mL), iodomethane (2.5
equivalents) was added. The reaction mixture was stirred at room temperature for 24 h. After
completion of reaction, the reaction mixture was concentrated in vacuo, washed thrice with diethyl
ether and freeze-dried to afford the product.
N,N,N-Trimethyl-3-(2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)propan-1-
aminium iodide (15a)
The titled compound was synthesised from N-(3-(Dimethylamino)propyl)-2-(4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide 11a (33 mg, 0.066 mmol) and iodomethane
(10 μL, 0.17 mmol) following general synthetic procedure E. The product was obtained as a yellow
sticky solid (40 mg, 94%); 1H NMR (600 MHz, DMSO-d6): δ 10.10 (bs, 1H, NH), 8.98 (t, J = 5.9 Hz, 1H,
NH), 8.03–7.97 (m, 2H, ArH), 7.68–7.64 (m, 2H, ArH), 7.59–7.56 (m, 1H, ArH), 7.52–7.48 (m, 2H, ArH),
7.43–7.39 (m, 1H, ArH), 3.39–3.30 (m, 4H, CH2), 3.21–3.16 (m, 2H, CH2), 3.06 (s, 9H, CH2), 2.02–1.95
(m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.39–1.15 (m, 10H, CH3), 0.83 (t, J = 7.1 Hz, 3H, CH3); 13C NMR
(150 MHz, DMSO-d6): δ 191.8 (CO), 163.7 (CO), 138.3 (ArC), 132.7 (ArCH), 130.1 (ArCH), 129.2 (ArC),
129.2 (ArCH), 128.0 (ArCH), 126.8 (ArC), 126.5 (ArCH), 126.2 (ArC), 122.9 (ArCH), 63.5 (CH2), 52.3
(CH3), 51.2 (CH2), 35.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.6 (CH2), 22.0
(CH2), 13.9 (CH3); IR (ATR): νmax 3396, 3032, 2925, 2854, 1647, 1582, 1508, 1483, 1394, 1330, 1265, 1195,
+
1140, 1076, 915, 841, 761, 698, 681, 617, 586, 564, 505 cm−1; HRMS (+ ESI): Found m/z 516.2892 [M] ,
C28H42N3O4S required 516.2891.
3-(2-(4’-Fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N,N-trimethylpropan-
1-aminium iodide (15b)
The titled compound was synthesised from N-(3-(dimethylamino)propyl)-2-(4’-fluoro-4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide 11b (32 mg, 0.062 mmol) and iodomethane
(10 μL, 0.16 mmol) following general synthetic procedure E. The product was obtained as a yellow
sticky solid (39 mg, 95%); 1H NMR (600 MHz, DMSO-d6): δ 10.07 (bs, 1H, NH), 8.97 (t, J = 6.0 Hz, 1H,
NH), 7.99–7.94 (m, 2H, ArH), 7.73–7.69 (m, 2H, ArH), 7.56 (d, J = 8.1 Hz, 1H, ArH), 7.35–7.30 (m, 2H,
ArH), 3.39–3.30 (m, 4H, CH2), 3.17 (t, J = 7.8 Hz, 2H, CH2), 3.07 (s, 9H, CH3), 2.02–1.95 (m, 2H, CH2),
1.71–1.62 (m, 2H, CH2), 1.39–1.14 (m, 10H, CH2), 0.82 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 191.7 (CO), 163.6 (CO), 162.1 (ArC), 137.4 (ArC), 135.3 (ArC), 134.8 (ArC), 132.6 (ArCH),

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130.0 (ArCH), 128.6 (ArCH), 127.2 (ArC), 123.1 (ArCH), 116.0 (ArCH), 63.5 (CH2), 52.3 (CH3), 51.2
(CH2), 35.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.6 (CH2), 22.0 (CH2), 13.9
(CH3); IR (ATR): νmax 3410, 2925, 2855, 2359, 1647, 1603, 1516, 1488, 1400, 1331, 1262, 1221, 1196, 1141,
+
1100, 1013, 915, 882, 828, 725, 670, 559, 520 cm−1; HRMS (+ ESI): Found m/z 534.2798 [M] ,
C28H41FN3O4S required 534.2796.
3-(2-(4’-Chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N,N-trimethylpropan-
1-aminium iodide (15c)
The titled compound was synthesised from 2-(4’-chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-
yl)-N-(3-(dimethylamino)propyl)-2-oxoacetamide 11c (35 mg, 0.065 mmol) and iodomethane (10 μL,
0.16 mmol) following general synthetic procedure E. The product was obtained as a yellow sticky
solid (41 mg, 93%); 1H NMR (600 MHz, DMSO-d6): δ 10.09 (bs, 1H, NH), 8.97 (bs, 1H, NH), 8.03-7.96
(m, 2H, ArH), 7.72-7.67 (m, 2H, ArH), 7.60-7.53 (m, 3H, ArH), 3.39–3.30 (m, 4H, CH2), 3.17 (t, J = 7.7
Hz, 2H, CH2), 3.07 (s, 9H, CH3), 2.02–1.95 (m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.38–1.14 (m, 10H,
CH2), 0.82 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 191.6 (CO), 163.6 (CO), 137.1
(ArC), 132.8 (ArC), 132.6 (ArCH), 130.0 (ArCH), 129.2 (ArC), 129.1 (ArCH), 128.3 (ArCH), 128.0 (ArC),
127.1 (ArC), 123.1 (ArCH), 63.5 (CH2), 52.3 (CH3), 51.2 (CH2), 35.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3
(CH2), 27.3 (CH2), 22.9 (CH2), 22.6 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3854, 3807, 3691, 3650,
3629, 2924, 2854, 2360, 1978, 1654, 1648, 1578, 1560, 1508, 1481, 1400, 1330, 1274, 1195, 1141, 1092, 1012,
915, 818, 762, 719, 697, 669, 542, 512, 460, 452, 444, 435, 428, 420 cm−1; HRMS (+ ESI): Found m/z 550.2503
[M] +, C28H41ClN3O4S required 550.2501.
N,N,N-Trimethyl-3-(2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamido)propan-1-
aminium iodide (15d)
The titled compound was synthesised from N-(3-(dimethylamino)propyl)-2-(5-(naphthalen-2-
yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamide 11d (35 mg, 0.063 mmol) and iodomethane (10 μL,
0.16 mmol) following general synthetic procedure E. The product was obtained as a yellow sticky
solid (40 mg, 90%); 1H NMR (600 MHz, DMSO-d6): δ 10.12 (bs, 1H, NH), 9.00 (t, J = 6.1 Hz, 1H, NH),
8.24 (d, J = 1.5 Hz, 1H, ArH), 8.17–8.13 (m, 2H, ArH), 8.06–8.00 (m, 2H, ArH), 7.98–7.95 (m, 1H, ArH),
7.84 (dd, J = 8.5, 1.9 Hz, 1H, ArH), 7.62 (d, J = 8.4 Hz, 1H, ArH), 7.59–7.54 (m, 2H, ArH), 3.41–3.33 (m,
4H, CH2), 3.19 (t, J = 7.7 Hz, 2H, CH2), 3.07 (s, 9H, CH3), 2.03–1.97 (m, 2H, CH2), 1.72–1.65 (m, 2H,
CH2), 1.38–1.30 (m, 2H, CH2), 1.26–1.15 (m, 8H, CH2), 0.82 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 191.7 (CO), 163.7 (CO), 137.4 (ArC), 136.3 (ArC), 135.6 (ArC), 133.2 (ArC), 132.9 (ArCH),
132.4 (ArC), 130.2 (ArCH), 128.8 (ArCH), 128.2 (ArCH), 127.6 (ArCH), 127.5 (ArC), 126.7 (ArCH),
126.5 (ArCH), 125.3 (ArCH), 124.7 (ArCH), 123.3 (ArCH), 63.5 (CH2), 52.3 (CH3), 51.2 (CH2), 35.9
(CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.6 (CH2), 22.0 (CH2), 13.9 (CH3); IR
(ATR): νmax 3422, 3051, 2924, 2853, 2360, 1642, 1492, 1466, 1396, 1329, 1263, 1234, 1202, 1189, 1142, 1088,
915, 892, 860, 816, 749, 720, 669, 593, 560, 516, 476, 428 cm−1; HRMS ( + ESI): Found m/z 566.3048 [M] +,
C32H44N3O4S required 566.3047.
3-(2-(5-Butyl-2-(octylsulfonamido)phenyl)-2-oxoacetamido)-N,N,N-trimethylpropan-1-aminium
iodide (15e)
The titled compound was synthesised from 2-(5-butyl-2-(octylsulfonamido)phenyl)-N-(3-
(dimethylamino)propyl)-2-oxoacetamide 11e (32 mg, 0.066 mmol) and iodomethane (10 μL, 0.17
mmol) following general synthetic procedure E. The product was obtained as a yellow sticky solid
(35 mg, 85%); 1H NMR (600 MHz, DMSO-d6): δ 9.95 (bs, 1H, NH), 8.88 (t, J = 6.0 Hz, 1H, NH), 7.55–
7.51 (m, 2H, ArH), 7.40–7.36 (m, 1H, ArH), 3.40–3.27 (m, 4H, CH2), 3.12–3.05 (m, 11H, CH2, CH3), 2.61
(t, J = 7.6 Hz, 2H, CH2), 2.01–1.94 (m, 2H, CH2), 1.66–1.59 (m, 2H, CH2), 1.58–1.50 (m, 2H, CH2), 1.35–
1.15 (m, 12H, CH2), 0.89 (t, J = 7.5 Hz, 3H, CH3), 0.84 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 192.3 (CO), 164.1 (CO), 139.1 (ArC), 135.9 (ArC), 134.6 (ArCH), 131.5 (ArCH), 126.9
(ArC), 122.7 (ArCH), 63.5 (CH2), 52.3 (CH3), 50.9 (CH2), 35.8 (CH2), 33.8 (CH2), 32.9 (CH2), 31.1 (CH2),

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28.3 (CH2), 28.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.6 (CH2), 22.0 (CH2), 21.7 (CH2), 13.9 (CH3), 13.7 (CH3);
IR (ATR): νmax 3424, 2954, 2925, 2855, 2358, 1670, 1577, 1529, 1492, 1466, 1397, 1328, 1233, 1178, 1141,
1074, 914, 837, 775, 723, 668, 553, 509, 424 cm−1; HRMS (+ ESI): Found m/z 496.3202 [M] +, C26H44N3O4S
required 496.3204.
3-(2-(4-(Butylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)-N,N,N-trimethylpropan-1-
aminium iodide (16a)
The titled compound was synthesised from 2-(4-(butylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-
(dimethylamino)propyl)-2-oxoacetamide 12a (34 mg, 0.076 mmol) and iodomethane (12 μL, 0.19
mmol) following general synthetic procedure E. The product was obtained as a yellow sticky solid
(26 mg, 58%); 1H NMR (600 MHz, DMSO-d6): δ 10.08 (bs, 1H, NH), 8.98 (t, J = 5.8 Hz, 1H, NH), 8.02–
7.98 (m, 2H, ArH), 7.68–7.64 (m, 2H, ArH), 7.60–7.56 (m, 1H, ArH), 7.52–7.47 (m, 2H, ArH), 7.43–7.39
(m, 1H, ArH), 3.39–3.30 (m, 4H, CH2), 3.21–3.16 (m, 2H, CH2), 3.07 (s, 9H, CH2), 2.02–1.95 (m, 2H,
CH2), 1.70–1.63 (m, 2H, CH2), 1.41–1.33 (m, 2H, CH3), 0.85 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 191.8 (CO), 163.7 (CO), 138.3 (ArC), 132.7 (ArCH), 130.1 (ArCH), 129.2 (ArC), 129.2
(ArCH), 127.9 (ArCH), 126.9 (ArC), 126.5 (ArCH), 126.3 (ArC), 123.0 (ArCH), 63.5 (CH2), 52.3 (CH3),
51.0 (CH2), 35.9 (CH2), 25.0 (CH2), 22.6 (CH2), 20.7 (CH2), 13.5 (CH3); IR (ATR): νmax 3195, 3032, 2959,
2872, 2359, 1979, 1644, 1582, 1508, 1482, 1452, 1394, 1329, 1268, 1195, 1140, 1076, 920, 842, 762, 699, 681,
616, 584, 536, 428 cm−1; HRMS (+ ESI): Found m/z 460.2264 [M] +, C24H34N3O4S required 460.2265.
General Synthetic Procedure F for Boc-Protected Glyoxamide Derivatives
To a solution of N-sulfonylisatin (1.0 equivalent) in dichloromethane (10 mL), N-Boc-1,3-
propandiamine (1.0 equivalent) in dichloromethane (5 mL) was added dropwise with stirring at 0 °C.
The reaction mixture was stirred at room temperature for 6 h. After completion of the reaction, water
was added to the reaction mixture and the product was extracted into dichloromethane (3 × 30 mL),
washed with brine, dried over anhydrous sodium sulphate and concentrated in vacuo to afford the
product.
tert-Butyl (3-(2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)propyl)carbamate (18a)
The titled compound was synthesised from 1-(octylsulfonyl)-5-phenylindoline-2,3-dione 8a
(0.32 g, 0.81 mmol) and N-Boc-1,3-propandiamine (0.15 g, 0.82 mmol) following general synthetic
procedure F. The product was obtained as a yellow solid (0.45 g, 97%); mp 127.3–127.4 °C; 1H NMR
(600 MHz, CDCl3): δ 10.48 (bs, 1H, NH), 8.75 (s, 1H, ArH), 7.88–7.81 (m, 2H, ArH), 7.71 (bs, 1H, NH),
7.59–7.55 (m, 2H, ArH), 7.47–7.43 (m, 2H, ArH), 7.39–7.35 (m, 1H, ArH), 4.81 (bs, 1H, NH), 3.48 (q, J
= 6.4 Hz, 2H, CH2), 3.27–3.21 (m, 2H, CH2), 3.21–3.16 (m, 2H, CH2), 1.85–1.78 (m, 2H, CH2), 1.78–1.73
(m, 2H, CH2), 1.44 (s, 9H, CH3), 1.42–1.34 (m, 2H, CH2), 1.30–1.16 (m, 8H, CH2), 0.85 (t, J = 7.2 Hz, 3H,
CH3); 13C NMR (150 MHz, CDCl3): δ 191.5 (CO), 162.9 (CO), 156.9 (CO), 141.1 (ArC), 139.1 (ArC), 135.7
(ArC), 135.1 (ArCH), 133.7 (ArCH), 129.2 (ArCH), 127.9 (ArCH), 126.9 (ArCH), 119.3 (ArC), 118.5
(ArCH), 79.9 (C), 52.8 (CH2), 37.3 (CH2), 36.4 (CH2), 31.8 (CH2), 30.2 (CH2), 29.1 (CH2), 29.0 (CH2), 28.5
(CH3), 28.2 (CH2), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3352, 3310, 2919, 2359, 1682, 1666,
1582, 1518, 1483, 1443, 1386, 1364, 1346, 1325, 1294, 1246, 1197, 1171, 1155, 1124, 1069, 1041, 1011, 977,
968, 906, 894, 851, 830, 804, 760, 741, 714, 684, 639, 609, 554, 537, 491, 453, 423 cm−1; HRMS (+ ESI):
Found m/z 596.2764 [M + Na] +, C30H43N3O6SNa required 596.2764.
tert-Butyl (3-(2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)propyl)
carbamate (18b)
The titled compound was synthesised from 5-(4-fluorophenyl)-1-(octylsulfonyl)indoline-2,3-
dione 8b (0.28 g, 0.67 mmol) and N-Boc-1,3-propandiamine (0.12 g, 0.67 mmol) following general
synthetic procedure F. The product was obtained as a yellow solid (0.38 g, 96%); mp 138.0–140.3 °C;
1H NMR (400 MHz, CDCl3): δ 10.46 (bs, 1H, NH), 8.72 (s, 1H, ArH), 7.85 (d, J = 8.7 Hz, 1H, ArH), 7.81–
7.69 (m, 2H, NH, ArH), 7.56–7.50 (m, 2H, ArH), 7.17–7.09 (m, 2H, ArH), 4.81 (t, J = 5.9 Hz, 1H, NH),

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3.47 (q, J = 6.4 Hz, 2H, CH2), 3.29–3.14 (m, 4H, CH2), 1.86–1.71 (m, 4H, CH2), 1.44 (s, 9H, CH3), 1.42–
1.33 (m, 2H, CH2), 1.31–1.18 (m, 8H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ
191.3 (CO), 162.8 (CO), 162.8 (ArC), 156.9 (CO), 141.1 (ArC), 135.2 (ArC), 134.9 (ArCH), 134.7 (ArC),
133.5 (ArCH), 128.6 (ArCH), 119.3 (ArC), 118.6 (ArCH), 116.1 (ArCH), 79.9 (C), 52.8 (CH2), 37.3 (CH2),
36.4 (CH2), 31.8 (CH2), 30.2 (CH2), 29.1 (CH2), 29.0 (CH2), 28.5 (CH3), 28.2 (CH2), 23.5 (CH2), 22.7 (CH2),
14.2 (CH3); IR (ATR): νmax 3357, 3313, 2923, 2856, 2303, 1887, 1680, 1645, 1519, 1487, 1388, 1344, 1247,
+
1155, 1070, 1011, 976, 906, 854, 826, 771 cm−1; HRMS (+ ESI): Found m/z 614.2670 [M + Na] ,
C30H42FN3O6SNa required 614.2671.
tert-Butyl (3-(2-(4’-chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamido)propyl)
carbamate (18c)
The titled compound was synthesised from 5-(4-chlorophenyl)-1-(octylsulfonyl)indoline-2,3-
dione 8c (0.30 g, 0.69 mmol) and N-Boc-1,3-propandiamine (0.13 g, 0.71 mmol) following general
synthetic procedure F. The product was obtained as a yellow solid (0.41 g, 97%); mp 134.7–135.1 °C;
1H NMR (600 MHz, CDCl3): δ 10.49 (bs, 1H, NH), 8.74 (s, 1H, ArH), 7.85 (d, J = 8.7 Hz, 1H, ArH), 7.78
(dd, J = 8.7, 2.3 Hz, 1H, ArH), 7.77 (bs, 1H, NH), 7.51–7.48 (m, 2H, ArH), 7.43–7.40 (m, 2H, ArH), 4.81
(bs, 1H, NH), 3.47 (q, J = 6.4 Hz, 2H, CH2), 3.27–3.22 (m, 2H, CH2), 3.20–3.16 (m, 2H, CH2), 1.84–1.72
(m, 4H, CH2), 1.44 (s, 9H, CH3), 1.41–1.34 (m, 2H, CH2), 1.29–1.18 (m, 8H, CH2), 0.85 (t, J = 7.2 Hz, 3H,
CH3); 13C NMR (150 MHz, CDCl3): δ 191.3 (CO), 162.7 (CO), 156.9 (CO), 141.3 (ArC), 137.5 (ArC), 134.8
(ArCH), 134.4 (ArC), 134.1 (ArC), 133.5 (ArCH), 129.3 (ArCH), 128.2 (ArCH), 119.3 (ArC), 118.6
(ArCH), 79.9 (C), 52.8 (CH2), 37.3 (CH2), 36.4 (CH2), 31.8 (CH2), 30.2 (CH2), 29.1 (CH2), 29.0 (CH2), 28.5
(CH3), 28.2 (CH2), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3352, 3311, 2922, 2855, 2359, 1979,
1682, 1665, 1643, 1581, 1519, 1483, 1443, 1388, 1364, 1345, 1246, 1197, 1155, 1138, 1093, 1070, 1041, 1010,
975, 907, 895, 882, 864, 817, 759, 741, 714, 685, 643, 609, 555, 537, 492, 471, 418 cm−1; HRMS (+ ESI):
Found m/z 630.2379 [M + Na] +, C30H42ClN3O6SNa required 630.2375.
tert-Butyl (3-(2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamido)propyl)
carbamate (18d)
The titled compound was synthesised from 5-(naphthalen-2-yl)-1-(octylsulfonyl)indoline-2,3-
dione 8d (0.31 g, 0.70 mmol) and N-Boc-1,3-propandiamine (0.13 g, 0.70 mmol) following general
synthetic procedure F. The product was obtained as a yellow solid (0.41 g, 95%); mp 78.9–80.0 °C; 1H
NMR (600 MHz, CDCl3): δ 10.51 (bs, 1H, NH), 8.87 (s, 1H, ArH), 8.01 (s, 1H, ArH), 7.99–7.83 (m, 5H,
ArH), 7.79–7.67 (m, 2H, NH, ArH), 7.55–7.46 (m, 2H, ArH), 4.84 (bs, 1H, NH), 3.49 (q, J = 6.5 Hz, 2H,
CH2), 3.30–3.16 (m, 4H, CH2), 1.88–1.72 (m, 4H, CH2), 1.44 (s, 9H, CH3), 1.46–1.34 (m, 2H, CH2), 1.31–
1.16 (m, 8H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3): δ 191.5 (CO), 162.9 (CO),
156.9 (CO), 141.1 (ArC), 136.3 (ArC), 135.6 (ArC), 135.4 (ArCH), 133.8 (ArCH), 133.7 (ArC), 132.9
(ArC), 128.9 (ArCH), 128.4 (ArCH), 127.8 (ArCH), 126.7 (ArCH), 126.4 (ArCH), 125.7 (ArCH), 125.0
(ArCH), 119.4 (ArC), 118.6 (ArCH), 79.9 (C), 52.8 (CH2), 37.3 (CH2), 36.5 (CH2), 31.8 (CH2), 30.2 (CH2),
29.1 (CH2), 29.0 (CH2), 28.5 (CH3), 28.2 (CH2), 23.6 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3346,
3055, 2925, 2855, 2285, 2081, 1911, 1683, 1636, 1572, 1497, 1466, 1393, 1365, 1336, 1247, 1140, 1012, 917,
890, 813, 747 cm−1; HRMS (+ ESI): Found m/z 646.2919 [M + Na] +, C34H45N3O6SNa required 646.2921.
General Synthetic Procedure G for Aminoglyoxamides
To a solution of Boc-protected glyoxamide (1.0 equivalent) in dichloromethane (10 mL), 4 M
HCl/dioxane (3 mL) was added. The reaction mixture was stirred at room temperature for 6 h. After
completion of reaction, the reaction mixture was concentrated in vacuo, washed thrice with diethyl
ether and dried under high vacuum to afford the product.
N-(3-Aminopropyl)-2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide hydrochloride
(19a)

Int. J. Mol. Sci. 2020, 21, 6789
27 of 38
The titled compound was synthesised from tert-butyl (3-(2-(4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamido)propyl)carbamate 18a (0.35 g, 0.61 mmol) following general
synthetic procedure G. The product was obtained as a yellow solid (0.20 g, 64%); mp 126.2–128.7 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.20 (bs, 1H, NH), 9.05 (t, J = 6.0 Hz, 1H, NH), 8.19–7.91 (m, 5H,
NH, ArH), 7.66–7.61 (m, 3H, ArH), 7.51 (t, J = 7.8 Hz, 2H, ArH), 7.41 (t, J = 7.2 Hz, 1H, ArH), 3.38–3.29
(m, 2H, CH2), 3.29–3.22 (m, 2H, CH2), 2.91–2.82 (m, 2H, CH2), 1.89–1.79 (m, 2H, CH2), 1.72–1.62 (m,
2H, CH2), 1.39–1.14 (m, 10H, CH2), 0.82 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 192.4
(CO), 163.8 (CO), 138.3 (ArC), 138.2 (ArC), 135.6 (ArC), 133.2 (ArCH), 130.5 (ArCH), 129.2 (ArCH),
127.9 (ArCH), 126.4 (ArCH), 124.5 (ArC), 121.7 (ArCH), 51.4 (CH2), 36.7 (CH2), 35.9 (CH2), 31.1 (CH2),
28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 26.9 (CH2), 22.9 (CH2), 22.0 (CH2), 14.2 (CH3); IR (ATR): νmax 3031,
2921, 2853, 2045, 1961, 1635, 1509, 1482, 1393, 1335, 1264, 1196, 1138, 1025, 917, 838, 759, 696 cm−1;
HRMS (+ ESI): Found m/z 474.2419 [M + H] +, C25H36N3O4S required 474.2421.
N-(3-Aminopropyl)-2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide
hydrochloride (19b)
The titled compound was synthesised from tert-butyl (3-(2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamido)propyl) carbamate 18b (0.34 g, 0.58 mmol) following general
synthetic procedure G. The product was obtained as a yellow solid (0.27 g, 90%); mp 155.2–157.8 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.19 (bs, 1H, NH), 9.05 (t, J = 5.9 Hz, 1H, NH), 8.14–7.93 (m, 5H,
NH, ArH), 7.72–7.65 (m, 2H, ArH), 7.65–7.59 (m, 1H, ArH), 7.34 (t, J = 8.9 Hz, 2H, ArH), 3.37–3.29 (m,
2H, CH2), 3.28–3.21 (m, 2H, CH2), 2.92–2.80 (m, 2H, CH2), 1.90–1.79 (m, 2H, CH2), 1.72–1.61 (m, 2H,
CH2), 1.38–1.14 (m, 10H, CH2), 0.82 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 192.3
(CO), 163.7 (CO), 162.1 (ArC), 138.2 (ArC), 134.7 (ArC), 134.6 (ArC), 133.0 (ArCH), 130.3 (ArCH), 128.5
(ArCH), 124.7 (ArC), 121.8 (ArCH), 116.0 (ArCH), 51.4 (CH2), 36.6 (CH2), 35.9 (CH2), 31.1 (CH2), 28.3
(CH2), 28.3 (CH2), 27.3 (CH2), 26.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3365, 3161,
2970, 2925, 2753, 2611, 2494, 2343, 2058, 1919, 1633, 1600, 1528, 1491, 1393, 1334, 1259, 1203, 1140, 1083,
+
1021, 919, 868, 823, 761 cm−1; HRMS (+ ESI): Found m/z 492.2325 [M + H] , C25H35FN3O4S required
492.2327.
N-(3-Aminopropyl)-2-(4’-chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide
hydrochloride (19c)
The titled compound was synthesised from tert-butyl (3-(2-(4’-chloro-4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamido)propyl) carbamate 18c (0.38 g, 0.62 mmol) following general
synthetic procedure G. The product was obtained as a yellow solid (0.28 g, 83%); mp 118.8–119.1 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.19 (bs, 1H, NH), 9.04 (t, J = 6.0 Hz, 1H, NH), 8.12–7.88 (m, 5H,
NH, ArH), 7.71–7.60 (m, 3H, ArH), 7.57 (d, J = 8.5 Hz, 2H, ArH), 3.38–3.29 (m, 2H, CH2), 3.28–3.21 (m,
2H, CH2), 2.92–2.81 (m, 2H, CH2), 1.89–1.79 (m, 2H, CH2), 1.72–1.61 (m, 2H, CH2), 1.39–1.13 (m, 10H,
CH2), 0.82 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 192.2 (CO), 163.7 (CO), 138.4
(ArC), 137.0 (ArC), 134.3 (ArC), 133.0 (ArCH), 132.8 (ArC), 130.4 (ArCH), 129.2 (ArCH), 128.3 (ArCH),
124.8 (ArC), 121.9 (ArCH), 51.5 (CH2), 36.7 (CH2), 35.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3
(CH2), 26.9 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3808, 2924, 2855, 2360, 2038, 1657,
1636, 1578, 1527, 1509, 1483, 1397, 1340, 1270, 1201, 1139, 1095, 1046, 1010, 919, 875, 815, 772, 700, 668,
596, 562, 494 cm−1; HRMS (+ ESI): Found m/z 508.2033 [M + H] +, C25H35ClN3O4S required 508.2031.
N-(3-Aminopropyl)-2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamide
hydrochloride (19d)
The titled compound was synthesised from tert-Butyl (3-(2-(5-(naphthalen-2-yl)-2-
(octylsulfonamido)phenyl)-2-oxoacetamido)propyl) carbamate 18d (0.37 g, 0.59 mmol) following
general synthetic procedure G. The product was obtained as a yellow solid (0.22 g, 67%); mp 85.2–
87.3 °C; 1H NMR (400 MHz, DMSO-d6): δ 10.23 (bs, 1H, NH), 9.07 (t, J = 5.9 Hz, 1H, NH), 8.22 (s, 1H,
ArH), 8.18–8.12 (m, 2H, ArH), 8.10–7.93 (m, 6H, NH, ArH), 7.81 (dd, J = 8.6, 1.8 Hz, 1H, ArH), 7.68

Int. J. Mol. Sci. 2020, 21, 6789
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(dd, J = 6.4, 2.8 Hz, 1H, ArH),7.60–7.52 (m, 2H, ArH), 3.40–3.31 (m, 2H, CH2), 3.29–3.22 (m, 2H, CH2),
2.92–2.82 (m, 2H, CH2), 1.92–1.82 (m, 2H, CH2), 1.74–1.63 (m, 2H, CH2), 1.39–1.14 (m, 10H, CH2), 0.81
(t, J = 7.0 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 192.3 (CO), 163.8 (CO), 138.2 (ArC), 135.6
(ArC), 133.2 (ArC), 133.2 (ArCH), 132.3 (ArC), 130.6 (ArCH), 128.9 (ArCH), 128.3 (ArCH), 127.6 (ArC),
127.6 (ArCH), 126.6 (ArCH), 126.4 (ArCH), 125.2 (ArC), 125.2 (ArCH), 124.6 (ArCH), 122.1 (ArCH),
51.4 (CH2), 36.7 (CH2), 35.9 (CH2), 31.1 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 26.9 (CH2), 22.9 (CH2),
22.0 (CH2), 13.9 (CH3); IR (ATR): νmax3312, 3176, 2921, 2852, 2321, 2050, 1922, 1635, 1494, 1463, 1396,
+
1333, 1264, 1200, 1138, 1016, 916, 812, 747 cm−1; HRMS ( + ESI): Found m/z 524.2579 [M + H] ,
C29H38N3O4S required 524.2578.
General Synthetic Procedure H for Boc-Protected Guanidine Glyoxamides
To
a solution of aminoglyoxamides (1.0 equivalent) and N,N’-di-Boc-1H-pyrazole-1-
carboxamidine (1.3 equivalents) in acetonitrile (10 mL), triethylamine (2.5 equivalents) in acetonitrile
(5 mL) was added dropwise with stirring at 0 °C under nitrogen atmosphere. The reaction mixture
was stirred at room temperature for 18 h. After completion of the reaction, the reaction mixture was
concentrated in vacuo. The product was purified by flash chromatography on silica using ethyl
acetate/n-hexane (1:4) as eluent to afford the product.
(E)-1-tert-Butyl-N-(N’-((tert-butyloxidanyl)carbonyl)-N-(3-(2-(4-(octylsulfonamido)-[1,1’-biphenyl]-
3-yl)-2-oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide (21a)
The titled compound was synthesised from N-(3-aminopropyl)-2-(4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamide hydrochloride 19a (0.16 g, 0.30 mmol), N,N’-di-Boc-1H-pyrazole-1-
carboxamidine (0.14 g, 0.45 mmol) and triethylamine (0.12 mL, 0.83 mmol) following general
synthetic procedure H. The product was obtained as a yellow solid (68 mg, 31%); mp 69.9–70.1 °C;
1H NMR (400 MHz, CDCl3): δ 11.47 (bs, 1H, NH), 10.62 (bs, 1H, NH), 8.68–8.50 (m, 3H, NH, ArH),
7.89–7.80 (m, 2H, ArH), 7.57 (d, J = 7.5 Hz, 2H, ArH), 7.45 (t, J = 7.9 Hz, 2H, ArH), 7.36 (t, J = 7.1 Hz,
1H, ArH), 3.62–3.52 (m, 2H, CH2), 3.46 (q, J = 6.4 Hz, 2H, CH2), 3.21–3.13 (m, 2H, CH2), 1.87–1.76 (m,
4H, CH2), 1.50 (s, 9H, CH3), 1.38 (s, 9H, CH3), 1.43–1.18 (m, 10H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C
NMR (150 MHz, CDCl3): δ 192.6 (CO), 163.7 (CO), 163.0 (CN), 157.4 (CO), 153.3 (CO), 141.2 (ArC),
139.1 (ArC), 135.6 (ArC), 135.0 (ArCH), 133.5 (ArCH), 129.1 (ArCH), 127.9 (ArCH), 127.0 (ArCH),
119.0 (ArC), 118.4 (ArCH), 83.8 (C), 79.9 (C), 52.8 (CH2), 37.3 (CH2), 35.9 (CH2), 31.8 (CH2), 30.1 (CH2),
29.1 (CH2), 29.0 (CH2), 28.4 (CH2), 28.3 (CH3), 28.2 (CH3), 23.6 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR):
νmax 3323, 2928, 2360, 1979, 1720, 1638, 1571, 1508, 1483, 1450, 1410, 1366, 1328, 1284, 1228, 1195, 1131,
1051, 1026, 978, 907, 855, 806, 760, 697, 681, 616, 587, 562, 537, 418 cm−1; HRMS (+ ESI): Found m/z
716.3684 [M + H] +, C36H54N5O8S required 716.3688.
(E)-1-tert-Butyl-N-(N’-((tert-butyloxidanyl)carbonyl)-N-(3-(2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide (21b)
The
titled
compound
was
synthesised
from
N-(3-aminopropyl)-2-(4’-fluoro-4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide hydrochloride 19b (0.24 g, 0.45 mmol),
N,N’-di-Boc-1H-pyrazole-1-carboxamidine (0.20 g, 0.64 mmol) and triethylamine (0.16 mL, 1.11 mmol)
following general synthetic procedure H. The product was obtained as a yellow solid (0.25, 77%); mp
62.8–65.0 °C; 1H NMR (400 MHz, CDCl3): δ 11.47 (bs, 1H, NH), 10.61 (bs, 1H, NH), 8.64 (t, J = 6.0 Hz,
1H, NH), 8.58 (bs, 1H, NH), 8.52 (d, J = 2.1 Hz, 1H, ArH), 7.85 (t, J = 7.9 Hz, 1H, ArH), 7.76 (dd, J = 8.7,
2.2 Hz, 1H, ArH), 7.56–7.49 (m, 2H, ArH), 7.13 (t, J = 8.6 Hz, 2H, ArH), 3.63–3.53 (m, 2H, CH2), 3.46
(q, J = 6.2 Hz, 2H, CH2), 3.20–3.13 (m, 2H, CH2), 1.86–1.76 (m, 4H, CH2), 1.50 (s, 9H, CH3), 1.38 (s, 9H,
CH3), 1.43–1.16 (m, 10H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ 192.3 (CO),
163.6 (CO), 162.8 (ArC), 162.7 (CN), 157.3 (CO), 153.3 (CO), 141.1 (ArC), 135.3 (ArC), 134.7 (ArCH),
134.7 (ArC), 133.3 (ArCH), 128.6 (ArCH), 119.1 (ArC), 118.5 (ArCH), 116.1 (ArCH), 83.9 (C), 80.1 (C),
52.8 (CH2), 37.3 (CH2), 35.9 (CH2), 31.8 (CH2), 30.0 (CH2), 29.1 (CH2), 29.0 (CH2), 28.4 (CH2), 28.2 (CH3),
28.2 (CH3), 23.5 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3324, 2926, 2855, 2322, 1890, 1720, 1638,

Int. J. Mol. Sci. 2020, 21, 6789
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1570, 1487, 1408, 1326, 1258, 1225, 1128, 1049, 906, 858, 800, 731, 669 cm−1; HRMS ( + ESI): Found m/z
734.3594 [M + H] +, C36H53FN5O8S required 734.3593.
(E)-1-tert-Butyl-N-(N’-((tert-butyloxidanyl)carbonyl)-N-(3-(2-(4’-chloro-4-(octylsulfonamido)-[1,1’-
biphenyl]-3-yl)-2-oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide (21c)
The
titled
compound
was
synthesised
from
N-(3-aminopropyl)-2-(4’-chloro-4-
(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide hydrochloride 19c (0.16 g, 0.29 mmol), N,N’-
di-Boc-1H-pyrazole-1-carboxamidine (0.12 g, 0.38 mmol) and triethylamine (0.10 mL, 0.72 mmol)
following general synthetic procedure H. The product was obtained as a yellow solid (0.14 g, 64%);
mp 77.1–77.4 °C; 1H NMR (400 MHz, CDCl3): δ 11.47 (bs, 1H, NH), 10.63 (bs, 1H, NH), 8.65 (t, J = 6.3
Hz, 1H, NH), 8.61–8.51 (m, 2H, NH, ArH), 7.86 (d, J = 8.7 Hz, 1H, ArH), 7.77 (dd, J = 8.7, 2.2 Hz, 1H,
ArH), 7.52–7.47 (m, 2H, ArH), 7.43–7.39 (m, 2H, ArH), 3.62–3.52 (m, 2H, CH2), 3.46 (q, J = 6.3 Hz, 2H,
CH2), 3.20–3.13 (m, 2H, CH2), 1.86–1.75 (m, 4H, CH2), 1.50 (s, 9H, CH3), 1.38 (s, 9H, CH3), 1.43–1.18
(m, 10H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3): δ 192.3 (CO), 163.5 (CO),
163.1 (CN), 157.5 (CO), 153.4 (CO), 141.5 (ArC), 137.6 (ArC), 134.7 (ArCH), 134.3 (ArC), 134.1 (ArC),
133.4 (ArCH), 129.3 (ArCH), 128.2 (ArCH), 118.9 (ArC), 118.4 (ArCH), 83.8 (C), 79.8 (C), 52.9 (CH2),
37.2 (CH2), 35.9 (CH2), 31.8 (CH2), 30.1 (CH2), 29.1 (CH2), 29.0 (CH2), 28.4 (CH2), 28.3 (CH3), 28.2 (CH3),
23.6 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3322, 2929, 1720, 1637, 1577, 1507, 1482, 1409, 1367,
1328, 1285, 1253, 1228, 1195, 1131, 1094, 1050, 1026, 1012, 979, 907, 856, 817, 770, 698, 657, 592, 562, 489,
419 cm−1; HRMS (+ ESI): Found m/z 772.3120 [M + Na] +, C36H52ClN5O8SNa required 772.3117.
(E)-1-tert-Butyl-N-(N’-((tert-butyloxidanyl)carbonyl)-N-(3-(2-(5-(naphthalen-2-yl)-2-
(octylsulfonamido)phenyl)-2-oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide (21d)
The titled compound was synthesised from N-(3-Aminopropyl)-2-(5-(naphthalen-2-yl)-2-
(octylsulfonamido)phenyl)-2-oxoacetamide hydrochloride 19d (0.18 g, 0.33 mmol), N,N’-di-Boc-1H-
pyrazole-1-carboxamidine (0.13 g, 0.40 mmol) and triethylamine (0.12 mL, 0.83 mmol) following
general synthetic procedure H. The product was obtained as a yellow solid (0.13 g, 52%); mp 71.6–
73.5 °C; 1H NMR (400 MHz, CDCl3): δ 11.47 (bs, 1H, NH), 10.66 (bs, 1H, NH), 8.71–8.53 (m, 3H, NH,
ArH), 8.01 (s, 1H, ArH), 7.98–7.84 (m, 5H, ArH), 7.71 (dd, J = 8.5, 1.7 Hz, 1H, ArH), 7.55–7.46 (m, 2H,
ArH), 3.64–3.54 (m, 2H, CH2), 3.48 (q, J = 6.2 Hz, 2H, CH2), 3.23–3.15 (m, 2H, CH2), 1.89–1.76 (m, 4H,
CH2), 1.50 (s, 9H, CH3), 1.39 (s, 9H, CH3), 1.44–1.17 (m, 10H, CH2), 0.85 (t, J = 7.1 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3): δ 192.6 (CO), 163.8 (CO), 162.8 (CN), 157.3 (CO), 153.3 (CO), 141.2 (ArC),
136.4 (ArC), 135.6 (ArC), 135.2 (ArC), 133.7 (ArCH), 133.7 (ArCH), 132.9 (ArC), 128.9 (ArCH), 128.3
(ArCH), 127.8 (ArCH), 126.7 (ArCH), 126.4 (ArCH), 125.7 (ArCH), 125.1 (ArCH), 119.2 (ArC), 118.5
(ArCH), 83.9 (C), 80.2 (C), 52.8 (CH2), 37.5 (CH2), 35.9 (CH2), 31.8 (CH2), 30.0 (CH2), 29.1 (CH2), 29.0
(CH2), 28.3 (CH2), 28.3 (CH3), 28.2 (CH3), 23.6 (CH2), 22.7 (CH2), 14.2 (CH3); IR (ATR): νmax 3323, 2928,
2360, 1979, 1720, 1638, 1571, 1508, 1483, 1450, 1410, 1366, 1328, 1284, 1228, 1195, 1131, 1051, 1026, 978,
907, 855, 806, 760, 697, 681, 616, 587, 562, 537, 418 cm−1; HRMS (+ ESI): Found m/z 766.3845 [M + H] +,
C40H56N5O8S required 766.3844.
General Synthetic Procedure I for Guanidinium Hydrochloride Salts
To a solution of Boc-protected guanidine glyoxamide (1.0 equivalent) in dichloromethane (1
mL), trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred at room temperature for
3 h. After completion of the reaction, the reaction mixture was concentrated in vacuo and washed
thrice with diethyl ether. To the residue in dichloromethane (1 mL), 4 M HCl/dioxane (1 mL) was
added. The reaction mixture was stirred at room temperature for 30 min. After completion of reaction,
the reaction mixture was concentrated in vacuo, washed thrice with diethyl ether and freeze-dried to
afford the product.
N-(3-Guanidinopropyl)-2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-oxoacetamide hydrochloride
(22a)

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The
titled
compound
was
synthesised
from
(E)-1-tert-Butyl-N-(N’-((tert-
butyloxidanyl)carbonyl)-N-(3-(2-(4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-
oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide 21a (40 mg, 0.056 mmol) following
1
general synthetic procedure I. The product was obtained as a yellow sticky solid (0.15 g, 50%); H
NMR (600 MHz, DMSO-d6): δ 10.20 (bs, 1H, NH), 9.01 (t, J = 5.7 Hz, 1H, NH), 8.03–7.99 (m, 2H, ArH),
7.69–7.61 (m, 4H, NH, ArH), 7.57–6.78 (m, 7H, NH, ArH), 3.30 (q, J = 6.5 Hz, 2H, CH2), 3.28–3.23 (m,
2H, CH2), 3.19 (q, J = 6.5 Hz, 2H, CH2), 1.79–1.63 (m, 4H, CH2), 1.37–1.29 (m, 2H, CH2), 1.26–1.15 (m,
8H, CH2), 0.82 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.6 (CO), 163.8 (CO), 156.9
(CN), 138.3 (ArC), 138.2 (ArC), 135.6 (ArC), 133.2 (ArCH), 130.5 (ArCH), 129.2 (ArCH), 127.9 (ArCH),
126.4 (ArCH), 124.5 (ArC), 121.6 (ArCH), 51.4 (CH2), 38.4 (CH2), 36.1 (CH2), 31.1 (CH2), 28.4 (CH2),
28.4 (CH2), 28.3 (CH2), 27.3 (CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3364, 3163, 2924,
2654, 2360, 1626, 1581, 1528, 1510, 1485, 1459, 1395, 1345, 1265, 1198, 1139, 1067, 924, 909, 849, 759, 683,
621, 587, 560, 530, 481, 424 cm−1; HRMS (+ ESI): Found m/z 516.2636 [M + H] +, C26H38N5O4S required
516.2639.
2-(4’-Fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-guanidinopropyl)-2-oxoacetamide
hydrochloride (22b)
The
titled
compound
was
synthesised
from
(E)-1-tert-Butyl-N-(N’-((tert-
butyloxidanyl)carbonyl)-N-(3-(2-(4’-fluoro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-
oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide 21b (0.10 g, 0.13 mmol) following
1
general synthetic procedure I. The product was obtained as a yellow sticky solid (49 mg, 61%); H
NMR (600 MHz, DMSO-d6): δ 10.19 (bs, 1H, NH), 9.01 (t, J = 5.9 Hz, 1H, NH), 8.00–7.96 (m, 2H, ArH),
7.74 (t, J = 5.8 Hz, 1H, NH), 7.71–7.66 (m, 2H, ArH), 7.64–7.60 (m, 1H, ArH), 7.58–6.80 (m, 6H, NH,
ArH), 3.30 (q, J = 6.7 Hz, 2H, CH2), 3.26–3.22 (m, 2H, CH2), 3.20 (q, J = 6.7 Hz, 2H, CH2), 1.77–1.71 (m,
2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.36–1.30 (m, 2H, CH2), 1.26–1.15 (m, 8H, CH2), 0.82 (t, J = 7.2 Hz,
3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.4 (CO), 163.7 (CO), 162.1 (ArC), 157.0 (CN), 138.2
(ArC), 134.7 (ArC), 134.6 (ArC), 133.0 (ArCH), 130.4 (ArCH), 128.5 (ArCH), 124.6 (ArC), 121.8 (ArCH),
116.0 (ArCH), 51.4 (CH2), 38.4 (CH2), 36.1 (CH2), 31.1 (CH2), 28.4 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3
(CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3313, 3155, 2923, 2853, 2292, 1910, 1639, 1487,
1396, 1330, 1195, 1137, 913, 826, 722 cm−1; HRMS (+ ESI): Found m/z 534.2544 [M + H] +, C26H37FN5O4S
required 534.2545.
2-(4’-Chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-N-(3-guanidinopropyl)-2-oxoacetamide
hydrochloride (22c)
The
titled
compound
was
synthesised
from
(E)-1-tert-butyl-N-(N’-((tert-
butyloxidanyl)carbonyl)-N-(3-(2-(4’-chloro-4-(octylsulfonamido)-[1,1’-biphenyl]-3-yl)-2-
oxoacetamido)propyl)carbamimidoyl)-1-oxidanecarboxamide 21c (91 mg, 0.12 mmol) following
1
general synthetic procedure I. The product was obtained as a yellow sticky solid (43 mg, 60%); H
NMR (600 MHz, DMSO-d6): δ 10.20 (bs, 1H, NH), 9.00 (t, J = 5.8 Hz, 1H, NH), 8.03–7.98 (m, 2H, ArH),
7.71–7.66 (m, 3H, NH, ArH), 7.63 (dd, J = 7.6, 1.5 Hz, 1H, ArH), 7.58–7.54 (m, 2H, ArH), 7.52–6.84 (bs,
4H, NH), 3.30 (q, J = 6.7 Hz, 2H, CH2), 3.27–3.22 (m, 2H, CH2), 3.19 (q, J = 6.6 Hz, 2H, CH2), 1.77–1.71
(m, 2H, CH2), 1.70–1.63 (m, 2H, CH2), 1.36–1.30 (m, 2H, CH2), 1.26–1.15 (m, 8H, CH2), 0.82 (t, J = 7.2
Hz, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 192.3 (CO), 163.7 (CO), 156.9 (CN), 138.5 (ArC), 137.1
(ArC), 134.2 (ArC), 133.0 (ArCH), 132.8 (ArC), 130.4 (ArCH), 129.2 (ArCH), 128.2 (ArCH), 124.7 (ArC),
121.8 (ArCH), 51.5 (CH2), 38.4 (CH2), 36.1 (CH2), 31.1 (CH2), 28.4 (CH2), 28.4 (CH2), 28.3 (CH2), 27.3
(CH2), 22.9 (CH2), 22.0 (CH2), 13.9 (CH3); IR (ATR): νmax 3164, 2925, 2854, 2360, 1640, 1534, 1507, 1481,
1397, 1332, 1273, 1197, 1139, 1093, 1012, 915, 818, 758, 697, 657, 508 cm−1; HRMS (+ ESI): Found m/z
550.2252 [M + H] +, C26H37ClN5O4S required 550.2249.
N-(3-Guanidinopropyl)-2-(5-(naphthalen-2-yl)-2-(octylsulfonamido)phenyl)-2-oxoacetamide
hydrochloride (22d)